Notably, KLK8 overexpression resulted in a significant loss of plakoglobin and VE-cadherin in the plasma membrane, whereas it caused marked nuclear translocation of plakoglobin in HCAECs (Figure ?(Figure9F).9F). In addition, transgenic overexpression of KLK8 led to interstitial and perivascular cardiac fibrosis, endothelial injury and EndMT in the heart. Adenovirus-mediated overexpression of KLK8 (Ad-KLK8) resulted in increases in endothelial cell damage, permeability and transforming growth factor (TGF)-1 release in HCAECs. KLK8 overexpression also induced EndMT in HCAECs, which was alleviated by a TGF-1-neutralizing antibody. A specificity protein-1 (Sp-1) consensus site was identified in the human KLK8 promoter and was found to mediate the high glucose-induced KLK8 expression. Mechanistically, it was identified that the vascular endothelial (VE)-cadherin/plakoglobin complex may associate with KLK8 in HCAECs. KLK8 cleaved the VE-cadherin extracellular domain, thus promoting plakoglobin nuclear translocation. Plakoglobin was required for KLK8-induced EndMT by cooperating with p53. KLK8 overexpression led to plakoglobin-dependent association of p53 with hypoxia inducible factor (HIF)-1, which further enhanced the transactivation effect of HIF-1 on the TGF-1 promoter. KLK8 also induced the binding of p53 with Smad3, subsequently promoting pro-EndMT reprogramming via the TGF-1/Smad signaling pathway in HCAECs. Thein vitroand findings further demonstrated that high glucose may promote plakoglobin-dependent cooperation of p53 with HIF-1 and Smad3, subsequently increasing the expression of TGF-1 and the pro-EndMT target genes of the TGF-1/Smad signaling pathway in a KLK8-dependent manner. Conclusions: The present findings uncovered a novel pro-EndMT mechanism during the pathogenesis of diabetic cardiac fibrosis via the upregulation of KLK8, and may contribute to the development of future KLK8-based therapeutic strategies for diabetic cardiomyopathy. 0.05, ** 0.01, *** 0.001, **** 0.0001. Expression of KLK8 in diabetic myocardium was then determined by immunohistochemistry staining and western LW6 (CAY10585) blotting. As shown in Figure ?Figure1B-C,1B-C, KLK8 staining was significantly increased in both cardiomyocytes and coronary endothelial cells in the myocardium of diabetic mice compared with the expression levels exhibited by the control group. As expected, Masson’s trichrome staining revealed a significant collagen deposition in both interstitial and perivascular regions in the diabetic myocardium (Figure ?(Figure1D-E).1D-E). Immunoblotting also confirmed the induction of KLK8 protein expression in the myocardium of diabetic mice (Figure ?(Figure11F). KLK8 deficiency attenuates diabetic cardiac fibrosis Mice with global deletion of KLK8 were then used to investigate whether KLK8 deficiency affects diabetes-associated cardiac fibrosis. As shown in Mouse monoclonal to ERBB3 Figure ?Figure2A,2A, the diabetes-induced upregulation of cardiac KLK8 was blunt in KLK8-deficient (KLK8-/-) mice. STZ-induced low insulin levels and hyperglycemia occurred in both KLK8-/- and KLK8+/+ mice, whereas the levels of insulin and blood glucose in non-diabetic mice were normal (Table ?(Table1,1, Figure S1). Under baseline conditions, KLK8-/- mice exhibited similar levels of body weight, total cholesterol (TC), triglyceride (TG), free fatty acid (FFA), low LW6 (CAY10585) density lipoprotein-cholesterol (LDL-C) and high density lipoprotein-cholesterol (HDL-C) to those found in their KLK8+/+ littermates (Figure S1). STZ-induced diabetic mice exhibited increased levels of TC, TG, FFA and LDL-C, as well as decreased body weight and HDL-C levels. The plasma levels of TC and FFA were significantly lower in KLK8-/- diabetic mice compared with those in KLK8+/+ diabetic mice. However, KLK8-/- diabetic mice exhibited similar levels of body weight, TG, LDL-C and HDL-C to those observed in KLK8+/+ diabetic mice. Under baseline conditions, KLK8-/- mice exhibited similar blood pressure as their KLK8+/+ littermates. Hyperglycemia did not increase blood pressure in the early stage (12 weeks) of the disease. However, LW6 (CAY10585) 24 weeks of diabetes mellitus led.