Differentiation of Cultured Human Epidermal Keratinocytes at High Cell Densities is Mediated by Endogenous Activation of the Protein Kinase C Signaling Pathway

Abstract

Normal human epidermal keratinocytes (NHEK) grown in serum-free medium on a plastic substrate spontaneously differentiate at high cell densities in vitro. Because protein kinase C (PKC) regulates murine keratinocyte differentiation triggered by a variety of stimuli, we examined the role of this signaling pathway in density-dependent activation of NHEK differentiation. Relative to subconfluent cultures, confluent NHEK expressed markedly higher levels of multiple differentiation markers assayed by immunoblotting, including keratin 1, loricrin, filaggrin, involucrin, TGK, and SPR-1. Expression of several of these markers continued to increase for several days after cells reached confluency. The total level of several PKC isoforms was not substantially altered in NHEK harvested at different cell densities, based on immunoblotting; however, subcellular fractionation revealed that PKCalpha underwent a redistribution to the particulate fraction in confluent and postconfluent NHEK cultures, suggesting that this isozyme was activated under these conditions and may be involved in triggering the terminal differentiation program. Supporting this concept, inhibition of PKC function using bryostatin 1 or GF 109203X blocked the induction of keratinocyte differentiation markers at high cell densities. These data suggest that endogenous activation of PKC is responsible for cell density-mediated stimulation of NHEK differentiation, establishing a critical role for this pathway in regulating human as well as murine keratinocyte differentiation.

Calcium-dependent Involucrin Expression Is Inversely Regulated by Protein Kinase C (PKC)α and PKCδ

“…Fig. 1B confirms that PKC␣, -␦, -⑀, -, and -are expressed in our model system, as has been reported elsewhere (10,14,29,(45)(46)(47)(48). Moreover, although the results cannot be regarded as quantitative, the film exposure times and protein loading densities required for visualization suggest that PKC␣ and -␦ are the most abundant isoforms.…”

“…PKC␦ Activity Is Required for Calcium-dependent Regulation of hINV Gene Expression-Because of their relative abundance, and the fact that they have been implicated as mediating differentiation-dependent regulation in keratinocytes (14,18,28,29,39,40,49), we focused on the PKC␦ and PKC␣ isoforms. We began by studying the role of PKC␦.…”

“…Keratinocytes express the PKC␣, -␦, -⑀, -, and -isoforms (10). These enzymes control a variety of signaling cascades and transcription factors and function as regulators of keratinocyte differentiation-dependent gene expression (11)(12)(13)(14)(15). In keratinocytes, PKC␣ and PKC␦ are abundant PKC isoforms that have been implicated as regulators of differentiation (16 -19).…”

PKCα induces differentiation through ERK1/2 phosphorylation in mouse keratinocytes

“…In the mouse keratinocytes, PKCα has been implicated in the induction of the late differentiation markers by calcium (Denning et al, 1995;Lee et al, 1997). Studies using pharmacological PKC inhibitors (Lee et al, 1998) and PKCα antisense oligonucleotides (Lee et al, 1997) have suggested that PKCα activation regulates the granular cell stage …”

Structural Organization and Regulation of the Small Proline-rich Family of Cornified Envelope Precursors Suggest a Role in Adaptive Barrier Function

“…SPRR1A, 1B, 3, 2A, and 2E do not respond to UV irradiation. The variability in SPRR2B, 2D, and 2F induction is likely because of small differences in cell density, which are difficult to control at the start of the experiment, but might affect gene expression (46). UV irradiation did not affect the expression of involucrin.…”