Protein kinase C isozymes regulate proliferation and high cell density-mediated differentiation in HaCaT keratinocytes Helga Papp 1 , Gabriella Czifra 1 , Jo´zsef La´za´ r 1 , Mo´nika Go¨nczi 1,2 , La´szlo´ Csernoch 1,2 , La´szlo´ Kova´ cs 1,2 and Tama´s Bı´ro´ 1,2 1 Department of Physiology and 2 Cell Physiology Research Group of the Hungarian Academy of Sciences, University of Debrecen, Medical and Health Science Center, Medical Faculty, Hungary Key words: human keratinocytes – HaCaT – differentiation – protein kinase C – isoen- zymes Tama ´s Bı´ro ´ MD, PhD, Department of Physiology, Hungarian Academy of Sciences, University of Debrecen, Medical and Health Science Center, Medical Faculty, Hungary, H-4012 Debrecen, Nagyerdei krt. 98. PO Box 22 Tel.: þ36 52 416 634 Fax: þ36 52 432 289 e-mail: biro@phys.dote.hu Accepted for publication 18 November 2002 Papp H, Czifra G, La´za´r J, Go¨ nczi M, Csernoch L, Kova´cs L, Bı´ro´ T. Protein kinase C isozymes regulate proliferation and high cell density-mediated differentiation in HaCaT keratinocytes. Exp Dermatol 2003:12: 811–824. # Blackwell Munksgaard, 2003 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 (PKCa, b, g,d,e,Z,y,z), which altered during proliferation and differentiation. The GF109203X compound, a selective PKC inhibitor, suppressed the expressions of the late (granular cell) differentiation markers involucrin (INV) and filaggrin (FIL), and the terminal marker keratinocytes-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. Introduction The epidermis, the most superficial layer of the skin, forms a major physical–chemical barrier to protect the organism against environmental fac- tors. The structure and the function of the epider- mis are maintained by a well-defined and -balanced program of proliferation and differen- tiation of the keratinocytes resulting in the basal, spinous, granular, and cornified layers (1). Once differentiation is initiated, the basal keratinocytes cease their proliferation and move toward the cor- nified layer. The process of differentiation can be investigated thoroughly by measuring the sequen- tial appearance of various differentiation markers representing different stages of differentiation. Namely, keratins (K) 5 and 14 representing the basal layer will be exchanged to K1 and K10 in the spinous layer (1). Later, in the granular layer, these intracellular proteins will be shifted to invo- lucrin (INV), filaggrin (FIL), and loricrin, which serve as substrates for the keratinocyte-specific transglutaminase-1 (TG) to form the chemically resistant cornified envelope structures in the term- inally differentiated keratinocytes (2–4). Protein kinase C (PKC) comprises a family of serine/threonine kinases that play central roles in the regulation of various cellular processes in Abbreviations: NHEK: normal human epidermal keratinocytes; PKC: protein kinase C; PMA: phorbol 12-myristate 13-acetate; DMSO: dimethyl sulfoxide; SDS: sodium dodecyl sulfate; PAGE: polyacrylamide gel electrophoresis; K: keratin; DMEM: Dulbecco’s modified eagle’s minimal essential medium; FCS: fetal calf serum; BrdU: bromo-deoxyuridine; PBS: phosphate-buffered saline; BCA: bicinchoninic acid. Experimental Dermatology 2003: 12: 811–824 Copyright # Blackwell Munksgaard 2003 Blackwell Munksgaard . Printed in Denmark EXPERIMENTAL DERMATOLOGY ISSN 0906-6705 811