epidermal-growth-factor and adenosine-3--5--cyclic-phosphorothioate

Treatment of serum-starved quiescent human cells with fetal bovine serum (FBS), epidermal growth factor (EGF), or the phorbol ester (12-O-tetradecanoylphorbol-13-acetate, TPA) activates the RAS-MAPK pathway which initiates a transcriptional program which drives cells toward proliferation. Stimulation of the RAS-MAPK pathway activates mitogen- and stress-activated kinases (MSK) 1 and 2, which phosphorylate histone H3 at S10 (H3S10ph) or S28 (H3S28ph) (nucleosomal response) located at the regulatory regions of immediate-early genes, setting in motion a series of chromatin remodeling events that result in transcription initiation. To investigate immediate-early genes regulated by the MSK, we have completed transcriptome analyses (RNA sequencing) of human normal fibroblast cells (CCD-1070Sk) stimulated with EGF or TPA ± H89, a potent MSK/PKA inhibitor. The induction of many immediate-early genes was independent of MSK activity. However, the induction of immediate-early genes attenuated with H89 also had reduced induction with the PKA inhibitor, Rp-cAMPS. Several EGF-induced genes, coding for transcriptional repressors, were further upregulated with H89 but not with Rp-cAMPS, suggesting a role for MSK in modulating the induction level of these genes.

Topics: Cell Line; Cyclic AMP; Epidermal Growth Factor; Fibroblasts; Gene Expression Profiling; Gene Expression Regulation; Genes, Immediate-Early; Humans; Isoquinolines; Mitogens; Reproducibility of Results; Ribosomal Protein S6 Kinases, 90-kDa; Sulfonamides; Tetradecanoylphorbol Acetate; Thionucleotides

Vasoactive intestinal peptide (VIP) is a neuropeptide with a broad range of biological activities in various tissues. Interactions of VIP and epidermal growth factor (EGF) are of particular interest for dermatology. They may be either co-mitogenic or inhibitory. HaCaT keratinocytes cultivated under serum-free conditions in vitro have been used to investigate the interactions of VIP and EGF. EGF was found to induce cell growth, whereas preincubation with VIP inhibited EGF-induced proliferation in a dose-dependent manner. Maximum growth inhibition was 46% (p < 0.01) at a VIP concentration of 10(-7) M. EGF-induced growth is mediated by tyrosine kinase (TK). Therefore we studied the effect of VIP on TK activity. Cells were incubated with VIP (10(-13)-10(-7) M) for 48 h and stimulated with EGF at a final concentration of 500 ng/ml. SDS-PAGE and Western blot with the antibody RC20H against TK were performed. We found a dose dependent decrease of EGF receptor TK activity. At VIP concentration of 10(-7) M a residual TK activity of 65% was detected. To investigate the possibly involved signal transduction pathways, we performed inhibition experiments with wortmannin, pertussis toxin, 2'5'diacylglycerol and adenosine-3':5'-mono-phosphorothioate. However, none of the inhibitors was effective in abolishing growth inhibition by VIP. VIP was shown to be growth inhibitory for human keratinocytes. The data suggest that EGF receptor TK is involved in signal transduction of VIP. Thus TK activity is a possible common target of both EGF- and VIP-induced cellular responses.

Topics: Androstadienes; Cell Division; Cells, Cultured; Culture Media, Serum-Free; Cyclic AMP; Diglycerides; Dose-Response Relationship, Drug; Drug Synergism; Epidermal Growth Factor; ErbB Receptors; Humans; Keratinocytes; Mitogens; Pertussis Toxin; Phosphorylation; Thionucleotides; Vasoactive Intestinal Peptide; Virulence Factors, Bordetella; Wortmannin