Phorbol ester binding and protein kinase C activity in normal and transformed human keratinocytes

Abstract

Normal keratinocytes, SV40-transformed keratinocytes (SVKi4), and various squamous carcinoma cell (XC) lines have been used as an in vitro model system to study the properties of phorbol ester receptor and protein kinase C expression during keratinocyte differentiation. The cell lines used exhibit a decreasing capacity to differentiate in the order of keratinocytes -SVK,,>SCC-12F2>SCC-15>SCC-4; moreover, all cell lines respond to a low external Ca*+ concentration by a decreased capacity to differentiate. Normal keratinocytes exhibited the highest number of phorbol ester receptors as compared to the other cell lines, while each individual cell line exhibited a higher number of phorbol ester receptors during growth under normal Ca*+ conditions as compared to cells grown under low Ca*+ conditions. The apparent dissociation constant (Kd) demonstrated only small variations in the various cell lines. In contrast, the cytoplasmic protein kinase C activity, was found to be higher in cells grown under low Ca*' conditions than in cells grown under normal Ca2+ conditions, indicating the absence of a causal relationship between cytoplasmic protein kinase C activity and phorbol ester receptor expression. Therefore the properties of protein kinase C have been determined in more detail in normal keratinocytes and SCC-15 cells. These studies revealed differences between protein kinase C properties from the two cell lines grown under normal and low Ca*+ conditions. The differences included the effect of phorbol 12-myristate 13-acetate (PMA) on the redistribution pattern of protein kinase C between the cytoplasmic and particulate fractions as well as the activating effect of diolein in vitro on protein kinase C activity, partly purified from particulate or cytoplasmic fractions. These observations demonstrate that the functional protein kinase C activity of keratinocytes is determined by various endogenous and exogenous activators and that these activators are modulated differently in various cell lines, under various growth conditions (low Ca*+ versus normal Ca2+). 0 1987 Academic Press, Inc.Mouse skin has been a main tool in the investigation of the tumor promoting activity of, in particular, the phorbol esters during multiple-stage carcinogenesis [l]. In addition to being well known as tumor promoters, these plant-derived cyclic diterpene compounds are known to modulate cellular differentiation and/or proliferation in a variety of cells and tissues (reviewed in [2]). The effects of phorbol esters on cellular differentiation are of particular interest since the regulation of cellular differentiation may be more important than the control of proliferation during the development of neoplasia. In skin, in contrast to many other tissues or organs, all three effects of phorbol esters have been demonstrated: tumor promotion, induction of terminal differentiation, and stimulation of cellular proliferation [3-51. A heterogeneous response is observed often in kera-' To whom reprint requests should be sent at

Cyclic strain stimulates isoform-specific PKC activation and translocation in cultured human keratinocytes

“…induced translocation of PKC from the cytosol to the particulate fraction in a manner analogous to that reported with PMAstimulation [Snoek et al, 1987]. Thus, the similarity between the effects of mechanical strain and chemical agonists on PKC signaling would suggest some similarities in distal transduction events.…”

“…Although the demonstration of strain-induced activation of PKC activity and translocation in keratinocytes is novel, the characteristics of the response have some similarity to previously described stimulation of keratinocytes by chemical agonists such as phorbol esters. First, the magnitude of strain-induced PKC activation in keratinocytes (184% maximal increase) was slightly less than the 200% increase previously reported for stimulation of PKC activity by PMA [Snoek et al, 1987]. Second, the onset of straininduced activation of PKC (5 min) is comparable to that previously described for PMA (5 min, Reynolds et al, 1994].…”

Binding and uptake of N‐nitrosonornicotine by oral epithelial cells

“…COULOMB et al (3) showed that increased serum concentration in the culture medium inhibits uptake of benzo[a]pyrene and benzo[e]pyrene by hamster embryo fibroblasts. Similarly, phorbol ester binding to cells is affected by such factors as stage of cell maturation, Ca' * content of the medium, and cell phospholipid composition (4)(5)(6). Other studies have shown that retinoic acid and NNN may decrease and increase, respectively, the binding of phorbol esters to oral epithelial celis and these correlated with changes in cell lipids (7,8).…”

Subcellular distribution of protein kinase C/phorbol ester receptors in differentiating mouse keratinocytes

“…The discovery by Nishizuka and colleagues that the phorbol ester receptor was identical to PKC (Castagna et al, 1982) strongly suggested that the direct activation of PKC by phorbol esters was responsible for some of the observed biological responses to these tumor promoters. Subsequent studies have demonstrated phorbol ester receptors in murine (Dunn and Blumberg, 1983;Solanki and Slaga, 1982;Delclos et al, 1980;Ashendell et al, 1983) and human (Chi& andKuroki, 1983;Greenebaumet al, 1983;Snoek et al, 1987) keratinocytes. In addition, Dunn et al (1985) have correlated differences in the binding affinities of the phorbol receptors to the state of murine keratinocyte differentiation, suggesting that the phorbol re-ceptoriPKC is involved in the control of keratinocyte differentiation.…”