Fer kinase sustains the activation level of ERK1/2 and increases the production of VEGF in hypoxic cells

Fer is a nuclear and cytoplasmic tyrosine kinase that is ubiquitously expressed in mammalian cells. Herein we show that Fer sustains a key signaling step in hypoxic cells. Knock-down of the Fer protein using a specific siRNA decreased the production of VEGF by the hypoxic cells. Conversely, ectopic expression of this kinase led to an elevated production of VEGF under hypoxia. At the molecular level, Fer was found to associate with ERK1/2 and this interaction was intensified under hypoxia. Moreover, Fer increased the activation levels of ERK1/2, and reducing the level of Fer, impaired the activation of ERK1/2 in hypoxic cells. Blocking the MEK-ERK1/2 signaling pathway with the MEK inhibitors U0126, or PD98059 led to the abrogation of ERK1/2 activity in hypoxic cells, an effect that was counteracted by Fer. Hence, Fer sustains the activation of ERK1/2 and increases the production of VEGF in hypoxic cells, without affecting the MEK-ERK signaling pathway.     

Growth-related signaling regulates activation of telomerase in regenerating hepatocytes

Although there have been many reports on the relationship between activation of telomerase and carcinogenesis, the role of telomerase in normal cellular growth is still unclear. In this study, we analyzed the relationship between upregulation of telomerase activity and cell cycle progression during the liver regeneration process by using an in vivo mouse two-thirds partial hepatectomy (PH) model as well as by using in vitro hepatocyte culture systems. Furthermore, we also investigated the effects of growth factors on telomerase activity during liver regeneration and the influence of MAPK pathway inhibitors (MEK inhibitors PD98059 and U0126; p38 MAPK inhibitor SB203580) on the telomerase activity of regenerating hepatocytes in vitro. An upregulation of the telomerase activity was found at 24 h after PH, and thereafter an increase in the S-phase fraction was observed at 36-48 h. There was no remarkable change in the telomere length after PH. Preoperative treatment with EGF and HGF increased the in vivo telomerase activity. In a hepatocyte primary culture, the upregulation of the telomerase activity required the presence of EGF, and this upregulation was accelerated by the addition of HGF. A remarkable activation of p44/42 MAPK was seen but no such activation of p38 MAPK was observed at 48 h after PH. Although SB203580 had no effect on the telomerase activity of regenerating hepatocytes, treatment with MEK inhibitors (PD 98059, U0126) significantly repressed the telomerase activity. In conclusion, the telomerase activity is upregulated before hepatocytes enter the S phase, and both EGF and HGF play important roles in this step. In addition, the activation of the p44/42 MAPK pathway seems to play an essential role in telomerase upregulation during the liver regeneration process.     

Differential effects of TGF-beta1 and BMP-4 on the hypoxic induction of cyclooxygenase-2 in human pulmonary artery smooth muscle cells

Chronic hypoxia-induced pulmonary hypertension results partly from proliferation of smooth muscle cells in small peripheral pulmonary arteries. Previously, we demonstrated that hypoxia modulates the proliferation of human peripheral pulmonary artery smooth muscle cells (PASMCs) by induction of cyclooxygenase-2 (COX-2) and production of antiproliferative prostaglandins. The transforming growth factor (TGF)-beta superfamily plays a critical role in the regulation of pulmonary vascular remodeling, although to date an interaction with hypoxia has not been examined. We therefore investigated the pathways involved in the hypoxic induction of COX-2 in peripheral PASMCs and the contribution of TGF-beta1 and bone morphogenetic protein (BMP)-4 in this response. In the present study, we demonstrate that hypoxia induces activation of p38MAPK, ERK1/2, and Akt in PASMCs and that these pathways are involved in the hypoxic regulation of COX-2. Whereas inhibition of p38(MAPK) or ERK1/2 activity suppressed hypoxic induction of COX-2, inhibition of the phosphoinositide 3-kinase pathway enhanced hypoxic induction of COX-2. Furthermore, exogenous TGF-beta1 induced COX-2 mRNA and protein expression, and our findings demonstrate that release of TGF-beta1 by PASMCs during hypoxia contributes to the hypoxic induction of COX-2 via the p38MAPK pathway. In contrast, BMP-4 inhibited the hypoxic induction of COX-2 by an MAPK-independent pathway. Together, these findings suggest that the TGF-beta superfamily is part of an autocrine/paracrine system involved in the regulation of COX-2 expression in the distal pulmonary circulation, and this modulates hypoxia-induced pulmonary vascular cell proliferation.     

Inhibitory effect of a naphthazarin derivative, S64, on heat shock factor (Hsf) activation and glutathione status following hypoxia

The presence of hypoxic cells in solid tumors has long been considered a problem in cancer treatment. Resistance of hypoxic cells to ionizing radiation and anticancer drugs has in part been attributed to changes in altered gene expression by hypoxia. We previously reported an activation of heat shock factor (Hsf) in murine tumor RIF cells following hypoxia and suggested that a subsequent accumulation of heat shock protein(s) (Hsp) is likely to contribute to the malignant progression of hypoxic tumor cells (Baek et al., 2001). In this study, we showed that hypoxia induced a DNA-binding activity of Hsf and activation of hsp70 gene expression in colon cancer Clone A cells, and that a naphthazarin derivative, S64, significantly inhibited the hypoxia-inducible hsp70 gene expression in Clone A cells. We also showed that S64 significantly reduced the cellular glutathione levels in this cell line. Considering the proposed effects of Hsp and glutathione on radiation and chemotherapy sensitivity, we suggest that the inhibitory effects of S64 on Hsf activation and cellular glutathione levels have potentially important clinical implications. We believe that the previously reported in vitro and in vivo anti-tumor effect of S64 (Song et al., 2000a, 2001) might be attributed, at least in part, to its effect on Hsf activation and/or glutathione depletion. We also believe that the detailed molecular mechanisms underlying the effects of S64 on Hsf and glutathione level following hypoxia deserve a more rigorous future study, the results of which could offer novel strategy to manipulate the resistance mechanisms of solid tumors.     

Susceptibility to cell death induced by blockade of MAPK pathway in human colorectal cancer cells carrying Ras mutations is dependent on p53 status

Constitutive activation of mitogen-activated protein kinase (MAPK) pathway is implicated in a variety of human malignancies especially those that carry Ras mutations and is currently exploited as a cancer therapeutic target. The variability of response by cancer cells to the inhibition of the Ras/MAPK pathway both in vivo and in vitro, however, suggests that the genetic background of the tumor cell may modulate the effectiveness of this directed therapeutic. In a panel of colorectal cancer cell lines that carry Ras mutations and have constitutively active MEK/MAPK, we found that inhibition of the MAPK upstream kinase MEK by the small molecular MEK inhibitor U0126 induced cell death only in p53 wild-type cells. By contrast, p53-deficient cells were not affected by blocking the MEK/MAPK pathway. Using isogenic colon cancer cell lines and RNA interference, we show that loss of p53 significantly reduces MAPK phosphorylation and renders cells resistant to U0126 treatment. These findings reveal a critical role for p53 in MAPK-driven cell survival and place p53 upstream in the control cascade of MAPK activity. The therapeutic implication of these observations is that MAPK inhibitors will be most beneficial as a therapeutic agent in p53 normal colon cancers where constitutively active MAPK resulting from a Ras mutation is required for cell survival.     

MEK1-independent activation of MAPK and MEK1-dependent activation of p70 S6 kinase by stem cell factor (SCF) in ovarian cancer cells

We discovered a stem cell factor (SCF)-triggered, MEK1-independent, and PI3K-dependent MAPK activation pathway in the Kit-expressing ovarian cancer cell line HEY. When we knocked down MEK1 with RNA interference (RNAi) to study the function of MEK1 on the proliferation and survival of ovarian cancer cells, we found that impaired cell growth still occurred after MEK1 expression had been suppressed, although MAPK activation remained intact. This suggests that there is MEK1-independent activation of MAPK in the SCF-induced ovarian cancer cell growth process, and that MEK1 still plays a crucial role in maintaining the malignant properties of ovarian cancer cells even when it fails to activate MAPK as expected.     

Raf-1/MEK/MAPK pathway is necessary for the G2/M transition induced by nocodazole

The dynamic balance between polymerization and depolymerization of microtubules is critical for cells to enter and exit mitosis, and drugs that disrupt this balance, such as taxol, colchicine, and nocodazole, arrest the cell cycle in mitosis. Although the Raf/MEK/MAPK pathway can be activated by these drugs, its role in mitosis has not been addressed. Here, we characterize activation of Raf/MEK/MAPK by nocodazole when mitosis is induced. We find that at early time points (up to 3 h) in nocodazole induction, Raf/MEK/MAPK is activated, and inhibition of MAPK activation by a MEK inhibitor, PD98059 or U0126, reduces the number of cells entering mitosis by creating a block at G(2). At later time points and in mitosis, activation of MEK/MAPK is severely inhibited, even though Raf-1 activity remains high and can be further increased by growth factor. This inhibition is reversed when cells are released from metaphase and enter G(0)/G(1) phase. In addition, we find that binding of Raf-1 to 14-3-3 is progressively induced by nocodazole, reaching a maximum in mitosis, and that this binding is necessary to maintain mitotic Raf-1 activity. Our present study indicates that activation of the Raf/MEK/MAPK pathway is necessary for the G(2)/M progression.     

Nuclear mitogen-activated protein kinase activation by protein kinase czeta during reoxygenation after ischemic hypoxia

We examined the upstream kinases for mitogen-activated protein kinase (MAPK) activation during ischemic hypoxia and reoxygenation using H9c2 cells derived from rat cardiomyocytes. Protein kinase C (PKC)zeta, an atypical PKC isoform mainly expressed in rat heart, has been shown to act as an upstream kinase of MAPK during ischemic hypoxia and reoxygenation by analyses with PKC inhibitors, antisense DNA, a dominant negative kinase defective mutant, and constitutively active mutants of PKCzeta. Immunocytochemical observations show PKCzeta staining in the nucleus during ischemic hypoxia and reoxygenation when phosphorylated MAPK is also detected in the nucleus. This nuclear localization of PKCzeta is inhibited by treatment with wortmannin, a phosphoinositide 3-kinase inhibitor that also inhibits MAPK activation in a dose-dependent manner. This is supported by the inhibition of MAPK phosphorylation by another blocker of phosphoinositide 3-kinase, LY294002. An upstream kinase of MAPK, MEK1/2, is significantly phosphorylated 15 min after reoxygenation and observed mainly in the nucleus, whereas it is present in the cytoplasm in serum stimulation. The phosphorylation of MEK is blocked by PKC inhibitors and phosphoinositide 3-kinase inhibitors, as observed in the case of MAPK phosphorylation. These observations indicate that PKCzeta, which is activated by phosphoinositide 3-kinase, induces MAPK activation through MEK in the nucleus during reoxygenation after ischemic hypoxia.     

The MAPK Pathway Signals Telomerase Modulation in Response to Isothiocyanate-Induced DNA Damage of Human Liver Cancer Cells

4-methylthiobutyl isothiocyanate (MTBITC), an aliphatic, sulphuric compound from Brassica vegetables, possesses in vitro and in vivo antitumor activity. Recently we demonstrated the potent growth inhibitory potential of the DNA damaging agent MTBITC in human liver cancer cells. Here we now show that MTBITC down regulates telomerase which sensitizes cells to apoptosis induction. This is mediated by MAPK activation but independent from production of reactive oxygen species (ROS). Within one hour, MTBITC induced DNA damage in cancer cells correlating to a transient increase in hTERT mRNA expression which then turned into telomerase suppression, evident at mRNA as well as enzyme activity level. To clarify the role of MAPK for telomerase regulation, liver cancer cells were pre-treated with MAPK-specific inhibitors prior to MTBITC exposure. This clearly showed that transient elevation of hTERT mRNA expression was predominantly mediated by the MAPK family member JNK. In contrast, activated ERK1/2 and P38, but not JNK, signalled to telomerase abrogation and consequent apoptosis induction. DNA damage by MTBITC was also strongly abolished by MAPK inhibition. Oxidative stress, as analysed by DCF fluorescence assay, electron spin resonance spectroscopy and formation of 4-hydroxynonenal was found as not relevant for this process. Furthermore, N-acetylcysteine pre-treatment did not impact MTBITC-induced telomerase suppression or depolarization of the mitochondrial membrane potential as marker for apoptosis. Our data therefore imply that upon DNA damage by MTBITC, MAPK are essential for telomerase regulation and consequent growth impairment in liver tumor cells and this detail probably plays an important role in understanding the potential chemotherapeutic efficacy of ITC.