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Vomhof-DeKrey EE, Stover AD, Labuhn M, Osman MR, Basson MD. Vil-Cre specific Schlafen 3 knockout mice exhibit sex-specific differences in intestinal differentiation markers and Schlafen family members expression levels. PLoS One 2021; 16:e0259195. [PMID: 34710177 PMCID: PMC8553116 DOI: 10.1371/journal.pone.0259195] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2021] [Accepted: 10/14/2021] [Indexed: 11/25/2022] Open
Abstract
The intestinal epithelium requires self-renewal and differentiation in order to function and adapt to pathological diseases such as inflammatory bowel disease, short gut syndrome, and ulcers. The rodent Slfn3 protein and the human Slfn12 analog are known to regulate intestinal epithelial differentiation. Previous work utilizing a pan-Slfn3 knockout (KO) mouse model revealed sex-dependent gene expression disturbances in intestinal differentiation markers, metabolic pathways, Slfn family member mRNA expression, adaptive immune cell proliferation/functioning genes, and phenotypically less weight gain and sex-dependent changes in villus length and crypt depth. We have now created a Vil-Cre specific Slfn3KO (VC-Slfn3KO) mouse to further evaluate its role in intestinal differentiation. There were increases in Slfn1, Slfn2, Slfn4, and Slfn8 and decreases in Slfn5 and Slfn9 mRNA expression that were intestinal region and sex-specific. Differentiation markers, sucrase isomaltase (SI), villin 1, and dipeptidyl peptidase 4 and glucose transporters, glucose transporter 1 (Glut1), Glut2, and sodium glucose transporter 1 (SGLT1), were increased in expression in VC-Slfn3KO mice based on intestinal region and were also highly female sex-biased, except for SI in the ileum was also increased for male VC-Slfn3KO mice and SGLT1 was decreased for both sexes. Overall, the variations that we observed in these VC-Slfn3KO mice indicate a complex regulation of intestinal gene expression that is sex-dependent.
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Affiliation(s)
- Emilie E. Vomhof-DeKrey
- Department of Surgery, School of Medicine and the Health Sciences, University of North Dakota, Grand Forks, ND, United States of America
- Department of Biomedical Sciences, School of Medicine and the Health Sciences, University of North Dakota, Grand Forks, ND, United States of America
| | - Allie D. Stover
- Department of Biomedical Sciences, School of Medicine and the Health Sciences, University of North Dakota, Grand Forks, ND, United States of America
| | - Mary Labuhn
- Department of Biomedical Sciences, School of Medicine and the Health Sciences, University of North Dakota, Grand Forks, ND, United States of America
| | - Marcus R. Osman
- Department of Biomedical Sciences, School of Medicine and the Health Sciences, University of North Dakota, Grand Forks, ND, United States of America
| | - Marc D. Basson
- Department of Surgery, School of Medicine and the Health Sciences, University of North Dakota, Grand Forks, ND, United States of America
- Department of Biomedical Sciences, School of Medicine and the Health Sciences, University of North Dakota, Grand Forks, ND, United States of America
- Department of Pathology, School of Medicine and the Health Sciences, University of North Dakota, Grand Forks, ND, United States of America
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Vomhof-DeKrey EE, Lansing JT, Darland DC, Umthun J, Stover AD, Brown C, Basson MD. Loss of Slfn3 induces a sex-dependent repair vulnerability after 50% bowel resection. Am J Physiol Gastrointest Liver Physiol 2021; 320:G136-G152. [PMID: 33237796 PMCID: PMC7864235 DOI: 10.1152/ajpgi.00344.2020] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/21/2020] [Revised: 11/13/2020] [Accepted: 11/20/2020] [Indexed: 02/07/2023]
Abstract
Bowel resection accelerates enterocyte proliferation in the remaining gut with suboptimal absorptive and digestive capacity because of a proliferation-associated decrease in functional differentiation markers. We hypothesized that although schlafen 3 (Slfn3) is an important regulator of enterocytic differentiation, Slfn3 would have less impact on bowel resection adaptation, where accelerated proliferation takes priority over differentiation. We assessed proliferation, cell shedding, and enterocyte differentiation markers from resected and postoperative bowel of wild-type (WT) and Slfn3-knockout (Slfn3KO) mice. Villus length and crypt depth were increased in WT mice and were even longer in Slfn3KO mice. Mitotic marker, Phh3+, and the proliferation markers Lgr5, FoxL1, and platelet-derived growth factor-α (PDGFRα) were increased after resection in male WT, but this was blunted in male Slfn3KO mice. Cell-shedding regulators Villin1 and TNFα were downregulated in female mice and male WT mice only, whereas Gelsolin and EGFR increased expression in all mice. Slfn3 expression increased after resection in WT mice, whereas other Slfn family members 1, 2, 5, 8, and 9 had varied expressions that were affected also by sex difference and loss of Slfn3. Differentiation markers sucrase isomaltase, Dpp4, Glut2, and SGLT1 were all decreased, suggesting that enterocytic differentiation effort is incompatible with rapid proliferation shift in intestinal adaptation. Slfn3 absence potentiates villus length and crypt depth, suggesting that the differentiating stimulus of Slfn3 signaling may restrain mucosal mass increase through regulating Villin1, Gelsolin, EGFR, TNFα, and proliferation markers. Therefore, Slfn3 may be an important regulator not only of "normal" enterocytic differentiation but also in response to bowel resection.NEW & NOTEWORTHY The differentiating stimulus of Slfn3 signaling restrains an increase in mucosal mass after bowel resection, and there is a Slfn3-sex interaction regulating differentiation gene expression and intestinal adaptation. This current study highlights the combinatory effects of gender and Slfn3 genotype on the gene expression changes that contribute to the adaptation in intestinal cellular milleu (i.e. villus and crypt structure) which are utilized to compensate for the stress-healing response that the animals display in intestinal adaptation.
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Affiliation(s)
- Emilie E Vomhof-DeKrey
- Departments of Surgery, Pathology, and Biomedical Sciences, School of Medicine and the Health Sciences, University of North Dakota, Grand Forks, North Dakota
| | - Jack T Lansing
- Departments of Surgery, Pathology, and Biomedical Sciences, School of Medicine and the Health Sciences, University of North Dakota, Grand Forks, North Dakota
- Department of Biology, University of North Dakota, Grand Forks, North Dakota
| | - Diane C Darland
- Department of Biology, University of North Dakota, Grand Forks, North Dakota
| | - Josey Umthun
- Departments of Surgery, Pathology, and Biomedical Sciences, School of Medicine and the Health Sciences, University of North Dakota, Grand Forks, North Dakota
- Department of Biology, University of North Dakota, Grand Forks, North Dakota
| | - Allie D Stover
- Departments of Surgery, Pathology, and Biomedical Sciences, School of Medicine and the Health Sciences, University of North Dakota, Grand Forks, North Dakota
| | - Christopher Brown
- Departments of Surgery, Pathology, and Biomedical Sciences, School of Medicine and the Health Sciences, University of North Dakota, Grand Forks, North Dakota
| | - Marc D Basson
- Departments of Surgery, Pathology, and Biomedical Sciences, School of Medicine and the Health Sciences, University of North Dakota, Grand Forks, North Dakota
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Vomhof-DeKrey EE, Umthun J, Basson MD. Loss of Schlafen3 influences the expression levels of Schlafen family members in ileum, thymus, and spleen tissue. PeerJ 2020; 8:e8461. [PMID: 32025381 PMCID: PMC6993753 DOI: 10.7717/peerj.8461] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2019] [Accepted: 12/26/2019] [Indexed: 12/11/2022] Open
Abstract
BACKGROUND The Schlafen (Slfn) family proteins are important for regulation of cell growth, cell differentiation and cell cycle progression. We sought to distinguish Slfn family expression in Slfn3 knockout (KO) mice after RNA sequencing analysis of Slfn3KO vs. wildtype (WT) mice revealed varying expressions of Slfn family in ileal mucosa. METHODS Quantitative PCR analysis of Slfn members was evaluated in ileal mucosa, thymus and spleen tissue since Slfn family members have roles in differentiating intestinal and immune cells. RESULTS Ileal mucosa of Slfn3KO mice displayed a decrease in Slfn3, 4, 8 and 9 while Slfn1 and 5 increased in mRNA expression vs. WT mice. Thymic tissue had a Slfn9 increase and a Slfn4 decrease while splenic tissue had a Slfn8 and Slfn9 increase in Slfn3KO mice vs. WT mice. These differential expressions of Slfn members could indicate a feedback regulatory mechanism within the Slfn family. Indeed, MATCH™ tool from geneXplain predicted that all Slfn members have regions in their promoters for the Kruppel-like factor-6 transcription factor. In addition, NFAT related factors, ING4, ZNF333 and KLF4 are also predicted to bind in up to 6 of the 8 Slfn promoters. This study further describes a possible autoregulatory mechanism amongst the Slfn family members which could be important in how they regulate the differentiation of various cell types.
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Affiliation(s)
- Emilie E. Vomhof-DeKrey
- Departments of Surgery, Pathology, and Biomedical Sciences, University of North Dakota, Grand Forks, ND, USA
| | - Josey Umthun
- Departments of Surgery, Pathology, and Biomedical Sciences, University of North Dakota, Grand Forks, ND, USA
| | - Marc D. Basson
- Departments of Surgery, Pathology, and Biomedical Sciences, University of North Dakota, Grand Forks, ND, USA
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Chaturvedi LS, Wang Q, More SK, Vomhof-DeKrey EE, Basson MD. Schlafen 12 mediates the effects of butyrate and repetitive mechanical deformation on intestinal epithelial differentiation in human Caco-2 intestinal epithelial cells. Hum Cell 2019; 32:240-250. [PMID: 30875077 PMCID: PMC6571040 DOI: 10.1007/s13577-019-00247-3] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2018] [Accepted: 03/02/2019] [Indexed: 12/12/2022]
Abstract
Intestinal epithelial differentiation may be stimulated by diverse pathways including luminal short-chain fatty acids and repetitive mechanical deformation engendered by villous motility and peristalsis. Schlafen 12 (SLFN12) is a cytosolic protein that stimulates sucrase-isomaltase (SI) expression. We hypothesized that two disparate differentiating stimuli, butyrate and repetitive deformation, would each stimulate SLFN12 expression in human Caco-2 intestinal epithelial cells and that increased SLFN12 expression would contribute to the differentiating activity of the human Caco-2 intestinal epithelial cells. We stimulated Caco-2 cells with 1-2 mM butyrate or repetitive mechanical deformation at 10 cycles/min at an average 10% strain, and measured SLFN12 and SI expression by qRT-PCR. Sodium butyrate enhanced SLFN12 expression at both 1 mM and 2 mM although SI expression was only significantly increased at 2 mM. Repetitive deformation induced by cyclic mechanical strain also significantly increased both SLFN12 and SI gene expression. Reducing SLFN12 by siRNA decreased basal, deformation-stimulated, and butyrate-stimulated SLFN12 levels, compared to control cells treated with non-targeting siRNA, although both deformation and butyrate were still able to stimulate SLFN12 expression in siRNA-treated cells compared to control cells treated with the same siRNA. This attenuation of the increase in SLFN12 expression in response to mechanical strain or butyrate was accompanied by parallel attenuation of SI expression. Butyrate stimulated SI-promoter activity, and reducing SLFN12 by siRNA attenuated butyrate-induced SI-promoter activity. These data suggest that SLFN12 mediates at least in part the stimulation by both butyrate and repetitive mechanical deformation of sucrase-isomaltase, a late stage differentiation marker in human intestinal epithelial cells.
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Affiliation(s)
- Lakshmi S Chaturvedi
- Departments of Surgery, Pathology, and Biomedical Sciences, School of Medicine and the Health Sciences, University of North Dakota, 1301 North Columbia Road, Stop 9037, Grand Forks, ND, 58202, USA
- Currently at Departments of Pharmaceutical Sciences and Biomedical Sciences-College of Pharmacy, Departments of Basic Sciences and Surgery-College of Medicine, California Northstate University, Elk Grove, CA, 95757, USA
| | - Qinggang Wang
- Departments of Surgery, Pathology, and Biomedical Sciences, School of Medicine and the Health Sciences, University of North Dakota, 1301 North Columbia Road, Stop 9037, Grand Forks, ND, 58202, USA
| | - Shyam K More
- Departments of Surgery, Pathology, and Biomedical Sciences, School of Medicine and the Health Sciences, University of North Dakota, 1301 North Columbia Road, Stop 9037, Grand Forks, ND, 58202, USA
| | - Emilie E Vomhof-DeKrey
- Departments of Surgery, Pathology, and Biomedical Sciences, School of Medicine and the Health Sciences, University of North Dakota, 1301 North Columbia Road, Stop 9037, Grand Forks, ND, 58202, USA
| | - Marc D Basson
- Departments of Surgery, Pathology, and Biomedical Sciences, School of Medicine and the Health Sciences, University of North Dakota, 1301 North Columbia Road, Stop 9037, Grand Forks, ND, 58202, USA.
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Vomhof-DeKrey EE, Lee J, Lansing J, Brown C, Darland D, Basson MD. Schlafen 3 knockout mice display gender-specific differences in weight gain, food efficiency, and expression of markers of intestinal epithelial differentiation, metabolism, and immune cell function. PLoS One 2019; 14:e0219267. [PMID: 31260507 PMCID: PMC6602453 DOI: 10.1371/journal.pone.0219267] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2019] [Accepted: 06/19/2019] [Indexed: 12/15/2022] Open
Abstract
Self-renewal and differentiation are essential for intestinal epithelium absorptive functioning and adaptation to pathological states such as short gut syndrome, ulcers, and inflammatory bowel disease. The rodent Slfn3 and its human analog Slfn12 are critical in regulating intestinal epithelial differentiation. We sought to characterize intestinal function in Slfn3 knockout (KO) mice. Male and female pair-fed Slfn3KO mice gained less weight with decreased food efficiency than wild type (WT) mice, with more pronounced effects in females. RNA sequencing performed on intestinal mucosa of Slfn3KO and WT mice showed gene ontology decreases in cell adhesion molecule signaling, tumor necrosis factor receptor binding, and adaptive immune cell proliferation/functioning genes in Slfn3KO mice, with greater effects in females. qPCR analysis of fatty acid metabolism genes, Pla2g4c, Pla2g2f, and Cyp3c55 revealed an increase in Pla2g4c, and a decrease in Pla2g2f in Slfn3KO females. Additionally, adipogenesis genes, Fabp4 and Lpl were decreased and ketogenesis gene Hmgcs2 was increased in female Slfn3KO mice. Sequencing did not reveal significant changes in differentiation markers, so qPCR was utilized. Slfn3KO tended to have decreased expression of intestinal differentiation markers sucrase isomaltase, dipeptidyl peptidase 4, villin 1, and glucose transporter 1 (Glut1) vs. WT males, although these trends did not achieve statistical significance unless data from several markers was pooled. Differentiation markers, Glut2 and sodium-glucose transporter 1 (SGLT1), did show statistically significant sex-dependent differences. Glut2 mRNA was reduced in Slfn3KO females, while SGLT1 increased in Slfn3KO males. Notch2 and Cdx2 were only increased in female Slfn3KO mice. Although Slfn3KO mice gain less weight and decreased food efficiency, their biochemical phenotype is more subtle and suggests a complex interplay between gender effects, Slfn3, and another regulatory pathway yet to be identified that compensates for the chronic loss of Slfn3.
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Affiliation(s)
- Emilie E. Vomhof-DeKrey
- Departments of Surgery, Pathology, and Biomedical Sciences, University of North Dakota School of Medicine and the Health Sciences, Grand Forks, ND, United States of America
| | - Jun Lee
- Departments of Surgery, Pathology, and Biomedical Sciences, University of North Dakota School of Medicine and the Health Sciences, Grand Forks, ND, United States of America
| | - Jack Lansing
- Departments of Surgery, Pathology, and Biomedical Sciences, University of North Dakota School of Medicine and the Health Sciences, Grand Forks, ND, United States of America
| | - Chris Brown
- Departments of Surgery, Pathology, and Biomedical Sciences, University of North Dakota School of Medicine and the Health Sciences, Grand Forks, ND, United States of America
| | - Diane Darland
- Department of Biology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND, United States of America
| | - Marc D. Basson
- Departments of Surgery, Pathology, and Biomedical Sciences, University of North Dakota School of Medicine and the Health Sciences, Grand Forks, ND, United States of America
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Deng Y, Cai Y, Huang Y, Yang Z, Bai Y, Liu Y, Deng X, Wang J. High SLFN11 expression predicts better survival for patients with KRAS exon 2 wild type colorectal cancer after treated with adjuvant oxaliplatin-based treatment. BMC Cancer 2015; 15:833. [PMID: 26525741 PMCID: PMC4631086 DOI: 10.1186/s12885-015-1840-6] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2015] [Accepted: 10/23/2015] [Indexed: 01/22/2023] Open
Abstract
Background SLFN11 was reported to be a predictive marker for DNA damage drugs. The study was to investigate whether SLFN11 expression is related to sensitivity to adjuvant oxaliplatin-based treatment in colorectal cancer. Methods A tissue microarray, made with specimens from consecutive 261 patients who received oxaliplatin based adjuvant chemotherapy, was stained with anti-SLFN11 antibody. The staining was dichotomized as high or low expression. SLFN11 expression was correlated to clinicopathological factors, KRAS exon 2 mutation and survival. Results SLFN11 high expression was found in 16.9 % of patients, and KRAS exon 2 mutation was detected in 32.2 % of patients. SLFN11 was expressed more common in well/moderate differentiation tumors(comparing to poor differentiation ones, 21 % v 4.9 %, P = 0.003) and stage II tumors(comparing to stage III tumors, 26.1 % v 11.4 %,p = 0.006). 23 out of 153 patients with KRAS exon 2 wild-type CRC had SLFN11 high expression, no death events was recorded in the 23 patients until last follow up. These patients had significantly better overall survival (OS) than those with SLFN11 low expression tumors (100 % vs 78.2 %, log rank P = 0.048). However, among patients with KRAS exon 2 mutant tumors, OS did not significantly differ between those with SLFN11 high and SLFN11 low tumors (Log rank P = 0.709). Conclusions SLFN11 expression predicts good better survival in colorectal cancer patients with KRAS exon 2 wild type who have received oxaliplatin based adjuvant chemotherapy.
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Affiliation(s)
- Yanhong Deng
- Department of Medical Oncology, Gastrointestinal Hospital, Sun Yat-sen Universtiy, Guangzhou, 510655, China. .,Department of Research Institute, Gastrointestinal Hospital, Sun Yat-sen Universtiy, Guangzhou, 510655, China.
| | - Yue Cai
- Department of Medical Oncology, Gastrointestinal Hospital, Sun Yat-sen Universtiy, Guangzhou, 510655, China.
| | - Yan Huang
- Department of Pathology, Gastrointestinal Hospital, Sun Yat-sen Universtiy, Guangzhou, 510655, China.
| | - Zihuan Yang
- Department of Research Institute, Gastrointestinal Hospital, Sun Yat-sen Universtiy, Guangzhou, 510655, China.
| | - Yang Bai
- Department of Research Institute, Gastrointestinal Hospital, Sun Yat-sen Universtiy, Guangzhou, 510655, China. .,Department of Colorectal Surgery, Gastrointestinal Hospital, Sun Yat-sen Universtiy, Guangzhou, 510655, China.
| | - Yanlu Liu
- Department of Medical Oncology, Gastrointestinal Hospital, Sun Yat-sen Universtiy, Guangzhou, 510655, China.
| | - Xiuping Deng
- Department of Pathology, Gastrointestinal Hospital, Sun Yat-sen Universtiy, Guangzhou, 510655, China.
| | - Jianping Wang
- Department of Research Institute, Gastrointestinal Hospital, Sun Yat-sen Universtiy, Guangzhou, 510655, China. .,Department of Colorectal Surgery, Gastrointestinal Hospital, Sun Yat-sen Universtiy, Guangzhou, 510655, China.
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Walsh MF, Hermann R, Lee JH, Chaturvedi L, Basson MD. Schlafen 3 Mediates the Differentiating Effects of Cdx2 in Rat IEC-Cdx2L1 Enterocytes. J INVEST SURG 2015; 28:202-207. [PMID: 26268420 PMCID: PMC4771065 DOI: 10.3109/08941939.2015.1005780] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
AIM Mature, differentiated enterocytes are essential for normal gut function and critical to recovery from pathological conditions. Little is known about the factors that regulate intestinal epithelial cell differentiation in the adult intestine. The transcription factor, Cdx2, involved in enterocytic differentiation, remains expressed in the adult. Since we have implicated Slfn3 in differentiation in vivo and in vitro, we examined whether it also mediated differentiation in the IEC-Cdx2-L1 cell model of differentiation. MATERIALS AND METHODS IEC-Cdx2-L1 cells, permanently transfected with Cdx2 under the control of isopropyl-β-D-thiogalactoside (IPTG), were stimulated to differentiate by 16-day exposure to IPTG. Transcript levels of Cdx2, Slfn 3, and villin were determined by quantitative reverse transcriptase-polymerase chain reaction of mRNA isolated from IPTG-treated and control cells. Slfn3 expression was lowered with specific siRNA to investigate the role of Slfn3 in Cdx2-driven villin expression in IPTG-differentiated cells. RESULTS Slfn3 and villin expression were significantly greater in IPTG-treated cells. Slfn3 siRNA lowered Slfn3 expression and abolished the IPTG-induced rise in villin expression (p < .05 by ANOVA); Cdx2 expression was unaffected by Slfn3 siRNA. DISCUSSION The data indicate that the presence of Slfn3 is required for Cdx2 to induce villin expression, and thus differentiation. However, Slfn3 must also promote differentiation of Cdx2 independently since IEC-6 cells that do not normally express Cdx2 can be differentiated by a variety of Slfn3-dependent mechanisms.
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Affiliation(s)
- Mary F Walsh
- Department of Surgery, College of Human Medicine, Michigan State University, East Lansing, Michigan, USA
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Chaturvedi L, Sun K, Walsh MF, Kuhn LA, Basson MD. The P-loop region of Schlafen 3 acts within the cytosol to induce differentiation of human Caco-2 intestinal epithelial cells. BIOCHIMICA ET BIOPHYSICA ACTA 2014; 1843:3029-3037. [PMID: 25261706 PMCID: PMC4487865 DOI: 10.1016/j.bbamcr.2014.09.017] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/05/2014] [Revised: 09/05/2014] [Accepted: 09/17/2014] [Indexed: 01/26/2023]
Abstract
Schlafen 3 (Slfn3) mediates rodent enterocyte differentiation in vitro and in vivo, required for intestinal function. Little is known about Schlafen protein structure-function relationships. To define the Slfn3 domain that promotes differentiation, we studied villin and sucrase isomaltase (SI) promoter activity in Slfn3-null human Caco-2BBE cells transfected with full-length rat Slfn3 DNA or truncated constructs. Confocal microscopy and Western blots showed that Slfn3 is predominantly cytosolic. Villin promoter activity, increased by wild type Slfn3, was further enhanced by adding a nuclear exclusion sequence, suggesting that Slfn3 does not affect transcription by direct nuclear action. We therefore sought to dissect the region in Slfn3 stimulating promoter activity. Since examination of the Slfn3 N-terminal region revealed sequences similar to both an aminopeptidase (App) and a divergent P-loop resembling those in NTPases, we initially divided Slfn3 into an N-terminal domain containing the App and P-loop regions, and a C-terminal region. Only the N-terminal construct stimulated promoter activity. Further truncation indicated that both the App and the smaller P-loop constructs enhanced promoter activity similarly to the N-terminal sequence. Point mutations within the N-terminal region (R128L, altering a critical active site residue in the App domain, and L212D, conserved in Schlafens but variable in P-loop proteins) did not affect activity. These results show that Slfn3 acts in the cytosol to trigger a secondary signal cascade that elicits differentiation marker expression and narrows the active domain to the third of the Slfn3 sequence homologous to P-loop NTPases, a first step in understanding its mechanism of action.
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Affiliation(s)
| | - Kelian Sun
- Department of Surgery, Michigan State University, East Lansing, MI, USA.
| | - Mary F Walsh
- Department of Surgery, Michigan State University, East Lansing, MI, USA.
| | - Leslie A Kuhn
- Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI, USA; Computer Science & Engineering, Michigan State University, East Lansing, MI, USA.
| | - Marc D Basson
- Department of Surgery, Michigan State University, East Lansing, MI, USA.
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Kovalenko PL, Yuan L, Sun K, Kunovska L, Seregin S, Amalfitano A, Basson MD. Regulation of epithelial differentiation in rat intestine by intraluminal delivery of an adenoviral vector or silencing RNA coding for Schlafen 3. PLoS One 2013; 8:e79745. [PMID: 24244554 PMCID: PMC3823574 DOI: 10.1371/journal.pone.0079745] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2013] [Accepted: 09/23/2013] [Indexed: 12/15/2022] Open
Abstract
Although we stimulate enterocytic proliferation to ameliorate short gut syndrome or mucosal atrophy, less effort has been directed at enterocytic differentiation. Schlafen 3 (Slfn3) is a poorly understood protein induced during IEC-6 enterocytic differentiation. We hypothesized that exogenous manipulation of Slfn3 would regulate enterocytic differentiation in vivo. Adenoviral vector coding for Slfn3 cDNA (Ad-GFP-Slfn3) or silencing RNA for Slfn3 (siSlfn3) was introduced intraluminally into rat intestine. We assessed Slfn3, villin, sucrase-isomaltase (SI), Dpp4, and Glut2 by qRT-PCR, Western blot, and immunohistochemistry. We also studied Slfn3 and these differentiation markers in atrophic defunctionalized jejunal mucosa and the crypt-villus axis of normal jejunum. Ad-GFP-Slfn3 but not Ad-GFP increased Slfn3, villin and Dpp4 expression in human Caco-2 intestinal epithelial cells. Injecting Ad-GFP-Slfn3 into rat jejunum in vivo increased mucosal Slfn3 mRNA three days later vs. intraluminal Ad-GFP. This Slfn3 overexpression was associated with increases in all four differentiation markers. Injecting siSlfn3 into rat jejunum in vivo substantially reduced Slfn3 and all four intestinal mucosal differentiation markers three days later, as well as Dpp4 specific activity. Endogenous Slfn3 was reduced in atrophic mucosa from a blind-end Roux-en-Y anastomosis in parallel with differentiation marker expression together with AKT and p38 signaling. Slfn3 was more highly expressed in the villi than the crypts, paralleling Glut2, SI and Dpp4. Slfn3 is a key intracellular regulator of rat enterocytic differentiation. Understanding how Slfn3 works may identify targets to promote enterocytic differentiation and maintain mucosal function in vivo, facilitating enteral nutrition and improving survival in patients with mucosal atrophy or short gut syndrome.
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Affiliation(s)
- Pavlo L. Kovalenko
- Department of Surgery, Michigan State University, East Lansing, Michigan, United States of America
| | - Lisi Yuan
- Department of Surgery, Michigan State University, East Lansing, Michigan, United States of America
- Research Service, John D. Dingell Veterans Affairs Medical Center, Detroit, Michigan, United States of America
| | - Kelian Sun
- Department of Surgery, Michigan State University, East Lansing, Michigan, United States of America
| | - Lyudmyla Kunovska
- Department of Surgery, Michigan State University, East Lansing, Michigan, United States of America
| | - Sergey Seregin
- Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, United States of America
| | - Andrea Amalfitano
- Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, United States of America
| | - Marc D. Basson
- Department of Surgery, Michigan State University, East Lansing, Michigan, United States of America
- Research Service, John D. Dingell Veterans Affairs Medical Center, Detroit, Michigan, United States of America
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Chaturvedi LS, Basson MD. Glucagonlike peptide 2 analogue teduglutide: stimulation of proliferation but reduction of differentiation in human Caco-2 intestinal epithelial cells. JAMA Surg 2013; 148:1037-1042. [PMID: 24068167 PMCID: PMC4574866 DOI: 10.1001/jamasurg.2013.3731] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
IMPORTANCE Short bowel syndrome occurs when a shortened intestine cannot absorb sufficient nutrients or fluids. Teduglutide is a recombinant analogue of human glucagonlike peptide 2 that reduces dependence on parenteral nutrition in patients with short bowel syndrome by promoting enterocytic proliferation, increasing the absorptive surface area. However, enterocyte function depends not only on the number of cells that are present but also on differentiated features that facilitate nutrient absorption and digestion. OBJECTIVE To test the hypothesis that teduglutide impairs human intestinal epithelial differentiation. DESIGN AND SETTING We investigated the effects of teduglutide in the modulation of proliferation and differentiation in human Caco-2 intestinal epithelial cells at a basic science laboratory. This was an in vitro study using Caco-2 cells, a human-derived intestinal epithelial cell line commonly used to model enterocytic biology. EXPOSURE Cells were exposed to teduglutide or vehicle control. MAIN OUTCOMES AND MEASURES We analyzed the cell cycle by bromodeoxyuridine incorporation or propidium iodide staining and flow cytometry and measured cell proliferation by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. We used quantitative reverse transcription-polymerase chain reaction to assay the expression of the enterocytic differentiation markers villin, sucrase-isomaltase, glucose transporter 2 (GLUT2), and dipeptidyl peptidase 4 (DPP-4), as well as that of the putative differentiation signals schlafen 12 (SLFN12) and caudal-related homeobox intestine-specific transcription factor (Cdx2). Villin promoter activity was measured by a luciferase-based assay. RESULTS The MTS assay demonstrated that teduglutide increased cell numbers by a mean (SD) of 10% (2%) over untreated controls at a maximal 500 nM (n = 6, P < .05). Teduglutide increased bromodeoxyuridine-positive cells vs untreated controls by a mean (SD) of 19.4% (2.3%) vs 12.0% (0.8%) (n = 6, P < .05) and increased the S-phase fraction by flow cytometric analysis. Teduglutide reduced the mean (SD) expression of villin by 29% (6%), Cdx2 by 31% (10%), DPP-4 by 15% (6%), GLUT2 by 40% (11%), SLFN12 by 61% (14%), and sucrase-isomaltase by 28% (8%) (n = 6, P < .05 for all). CONCLUSIONS AND RELEVANCE Teduglutide increased Caco-2 proliferation but tended to inhibit intestinal epithelial differentiation. The effects of mitogenic stimulation with teduglutide in patients with short bowel syndrome might be greater if the more numerous teduglutide-treated cells could be stimulated toward a more fully differentiated phenotype.
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Affiliation(s)
- Lakshmi S Chaturvedi
- Department of Surgery, College of Human Medicine, Michigan State University, East Lansing2Research Service, John D. Dingell Veterans Affairs Medical Center, Detroit, Michigan3Department of Anesthesiology, Wayne State University, Detroit, Michigan
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Kovalenko PL, Basson MD. The correlation between the expression of differentiation markers in rat small intestinal mucosa and the transcript levels of schlafen 3. JAMA Surg 2013; 148:1013-1019. [PMID: 24005468 PMCID: PMC4590985 DOI: 10.1001/jamasurg.2013.3572] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
IMPORTANCE The normal absorptive function and structural maintenance of the intestinal mucosa depend on a constant process of proliferation of enterocytic stem cells followed by progressive differentiation toward a mature phenotype. The mechanisms that govern enterocytic differentiation in the mucosa of the small intestine are poorly understood. OBJECTIVE To determine whether schlafen 3 (but not other schlafen proteins) act in vivo and whether its effects are limited to the small intestine. We have previously demonstrated in nonmalignant rat intestinal IEC-6 cells that schlafen 3 levels correlate with the expression of various differentiation markers in vitro in response to differentiation stimuli. DESIGN Randomized controlled experiment. SETTING Animal science laboratory. PARTICIPANTS Male Sprague-Dawley rats 8 to 13 weeks old. MAIN OUTCOMES AND MEASURES Messenger RNA (mRNA) from jejunal and colonic mucosa was isolated, and transcript levels of schlafen proteins 1, 2, 3, 4, 5, 13, and 14; sucrase isomaltase (SI); dipeptidyl peptidase 4 (Dpp4); glucose transporter type 2 (Glut2); and villin were measured by quantitative reverse transcriptase-polymerase chain reaction. We tested parallel variations in protein levels by Western blotting and Dpp4 enzyme activity. RESULTS The transcript level of schlafen 3 (Slfn3) correlated with the levels of the differentiation markers SI, Dpp4, Glut2, and villin. However, the expression of schlafen proteins 1, 2, 4, 5, 13, and 14 did not correlate with the expression of the differentiation markers. The mucosal mRNA levels of Slfn3, SI, Glut2, and Dpp4 were all substantially higher in the rat jejunum than in colonic mucosa by a mean (SE) factor of 51.0 (13.2) for 6 rats (P < .05), 599 (99) for 8 rats (P < .01), 12.5 (5.5) for 8 rats (P < .01), and 14.0 (3.9) for 8 rats (P < .01), respectively. In IEC-6 cells, infection with adenovirus-expressing GFP-tagged Slfn3 significantly increased Slfn3 expression and Dpp4-specific activity compared with GFP-expressing virus (in 6 rats; P < .05). CONCLUSIONS AND RELEVANCE Taken together with our previous in vitro observations, the results suggest that small intestinal enterocytic epithelial differentiation in rats may be regulated by Slfn3 in vivo, as in vitro, and that these effects may be specific to the small intestinal enterocytic phenotype as opposed to that of the mature colonocyte. Slfn3 human orthologs may be targeted to stimulate intestinal differentiation in patients with short bowel syndrome.
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Mavrommatis E, Fish EN, Platanias LC. The schlafen family of proteins and their regulation by interferons. J Interferon Cytokine Res 2013; 33:206-10. [PMID: 23570387 DOI: 10.1089/jir.2012.0133] [Citation(s) in RCA: 117] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
The Schlafen (SLFN) family of proteins includes several mouse and human members. There is emerging evidence that members of this family of proteins are involved in important functions, such as the control of cell proliferation, induction of immune responses, and the regulation of viral replication. These proteins span across all species with great diversity, with 10 murine and 5 human isoforms. Recent work has established that mouse and human SLFN proteins are regulated by interferons (IFNs). Several Slfn genes were shown to be induced as classical interferon-stimulated genes, and emerging evidence suggests that these proteins play important roles in the growth inhibitory and antineoplastic effects of IFNs. In the current review, the known properties of mouse and human SLFNs are reviewed, and the implications of their emerging functions are discussed.
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Affiliation(s)
- Evangelos Mavrommatis
- Robert H. Lurie Comprehensive Cancer Center, Northwestern University Medical School, Chicago, IL 60611, USA
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Shaw D, Gohil K, Basson MD. Intestinal mucosal atrophy and adaptation. World J Gastroenterol 2012; 18:6357-6375. [PMID: 23197881 PMCID: PMC3508630 DOI: 10.3748/wjg.v18.i44.6357] [Citation(s) in RCA: 89] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/06/2012] [Revised: 11/06/2012] [Accepted: 11/14/2012] [Indexed: 02/06/2023] Open
Abstract
Mucosal adaptation is an essential process in gut homeostasis. The intestinal mucosa adapts to a range of pathological conditions including starvation, short-gut syndrome, obesity, and bariatric surgery. Broadly, these adaptive functions can be grouped into proliferation and differentiation. These are influenced by diverse interactions with hormonal, immune, dietary, nervous, and mechanical stimuli. It seems likely that clinical outcomes can be improved by manipulating the physiology of adaptation. This review will summarize current understanding of the basic science surrounding adaptation, delineate the wide range of potential targets for therapeutic intervention, and discuss how these might be incorporated into an overall treatment plan. Deeper insight into the physiologic basis of adaptation will identify further targets for intervention to improve clinical outcomes.
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