Modulation of growth and differentiation in normal human keratinocytes by transforming growth factor‐β

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Abstract
The effect of transforming growth factor‐type β 1(TGF‐β) on the growth and differentiation of normal human skin keratinocytes cultured in serum‐free medium was investigated. TGF‐β markedly inhibited the growth of keratinocytes at the concentrations >2 ng/ml under low Ca2+ conditions (0.1 mM). Growth inhibition was accompanied by changes in cell functions related to proliferation. Remarkable inhibition of DNA synthesis was demonstrated by the decrease of [3H]thymidine incorporation. The decrease of [3H]thymidine incorporation was observed as early as 3 hr after addition of TGF‐β. TGF‐β also decreased c‐myc messenger RNA (mRNA) expression 30 min after addition of TGF‐β. This rapid reduction of c‐myc mRNA expression by TGF‐β treatment is possibly one of the main factors in the process of TGF‐β‐induced growth inhibition of human keratinocytes. Since growth inhibition and induction of differentiation are closely related in human keratinocytes, the growth‐inhibitory effect of TGF‐β under high Ca2+ conditions (1.8 mM Ca2+ differentiation‐promoting culture environment) was examined. TGF‐β inhibited the growth of keratinocytes under high Ca2+ conditions in the same manner as under low Ca2+ conditions, suggesting that it is a strong growth inhibitor in both low and high Ca2+ environments. The induction of keratinocyte differentiation was evaluated by measuring involucrin expression and cornified envelope formation: TGF‐β at 20 ng/ml increased in‐volucrin expression from 9.3% to 18.8% under high Ca2+ conditions, while it decreased involucrin expression from 7.0% to 3.3% under low Ca2+ conditions. Cornified envelope formation was modulated in a similar way by addition of TGF‐β: TGF‐β at 20 ng/ml decreased cornified envelope formation by 53% under low Ca2+ conditions, while it enhanced cornified envelope formation by 30.7% under high Ca2+ conditions. Thus, the effect of TGF‐β on keratinocyte differentiation is Ca2+ dependent. It enhances differentiation of human keratinocytes under high Ca2+ conditions, but inhibits differentiation under low Ca2+ conditions. Taken together, there is a clear discrepancy between TGF‐β effects on growth inhibition and induction of differentiation in human keratinocytes. These data indicate that growth inhibition of human keratinocytes by TGF‐β is direct and not induced by differentiation.