The effects of IL-4 on the proliferation of normal human keratinocytes in vitro were studied. Proliferation of keratinocytes was stimulated by IL-4 and neutralized by the treatment of anti-IL-4 antibody. Anti-IL-6 antibody did not inhibit the proliferation of normal human keratinocytes, suggesting that the IL-4 is directly responsible for the keratinocyte proliferation. The IL-4 significantly induced cell cycle progression from G0/G1 to S phase. The keratinocyte proliferation by IL-4 appeared to be mediated through a growth control gene, c-myc proto-oncogene. The c-myc mRNA expression was significantly increased after the IL-4 treatment of keratinocytes, suggesting that c-myc gene expression plays a key role in the control of keratinocyte proliferation. The signal transduction pathways induced by IL-4 in the keratinocytes were also studied using chemicals that can intervene in certain signal transduction events. Genistein, a tyrosine kinase inhibitor, suppressed c-myc mRNA expression induced by IL-4. H7, a serine/threonine kinase inhibitor, and okadaic acid, a protein phosphatase 1 and 2A inhibitor, however, did not block the c-myc gene expression. These results suggest that IL-4 stimulates the proliferation of keratinocytes in vitro by promoting a cell cycle transition from G0/G1 to S phase. The induction of c-myc after IL-4 treatment implicates an important role of c-myc in the proliferation of keratinocytes.
The data also suggest an important role of tyrosine kinases on the IL-4-induced proliferation of keratinocytes. Tyrosine phosphorylation and dephosphorylation of cellular proteins have been suggested to play important roles in cellular signaling and many other cellular activities. After the search of protein tyrosine phosphatases expressed in human keratinocytes, a human homologue of the murine R-PTP-k (human R-PTP-k) has been cloned. Amongst the several overlapping cDNA clones, one clone was found to encode a full length of human R-PTP-k. The human R-PTP-$\kappa$ gene was located in the chromosome 6. Northern blot analysis from several human tissues revealed that the human R-PTP-$\kappa$ mRNA of $\sim 7$kb in length was predominantly expressed in spleen, prostate, and ovary. Lower levels of R-PTP-$\kappa$ transcripts were detected in tissues such as thymus, testis, small intestine, and colon. Interestingly, the expression of R-PTP-$\kappa$ was not detectable in peripheral blood lymphocytes. It was found that human R-PTP-$\kappa$ was expressed in epidermal cells and cell lines including human normal keratinocytes, HaCaT cell, and A431 cell.
In an attempt to find a regulator of human R-PTP-k gene expression, various regulators of the keratinocyte growth were treated in an immortalized human keratinocyte cell line (HaCaT). The treatment of TGF-β1 to the HaCaT cells markedly increased the expression of R-PTP-$\kappa$ mRNA. The induction of R-PTP-$\kappa$ mRNA expression was observed at a dose as low as 0.02 ng/ml TGF-β1 and reached a peak at 2 ng/ml of TGF-β1 in 6 h after treatment. To elucidate the signal transduction pathways induced by TGF-β1, effects of the inhibitors of protein kinases or phosphatases were examined on the expression of the R-PTP-$\kappa$ gene. Sodium orthovanadate, a tyrosine phosphatase inhibitor, fully suppressed the R-PTP-$\kappa$ mRNA expression induced by TGF-β1, but genistein, a tyrosine kinase inhibitor, did not affect the R-PTP-$\kappa$ gene expression. A serine/threonine kinase inhibitor, H7, was found to exert an inhibitory effect on the TGF-β1 induction of R-PTP-$\kappa$ gene expression. These studies suggest that TGF-β1 inhibits the human keratinocyte proliferation $\It{in vitro}$ through the signal transduction pathway that could also induce the R-PTP-$\kappa$ gene expression.