Colonic PDGFRα Overexpression Accompanied Forkhead Transcription Factor FOXO3 Up-Regulation in STZ-Induced Diabetic Mice.

Background: Colonic transit disorder-induced constipation is a major complication in diabetic patients. PDGFRα+ (platelet-derived growth factor receptor α-positive) cells play critical roles in the inhibitory regulation of colonic motility, and FOXO3 (forkhead transcription factor 3) has a broad range of biological functions. The present study was designed to investigate the relationship between FOXO3 and PDGFRα+ cell proliferation in streptozotocin (STZ)- induced diabetic mice. Methods: The major experimental techniques used in this paper are immunohistochemistry, quantitative RT-RCR and Western blotting for the evaluation of specific protein expression; ChIP assay for identifying the interaction between FOXO3 protein and the PDGFRα promotor; and lentiviral transfection for the overexpression of short hairpin RNAs (shRNAs) to down-regulate FOXO3. Results: In proximal colonic smooth muscle tissue of STZinduced diabetic mice, there was a significant increase in PDGFRα and Ki67 immunoreactivity. PDGFRa mRNA and protein expression levels were both significantly increased in colonic smooth muscle tissue, but PDGFRb expression was unchanged. Meanwhile, the expression of PDGF ligands, including both PDGFα and PDGFβ, was significantly increased in diabetic colonic smooth muscle tissue. In whole cell and nuclear extracts, the expression of FOXO3 protein was also significantly increased; however, the expression of P-FOXO3 (phosphorylated FOXO3) protein was significantly decreased. When NIH cells were incubated with 50 mmol/L glucose for 12 h, 24 h and 48 h, the expression of PDGFRa significantly increased, and in whole cell and nuclear extracts, the expression of FOXO3 protein was significantly increased. However, the expression of P-FOXO3 protein was significantly decreased. FOXO3 could bind to a site on the PDGFRα promoter, and the basal expression of PDGFRa was significantly reduced when endogenous FOXO3 expression was knocked down with FOXO3 short hairpin RNA (shRNA) in NIH cells. The expression of phosphorylated Akt was significantly down-regulated in diabetic colonic muscle tissue. Conclusions: These results suggest that diabetes-induced colonic PDGFRα⁺ cell proliferation is mediated by FOXO3 up-regulation. FOXO3 up-regulation may be induced by inhibiting the PI3K/Akt signaling pathway in STZ-induced diabetic mice. PDGFRα⁺ cell proliferation could be a new target for clinical therapy of diabetes-induced colonic transit disorder. [ABSTRACT FROM AUTHOR]