CYCLIC STRAIN STIMULATES ISOFORM-SPECIFIC PKC ACTIVATION AND TRANSLOCATION IN CULTURED HUMAN KERATINOCYTES

Previous studies have demonstrated that cyclic strain induces keratino cyte proliferative and morphological changes. Since protein kinase C ( PKC) is known to play an important role in the regulation of keratinoc yte growth and differentiation, the objective of this study was to det ermine the role of the PKC signaling pathway as a mediator of strain m odulation of the keratinocyte phenotype. In particular, we tested the following specific hypotheses: (1) cyclic strain stimulates PKC activi ty and translocation, (2) cyclic strain activates PKC in an isoform-sp ecific manner, and (3) PKC mediates the strain activated proliferative and morphological response in cultured human keratinocytes. To test t hese hypotheses, keratinocytes were subjected to vacuum-generated cycl ic strain (10% average strain), followed by measurement of PKC activit y, PKC isoform distribution by Western blot analysis and confocal micr oscopy, and examination of the effect of PKC inhibitors (calphostin C and staurosporine) on strain induced proliferative and morphological c hanges. We observed stimulation of PKC activity (62.3 +/- 5.1% increas e) coupled with translocation of PKC from the cytosolic to the membran e fraction in keratinocytes subjected to acute cyclic strain. Cyclic s train also caused translocation of PKC alpha and delta, but not zeta i soforms, from the cytosolic to the membrane fraction as demonstrated b y both Western blot analysis and confocal microscopy. PKC beta was not detected in these cells. PKC inhibitors, calphostin C (10 nM), and st aurosporine (5 nM), inhibited strain-induced PKC activation and kerati nocyte proliferation, but did not block the effects of strain on cellu lar morphology or alignment. We conclude that these data support our h ypothesis that cyclic strain stimulates PKC activity and translocation in an isoform-specific manner in cultured human keratinocytes. Moreov er, our studies with PKC inhibitors support the hypothesis that strain -induced changes in the keratinocyte phenotype may be selectively modu lated by PKC. (C) 1997 Wiley-Liss, Inc.