Non-receptor tyrosine kinase proline-rich protein tyrosine kinase 2 (Pyk2) functions as an integrator of multiple signaling pathways involved in the regulation of fundamental cellular processes. Pyk2 expression, regulation, and functions in skin have not been examined. Here we investigated the expression and subcellular localization of Pyk2 in human epidermis and in primary human keratinocytes, and studied the mechanisms of Pyk2 activation by differentiation-inducing stimuli, and the role of Pyk2 as a regulator of keratinocyte differentiation. We demonstrate that Pyk2 is abundantly expressed in skin keratinocytes. Notably, the endogenous Pyk2 protein is predominantly localized in keratinocyte nuclei throughout all layers of healthy human epidermis, and in cultured human keratinocytes. Pyk2 is activated by treatment with keratinocyte-differentiating agents, 12-O-tetradecanoylphorbol-13-acetate and calcium via a mechanism that requires intracellular calcium release and functional protein kinase C (PKC) and Src activities. Particularly, differentiation-promoting PKC delta and PKC eta elicit Pyk2 activation. Our data show that Pyk2 increases promoter activity and endogenous protein levels of involucrin, a marker of keratinocyte terminal differentiation. This regulation is associated with increased expression of Fra-1 and JunD, activator protein-1 transcription factors known to be required for involucrin expression. Altogether, these results provide insights into Pyk2 signaling in epidermis and reveal a novel role for Pyk2 in regulation of keratinocyte differentiation.

Urocortin (Ucn1), a member of the corticotrophin-releasing hormone (CRH) family, has been reported to participate in inflammation. The increased expression of intercellular adhesion molecule 1 (ICAM1) plays important roles in inflammation and immune responses. Our previous results demonstrated that Ucn1 significantly enhanced the expression of ICAM1. However, the underlying mechanisms are still unknown. The purpose of this study is to investigate the detailed mechanisms of Ucn1-induced upregulation of ICAM1. Here, we characterized the mechanisms of Ucn1 usage to regulate ICAM1 expression in human umbilical vein endothelial cells (HUVECs). Our data revealed that Ucn1 increased ICAM1 and cyclooxygenase 2 (COX2) expressions in a time-dependent manner via CRH receptor 2 (CRHR2). In addition, COX2 was involved in ICAM1 upregulation. Furthermore, Ucn1 could increase the expression and phosphorylation of cytosolic phospholipases A2 (cPLA2) in a time-dependent manner via CRHR2 and CRHR1. Moreover, ablation of cPLA2 by the inhibitor pyrrophenone or siRNA attenuated the ICAM1 increase induced by Ucn1. In addition, nuclear factor kB (NF-kB) was activated, indicated by the increase in nuclear p65NF-kB expression and phosphorylation of p65NF-kB, depending on cPLA2 and CRHR2 activation. Pyrrolidinedithiocarbamic acid, an inhibitor of NF-kB, abolished the elevation of ICAM1 but not COX2. Also, Ucn1 increased the production of prostaglandin E 2 (PGE 2 ) which further activated protein kinase A (PKA)-CREB pathways dependent of cPLA2 via CRHR2. Moreover, the increase in NF-kB phosphorylation was not affected by the selective COX2 inhibitor NS-398 or the PKA inhibitor H89. In conclusion, these data indicate that Ucn1 increase the ICAM1 expression via cPLA2-NF-kB and cPLA2-COX2-PGE 2 -PKA-CREB pathways by means of CRHR2.