Protein kinase C isozymes regulate proliferation and high cell density‐mediated differentiation in HaCaT keratinocytes

Abstract

Protein kinase C (PKC) isoforms play pivotal roles in the regulation of differentiation of normal human epidermal keratinocytes (NHEK). In this study, we investigated the participation of the PKC system in the proliferation and high cell density-induced differentiation of the human immortalized keratinocyte line HaCaT. HaCaT keratinocytes possessed a characteristic PKC isoform pattern (PKC alpha, beta, gamma, delta, epsilon, eta, theta, zeta), which altered during proliferation and differentiation. The GF109203X compound, a selective PKC inhibitor, suppressed the expressions of the lat (granular cell) differentiation markers involucrin (INV) and filaggrin (FIL), and the terminal marker keratinocyte-specific transglutaminase-1 (TG), but did not affect the level of the early (spinous cell) marker keratin 10 (K10) and cellular proliferation. Phorbol 12-myristate 13-acetate (PMA), an activator of PKC, inhibited proliferation, elevated intracellular calcium concentration, decreased the expression of K10, and increased the expressions of INV, FIL, and TG. These data indicate that the endogenous activation of PKC regulates the expressions of the late differentiation markers, and that the exogenous activation of PKC by PMA results in the induction of terminal differentiation. Because the cellular effects of PMA were accompanied by differential down-regulations of the sensitive PKC isoforms in proliferating and differentiating cultures, our findings argue for the differential roles of the existing PKC isoforms in the regulation of cellular proliferation and high cell density-induced differentiation of HaCaT cells.

Opposite roles of protein kinase C isoforms in proliferation, differentiation, apoptosis, and tumorigenicity of human HaCaT keratinocytes

“…All primary antibodies against PKC isoenzymes were developed in rabbits and were shown to react specifically with the given PKC isoforms [19]. Anti-PKCa, b, and e were from Sigma (St. Louis, Mo.…”

“…To assess equal loading, nitrocellulose membranes were stripped in 200 ml of 50 mM Tris-HCl buffer (pH 7.5) containing 2 % SDS and 0.1 b-mercaptoethanol at 65°C for 1 h and were reprobed with a mouse anti-cytochrome c antibody (Santa Cruz) followed by a similar visualization procedure as described above. When applicable, immunoblots were subjected to densitometric analysis as described previously [19] and normalized densitometric values of the individual lanes of several independent experiments were then determined.…”

“…For example, PKCa and d were reported to be key components regulating the differentiation of NHEKs [10 -15], and PKCe has been implicated in promoting skin tumor development in transgenic mice [12]. We have previously shown that immortalized HaCaT keratinocytes [16], which are very often used to model certain properties of NHEKs and to assess the processes of immortalization, transformation, and tumor progression in human skin [17,18], possess a similar (although not identical) PKC system to that of NHEKs [19]. Namely, HaCaT cells express several PKC isoforms, the pattern of which alters with differentiation, and respond with cessation of proliferation and induction of differentiation upon the application of the PKC activator phorbol esters [19].…”

UV-B induced alteration in purinergic receptors and signaling on HaCaT keratinocytes

“…Mock treated samples were handled the same way as irradiated ones. Untreated controls cells were taken up at the time of the experiment, while UV-B irradiated and mock treated cells were supplied with 37 °C culture media and were used for experiments at 8,12,16,24,30,36 and 48 h as indicated.…”

“…To determine the expression of different purinoreceptors in cultured cells, the Western blot technique was applied [16]. …”