Protein Kinase D Is Implicated in the Reversible Commitment to Differentiation in Primary Cultures of Mouse Keratinocytes

• Published in: The Journal of biological chemistry 2010-07, Vol.285 (30), p.23387-23397
• Main Authors: Jadali, Azadeh, Ghazizadeh, Soosan
• Format: Article
• Language: English
• Subjects: Animals; Calcium - metabolism; Calcium - pharmacology; Carbazoles - pharmacology; Cell Biology; Cell Cycle; Cell Cycle - drug effects; Cell Differentiation - drug effects; Cell Proliferation - drug effects; Cells, Cultured; Dedifferentiation; Differentiation; Dose-Response Relationship, Drug; Enzyme Activation; Epidermal Cells; Epidermis - drug effects; Epidermis - enzymology; Epithelial Cell; ERK; Extracellular Space - drug effects; Extracellular Space - metabolism; Keratinocytes; Keratinocytes - cytology; Keratinocytes - drug effects; Keratinocytes - enzymology; Kinetics; MAP Kinase Signaling System - drug effects; Mice; Protein Kinase C (PKC); Protein Kinase C - antagonists & inhibitors; Protein Kinase C - metabolism; Protein Kinase D; Signal Transduction
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M110.105619

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 Ca2+-induced differentiation. Lowering Ca2+ 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 PKCα, 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 Ca2+ 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 Ca2+-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.