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Results for “Signaling pathways”

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PubMed2006

Class I and III phosphatidylinositol 3'-kinase play distinct roles in TLR signaling pathway.

PI3K involvement has been implicated in the TLR signal pathway. However, the precise roles of the different classes of PI3K in the pathway remain elusive. In this study, we have explored the functions of class I and class III PI3K in the TLR signal pathway using specific kinase mutants and PI3K lipid products. Our results reveal that class III PI3K specifically regulates CpG oligodeoxynucleotide (ODN)-induced cytokine and NO production as well as NF-kappaB activation, whereas class I PI3K regulates both CpG ODN- and LPS-induced IL-12 production and NF-kappaB activation. Additional studies of CpG ODN uptake with flow cytometric analysis show that class III PI3K, but not class I, regulates cellular CpG ODN uptake. Furthermore, experiments with MyD88-overexpressing fibroblast cells transfected with dominant-negative mutants of PI3K demonstrate that class III PI3K regulates CpG ODN-mediated signaling upstream of MyD88, while class I PI3K regulation is downstream of MyD88. These results suggest that class I and class III PI3K play distinct roles in not only the uptake of CpG ODN, but also responses elicited by CpG ODN and LPS.

Adaptor Proteins, Signal Transducing↗

PubMed1999

A novel human STE20-related protein kinase, HGK, that specifically activates the c-Jun N-terminal kinase signaling pathway.

The yeast serine/threonine kinase STE20 activates a signaling cascade that includes STE11 (mitogen-activated protein kinase kinase kinase), STE7 (mitogen-activated protein kinase kinase), and FUS3/KSS1 (mitogen-activated protein kinase) in response to signals from both Cdc42 and the heterotrimeric G proteins associated with transmembrane pheromone receptors. Using degenerate polymerase chain reaction, we have isolated a human cDNA encoding a protein kinase homologous to STE20. This protein kinase, designated HPK/GCK-like kinase (HGK), has nucleotide sequences that encode an open reading frame of 1165 amino acids with 11 kinase subdomains. HGK was a serine/threonine protein kinase that specifically activated the c-Jun N-terminal kinase (JNK) signaling pathway when transfected into 293T cells, but it did not stimulate either the extracellular signal-regulated kinase or p38 kinase pathway. HGK also increased AP-1-mediated transcriptional activity in vivo. HGK-induced JNK activation was inhibited by the dominant-negative MKK4 and MKK7 mutants. The dominant-negative mutant of TAK1, but not MEKK1 or MAPK upstream kinase (MUK), strongly inhibited HGK-induced JNK activation. TNF-alpha activated HGK in 293T cells, as well as the dominant-negative HGK mutants, inhibited TNF-alpha-induced JNK activation. These results indicate that HGK, a novel activator of the JNK pathway, may function through TAK1, and that the HGK --> TAK1 --> MKK4, MKK7 --> JNK kinase cascade may mediate the TNF-alpha signaling pathway.

Amino Acid Sequence↗

PubMed2003

Reciprocal modulation of Toll-like receptor-4 signaling pathways involving MyD88 and phosphatidylinositol 3-kinase/AKT by saturated and polyunsaturated fatty acids.

Toll-like receptor-4 (TLR4) can be activated by nonbacterial agonists, including saturated fatty acids. However, downstream signaling pathways activated by nonbacterial agonists are not known. Thus, we determined the downstream signaling pathways derived from saturated fatty acid-induced TLR4 activation. Saturated fatty acid (lauric acid)-induced NFkappaB activation was inhibited by a dominant-negative mutant of TLR4, MyD88, IRAK-1, TRAF6, or IkappaBalpha in macrophages (RAW264.7) and 293T cells transfected with TLR4 and MD2. Lauric acid induced the transient phosphorylation of AKT. LY294002, dominant-negative (DN) phosphatidylinositol 3-kinase (PI3K), or AKT(DN) inhibited NFkappaB activation, p65 transactivation, and cyclooxygenase-2 (COX-2) expression induced by lauric acid or constitutively active (CA) TLR4. AKT(DN) blocked MyD88-induced NFkappaB activation, suggesting that AKT is a MyD88-dependent downstream signaling component of TLR4. AKT(CA) was sufficient to induce NFkappaB activation and COX-2 expression. These results demonstrate that NFkappaB activation and COX-2 expression induced by lauric acid are at least partly mediated through the TLR4/PI3K/AKT signaling pathway. In contrast, docosahexaenoic acid (DHA) inhibited the phosphorylation of AKT induced by lipopolysaccharide or lauric acid. DHA also suppressed NFkappaB activation induced by TLR4(CA), but not MyD88(CA) or AKT(CA), suggesting that the molecular targets of DHA are signaling components upstream of MyD88 and AKT. Together, these results suggest that saturated and polyunsaturated fatty acids reciprocally modulate the activation of TLR4 and its downstream signaling pathways involving MyD88/IRAK/TRAF6 and PI3K/AKT and further suggest the possibility that TLR4-mediated target gene expression and cellular responses are also differentially modulated by saturated and unsaturated fatty acids.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Activation of the STAT signaling pathway can cause expression of caspase 1 and apoptosis.

Protein tyrosine kinases activate the STAT (signal transducer and activator of transcription) signaling pathway, which can play essential roles in cell differentiation, cell cycle control, and development. However, the potential role of the STAT signaling pathway in the induction of apoptosis remains unexplored. Here we show that gamma interferon (IFN-gamma) activated STAT1 and induced apoptosis in both A431 and HeLa cells, whereas epidermal growth factor (EGF) activated STAT proteins and induced apoptosis in A431 but not in HeLa cells. EGF receptor autophosphorylation and mitogen-activated protein kinase activation in response to EGF were similar in both cell lines. The breast cancer cell line MDA-MB-468 exhibited a similar response to A431 cells, i.e., STAT activation and apoptosis correlatively resulted from EGF or IFN-gamma treatment. In addition, in a mutant A431 cell line in which STAT activation was abolished, no apoptosis was induced by either EGF or IFN-gamma. We further demonstrated that both EGF and IFN-gamma induced caspase 1 (interleukin-1beta converting enzyme [ICE]) gene expression in a STAT-dependent manner. IFN-gamma was unable to induce ICE gene expression and apoptosis in either JAK1-deficient HeLa cells (E2A4) or STAT1-deficient cells (U3A). However, ICE gene expression and apoptosis were induced by IFN-gamma in U3A cells into which STAT1 had been reintroduced. Moreover, both EGF-induced apoptosis and IFN-gamma-induced apoptosis were effectively blocked by Z-Val-Ala-Asp-fluoromethylketone (ZVAD) in all the cells tested, and studies from ICE-deficient cells indicated that ICE gene expression was necessary for IFN-gamma-induced apoptosis. We conclude that activation of the STAT signaling pathway can induce apoptosis through the induction of ICE gene expression.

Apoptosis↗

PubMed2005

Disruption of the gamma-interferon signaling pathway at the level of signal transducer and activator of transcription-1 prevents immune destruction of beta-cells.

beta-cells under immune attack are destroyed by the aberrant activation of key intracellular signaling cascades. The aim of the present study was to evaluate the contribution of the signal transducer and activator of transcription (STAT)-1 pathway for beta-cell apoptosis by studying the sensitivity of beta-cells from STAT-1 knockout (-/-) mice to immune-mediated cell death in vitro and in vivo. Whole islets from STAT-1-/- mice were completely resistant to interferon (IFN)-gamma (studied in combination with interleukin [IL]-1beta)-mediated cell death (92 +/- 4% viable cells in STAT-1-/- mice vs. 56 +/- 3% viable cells in wild-type controls, P < or = 0.001) and had preserved insulin release after exposure to IL-1beta and IFN-gamma. Moreover, analysis of cell death in cytokine-exposed purified beta-cells confirmed that protection was due to absence of STAT-1 in the beta-cells themselves. Deficiency of STAT-1 in islets completely prevented cytokine-induced upregulation of IL-15, interferon inducible protein 10, and inducible nitric oxide synthase transcription but did not interfere with monocyte chemoattractant protein 1 and macrophage inflammatory protein 3alpha expression. In vivo, STAT-1-/- mice were partially resistant to development of diabetes after multiple low-dose streptozotocin injections as reflected by mean blood glucose at 12 days after first injection (159 +/- 28 vs. 283 +/- 81 mg/dl in wild-type controls, P < or = 0.05) and diabetes incidence at the end of the follow-up period (39 vs. 73% in wild-type controls, P < or = 0.05). In conclusion, the present results indicate that STAT-1 is a crucial transcription factor in the process of IFN-gamma-mediated beta-cell death and the subsequent development of immune-mediated diabetes.

Animals↗

PubMed2006

Mechanisms of disease: signaling pathways and immunobiology of inflammatory myopathies.

The signaling pathways involved in the immunobiology of polymyositis, dermatomyositis, and inclusion-body myositis are outlined in this Review, which is based on research performed during the past 10 years. In dermatomyositis, the complement cascade is activated and the expression of cytokines and chemokines is upregulated. In polymyositis and inclusion-body myositis, autoinvasive CD8+ T cells are clonally expanded. This T-cell subset possesses conserved amino-acid sequences in complementarity-determining region 3 of the T-cell receptor and, via the perforin pathway, exerts a myotoxic effect on muscle fibers that express major histocompatibility complex (MHC) class I molecules. In all inflammatory myopathies, molecules associated with T-cell transmigration and cytokine signaling, as well as chemokines and their receptors, are strongly expressed by endothelial and inflammatory cells. Early in the pathogenesis of polymyositis and inclusion-body myositis, expression of MHC class I molecules on muscle fibers is upregulated, even in the absence of autoinvasive CD8+ T cells. Emerging data indicate that such continuous upregulation of the expression of MHC class I molecules on muscle fibers leads to an endoplasmic reticulum stress response, intracellular accumulation of misfolded glycoproteins, and activation of nuclear factor kappaB pathways, which can further stimulate formation of MHC class I-CD8 complexes, resulting in a self-sustaining inflammatory response. Advances in our understanding of the signaling pathways involved in the pathogenesis of these inflammatory myopathies are expected to result in the identification of novel therapeutic targets for these diseases.

Disease Progression↗

PubMed2006

The in vitro effects of resistin on the innate immune signaling pathway in isolated human subcutaneous adipocytes.

CONTEXT: Obesity-associated inflammation is a contributory factor in the pathogenesis of type 2 diabetes mellitus (T2DM); the mechanisms underlying the progression to T2DM are unclear. The adipokine resistin has demonstrated proinflammatory properties in relation to obesity and T2DM. OBJECTIVES: The objectives of this study were to characterize resistin expression in human obesity and address the role of resistin in the innate immune pathway; to examine the influence of lipopolysaccharide, recombinant human resistin (rhResistin), insulin, and rosiglitazone in human adipocytes; and, finally, to analyze the effect of rhResistin on the expression of components of the nuclear factor-kappaB pathway and insulin signaling cascade. METHODS: Abdominal sc adipose tissue was obtained from patients undergoing elective liposuction surgery (n = 35; age, 36-49 yr; body mass index, 26.5 +/- 5.9 kg/m2). Isolated adipocytes were cultured with rhResistin (10-50 ng/ml). The level of cytokine secretion from isolated adipocytes was examined by ELISA. The effect of rhResistin on protein expression of components of the innate immune pathway was examined by Western blot. RESULTS: In vitro studies demonstrated that antigenic stimuli increase resistin secretion (P < 0.001) from isolated adipocytes. Proinflammatory cytokine levels were increased in response to rhResistin (P < 0.001); this was attenuated by rosiglitazone (P < 0.01). When examining components of the innate immune pathway, rhResistin stimulated Toll-like receptor-2 protein expression. Similarly, mediators of the insulin signaling pathway, phosphospecific c-Jun NH2-terminal kinase (JNK) 1 and JNK2, were up-regulated in response to rhResistin. CONCLUSION: Resistin may participate in more than one mechanism to influence proinflammatory cytokine release from human adipocytes, potentially via the integration of nuclear factor-kappaB and JNK signaling pathways.

Adipocytes↗

PubMed2004

[Design of new anti-tumor agents interrupting deregulated signaling pathways induced by tyrosine kinase proteins. Inhibition of protein-protein interaction involving Grb2].

Cellular signaling pathways induced by growth-factor receptors are frequently deregulated in cancer. Anti-tumor agents that inhibit their enzymatic tyrosine kinase activity have been designed and are now used in human chemotherapy. We propose here an alternative way to interrupt over-expressed signaling by inhibiting protein-protein interactions that involve either the over-expressed proteins or proteins located downstream. The adaptor protein Grb2 over-expressed in connection with HER2/ErbB2/neu in Ras signaling pathway was chosen as a target. Peptides with very high affinity for Grb2 were rationally designed from structural data. Their capacity to interrupt the signaling pathway, their anti-proliferative activity as well as their potential anti-tumor properties are described.

Adaptor Proteins, Signal Transducing↗

PubMed2001

[Gq/G11-mediated cellular signal pathway and its significance for cardiac diseases].

Transmembrane and intracellular signalling pathways are at the center of many cellular regulatory processes. One of the most ubiquitous transmembrane signalling mechanisms involves heterotrimeric guaninnucleotide binding proteins (G-proteins). Members of the Gq/11-family of G-proteins couple receptors to beta-isoforms of phospholipase C. During recent years the establishment of various transgenic animal models has provided new insights into the physiological and pathological role of this signalling pathway. This overview summarizes recent data on the role of Gq/11-mediated signalling processes in platelet activation and myocardial hypertrophy. The increasing knowledge on the molecular mechanisms underlying these processes may lead to new preventive and therapeutic options for the management of coronary artery disease or heart failure.

Animals↗

PubMed2000

A misexpression screen identifies genes that can modulate RAS1 pathway signaling in Drosophila melanogaster.

Differentiation of the R7 photoreceptor cell is dependent on the Sevenless receptor tyrosine kinase, which activates the RAS1/mitogen-activated protein kinase signaling cascade. Kinase suppressor of Ras (KSR) functions genetically downstream of RAS1 in this signal transduction cascade. Expression of dominant-negative KSR (KDN) in the developing eye blocks RAS pathway signaling, prevents R7 cell differentiation, and causes a rough eye phenotype. To identify genes that modulate RAS signaling, we screened for genes that alter RAS1/KSR signaling efficiency when misexpressed. In this screen, we recovered three known genes, Lk6, misshapen, and Akap200. We also identified seven previously undescribed genes; one encodes a novel rel domain member of the NFAT family, and six encode novel proteins. These genes may represent new components of the RAS pathway or components of other signaling pathways that can modulate signaling by RAS. We discuss the utility of gain-of-function screens in identifying new components of signaling pathways in Drosophila.

Amino Acid Sequence↗

PubMed2007

Activation of the mTOR signaling pathway in renal clear cell carcinoma.

PURPOSE: We determined the frequency of mTOR/p70S6 kinase signaling pathway activation in clear cell renal cell carcinoma. MATERIALS AND METHODS: Phospho-S6 (Ser235/236) and phospho-mTOR (Ser2448) staining was performed on renal tumor tissue microarrays containing 29 clear cell renal cell carcinomas. Mutational analysis of Rheb and RhebL1 was performed on DNA from phospho-mTOR/phospho-S6 positive clear cell renal cell carcinoma. The 3 clear cell renal cell carcinoma derived cell lines A498, 786-O and Caki1 were also assessed for mTOR activation and the effect of the mTOR inhibitor rapamycin (Biomol) on proliferation. RESULTS: Moderate or strong phospho-S6 immunoreactivity was found in 17 of 29 clear cell carcinomas (59%), of which 14 were also moderately/strongly positive for phospho-mTOR (Ser2448). We hypothesized that this activation of the mTOR signaling pathway in clear cell renal cell carcinoma could reflect mutational activation of Rheb or RhebL1, which are Ras family members that directly activate mTOR. However, no mutations in exons 3 and 4 (homologous sites of Ras activating mutations) in Rheb or RhebL1 were identified. Two of 3 renal clear cell carcinoma derived cell lines also showed inappropriate S6 hyperphosphorylation. Treatment of all 3 cell lines with rapamycin significantly decreased S6 phosphorylation and proliferation. CONCLUSIONS: The mTOR/p70S6 kinase signaling pathway is activated in most clear cell renal cell carcinomas. Moreover, the growth of renal clear cell carcinoma derived cell lines is inhibited by rapamycin. This is especially significant in light of new agents such as CCI-779, an ester of rapamycin and inhibitor of mTOR, which has shown promise in the treatment of renal carcinoma.

Carcinoma, Renal Cell↗

PubMed2004

Myocardium tolerant to an adenosine-dependent ischemic preconditioning stimulus can still be protected by stimuli that employ alternative signaling pathways.

Clinical studies on cardioprotection by preinfarct angina are ambiguous, which may involve development of tolerance to repeated episodes of ischemia. Not all preconditioning stimuli use identical signaling pathways, and because patients likely experience varying numbers of episodes of preinfarct angina of different degrees and durations, it is important to know whether myocardium tolerant to a particular preconditioning stimulus can still be protected by stimuli employing alternative signaling pathways. We tested the hypothesis that development of tolerance to a particular stimulus does not affect cardioprotection by stimuli that employ different signaling pathways. Anesthetized rats underwent classical, remote or pharmacological preconditioning. Infarct size (IS), produced by a 60-min coronary artery occlusion (CAO), was determined after 120 min of reperfusion. Preconditioning by two 15-min periods of CAO (2CAO15, an adenosine-dependent stimulus) limited IS from 69 +/- 2% to 37 +/- 6%, but when 2CAO15 was preceded by 4CAO15, protection by 2CAO15 was absent (IS = 68 +/- 1%). This development of tolerance coincided with a loss of cardiac interstitial adenosine release, whereas two 15-min infusions of adenosine (200 microg/min i.v.) still elicited cardioprotection (IS = 40 +/- 4%). Furthermore, cardioprotection was produced when 4CAO15 was followed by the adenosine-independent stimulus 3CAO3 (IS = 50 +/- 8%) or the remote preconditioning stimulus of two 15-min periods of mesenteric artery occlusion (IS = 49 +/- 6%). In conclusion, development of tolerance to cardioprotection by an adenosine-dependent preconditioning stimulus still allows protection by pharmacological or ischemic stimuli intervention employing different signaling pathways.

Adaptation, Physiological↗

PubMed2002

Decreased Smad4 expression in the transforming growth factor-beta signaling pathway during progression of esophageal squamous cell carcinoma.

BACKGROUND: Transforming growth factor-beta (TGF-beta) has antiproliferative effects in various cells, and inactivation of the TGF-beta signaling pathway contributes to tumor progression or development. Smad4, a tumor suppressor gene, is a central mediator in the signaling pathways of the TGF-beta superfamily. This study was undertaken to clarify the correlation between Smad4 expression and the clinicopathologic characteristics of patients with esophageal squamous cell carcinoma (SCC). The authors also investigated the expression of components of the TGF-beta signaling pathway in seven established cell lines derived from esophageal SCC. METHODS: Immunohistochemistry for Smad4 using monoclonal anti-Smad4 antibody was performed on surgical specimens obtained from 80 patients with esophageal SCC. In seven cell lines, the authors examined the expression of components of the TGF-beta signaling pathway using Western and Northern blot analyses. RESULTS: There was a significant inverse correlation between Smad4 expression and both depth of invasion (P = 0.0008) and pathologic stage (P = 0.0079). The expression of Smad4 proteins could be detected in five of seven cell lines. The expression of TGF-beta type II receptor protein was decreased in two of seven cell lines, and the expression of both Smad2 and Smad3 proteins was decreased in only one cell line. The level of expression of Smad4 mRNA did not differ dramatically between cell lines and was not correlated with the quantity of Smad4 protein. CONCLUSIONS: In this study, the expression of Smad4 protein appeared to be correlated with the depth of invasion of esophageal SCC. The loss of Smad4 expression was not regulated at the level of transcription.

DNA-Binding Proteins↗

PubMed1997

Dominant enhancers of Egfr in Drosophila melanogaster: genetic links between the Notch and Egfr signaling pathways.

The Drosophila epidermal growth factor receptor (EGFR) is a key component of a complex signaling pathway that participates in multiple developmental processes. We have performed an F1 screen for mutations that cause dominant enhancement of wing vein phenotypes associated with mutations in Egfr. With this screen, we have recovered mutations in Hairless (H), vein, groucho (gro), and three apparently novel loci. All of the E(Egfr)s we have identified show dominant interactions in transheterozygous combinations with each other and with alleles of N or Su(H), suggesting that they are involved in cross-talk between the N and EGFR signaling pathways. Further examination of the phenotypic interactions between Egfr, H, and gro revealed that reductions in Egfr activity enhanced both the bristle loss associated with H mutations, and the bristle hyperplasia and ocellar hypertrophy associated with gro mutations. Double mutant combinations of Egfr and gro hypomorphic alleles led to the formation of ectopic compound eyes in a dosage sensitive manner. Our findings suggest that these E(Egfr)s represent links between the Egfr and Notch signaling pathways, and that Egfr activity can either promote or suppress Notch signaling, depending on its developmental context.

Animals↗

PubMed2005

Cross-talk between the TGFbeta and Wnt signaling pathways in murine embryonic maxillary mesenchymal cells.

The transforming growth factor beta (TGFbeta) and Wnt signaling pathways play central roles regulating embryogenesis and maintaining adult tissue homeostasis. TGFbeta mediates its cellular effects through types I and II cell surface receptors coupled to the nucleocytoplasmic Smad proteins. Wnt signals via binding to a cell surface receptor, Frizzled, which in turn activates intracellular Dishevelled, ultimately leading to stabilization and nuclear translocation of beta-catenin. Previous studies have demonstrated several points of cross-talk between the TGFbeta and Wnt signaling pathways. In yeast two-hybrid and GST-pull down assays, Dishevelled-1 and Smad 3 have been shown to physically interact through the C-terminal one-half of Dishevelled-1 and the MH2 domain of Smad 3. The current study demonstrates that co-treatment of murine embryonic maxillary mesenchyme (MEMM) cells with Wnt-3a and TGFbeta leads to enhanced reporter activity from TOPflash, a Wnt-responsive reporter plasmid. Transcriptional cooperation between TGFbeta and Wnt did not require the presence of a Smad binding element, nor did it occur when a TGFbeta-responsive reporter plasmid (p3TP-lux) was transfected. Overexpression of Smad 3 further enhanced the cooperation between Wnt and TGFbeta while overexpression of dominant-negative Smads 2 and 3 inhibited this effect. Co-stimulation with TGFbeta led to greater nuclear translocation of beta-catenin, providing explanation for the effect of TGFbeta on Wnt-3a reporter activity. Wnt-3a exerted antiproliferative activity in MEMM cells, similar to that exerted by TGFbeta. In addition, Wnt-3a and TGFbeta in combination led to synergistic decreases in MEMM cell proliferation. These data demonstrate a functional interaction between the TGFbeta and Wnt signaling pathways and suggest that Wnt activation of the canonical pathway is an important mediator of MEMM cell growth.

Active Transport, Cell Nucleus↗

PubMed2007

Prostaglandin F2alpha inhibits adipocyte differentiation via a G alpha q-calcium-calcineurin-dependent signaling pathway.

Prostaglandin F2alpha (PGF2alpha) is a potent physiological inhibitor of adipocyte differentiation, however the specific signaling pathways and molecular mechanisms involved in mediating its anti-adipogenic effects are not well understood. In the current study, we now provide evidence that PGF2alpha inhibits adipocyte differentiation via a signaling pathway that requires heterotrimeric G-protein G alpha q subunits, the elevation of the intracellular calcium concentration ([Ca2+]i), and the activation of the Ca2+/calmodulin-regulated serine/threonine phosphatase calcineurin. We show that while this pathway acts to inhibit an early step in the adipogenic cascade, it does not interfere with the initial mitotic clonal expansion phase of adipogenesis, nor does it affect either the expression, DNA binding activity or differentiation-induced phosphorylation of the early transcription factor C/EBPbeta. Instead, we find that PGF2alpha inhibits adipocyte differentiation via a calcineurin-dependent mechanism that acts to prevent the expression of the critical pro-adipogenic transcription factors PPARgamma and C/EBPalpha. Furthermore, we demonstrate that the inhibitory effects of PGF2alpha on both the expression of PPARgamma and C/EBPalpha and subsequent adipogenesis can be attenuated by treatment of preadipocytes with the histone deacetylase (HDAC) inhibitor trichostatin A. Taken together, these results indicate that PGF2alpha inhibits adipocyte differentiation via a G alpha q-Ca2+-calcineurin-dependent signaling pathway that acts to block expression of PPARgamma and C/EBPalpha by a mechanism that appears to involves an HDAC-sensitive step.

3T3-L1 Cells↗

PubMed2006

[nm23-H1 gene inhibits lung cancer cell invasion through down-regulation of PKC signal pathway].

OBJECTIVE: To study the molecular mechanisms of nm23-H1 for regulating PKC signal pathway before and after transfection with nm23-H1 gene. METHODS: Using Western-blot, Boyden-chamber, MTT and laser scanning confocal microscopy (LSCM) techniques to detect the distribution of PKC in cytosol and plasma membrane, changes of invasion and proliferation activity, PKC translocation status and changes of intracellular Ca(2+) concentration among different human pulmonary carcinoma cells with transfected or untransfected nm23-H1 gene, and changes of the three cell lines after treatment with Calphostin C, a PKC inhibitor. RESULTS: (1) The expression of PKCalpha, PKCbeta II on L9981 and L9981-pLXSN cell membrane, which was in activated status, was remarkably higher than those in L9981-nm23-H1 cell line (P < 0.001). The expression of PKCalpha, PKCbeta II in cytosol in L9981 and L9981-pLXSN cell lines, which was in inactivated status, was lower than those in L9981-nm23-H1 cell line (P < 0.001). It means that the PKC signal pathway was activated in L9981 and L9981-pLXSN cell lines. (2) PKCalpha and PKCbeta II mainly located in nuclei and perinuclear area in L9981 and L9981-pLXSN cells, which were in active status, and the Ca(2+) concentration in these cells was obviously higher than that in L9981-nm23-H1 cell line (P < 0.01). In L9981-nm23-H1 cell line, which was transfected with nm23-H1 gene, PKCalpha and PKCbeta II mainly located in soluble cytosolic section, in an inactive status. (3) The invasion and proliferation ability of L9981 and L9981-pLXSN lung cancer cells was higher than that of L9981-nm23-H1 cell line (P < 0.001). There was no statistically significant difference between L9981 and L9981-pLXSN cell lines (P > 0.05). (4) After treated with PKC inhibitor Calphstin C, the expression of PKC and PKCbeta II in membrane in L9981 and L9981-pLXSN cell lines was down-regulated (P < 0.001), PKCalpha and PKCbeta II were mainly located in cytosolic area, mainly in an inactive status, and the Ca(2+) concentration was found to be decreased in all the three cell lines. The invasion and proliferation ability of the three lung cancer cell lines were obviously decreasing (P < 0.001). However, the invasion and proliferation ability of L9981-nm23-H1 lung cancer cell line was still lower than that of L9981 and L9981-pLXSN lung cancer cell lines (P < 0.001). There was also no significant difference between L9981 and L9981-pLXSN cell lines (P > 0.05). CONCLUSION: The results of this study suggest that nm23-H1 gene might inhibit the invasion and metastasis of lung cancer cells by down-regulating PKC signaling pathway. The Ca(2+) in cells might be involved in this process.

Calcium↗

PubMed2002

Autocrine activation of the IGF-I signaling pathway in mesangial cells isolated from diabetic NOD mice.

Mesangial cells isolated from NOD mice after the onset of diabetes have undergone a stable phenotypic change. This phenotype is characterized by increased expression of IGF-I and downregulation of collagen degradation, which is associated with decreased MMP-2 activity. Here, we investigated the IGF-I signaling pathway in mesangial cells isolated from NOD mice before (nondiabetic NOD mice [ND-NOD]) and after (diabetic NOD mice [D-NOD]) the onset of diabetes. We found that the IGF-I signaling pathway in D-NOD cells was activated by autocrine IGF-I. They had phosphorylation of the IGF-I receptor beta-subunit, phosphorylation of insulin receptor substrate (IRS)-1, and association of the p85 subunit (phosphatidylinositol 3-kinase [PI3K]) with the IGF-I receptor and IRS-1 in D-NOD cells in the basal state. This was also associated with increased phosphorylation of ERK2 in D-NOD mesangial cells. Inhibiting autocrine IGF-I from binding to its receptor using an IGF-I-neutralizing antibody or inhibiting IGF-I signaling pathways using a specific PI3K inhibitor or a specific mitogen-activated protein kinase/extracellular response kinase kinase inhibitor decreased phosphorylated ERKs in D-NOD cells. Importantly, this was associated with increased MMP-2 activity. The addition of exogenous IGF-I to ND-NOD activated signal transduction. Therefore, we conclude that the IGF-I signaling pathway is intact in both D-NOD and ND-NOD cells. However, the phenotypic change in D-NOD cells is associated with constitutive activation of the IGF-I signaling pathways, which may participate in the development and progression of diabetic glomerulosclerosis.

Analysis of Variance↗