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Aljubran F, Schumacher K, Graham A, Gunewardena S, Marsh C, Lydic M, Holoch K, Nothnick WB. Uterine cyclin A2-deficient mice as a model of female early pregnancy loss. J Clin Invest 2024; 134:e163796. [PMID: 39264721 PMCID: PMC11563677 DOI: 10.1172/jci163796] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2023] [Accepted: 09/06/2024] [Indexed: 09/14/2024] Open
Abstract
Proper action of the female sex steroids 17β-estradiol (E2) and progesterone (P4) on the endometrium is essential for fertility. Beyond its role in regulating the cell cycle, cyclin A2 (CCNA2) also mediates E2 and P4 signaling in vitro, but a potential role in modulating steroid action for proper endometrial tissue development and function is unknown. To fill this gap in our knowledge, we examined human endometrial tissue from fertile and infertile cisgender women for CCNA2 expression and correlated this with pregnancy outcome. Functional assessment of CCNA2 was validated in vivo using a conditional Ccna2 uterine-deficient mouse model, while in vitro function was assessed using human cell culture models. We found that CCNA2 expression was significantly reduced in endometrial tissue, specifically the stromal cells, from women undergoing in vitro fertilization who failed to achieve pregnancy. Conditional deletion of Ccna2 from mouse uterine tissue resulted in an inability to achieve pregnancy, which appeared to be due to alterations in the process of decidualization, which was confirmed using in vitro models. From these studies, we conclude that CCNA2 expression during the proliferative/regenerative stage of the menstrual cycle allows for proper steroid responsiveness, decidualization, and pregnancy. When CCNA2 expression levels are insufficient, there is impaired endometrial responsiveness, aberrant decidualization, and loss of pregnancy.
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Affiliation(s)
| | | | | | | | - Courtney Marsh
- Department of Cell Biology and Physiology
- Department of Obstetrics and Gynecology
- Center for Reproductive Sciences
| | - Michael Lydic
- Department of Obstetrics and Gynecology
- Center for Reproductive Sciences
| | | | - Warren B. Nothnick
- Department of Cell Biology and Physiology
- Department of Obstetrics and Gynecology
- Center for Reproductive Sciences
- Department of Cancer Biology
- Institute for Reproductive and Developmental Sciences, University of Kansas Medical Center, Kansas City, Kansas, USA
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Schalich KM, Koganti PP, Castillo JM, Reiff OM, Cheong SH, Selvaraj V. The uterine secretory cycle: recurring physiology of endometrial outputs that setup the uterine luminal microenvironment. Physiol Genomics 2024; 56:74-97. [PMID: 37694291 DOI: 10.1152/physiolgenomics.00035.2023] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2023] [Revised: 09/08/2023] [Accepted: 09/08/2023] [Indexed: 09/12/2023] Open
Abstract
Conserved in female reproduction across all mammalian species is the estrous cycle and its regulation by the hypothalamic-pituitary-gonadal (HPG) axis, a collective of intersected hormonal events that are crucial for ensuring uterine fertility. Nonetheless, knowledge of the direct mediators that synchronously shape the uterine microenvironment for successive yet distinct events, such as the transit of sperm and support for progressive stages of preimplantation embryo development, remain principally deficient. Toward understanding the timed endometrial outputs that permit luminal events as directed by the estrous cycle, we used Bovidae as a model system to uniquely surface sample and study temporal shifts to in vivo endometrial transcripts that encode for proteins destined to be secreted. The results revealed the full quantitative profile of endometrial components that shape the uterine luminal microenvironment at distinct phases of the estrous cycle (estrus, metestrus, diestrus, and proestrus). In interpreting this comprehensive log of stage-specific endometrial secretions, we define the "uterine secretory cycle" and extract a predictive understanding of recurring physiological actions regulated within the uterine lumen in anticipation of sperm and preimplantation embryonic stages. This repetitive microenvironmental preparedness to sequentially provide operative support was a stable intrinsic framework, with only limited responses to sperm or embryos if encountered in the lumen within the cyclic time period. In uncovering the secretory cycle and unraveling realistic biological processes, we present novel foundational knowledge of terminal effectors controlled by the HPG axis to direct a recurring sequence of vital functions within the uterine lumen.NEW & NOTEWORTHY This study unravels the recurring sequence of changes within the uterus that supports vital functions (sperm transit and development of preimplantation embryonic stages) during the reproductive cycle in female Ruminantia. These data present new systems knowledge in uterine reproductive physiology crucial for setting up in vitro biomimicry and artificial environments for assisted reproduction technologies for a range of mammalian species.
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Affiliation(s)
- Kasey M Schalich
- Department of Animal Science, College of Agriculture and Life Sciences, Cornell University, Ithaca, New York, United States
| | - Prasanthi P Koganti
- Department of Animal Science, College of Agriculture and Life Sciences, Cornell University, Ithaca, New York, United States
| | - Juan M Castillo
- Department of Clinical Sciences, Veterinary College, Cornell University, Ithaca, New York, United States
| | - Olivia M Reiff
- Department of Animal Science, College of Agriculture and Life Sciences, Cornell University, Ithaca, New York, United States
| | - Soon Hon Cheong
- Department of Clinical Sciences, Veterinary College, Cornell University, Ithaca, New York, United States
| | - Vimal Selvaraj
- Department of Animal Science, College of Agriculture and Life Sciences, Cornell University, Ithaca, New York, United States
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Moon S, Hwang S, Kim B, Lee S, Kim H, Lee G, Hong K, Song H, Choi Y. Hippo Signaling in the Endometrium. Int J Mol Sci 2022; 23:ijms23073852. [PMID: 35409214 PMCID: PMC8998929 DOI: 10.3390/ijms23073852] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2022] [Revised: 03/22/2022] [Accepted: 03/22/2022] [Indexed: 01/27/2023] Open
Abstract
The uterus is essential for embryo implantation and fetal development. During the estrous cycle, the uterine endometrium undergoes dramatic remodeling to prepare for pregnancy. Angiogenesis is an essential biological process in endometrial remodeling. Steroid hormones regulate the series of events that occur during such remodeling. Researchers have investigated the potential factors, including angiofactors, involved in endometrial remodeling. The Hippo signaling pathway discovered in the 21st century, plays important roles in various cellular functions, including cell proliferation and cell death. However, its role in the endometrium remains unclear. In this review, we describe the female reproductive system and its association with the Hippo signaling pathway, as well as novel Hippo pathway genes and potential target genes.
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Abnormal expression of connective tissue growth factor and its correlation with fibrogenesis in adenomyosis. Reprod Biomed Online 2020; 42:651-660. [PMID: 33431336 DOI: 10.1016/j.rbmo.2020.11.002] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2020] [Revised: 10/28/2020] [Accepted: 11/09/2020] [Indexed: 11/20/2022]
Abstract
RESEARCH QUESTION Does connective tissue growth factor (CTGF) expression relate to adenomyotic fibrosis and determine the correlation between fibrosis with adenomyosis-associated dysmenorrhoea? DESIGN Protein and mRNA expression of CTGF was detected by Western blots and real-time quantitative polymerase chain reaction in the endometrium of the control group and the eutopic and ectopic endometrium of the adenomyosis group. Collagen fibres and type I collagen in the myometrium were detected by immunohistochemistry and Masson's trichrome staining, and the correlations of CTGF protein and mRNA levels with the degree of fibrosis were analysed. Furthermore, the relationship between the severity of dysmenorrhoea and the degree of fibrosis was determined, and the correlation between uterus size and the degree of fibrosis was also analysed. RESULTS Levels of CTGF mRNA and protein were significantly higher in patients with adenomyosis than in controls, and CTGF mRNA and protein expression in adenomyosis was positively correlated with fibrosis severity (r = 0.57, P < 0.001 and r = 0.39, P = 0.012), which correlated positively with dysmenorrhoea and uterus size (r = 0.42 and r = 0.6, P < 0.002). CONCLUSIONS Increased CTGF may contribute to the occurrence and fibrogenic progression of adenomyosis and may play an important role in dysmenorrhoea. The present study may provide ideas for treating adenomyosis-associated dysmenorrhoea.
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Akizawa H, Yanagawa Y, Nagano M, Bai H, Takahashi M, Kawahara M. Significance of CCN2 expression in bovine preimplantation development. Anim Sci J 2018; 90:49-54. [PMID: 30358017 DOI: 10.1111/asj.13126] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2018] [Revised: 08/10/2018] [Accepted: 09/27/2018] [Indexed: 11/30/2022]
Abstract
In mammalian preimplantation development, the first cell lineage segregation occurs during the blastocyst stage, when the inner cell mass and trophectoderm (TE) differentiate. Species-specific analyses are essential to elucidate the molecular mechanisms that underlie this process, since they differ between various species. We previously showed that the reciprocal regulation of CCN2 and TEAD4 is required for proper TE differentiation in bovine blastocysts; however, the function of CCN2 during early embryogenesis has remained otherwise elusive. The present study assessed the spatiotemporal expression dynamics of CCN2 in bovine embryos, and evaluated how changes to CCN2 expression (using a CCN2 knockdown (KD) blastocyst model) regulate the expression of pluripotency-related genes such as OCT4 and NANOG. The conducted quantitative PCR analysis revealed that CCN2 mRNA was expressed in bovine oocytes (at the metaphase stage of their second meiosis) and embryos. Similarly, immunostaining detected both cytoplasmic and nuclear CCN2 at all analyzed oocyte and embryonic stages. Finally, both OCT4 and NANOG expression levels were shown to be significantly reduced in CCN2 KD blastocysts. Together, these results demonstrate that bovine CCN2 exhibits unique expression patterns during preimplantation development, and is required for the proper expression of key regulatory genes in bovine blastocysts.
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Affiliation(s)
- Hiroki Akizawa
- Laboratory of Animal Breeding and Reproduction, Research Faculty of Agriculture, Hokkaido University, Sapporo, Japan
| | - Yojiro Yanagawa
- Laboratory of Theriogenology, Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan
| | - Masashi Nagano
- Laboratory of Theriogenology, Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan
| | - Hanako Bai
- Laboratory of Animal Breeding and Reproduction, Research Faculty of Agriculture, Hokkaido University, Sapporo, Japan
| | - Masashi Takahashi
- Laboratory of Animal Breeding and Reproduction, Research Faculty of Agriculture, Hokkaido University, Sapporo, Japan
| | - Manabu Kawahara
- Laboratory of Animal Breeding and Reproduction, Research Faculty of Agriculture, Hokkaido University, Sapporo, Japan
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Akizawa H, Kobayashi K, Bai H, Takahashi M, Kagawa S, Nagatomo H, Kawahara M. Reciprocal regulation of TEAD4 and CCN2 for the trophectoderm development of the bovine blastocyst. Reproduction 2018; 155:563-571. [DOI: 10.1530/rep-18-0043] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2018] [Accepted: 04/16/2018] [Indexed: 12/12/2022]
Abstract
The first segregation at the blastocyst stage is the symmetry-breaking event to characterize two cell components; namely, inner cell mass (ICM) and trophectoderm (TE). TEA domain transcription factor 4 (TEAD4) is a well-known regulator to determine TE properties of blastomeres in rodent models. However, the roles of bovine TEAD4 in blastocyst development have been unclear. We here aimed to clarify the mechanisms underlining TE characterization by TEAD4 in bovine blastocysts. We first found that theTEAD4mRNA expression level was greater in TE than in ICM, which was further supported by TEAD4 immunofluorescent staining. Subsequently, we examined the expression patterns of TE-expressed genes;CDX2,GATA2andCCN2, in theTEAD4-knockdown (KD) blastocysts. These expression levels significantly decreased in theTEAD4KD blastocysts compared with controls. Of these downregulated genes, theCCN2expression level decreased the most. We further analyzed the expression levels of TE-expressed genes;CDX2,GATA2andTEAD4in theCCN2KD blastocysts. Strikingly, theCCN2KD blastocysts showed the downregulation ofCDX2,GATA2andTEAD4. Furthermore, the ratio of TE-to-ICM cell numbers in theCCN2KD blastocysts significantly decreased compared to controls. To our knowledge, this is the first study showing the regulation ofCCN2expression thoroughTEAD4in mammalian embryos. Not only that, this study also provides evidence that reciprocal regulation ofTEAD4andCCN2is required for TE development with appropriate gene expression in bovine blastocysts.
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Seleem AA, Sultan ARS, Said A, Shahat MM, Moustafa MA. Localization of connective tissue growth factor (CTGF) and transforming growth factor beta-2 (TGF-β2) during eye development of four species of birds. J Histotechnol 2018. [DOI: 10.1080/01478885.2018.1475861] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/16/2022]
Affiliation(s)
- Amin A. Seleem
- Biology Department, Faculty of Science and Arts, Taibah University, Allula, Kingdom of Saudi Arabia
- Zoology Department, Faculty of Science, Sohag University, Sohag, Egypt
| | | | - Ahmed Said
- Zoology Department, Faculty of Science, Al-Azhar University, Assiut, Egypt
| | - Mohamed M. Shahat
- Zoology Department, Faculty of Science, Al-Azhar University, Assiut, Egypt
| | - Mohsen A. Moustafa
- Zoology Department, Faculty of Science, Al-Azhar University, Assiut, Egypt
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8
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Latifi Z, Fattahi A, Ranjbaran A, Nejabati HR, Imakawa K. Potential roles of metalloproteinases of endometrium-derived exosomes in embryo-maternal crosstalk during implantation. J Cell Physiol 2017; 233:4530-4545. [PMID: 29115666 DOI: 10.1002/jcp.26259] [Citation(s) in RCA: 34] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2017] [Accepted: 10/09/2017] [Indexed: 12/15/2022]
Abstract
During embryo implantation, crosstalk between the endometrial epithelium and the blastocyst, especially the trophoblasts, is a prerequisite for successful implantation. During this crosstalk, various molecular and functional changes occur to promote synchrony between the embryo and the endometrium as well as the uterine cavity microenvironment. In the past few years, growing evidence has shown that endometrium-derived exosomes play pivotal roles in the embryonic-maternal crosstalk during implantation, although the exact mechanism of this crosstalk has yet to be determined. The presence of metalloproteinases has been reported in endometrium-derived exosomes, implying the importance of these enzymes in exosome-based crosstalk. Thus, in this review, we describe the potential roles of the metalloproteinases of endometrium-derived exosomes in promoting embryo attachment and implantation. This study could provide a better understanding of the potential roles of exosomal metalloproteinases in embryo implantation and pave the way for developing novel exosome-based regulatory agents to support early pregnancy.
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Affiliation(s)
- Zeinab Latifi
- Animal Resource Science Center, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Ibaraki, Japan.,Stem Cell and Regenerative Medicine Institute, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Amir Fattahi
- Department of Reproductive Biology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Ali Ranjbaran
- Women's Reproductive Health Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Hamid Reza Nejabati
- Women's Reproductive Health Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Kazuhiko Imakawa
- Animal Resource Science Center, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Ibaraki, Japan
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Kannampuzha-Francis J, Tribulo P, Hansen PJ. Actions of activin A, connective tissue growth factor, hepatocyte growth factor and teratocarcinoma-derived growth factor 1 on the development of the bovine preimplantation embryo. Reprod Fertil Dev 2017; 29:1329-1339. [DOI: 10.1071/rd16033] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2016] [Accepted: 04/20/2016] [Indexed: 12/24/2022] Open
Abstract
The reproductive tract secretes bioactive molecules collectively known as embryokines that can regulate embryonic growth and development. In the present study we tested four growth factors expressed in the endometrium for their ability to modify the development of the bovine embryo to the blastocyst stage and alter the expression of genes found to be upregulated (bone morphogenetic protein 15 (BMP15) and keratin 8, type II (KRT8)) or downregulated (NADH dehydrogenase 1 (ND1) and S100 calcium binding protein A10 (S100A10)) in embryos competent to develop to term. Zygotes were treated at Day 5 with 0.01, 0.1 or 1.0 nM growth factor. The highest concentration of activin A increased the percentage of putative zygotes that developed to the blastocyst stage. Connective tissue growth factor (CTGF) increased the number of cells in the inner cell mass (ICM), decreased the trophectoderm : ICM ratio and increased blastocyst expression of KRT8 and ND1. The lowest concentration of hepatocyte growth factor (HGF) reduced the percentage of putative zygotes becoming blastocysts. Teratocarcinoma-derived growth factor 1 increased total cell number at 0.01 nM and expression of S100A10 at 1.0 nM, but otherwise had no effects. Results confirm the prodevelopmental actions of activin A and indicate that CTGF may also function as an embryokine by regulating the number of ICM cells in the blastocyst and altering gene expression. Low concentrations of HGF were inhibitory to development.
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Abstract
Gingival overgrowth occurs mainly as a result of certain anti-seizure, immunosuppressive, or antihypertensive drug therapies. Excess gingival tissues impede oral function and are disfiguring. Effective oral hygiene is compromised in the presence of gingival overgrowth, and it is now recognized that this may have negative implications for the systemic health of affected patients. Recent studies indicate that cytokine balances are abnormal in drug-induced forms of gingival overgrowth. Data supporting molecular and cellular characteristics that distinguish different forms of gingival overgrowth are summarized, and aspects of gingival fibroblast extracellular matrix metabolism that are unique to gingival tissues and cells are reviewed. Abnormal cytokine balances derived principally from lymphocytes and macrophages, and unique aspects of gingival extracellular matrix metabolism, are elements of a working model presented to facilitate our gaining a better understanding of mechanisms and of the tissue specificity of gingival overgrowth.
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Affiliation(s)
- P C Trackman
- Boston University Goldman School of Dental Medicine, Department of Periodontology and Oral Biology, Division of Oral Biology, Boston, MA 02118, USA.
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11
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Maillo V, de Frutos C, O'Gaora P, Forde N, Burns GW, Spencer TE, Gutierrez-Adan A, Lonergan P, Rizos D. Spatial differences in gene expression in the bovine oviduct. Reproduction 2016; 152:37-46. [PMID: 27069007 DOI: 10.1530/rep-16-0074] [Citation(s) in RCA: 41] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2016] [Accepted: 04/08/2016] [Indexed: 12/19/2022]
Abstract
The aim of this study was to compare the transcriptome of the oviductal isthmus of pregnant heifers with that of cyclic heifers as well as to investigate spatial differences between the transcriptome of the isthmus and ampulla of the oviduct in pregnant heifers. After synchronizing crossbred beef heifers, those in standing oestrus (=Day 0) were randomly assigned to cyclic (non-bred, n=6) or pregnant (artificially inseminated, n=11) groups. They were slaughtered on Day 3 and both oviducts from each animal were isolated and cut in half to separate ampulla and isthmus. Each portion was flushed to confirm the presence of an oocyte/embryo and was then opened longitudinally and scraped to obtain epithelial cells which were snap-frozen. Oocytes and embryos were located in the isthmus of the oviduct ipsilateral to the corpus luteum Microarray analysis of oviductal cells revealed that proximity to the corpus luteum did not affect the transcriptome of the isthmus, irrespective of pregnancy status. However, 2287 genes were differentially expressed (P<0.01) between the ampulla and isthmus of the oviduct ipsilateral to the corpus luteum in pregnant animals. Gene ontology revealed that the main biological processes overrepresented in the isthmus were synthesis of nitrogen, lipids, nucleotides, steroids and cholesterol as well as vesicle-mediated transport, cell cycle, apoptosis, endocytosis and exocytosis, whereas cell motion, motility and migration, DNA repair, calcium ion homeostasis, carbohydrate biosynthesis, and regulation of cilium movement and beat frequency were overrepresented in the ampulla. In conclusion, large differences in gene expression were observed between the isthmus and ampulla of pregnant animals at Day 3 after oestrus.
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Affiliation(s)
- Veronica Maillo
- Departamento de Reproduccion AnimalInstituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria (INIA), Madrid, Spain
| | - Celia de Frutos
- Departamento de Reproduccion AnimalInstituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria (INIA), Madrid, Spain
| | - Peadar O'Gaora
- School of Biomolecular and Biomedical SciencesUniversity College Dublin, Belfield, Dublin 4, Ireland
| | - Niamh Forde
- Division of Reproduction and Early DevelopmentLeeds Institute of Cardiovascular and Molecular Medicine, School of Medicine, University of Leeds, Clarendon Way, Leeds, UK
| | - Gregory W Burns
- Division of Animal Sciences and Department of Obstetrics, Gynecology and Women's HealthUniversity of Missouri, Columbia, Missouri
| | - Thomas E Spencer
- Division of Animal Sciences and Department of Obstetrics, Gynecology and Women's HealthUniversity of Missouri, Columbia, Missouri
| | - Alfonso Gutierrez-Adan
- Departamento de Reproduccion AnimalInstituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria (INIA), Madrid, Spain
| | - Patrick Lonergan
- School of Agriculture and Food ScienceUniversity College Dublin, Belfield, Dublin 4, Ireland
| | - Dimitrios Rizos
- Departamento de Reproduccion AnimalInstituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria (INIA), Madrid, Spain
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12
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Zhao Y, Li Q, Katzenellenbogen BS, Lau LF, Taylor RN, Bagchi IC, Bagchi MK. Estrogen-induced CCN1 is critical for establishment of endometriosis-like lesions in mice. Mol Endocrinol 2015; 28:1934-47. [PMID: 25321413 DOI: 10.1210/me.2014-1080] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
Endometriosis is a prevalent gynecological disorder in which endometrial tissue proliferates in extrauterine sites, such as the peritoneal cavity, eventually giving rise to painful, invasive lesions. Dysregulated estradiol (E) signaling has been implicated in this condition. However, the molecular mechanisms that operate downstream of E in the ectopic endometrial tissue are unknown. To investigate these mechanisms, we used a mouse model of endometriosis. Endometrial tissue from donor mice was surgically transplanted on the peritoneal surface of immunocompetent syngeneic recipient mice, leading to the establishment of cystic endometriosis-like lesions. Our studies revealed that treatment with E led to an approximately 3-fold increase in the lesion size within a week of transplantation. E also caused a concomitant stimulation in the expression of connective tissue growth factor/Cyr61/Nov (CCN1), a secreted cysteine-rich matricellular protein, in the lesions. Interestingly, CCN1 is highly expressed in human ectopic endometriotic lesions. To address its role in endometriosis, endometrial tissue from Ccn1-null donor mice was transplanted in wild-type recipient mice. The resulting ectopic lesions were reduced up to 75% in size compared with wild-type lesions due to diminished cell proliferation and cyst formation. Notably, loss of CCN1 also disrupted the development of vascular networks in the ectopic lesions and reduced the expression of several angiogenic factors, such as vascular endothelial growth factor-A and vascular endothelial growth factor-C. These results suggest that CCN1, acting downstream of E, critically controls cell proliferation and neovascularization, which support the growth and survival of endometriotic tissue at ectopic sites. Blockade of CCN1 signaling during the early stages of lesion establishment may provide a therapeutic avenue to control endometriosis.
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Affiliation(s)
- Yuechao Zhao
- Departments of Molecular and Integrative Physiology (Y.Z., B.S.K., M.K.B.) and Comparative Biosciences (Q.L., I.C.B.), University of Illinois at Urbana-Champaign, Urbana, Illinois 61801; Department of Biochemistry and Molecular Genetics (L.F.L.), University of Illinois College of Medicine, Chicago, Illinois 60637; and Department of Obstetrics and Gynecology (R.N.T.), Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157
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13
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Remst DFG, Blaney Davidson EN, van der Kraan PM. Unravelling osteoarthritis-related synovial fibrosis: a step closer to solving joint stiffness. Rheumatology (Oxford) 2015; 54:1954-63. [PMID: 26175472 DOI: 10.1093/rheumatology/kev228] [Citation(s) in RCA: 79] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2014] [Indexed: 01/01/2023] Open
Abstract
Synovial fibrosis is often found in OA, contributing heavily to joint pain and joint stiffness, the main symptoms of OA. At this moment the underlying mechanism of OA-related synovial fibrosis is not known and there is no cure available. In this review we discuss factors that have been reported to be involved in synovial fibrosis. The aim of the study was to gain insight into how these factors contribute to the fibrotic process and to determine the best targets for therapy in synovial fibrosis. In this regard, the following factors are discussed: TGF-β, connective tissue growth factor, procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2, tissue inhibitor of metalloproteinase 1, A disintegrin and metalloproteinase domain 12, urotensin-II, prostaglandin F2α and hyaluronan.
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Affiliation(s)
- Dennis F G Remst
- Radboud University Medical Center, Experimental Rheumatology, Nijmegen, The Netherlands
| | | | - Peter M van der Kraan
- Radboud University Medical Center, Experimental Rheumatology, Nijmegen, The Netherlands
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14
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Tan JTM, McLennan SV, Williams PF, Rezaeizadeh A, Lo LWY, Bonner JG, Twigg SM. Connective tissue growth factor/CCN-2 is upregulated in epididymal and subcutaneous fat depots in a dietary-induced obesity model. Am J Physiol Endocrinol Metab 2013; 304:E1291-302. [PMID: 23571711 DOI: 10.1152/ajpendo.00654.2012] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
Connective tissue growth factor (CTGF), also known as CCN-2, is a cysteine-rich secreted protein that is involved in a range of biological processes, including regulation of cell growth and differentiation. Our previous in vitro studies have shown that CCN-2 inhibits adipocyte differentiation, although whether CCN-2 is regulated in vivo in adipogenesis is undetermined and was investigated in this study. C57BL/6 male mice were fed either standard laboratory chow (ND) or a diet high in fat (HFD; 45% fat) for 15 or 24 wk. HFD animals that gained >5 g in weight (termed HFD-fat) were insulin resistant and were compared with HFD-fed animals, which failed to gain weight (termed HFD-lean). HFD-fat mice had significantly increased CCN-2 mRNA levels in both the subcutaneous and epididymal fat pads, whereas CCN-2 mRNA was not induced in the epididymal site in HFD-lean mice. Also in HFD-fed animals, epididymal CCN-2 mRNA correlated positively with key genes involved in adipocyte differentiation, adiponectin and PPARγ (P < 0.001 and P < 0.002, respectively). Additionally, epididymal CCN-2 mRNA correlated positively with two markers of tissue turnover, PAI-1 in HFD-fat mice only and TIMP-1, but only in the HFD-lean mice. Collectively, these findings suggest that CCN-2 plays a role in adipocyte differentiation in vivo and thus in the pathogenesis of obesity linked with insulin resistance.
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Affiliation(s)
- Joanne T M Tan
- Sydney Medical School, The University of Sydney, Sydney, New South Wales, Australia; and
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Abstract
Various physiologically relevant processes are regulated by the interaction of the receptor tyrosine kinase (c-Kit) and its ligand stem cell factor (SCF), with SCF known to be the most important growth factor for mast cells (MCs). In spite of their traditional role in allergic disorders and innate immunity, MCs have lately emerged as versatile modulators of a variety of physiologic and pathologic processes. Here we show that MCs are critical for pregnancy success. Uterine MCs presented a unique phenotype, accumulated during receptivity and expanded upon pregnancy establishment. KitW-sh/W-sh mice, whose MC deficiency is based on restricted c-Kit gene expression, exhibited severely impaired implantation, which could be completely rescued by systemic or local transfer of wild-type bone marrow-derived MCs. Transferred wild-type MCs favored normal implantation, induced optimal spiral artery remodeling and promoted the expression of MC proteases, transforming growth factor-β and connective tissue growth factor. MCs contributed to trophoblast survival, placentation and fetal growth through secretion of the glycan-binding protein galectin-1. Our data unveil unrecognized roles for MCs at the fetomaternal interface with critical implications in reproductive medicine.
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Yang H, Choi KC, Jung EM, An BS, Hyun SH, Jeung EB. Expression and regulation of sodium/calcium exchangers, NCX and NCKX, in reproductive tissues: do they play a critical role in calcium transport for reproduction and development? ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2013; 961:109-21. [PMID: 23224874 DOI: 10.1007/978-1-4614-4756-6_10] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
Plasma membrane sodium/calcium (Na(+)/Ca(2+)) exchangers are an important component of intracellular calcium [Ca(2+)](i) homeostasis and electrical conduction. Na(+)/Ca(2+) exchangers, NCX and NCKX, play a critical role in the transport of one [Ca(2+)](i) and potassium ion across the cell membrane in exchange for four extracellular sodium ions [Na(+)](e). Mammalian plasma membrane Na(+)/Ca(2+) exchange proteins are divided into two families: one in which Ca(2+) flux is dependent only on sodium (NCX1-3) and another in which Ca(2+) flux is also dependent on potassium (NCKX1-4). Both molecules are capable of forward- and reverse-mode exchange. In cells and tissues, Na(+)/Ca(2+) (and K(+)) gradients localize to the cell membrane; thus, the exchangers transport ions across a membrane potential. Uterine NCKX3 has been shown to be involved in the regulation of endometrial receptivity by [Ca(2+)](i). In the uterus and placenta, NCKX3 expression is regulated by the sex steroid hormone estrogen (E2) and hypoxia stress, respectively. In this chapter, we described the expression and regulation of these proteins for reproductive functions in various tissues including uterus, placenta, and kidney of humans and rodents. Evidence to date suggests that NCKX3 and NCX1 may be regulated in a tissue-specific manner. In addition, we focused on the molecular mechanism involved in the regulation of NCKX3 and NCX1 in mammals, based upon our recent results and those of others.
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Affiliation(s)
- Hyun Yang
- College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea
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17
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Remst DFG, Blaney Davidson EN, Vitters EL, Blom AB, Stoop R, Snabel JM, Bank RA, van den Berg WB, van der Kraan PM. Osteoarthritis-related fibrosis is associated with both elevated pyridinoline cross-link formation and lysyl hydroxylase 2b expression. Osteoarthritis Cartilage 2013; 21:157-64. [PMID: 23069856 DOI: 10.1016/j.joca.2012.10.002] [Citation(s) in RCA: 53] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/29/2012] [Revised: 09/05/2012] [Accepted: 10/04/2012] [Indexed: 02/02/2023]
Abstract
OBJECTIVE Fibrosis is a major contributor to joint stiffness in osteoarthritis (OA). We investigated several factors associated with the persistence of transforming growth factor beta (TGF-β)-induced fibrosis and whether these factors also play a role in OA-related fibrosis. DESIGN Mice were injected intra-articularly (i.a.) with an adenovirus encoding either TGF-β or connective tissue growth factor (CTGF). In addition, we induced OA by i.a. injection of bacterial collagenase into the right knee joint of C57BL/6 mice. mRNA was isolated from the synovium for Q-PCR analysis of the gene expression of various extracellular matrix (ECM) components, ECM degraders, growth factors and collagen cross-linking-related enzymes. Sections of murine knee joints injected with Ad-TGF-β or Ad-CTGF or from experimental OA were stained for lysyl hydroxylase 2 (LH2). The number of pyridinoline cross-links per triple helix collagen in synovium biopsies was determined with high-performance liquid chromatography (HPLC). RESULTS Expression of collagen alpha-1(I) chain precursor (Col1a1), tissue inhibitor of metalloproteinases 1 (TIMP1) and especially procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2b (Plod2b) were highly upregulated by TGF-β but not by CTGF. Elevated expression of Plod2b mRNA was associated with high lysyl hydroxylase 2 (LH2) protein staining after TGF-β overexpression and in experimental OA. Furthermore, in experimental OA the number of hydroxypyridinoline cross-links was significant increased compared to control knee joints. CONCLUSIONS Our data show that elevated LH2b expression is associated with the persistent nature of TGF-β-induced fibrosis. Also in experimental OA, LH2b expression as well as the number of hydroxypyridinoline cross-link were significantly upregulated. We propose that LH2b, and the subsequent increase in pyridinoline cross-links, is responsible for the persistent fibrosis in experimental OA.
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Affiliation(s)
- D F G Remst
- Rheumatology Research and Advanced Therapeutics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
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Robinson PM, Smith TS, Patel D, Dave M, Lewin AS, Pi L, Scott EW, Tuli SS, Schultz GS. Proteolytic processing of connective tissue growth factor in normal ocular tissues and during corneal wound healing. Invest Ophthalmol Vis Sci 2012; 53:8093-103. [PMID: 23139278 DOI: 10.1167/iovs.12-10419] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023] Open
Abstract
PURPOSE Connective tissue growth factor (CTGF) is a fibrogenic cytokine that is up-regulated by TGF-β and mediates most key fibrotic actions of TGF-β, including stimulation of synthesis of extracellular matrix and differentiation of fibroblasts into myofibroblasts. This study addresses the role of proteolytic processing of CTGF in human corneal fibroblasts (HCF) stimulated with TGF-β, normal ocular tissues and wounded corneas. METHODS Proteolytic processing of CTGF in HCF cultures, normal animal eyes, and excimer laser wounded rat corneas were examined by Western blot. The identity of a 21-kDa band was determined by tandem mass spectrometry, and possible alternative splice variants of CTGF were assessed by 5' Rapid Amplification of cDNA Ends (RACE). RESULTS HCF stimulated by TGF-β contained full length 38-kDa CTGF and fragments of 25, 21, 18, and 13 kDa, while conditioned medium contained full length 38- and a 21-kDa fragment of CTGF that contained the middle "hinge" region of CTGF. Fragmentation of recombinant CTGF incubated in HCF extracts was blocked by the aspartate protease inhibitor, pepstatin. Normal mouse, rat, and rabbit whole eyes and rabbit ocular tissues contained abundant amounts of C-terminal 25- and 21-kDa fragments and trace amounts of 38-kDa CTGF, although no alternative transcripts were detected. All forms of CTGF (38, 25, and 21 kDa) were detected during healing of excimer ablated rat corneas, peaking on day 11. CONCLUSIONS Proteolytic processing of 38-kDa CTGF occurs during corneal wound healing, which may have important implications in regulation of corneal scar formation.
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Affiliation(s)
- Paulette M Robinson
- Department of Obstetrics and Gynecology, Institute for Wound Research, University of Florida, Gainesville, Florida 32610-0294, USA
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19
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Myers RB, Rwayitare K, Richey L, Lem J, Castellot JJ. CCN5 Expression in mammals. III. Early embryonic mouse development. J Cell Commun Signal 2012; 6:217-23. [PMID: 22926930 DOI: 10.1007/s12079-012-0176-0] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2012] [Accepted: 08/10/2012] [Indexed: 01/24/2023] Open
Abstract
CCN proteins play crucial roles in development, angiogenesis, cell motility, matrix turnover, proliferation, and other fundamental cell processes. Early embryonic lethality in CCN5 knockout and over-expressing mice led us to characterize CCN5 distribution in early development. Previous papers in this series showed that CCN5 is expressed widely in mice from E9.5 to adult; however, its distribution before E9.5 has not been studied. To fill this gap in our knowledge of CCN5 expression in mammals, RT-PCR was performed on preimplantation murine embryos: 1 cell, 2 cell, 4 cell, early morula, late morula, and blastocyst. CCN5 mRNA was not detected in 1, 2, or 4 cell embryos. It was first detected at the early morula stage and persisted to the preimplantation blastocyst stage. Immunohistochemical staining showed widespread CCN5 expression in post-implantation blastocysts (E4.5), E5.5, E6.5, and E7.5 stage embryos. Consistent with our previous study on E9.5 embryos, this expression was not limited to a particular germ layer or cell type. The widespread distribution of CCN5 in early embryos suggests a crucial role in development.
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Affiliation(s)
- Ronald B Myers
- Program in Cell, Molecular, and Developmental Biology, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, MA, USA
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20
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Fujii M, Nakanishi H, Toyoda T, Tanaka I, Kondo Y, Osada H, Sekido Y. Convergent signaling in the regulation of connective tissue growth factor in malignant mesothelioma: TGFβ signaling and defects in the Hippo signaling cascade. Cell Cycle 2012; 11:3373-9. [PMID: 22918238 PMCID: PMC3466546 DOI: 10.4161/cc.21397] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
Malignant mesothelioma (MM) is a neoplasm that arises from serosal surfaces of the pleural, peritoneal and pericardial cavities with worldwide incidence, much of which is caused by asbestos exposure. Patients suffer from pain and dyspnea due to direct invasion of the chest wall, lungs and vertebral or intercostal nerves by masses of thick fibrotic tumors. Although there has been recent progress in the clinical treatment, current therapeutic approaches do not provide satisfactory results. Therefore, development of a molecularly targeted therapy for MM is urgently required. Our recent studies suggest that normal mesothelial and MM cell growth is promoted by TGFβ, and that TGFβ signaling together with intrinsic disturbances in neurofibromatosis type 2 (NF2) and Hippo signaling cascades in MM cells converges upon further expression of connective tissue growth factor (CTGF). The formation of a YAP-TEAD4-Smad3-p300 complex on the specific CTGF promoter site with an adjacent TEAD and Smad binding motif is a critical and synergistic event caused by the dysregulation of these two distinct cascades. Furthermore, we demonstrated the functional importance of CTGF through the mouse studies and human histological analyses, which may elucidate the clinical features of MM with severe fibrosis in the thoracic cavity.
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Affiliation(s)
- Makiko Fujii
- Division of Molecular Oncology, Aichi Cancer Center Research Institute, Nagoya, Japan.
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21
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Hall-Glenn F, Lyons KM. Roles for CCN2 in normal physiological processes. Cell Mol Life Sci 2011; 68:3209-17. [PMID: 21858450 PMCID: PMC3670951 DOI: 10.1007/s00018-011-0782-7] [Citation(s) in RCA: 99] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2011] [Revised: 07/19/2011] [Accepted: 07/19/2011] [Indexed: 12/13/2022]
Abstract
CCN2, also known as connective tissue growth factor, is a member of the CCN (CCN1-6) family of modular matricellular proteins. Analysis of CCN2 function in vivo has focused primarily on its key role as a mediator of excess ECM synthesis in multiple fibrotic diseases. However, CCN2 and related family members are widely expressed during development. Recent studies using new genetic models are revealing that CCN2 has essential roles in the development of many tissues. This review focuses on current and emerging data on CCN2 and its functions in chondrogenesis and angiogenesis, and on new studies showing that CCN2 has essential functions during embryonic and postnatal development in a number of epithelial tissues.
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Affiliation(s)
- Faith Hall-Glenn
- Department of Molecular, Cell and Developmental Biology, UCLA/Orthopaedic Hospital Department of Orthopaedic Surgery, University of California, 510 Orthopaedic Hospital Research Center, 615 Charles E Young Drive South, Los Angeles, CA 90095 USA
| | - Karen M. Lyons
- Department of Molecular, Cell and Developmental Biology, UCLA/Orthopaedic Hospital Department of Orthopaedic Surgery, University of California, 510 Orthopaedic Hospital Research Center, 615 Charles E Young Drive South, Los Angeles, CA 90095 USA
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22
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Nagashima T, Kim J, Li Q, Lydon JP, DeMayo FJ, Lyons KM, Matzuk MM. Connective tissue growth factor is required for normal follicle development and ovulation. Mol Endocrinol 2011; 25:1740-59. [PMID: 21868453 DOI: 10.1210/me.2011-1045] [Citation(s) in RCA: 72] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Connective tissue growth factor (CTGF) is a cysteine-rich protein the synthesis and secretion of which are hypothesized to be selectively regulated by activins and other members of the TGF-β superfamily. To investigate the in vivo roles of CTGF in female reproduction, we generated Ctgf ovarian and uterine conditional knockout (cKO) mice. Ctgf cKO mice exhibit severe subfertility and multiple reproductive defects including disrupted follicle development, decreased ovulation rates, increased numbers of corpus luteum, and smaller but functionally normal uterine horns. Steroidogenesis is disrupted in the Ctgf cKO mice, leading to increased levels of serum progesterone. We show that disrupted follicle development is accompanied by a significant increase in granulosa cell apoptosis. Moreover, despite normal cumulus expansion, Ctgf cKO mice exhibit a significant decrease in oocytes ovulated, likely due to impaired ovulatory process. During analyses of mRNA expression, we discovered that Ctgf cKO granulosa cells show gene expression changes similar to our previously reported granulosa cell-specific knockouts of activin and Smad4, the common TGF-β family intracellular signaling protein. We also discovered a significant down-regulation of Adamts1, a progesterone-regulated gene that is critical for the remodeling of extracellular matrix surrounding granulosa cells of preovulatory follicles. These findings demonstrate that CTGF is a downstream mediator in TGF-β and progesterone signaling cascades and is necessary for normal follicle development and ovulation.
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Affiliation(s)
- Takashi Nagashima
- Department of Pathology & Immunology, Baylor College of Medicine, Houston, Texas 77030, USA
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23
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Lemons AR, Naz RK. Contraceptive vaccines targeting factors involved in establishment of pregnancy. Am J Reprod Immunol 2011; 66:13-25. [PMID: 21481058 DOI: 10.1111/j.1600-0897.2011.01001.x] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
Current methods of contraception lack specificity and are accompanied with serious side effects. A more specific method of contraception is needed. Contraceptive vaccines can provide most, if not all, the desired characteristics of an ideal contraceptive. This article reviews several factors involved in the establishment of pregnancy, focusing on those that are essential for successful implantation. Factors that are both essential and pregnancy-specific can provide potential targets for contraception. Using database search, 76 factors (cytokines/chemokines/growth factors/others) were identified that are involved in various steps of the establishment of pregnancy. Among these factors, three, namely chorionic gonadotropin (CG), leukemia inhibitory factor (LIF), and pre-implantation factor (PIF), are found to be unique and exciting molecules. Human CG is a well-known pregnancy-specific protein that has undergone phase I and phase II clinical trials, in women, as a contraceptive vaccine with encouraging results. LIF and PIF are pregnancy-specific and essential for successful implantation. These molecules are intriguing and may provide viable targets for immunocontraception. A multiepitope vaccine combining factors/antigens involved in various steps of the fertilization cascade and pregnancy establishment may provide a highly immunogenic and efficacious modality for contraception in humans.
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Affiliation(s)
- Angela R Lemons
- Reproductive Immunology and Molecular Biology Laboratories, Department of Obstetrics and Gynecology, School of Medicine, West Virginia University, 1 Medical Center Drive, Morgantown, WV 26506-9186, USA
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24
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Yang H, Kim TH, Lee HH, Choi KC, Jeung EB. Distinct expression of the calcium exchangers, NCKX3 and NCX1, and their regulation by steroid in the human endometrium during the menstrual cycle. Reprod Sci 2011; 18:577-85. [PMID: 21321244 DOI: 10.1177/1933719110396229] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Plasma membrane sodium/calcium exchangers are an important component of intracellular calcium homeostasis and electrical conduction. The potassium-dependent sodium/calcium exchangers NCKX3 (gene SLC24A3) and NCX1 (gene SLC8A1) play a critical role in the transport of intracellular calcium across the cell membrane in exchange for extracellular sodium ions. NCKX3 and NCX1 transcripts are most abundant in the brain and smooth muscle, but many other tissues, particularly the uterus, aorta, and intestine, also express this gene at lower levels. However, the expression patterns and physiological roles of NCKX3 and NCX1 in the human endometrium during the menstrual cycle are unknown. Thus, we examined the endometrial expression of NCKX3 and NCX1 messenger RNA (mRNA) and protein throughout the different phases of the menstrual cycle. Endometrial expression of NCKX3 mRNA and protein was increased 1.5- to 2.5-fold during the early-proliferative, mid-proliferative, and early-secretory phases compared with the other phases; however, no significant alteration in NCX1 expression level was observed. The effects of the sex-steroid hormones, 17β-estradiol (E2) and progesterone (P4), on the expression of NCKX3 and NCX1 in Ishikawa cells was also investigated. NCKX3 expression was significantly increased by E2 (10(-8) mol/L). However, the expression of NCX1 was not affected by E2 and P4. Subsequent immunohistochemical analysis revealed that the uterine NCKX3 and NCX1 proteins were abundantly localized in the cytoplasm of luminal and glandular epithelial cells throughout the menstrual cycle. Taken together, these results indicate that NCKX3 is abundantly expressed within the human endometrium at the transcriptional and translational levels, and its level appears to be regulated by a steroid hormone, in particular, E2 during the human menstrual cycle.
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Affiliation(s)
- Hyun Yang
- Laboratory of Veterinary Biochemistry and Molecular Biology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea
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25
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Waddell JM, Evans J, Jabbour HN, Denison FC. CTGF expression is up-regulated by PROK1 in early pregnancy and influences HTR-8/Svneo cell adhesion and network formation. Hum Reprod 2010; 26:67-75. [PMID: 21098624 PMCID: PMC3005999 DOI: 10.1093/humrep/deq294] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
BACKGROUND Prokineticin-1 (PROK1) and connective tissue growth factor (CTGF) are expressed in human endometrium and first-trimester decidua and have individually been proposed to have roles in implantation and placentation. We have recently demonstrated that CTGF may be a target gene for PROK1 in gene array analysis of a prokineticin receptor-1 stably transfected Ishikawa endometrial epithelial cell line (PROKR1-Ishikawa). The first aim of the study was to determine the effect of PROK1 on CTGF expression in PROKR1-Ishikawa cells and first-trimester decidua samples. Secondly, the effect of CTGF on trophoblast-derived HTR-8/SVneo cell adhesion and network formation was investigated. METHODS AND RESULTS Real-time qPCR showed that CTGF expression is elevated in first-trimester decidua compared with non-pregnant endometrium. In decidua, CTGF co-localized with PROKR1 to the glandular epithelium and a subset of stromal cells. PROK1 increased CTGF mRNA and protein expression in PROKR1-Ishikawa cells and first-trimester human decidua (8–12 weeks gestation). Knock down of endogenous PROK1 using micro RNA constructs targeted at PROK1, resulted in decreased expression of CTGF mRNA and protein in decidua. Inhibitors of specific cell signalling molecules demonstrated that PROK1 regulates CTGF expression via the Gq, phospholipase C (PLC), cSrc, epidermal growth factor receptor (EGFR), mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) kinase pathway activation. Treatment of trophoblast-derived HTR-8/Svneo cells with 1 µg/ml CTGF significantly increased adhesion to collagen IV, and differentiation of the cells into tube-like structures in matrigel. CONCLUSIONS CTGF expression in early pregnancy decidua is regulated by PROK1, via activation of the Gq, PLC, cSrc, EGFR, MAPK/ERK kinase pathway. CTGF in turn may contribute to the regulation of trophoblast conversion of maternal spiral arteries.
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Affiliation(s)
- Jennifer M Waddell
- Medical Research Council, Human Reproductive Sciences Unit, The Queen's Medical Research Institute, Edinburgh, UK
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Forde N, Spencer TE, Bazer FW, Song G, Roche JF, Lonergan P. Effect of pregnancy and progesterone concentration on expression of genes encoding for transporters or secreted proteins in the bovine endometrium. Physiol Genomics 2010; 41:53-62. [DOI: 10.1152/physiolgenomics.00162.2009] [Citation(s) in RCA: 75] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022] Open
Abstract
The objective of this study was to determine the temporal and spatial expression patterns of genes encoding transporters, as well as selected secreted proteins that may be regulated by progesterone (P4) and/or the presence of the conceptus in the bovine endometrium. Estrus-synchronized beef heifers were randomly assigned to either: 1) pregnant, high P4; 2) pregnant, normal P4; 3) cyclic, high P4; or 4) cyclic, normal P4. Uteri were collected on days 5, 7, 13, and 16 of the estrous cycle or pregnancy. Localization of mRNAs for ANPEP, CTGF, LPL, LTF, and SLC5A1 in the uteri was determined by radioactive in situ hybridization, and expression quantified in the endometria by quantitative real-time PCR. ANPEP localized to luminal (LE) and superficial glandular (sGE) epithelia of all heifers on days 5 and 7 only. SLC5A1 mRNA was detected in the LE and sGE on days 13 and 16 in all heifers, and expression increased on day 16 in pregnant groups. CTGF localized weakly to the LE and GE on days 5 and 7 but increased on days 13 and 16 with an increase ( P < 0.05) in CTGF expression in high P4 ( day 7) and pregnant heifers ( day 16). Both LPL and LTF localized to the GE only on days 5 and 7. In conclusion we have characterized the temporal expression pattern of these genes and modulation of their transcript abundance by P4 ( CTGF, LPL) and/or the conceptus ( CTGF, SLC5A1) likely modifies the uterine microenvironment, enhancing histotroph composition and contributing to advanced conceptus elongation.
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Affiliation(s)
- N. Forde
- School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Belfield, Dublin, Ireland; and
| | - T. E. Spencer
- Centre for Animal Biotechnology and Genomics, Department of Animal Science, Texas A&M University, College Station, Texas
| | - F. W. Bazer
- Centre for Animal Biotechnology and Genomics, Department of Animal Science, Texas A&M University, College Station, Texas
| | - G. Song
- Centre for Animal Biotechnology and Genomics, Department of Animal Science, Texas A&M University, College Station, Texas
| | - J. F. Roche
- School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Belfield, Dublin, Ireland; and
| | - P. Lonergan
- School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Belfield, Dublin, Ireland; and
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Liu J, Xu W, Sun T, Wang F, Puscheck E, Brigstock D, Wang QT, Davis R, Rappolee DA. Hyperosmolar stress induces global mRNA responses in placental trophoblast stem cells that emulate early post-implantation differentiation. Placenta 2008; 30:66-73. [PMID: 19036436 DOI: 10.1016/j.placenta.2008.10.009] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/09/2008] [Revised: 10/10/2008] [Accepted: 10/13/2008] [Indexed: 01/13/2023]
Abstract
Hyperosmolar stress acts in two ways on the implanting embryo and its major constituent, placental trophoblast stem cells (TSC). Stress causes homeostasis that slows development with lesser cell accumulation, increased cell cycle arrest, and apoptosis. Stress may also cause placental differentiation at implantation. To test for the homeostatic and differentiation-inducing consequences of stress, TSC were exposed to hyperosmolar stress for 24 h and tested using whole mouse genome arrays and Real-time quantitative (Q)PCR. At 0.5 h, all 31 highly changing mRNA (>1.5-fold compared with unstressed TSC) decreased, but by 24 h 158/288 genes were upregulated. Many genes upregulated at 24 h were near baseline levels in unstressed TSC, suggesting new transcription. Thus few genes change during the early stress response, but by 24 h TSC have adapted to start new transcription with large gene sets. Types of genes upregulated at 24 h included homeostatic genes regulating growth and DNA damage induced (GADD45beta/gamma), activator protein (AP)-1 (junB/junC/ATF3/4), heat shock proteins (HSP22/68), and cyclin-dependent kinase inhibitor [CDKI; p15, p21]. But, stress also induced transcription factors that mediate TSC differentiation to trophoblast giant cells (TGC) (Stra13, HES1, GATA-binding2), placental hormones [proliferin, placental lactogen (PL)1, prolactin-like protein (PLP)M], and extracellular matrix genes (CCN1/2). Transcription factors for later placental cell lineages, spongiotrophoblast (MASH2, TPBPalpha) and syncytiotrophoblast (GCM1, TEF5) and placental hormones (PLPA, PLII) were not induced by 24 h stress. Thus stress induced the temporal and spatial placental differentiation normal after implantation. Although differentiation was induced, markers of TSC stemness such as inhibitor of differentiation (ID)2 remained at 100% of levels of unstressed TSC, suggesting that retained mRNA might mediate dedifferentiation were stress to subside.
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Affiliation(s)
- J Liu
- C.S. Mott Center for Human Growth and Development, Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI 48201, USA
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Rimon E, Chen B, Shanks AL, Nelson DM, Sadovsky Y. Hypoxia in human trophoblasts stimulates the expression and secretion of connective tissue growth factor. Endocrinology 2008; 149:2952-8. [PMID: 18292194 PMCID: PMC2408813 DOI: 10.1210/en.2007-1099] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
The mechanisms underlying cellular injury when human placental trophoblasts are exposed to hypoxia are unclear. Connective tissue growth factor (CTGF) mediates cell injury and fibrosis in diverse tissues. We hypothesized that hypoxia enhances the production of CTGF in primary term human trophoblasts. Using cultured term primary human trophoblasts as well as villous biopsies from term human placentas, we showed that CTGF protein is expressed in trophoblasts. When compared with cells cultured in standard conditions (FiO2 = 20%), exposure of primary human trophoblasts to low oxygen concentration (FiO2 = 8% or <or= 1%) enhanced the expression of CTGF mRNA in a time-dependent manner, with a significant increase in CTGF levels after 16 h (2.7 +/- 0.7-fold; P < 0.01), reaching a maximum of 10.9 +/- 3.2-fold at 72 h. Whereas exposure to hypoxia had no effect on cellular CTGF protein levels, secretion of CTGF to the medium was increased after 16 h in hypoxia and remained elevated through 72 h. The increase in cellular CTGF transcript levels and CTGF protein secretion was recapitulated by exposure of trophoblasts to agents that enhance the activity of hypoxia-inducible factor (HIF)1alpha, including cobalt chloride or the proline hydroxylase inhibitor dimethyloxaloylglycine, and attenuated using the HIF1alpha inhibitor 2-methoxyestradiol. Although all TGFbeta isoforms stimulated the expression of CTGF in trophoblasts, only the expression of TGFbeta1 mRNA was enhanced by hypoxia. We conclude that hypoxia increases cellular CTGF mRNA levels and CTGF protein secretion from cultured trophoblasts, likely in a HIF1alpha-dependent manner.
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Affiliation(s)
- Eli Rimon
- Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, Missouri 63110, USA
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Shi-Wen X, Leask A, Abraham D. Regulation and function of connective tissue growth factor/CCN2 in tissue repair, scarring and fibrosis. Cytokine Growth Factor Rev 2008; 19:133-44. [PMID: 18358427 DOI: 10.1016/j.cytogfr.2008.01.002] [Citation(s) in RCA: 284] [Impact Index Per Article: 16.7] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
Affiliation(s)
- Xu Shi-Wen
- Centre for Rheumatology, Department of Medicine, Hampstead Campus, University College London, Rowland Hill Street, London NW3 2PF, UK
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30
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CCN5 Expression in mammals. II. Adult rodent tissues. J Cell Commun Signal 2007; 1:145-58. [PMID: 18481204 DOI: 10.1007/s12079-007-0013-z] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2007] [Accepted: 09/25/2007] [Indexed: 12/31/2022] Open
Abstract
CCN5 is a secreted heparin- and estrogen-regulated matricellular protein that inhibits vertebrate smooth muscle cell proliferation and motility. CCN5 is expressed throughout murine embryonic development in most organs and tissues. However, after embryonic development is complete, we hypothesized that CCN5 distribution would be largely restricted to small set of tissues, including smooth muscle cells of the arteries, uterus, airway, and digestive tract. Because CCN5 inhibits proliferation of smooth muscle cells in vitro, it might function to prevent excessive growth in vivo. In contrast, another member of the CCN family, CCN2, promotes smooth muscle cell proliferation in vitro, and thus it was expected that its expression levels would be low in uninjured normal adult tissues. Frozen sections from adult tissues and organs were analyzed immunohistochemically using anti-CCN5 and anti-CCN2 antibodies. Both proteins were detected in arteries, the uterus, bronchioles, and the digestive tract as expected, and also in many other tissues including the pancreas, spleen, liver, skeletal muscle, ovary, testis, thymus, brain, olfactory epithelium, and kidney. CCN5 and CCN2 protein was found in smooth muscle, endothelial cells, epithelial cells, skeletal muscle, cells of the nervous system, and numerous other cell types. In many cells, both CCN5 and CCN2 was present in the nucleus. Rather than having opposite patterns of localization, CCN5 and CCN2 often had similar sites of expression. The wide distribution of both CCN5 and CCN2 suggests that both proteins have additional biological functions beyond those previously identified in specific cellular and pathological models.
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Jones JA, Gray MR, Oliveira BE, Koch M, Castellot JJ. CCN5 expression in mammals : I. Embryonic and fetal tissues of mouse and human. J Cell Commun Signal 2007; 1:127-43. [PMID: 18481203 PMCID: PMC2275877 DOI: 10.1007/s12079-007-0012-0] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2007] [Accepted: 09/06/2007] [Indexed: 02/08/2023] Open
Abstract
The six proteins of the CCN family have important roles in development, angiogenesis, cell motility, proliferation, and other fundamental cell processes. To date, CCN5 distribution in developing rodents and humans has not been mapped comprehensively. CCN5 strongly inhibits adult smooth muscle cell proliferation and motility. Its anti-proliferative action predicts that CCN5 would not be present in developing tissues until the proliferation phase of tissue morphogenesis is complete. However, estrogen induces CCN5 expression in epithelial and smooth muscle cells, suggesting that CCN5 might be widely expressed in embryonic tissues exposed to high levels of estrogen. 9-16 day murine embryos and fetuses and 3-7 month human fetal tissues were analyzed by immunohistochemistry. CCN5 was detected in nearly all developing tissues. CCN5 protein expression was initially present in most tissues, and at later times in development tissue-specific expression differences were observed. CCN5 expression was particularly strong in vascular tissues, cardiac muscle, bronchioles, myotendinous junctions, and intestinal smooth muscle and epithelium. CCN5 expression was initially absent in bone cartilaginous forms but was increasingly expressed during bone endochondral ossification. Widespread CCN5 mRNA expression was detected in GD14.5 mice. Although CCN2 and CCN5 protein expression patterns in some adult pathologic conditions are inversely expressed, this expression pattern was not found in developing mouse and human tissues. The widespread expression pattern of CCN5 in most embryonic and fetal tissues suggests a diverse range of functions for CCN5.
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Affiliation(s)
- Jennifer A. Jones
- Program in Cell, Molecular, and Developmental Biology, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, MA USA
| | - Mark R. Gray
- Department of Anatomy and Cell Biology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111 USA
| | - Beatriz Enes Oliveira
- Program in Cell, Molecular, and Developmental Biology, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, MA USA
- Instituto de Ciencias Biomedicas Abel Salazar, 4099-003 Porto, Portugal
| | - Manuel Koch
- Center for Biochemistry, Department of Dermatology, Medical Faculty, University of Cologne, Cologne, Germany
| | - John J. Castellot
- Program in Cell, Molecular, and Developmental Biology, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, MA USA
- Department of Anatomy and Cell Biology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111 USA
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Isbert C, Ritz JP, Roggan A, Schuppan D, Ajubi N, Buhr HJ, Hohenberger W, Germer CT. Laser-induced thermotherapy (LITT) elevates mRNA expression of connective tissue growth factor (CTGF) associated with reduced tumor growth of liver metastases compared to hepatic resection. Lasers Surg Med 2007; 39:42-50. [PMID: 17111416 DOI: 10.1002/lsm.20448] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
Abstract
BACKGROUND AND OBJECTIVES Proliferation and synthesis of hepatocellular tissue after tissue damage are promoted by specific growth factors such as hepatic tissue growth factor (HGF) and connective growth factor (CTGF). Laser-induced thermotherapy (LITT) for the treatment of liver metastases is deemed to be a parenchyma-saving procedure compared to hepatic resection. The aim of this study was to compare the impact of LITT and hepatic resection on intrahepatic residual tumor tissue and expression levels of mRNA HGF/CTGF within liver and tumor tissue. STUDY DESIGN/MATERIALS AND METHODS Two independent adenocarcinomas (CC531) were implanted into 75 WAG rats, one in the right (untreated tumor) and one in the left liver lobe (treated tumor). The left lobe tumor was treated either by LITT or partial hepatectomy. The control tumor was submitted to in-situ hybridization of HGF and CTGF 24-96 hours and 14 days after intervention. RESULTS Volumes of the untreated tumors prior to intervention were 38+/-8 mm(3) in group I (laser), 39 +/- 7 mm(3) in group II (resection), and 42 +/- 12 mm(3) in group III (control) and did not differ significantly (P > 0.05). Fourteen days after the intervention the mean tumor+/-SEM volume of untreated tumor in group I (laser) [223 +/- 36] was smaller than in group II (resection) [1233.28 +/- 181.52; P < 0.001], and in group III (control) [978.92 +/- 87.57; P < 0.003]. Forty-eight hours after the intervention intrahepatic mRNA expression level of HGF in group II (resection) was almost twofold higher than in group I (laser) [7.2 +/- 1.0 c/mf vs. 3.9 +/- 0.4 c/mf; P<0.01]. Fourteen days after the intervention intrahepatic mRNA expression level of CTGF in group I (laser) was higher than in group II (resection) [13.89 +/- 0.77 c/mf vs. 9.09 +/- 0.78 c/mf; P < 0.003]. CONCLUSIONS LITT leads to a decrease of residual tumor growth in comparison to hepatic resection. Accelerated tumor growth after hepatic resection is associated with higher mRNA level of HGF and reduced tumor growth after LITT with higher mRNA level of CTGF. The increased CTGF-mediated regulation of ECM may cause reduced residual tumor growth after LITT.
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Affiliation(s)
- Christoph Isbert
- Department of Surgery, Klinikum Nuernberg Nord, D-90419 Nuernberg, Germany.
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Klein C, Bauersachs S, Ulbrich SE, Einspanier R, Meyer HHD, Schmidt SEM, Reichenbach HD, Vermehren M, Sinowatz F, Blum H, Wolf E. Monozygotic Twin Model Reveals Novel Embryo-Induced Transcriptome Changes of Bovine Endometrium in the Preattachment Period1. Biol Reprod 2006; 74:253-64. [PMID: 16207835 DOI: 10.1095/biolreprod.105.046748] [Citation(s) in RCA: 129] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/01/2022] Open
Abstract
Initiation and maintenance of pregnancy are critically dependent on an intact embryo-maternal communication in the preimplantation period. To get new insights into molecular mechanisms underlying this complex dialog, a holistic transcriptome study of endometrium samples from Day 18 pregnant vs. nonpregnant twin cows was performed. This genetically defined model system facilitated the identification of specific conceptus-induced changes of the endometrium transcriptome. Using a combination of subtracted cDNA libraries and cDNA array hybridization, 87 different genes were identified as upregulated in pregnant animals. Almost one half of these genes are known to be stimulated by type I interferons. For the ISG15ylation system, which is assumed to play an important role in interferon tau (IFNT) signaling, mRNAs of four potential components (IFITM1, IFITM3, HSXIAPAF1, and DTX3L) were found at increased levels in addition to ISG15 and UBE1L. These results were further substantiated by colocalization of these mRNAs in the endometrium of pregnant animals shown by in situ hybridization. A functional classification of the identified genes revealed several different biological processes involved in the preparation of the endometrium for the attachment and implantation of the embryo. Specifically, elevated transcript levels were found for genes involved in modulation of the maternal immune system, genes relevant for cell adhesion, and for remodeling of the endometrium. This first systematic study of maternal transcriptome changes in response to the presence of an embryo on Day 18 of pregnancy in cattle is an important step toward deciphering the embryo-maternal dialog using a systems biology approach.
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Affiliation(s)
- Claudia Klein
- Institute of Molecular Animal Breeding and Biotechnology, Laboratory for Functional Genome Analysis, Gene Center, Ludwig-Maximilians University, 81377 Munich, Germany
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Abstract
The CCN family currently comprises six members (CCN1-6) that regulate diverse cell functions, including mitogenesis, adhesion, apoptosis, extracellular matrix (ECM) production, growth arrest, and migration. These properties can result in a multiplicity of effects during development, differentiation, wound healing, and disease states, such as tumorigenesis and fibrosis. CCN proteins have emerged as major regulators of chondrogenesis, angiogenesis, and fibrogenesis. CCN proteins are mosaic in nature and consist of up to four structurally conserved modules, at least two of which are involved in binding to cell surfaces via molecules that include integrins, heparan sulfate proteoglycans, and low-density lipoprotein receptor-related protein. CCN proteins use integrins as signal transducing receptors to regulate context-dependent responses in individual cell types. The involvement of integrins in mediating CCN signaling allows for considerable plasticity in response because some effects are specific for certain integrin subtypes and integrin signaling is coordinated with other signaling pathways in the cell. In addition to their own biological properties, CCN proteins regulate the functions of other bioactive molecules (e.g., growth factors) via direct binding interactions. CCN molecules demonstrate complex multifaceted modes of action and regulation and have emerged as important matricellular regulators of cell function.
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Affiliation(s)
- Amy W Rachfal
- Center for Cell and Vascular Biology, Children's Research Institute, Columbus, Ohio 43205, USA
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35
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Zeng ZJ, Yang LY, Ding X, Wang W. Expressions of cysteine-rich61, connective tissue growth factor and Nov genes in hepatocellular carcinoma and their clinical significance. World J Gastroenterol 2004; 10:3414-8. [PMID: 15526358 PMCID: PMC4576220 DOI: 10.3748/wjg.v10.i23.3414] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
AIM: To investigate the expression of cysteine-rich61 (Cyr61), connective tissue growth factor (CTGF) and nephroblastoma overexpressed gene (Nov) in hepatocellular carcinoma (HCC), and to evaluate the relationship between Cyr61, CTGF and Nov genes expression with invasion and metastasis of HCC.
METHODS: Thirty-one HCC specimens were divided into small hepatocellular carcinoma (SHCC), nodular hepatocellular carcinoma (NHCC), solitary large hepatocellular carcinoma (SLHCC) according to their diameter and number of nodes. Reverse transcription polymerse chain reaction (RT-PCR) was used to detect the mRNA expression levels of Cyr61, CTGF and Nov genes in 31 resected specimens of hepatocellular carcinoma and para-cancerous normal liver tissues semi-quantitatively and the relation between their expression levels and clinical pathological parameters were compared.
RESULTS: The expressions of Cyr61 and CTGF mRNA in carcinoma tissues were significantly higher than those in para-cancerous normal liver tissues (P < 0.01). The expressions of Cyr61 and CTGF mRNA in HCC with venous invasion were higher than those in HCC without venous invasion. CTGF expression in HCC Edmondson’s grade III-IV was significantly higher than that in HCC Edmondson’s grade I-II (P = 0.022). There was no obvious correlation between Nov mRNA and clinical-pathological features. Compared to NHCC, SLHCC had better cell differentiation, easier capsule formation, less microscopic venous invasion, milder liver cirrhosis. The expressions of Cyr61 and CTGF mRNA in NHCC were significantly higher than those in SLHCC and SHCC.
CONCLUSION: Cyr61 and CTGF genes may play an important role in hepatocellular carcinogenesis and correlate with recurrence and metastasis of hepatocellular carcinoma. SLHCC has better biological behaviors than NHCC.
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Affiliation(s)
- Zhi-Jun Zeng
- Liver Cancer Laboratory, Department of Surgery, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China
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36
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Yamamoto Y, Oelgeschläger M. Regulation of bone morphogenetic proteins in early embryonic development. Naturwissenschaften 2004; 91:519-34. [PMID: 15517134 DOI: 10.1007/s00114-004-0575-z] [Citation(s) in RCA: 57] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Bone morphogenetic proteins (BMPs), a large subgroup of the TGF-beta family of secreted growth factors, control fundamental events in early embryonic development, organogenesis and adult tissue homeostasis. The plethora of dose-dependent cellular processes regulated by BMP signalling demand a tight regulation of BMP activity. Over the last decade, a number of proteins have been identified that bind BMPs in the extracellular space and regulate the interaction of BMPs with their cognate receptors, including the secreted BMP antagonist Chordin. In the early vertebrate embryo, the localized secretion of BMP antagonists from the dorsal blastopore lip establishes a functional BMP signalling gradient that is required for the determination of the dorsoventral - or back to belly - body axis. In particular, inhibition of BMP activity is essential for the formation of neural tissue in the development of vertebrate and invertebrate embryos. Here we review recent studies that have provided new insight into the regulation of BMP signalling in the extracellular space. In particular, we discuss the recently identified Twisted gastrulation protein that modulates, in concert with metalloproteinases of the Tolloid family, the interaction of Chordin with BMP and a family of proteins that share structural similarities with Chordin in the respective BMP binding domains. In addition, genetic and functional studies in zebrafish and frog provide compelling evidence that the secreted protein Sizzled functionally interacts with the Chd-BMP pathway, despite being expressed ventrally in the early gastrula-stage embryo. These intriguing discoveries may have important implications, not only for our current concept of early embryonic patterning, but also for the regulation of BMP activity at later developmental stages and tissue homeostasis in the adult.
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Affiliation(s)
- Yukiyo Yamamoto
- Department of Developmental Biology, Max-Planck Institute of Immunobiology, Stübeweg 51, 79108, Freiburg, Germany
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37
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Leask A, Abraham DJ. The role of connective tissue growth factor, a multifunctional matricellular protein, in fibroblast biology. Biochem Cell Biol 2004; 81:355-63. [PMID: 14663501 DOI: 10.1139/o03-069] [Citation(s) in RCA: 209] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Connective tissue growth factor (CTGF, CCN2), a member of the CCN family of proteins, is a cysteine-rich proadhesive matricellular protein that plays an essential role in the formation of blood vessels, bone, and connective tissue. As expression of this protein is potently induced by transforming growth factor-beta (TGFbeta), it has been hypothesized that CTGF mediates several of the downstream actions of TGFbeta. In particular, CTGF is profibrotic, as CTGF is overexpressed in fibrotic disease and synergizes with TGFbeta to promote sustained fibrosis in vivo. Over the last several years, key data regarding the developmental role and structure and function relationship of CTGF have emerged. In addition, increased information concerning the mechanisms underlying the control of CTGF expression in normal and fibrotic cells and the signal transduction pathways through which CTGF acts on cells has been uncovered. This review summarizes the current state of knowledge regarding CTGF biology.
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Affiliation(s)
- Andrew Leask
- Center for Rheumatology, Department of Medicine, Royal Free, University College London, Rowland Hill Sreet, London NW3 @PF, U.K.
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Leask A, Denton CP, Abraham DJ. Insights into the molecular mechanism of chronic fibrosis: the role of connective tissue growth factor in scleroderma. J Invest Dermatol 2004; 122:1-6. [PMID: 14962082 DOI: 10.1046/j.0022-202x.2003.22133.x] [Citation(s) in RCA: 101] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
Connective tissue growth factor (CCN2), a member of the CCN family of proteins, is a cysteine-rich matricellular protein. Connective tissue growth factor is not normally expressed in dermal fibroblasts unless induced. The most potent inducer of connective tissue growth factor thus far identified is transforming growth factor beta. Connective tissue growth factor, however, is constitutively overexpressed by fibroblasts present in skin fibrotic lesions, including scleroderma. The overexpression of connective tissue growth factor present in fibrotic lesions contributes to the phenotype of scleroderma in that connective tissue growth factor promotes matrix deposition, and fibroblast adhesion and proliferation. In animal models, whereas either transforming growth factor beta or connective tissue growth factor alone produce only a transient fibrotic response, connective tissue growth factor and transforming growth factor beta act together to promote sustained fibrosis. Thus the constitutive overexpression of connective tissue growth factor by fibroblasts present in fibrotic lesions would be expected to contribute directly to chronic, persistent fibrosis. This review discusses recent information regarding insights into connective tissue growth factor biology and, using scleroderma as a model system, the part connective tissue growth factor might play in fibrotic disease.
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Affiliation(s)
- Andrew Leask
- Center for Rheumatology, Royal Free and University College Medical School, University College London, Royal Free Campus, London, UK.
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Deininger MH, Winkler S, Kremsner PG, Meyermann R, Schluesener HJ. Angiogenic proteins in brains of patients who died with cerebral malaria. J Neuroimmunol 2003; 142:101-11. [PMID: 14512169 DOI: 10.1016/s0165-5728(03)00250-9] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
In cerebral malaria (CM), microvascular activation accompanies blood-brain barrier dysfunction which in turn represents the pathophysiological basis of neurological impairments in affected patients. To dissect the molecular basis of this process, we analyzed localization of proangiogenic vascular endothelial growth factor (VEGF), its receptor vascular endothelial growth factor receptor-1 (VEGFR-1, Flt-1), of downstream VEGF effectors matrix-metalloproteinase-1 (MMP-1) and connective tissue growth factor (CTGF), and of VEGF-interacting antiangiogenic thrombospondin-1 and -independent angiostatin in brains of patients who died with CM and controls by immunohistochemistry and Western blotting experiments. Most prominently, we detected more VEGF(+) astrocytes in CM patients and deposition of Flt-1 in Dürck's granulomas. MMP-1 and thrombospondin-1 accumulated in macrophages/microglial cells in Dürck's granulomas. In one CM patient, massive amounts of CTGF were detected as perivascular paracellular deposits. Angiostatin was observed in the serum of 2/7 control but in no CM patients. These data demonstrate the activation of the proangiogenic VEGF signaling cascade in patients with CM, probably reflecting compensatory mechanisms of general and focal brain hypoxia observed in these patients.
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Affiliation(s)
- Martin H Deininger
- Institute of Brain Research, University of Tuebingen, Medical School, Calwer Str. 3, D-72076 Tübingen, Germany.
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40
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Safadi FF, Xu J, Smock SL, Kanaan RA, Selim AH, Odgren PR, Marks SC, Owen TA, Popoff SN. Expression of connective tissue growth factor in bone: its role in osteoblast proliferation and differentiation in vitro and bone formation in vivo. J Cell Physiol 2003; 196:51-62. [PMID: 12767040 DOI: 10.1002/jcp.10319] [Citation(s) in RCA: 148] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023]
Abstract
Connective tissue growth factor (CTGF) is a secreted, extracellular matrix-associated signaling protein that regulates diverse cellular functions. In vivo, CTGF is expressed in many tissues with highest levels in the kidney and brain. The purpose of this study was twofold; first, to localize CTGF in normal bone in vivo during growth and repair, and second, to examine CTGF expression and function in primary osteoblast cultures in vitro and test its effect on bone formation in vivo. Northern and Western blot analyses confirmed that CTGF is expressed in normal long bones during the period of growth or modeling. In situ hybridization and immunohistochemical analysis demonstrated intense staining for CTGF mRNA and protein in osteoblasts lining metaphyseal trabeculae. Examination of CTGF expression in the fracture callus demonstrated that it was primarily localized in osteoblasts lining active, osteogenic surfaces. In primary osteoblast cultures, CTGF mRNA levels demonstrated a bimodal pattern of expression, being high during the peak of the proliferative period, abating as the cells became confluent, and increasing to peak levels and remaining high during mineralization. This pattern suggests that CTGF may play a role in osteoblast proliferation and differentiation as previously demonstrated for fibroblasts and chondrocytes. Treatment of primary osteoblast cultures with anti-CTGF neutralizing antibody caused a dose-dependent inhibition of nodule formation and mineralization. Treatment of primary osteoblast cultures with recombinant CTGF (rCTGF) caused an increase in cell proliferation, alkaline phosphatase activity, and calcium deposition, thereby establishing a functional connection between CTGF and osteoblast differentiation. In vivo delivery of rCTGF into the femoral marrow cavity induced osteogenesis that was associated with increased angiogenesis. This study clearly shows that CTGF is important for osteoblast development and function both in vitro and in vivo.
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Affiliation(s)
- Fayez F Safadi
- Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA
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Yu J, Prado GN, Taylor L, Pal-Ghosh R, Polgar P. Hybrid formation between the intracellular faces of the bradykinin B2 and angiotensin II AT1 receptors and signal transduction. Int Immunopharmacol 2002; 2:1807-22. [PMID: 12489795 DOI: 10.1016/s1567-5769(02)00177-7] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
Most frequently, the physiologic functions of the angiotensin II (Ang II) type 1 receptor (AT1R) and bradykinin B2 receptor (BKB2R) are antagonistic, particularly with respect to the regulation of vascular tone. Despite major differences in their physiologic actions, the receptors share sequence similarities. Both link to Galpha(i) and Galpha(q) and transduce very similar signal paths, not only those relating to the traditional G-protein associated second messengers, but also those involved in transactivation mechanisms involving receptor tyrosine kinases. With respect to these paths, some differences in signaling may be accounted for by cell type specificity. However, alternative signal cascades for these two receptors are becoming increasingly evident. One such is the recruitment of signaling molecules upon receptor translocation and internalization. The AT1R translocates into clathrin-coated pits and internalizes upon recruitment of beta-arrestin 2 which then recruits ASK1 and JNK3. The BKB2R translocates and internalizes mainly via caveolae. Another signaling divergence may be due to the direct activation of small G-proteins by both receptors. AT1R activates the RhoA, Rac1, Cdc42 while BKB2R couples only with Rac1 and Cdc42. Both receptors may serve as docking stations for intracellular proteins. One such example is the YIPP motif within the C-terminus of the ATIR which associates with the JAK/STAT pathway. Another potential alternative is the activation of tyrosine/serine kinase phosphatases by BK. This mechanism may directly oppose some of the protein tyrosine/ serine kinase paths activated by AT1R. These alternative mechanisms in sum are potentially responsible for the diversion in signal transduction between these two receptors. Regardless of the route of action, our results suggest that in Rat-1 fibroblasts stably transfected with BKB2R, BK slightly decreases connective tissue growth factor (CTGF) mRNA level while in ATIR transfected cells Ang II increases CTGF mRNA markedly. To determine whether mutant hybrids can be formed between these two receptors which encompass some of the function of the donor receptor but bind the ligand of the recipient receptor, a series of hybrids were formed with BKB2R the recipient and AT1R the donor receptor. Some of these hybrids show resistance to exchanges with the AT1R and form receptors which either do not bind (IC1 exchanges) or demonstrate poor function but normal internalization (proximal C-terminus exchanges). However, other hybrids have proven very functional. For example, the IC2, IC3 and distal C-terminus of the BKB2R IC face can be replaced simultaneously with the AT1R resulting in an hybrid which binds BK, continues to signal, is internalized and resensitized. Formation of this and other less extensive hybrids is discussed. Some of these hybrids possess the capacity to function as the AT1R as exemplified by their ability to upregulate CTGF expression as wild-type (WT) AT1R.
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MESH Headings
- Amino Acid Sequence
- Animals
- Cells, Cultured
- Humans
- Molecular Sequence Data
- Mutation
- Receptor, Angiotensin, Type 1
- Receptor, Bradykinin B2
- Receptors, Angiotensin/genetics
- Receptors, Angiotensin/metabolism
- Receptors, Angiotensin/physiology
- Receptors, Bradykinin/genetics
- Receptors, Bradykinin/metabolism
- Receptors, Bradykinin/physiology
- Signal Transduction/genetics
- Signal Transduction/physiology
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Affiliation(s)
- Jun Yu
- Department of Biochemistry, Boston University School of Medicine, 80 East Concord Street, Boston, MA 02118, USA
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Moussad EEDA, Rageh MAE, Wilson AK, Geisert RD, Brigstock DR. Temporal and spatial expression of connective tissue growth factor (CCN2; CTGF) and transforming growth factor beta type 1 (TGF-beta1) at the utero-placental interface during early pregnancy in the pig. Mol Pathol 2002; 55:186-92. [PMID: 12032230 PMCID: PMC1187172 DOI: 10.1136/mp.55.3.186] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/04/2022]
Abstract
AIMS To determine the localisation and distribution of connective tissue growth factor (CCN2; CTGF) and transforming growth factor beta type 1 (TGF-beta1) in uterine tissues from cycling and early pregnant pigs. METHODS In situ hybridisation and immunohistochemistry were used to localise CCN2 (CTGF) or TGF-beta1 in uteri obtained from gilts on days 0, 5, 10, 12, 15, and 18 of the oestrous cycle or days 10, 12, 14, 16, 17, and 21 of gestation. RESULTS In cycling animals, CCN2 (CTGF) mRNA and protein were abundant in luminal epithelial cells (LECs) and glandular epithelial cells (GECs), with lesser amounts in stromal fibroblasts and little or none in endothelial cells. A similar pattern of staining was seen up to day 10 of pregnancy, except that overall staining intensities for CCN2 (CTGF) mRNA or protein were higher and that stromal and endothelial cells were CCN2 (CTGF) positive. However, on days 12-17 there was a striking decrease in the amount of CCN2 (CTGF) in LECs at the utero-conceptus interface, which was associated with maternal stromal matrix reorganisation and the onset of subepithelial neovascularisation. This differential distribution of CCN2 (CTGF) was localised to those LECs that were in close proximity to or in apposition with trophoblast cells. This decrease in CCN2 (CTGF) staining was transient in nature and high amounts of CCN2 (CTGF) were again apparent in LECs on days 17-21, when endometrial neovascularisation and matrix remodelling were complete. The expression of uterine TGF-beta1 was comparable to that of CCN2 (CTGF) at most stages of the oestrous cycle or early pregnancy. Pre-elongation blastocysts recovered on day 10 were positive for both CCN2 (CTGF) and TGF-beta1 in the extra-embryonic trophectoderm, endoderm, and inner cell mass. On day 12, trophectoderm expressed low amounts of TGF-beta1 mRNA and non-detectable amounts of TGF-beta1 protein or CCN2 (CTGF) mRNA or protein. By days 17-21, the expression of both growth factors in the extra-embyronic/placental membranes increased and frequently exceeded that seen in LECs. CONCLUSIONS The pattern of CCN2 (CTGF) production during the initial attachment phase supports a role for this factor in stromal remodelling and neovascularisation, although alternative functions at later stages such as epithelial-epithelial interactions are also possible. In most major cell types in the uterus or utero-placental unit, CCN2 (CTGF) expression was highly correlated with that of TGF-beta(1), indicating that CCN2 (CTGF) may mediate some of the functions of TGF-beta in the reproductive tract during the oestrous cycle and pregnancy. The data further highlight epithelium as an important source of CCN2 (CTGF) in the regulation of uterine function.
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Affiliation(s)
- E E-D A Moussad
- Department of Surgery, Children's Research Institute, Children's Hospital and The Ohio State University, Columbus OH 43205, USA
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Quan T, He T, Kang S, Voorhees JJ, Fisher GJ. Connective tissue growth factor: expression in human skin in vivo and inhibition by ultraviolet irradiation. J Invest Dermatol 2002; 118:402-8. [PMID: 11874477 DOI: 10.1046/j.0022-202x.2001.01678.x] [Citation(s) in RCA: 58] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Connective tissue growth factor, which is induced by transforming growth factor beta, has been reported to mediate the stimulatory actions of transforming growth factor beta on type I procollagen synthesis. Connective tissue growth factor is expressed in fibrotic disease such as scleroderma, where it is believed to promote abnormal deposition of collagen. Connective tissue growth factor expression has not been described in normal human skin or cultured skin cells, however. We report here that connective tissue growth factor mRNA is constitutively expressed in normal human skin. In situ hybridization demonstrated that connective tissue growth factor mRNA was expressed in keratinocytes throughout the epidermis and in dermal cells. Quantitative real-time reverse transcription polymerase chain reaction revealed that the level of connective tissue growth factor mRNA in the epidermis and dermis of normal human skin was comparable to the level of housekeeping gene 36B4. Ultraviolet irradiation (2 minimal erythema dose, UVB/A2 source) reduced connective tissue growth factor mRNA expression throughout the epidermis and dermis in normal human skin in vivo. Connective tissue growth factor mRNA was reduced (30%) within 4 h post ultraviolet irradiation, and remained reduced (50%) 8-24 h post ultraviolet. Connective tissue growth factor mRNA and protein were also constitutively highly expressed in normal cultured human skin keratinocytes and fibroblasts. Ultraviolet irradiation of cultured normal human skin fibroblasts resulted in a time-dependent inhibition of connective tissue growth factor mRNA expression. At 24 h post ultraviolet, connective tissue growth factor mRNA expression was reduced 80%. Transforming growth factor beta1 rapidly induced connective tissue growth factor mRNA levels (5-fold within 4 h) in skin fibroblasts, but not keratinocytes, and this induction was attenuated 80% by ultraviolet irradiation. Electrophoretic mobility shift assays demonstrated that ultraviolet irradiation reduced protein binding to the transforming growth factor beta/Smad responsiveness elements in the connective tissue growth factor gene promoter, in human skin in vivo and human skin fibroblasts. Constitutive expression of connective tissue growth factor in normal human skin suggests that it is a physiologic regulator of procollagen synthesis. Ultraviolet reduction of connective tissue growth factor expression may contribute to reduced procollagen synthesis observed in ultraviolet-irradiated normal human skin and human skin fibroblasts.
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Affiliation(s)
- Taihao Quan
- Department of Dermatology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0609, USA
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Segarini PR, Nesbitt JE, Li D, Hays LG, Yates JR, Carmichael DF. The low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor is a receptor for connective tissue growth factor. J Biol Chem 2001; 276:40659-67. [PMID: 11518710 DOI: 10.1074/jbc.m105180200] [Citation(s) in RCA: 158] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Connective tissue growth factor (CTGF) expression is regulated by transforming growth factor-beta (TGF-beta) and strong up-regulation occurs during wound healing; in situ hybridization data indicate that there are high levels of CTGF expression in fibrotic lesions. Recently the binding parameters of CTGF to both high and lower affinity cell surface binding components have been characterized. Affinity cross-linking and SDS-polyacrylamide gel electrophoresis analysis demonstrated the binding of CTGF to a cell surface protein with a mass of approximately 620 kDa. We report here the purification of this protein by affinity chromatography on CTGF coupled to Sepharose and sequence information obtained by mass spectroscopy. The binding protein was identified as the multiligand receptor, low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor (LRP). The identification of LRP as a receptor for CTGF was validated by several studies: 1) binding competition with many ligands that bind to LRP, including receptor-associated protein; 2) immunoprecipitation of CTGF-receptor complex with LRP antibodies; and 3) cells that are genetically deficient for LRP were unable to bind CTGF. Last, CTGF is rapidly internalized and degraded and this process is LRP-dependent. In summary, our data indicate that LRP is a receptor for CTGF, and may play an important role in mediating CTGF biology.
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Affiliation(s)
- P R Segarini
- FibroGen, Inc., South San Francisco, California 94080, USA
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Wahab NA, Brinkman H, Mason RM. Uptake and intracellular transport of the connective tissue growth factor: a potential mode of action. Biochem J 2001; 359:89-97. [PMID: 11563972 PMCID: PMC1222124 DOI: 10.1042/0264-6021:3590089] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Connective tissue growth factor (CTGF) is a secreted cysteine-rich protein now considered as an important effector molecule in both physiological and pathological processes. An increasing amount of evidence indicates that CTGF plays a key role in the pathogenesis of different fibrotic disorders including diabetic nephropathy. However, the molecular mechanisms by which CTGF exerts its effects are not known. Here we provide the first evidence for the existence of an intracellular transport pathway for the growth factor in human mesangial cells. Our results demonstrate that CTGF is internalized from the cell surface in endosomes and accumulates in a juxtanuclear organelle from which the growth factor is then translocated into the cytosol. In the cytosol CTGF is phosphorylated by protein kinase C and PMA treatment can enhance this phosphorylation. Phosphorylated CTGF may have an important role in the cytosol, but it is also translocated into the nucleus where it may directly affect transcription.
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Affiliation(s)
- N A Wahab
- Cell and Molecular Biology Section, Division of Biomedical Sciences, Imperial College School of Medicine, Sir Alexander Fleming Building, Exhibition Road, South Kensington, London SW7 2AZ, UK
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Rageh MA, Moussad EE, Wilson AK, Brigstock DR. Steroidal regulation of connective tissue growth factor (CCN2; CTGF) synthesis in the mouse uterus. Mol Pathol 2001; 54:338-46. [PMID: 11577177 PMCID: PMC1187092 DOI: 10.1136/mp.54.5.338] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
AIMS To determine mechanisms regulating the production of connective tissue growth factor (CCN2; CTGF) and transforming growth factor beta1 (TGF-beta1) in the mouse uterus. METHODS In situ hybridisation and immunohistochemistry were used to localise CCN2 (CTGF) and TGF-beta1 in uteri from sexually mature female mice that had either been (1) mated with sterile males to induce pseudopregnancy or (2) ovariectomised (OVX) and administered estradiol-17beta (E2) or progesterone (P4), either alone or in combination. Uteri collected on days 0.5, 1.5, 2.5, 3.5, 4.5, or 5.5 of pseudopregnancy or at one, three, six, 12, or 24 hours after steroid administration were fixed, sectioned, and incubated with specific riboprobes or antibodies to permit detection and localisation of mRNA or protein for CTGF and TGF-beta1. RESULTS On days 0.5-2.5 of pseudopregnancy, CCN2 (CTGF) and TGF-beta1 were principally colocalised to uterine epithelial cells, with much smaller amounts in the stroma. On days 3.5-4.5, there was a reduction of CCN2 (CTGF) and TGF-beta1 in the epithelium but an increase in stromal and endothelial cells, corresponding to a period of extracellular matrix remodelling and neovascularisation within the endometrium. In OVX mice, epithelial cells were weakly positive for both CCN2 (CTGF) and TGF-beta1 in the absence of steroid hormones. Epithelial CTGF mRNA production were strongly but transiently stimulated in OVX mice cells by E2. These effects were antagonised by P4, which itself transiently stimulated epithelial CCN2 (CTGF) production, although less robustly than E2. CTGF and TGF-beta1 protein amounts were high in epithelial cells throughout steroid treatment and were increased in the stroma, where they were relatively long lived. Stromal CCN2 (CTGF) and TGF-beta1 were lower after co-administration of E2 and P4 than in response to each hormone individually. Although ccn2 (ctgf) is a TGF-beta1 inducible gene in other systems, and both growth factors were often co-localised in uterine tissues in these studies, several treatment regimens resulted in high amounts of TGF-beta1 protein in stromal cells without the concomitant production of ccn2 (ctgf) mRNA. CONCLUSIONS Maternal factors are principal cues for CCN2 (CTGF) and TGF-beta1 production in the uterus because (1) their expression during pseudopregnancy is comparable to that seen in pregnancy and (2) they are regulated by ovarian steroids. TGF-beta dependent and independent mechanisms of ccn2 (ctgf) gene transcription exist in the uterus that are variably regulated by steroid hormones. Collectively, the data support a role for CCN2 (CTGF) in mediating the effects of steroid hormones and TGF-beta on endometrial function.
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Affiliation(s)
- M A Rageh
- Department of Surgery, The Ohio State University, and Children's Research Institute, Columbus, Ohio 43205, USA
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Gupta S, Clarkson MR, Duggan J, Brady HR. Connective tissue growth factor: potential role in glomerulosclerosis and tubulointerstitial fibrosis. Kidney Int 2000; 58:1389-99. [PMID: 11012874 DOI: 10.1046/j.1523-1755.2000.00301.x] [Citation(s) in RCA: 216] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
Transforming growth factor beta (TGF-beta) is a pivotal driver of glomerulosclerosis and tubulointerstitial fibrosis in renal diseases. Because TGF-beta also plays important anti-inflammatory and antiproliferative roles in mammalian systems, there has been a recent drive to elucidate downstream mediators of TGF-beta's pro-fibrotic effects with the ultimate goal of developing new anti-fibrotic strategies for treatment of chronic diseases. Connective tissue growth factor (CTGF) belongs to the CCN family of immediate early response genes. Several lines of evidence suggest that CTGF is an important pro-fibrotic molecule in renal disease and that CTGF contributes to TGF-beta bioactivity in this setting. CTGF expression is increased in the glomeruli and tubulointerstium in a variety of renal disease in association with scarring and sclerosis of renal parenchyma. In model systems in vitro, mesangial cell CTGF expression is induced by high extracellular glucose, cyclic mechanical strain and TGF-beta. Recombinant human CTGF augments the production of fibronectin and type IV collagen by mesangial cells and the effects of high glucose on mesangial cell CTGF expression and matrix production are attenuated, in part, by anti-TGF-beta antibody. In aggregate, these observations identify CTGF as an attractive therapeutic target in fibrotic renal diseases.
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Affiliation(s)
- S Gupta
- Department of Medicine and Therapeutics, Mater Misericordiae Hospital, University College Dublin, Dublin, Ireland
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Abstract
Connective tissue growth factor (CTGF) is a member of the recently described CCN gene family which contains CTGF itself, cyr61, nov, elm1, Cop1, and WISP-3. CTGF is transcriptionally activated by several factors although its stimulation by transforming growth factor beta (TGF-beta) has attracted considerable attention. CTGF acts to promote fibroblast proliferation, migration, adhesion, and extracellular matrix formation, and its overproduction is proposed to play a major role in pathways that lead to fibrosis, especially those that are TGF-beta-dependent. This includes fibrosis of major organs, fibroproliferative diseases, and scarring. CTGF also appears to play a role in the extracellular matrix remodeling that occurs in normal physiological processes such as embryogenesis, implantation, and wound healing. However, recent advances have shown that CTGF is involved in diverse autocrine or paracrine actions in several other cell types such as vascular endothelial cells, epithelial cells, neuronal cells, vascular smooth muscle cells, and cells of supportive skeletal tissues. Moreover, in some circumstances CTGF has negative effects on cell growth in that it can be antimitotic and apoptotic. In light of these discoveries, CTGF has been implicated in a diverse variety of processes that include neovascularization, transdifferentiation, neuronal scarring, atherosclerosis, cartilage differentiation, and endochondral ossification. CTGF has thus emerged as a potential important effector molecule in both physiological and pathological processes and has provided a new target for therapeutic intervention in fibrotic diseases.
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Affiliation(s)
- E E Moussad
- Department of Surgery, Children's Hospital and Ohio State University, Columbus, Ohio 43205, USA
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Shi-wen X, Pennington D, Holmes A, Leask A, Bradham D, Beauchamp JR, Fonseca C, du Bois RM, Martin GR, Black CM, Abraham DJ. Autocrine overexpression of CTGF maintains fibrosis: RDA analysis of fibrosis genes in systemic sclerosis. Exp Cell Res 2000; 259:213-24. [PMID: 10942593 DOI: 10.1006/excr.2000.4972] [Citation(s) in RCA: 148] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
We have used representational difference analysis (RDA) to identify up-regulated genes in skin fibroblasts from fibrotic lesions obtained from patients with systemic sclerosis (scleroderma). RDA of cDNA libraries derived from fibroblasts from involved and uninvolved skin detected several differentially expressed genes. One such gene consistently up-regulated in scleroderma cells coded for human connective tissue growth factor (CTGF). Other studies described here show that the CTGF protein is readily detected in cultures of systemic sclerosis fibroblasts but was not detected in comparable normal cells. High levels of CTGF are also evident in biological fluids from patients with systemic sclerosis. TGFbeta stimulates CTGF production in both normal and systemic sclerosis fibroblasts with the latter found to be higher producers. Moreover, an analysis of constitutive and TGFbeta-induced CTGF gene activation showed altered and elevated transcriptional responses in systemic sclerosis cells compared with controls. CTGF stimulated a two- to threefold increase in proalpha1(I) collagen and fibronectin synthesis by both dermal and lung fibroblasts in culture and promoted significant matrix remodeling of fibroblast-populated three-dimensional collagen lattices. A direct relation between the overexpression of CTGF and elevated collagen synthesis was suggested by the observation that transfection of a CMV-CTGF cDNA construct and protein expression in fibroblasts increased the transcription of a Col 1alpha2 promoter-reporter construct to levels seen in systemic sclerosis fibroblasts. Using Col 1alpha2 promoter deletion constructs the CTGF responsive element was localized to the first 379 bp upstream of the transcriptional start site. These data indicate that there is an overexpression of CTGF in the systemic sclerosis cells, probably due to increased gene transcription, and suggest that the dysregulation of CTGF production is an important factor in fibroblast activation and the excessive deposition of collagen in systemic sclerosis.
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Affiliation(s)
- X Shi-wen
- Centre for Rheumatology, Royal Free and University College Medical School, University College London (Royal Free Campus), Rowland Hill Street, London, NW3 2PF, United Kingdom
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Binart N, Helloco C, Ormandy CJ, Barra J, Clément-Lacroix P, Baran N, Kelly PA. Rescue of preimplantatory egg development and embryo implantation in prolactin receptor-deficient mice after progesterone administration. Endocrinology 2000; 141:2691-7. [PMID: 10875275 DOI: 10.1210/endo.141.7.7568] [Citation(s) in RCA: 77] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
PRL, a hormone secreted essentially by the pituitary and other extrapituitary sources such as decidua, has been attributed regulatory roles in reproduction and cell growth in mammals. These effects are mediated by a membrane PRL receptor belonging to the cytokine receptor superfamily. Null mutation of the PRL receptor gene leads to female sterility due to a severely compromised preimplantation development and a complete failure of the implantation of the few embryos reaching the blastocyst stage, strongly implicating PRL in the maternal control of implantation. We measured the hormonal status of -/- mice, which confirmed that the corpus luteum is unable to produce progesterone. Progesterone administration to -/- mice completely rescued the development of preimplantatory eggs and embryo implantation. Pregnancy could be maintained to 19.5 days postcoitum, with about 22% of resulting embryos reaching adulthood. Although progesterone and perhaps PRL appear to facilitate mouse preembryo development throughout the preimplantation stages, other factors as well as a possible direct effect of PRL on the uterus are probably necessary to fully maintain pregnancy. Finally, reduced ductal side-branching in the mammary gland can be rescued by progesterone treatment, but females exhibit reduced alveolar formation. Our model establishes the PRL receptor as a key regulator of reproduction and provides novel insights into the function of lactogenic hormones and their receptor.
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Affiliation(s)
- N Binart
- INSERM, U-344, Endocrinologie Moléculaire, Faculté de Médecine Necker, Paris, France.
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