epidermal-growth-factor and sapropterin

Recently we demonstrated a strong expression of inducible nitric oxide synthase (iNOS) and GTP-cyclohydrolase I (GTP-CH I) in the basal keratinocytes of the epidermis adjacent to the wound and of the hyperproliferative epithelium during wound healing. To identify possible mediators of iNOS and GTP-CH I expression during this process, we analyzed the regulation of iNOS and GTP-CH I expression in cultured human keratinocytes. We found a large and long lasting coinduction of iNOS and GTP-CH I expression upon simultaneous treatment of quiescent cells with inflammatory cytokines interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma, but not with serum growth factors. The stimulatory effect of interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma is strongly synergistic on iNOS and GTP-CH I expression, because these factors alone stimulated GTP-CH I expression, although to a much lesser extent. Furthermore, iNOS mRNA levels are not influenced at all by stimulation with IL-1beta and revealed only a weak induction after treatment with tumor necrosis factor-alpha and interferon-gamma. Induction of iNOS and GTP-CH I gene expression upon cytokine and interferon-gamma exposure is independent of de novo protein synthesis. Because these cytokines are present at the wound site, they might be responsible for iNOS and GTP-CH I induction during cutaneous repair. Serum, which is released upon hemorrhage, is likely to play no stimulatory role in iNOS and GTP-CH I induction during wound healing.

Topics: Antioxidants; Biopterins; Cell Line; Cytokines; Enzyme Induction; Epidermal Growth Factor; GTP Cyclohydrolase; Humans; Interferon-gamma; Interleukin-1; Keratinocytes; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Platelet-Derived Growth Factor; RNA, Messenger; Skin Physiological Phenomena; Tumor Necrosis Factor-alpha; Up-Regulation; Wound Healing

Epidermal growth factor and nerve growth factor increased the proliferation of rat phaeochromocytoma PC12 cells through obligatory elevation of intracellular (6R)-tetrahydrobiopterin (BH4). Epidermal growth factor and nerve growth factor increased intracellular BH4 by inducing GTP-cyclohydrolase, the rate-limiting enzyme in BH4 biosynthesis. Specific inhibitors of BH4 biosynthesis prevented growth factor-induced increases in BH4 levels and proliferation. The induction of GTP cyclohydrolase, BH4 and cellular proliferation by nerve growth factor was mediated by cAMP. Elevation of BH4 biosynthesis occurred downstream from cAMP in the cascade used by nerve growth factor to increase proliferation. Thus, intracellular BH4 is an essential mediator of the proliferative effects of epidermal growth factor and nerve growth factor in PC12 cells.

Topics: Animals; Biopterins; Cell Division; Cyclic AMP; DNA Fragmentation; Epidermal Growth Factor; Nerve Growth Factors; PC12 Cells; Rats

The regulation of catecholamine and tetrahydrobiopterin synthesis was investigated in cultured rat pheochromocytoma PC12 cells following treatments with nerve growth factor (NGF), epidermal growth factor (EGF) and interferon-gamma (IFN-gamma). NGF and EGF, but not IFN-gamma, caused an increase after 24 h in the levels of BH4 and catecholamines, and the activities of tyrosine hydroxylase and GTP cyclohydrolase, the rate-limiting enzymes in catecholamine and BH4 synthesis, respectively. Actinomycin D, a transcriptional inhibitor, blocked treatment-induced elevations in tyrosine hydroxylase and GTP cyclohydrolase activities. NGF, EGF or IFN-gamma did not affect the activity of sepiapterin reductase, the final enzyme in BH4 biosynthesis. Rp-cAMP, an inhibitor of cAMP-mediated responses, blocked the induction of tyrosine hydroxylase by NGF or EGF; inhibition of protein kinase C partially blocked the EGF effect, but not the NGF effect, NGF also induced GTP cyclohydrolase in a cAMP-dependent manner, while the EGF effect was not blocked by Rp-cAMP or protein kinase C inhibitors. Sphingosine induced GTP cyclohydrolase in a protein kinase C-independent manner without affecting tyrosine hydroxylase activity. Our results suggest that both tyrosine hydroxylase and GTP cyclohydrolase are induced in a coordinate and transcription-dependent manner by NGF and EGF, while conditions exist where the induction of tyrosine hydroxylase and GTP cyclohydrolase is not coordinately regulated.

Topics: Animals; Biopterins; Cells, Cultured; Epidermal Growth Factor; Interferon-gamma; Nerve Growth Factors; PC12 Cells; Rats; Time Factors; Tyrosine 3-Monooxygenase