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

Abstract

Several studies have suggested that murine and human keratinocytes respond differently to phorbol 12-myristate 13-acetate (PMA). Using an in vitro assay, we found that in contrast to its effect on murine skin, PMA did not induce ornithine decarboxylase (ODC) activity in human skin biopsies. To explore the signalling induced by PMA and to determine whether an in vitro culture system could be used to predict biological activity of retinoids in human keratinocytes, we studied a simian virus 40 (SV40)-transformed human keratinocyte cell line. Epidermal growth factor (EGF) stimulates ODC activity and increases the steady-state level of ODC mRNA in a dose- and time-dependent manner in these cells [Prystowsky, Clevenger and Zheng (1993) Exp. Dermatol. 2, 125–132]. In this report, 10-10 M–10-7 M PMA induced ODC mRNA and enzyme synthesis at 7 h, but did not significantly induce ODC activity and inhibited the EGF induction of ODC activity. To explore the mechanism whereby PMA interferred with EGF signalling, the effect of PMA on EGF binding to its cell-surface receptor was studied; acute treatment with PMA (within 7 h) decreased EGF binding to 41–57% of the baseline level. In contrast, chronic treatment with PMA (24 h) increased EGF binding to 156% of the baseline level and was associated with an increase in quantity of EGF receptor protein. Protein kinase C (PKC) activation correlated with the acute decrease in EGF binding following PMA treatment. In summary, PMA induced ODC mRNA and ODC enzyme synthesis, while steady-state levels of immunoprecipitable ODC enzyme protein and ODC activity were not increased, demonstrating possible increased turnover of ODC enzyme protein. Additionally, PMA inhibited the induction of ODC by EGF through decreased EGF binding, possibly mediated by PKC activation. Finally treatment of the keratinocytes with retinoids including etretinate, Ro13-7410, etarotene, Ro40-8757, 13-cis-retinoic acid, and acitretin blocked the PMA induction of ODC mRNA, suggesting this in vitro model could be a valuable screening assay for predicting biological activity in humans.

The effects of 17β‐oestradiol, testosterone and tamoxifen on the development of papillomata in Catostomus commersoni

“…ODC activity was elevated in proliferating, newly developed and wild-type tumours but was suppressed or reduced in regressing tumours. These are in accordance with mammalian studies where depressed PKC activity is concurrent with increased ODC activity in proliferating or steady-state tumours; and where, in normal tissues, high levels of PKC are concurrent with minimal ODC activity (Verma et al, 1996;Xue et al, 1996). Tamoxifen was effective in preventing the development papillomata and inhibited the growth of existing neoplasms; sunflower oil also appeared to provide some chemotherapeutic benefits.…”

“…PKC and ODC are differentially regulated in a time and dose-dependant manner (Verma et al, 1996;Xue et al, 1996). Mitogenic stimulation induces a rapid, but very transient, increase in available PKC that precedes a rapid and sustained drop in PKC levels as the enzyme is depleted from the cellular pools and translocated to the cellular membrane (Butler et al, 1994;Verma et al, 1996).…”

Growth-inhibitory effects of the chemopreventive agent indole-3-carbinol are increased in combination with the polyamine putrescine in the SW480 colon tumour cell line

“…ODC activity can be regulated via many signalling pathways, including the epidermal growth factor receptor, PI3K, and estrogen receptor pathways [41-44], which are potential targets for the action of chemopreventive agents such as I3C. We have shown, for example, that I3C can inhibit the PI3K/ protein kinase B pathway in the MDA MB468 breast tumour cell line, but not in the normal derived HBL100 line [67], but this did not correlate with the ability of this agent to inhibit ODC activity in those cell lines (Howells L.M.…”

“…The ODC promoter has been shown in various species to be regulated by a number of transcription factors, including the Wilms' tumour suppressor WT1 [37-40] and references therein. ODC activity can also be modulated in response to many signalling pathways including the epidermal growth factor receptor, phospho-inositide 3-kinase (PI3K), and estrogen receptor pathways [41-44]. …”

Human Melanocytes Do Not Express EGF Receptors

“…Additionally, the results may have been biased by inclusion of TPA in the culture medium, as is commonly employed for melanocyte cultivation (Eisinger et al, 1985;Herlyn et al, 1987;Pittelkow and Shipley, 1989;Halaban, 1994Halaban, , 2000. TPA can alter EGFR expression and ligand binding, resulting in an increase in EGF-mediated signaling and function (Matsui et al, 1993;Sedlak et al, 1994;Xue et al, 1996). For that reason, for this study, some experiments were performed on human melanocytes cultivated in the absence of TPA.…”