Protein kinase C in normal human epidermal keratinocytes during proliferation and calcium-induced differentiation.
Recent evidence has implicated protein kinase C (PKC) in the etiology of hyperproliferative diseases such as psoriasis and non-melanoma skin cancer. In this study, PKC activity, immunoreactive protein, and phorbol ester-binding kinetics were examined in primary cultures of normal human epidermal keratinocytes (NHEK) in order to elucidate the relationship between PKC and NHEK proliferation and differentiation. NHEK were maintained in a proliferative phase in serum-free low-calcium (0.15 mM) medium, and then were exposed to high calcium (1.6 mM) in order to stimulate growth arrest and differentiation. Staurosporine was inhibitory to Ca(++)-induced differentiation. Scatchard analysis of phorbol binding indicated that exposure to high calcium for 24 h increased the number of binding sites (Bmax) by fivefold. In correlation with the ligand-binding results, PKC activity was extremely low in proliferating (low-calcium) NHEK compared to differentiating cells (high calcium). When assayed after 24, 48, and 72 h, high calcium induced tenfold or greater increases in Ca++/phospholipid-dependent phosphotransferase activity. Immunoblot analysis of NHEK PKC using antibodies directed against the hinge region of PKC alpha/beta also indicated that exposure to high calcium resulted in higher levels of immunoreactive protein. Therefore, PKC in NHEK appears to be upregulated under conditions of Ca(++)-induced growth arrest and differentiation. In addition, NHEK and other human skin cell particulate fractions contain a protein of approximately 116 kDa that is highly immunoreactive to an antibody to PKC alpha/beta, which coelutes from DEAE-sephacel under the same buffer conditions as the 80-kDa PKC.
Matsui
M S
MS
Department of Dermatology, Columbia University, NY, NY 10032.
Chew
S L
SL
DeLeo
V A
VA
D000328:Adult; D002118:Calcium / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects*; D002455:Cell Division; D006801:Humans; D015151:Immunoblotting; D015603:Keratinocytes / Q000166:cytology / Q000201:enzymology*; D008024:Ligands; D008565:Membrane Proteins / Q000276:immunology; D011493:Protein Kinase C / Q000032:analysis* / Q000378:metabolism
D008024:Ligands; D008565:Membrane Proteins; D011493:Protein Kinase C; D002118:Calcium