Protein kinase D1 deficiency promotes differentiation in epidermal keratinocytes

Abstract

Background Protein kinase D (PKD or PKD1) is a serine/threonine protein kinase that has been shown to play a role in a variety of cellular processes; however, the function of PKD1 in the skin has not been fully investigated. The balance between proliferation and differentiation processes in the predominant cells of the epidermis, the keratinocytes, is essential for normal skin function. Objective To investigate the effect of PKD1 deficiency on proliferation and differentiation of epidermal keratinocytes. Methods We utilized a floxed PKD1 mouse model such that infecting epidermal keratinocytes derived from these mice with an adenovirus expressing Cre-recombinase allowed us to determine the effect of PKD1 gene loss in vitro. Proliferation and differentiation were monitored using qRT-PCR, Western blot, transglutaminase activity assays, [3H]thymidine incorporation into DNA and cell cycle analysis. Results A significant decrease in PKD1 mRNA and protein levels was achieved in adenoviral Cre-recombinase-infected cells. Deficiency of PKD1 resulted in significant increases in the mRNA and protein expression of various differentiation markers such as loricrin, involucrin, and keratin 10 either basally and/or upon stimulation of differentiation. PKD1-deficient keratinocytes also showed an increase in transglutaminase expression and activity, indicating an anti-differentiative role of PKD1. Furthermore, the PKD1-deficient keratinocytes exhibited decreased proliferation. However, PKD1 loss had no effect on stem cell marker expression. Conclusions Cre-recombinase-mediated knockdown represents an additional approach demonstrating that PKD1 is an anti-differentiative, pro-proliferative signal in mouse keratinocytes.

Regulation of the Glycerol Transporter, Aquaporin-3, by Histone Deacetylase-3 and p53 in Keratinocytes

“…Keratinocytes were prepared from transgenic neonatal mice in which the HDAC3 gene is flanked by loxP sites (McGee-Lawrence et al, 2015). These cells were then infected with GFP-expressing (control) adenovirus and adenovirus expressing Cre-recombinase (to delete the HDAC3 gene), as in (Choudhary et al, 2014). Infection with Cre-expressing adenovirus significantly decreased HDAC3 protein expression in the floxed keratinocytes, reducing protein levels to about 25–30% of the GFP- expressing control cells (Figure 4a, upper and lower panels).…”

Deletion of protein kinase D1 in osteoprogenitor cells results in decreased osteogenesis in vitro and reduced bone mineral density in vivo

“…Isolated BMMSCs derived from PRKD1 floxed mice cells were infected with GFP-expressing (control) adenovirus and adenovirus expressing Cre recombinase (and GFP) to delete the PRKD1 gene as performed previously in epidermal keratinocytes (Choudhary et al, 2014). These cells were then cultured for 1–2 days before stimulation with the indicated reagents or transfer into an osteogenic medium as described below.…”

Exploring Skin Wound Healing Models and the Impact of Natural Lipids on the Healing Process

“…Many of these PKC isoenzymes are expressed in the skin and several are known to be involved in regulating keratinocyte proliferation and/or differentiation [122,123]. Diacylglycerol also activates protein kinase D (PKD), for which the evidence indicates a pro-proliferative effect [124][125][126]. Diacylglycerol can also be produced by the lipin-mediated dephosphorylation of the phosphatidic acid produced by phospholipase D activity [127,128].…”