Endothelial protein C receptor and protease-activated receptor-1 mediate induction of a wound-healing phenotype in human keratinocytes by activated protein C.
ABSTRACT Activated protein C (APC) is a natural anticoagulant and inhibitor of inflammation that can stimulate keratinocyte wound repair in vitro and promote wound healing in vivo. The signaling mechanisms, however, are unknown and a keratinocyte receptor for APC has not been identified. Here, we show that cultured human keratinocytes from neonatal foreskins express the endothelial protein C receptor (EPCR). EPCR was also strongly expressed by lower epidermal layers of neonatal foreskin as determined by immunohistochemistry. In cultured keratinocytes, EPCR expression was upregulated by the addition of APC and inhibited by tumor necrosis factor-alpha. Addition of APC stimulated cell proliferation, production of matrix metalloproteinase-2, activation of ERK and p38 kinase signaling pathways, and expression of protease-activated receptor (PAR)-1. A monoclonal antibody, RCR252, which blocks APC binding to EPCR, or a blocking antibody to PAR-1, abolished APC's effects on keratinocytes. In summary, this study demonstrates that EPCR, a major receptor of protein C pathway, is expressed by human keratinocytes, and facilitates APC's function on keratinocytes via activation of PAR-1 pathway. Our findings highlight a possible new role for the protein C pathway in skin physiology and help elucidate the mechanisms of action by which APC promotes wound healing.
Article: Low Circulating Protein C Levels Are Associated with Lower Leg Ulcers in Patients with Diabetes.[show abstract] [hide abstract]
ABSTRACT: Activated protein C (APC) promotes angiogenesis and reepithelialisation and accelerates healing of diabetic ulcers. The aim of this study was to determine the relationship between the incidence of lower leg ulcers and plasma levels of APC's precursor, protein C (PC), in diabetic patients. Patients with diabetes who had a lower leg ulcer(s) for >6 months (n = 36) were compared with age-, type of diabetes-, and sex-matched subjects with diabetes but without an ulcer (n = 36, controls). Total PC was assessed using a routine PC colorimetric assay. There was a significantly (P < 0.001) lower level of plasma PC in patients with ulcers (103.3 ± 22.7, mean ± SD) compared with control (127.1 ± 34.0) subjects, when corrected for age and matched for gender and type of diabetes. Ulcer type (neuropathic, ischaemic, or mixed) was not a significant covariate for plasma PC levels (P = 0.35). There was no correlation between PC levels and gender, type of diabetes, HbA1c, or C-reactive protein in either group. In summary, decreased circulating PC levels are associated with, and may predispose to, lower leg ulceration in patients with diabetes.BioMed research international. 01/2013; 2013:719570.
Article: Adenosine regulates thrombomodulin and endothelial protein C receptor expression in folliculostellate cells of the pituitary gland.[show abstract] [hide abstract]
ABSTRACT: Adenosine stimulates the release of interleukin 6 (IL-6) and vascular endothelial growth factor from folliculostellate cells of the anterior pituitary gland indicating that such cells are also involved in the communication between the immune and endocrine systems during stress and inflammation. In order to understand the precise actions of adenosine on folliculostellate cells, DNA microarray analysis was used to determine global changes in gene expression. Hierarchical clusters revealed, of the genes that had altered expression, the majority were suppressed and many, such as B cell translocation gene 2 and cyclin-dependent kinase inhibitor 2b were related to cell cycle arrest or inhibition of proliferation. Several of the up-regulated genes were associated with cytokine signalling or membrane receptor activity. The most notable of these being IL-6, sulfiredoxin 1, endothelial protein C receptor (EPCR) and thrombomodulin (THBD) which can all play a role in controlling inflammation. The EPCR and THBD pathway is well known in anti-coagulation but also has anti-inflammatory and anti-apoptotic properties. Up-regulation of EPCR and THBD in folliculostellate cells was confirmed by qRT-PCR and western blotting analysis and their expression were also demonstrated in many of the hormone-secreting cells of the anterior pituitary gland. Our findings suggest that adenosine can stimulate expression of stress and inflammation related genes from folliculostellate cells of the anterior pituitary gland. These genes include EPCR and THBD, neither of which has been previously identified in the pituitary gland.Purinergic Signalling 10/2009; 6(1):19-29. · 3.16 Impact Factor
Article: Activated protein C differentially regulates both viability and differentiation of osteoblasts mediated by bisphosphonates.[show abstract] [hide abstract]
ABSTRACT: Activated protein C (APC) is a cytoprotective anticoagulant that can promote cutaneous healing. We examined the effect of APC on viability and differentiation of the osteoblastic line, MG63, in the presence and absence of bisphosphonates (BPs). Osteoblasts were cultured and treated for 24 or 48 h with Alendronate (Aln), Zoledronate (Zol) or Pamidronate (Pam) at concentrations ranging from 10(-4) to 10(-6) M. Cell differentiation was measured using type 1 collagen production, Alizarin red staining and alkaline phosphatase activity, whereas cell viability was assessed using MTT and crystal violet assays. All three BPs induced MG63 cell death in a dose- and time-dependent manner. Pam- and Zol-related cell death was prevented by APC treatment; however, cell death induced by Aln was accelerated by APC. APC induced MG63 cell differentiation that was enhanced by Aln, but inhibited by Pam or Zol. Endothelial protein C receptor (EPCR) was expressed by MG63 cells and mediated the protective effect of APC on Zol-induced viability. In summary, we have demonstrated that (1) APC favorably regulates MG63 viability and differentiation toward bone growth, (2) APC differentially regulates the effects of specific BPs and (3) at least part of the effects of APC is mediated through EPCR. These findings highlight the potential importance of the PC pathway in bone physiology and provide strong evidence that APC may influence bone cells and has potential to be a therapeutic drug for bone regeneration, depending on concurrent BP treatment.Experimental & molecular medicine. 01/2013; 45:e9.