• Humans
• Gastrointestinal Neoplasms/genetics
• Single-Cell Analysis
• Neural Networks, Computer

• AMP-18
• Cancer disease
• DNA- repair
• Gene polymorphism
• Reactive oxygen species

• Humans
• Proteomics
• DNA, Mitochondrial
• Incidence
• Genomics
• Neoplasms/genetics

• CAPN8
• immunotherapy
• prognosis
• thyroid cancer
• tumor immune microenvironment

• Humans
• Thyroid Neoplasms/genetics
• Tumor Microenvironment
• Prognosis
• Immunotherapy
• DNA Copy Number Variations

• Heterorhabditis
• Photorhabdus
• anti-inflammatory and anticancer
• antimicrobial
• antiprotozoal
• nematodes

• Council of Scientific and Industrial Research, India

• Bioinformatics analysis
• calpains
• immune infiltration
• pancreatic cancer
• prognosis

• Machado Joseph disease
• calpain
• polyglutamine
• spinocerebellar ataxia-3
• zebrafish

• Animals
• Antineoplastic Agents/pharmacology
• Antineoplastic Agents/therapeutic use
• Ataxin-3/drug effects
• Disease Models, Animal
• Glycoproteins/metabolism
• Neurodegenerative Diseases/drug therapy
• Zebrafish

• CAPN
• CAPNS1
• CAST
• calpain
• calpastatin
• cancer

Helicobacter pylori causes persistent infection in the gastric epithelium of more than half of the world’s population, leading to the development of severe complications such as peptic ulcer diseases, gastric cancer, and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Several virulence factors, including cytotoxin-associated gene A (CagA), which is translocated into the gastric epithelium via the type 4 secretory system (T4SS), have been indicated to play a vital role in disease development. Although infection with strains harboring the East Asian type of CagA possessing the EPIYA-A, -B, and -D sequences has been found to potentiate cell proliferation and disease pathogenicity, the exact mechanism of CagA involvement in disease severity still remains to be elucidated. Therefore, we discuss the possible role of CagA in gastric pathogenicity.

• CagA
• H. pylori
• cag pathogenicity island
• gastric cancer
• virulence factor

• Antigens, Bacterial/chemistry
• Antigens, Bacterial/genetics
• Antigens, Bacterial/metabolism
• Bacterial Proteins/chemistry
• Bacterial Proteins/genetics
• Bacterial Proteins/metabolism
• Gastric Mucosa/metabolism
• Gastric Mucosa/microbiology
• Helicobacter Infections/genetics
• Helicobacter Infections/microbiology
• Helicobacter pylori/pathogenicity
• Humans
• Peptic Ulcer/metabolism
• Peptic Ulcer/microbiology
• Phosphorylation
• Stomach Neoplasms/metabolism
• Stomach Neoplasms/microbiology
• Type IV Secretion Systems/genetics
• Tyrosine/metabolism
• Virulence/genetics
• Virulence Factors/genetics

• Exosome
• GKN1
• Gastric cancer
• Ras signaling
• Uptake

• Helicobacter pylori
• AU-rich element RNA-binding factor 1
• CagA
• gastric cancer
• gastrokine 1

Early detection of cancer provides effective treatment and saves lives. The objective of this study was to determine whether serum gastrokine 1 (GKN1) protein is a gastric cancer‐specific diagnostic biomarker. The serum concentration of GKN1 in healthy individuals (median: 6.34 ng/μL, interquartile range (IQR): 5.66‐7.54 ng/μL) was significantly higher compared with the levels in gastric cancer patients (median: 3.48 ng/μL, IQR: 2.90‐4.11 ng/μL; P < .0001). At the optimum cutoff (4.94 ng/μL) of serum GKN1 protein, the sensitivity and specificity were 91.2% and 96.0%, respectively, for gastric cancer. Using serum GKN1 protein as the diagnostic reference, the ROC curve showed a satisfactory diagnostic efficacy with an AUC value of 0.9954 (95% CI 0.9919‐0.9988) and Youden index of 0.8740. In addition, the diagnostic accuracy of the serum GKN1 protein at the optimum cutoff was 0.9675. Interestingly, serum GKN1 concentrations in patients with advanced gastric cancer (AGC; median: 3.11 ng/μL, IQR: 2.72‐3.72 ng/μL) were lower than in patients with early gastric cancer (EGC; median: 4.31 ng/μL, IQR: 3.88‐4.88 ng/μL). The diagnostic accuracies at the optimum serum GKN1 cutoff were 0.8912 and 0.9589 for EGC and AGC, respectively. Furthermore, the serum GKN1 concentrations robustly discriminated the patients with gastric cancer from the patients with colorectal, liver, lung, breast, pancreatic, ovary, and prostatic cancers with AUC values greater than 0.94. These data suggest that serum GKN1 is a promising and highly specific diagnostic biomarker for the prompt detection of early and advanced gastric cancers.

• GKN1
• biomarker
• gastric cancer
• liquid biopsy

• Aged
• Case-Control Studies
• Cell Line, Tumor
• Cell Proliferation
• Down-Regulation
• Early Detection of Cancer
• Female
• Gene Expression Regulation, Neoplastic
• Humans
• Male
• Middle Aged
• Neoplasm Staging
• Peptide Hormones/blood
• Sensitivity and Specificity
• Stomach Neoplasms/blood
• Stomach Neoplasms/diagnosis
• Stomach Neoplasms/pathology

• National Research Foundation of Korea
• Ministry of Education, Science and Technology

Alternative splicing (AS) is an important mRNA maturation step that allows increased variability and diversity of proteins in eukaryotes. AS is dysregulated in numerous diseases, and its implication in the carcinogenic process is well known. However, progress in understanding how oncogenic viruses modulate splicing, and how this modulation is involved in viral oncogenicity has been limited. Epstein-Barr virus (EBV) is involved in various cancers, and its EBNA1 oncoprotein is the only viral protein expressed in all EBV malignancies.

In the present study, the ability of EBNA1 to modulate the AS of cellular genes was assessed using a high-throughput RT-PCR approach to examine AS in 1238 cancer-associated genes. RNA immunoprecipitation coupled to RNA sequencing (RIP-Seq) assays were also performed to identify cellular mRNAs bound by EBNA1.

Upon EBNA1 expression, we detected modifications to the AS profiles of 89 genes involved in cancer. Moreover, we show that EBNA1 modulates the expression levels of various splicing factors such as hnRNPA1, FOX-2, and SF1. Finally, RNA immunoprecipitation coupled to RIP-Seq assays demonstrate that EBNA1 immunoprecipitates specific cellular mRNAs, but not the ones that are spliced differently in EBNA1-expressing cells.

The EBNA1 protein can modulate the AS profiles of numerous cellular genes. Interestingly, this modulation protein does not require the RNA binding activity of EBNA1. Overall, these findings underline the novel role of EBNA1 as a cellular splicing modulator.

The online version of this article (10.1186/s12985-019-1137-5) contains supplementary material, which is available to authorized users.

• Alternative splicing
• EBNA1
• Epstein-Barr virus
• High-throughput RT-PCR
• RIP-sequencing
• Splicing factors
• Virus-host interaction

Hirschsprung disease is a birth defect characterized by complete absence of neuronal ganglion cells from a portion of the intestinal tract. To uncover genetic variants contributing to HSCR, we performed whole exome sequencing on seven members of an HSCR family. With the minor allele frequency (MAF) calculated by gnomAD, we finally filtered a total of 1,059 rare variants in this family (MAF < 0.1%). With the mode of inheritance and pathogenicity scores by bioinformatics tools, we identified an in-frameshift variant p.Phe147del in RET as the disease-causing variant. Further analysis revealed that the in-frameshift variant may function by disrupting the glycosylation of RET protein. To our knowledge, this is the first study to report the in-frameshift variant p.Phe147del in RET responsible for heritable HSCR.

• Hirschsprung disease
• RET variant
• bioinformatics
• minor allele frequencies
• whole exome sequencing

• Exosome
• GKN1
• Gastric cancer
• Theragnosis

• Ascites
• Biomarkers
• Extracellular vesicles
• Ovarian cancer
• Peritoneal fluids

• Aged
• Aged, 80 and over
• Ascites/pathology
• Ascitic Fluid/metabolism
• Biomarkers, Tumor
• Cell Line, Tumor
• Cell-Free Nucleic Acids
• Extracellular Vesicles/genetics
• Female
• High-Throughput Nucleotide Sequencing
• Humans
• Liquid Biopsy
• MicroRNAs/genetics
• Middle Aged
• Neoplasm Grading
• Neoplasm Staging
• Ovarian Neoplasms/genetics
• Ovarian Neoplasms/pathology
• RNA, Messenger/genetics
• Real-Time Polymerase Chain Reaction
• Reproducibility of Results

• calcium
• calpain
• dimerization
• non-proteolytic function
• protease
• protease complex
• proteolysis

• Calpain2
• GAS2
• GAS2DN
• RNAi
• immunoprecipitation

• Biomarkers, Tumor/metabolism
• Calpain/metabolism
• Cell Line, Tumor
• Cell Proliferation
• Humans
• Jurkat Cells
• Leukemia/metabolism
• Leukemia/pathology
• Microfilament Proteins/metabolism
• Signal Transduction

• AGR2
• AGR3
• Cancer
• PDI family

The calpains are intracellular cysteine proteases that function in a variety of important cellular functions, including signalling, motility, apoptosis and survival. In breast cancer high calpain-1 and calpain-2 expression has been associated with adverse clinical outcome. Calpain-9 was thought to be exclusively expressed in the digestive tract; however recent studies have shown that this protein is also expressed in breast tissue.

We investigated the expression of calpain-9 in a large cohort of early stage breast cancer patients (n = 783) using immunohistochemistry on a tissue microarray. Patients had long-term follow-up information available for analysis.

Low expression of calpain-9 was associated with patients over 40 years of age (P = 0.025), smaller tumour size (P = 0.001), lower tumour stage (P = 0.009), a more favourable Nottingham Prognostic Index value (P = 0.002) and positive oestrogen receptor status (P = 0.014). Calpain-9 expression was not associated with survival in the total patient cohort, however low calpain-9 expression was associated with adverse survival in patients who received endocrine therapy (P = 0.033), which remained significant in multivariate Cox regression analysis accounting for potential confounding factors (hazard ratio (HR) = 0.56, 95% confidence interval (95% CI) = 0.36-0.89, P = 0.013). Low calpain-9 expression was also associated with adverse survival in patients with an intermediate Nottingham Prognostic Index value (P = 0.009), and remained so in multivariate analysis (HR = 0.54, 95% CI = 0.36-0.82, P = 0.003).

This study suggests that calpain-9 may play a role in breast cancer and that low expression is associated with poorer patient clinical outcome following endocrine therapy. Validation studies are warranted as determining expression of calpain-9 may provide important prognostic information.

Homeostatic imbalance between cell proliferation and death in gastric mucosal epithelia may lead to gastritis and gastric cancer. Despite abundant gastrokine 1 (GKN1) expression in the normal stomach, the loss of GKN1 expression is frequently detected in gastric mucosa infected with Helicobacter pylori, as well as in intestinal metaplasia and gastric cancer tissues, suggesting that GKN1 plays an important role in gastric mucosal defense, and the gene functions as a gastric tumor suppressor. In the stomach, GKN1 is involved in gastric mucosal inflammation by regulating cytokine production, the nuclear factor-κB signaling pathway, and cyclooxygenase-2 expression. GKN1 also inhibits the carcinogenic potential of H. pylori protein CagA by binding to it, and up-regulates antioxidant enzymes. In addition, GKN1 reduces cell viability, proliferation, and colony formation by inhibiting cell cycle progression and epigenetic modification by down-regulating the expression levels of DNMT1 and EZH2, and DNMT1 activity, and inducing apoptosis through the death receptor-dependent pathway. Furthermore, GKN1 also inhibits gastric cancer cell invasion and metastasis via coordinated regulation of epithelial mesenchymal transition-related protein expression, reactive oxygen species production, and PI3K/Akt signaling pathway activation. Although the modes of action of GKN1 have not been clearly described, recent limited evidence suggests that GKN1 acts as a gastric-specific tumor suppressor. This review aims to discuss, comment, and summarize the recent progress in the understanding of the role of GKN1 in gastric cancer development and progression.

• GKN1 protein
• Gastritis
• Homeostasis
• Stomach neoplasms
• Tumor suppressor gene

Calpains are ubiquitous non-lysosomal Ca²⁺-dependent cysteine proteases also present in myocardial cytosol and mitochondria. Numerous experimental studies reveal an essential role of the calpain system in myocardial injury during ischemia, reperfusion and postischemic structural remodelling. The increasing Ca²⁺-content and Ca²⁺-overload in myocardial cytosol and mitochondria during ischemia and reperfusion causes an activation of calpains. Upon activation they are able to injure the contractile apparatus and impair the energy production by cleaving structural and functional proteins of myocytes and mitochondria. Besides their causal involvement in acute myocardial dysfunction they are also involved in structural remodelling after myocardial infarction by the generation and release of proapoptotic factors from mitochondria. Calpain inhibition can prevent or attenuate myocardial injury during ischemia, reperfusion, and in later stages of myocardial infarction.

• Calpain
• Calpain inhibition
• Calcium overload
• Myocardial injury
• Ischemia
• Reperfusion
• Myocardial infarction
• Remodelling

• Apoptosis
• Calpain
• Migration
• Proliferation
• Survival

• E-cadherin
• Gastrokine-1
• Gestational trophoblastic neoplasias
• JEG-3
• MMP-9
• Placenta
• Trefoil factor

• 1000 Genomes data
• BRICHOS domain
• population genetics
• selective sweeps

• linkage analysis
• genetic association
• common variation
• keratoconus
• calpains
• calpastatin

• Calcium-Binding Proteins/genetics
• Case-Control Studies
• Chromosome Mapping
• Cysteine Proteinase Inhibitors/genetics
• Female
• Genetic Association Studies
• Genetic Linkage
• Genetic Predisposition to Disease
• Genotype
• Genotyping Techniques
• Humans
• Keratoconus/genetics
• Male
• Microsatellite Repeats
• Middle Aged
• Pedigree
• Polymorphism, Single Nucleotide

• UL1 RR033176 NCRR NIH HHS
• UL1 TR000124 NCATS NIH HHS
• R01 EY009052 NEI NIH HHS
• R01-09,052 PHS HHS
• UL1RR033176 NCRR NIH HHS
• UL1TR000124 NCATS NIH HHS

Conventional calpains are ubiquitous cysteine proteases whose activity is promoted by calcium signaling and specifically limited by calpastatin. Calpain expression has been shown to be increased in human malignant cells, but the contribution of the calpain/calpastatin system in tumorigenesis remains unclear. It may play an important role in tumor cells themselves (cell growth, migration, and a contrario cell death) and/or in tumor niche (tissue infiltration by immune cells, neo-angiogenesis). In this study, we have used a mouse model of melanoma as a tool to gain further understanding of the role of calpains in tumor progression. To determine the respective importance of each target, we overexpressed calpastatin in tumor and/or host in isolation. Our data demonstrate that calpain inhibition in both tumor and host blunts tumor growth, while paradoxically increasing metastatic dissemination to regional lymph nodes. Specifically, calpain inhibition in melanoma cells limits tumor growth in vitro and in vivo but increases dissemination by amplifying cell resistance to apoptosis and accelerating migration process. Meanwhile, calpain inhibition restricted to host cells blunts tumor infiltration by immune cells and angiogenesis required for antitumor immunity, allowing tumor cells to escape tumor niche and disseminate. The development of highly specific calpain inhibitors with potential medical applications in cancer should take into account the opposing roles of the calpain/calpastatin system in initial tumor growth and subsequent metastatic dissemination.

• Animals
• Calcium-Binding Proteins/genetics
• Calcium-Binding Proteins/metabolism
• Calpain/genetics
• Calpain/metabolism
• Cell Line, Tumor
• Disease Models, Animal
• Male
• Melanoma/genetics
• Melanoma/metabolism
• Melanoma/pathology
• Mice
• Mice, Inbred C57BL
• Mice, Transgenic

Gastrokine-1 (GKN1), a secreted protein, is specifically expressed in gastric mucosa to protect and maintain the integrity of gastric epithelium. The present study investigated differential expression of GKN1 in normal, precancerous, and cancerous gastric tissues, and explored the biological functions of GKN1 protein in gastric cancer cells.

RT-PCR, Western blot, and immunohistochemistry were performed to detect GKN1 expression in normal, precancerous, cancerous gastric tissues and seven gastric cancer cell lines. Gene transfection was used to restore GKN1 expression in gastric cancer AGS cells. Phenotypic changes (i.e., cell viability, apoptosis, cell cycle modulation, and sensitivity of gastric cancer cells to fluorouracil (5-FU)) were assayed in the transfected cells. DNA microarrays were used to analyze expression changes of apoptosis-related genes.

Significant downregulation or absence of GKN1 expression in seven gastric cancer cell lines were detected and progressive decrease of GKN1 expression from normal mucosa, precancerous tissue, to cancer tissues was observed. Moreover, restoration of GKN1 expression suppressed gastric cancer cell viability and induced the cells to undergo apoptosis. GKN1 expression also enhanced tumor cell sensitivity to 5-FU treatment. Moreover, it was found that GKN1 expression in AGS cells modulated expression of 19 apoptosis-related genes.

Expression of GKN1 is progressively lost from normal mucosa, precancerous to cancerous gastric tissues, while restoration of GKN1 expression induces gastric cancer cells to undergo apoptosis, and enhances sensitivity of gastric cancer cells to 5-FU-induced apoptosis.

The calpains are physiologically important Ca²⁺-activated regulatory proteases, which are divided into typical or atypical sub-families based on constituent domains. Both sub-families are present in mammals, but our understanding of calpain function is based primarily on typical sub-family members. Here, we take advantage of the model organism Caenorhabditis elegans, which expresses only atypical calpains, to extend our knowledge of the phylogenetic evolution and function of calpains. We provide evidence that a typical human calpain protein with a penta EF hand, detected using custom profile hidden Markov models, is conserved in ancient metazoans and a divergent clade. These analyses also provide evidence for the lineage-specific loss of typical calpain genes in C. elegans and Ciona, and they reveal that many calpain-like genes lack an intact catalytic triad. Given the association between the dysregulation of typical calpains and human degenerative pathologies, we explored the phenotypes, expression profiles, and consequences of inappropriate reduction or activation of C. elegans atypical calpains. These studies show that the atypical calpain gene, clp-1, contributes to muscle degeneration and reveal that clp-1 activity is sensitive to genetic manipulation of [Ca²⁺]_i. We show that CLP-1 localizes to sarcomeric sub-structures, but is excluded from dense bodies (Z-disks). We find that the muscle degeneration observed in a C. elegans model of dystrophin-based muscular dystrophy can be suppressed by clp-1 inactivation and that nemadipine-A inhibition of the EGL-19 calcium channel reveals that Ca²⁺ dysfunction underlies the C. elegans MyoD model of myopathy. Taken together, our analyses highlight the roles of calcium dysregulation and CLP-1 in muscle myopathies and suggest that the atypical calpains could retain conserved roles in myofilament turnover.

Calpains are calcium activated non-lysosomal proteases that cleave proteins with exquisite selectivity. Proteins can be activated by calpain cleavage, because they are released from inhibitory constraints, or they can be targeted for further degradation to facilitate their normal physiological turnover or to promote cellular remodelling. Inappropriate calpain activity can lead to degenerative pathologies and cancers. Our understanding of calpain function is based primarily on typical calpains, which carry EF hand motifs that bind Ca²⁺ or mediate dimerization; however, typical and atypical calpains, which lack EF hand motifs, are both present in mammals. Hence, any therapeutic intervention designed to suppress degenerative conditions, particularly those caused by elevated Ca²⁺ levels, should also consider the potential involvement of atypical calpains. We have taken advantage of the model organism C. elegans, which only encodes atypical calpain proteins, to gain an understanding of the evolution and activities of these proteins. We show that the CLP-1 atypical calpain is normally expressed in muscle and localizes to sarcomeric sub-structures. We find that CLP-1 contributes to the muscle degeneration observed in a model of Duchenne muscular dystrophy. Our studies also highlight the importance of calcium dysregulation in promoting CLP-1 activity and muscle degeneration.

AIM: To search for and validate differentially expressed proteins in patients with gastric adenocarcinoma.

METHODS: We used two-dimensional gel electrophoresis and mass spectrometry to search for differentially expressed proteins in patients with gastric adenocarcinoma. A set of proteins was validated with immunoblotting.

RESULTS: We identified 30 different proteins involved in various biological processes: metabolism, development, death, response to stress, cell cycle, cell communication, transport, and cell motility. Eight proteins were chosen for further validation by immunoblotting. Our results show that gastrokine-1, 39S ribosomal protein L12 (mitochondrial precursor), plasma cell-induced resident endoplasmic reticulum protein, and glutathione S-transferase mu 3 were significantly underexpressed in gastric adenocarcinoma relative to adjacent non-tumor tissue samples. On the other hand, septin-2, ubiquitin-conjugating enzyme E2 N, and transaldolase were significantly overexpressed. Translationally controlled tumor protein was shown to be differentially expressed only in patients with cancer of the gastric cardia/esophageal border.

CONCLUSION: This work presents a set of possible diagnostic biomarkers, validated for the first time. It might contribute to the efforts of understanding gastric cancer carcinogenesis.

• Two-dimensional gel electrophoresis
• Mass spectrometry
• Gastric adenocarcinoma
• Proteome
• Biomarkers

• Apoptosis/genetics
• Caspase 3/metabolism
• Cell Line, Tumor
• Cell Transformation, Neoplastic/genetics
• Cell Transformation, Neoplastic/metabolism
• Fas Ligand Protein/biosynthesis
• Fas Ligand Protein/genetics
• Fas Ligand Protein/metabolism
• Gene Expression Regulation, Neoplastic/physiology
• Humans
• Peptide Hormones/biosynthesis
• Peptide Hormones/genetics
• Peptide Hormones/physiology
• Signal Transduction/genetics
• Stomach Neoplasms/genetics
• Stomach Neoplasms/metabolism
• Stomach Neoplasms/pathology
• fas Receptor/biosynthesis
• fas Receptor/genetics
• fas Receptor/physiology

• Calpain/physiology
• Cell Death
• Cell Movement
• Humans
• Neoplasms/etiology
• Neoplasms/pathology
• Neoplasms/therapy
• Signal Transduction

• D20453 Biotechnology and Biological Sciences Research Council
• Cancer Research UK

• Amino Acid Sequence
• Animals
• Binding Sites
• Calcium/metabolism
• Calcium/pharmacology
• Calpain/chemistry
• Calpain/metabolism
• Catalytic Domain
• Chromatography, Gel
• Electrophoresis, Polyacrylamide Gel
• Enzyme Activation/drug effects
• Hydrophobic and Hydrophilic Interactions
• Kinetics
• Models, Biological
• Models, Molecular
• Molecular Sequence Data
• Molecular Weight
• Protein Multimerization/drug effects
• Protein Structure, Tertiary
• Protein Subunits/chemistry
• Protein Subunits/metabolism
• Rats
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

• 74681 Canadian Institutes of Health Research

• calpains
• cancer
• migration
• invasion
• apoptosis

• Animals
• Antineoplastic Agents/pharmacology
• Apoptosis/drug effects
• Calpain/drug effects
• Calpain/metabolism
• Cell Survival/drug effects
• Drug Delivery Systems
• Humans
• Neoplasms/drug therapy
• Neoplasms/physiopathology
• Neovascularization, Pathologic/drug therapy
• Neovascularization, Pathologic/physiopathology

• R01 GM069668-07 NIGMS NIH HHS
• R01 GM063569-06 NIGMS NIH HHS
• R01 GM063569 NIGMS NIH HHS
• R01 GM069668-08 NIGMS NIH HHS
• R01 GM063569-07 NIGMS NIH HHS
• R01 GM069668 NIGMS NIH HHS
• R56 GM063569-05A1 NIGMS NIH HHS
• R56 GM063569 NIGMS NIH HHS
• R01 GM069668-06 NIGMS NIH HHS

Calpains constitute a superfamily of Ca²⁺-dependent cysteine proteases, indispensable for various cellular processes. Among the 15 mammalian calpains, calpain 8/nCL-2 and calpain 9/nCL-4 are predominantly expressed in the gastrointestinal tract and are restricted to the gastric surface mucus (pit) cells in the stomach. Possible functions reported for calpain 8 are in vesicle trafficking between ER and Golgi, and calpain 9 are implicated in suppressing tumorigenesis. These highlight that calpains 8 and 9 are regulated differently from each other and from conventional calpains and, thus, have potentially important, specific functions in the gastrointestinal tract. However, there is no direct evidence implicating calpain 8 or 9 in human disease, and their properties and physiological functions are currently unknown. To address their physiological roles, we analyzed mice with mutations in the genes for these calpains, Capn8 and Capn9. Capn8^−/− and Capn9^−/− mice were fertile, and their gastric mucosae appeared normal. However, both mice were susceptible to gastric mucosal injury induced by ethanol administration. Moreover, the Capn8^−/− stomach showed significant decreases in both calpains 9 and 8, and the same was true for Capn9^−/−. Consistent with this finding, in the wild-type stomach, calpains 8 and 9 formed a complex we termed “G-calpain,” in which both were essential for activity. This is the first example of a “hybrid” calpain complex. To address the physiological relevance of the calpain 8 proteolytic activity, we generated calpain 8:C105S “knock-in” (Capn8^CS/CS) mice, which expressed a proteolytically inactive, but structurally intact, calpain 8. Although, unlike the Capn8^−/− stomach, that of the Capn8^CS/CS mice expressed a stable and active calpain 9, the mice were susceptible to ethanol-induced gastric injury. These results provide the first evidence that both of the gastrointestinal-tract-specific calpains are essential for gastric mucosal defense, and they point to G-calpain as a potential target for gastropathies caused by external stresses.

The continuous or improper ingestion of irritants, including alcohol, nonsteroidal anti-inflammatory drugs (NSAIDs), and Helicobacter pylori, often leads to serious gastropathies, affecting a wide range of people. A complex gastric defense system helps protect against these threats, for example by secreting mucus. Here we report that two gastrointestinal-tract-specific calpains, calpain 8/nCL-2 and calpain 9/nCL-4, are involved in the mucosal defense against stress-induced gastropathies. Calpains are Ca²⁺-dependent cytosolic proteases that are indispensable for various cellular processes. Improper calpain activities can result in death or serious disorders, such as muscular dystrophies and lissencephaly, although no role for calpains in gastrointestinal diseases has been reported. Here we show that mice with mutations in the genes for calpains 8 and 9 are susceptible to alcohol-induced gastric injury. Moreover, these calpains form a stable complex, in which both molecules are essential for activity. Thus, human calpains 8 and 9 may contribute to the stomach's susceptibility to stress caused by irritants such as alcohol. Indeed, some reported human single nucleotide polymorphisms (SNPs) in these calpains are predicted to compromise their proteolytic activity. Our mutant mice provide unique animal models for potential human gastropathies caused by such SNPs.