Opposite long-term regulation of c-Myc and p27Kip1 through overactivation of Raf-1 and the MEK/ERK module in proliferating human choroidal melanoma cells.

Although there is no current evidence for ras gene mutation in choroidal melanoma, there is an increasing body of evidence indicating that deregulated intracellular signalling pathways are involved in choroidal melanoma pathogenesis. The various components of the linear Raf/MEK/ERK signalling pathway have been implicated in various tumours. We therefore investigated the role of Raf-1 and the MEK/ERK module in the proliferation of human normal choroidal melanocytes (NCM) and cells from the ocular choroidal melanoma (OCM-1) cell line. OCM-1 cells proliferated four times faster than NCM. High basal activation of the MEK/ERK module was observed in unstimulated OCM-1 cells, whereas rapid and persistent activation was detected after serum stimulation, throughout the 24-h period of culture. In contrast, the activation of MEK/ERK was barely detectable in unstimulated NCM and occurred late (6 h) after the stimulation of cell proliferation. Inhibition of Raf-1 and MEK1/2 activation by pharmacological approaches and of the production of Raf-1 and ERK1/2 by antisense oligonucleotide approaches demonstrated that Raf-1 and the MEK/ERK module controlled proliferation in OCM-1 cells, but not in NCM. OCM-1 cells produced very low levels of p27Kip1, whereas NCM produced constant, high levels of p27Kip1. The inhibition of Raf-1 or MEK1/2 induced a large increase in p27Kip1 in OCM-1 cells, associated with an arrest of cell proliferation. Levels of c-Myc production were high and constant in OCM-1 cells and low in NCM, in contrast to what was observed for p27Kip1. The inhibition of both Raf-1 and MEK1/2 induced a decrease in c-Myc production and downregulated c-Myc activity by preventing c-Myc phosphorylation in OCM-1 cells. We conclude that Raf-1 and the MEK/ERK module control the production of both p27Kip1 and c-Myc, and the activation of c-Myc for OCM-1 cell proliferation.

Activation and role of MAP kinase-dependent pathways in retinal pigment epithelium cells: JNK1, P38 kinase, and cell death.

PURPOSE: Retinal pigment epithelial (RPE) cell death is an important step in the pathogenesis of ocular diseases. JNK1 and P38 kinase, two stress-activated kinases, play key roles relaying stress signals leading to cell death through cyclin D1 and c-Myc. Recently, stress-activated kinases have been shown to regulate cell proliferation. In the current study, the involvement of the JNK1 and P38 kinase signaling pathways in RPE cell proliferation and death was investigated. METHODS: RPE cell proliferation was stimulated with 10% fetal calf serum (FCS). Activation of the JNK1 and P38 kinase cascades and their potential targets was detected by Western blot analysis. Pharmacologic inhibitors and activators, and antisense oligodeoxynucleotides (ODN) directed against the stress kinases were used to analyze the signaling involved in RPE cell death. RESULTS: P38 and JNK1 and their respective upstream activating kinases, MKK3/6 and -4, were all transiently activated in FCS-stimulated RPE cell cultures. Ras controlled only the activation of JNK1, whereas Rho transmitted the activation of both JNK1 and P38, suggesting parallel signaling pathways and cross talk between the two kinases. Pharmacologic inhibition of JNK1 did not affect cell proliferation in FCS-stimulated cells. Inactivation of P38 kinase and antisense ODN-induced downregulation of P38 kinase also had no affect on cell proliferation. Long-term, high-level activation of JNK1 and P38 kinase occurred during serum depletion-induced RPE cell death. Overactivation of JNK1 and P38 kinase was also observed during pharmacologically induced cell death, suggesting that this process is common to RPE cell-death-signaling pathways induced by various stress stimuli. Cell death mediated by the overactivation of JNK1 and P38 kinase was cyclin D1- and c-Myc-independent. CONCLUSIONS: The inhibition of JNK1 or P38 kinase had no effect on FCS-stimulated proliferation of RPE cells, whereas the overactivation of these two enzymes was involved in RPE cell death in FCS-depleted cultures. Parallel upstream signaling pathways and cross talk between the two kinases suggest that the regulation of signaling in RPE cell death is complex.

Activation and role of MAP kinase-dependent pathways in retinal pigment epithelial cells: ERK and RPE cell proliferation.

PURPOSE: Retinal pigment epithelial (RPE) cell proliferation plays a key role in the pathogenesis of ocular diseases involving the posterior segment. Mitogen-activated protein kinases (MAPKs) are involved in the control of cell proliferation. This study was conducted to investigate the involvement of the extracellular signal-regulated kinase (ERK) pathway, the major MAPK pathway implicated in cell growth during the induction of RPE cell proliferation. METHODS: RPE cell proliferation was stimulated with 10% fetal calf serum (FCS). Activation of the Ras/Raf/MAP kinase-ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway was detected by Western blot analysis and immunochemistry with specific anti-phosphosignaling protein antibodies. Pharmacologic and antisense (AS) oligonucleotide (ODN) strategies were used to analyze the ERK signaling involved in serum-induced cell proliferation. RESULTS: FCS (10%) induced more vigorous RPE cell proliferation than did FGF2, VEGF, platelet-derived growth factor (PDGF), or epidermal growth factor (EGF), alone or in combination. Pharmacologic inhibition of Ras and Raf-1 reduced cell proliferation by 67% to 100% and by 62% to 79%, respectively, demonstrating that activation of the Ras/Raf-1 pathway was essential for FCS-induced RPE cell proliferation. MEK1/2, ERK2, and P90 ribosomal S6 kinase (P90(RSK)), the kinases downstream from ERK2, were strongly activated during cell proliferation. Pharmacologic inhibition of MEK1/2 abolished activation of ERK2, but reduced cell proliferation by only 32%, showing that MEK/ERK participates in the signaling involved in RPE cell growth. Both inhibition of ERK2 activation, which reduced cyclin D1 production, and inhibition of cyclin D1 by AS ODN decreased cell proliferation, suggesting that RPE cell proliferation is mediated by cyclin D1 through ERK2. CONCLUSIONS: The requirement for Ras and the regulatory role of ERK2 in cyclin D1 production and in cell proliferation suggest that the Ras/Raf/MEK/ERK pathway plays a key role in the control of RPE cell proliferation. These data may have important implications for the development of more selective methods for the inhibition of retinal proliferation.

cAMP inhibits the proliferation of retinal pigmented epithelial cells through the inhibition of ERK1/2 in a PKA-independent manner.

Retinal pigmented epithelial (RPE) cell integrity is critical to the maintenance of retina functions and RPE cells do not proliferate in adults. The activation of RPE results in cell proliferation which may be associated with proliferative retinopathy and choroidal melanoma. Mitogen-activated protein kinase (MAPK) is believed to be a key participant in the response to mitogenic stimuli. We therefore investigated the involvement of the extracellular signal-regulated protein kinase (ERK) 1 and 2 during the induction of RPE cell proliferation. After foetal calf serum (FCS) stimulation activation of the Ras/Raf/ERK signalling pathway was detected by Western blotting and immunochemistry, with specific anti-phosphosignalling protein antibodies. Pharmacological and antisense (AS) oligonucleotide (ODN) strategies were used to analyse the signalling involved in FCS-induced RPE cell proliferation. Activation of the small G protein Ras and, to a lesser extent of Raf-1, the kinase directly downstream from Ras, was necessary to FCS-induced cell proliferation. MEK1/2 and ERK1/2 were activated during cell proliferation. Inhibition of MEK1/2 with UO 126 completely abolished ERK1/2 activation and reduced cell proliferation by 33-43%. ERK1/2 depletion by an AS ODN approach reduced cell proliferation by 27-33%, confirming the role of ERK1/2 in the FCS stimulation of RPE cells. We also investigated the role of PKA/cAMP, one of the major inhibitory pathways of ERK1/2. PKA blockade did not modify ERK1/2 activation or cell proliferation. In contrast, agents that increased cAMP concentration, abolished RPE proliferation, and MEK/ERK activation. Moreover, inhibition of the cAMP-activated small G protein Rap1, partially reversed the inhibitory effects of cAMP on cell proliferation and MEK/ERK activation. The requirement for Ras and ERK1/2, the lack of ERK1/2 regulation by PKA and the cAMP/Rap1 counter-regulatory pathway for ERK-mediated cell proliferation suggest complex regulation of signalling in RPE cells. These data may have important implications for the development of more selective models for retinal anti-proliferative therapies.