title: Protein kinase D is implicated in the reversible commitment to differentiation in primary cultures of mouse keratinocytes.
abstract: Although commitment to epidermal differentiation is generally considered to be irreversible, differentiated keratinocytes (KCs) have been shown to maintain a regenerative potential and to reform skin epithelia when placed in a suitable environment. To obtain insights into the mechanism of reinitiation of this proliferative response in differentiated KCs, we examined the reversibility of commitment to Ca(2+)-induced differentiation. Lowering Ca(2+) concentration to micromolar levels triggered culture-wide morphological and biochemical changes, as indicated by derepression of cyclin D1, reinitiation of DNA synthesis, and acquisition of basal cell-like characteristics. These responses were inhibited by Goedecke 6976, an inhibitor of protein kinase D (PKD) and PKCalpha, but not with GF109203X, a general inhibitor of PKCs, suggesting PKD activation by a PKC-independent mechanism. PKD activation followed complex kinetics with a biphasic early transient phosphorylation within the first 6 h, followed by a sustained and progressive phosphorylation beginning at 24 h. The second phase of PKD activation was followed by prolonged ERK1/2 signaling and progression to DNA synthesis in response to the low Ca(2+) switch. Specific knockdown of PKD-1 by RNA interference or expression of a dominant negative form of PKD-1 did not have a significant effect on normal KC proliferation and differentiation but did inhibit Ca(2+)-mediated reinitiation of proliferation and reversion in differentiated cultures. The present study identifies PKD as a major regulator of a proliferative response in differentiated KCs, probably through sustained activation of the ERK-MAPK pathway, and provides new insights into the process of epidermal regeneration and wound healing.
authors.affiliation: Graduate Program in Molecular and Cellular Biology, Stony Brook University, Stony Brook, New York 11794, USA.
authors.forename: Azadeh
authors.initials: A
authors.lastname: Jadali
authors.forename: Soosan
authors.initials: S
authors.lastname: Ghazizadeh
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002227:Carbazoles / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D000078404:Epidermal Cells; D004817:Epidermis / Q000187:drug effects / Q000201:enzymology; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D015603:Keratinocytes / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D020935:MAP Kinase Signaling System / Q000187:drug effects; D051379:Mice; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D002227:Carbazoles; C081021:Go 6976; C089212:protein kinase D; D011493:Protein Kinase C; D002118:Calcium
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