Modulation of mRNA levels during human keratinocyte differentiation

Abstract

Cultures of human keratinocytes provide an excellent model system in which to study differentiation. Using the phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) and calcium, two agents known to induce keratinocyte differentiation in vitro, we examined the expression of the genes encoding c-fos, c-myc, and c-jun; involucrin, a protein precursor of the keratinocyte cornified envelope; and L-7, a ribosomal protein. Overall, at the doses studied, TPA induced a more rapid and profound differentiation than did calcium, as evaluated by culture morphology and northern blot analysis. Our studies showed a constant low level of c-fos and c-jun expression in unstimulated cells with no significant change after addition of either TPA or calcium except when transcript breakdown was inhibited by cycloheximide. The c-myc proto-oncogene, known to have a high constitutive expression in actively proliferating cells, was strongly downregulated by TPA, but calcium had no effect over a 32 hour period, consistent with the greater growth inhibition of TPA in this system. Involucrin was induced about ninefold by both TPA and calcium as early as 8 hours after stimulation, suggesting transcriptional regulation of this gene during differentiation. L-7, recently demonstrated to be downregulated in late passage human fibroblasts in an in vitro model of senescence, was also strongly downregulated by either TPA or calcium, consistent with an interrelationship between the basic cellular processes of aging and differentiation. These finding expand our knowledge of the differentiation process in human keratinocytes.

Mitogenic Signaling of Insulin-like Growth Factor I in MCF-7 Human Breast Cancer Cells Requires Phosphatidylinositol 3-Kinase and Is Independent of Mitogen-activated Protein Kinase

“…Growth inhibition and induction of differentiation by TPA have also been documented in other cell types, e.g. keratinocytes (21,54) and melanoma-derived cells (20,55). In contrast, TPA has been shown to stimulate the growth of several other cell types, e.g.…”

Bricks and mortar of the epidermal barrier

“…by calcitriol (Su et al, 1994), corticosteroids (Cline and Rice, 1983), phorbol esters (Takahashi and Iizuka, 1993), and Ca 2 + ( Younus and Gilchrest, 1992), by regulatory elements located within about the first 2.5 kb upstream of the cap site (Welter et al, 1995;Lopez-Bayghen e t a l., 1996;Welter et al, 1996;Crish et al, 1998). CEs from 'dry' epithelia such as human foreskin epidermis contain about 5% involucrin .…”

c-Myc promotes differentiation of human epidermal stem cells

“…Down-regulation of c-Myc accompanies differentiation of a range of cell types and ectopic expression of c-Myc has been shown to inhibit differentiation in a variety of in vitro models (DePinho et al 1991;Henriksson and Lü scher 1996). We and others have shown that the level of c-Myc mRNA decreases when keratinocytes undergo terminal differentiation (Younus and Gilchrest 1992;Yaar et al 1993;Gandarillas and Watt 1995;Hurlin et al 1995b). TGF␤ 1 is thought to inhibit keratinocyte proliferation by downregulating c-Myc (Pietenpol et al 1990;Alexandrow et al 1995) and c-Myc antisense inhibits keratinocyte growth (Hashiro et al 1991).…”