PTP1B in Calpain-dependent Feedback Regulation of VEGFR2 in Endothelial Cells: Implication in VEGF-dependent Angiogenesis and Diabetic Wound Healing

The VEGF/VEGFR2/Akt/eNOS/NO pathway is essential to VEGF-induced angiogenesis. We have previously discovered a novel role of calpain in mediating VEGF-induced PI3K/AMPK/Akt/eNOS activation through Ezrin. Here, we sought to identify possible feedbak regulation of VEGFR2 by calpain via its substrate protein phosphotyrosine phosphatase 1B (PTP1B), and the relevance of this pathway to VEGF-induced angiogenesis, especially in diabetic wound healing. Over-expression of PTP1B inhibited VEGF-induced VEGFR2 and Akt phosphorylation in bovine aortic endothelial cells, while PTP1B siRNA increased both, implicating the negative regulation of VEGFR2 by PTP1B. Calpain inhibitor ALLN induced VEGFR2 activation, which can be completely blocked by PTP1B. Calpain activation induced by overexpression or Ca/A23187 resulted in PTP1B cleavage, which can be blocked by ALLN. Moreover, calpain activation inhibited VEGFinduced VEGFR2 phosphorylation, which can be restored by PTP1B siRNA. These data implicate calpain/PTP1B negative feedback regulation of VEGFR2, in addition to the primary signaling pathway of VEGF/VEGFR2/calpain/PI3K/AMPK/Akt/eNOS. We next examined a potential role of PTP1B in VEGF-induced angiogenesis. Endothelial cells transfected with PTP1B siRNA showed faster wound closure in response to VEGF. Aortic discs isolated from PTP1B siRNA transfected mice also had augmented endothelial outgrowth. Importantly, PTP1B inhibition and/or calpain overexpression significantly accelerated wound healing in STZ-induced diabetic mice. In conclusion, our data for the first time demonstrate a calpain/PTP1B/VEGFR2 negative feedback loop in the regulation of VEGF-induced angiogenesis. Modulation of local PTP1B and/or calpain activities may prove beneficial in the treatment of impaired wound healing in diabetes. INTRODUCTION Vascular endothelial growth factor (VEGF) is one of the main initiators of http://www.jbc.org/cgi/doi/10.1074/jbc.M116.766832 The latest version is at JBC Papers in Press. Published on November 21, 2016 as Manuscript M116.766832 Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on Jauary 8, 2018 hp://w w w .jb.org/ D ow nladed from Calpain/PTP1B axis in VEGFR2 regulation and angiogenesis 2 angiogenesis. Others and we have previously shown that calpain activity is increased within 10 min of VEGF stimulation of endothelial cells, which can be inhibited by antagonist of VEGF receptor (1-4). In addition, we have demonstrated an important role of calpain in mediating VEGFinduced PI3K/AMPK/Akt activation and consequent eNOS phosphorylation and nitric oxide (NO) production (1). Treatments with calpain 2 siRNA or inhibitors of calpain, Calpeptin or ALLN, decreased VEGF-induced Akt phosphorylation and eNOS/NO activation (1). However, whether there is any potential feedback regulation of VEGF/VEGF receptor 2 (VEGFR2) signaling by calpain remains unknown. Protein phosphotyrosine phosphatase 1B (PTP1B) is a non-receptor phospho-tyrosine protein phosphatase. Previous studies have shown that PTP1B is cleaved by calpain at the C terminus, resulting in enhanced phosphatase activity (5-7). Furthermore, PTP1B binds to and dephosphorylates VEGFR2 through direct proteinprotein interaction (8). These studies suggest a possible calpain/PTP1B regulation on VEGFR2 phosphorylation. It has been reported that mice overexpressing calpastatin, a natural calpain inhibitor, have impaired angiogenesis and delayed wound healing (4,9). On the other hand, there is evidence showing that PTP1B serves as a negative regulator of VEGF-activated ERK signaling in endothelial cells (8,10). These observations implicate that calpain/PTP1B may be involved in the regulation of VEGF-induced angiogenesis. Therefore, the present study was designed to identify potential calpain/PTP1B-dependent regulation of VEGFR2 and their roles in angiogenesis. It is known that impaired angiogenesis leads to delayed wound healing and diabetic foot ulcers, which affect 15% of diabetic patients and claim 84% of diabetesrelated lower limb amputations (11). We therefore investigated potential impact of PTP1B on diabetic