Protein kinase D is implicated in the reversible commitment to differentiation in primary cultures of mouse keratinocytes - PubMed (original) (raw)

Reinitiation of DNA synthesis and reversal of differentiation in differentiated epidermal cultures exposed to low Ca2+ conditions. A, schematic representation of the culture conditions used in this study. Primary cultures of KCs grown in 0.05 m

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Ca2+ to confluence (P) were exposed to 1.2 m

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Ca2+ for 3 days to induce differentiation (D). Differentiated cultures were reverted by subsequent exposure to 0.05 m

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Ca2+-containing medium for 7 days (R). B, phase-contrast images showing morphology of KCs in proliferative stage (P), 3 days after Ca2+-induced differentiation (D3), and in reverted cultures after 2 (R2), 4 (R4), and 7 days (R7). Some of the 7-day cultures were induced to differentiate for a second time (R7D3). Bar, 50 μm. C, proliferation rates in various culture conditions were measured by either BrdUrd labeling (6-h pulse) or [3H]thymidine incorporation (16-h pulse) as described under “Experimental Procedures.” Values are expressed as mean ± S.D. (error bars) of three independent experiments quantified in a total of nine different samples. *, p < 0.001 when comparing differentiation day 3 to either proliferative stage or reverted cultures. D, quantitative RT-PCR analysis of the transcript levels of p63, keratin 10 (K10), INV, filaggrin (FIL), and loricrin (LOR) in KCs at proliferative stage, 3 days after Ca2+-induced differentiation, and in reverted cultures after 2, 4, and 7 days. Transcript levels were normalized to phosphoglycerate kinase. E, protein lysates of KCs at proliferative stage, 3 days after Ca2+-induced differentiation, and in reverted cultures after 2, 4, and 7 days were analyzed by immunoblotting for the indicated proteins. Actin was used as a loading control.