MAP Kinase-dependent Pathway Research Articles

TLR2 Activation by Porphyromonas gingivalis Requires Both PPAD Activity and Fimbriae.

Porphyromonas gingivalis, a keystone oral pathogen implicated in development and progression of periodontitis, may also contribute to the pathogenicity of diseases such as arthritis, atherosclerosis, and Alzheimer’s. P. gingivalis is a master manipulator of host immune responses due to production of a large variety of virulence factors. Among these, P. gingivalis peptidilarginine deiminase (PPAD), an enzyme unique to P. gingivalis, converts C-terminal Arg residues in bacterium- and host-derived proteins and peptides into citrulline. PPAD contributes to stimulation of proinflammatory responses in host cells and is essential for activation of the prostaglandin E2 (PGE2) synthesis pathway in gingival fibroblasts. Since P. gingivalis is recognized mainly by Toll-like receptor-2 (TLR2), we investigated the effects of PPAD activity on TLR2-dependent host cell responses to P. gingivalis, as well as to outer membrane vesicles (OMVs) and fimbriae produced by this organism. Using reporter cell lines, we found that PPAD activity was required for TLR2 activation by P. gingivalis cells and OMVs. We also found that fimbriae, an established TLR2 ligand, from wild-type ATCC 33277 (but not from its isogenic PPAD mutant) enhanced the proinflammatory responses of host cells. Furthermore, only fimbriae from wild-type ATCC 33277, but not from the PPAD-deficient strains, induced cytokine production and stimulated expression of genes within the PGE2 synthesis pathway in human gingival fibroblasts via activation of the NF-ĸB and MAP kinase-dependent signaling pathways. Analysis of ten clinical isolates revealed that type I FimA is preferable for TLR2 signaling enhancement. In conclusion, the data strongly suggest that both PPAD activity and fimbriae are important for TLR2-dependent cell responses to P. gingivalis infection.

Src tyrosine kinase mediates endothelin-1-induced early growth response protein-1 expression via MAP kinase-dependent pathways in vascular smooth muscle cells.

We have previously demonstrated that the non-receptor protein tyrosine kinase(NR-PTK) c-Src is an upstream regulator of endothelin-1(ET-1) and angiotensinII-induced activation of protein kinaseB(PKB) signaling in vascular smooth muscle cells(VSMCs). We have also demonstrated that ET-1 potently induces the expression of the early growth response protein-1(Egr-1), a zinc finger transcription factor that is overexpressed in models of vascular diseases, such as atherosclerosis. However, the involvement of c-Src in ET-1‑induced Egr-1 expression has not yet been investigated and its role in mitogen-activated protein kinase(MAPK) signaling remains controversial. Therefore, the aim of the present study was to examine the role of c-Src in the ET-1-induced phosphorylation of extracellular signal-regulated kinase(ERK)1/2, c-Jun N-terminal kinase(JNK) and p38MAPK, 3key members of the MAPK family and in the regulation of Egr-1 expression in rat aortic A10VSMCs. ET-1 rapidly induced the phosphorylation of MAPKs, as well as the expression of Egr-1; however, treatment of the VSMCs with PP2, a specific pharmacological inhibitor of c-Src, dose-dependently reduced the phosphorylation of the 3MAPKs and the expression of Egr-1 induced by ET-1. Furthermore, in mouse embryonic fibroblasts(MEFs) deficient in c-Src(SYF), the ET-1-induced Egr-1 expression and MAPK phosphorylation were significantly suppressed, as compared to MEFs expressing normal Src levels. These results suggest that c-Src plays a critical role in mediating ET-1-induced MAPK phosphorylation and Egr-1 expression in VSMCs.

Oncogenic K-ras confers SAHA resistance by up-regulating HDAC6 and c-myc expression.

Histone deacetylase inhibitors (HDIs) represent a new class of anticancer drugs. Suberoylanilide hydroxamic acid (SAHA), the first HDI approved for the treatment of cutaneous T cell lymphoma (CTCL), is currently being tested in clinical trials for other cancers. However, SAHA has been ineffective against solid tumors in many clinical trials. A better understanding of molecular mechanisms of SAHA resistance may provide the basis for improved patient selection and the enhancement of clinical efficacy. Here we demonstrate that oncogenic K-ras contributes to SAHA resistance by upregulating HDAC6 and c-myc expression. We find that the high levels of HDAC6 expression are associated with activated K-ras mutant in colon cancer patients. And expressions of HDAC6 and c-myc are increased in fibroblasts transformed with activated K-ras. Surprisingly, we find that activated K-ras transformed cells are more resistant to SAHA inhibition on cell growth and anchorage-independent colony formation. We show that a K-ras inhibitor sensitizes K-ras mutated lung cancer cells to SAHA induced growth inhibition. We also find that mutant K-ras induces HDAC6 expression by a MAP kinase dependent pathway. Our study suggests that combined treatment with SAHA and K-ras inhibitors may represent an effective strategy to overcome SAHA resistance.

Activation of RANKL and MMP Production by Endothelin Signaling in Cultured Periodontal Fibroblasts

Periodontitis is a group of inflammatory diseases that affect the tissues surrounding and supporting the teeth. These diseases progressively cause the loss of the alveolar bone and the periodontal ligaments, which can lead to dental exfoliation. Endothelin signaling via the activation of the endothelin‐A receptor (Ednra) by endothelin‐1 (Edn1) is a pathway that may play a role in the disease since the expression of the receptor and ligand is elevated in the periodontal tissues of patients with periodontitis. Using cultured primary periodontal fibroblasts (HPFs), we investigated whether Ednra activation by Edn1 alters the expression of factors involved in the destruction of the periodontium. HPFs were obtained from the extracted teeth of consenting healthy non‐smoking patients with institutional board review approval. The HPFs were treated with 20 and 100nM Edn1for 6 and 24 hours. The osteoclast‐activating factor RANKL and members of the matrix metalloproteinase (MMP) family were examined by western blotting. Edn1 treatment stimulated the production of MMP1, MMP8 and RANKL in a dose‐ and time‐dependent manner; blocking Ednra function with the specific antagonist TBC3214 inhibited the response. Stimulation of Ednra by Edn1 activated phospholipase C. In turn the enzyme activated the ERK MAP kinase‐dependent pathway to increase the MMP1 protein levels, and also activated an independent pathway for RANKL, likely involving NFAT activity. These results support the theory that endothelin signaling, <em style=”mso‐bidi‐font‐style: normal; via the activation of Ednra, stimulates the destruction of the periodontal tissues associated with periodontitis by promoting the expression of MMPs and RANKL.

AB0203 The role of estrogens on the production of IL-21 in patients with systemic lupus erythematosus

BackgroundSystemic lupus erythematosus (SLE) is an autoimmune disease in which various organs and tissues are damaged through abnormal immune responses mediated by tissue-binding autoantibodies. Though, the exact pathogenic mechanism is...

Leucine Facilitates Insulin Signaling through a Gαi Protein-dependent Signaling Pathway in Hepatocytes

In this study, we addressed the direct effect of leucine on insulin signaling. In investigating the associated mechanisms, we found that leucine itself does not activate the classical Akt- or ERK1/2 MAP kinase-dependent signaling pathways but can facilitate the insulin-induced phosphorylations of Akt(473) and ERK1/2 in a time- and dose-dependent manner in cultured hepatocytes. The leucine-facilitated insulin-induced phosphorylation of Akt at residue 473 was not affected by knocking down the key component of mTORC1 or -2 complexes but was blocked by inhibition of c-Src (PP2), PI3K (LY294002), Gαi protein (pertussis toxin or siRNA against Gαi1 gene, or β-arrestin 2 (siRNA)). Similarly, the leucine-facilitated insulin activation of ERK1/2 was also blunted by pertussis toxin. We further show that leucine facilitated the insulin-mediated suppression of glucose production and expression of key gluconeogenic genes in a Gαi1 protein-dependent manner in cultured primary hepatocytes. Together, these results show that leucine can directly facilitate insulin signaling through a Gαi protein-dependent intracellular signaling pathway. This is the first evidence showing that macronutrients like amino acid leucine can facilitate insulin signaling through G proteins directly.

Activation of cannabinoid receptor 2 reduces inflammation in acute experimental pancreatitis via intra-acinar activation of p38 and MK2-dependent mechanisms

The endocannabinoid system has been shown to mediate beneficial effects on gastrointestinal inflammation via cannabinoid receptors 1 (CB(1)) and 2 (CB(2)). These receptors have also been reported to activate the MAP kinases p38 and c-Jun NH(2)-terminal kinase (JNK), which are involved in early acinar events leading to acute pancreatitis and induction of proinflammatory cytokines. Our aim was to examine the role of cannabinoid receptor activation in an experimental model of acute pancreatitis and the potential involvement of MAP kinases. Cerulein pancreatitis was induced in wild-type, CB(1)-/-, and MK2-/- mice pretreated with selective cannabinoid receptor agonists or antagonists. Severity of pancreatitis was determined by serum amylase and IL-6 levels, intracellular activation of pancreatic trypsinogen, lung myeloperoxidase activity, pancreatic edema, and histological examinations. Pancreatic lysates were investigated by Western blotting using phospho-specific antibodies against p38 and JNK. Quantitative PCR data, Western blotting experiments, and immunohistochemistry clearly show that CB(1) and CB(2) are expressed in mouse pancreatic acini. During acute pancreatitis, an upregulation especially of CB(2) on apoptotic cells occurred. The unselective CB(1)/CB(2) agonist HU210 ameliorated pancreatitis in wild-type and CB(1)-/- mice, indicating that this effect is mediated by CB(2). Furthermore, blockade of CB(2), not CB(1), with selective antagonists engraved pathology. Stimulation with a selective CB(2) agonist attenuated acute pancreatitis and an increased activation of p38 was observed in the acini. With use of MK2-/- mice, it could be demonstrated that this attenuation is dependent on MK2. Hence, using the MK2-/- mouse model we reveal a novel CB(2)-activated and MAP kinase-dependent pathway that modulates cytokine expression and reduces pancreatic injury and affiliated complications.

Protease-activated receptor-2 induces expression of vascular endothelial growth factor and cyclooxygenase-2 via the mitogen-activated protein kinase pathway in gastric cancer cells

Protease-activated receptor-2 (PAR-2) has shown strong pro-angiogenesis activity physiologically and pathologically. This study aimed to explore PAR-2 regulation of pro-angiogenesis gene expression and the underlying molecular pathways in gastric cancer cells. MKN28 human gastric cancer cells were treated with trypsin, a PAR-2 activator, and subjected to real-time reverse transcription polymerase chain reaction (qRT-PCR), western blotting and ELISA for gene expression analyses. ERK1/2 phosphorylation and p38 MAP kinase inhibitors (PD98059 and SB203580, respectively) were used to block their gene activities. PAR-2 mRNA and protein were expressed in MKN-28 cells and activated by trypsin treatment. Trypsin-activated PAR-2 protein significantly enhanced expression of vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2) mRNA and protein in gastric cancer cells in a dose- and time-dependent manner. PAR-2 activation also induced the phosphorylation of ERK1/2 and p38 MAP kinase, but the ERK1/2 and p38 inhibitors blocked the activated PAR-2-induced VEGF and COX-2 expression in gastric cancer cells. PAR-2-induced expression of VEGF and COX-2 mRNA and protein in gastric cancer MKN28 cells was mediated by activation of an ERK1/2- and p38 MAP kinase-dependent pathway. Thus, PAR-2 may serve as a promising target for anti-angiogenesis therapy to treat gastric cancer.

Stimulation of apical Cl−/HCO3−(OH−) exchanger, SLC26A3 by neuropeptide Y is lipid raft dependent

Neuropeptide Y (NPY), an important proabsorptive hormone of the gastrointestinal tract has been shown to inhibit chloride secretion and stimulate NaCl absorption. However, mechanisms underlying the proabsorptive effects of NPY are not fully understood. The present studies were designed to examine the direct effects of NPY on apical Cl⁻/HCO₃⁻(OH⁻) exchange activity and the underlying mechanisms involved utilizing Caco2 cells. Our results showed that NPY (100 nM, 30 min) significantly increased Cl⁻/HCO₃⁻(OH⁻) exchange activity (∼2-fold). Selective NPY/Y1 or Y2 receptor agonists mimicked the effects of NPY. NPY-mediated stimulation of Cl⁻/HCO₃⁻(OH⁻) exchange activity involved the ERK1/2 MAP kinase-dependent pathway. Cell surface biotinylation studies showed that NPY does not alter DRA (apical Cl⁻/HCO₃⁻(OH⁻) exchanger) surface expression, ruling out the involvement of membrane trafficking events. Interestingly, DRA was found to be predominantly expressed in the detergent-insoluble (DI) and low-density fractions (LDF) of human colonic apical membrane vesicles (AMVs) representing lipid rafts. Depletion of membrane cholesterol by methyl-β-cyclodextrin (MβCD, 10 mM, 1 h) remarkably decreased DRA expression in the DI fractions. Similar results were obtained in Triton-X 100-treated Caco2 plasma membranes. DRA association with lipid rafts in the DI and LDF fractions of Caco2 cells was significantly enhanced (∼45%) by NPY compared with control. MβCD significantly decreased Cl⁻/HCO₃⁻(OH⁻) exchange activity in Caco2 cells as measured by DIDS- or niflumic acid-sensitive ³⁶Cl⁻ uptake (∼50%). Our results demonstrate that NPY modulates Cl⁻/HCO₃⁻(OH⁻) exchange activity by enhancing the association of DRA with lipid rafts, thereby resulting in an increase in Cl⁻/HCO₃⁻(OH⁻) exchange activity. Our findings suggest that the alteration in the association of DRA with lipid rafts may contribute to the proabsorptive effects of NPY in the human intestine.

Bluetongue virus infection: Activation of the MAP kinase-dependent pathway is required for apoptosis

Bluetongue virus (BTV) is a double-stranded RNA virus that induces apoptosis both in mammalian cell cultures and in target tissues. Based on information that members of the mitogen-activated protein kinase family (MAPKs) are mediators of apoptosis, we have examined in detail the MAPK-dependent apoptosis in BTV infection. Previously, we have shown that apoptosis in BTV infection requires the participation of mitochondrial apoptotic pathways. In addition, we demonstrated that NF-κB is activated and that its inhibition substantially reduces cellular apoptosis. For the first time, here we demonstrated the activation of MAPKs after BTV infection. Moreover, by pre-treatment with MAPK inhibitors, c-Jun N-terminal kinases (JNKs) and p38 MAPK, but not extracellular signal-related kinase (ERK), significantly decreased the induction of apoptosis. JNK and p38 activation regulated the cytochrome c released from mitochondria and caspase 3 activation. These results strengthen the understanding of BTV infection and contribute to our previous data confirming that BTV infection induces robust apoptosis in mammalian cells and is likely to play a primary role in BTV pathophysiology.

Mechanisms underlying modulation of monocarboxylate transporter 1 (MCT1) by somatostatin in human intestinal epithelial cells

Somatostatin (SST), an important neuropeptide of the gastrointestinal tract has been shown to stimulate sodium chloride absorption and inhibit chloride secretion in the intestine. However, the effects of SST on luminal butyrate absorption in the human intestine have not been investigated. Earlier studies from our group and others have shown that monocarboxylate transporter (MCT1) plays an important role in the transport of butyrate in the human intestine. The present studies were undertaken to examine the effects of SST on butyrate uptake utilizing postconfluent human intestinal epithelial Caco2 cells. Apical SST treatment of Caco-2 cells for 30-60 min significantly increased butyrate uptake in a dose-dependent manner with maximal increase at 50 nM ( approximately 60%, P < 0.05). SST receptor 2 agonist, seglitide, mimicked the effects of SST on butyrate uptake. SST-mediated stimulation of butyrate uptake involved the p38 MAP kinase-dependent pathway. Kinetic studies demonstrated that SST increased the maximal velocity (V(max)) of the transporter by approximately twofold without any change in apparent Michaelis-Menten constant (K(m)). The higher butyrate uptake in response to SST was associated with an increase in the apical membrane levels of MCT1 protein parallel to a decrease in the intracellular MCT1 pool. MCT1 has been shown to interact specifically with CD147 glycoprotein/chaperone to facilitate proper expression and function of MCT1 at the cell surface. SST significantly enhanced the membrane levels of CD147 as well as its association with MCT1. This association was completely abolished by the specific p38 MAP kinase inhibitor, SB203580. Our findings demonstrate that increased MCT1 association with CD147 at the apical membrane in response to SST is p38 MAP kinase dependent and underlies the stimulatory effects of SST on butyrate uptake.

Constitutively active erythropoietin receptor expression in breast cancer cells promotes cellular proliferation and migration through a MAP-kinase dependent pathway

The role of erythropoietin receptor (EpoR) expression in tumor cells and the potential of EpoR-mediated signaling to contribute to cellular proliferation and invasiveness require further characterization. To determine whether EpoR expression and activation in tumor cells modulates intracellular signal transduction to promote cellular proliferation and migration, we employed a novel experimental model using human breast cancer cells engineered to stably express a constitutively active EpoR-R129C variant. EpoR-R129C expression resulted in increased cellular proliferation and migration of breast cancer cells and these effects were associated with significantly increased Epo-induced phosphorylation of ERK1/2, AKT and c-Jun-NH2-kinase (SAPK/JNK) proteins. Expression of the constitutively active EpoR-R129C receptor promoted the proliferation and migration of breast cancer cells via activation of ERK- and SAPK/JNK-dependent signaling pathways, respectively. These findings suggest that EpoR over-expression and activation in breast cancer cells has the potential to contribute to tumor progression by promoting the proliferation and invasiveness of the neoplastic cells.

Oleic acid prevents apoptotic cell death induced by trans10, cis12 isomer of conjugated linoleic acid via p38 MAP kinase dependent pathway

Our previous study indicated that oleic acid prevented apoptotic cell death induced by trans10, cis12 (t10, c12)-conjugated linoleic acid in rat hepatoma dRLh-84 cells. The intracellular mechanism of action oleic acid is still unknown. Here, we showed that p38 mitogen-activated protein kinase (MAPK) inhibition using its specific inhibitor SB203580 cancelled the ameliorative effect of oleic acid on the cytotoxicity of t10, c12-conjugated linoleic acid. In addition, SubG1 cell population analysis showed that p38 MAPK played an essential role in the prevention of apoptotic cell death by oleic acid. In fact, p38 phosphorylation level was upregulated in cells treated with oleic acid irrespective of t10, c12-conjugated linoleic acid stimulation. Interestingly, t10, c12-conjugated linoleic acid increased intracellular triglyceride accumulation. However, oleic acid completely inhibited this effect. These observations indicated the involvement of blockade of a p38 MAPK pathway in the ameliorative effect of oleic acid on apoptosis induced by t10, c12-conjugated linoleic acid.

Connexin Hemichannel Composition Determines the FGF-1–induced Membrane Permeability and Free [Ca2+]iResponses

Cell surface hemichannels (HCs) composed of different connexin (Cx) types are present in diverse cells and their possible role on FGF-1-induced cellular responses remains unknown. Here, we show that FGF-1 transiently (4-14 h, maximal at 7 h) increases the membrane permeability through HCs in HeLa cells expressing Cx43 or Cx45 under physiological extracellular Ca(2+)/Mg(2+) concentrations. The effect does not occur in HeLa cells expressing HCs constituted of Cx26 or Cx43 with its C-terminus truncated at aa 257, or in parental nontransfected HeLa cells. The increase in membrane permeability is associated with a rise in HC levels at the cell surface and a proportional increase in HC unitary events. The response requires an early intracellular free Ca(2+) concentration increase, activation of a p38 MAP kinase-dependent pathway, and a regulatory site of Cx subunit C-terminus. The FGF-1-induced rise in membrane permeability is also associated with a late increase in intracellular free Ca(2+) concentration, suggesting that responsive HCs allow Ca(2+) influx. The cell density of Cx26 and Cx43 HeLa transfectants cultured in serum-free medium was differentially affected by FGF-1. Thus, the FGF-1-induced cell permeabilization and derived consequences depend on the Cx composition of HCs.

S100A8/A9 at low concentration promotes tumor cell growth via RAGE ligation and MAP kinase-dependent pathway

The complex formed by two members of the S100 calcium-binding protein family, S100A8/A9, exerts apoptosis-inducing activity against various cells, especially tumor cells. Here, we present evidence that S100A8/A9 also has cell growth-promoting activity at low concentrations. Receptor of advanced glycation end product (RAGE) gene silencing and cotreatment with a RAGE-specific blocking antibody revealed that this activity was mediated via RAGE ligation. To investigate the signaling pathways, MAPK phosphorylation and NF-kappaB activation were characterized in S100A8/A9-treated cells. S100A8/A9 caused a significant increase in p38 MAPK and p44/42 kinase phosphorylation, and the status of stress-activated protein kinase/JNK phosphorylation remained unchanged. Treatment of cells with S100A8/A9 also enhanced NF-kappaB activation. RAGE small interfering RNA pretreatment abrogated the S100A8/A9-induced NF-kappaB activation. Our data indicate that S100A8/A9-promoted cell growth occurs through RAGE signaling and activation of NF-kappaB.

3-Amino-1,4-dimethyl-5H-pyrido[4,3- b]indole (Trp-P-1) attenuates LPS-induced IL-8 expression by decreasing mRNA stability in THP-1 cells

Heterocyclic amines (HCAs), such as Trp-P-1, PhIP, and IQ, are notorious food-born carcinogens. This study examined the inhibitory effects of HCAs on the expression of interleukin-8 (IL-8), which is an important chemokine for the initiation of innate immune responses that function by recruiting immune cells to inflamed sites. Among HCA types tested, only Trp-P-1 showed a robust inhibition of the lipopolysaccharide (LPS)-induced IL-8 expression at both mRNA and protein levels in a human monocytic cell-line, THP-1. However, Trp-P-1 did not inhibit the DNA-binding abilities of the transcription factors NF-κB, AP-1, and NF-IL6, all of which are known to regulate IL-8 transcription. Meanwhile, treatment with actinomycin D facilitated the Trp-P-1-induced down-regulation of IL-8 expression in LPS-stimulated THP-1 cells, implying that the inhibition might be due to a decrease in the stability of IL-8 mRNA rather than an attenuation of IL-8 transcription. Furthermore, Trp-P-1 also inhibited phosphorylation of p38 MAP kinase, which is involved in the regulation of IL-8 mRNA stability. Exogenous addition of ionomycin rescued both the IL-8 protein levels and phosphorylation of p38 MAP kinase inhibited by Trp-P-1. Collectively, these results suggest that Trp-P-1 suppresses LPS-induced IL-8 production in human monocytic cells through down-regulation of an intracellular calcium/p38 MAP kinase-dependent pathway, leading to the reduction of IL-8 mRNA stability.

Pathobiological role of advanced glycation endproducts via mitogen-activated protein kinase dependent pathway in the diabetic vasculopathy

Advanced glycation endproducts (AGEs) have been reported to play a role in neointimal formation and increase the rate of in-stent restenosis (ISR) in the diabetic coronary artery disease patients treated with stents, but the potential pathogenic mechanisms of AGEs in vascular smooth muscle cell proliferation remain unclear. We sought to determine the AGEs related pathobiological mechanism of diabetic vasculopathy. Rat aortic smooth muscle cell (RAoSMC) culture was done with different concentrations of AGEs and proliferation was assessed. Immunohistochemistry for receptor of AGEs (RAGE) was performed with human carotid atheroma. Western blotting was performed to assess the activation of MAP kinase system in the cultured RAoSMC. AGEs increased RAoSMC proliferation and were associated with increased phosphorylation of ERK and p38 kinase by time and dose dependent manner. The MAP kinase activity was decreased by RNA interference for RAGE. AGEs stimulation increased reactive oxygen species (ROS) generation in cultured RAoSMC. From this study it is concluded that AGEs played a key role in RAoSMC proliferation via MAP kinase dependent pathways. Activation of vascular smooth muscle cell (VSMC) proliferation by MAP kinase system and increased formation of ROS may be the possible mechanisms of AGEs induced diabetic vasculopathy.

PI3 kinase and direct S-nitrosation are involved in down-regulation of apoptosis signal-regulating kinase 1 during LPS-induced Toll-like receptor 4 signalling

Toll-like receptor 4 (TLR4) is the human pattern recognition receptor that detects lipopolysaccharide (LPS) shared by Gram-negative bacteria. TLR4 is expressed in different cell types including myeloid cells, the key effectors of innate immune reactions. Apoptosis signal-regulating kinase 1 (ASK1), the upstream kinase of MAP kinase-dependent apoptotic pathway has recently been found to be selectively required for p38 MAP kinase activation/cytokine production during TLR4 signalling. However, the activity of this enzyme has to be down-regulated to protect the cells against apoptosis. In the present study we have found that inhibition of PI3 kinase by LY294002 in THP-1 cells exposed to LPS attenuated down-regulation of ASK1 activity followed by programmed cell death. In addition, nitric oxide produced in response to exposure of THP-1 cells to LPS was found to S-nitrosate and therefore, down-regulate ASK1 activity.

Mechanical stress activates xanthine oxidoreductase through MAP kinase-dependent pathways

Mechanical stress activates xanthine oxidoreductase through MAP kinase-dependent pathways

IGF regulation of neutral amino acid transport in the BeWo choriocarcinoma cell line (b30 clone): Evidence for MAP kinase-dependent and MAP kinase-independent mechanisms

Objective IGF-1 and IGF-1 receptors are major determinants of fetal growth and are expressed primarily on the maternal-facing surface of the syncytiotrophoblast cell membrane in the human placenta. IGF-1 regulates fetal growth, in part, by regulating amino acid transport across the placenta. The objective of these studies was to study the role of IGF-1 and its signaling pathway in regulating neutral amino acid transport in a human trophoblast cell culture model. Design The regulation of neutral amino acid transport by IGF-1 was studied in cultured BeWo b30 choriocarcinoma cells using the non-metabolizing amino acid analog, [ 3H]-α-aminoisobutyric acid (AIB). Transport in the absence of Na was used to distinguish system L from total AIB transport. Similarly, Na-dependent transport in the presence of excess methyl-AIB (MeAIB) permitted discrimination of systems A (MeAIB-sensitive) and ASC (MeAIB-insensitive). Specific inhibitors of intracellular signaling pathways were then used to determine the signaling pathway utilized by IGFs to regulate each amino acid transport system. Specificity of inhibition was assessed using specific markers of p70 S6 kinase activity and MAP kinase activation. Results Maximal stimulating concentrations of IGF-I (100 ng/ml) stimulated AIB transport by 30–40% exclusively through system A. Wortmannin (100 nM), an inhibitor of PI-3-kinase activity, inhibited all IGF-I-stimulated transport. Rapamycin (100 ng/ml), an inhibitor of p70 S6 kinase, and bisindolylmaleimide, an inhibitor of protein kinase C (PKC), had no effect. PD-098059 (50 μM), an inhibitor of MAP kinase activation, inhibited 20–30% of basal AIB transport but did not inhibit IGF-I-stimulated transport under the conditions studied. IGF-1 did not increase steady state mRNA levels of the system A transporters, SNAT1 and SNAT2, suggesting IGF-1 stimulates transport via post-transcriptional mechanisms. Conclusions These data demonstrate that IGF-I stimulates neutral amino acid transport system A by a PI3-kinase dependent, post-transcriptional pathway in the BeWo b30 cell line. Additionally, system A activity appear to be sensitive to MAP kinase-dependent pathways not regulated by IGFs.