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SONIC HEDGEHOG SIGNALING IS CRITICAL FOR GOBLET AND PANETH CELLS TERMINAL DIFFENTIATION AND PROTECT AGAINST INTESTINAL INFLAMMATION
J Gagné Sansfaçon, D Ménard, N PerreaultUniversité de Sherbrooke, Sherbrooke, QC
Aims: Hedgehogs (Hh) are morphogenes known to regulate numerous cell processes in various tissues. In the gastrointestinal tract, Sonic (Shh) and Indian (Ihh) hedgehog proteins have been shown to play roles in morphogenesis and cancer. Complete abrogation of Hh signaling revealed abnormal epithelial cell proliferation and defective enterocytes and myofibroblasts specification leading to mucosal inflammation with age. Also, it has been shown that loss of Ihh alone leads to increase epithelial proliferation, excessive deposition of type IV collagen and fibronectin. However, the specific role of Shh on intestinal mucosal homeostasis in the adult has not yet been explored. We propose to specifically address the molecular relevance of the Shh on intestinal homeostasis and mucosal integrity during intestinal inflammation.
Methods: With the use of the Cre/loxP system, we have generated a mouse with the Shh gene deleted exclusively in the small and large intestinal epithelium. Histological analysis was performed with HandE staining. Proliferation was analyzed by immunofluorescence. Cell specification of the main four cell types were analyzed with specific cellular staining (alcian blue and Periodic-Acid Schiff), immunostaining, Western blot, qPCR and ultrastructural analyses. The epithelial-specific Shh-null and control mice were subjected to dextran sulfate sodium induced experimental colitis and their clinical and histological symptoms were assessed.
Results: Macroscopic, histological and proliferation analyses showed no important morphological abnormalities and deregulation in mutant mice. No modulation was observed in enterocyte and enteroendocrine cells specification. Alcian blue and PAS staining revealed a significant decrease of 17% in goblets cells number and the mucin glycosylation process was altered according to the complete loss of UEA marker in mutant mice. Immunohistological analysis of Paneth cells revealed a significant 27% decrease of this cell population. Reduction in lysozyme and cryptin proteins expression was confirmed in the mutant mice. Electron microscopy analysis showed Paneth cells with a reduction in granules size and loose endoplasmic reticulum. Since the latter observation can be related to autophagy we performed a qPCR analysis of ATG16L and showed a significant decrease in its mRNA. Finally, the reduction in mucus protection coat and antimicrobial agents seen in mutant mice suggested a possible involvement in inflammatory response. Experimental colitis demonstrated increased susceptibility to the disease in Shh mutant mice.
Conclusions: Our results have highlighted an important role played by Shh signaling in goblet and Paneth cells specification and autophagy. Thus, Shh might play a protecting role in immune defense and inflammatory diseases.