Association of protein kinase C activation with induction of ornithine decarboxylase in murine but not human keratinocyte cultures

Abstract

The goal of this study was to compare the response of mouse epidermal keratinocytes (MEKs) and human epidermal keratinocytes (HEKs) to 12-O-tetradecanoylphorbol-13-acetate (TPA) with respect to the activation and downregulation of protein kinase C (PKC), the expression of c-jun and c-fos, and the expression and induction of ornithine decarboxylase (ODC) activity. Keratinocytes from adult CD-1 mice and from discarded adult human skin were grown in primary culture in a high-calcium serum-free medium that supported proliferation and differentiation. Immunoblotting of freshly isolated and cultured MEKs and HEKs for isozymes of protein kinase C revealed that fresh HEKs contained PKC alpha, PKC beta, and PKC delta; no PKC gamma, PKC epsilon, or PKC zeta were detected. In fresh MEKs, PKC alpha, PKC beta, PKC delta, and PKC zeta were observed, but not PKC gamma or PKC epsilon. After 2 wk in culture, the isozyme profiles of MEKs and HEKs were similar except that PKC gamma was noticeably present in HEK cultures. Activation of partially purified total PKC by TPA was similar in freshly isolated and cultured MEKs and HEKs, indicating that the two species were similar in this regard and that 2 wk of culture did not alter this characteristic. When MEK and HEK cultures were treated with TPA for 3 h, less than 30% of the control level of PKC activity was detected, indicating that TPA-induced downregulation of PKC was similar in MEKs and HEKs. After treatment with TPA, MEK cultures produced a large induction of both c-jun and c-fos mRNA by 60 min, as determined by northern blot analysis, and a large induction of ODC mRNA and enzyme activity by 6 h. TPA treatment of cultured HEKs, however, did not induce ODC activity; in fact, less activity, compared with that of control cultures, was observed. Northern blot analysis also revealed no increase in c-jun, c-fos, and ODC mRNA in HEKs. However, c-jun and c-fos mRNA and both ODC mRNA and enzyme activity were induced in HEKs fed growth factors after several days of deprivation. This suggests that the lack of ODC induction by TPA in HEKs is probably due to species differences in downstream steps in PKC signal transduction.

Phorbol 12-myristate 13-acetate inhibits epidermal growth factor signalling in human keratinocytes, leading to decreased ornithine decarboxylase activity

“…In contrast to our results, Fisher and colleagues noted no increase in either ODC mRNA or ODC enzyme activity in normal human epidermal keratinocytes, whereas both were increased in murine epidermal keratinocyte cultures [9]. Hickok and colleagues reported that PMA caused a marked decrease in ODC enzyme activity with no detectable effect on ODC mRNA levels in cultured normal and immortalized, but not tumorigenic, human keratinocytes.…”

“…The observed lack of increased ODC activity in normal [3,9] or transformed human keratinocytes is consistent with the reported effect that PMA treatment has on inducing human keratinocyte differentiation [30]. PMA has been shown to induce differen-tiation in the same SV40-transformed keratinocytes studied here in parallel to an inhibition in growth [31] and in human papillomavirus (HPV) type 16 immortalized human keratinocytes [32].…”

“…Although it has previously been reported that PMA induces ODC in human skin biopsy specimens [7,25], recent reports suggest that ODC activity is down-regulated by PMA and UV B radiation in human keratinocytes [9,10,14] in contrast to rodentderived cells where both PMA and UV radiation induce ODC activity [26,27]. Furthermore, in our preliminary studies (results not presented) we were unable to demonstrate increased ODC activity following incubation of human skin with PMA.…”

“…In preliminary studies, we found that while epidermal growth factor (EGF) induced ODC activity in both murine and human punch biopsies, PMA induced ODC activity in murine but not human skin biopsies. Thus, from these and other comparative studies reported by others, an important species difference is evident in phorbol ester signalling that leads to ODC induction [8][9][10]. The studies described here were undertaken to define in greater detail the signalling effects of phorbol esters in human skin cells.…”

“…In the SV40-transformed cells utilized in the studies reported here PMA induced ODC mRNA in quiescent cells (growth factor-deprived) and inhibited the induction of ODC activity in EGF-stimulated cells. It is likely that the presence of EGF or other mitogens in the growth medium utilized in the human keratinocyte studies alluded to above [9,10] was at least partially responsible for the observation of decreased ODC activity in cultures treated with PMA in growth medium compared with cultures treated with growth medium alone. Additionally, the transformed state of the Z114 cells studied here may have altered the signalling response to PMA ; a study of PMA responsiveness in parental 10T1\2 fibroblasts and H-ras-transformed cells revealed that PMA induced ODC mRNA only in the transformed cells and not in the parental cell line [29].…”