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” (Capn8CS/CS) mice, which expressed a proteolytically inactive, but structurally intact, calpain 8. Although, unlike the Capn8−/− stomach, that of the Capn8CS/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.