Retinyl acetate mediates autocrine proliferation and wound healing of keratinocytes through a c-neu (erbB-2)-like receptor

Pleiotropic effects of retinoids on cell growth signal transduction and receptor-mediated gene regulation are well documented.1,2 The use of chemically-defined serum free medium (SFM) has identified two protein growth factors, EGF and insulin, that are required for normal human keratinocytes (NHK) proliferation in low calcium (<1mM) SFM medium.3,4 The role of retinoids in regulating proliferation of normal human keratinocytes is less well-understood. For example, all-trans retinoic acid (t-RA) stimulates proliferation in essential fatty acid-supplemented keratinocytes.5 Retinoic acid (t-RA, 10-7M) treatment inhibited proliferation of HaCaT cells adapted to serumfree DME/Hams F12 medium, while retinol (ROL, 10-7M) did not, but overall growth in this SFM was much curtailed.6 By contrast, a panel of retinoids including t-RA, ROL, 13-cis RA, all inhibited the clonal growth of NHK in SFM supplemented with EGF and insulin in rapidly proliferating NHK.7 The strength of inhibition was linearly correlated with their ability to suppress both ornithine decarboxylase enzyme induction by tumor promoter and papilloma formation in the mouse skin model of tumorigenesis. 7 By contrast, t-RA stimulates growth-arrested adult keratinocytes in protein growth factor-deficient SFM.8 These conflicting effects appear to involve differing growth media conditions. Verani et al.9 also reported that induced alterations in membrane intracellular calcium ion fluctuations appear to underlie retinoid growth stimulation.9 Retinoid stimulation of growth-arrested NHK was also found to involve autocrine production of a heparinbonding EGF (hb-EGF), and activation of erbB receptors10 present on suprabasal cells. Moreover, autocrine stimulation via these signaling pathways appears to underlie retinoid-induced epidermal hyperplasia.10 Recently, t-RA was reported to inhibit the expression of the erbB (c-neu) receptor and other proto-oncogenes in several different epidermoid carcinoma cell lines.11 In addition, t-RA reverses the super-induction by alcohol of aryl hydrocarbon hydrylase induced by benzo(α) pyrene in SFM culture of NHK.12 Retinoids also have profound effects on epidermal keratinocyte differentiation in many different tumor cells lines.13 Retinyl acetate (RetAc) is a naturally occurring fatty acid ester of retinol, and is less toxic than t-RA. It is stored in human liver and is involved to metabolic conversion to vitamin A (retinol). Here, we explore the effect of RetAc on the clonal growth of NHK and HaCaT cells treated with different combinations of EGF and insulin. We established that serum can be dispensed with by culturing HaCaT keratinocytes in SFM supplemented with EGF and insulin. This allowed us to conduct detailed clonal assay to determine their minimal growth factor requirements. Clonal growth assay studies also examined the effect of RetAc. Unlike t-RA,7 we report that RetAc stimulates HaCaT clonal growth at physiological levels. We further explored the underlying biochemical events that reveal an autocrine signaling pathway and possible involvement of the c-neu (erbB-2) cytoplasmic receptor and a treatment-induced translocation to focal membrane areas. Finally, we examined the effect