Regulation of Protein Kinase D During Differentiation and Proliferation of Primary Mouse Keratinocytes

Abstract

Diseased skin often exhibits a deregulated program of the keratinocyte maturation necessary for epidermal stratification and function. Protein kinase D (PKD), a serine/threonine kinase, is expressed in proliferating keratinocytes, and PKD activation occurs in response to mitogen stimulation in other cell types. We have proposed that PKD functions as a pro-proliferative and/or anti-differentiative signal in keratinocytes and hypothesized that differentiation inducers will downmodulate PKD to allow differentiation to proceed. Thus, changes in PKD levels, autophosphorylation, and activity were analyzed upon stimulation of differentiation and proliferation in primary mouse keratinocytes. Elevated extracellular calcium and acute 12-O-tetradecanoylphorbol-13-acetate (TPA) treatments induced differentiation and triggered a downmodulation of PKD levels, autophosphorylation at serine 916, and activity. Chronic TPA treatment stimulated proliferation and resulted in a recovery of PKD levels, autophosphorylation, and activity. Immunohistochemical analysis demonstrated PKD localization predominantly in the proliferative basal layer of mouse epidermis. Co-expression studies revealed a pro-proliferative, anti-differentiative effect of PKD on keratinocyte maturation as monitored by increased and decreased promoter activities of keratin 5, a proliferative marker, and involucrin, a differentiative marker, respectively. This work describes the inverse regulation of PKD during keratinocyte differentiation and proliferation and the pro-proliferative/anti-differentiative effects of PKD co-expression on keratinocyte maturation.

Protein kinase D1 deficiency promotes differentiation in epidermal keratinocytes

“…Similar to our results, siRNA-mediated PKD1 knockdown in human keratinocytes resulted in increased involucrin mRNA expression [24]. Also, we have previously reported that PKD overexpression with an involucrin promoter reporter constructs results in decreased promoter activity [21]. …”

“…2B) levels. These results are in line with the fact that in the epidermis loricrin is expressed in the upper granular layer where the calcium concentration is high [48] and where PKD1 levels are low [21, 49]. However, basally loricrin expression was not affected by the presence or absence of PKD1.…”

“…There is no PKD1-specific antibody available; the antibody we used recognizes both PKD1 and PKD2. Based on our previous results in keratinocytes [18, 35], the uppermost band represents phosphorylated, activated forms of PKD1 and PKD2, while the middle and lowermost band represents unphosphorylated PKD1 and PKD2, respectively [18, 21]. …”

“…However, WT-PKD overexpression showed no significant effect on expression of these differentiation markers. This may be due to the fact that under basal growth conditions keratinocytes exhibit partial activation of PKD, as observed by its detectable autophosphorylation under such conditions [21]; thus, PKD may already be suppressing the expression of these markers basally. Consistent with this idea, mutant PKD overexpression likely released endogenous PKD-mediated suppression of these differentiation markers to result in their increased expression in wild-type keratinocytes.…”

“…Increased PKD levels in mouse epidermal carcinomas [19], decreased DNA synthesis [19, 20] and increased differentiation upon inhibition of its activity [20], and increased DNA synthesis [18] and promoter activity of keratin 5, a basal layer marker, [21] upon PKD overexpression all suggest the pro-proliferative role of PKD in keratinocytes. Reversal of calcium-differentiated keratinocytes to a proliferative basal-like phenotype upon switching of the cells to a low calcium medium has also been described [22], and this low calcium switch-induced mitogenic response is dependent upon PKD signaling.…”