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.

The coiled-coil domain-containing delta-interacting protein A (DIPA) is a transcription factor implicated in developmental regulation. DIPA is the first protein discovered to selectively interact with the p120-catenin (p120) isoform 1, an alternatively spliced form of p120 expressed preferentially in mesenchymal cells. Although a small fraction of p120 can be observed in the nucleus under certain circumstances, the vast majority of it associates with classical cadherins at adherens junctions. We observed for the first time that a discrete fraction of DIPA exists at cell-cell junctions, in addition to its predominantly nuclear localization. Thus, the p120-DIPA interaction may regulate cell signaling and/or transcriptional events, as has been described previously for b-catenin and the LEF/TCF transcription factor family. To facilitate further study of DIPA and to determine the physiological relevance of its interaction with p120, we have generated and characterized a panel of five DIPAspecific monoclonal antibodies (MAbs) that function in immunoblotting, immunoprecipitation, and immunofluorescence assays.

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Gap junctions mediate cell-to-cell electrical coupling required for synchronized cardiac contraction. They also serve as conduits for exchange of small signaling molecules. Reduction in connexin43 (Cx43), the major gap junction protein in ventricular tissue, likely underlies malignant arrhythmias in infarcted hearts. However, little is known about the role of reduced Cx43 expression in infarct healing. This study was performed to test the hypothesis that reduced expression of Cx43 influences cardiac fibroblast function and wound healing after myocardial infarction (MI). Methods: The left anterior coronary artery was ligated in wild-type (WTs) and Cx43-deficient (Cx43 +/− ) mice studied 6 d later. We quantified infarct size by Masson’s trichrome staining; cell proliferation by bromodeoxyuridine staining; matrix metallo-proteinase (MMP) activity in the noninfarcted, border and infarct regions (IZ) by zymography; myofibroblasts by α-smooth muscle actin (αSMA) immunostaining; and collagen deposition by Picrosirius red staining. Results: 6 d post-MI mean infarct size by trichrome staining was not different in Cx43 +/− (46±4%) vs WTs (46±5%). However, significant differences were observed in the Cx43 +/− infarcts. The area of unresorbed necrotic myocardium in the IZ was larger (p<0.01) in Cx43 +/− (40±11%, n=12) vs WTs (17±8%, n=6). Fibroblast proliferation in the IZ was increased (p<0.05) in Cx43 +/− (6.1±0.5%, n=4) vs WTs (4.1±0.7%, n=5), and activated MMP-2 was increased even more (p< 0.05) in the IZ of Cx43 +/− (40±6 relative density units, n=5) vs WTs (29±4 units, n=3) consistent with enhanced fibroblast proliferation. On the other hand, transformation to myofibroblasts in the IZ was diminished as reflected by reduced (p<0.01) αSMA-positive immunostaining in Cx43 +/− (32±16%, n=6) vs WTs (57±5%, n=12), and collagen deposition in the IZ was less (p<0.01) in Cx43 +/− (36±6%, n=6) vs WTs (50±13%, n=8) consistent with fewer myofibroblasts. Conclusion: Reduced Cx43 expression increases fibroblast proliferation and MMP activity, and reduces myofibro-blasts and collagen deposition indicating that remodeling of Cx43 can influence fibroblast function and delay scar formation and cardiac wound healing after MI.