Knockdown of PKD1 in normal human epidermal keratinocytes increases mRNA expression of keratin 10 and involucrin: early markers of keratinocyte differentiation

Abstract

Subconfluent normal human keratinocytes exhibit autonomous (autocrine growth factor driven) proliferation and express the specific markers for keratinocyte proliferation K5 (keratin 5) and K14 (keratin 14). Utilizing this model the effects of PKD1 (Protein kinase D1) knockdown on activation of differentiation was studied. siRNA approach was applied to achieve specific knockdown of PKD1 and the mRNA levels of different keratinocyte markers -- K14 and PCNA (markers of basal proliferating keratinocytes), involucrin and K10 (early differentiation markers) were analyzed. Treatment of cultured keratinocytes with siRNA for PKD1 resulted in reduction of mRNA levels of PKD1, altered cell phenotype and promotion of keratinocyte differentiation, demonstrated by increased expression of involucrin and K10 mRNAs. No significant changes in K14 mRNA expression levels were detected, but the expression of PCNA mRNA was markedly diminished. This study was the first to show that mRNA expression of PKD1 in subconfluent normal human keratinocytes is very low, the PKD1 mRNA levels were more than 8-fold lower than the same ones in hTert keratinocytes. These findings suggest antidifferentiative role of PKD1 in normal human keratinocytes, contrary to the prodiferentiative role of PKD1 in human hTert keratinocytes. We came to the conclusion that there are differences between transduction pathways involving PKD1 in primary human keratinocyte cultures and these in immortalized hTert keratinocytes.

Protein kinase D1 deficiency promotes differentiation in epidermal keratinocytes

“…This increase was observed in PKD1-deficient keratinocytes under basal as well as differentiating conditions. 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].…”

“…These results confirm that PKD1 is an anti-differentiative and pro-proliferative signaling enzyme in mouse keratinocytes, similar to human keratinocytes [24], but are unique in that they were obtained with the use of the floxed PKD1 mouse model rather than overexpression [40] or RNA interference [24], with the attendant caveats of these methods. Thus, in this study, we used PKD1-floxed mice that possess loxP sites flanking exons 12 through 14 of the PKD1 gene, which encodes part of the catalytic domain.…”

“…Therefore, the different keratinocyte cultures derived from floxed PKD1 versus PKD1 knockout animals may not be entirely comparable. On the other hand, in a previous study using siRNA to knock down PKD1 in human keratinocytes, Ivanova and coworkers were unable to show an effect on PKD1 at the protein level [24]. In addition, siRNA technology has a risk of off-target effects [41, 42].…”

“…In addition, possible compensatory effects related to the loss of PKD1 in utero could not be excluded. On the other hand, Ivanova and coworkers have shown that siRNA-mediated down-regulation of PKD1 decreases human keratinocyte proliferation, although these authors failed to demonstrate PKD1 knockdown at the protein level [24]. Thus, all of the strategies used to date have both benefits and the potential for artifacts, and the results from multiple approaches can only strengthen the evidence for PKD1’s role in keratinocytes.…”

Opposing Growth Regulatory Roles of Protein Kinase D Isoforms in Human Keratinocytes

“…KCs-Previous studies have indicated that PKD1 is expressed in human skin and is up-regulated in hyperproliferative skin disorders (4,20). To further investigate the role of PKD1 in human epidermis, we used lentivirus-mediated shRNA to knockdown PKD1 in NHKCs.…”