Protein kinase C mediates up-regulation of urokinase and its receptor in the migrating keratinocytes of wounded cultures, but urokinase is not required for movement across a substratum in vitro

Abstract

Both in cell culture and in vivo, keratinocytes that are migrating in response to a wound express enhanced levels of both urokinase-type plasminogen activator (uPA) and the uPA cell surface receptor (uPA-R). To explore the mechanism of this up-regulation, keratinocyte cultures were treated proir to wounding with a variety of metabolic and growth factor inhibitors in order to evaluate their effect on uPA and uPA-R expression. Actinomycin D and cycloheximide inhibited the up-regulation of both uPA and uPA-R, as determined by immunohistochemistry, indicating that RNA and protein syntheses are required for their induction in migrating keratinocytes. Neither removal of protein growth factors from the medium nor addition of inhibitory antibodies to a number of growth factors depressed uPA or uPA-R induction; these findings suggest that a variety of exogenous or endogenous growth factors [i.e., basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), amphiregulin, and tumor necrosis factor-alpha (TNF-alpha) do not have a critical role in the induction of uPA or uPA-R. In contrast, when protein kinase C (PKC) was either down-regulated with bryostatin 5 or inhibited with Ro31-8220 or staurosporine, the expression of both uPA and uPA-R was greatly decreased in migrating keratinocytes. Furthermore, pharmacologic activation of PKC enhanced uPA levels in non-wounded cultures. These data suggest that the enhanced expression of uPA and uPA-R in migrating keratinocytes is mediated by selective activation of PKC in these cells, perhaps secondary to alterations in the cytoskeleton induced by wounding. To test the requirement for uPA during keratinocyte migration in vitro, the extent of migration was quantified in the presence and absence of a variety of inhibitors in the wounded culture model. Migration was not altered by actinomycin D, cycloheximide, any of the above growth factor inhibitors, anti-uPA antibodies, a variety of inhibitors of uPA or plasmin enzymatic activity, or exogenous uPA. The independence of keratinocyte migration in vitro from uPA was further suggested by experiments which combined the phagokinetic assay of migration and the zymographic assay for pericellular uPA activity; no relationship was observed between pericellular uPA activity and the motility of individual cells.

Two distinct signaling pathways in hair cycle induction: Stat3-dependent and -independent pathways

“…It is not known which isotype of PKC is involved in the PKC-dependent migration, because GF109203X at the concentration used in the present study (5 M) attenuates the PKC activation of various subfamilies found in keratinocytes (24,25). Activation of PKC has been reported to play an important role in keratinocyte migration through enhancement of the specific integrin-mediated mo- tility (26), microfilament organization (27), or up-regulation of proteinases such as metalloproteinases (28) and urokinase (29). Requirement of PI3K activation for the Stat3-independent PKC-dependent signaling pathway toward migration was clearly demonstrated by in vitro studies by using wortmannin, a specific inhibitor of PI3K (Fig.…”

Molecular Disruption of Breast Tumor Angiogenesis

“…Although both keratinocytes and endothelial cells express increased levels of uPA and PAI-1 in response to monolayer wounding, uPA is apparently not required for keratinocyte locomotion in vitro, whereas occupancy of uPAR by uPA, but not uPA catalytic activity, facilitates wound-responsive endothelial 278 PROVIDENCE ET AL. cell motility (Pepper et al, 1987;Sato and Rifkin, 1988;Okedon et al, 1992;Ando and Jensen, 1996). Present data indicate that PAI-1 expression is an early response to injury and necessary for normal rat kidney-derived epithelial cells to effectively repair monolayer wounds.…”

“…In vitro migration into scrape-denuded areas is accomplished by the lateral movement of surviving cells across relatively uncomplicated substrates (e.g., Pepper et al, 1987;Sato and Rifkin, 1988;Ando and Jensen, 1996) unlike in vivo transit through fibrin-rich barriers or a provisional wound matrix Yamada and Clark, 1995). Despite these considerable physiologic differences, certain commonalites are evident between the two models.…”

“…uPAuPAR interactions induce c-fos gene expression (Dumler et al, 1994), whereas pro-uPA binding to the u-PAR has been reported to inhibit cell cycle progression of HL-60 cells (Howell et al, 1994), explaining, perhaps, the delay in proliferation by cells of the migratory front. The role of uPA in migration across a denuded zone may be cell type related (Pepper et al, 1987;Sato and Rifkin, 1988;Ando and Jensen, 1996) and appears complicated by differential utilization of uPA/uPAR vs. vitronectin/integrin targets as PAI-1-sensitive motors (e.g., Chapman, 1997;Loskutoff et al, 1999). Regardless of the precise mechanism(s) involved, the present findings, using the complementary approaches of molecular genetic targeting of PAI-1 expression and rescue of a repair-deficient phenotype, strongly suggest that PAI-1 regulates renal epithelial cell motility in response to monolayer wounding.…”

Transcriptional Regulation of the Urokinase Receptor (u-PAR) – A Central Molecule of Invasion and Metastasis

“…Although altered mRNA stability and receptor recycling may be involved, the amounts of u-PAR are controlled mainly at the transcriptional level in malignancies such as colon cancer (Lund et al, 1995;Lengyel et al, 1996;Shetty et al, 1997;Gum et al, 1998). Altered transcription of the gene is the main mediator of u-PAR gene expression brought about by, for example, epidermal growth factor (EGF; Boyd, 1989), basic fibroblast growth factor (FGF; Mignatti et al, 1991), vascular endothelial growth factor (VEGF; Mandriota et al, 1995), transforming growth factor β type 1 (TGF-β1; Lund et al, 1995), phorbol 12-myristate 13-acetate (PMA; Lengyel et al, 1996), IFN-α or IFN-γ, (Wu et al, 2002), protein kinase C (PKC; Ando et al, 1996), protein kinase A (PKA)/c-AMP (Langer et al, 1993;Li et al, 1995), the MAPK- (Lengyel et al, 1997) and the JNK-pathway (Gum et al, 1998).…”