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Results for “Signaling pathways”

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PubMed2002

Assay and screening methods for bioactive substances based on cellular signaling pathways.

Assay and screening methods for bioactive substances based on cellular signaling pathways are presented. Examples include: (1) intracellular protein phosphorylation and protein-protein interaction, (1-i) a new assay method for evaluating chemical selectivity of agonists for insulin signaling pathways based on agonist-induced phosphorylation of a target peptide, (1-ii) an SPR-based screening method for agonist selectivity for insulin signaling pathways based on the binding of phosphotyrosine to its specific binding protein, (1-iii) a fluorescent indicator for tyrosine phosphorylation-based insulin signaling pathways, and (1-iv) split luciferase as an optical probe for detecting protein-protein interactions in mammalian cells based on protein splicing; (2) a screening method for antigen-specific IgE using mast cells based on intracellular calcium signaling; (3) a screening method for substrates of multidrug resistance-associated protein (MRP); and (4) fluorescent indicators for cyclic GMP based on cyclic GMP-dependent protein kinase Ialpha and green fluorescent proteins.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2002

Linking beta-catenin to androgen-signaling pathway.

The androgen-signaling pathway is important for the growth and progression of prostate cancer cells. The growth-promoting effects of androgen on prostate cells are mediated mostly through the androgen receptor (AR). There is increasing evidence that transcription activation by AR is mediated through interaction with other cofactors. beta-Catenin plays a critical role in embryonic development and tumorigenesis through its effects on E-cadherin-mediated cell adhesion and Wnt-dependent signal transduction. Here, we demonstrate that a specific protein-protein interaction occurs between beta-catenin and AR. Unlike the steroid hormone receptor coactivator 1 (SRC1), beta-catenin showed a strong interaction with AR but not with other steroid hormone receptors such as estrogen receptor alpha, progesterone receptor beta, and glucocorticoid receptor. The ligand binding domain of AR and the NH(2) terminus combined with the first six armadillo repeats of beta-catenin were shown to be necessary for the interaction. Through this specific interaction, beta-catenin augments the ligand-dependent activity of AR in prostate cancer cells. Moreover, expression of E-cadherin in E-cadherin-negative prostate cancer cells results in redistribution of the cytoplasmic beta-catenin to the cell membrane and reduction of AR-mediated transcription. These data suggest that loss of E-cadherin can elevate the cellular levels of beta-catenin in prostate cancer cells, which may directly contribute to invasiveness and a more malignant tumor phenotype by augmenting AR activity during prostate cancer progression.

Androgens↗

PubMed1998

Pyk2 is a downstream mediator of the IL-2 receptor-coupled Jak signaling pathway.

Many cytokines transmit signals to the cell interior through activation of receptor-associated, Janus family protein tyrosine kinases (Jak PTKs). The interleukin-2 receptor (IL-2R) is associated with the Jak1 and Jak3 PTKs, and ligand-induced activation of these PTKs is essential for lymphocyte proliferation. Here, the nonreceptor PTK, Pyk2, was found to be activated following IL-2 stimulation in a Jak-dependent manner. Furthermore, physical association was detected between endogenous Pyk2 and Jak3, and a dominant interfering mutant of Pyk2 inhibited IL-2-induced cell proliferation without affecting Stat5 activation. Collectively, these results suggest that Pyk2 is a newly identified component of the Jak-mediated IL-2 signaling pathway.

Binding Sites↗

PubMed2000

Dissection of Ras-dependent signaling pathways controlling aggressive tumor growth of human fibrosarcoma cells: evidence for a potential novel pathway.

Activation of multiple signaling pathways is required to trigger the full spectrum of in vitro and in vivo phenotypic traits associated with neoplastic transformation by oncogenic Ras. To determine which of these pathways are important for N-ras tumorigenesis in human cancer cells and also to investigate the possibility of cross talk among the pathways, we have utilized a human fibrosarcoma cell line (HT1080), which contains an endogenous mutated allele of the N-ras gene, and its derivative (MCH603c8), which lacks the mutant N-ras allele. We have stably transfected MCH603c8 and HT1080 cells with activating or dominant-negative mutant cDNAs, respectively, of various components of the Raf, Rac, and RhoA pathways. In previous studies with these cell lines we showed that loss of mutant Ras function results in dramatic changes in the in vitro phenotypic traits and conversion to a weakly tumorigenic phenotype in vivo. We report here that only overexpression of activated MEK contributed significantly to the conversion of MCH603c8 cells to an aggressive tumorigenic phenotype. Furthermore, we have demonstrated that blocking the constitutive activation of the Raf-MEK, Rac, or RhoA pathway alone is not sufficient to block the aggressive tumorigenic phenotype of HT1080, despite affecting a number of in vitro-transformed phenotypic traits. We have also demonstrated the possibility of bidirectional cross talk between the Raf-MEK-ERK pathway and the Rac-JNK or RhoA pathway. Finally, overexpression of activated MEK in MCH603c8 cells appears to result in the activation of an as-yet-unidentified target(s) that is critical for the aggressive tumorigenic phenotype.

Animals↗

PubMed1998

Nck-2, a novel Src homology2/3-containing adaptor protein that interacts with the LIM-only protein PINCH and components of growth factor receptor kinase-signaling pathways.

Many of the protein-protein interactions that are essential for eukaryotic intracellular signal transduction are mediated by protein binding modules including SH2, SH3, and LIM domains. Nck is a SH3- and SH2-containing adaptor protein implicated in coordinating various signaling pathways, including those of growth factor receptors and cell adhesion receptors. We report here the identification, cloning, and characterization of a widely expressed, Nck-related adaptor protein termed Nck-2. Nck-2 comprises primarily three N-terminal SH3 domains and one C-terminal SH2 domain. We show that Nck-2 interacts with PINCH, a LIM-only protein implicated in integrin-linked kinase signaling. The PINCH-Nck-2 interaction is mediated by the fourth LIM domain of PINCH and the third SH3 domain of Nck-2. Furthermore, we show that Nck-2 is capable of recognizing several key components of growth factor receptor kinase-signaling pathways including EGF receptors, PDGF receptor-beta, and IRS-1. The association of Nck-2 with EGF receptors was regulated by EGF stimulation and involved largely the SH2 domain of Nck-2, although the SH3 domains of Nck-2 also contributed to the complex formation. The association of Nck-2 with PDGF receptor-beta was dependent on PDGF activation and was mediated solely by the SH2 domain of Nck-2. Additionally, we have detected a stable association between Nck-2 and IRS-1 that was mediated primarily via the second and third SH3 domain of Nck-2. Thus, Nck-2 associates with PINCH and components of different growth factor receptor-signaling pathways via distinct mechanisms. Finally, we provide evidence indicating that a fraction of the Nck-2 and/or Nck-1 proteins are associated with the cytoskeleton. These results identify a novel Nck-related SH2- and SH3-domain-containing protein and suggest that it may function as an adaptor protein connecting the growth factor receptor-signaling pathways with the integrin-signaling pathways.

3T3 Cells↗

PubMed2002

JNK phosphorylation and activation of BAD couples the stress-activated signaling pathway to the cell death machinery.

The c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in mediating apoptosis in the developing and mature organism. The JNK signaling pathway is thought to induce apoptosis via transcription-dependent and transcription-independent mechanisms that remain to be elucidated. In this study, we report a novel mechanism by which the JNK signaling pathway directly activates a component of the cell death machinery. We have found that JNK catalyzes the phosphorylation of the BH3-only protein BAD at the distinct site of serine 128 in vitro. Activation of the JNK signaling pathway induces the BAD serine 128 phosphorylation in vivo, including in primary granule neurons of the developing rat cerebellum. The JNK-induced BAD serine 128 phosphorylation promotes the apoptotic effect of BAD in primary neurons by antagonizing the ability of growth factors to inhibit BAD-mediated apoptosis. These findings indicate that BAD is a novel substrate of JNK that links the stress-activated signaling pathway to the cell death machinery.

3T3 Cells↗

PubMed1999

Regulation of glycogen synthase in rat hepatocytes. Evidence for multiple signaling pathways.

We examined the signaling pathways regulating glycogen synthase (GS) in primary cultures of rat hepatocytes. The activation of GS by insulin and glucose was completely reversed by the phosphatidylinositol 3-kinase inhibitor wortmannin. Wortmannin also inhibited insulin-induced phosphorylation and activation of protein kinase B/Akt (PKB/Akt) as well as insulin-induced inactivation of GS kinase-3 (GSK-3), consistent with a role for the phosphatidylinositol 3-kinase/PKB-Akt/GSK-3 axis in insulin-induced GS activation. Although wortmannin completely inhibited the significantly greater level of GS activation produced by the insulin-mimetic bisperoxovanadium 1,10-phenanthroline (bpV(phen)), there was only minimal accompanying inhibition of bpV(phen)-induced phosphorylation and activation of PKB/Akt, and inactivation of GSK-3. Thus, PKB/Akt activation and GSK-3 inactivation may be necessary but are not sufficient to induce GS activation in rat hepatocytes. Rapamycin partially inhibited the GS activation induced by bpV(phen) but not that effected by insulin. Both insulin- and bpV(phen)-induced activation of the atypical protein kinase C (zeta/lambda) (PKC (zeta/lambda)) was reversed by wortmannin. Inhibition of PKC (zeta/lambda) with a pseudosubstrate peptide had no effect on GS activation by insulin, but substantially reversed GS activation by bpV(phen). The combination of this inhibitor with rapamycin produced an additive inhibitory effect on bpV(phen)-mediated GS activation. Taken together, our results indicate that the signaling components mammalian target of rapamycin and PKC (zeta/lambda) as well as other yet to be defined effector(s) contribute to the modulation of GS in rat hepatocytes.

Animals↗

PubMed1998

Bradykinin causes contraction in rat uterus through the same signal pathway as oxytocin.

The signal pathway for bradykinin-induced contraction of the uterine smooth muscle was investigated by comparing the effect of blocking agents on bradykinin and oxytocin induced contractions of the isolated rat uterus in organ bath. The phospholipase C inhibitor U-73,122 abolished the effect of both bradykinin and oxytocin. Inhibition of non-voltage-dependent Ca2+ influx by SK & F 96,365 reduced the contraction induced by both agonists to about 20% of control. The tissues failed to contract when they were exposed to bradykinin or oxytocin in Ca(2+)-free Krebs-Henseleit buffer with 2 mM EDTA. Both bradykinin and oxytocin induced further contraction when the tissues were partially depolarized and partially contracted by 30 mM KCl. These observations suggest that bradykinin, like oxytocin, activates phospholipase C which generates IP3 with a subsequent release of Ca2+ from intracellular stores followed by store-operated Ca2+ influx. Thus, membrane potential independent steps appear to be important in bradykinin-induced contraction in the rat uterus.

Animals↗

PubMed2003

Abnormal PI3 kinase/Akt signal pathway in vagal afferent neurons and vagus nerve of streptozotocin-diabetic rats.

The PI3 (phosphatidylinositol-3) kinase/Akt (protein kinase B) signal pathway is involved in the molecular signaling that regulates retrograde axonal transport of neurotrophins in the nervous system. Previous work showed that a reduced retrograde axonal transport of endogenous nerve growth factor (NGF) and neurotrophin-3 (NT-3) in the vagus nerve of diabetic rats occurred in the presence of normal production of neurotrophins and neurotrophin receptors. To assess the potential involvement of an impaired PI3 kinase/Akt signal pathway in the diabetes-induced reduction in retrograde axonal transport of neurotrophins in the vagus nerve, we characterized diabetes-induced changes in the PI3 kinase/Akt signal pathway in the vagus nerve and vagal afferent neurons. Control and streptozotocin (STZ)-induced diabetic rats with a duration of 16 weeks, kinase assays, Western blotting, and immunocytochemistry were used to show that diabetes resulted in alterations in activity and protein expression of the PI3 kinase/Akt signal pathway in the vagus nerve and vagal afferent neurons. Diabetes caused a significant decrease in enzymatic activity of PI3 kinase and Akt (52 and 36% of control, respectively) in the vagus nerve. The reduced enzymatic activity was not associated with decreased protein expression of the p85 subunit of PI3 kinase, Akt and phosphorylation of Akt (ser473). In contrast, there was a significant increase in the phosphorylation of p70s6 kinase (thr421/ser424) along with a normal protein expression of p70s6 kinase in the vagus nerve of diabetic rats. However, diabetes induced an overall decrease in immunoreactivity of the p85 subunit of PI3 kinase, phospho-Akt (ser473) and phospho-p70s6/p85s6 kinase (thr421/ser424) in vagal afferent neurons. Thus, impaired PI3 kinase/Akt signal pathway may partly account for the reduced retrograde axonal transport of neurotrophins in the vagus nerve of STZ-induced diabetic rats.

Animals↗

PubMed2002

beta1 and beta2 integrins activate different signalling pathways in monocytes.

Integrin-mediated signals play an important but poorly understood role in regulating many leucocyte functions. In monocytes and macrophages, integrins of the beta2 subfamily are involved in cell-cell interactions that are important for migration of the cells through the endothelium and also for phagocytosis. On the other hand, in the same cells, beta1 integrin-mediated adhesion to extracellular matrix proteins results in a strong induction of immediate early genes that are important in inflammation. To investigate the signalling pathways from these two types of integrin in monocytic cells, THP-1 cells were selectively stimulated via beta1 or beta2 integrins by cross-linking each type of receptor with specific monoclonal antibodies or their natural ligands. The involvement of extracellular signal-regulated kinase (ERK), Syk and phosphoinositide 3-kinase (PI-3K) was then analysed. Nuclear factor kappaB (NF-kappaB) activation was also detected in THP-1 cells transiently transfected with an NF-kappaB-driven luciferase reporter gene. We found that binding of both types of integrin to their natural ligands activated ERK in a Syk- and PI-3K-dependent manner. Yet, cross-linking of integrins by anti-beta1 antibodies caused activation of ERK while that by anti-beta2 antibodies did not. Also both types of integrin activated NF-kappaB. However, PI-3K was required for beta1 integrin-, but not beta2 integrin-, mediated NF-kappaB activation. In addition, inhibition of PI-3K with wortmannin and LY294002 blocked beta1 integrin-mediated NF-kappaB activation, but did not affect that mediated by beta2 integrin. These data suggest that distinct integrins activate different signalling pathways in monocytic cells.

Androstadienes↗

PubMed2004

[Effect of curcumin on STAT5 signaling pathway in primary CML cells].

To study the effect of curcumin on signaling pathway of signal transducers and activators of transcription (STAT5) in primary newly-diagnosed chronic myelocytic leukemia (CML) cells, and to explore the clinical significance of curcumin in the treatment of primary CML cells, the cells were randomly divided into 3 groups: normal control group, CML cells group, and curcumin group; the cellular proliferation was assayed by MTT test; the expression of cellular STAT5 mRNA in CML cells was detected by RT-PCR; the activation of STAT5 in CML cell was detected by electrophoretic mobility shift assay (EMSA). The results showed that the cellular proliferation of curcumin group (OD value 0.640 +/- 0.073) was decreased, as compared with that of the CML cells group (OD value 0.856 +/- 0.083, P <0.01). The expression levels of STAT5 mRNA in CML cells group (integral ratio of OD 1.782 +/- 0.156) were significantly greater than that in the normal control group (integral ratio of OD 0.289 +/- 0.025, P <0.01). The expression of STAT5 mRNA in curcumin group (integral ratio of OD 1.398 +/- 0.126) was significantly decreased as compared with that in the CML cells group (P <0.01). The activation of STAT5 was significantly increased in CML cells group (gray value 5323.375 +/- 515.640) as compared with that in the normal control group (gray value 2943.000 +/- 273.377, P <0.01). The activation of STAT5 of curcumin group (gray value 4331.750 +/- 398.035) was significantly decreased as compared with that of CML cells group (P <0.01). It is concluded that the cellular proliferation and the expression of STAT5 mRNA are increased in the primary CML cells. The activation of STAT5 in primary CML cells is markedly enhanced. STAT5 signaling pathway may be involved in the proliferation of primary CML cells. Curcumin can inhibit the cellular proliferation and the expression of STAT5 mRNA, and down-regulate the activation of STAT5 in primary CML cells. Curcumin may be used in treatment of leukemia.

Adult↗

PubMed2003

Nordihydroguaiaretic acid, an antioxidant, inhibits transforming growth factor-beta activity through the inhibition of Smad signaling pathway.

Transforming growth factor-beta (TGF-beta) and its family are potent and multi-functional cytokines that affect various fundamental biological events. TGF-beta has a unique signaling pathway that is carried by Smad family, and many recent studies showed the extensive crosstalk between Smad pathway and other signaling pathway. There were also clear evidences for the involvement of oxidative events in TGF-beta signaling pathway. To elucidate the role of oxidative events in carrying TGF-beta signals, we examined the effect of various antioxidants on TGF-beta activities in osteoblastic cell line. Among the examined compounds, we found nordihydroguaiaretic acid (NDGA) has a unique and strong inhibitory effect on various TGF-beta activities. Since the majority of TGF-beta activities are mediated by Smad, we questioned whether NDGA blocks the Smad signaling pathway. The result showed that NDGA inhibits the translocation of Smad2 to the nucleus. Further study revealed the strong inhibitory effect of NDGA on the phosphorylation of Smad2. This result may be important for designing chemical modulators of TGF-beta and its family related events and may provide new insights into the action mechanism of antioxidant.

Active Transport, Cell Nucleus↗

PubMed2005

Hedgehog signaling pathway and gastric cancer.

Hedgehog, WNT, FGF and BMP signaling pathways network together during embryogenesis, tissue regeneration, and carcinogenesis. Aberrant activation of Hedgehog signaling pathway leads to pathological consequences in a variety of human tumors, such as gastric cancer and pancreatic cancer. Endoscopic mucosal resection (EMR), endoscopic submucosal dissection (ESD), surgical gastrectomy and chemotherapy are therapeutic options for gastric cancer; however, prognosis of advanced gastric cancer patient is still poor. Here, Hedgehog signaling pathway in human gastric cancer and its clinical applications will be reviewed. Human SHH, IHH, DHH (Hedgehog homologs), HHAT (Hedgehog acyltransferase), HHIP (Hedgehog-interacting protein), DISP1, DISP2, DISP3 (Dispatched homologs), PTCH1, PTCH2 (Patched homologs), SMO (Smoothened homolog), KIF27, KIF7 (Costal-2 homologs), STK36 (Fused homolog), SUFU (SuFu homolog), DZIP1 (Iguana homolog), GLI1, GLI2 and GLI3 (Cubitus interruptus homologs) are implicated in the Hedgehog signaling. PTCH1, FOXM1 and CCND2 are direct transcriptional targets of Hedgehog signaling. Hedgehog signaling activation leads to cell proliferation through cell cycle regulation. SHH regulates growth and differentiation within gastric mucosa through autocrine loop and FOXL1-mediated epithelial-mesenchymal interaction. SHH is implicated in stem/progenitor cell restitution of damaged gastric mucosa during chronic infection with Helicobacter pylori. SHH up-regulation, IHH upregulation and HHIP down-regulation lead to aberrant activation of Hedgehog signaling through PTCH1 to GLI1 in gastric cancer. Small molecule compounds targeted to SMO (KADD-cyclopamine, SANT1-4, Cur61414) as well as humanized anti-SHH antibodies are potent anti-cancer drugs for gastric cancer. Cocktail of Hedgehog inhibitors would be developed as novel therapeutics for gastric cancer. Single nucleotide polymorphism (SNP) and copy number polymorphism (CNP) of Hedgehog signaling genes would be utilized for genetic screening of gastric cancer, while cDNA-PCR, microarray and ELISA detecting aberrant Hedgehog signaling activation would be utilized for therapeutic optional choice. Genetic screening and precise selection of therapeutic options would contribute to the realization of personalized medicine.

Animals↗

PubMed1999

NGF activates similar intracellular signaling pathways in vascular smooth muscle cells as PDGF-BB but elicits different biological responses.

The signaling pathways that regulate smooth muscle cell migration and proliferation are incompletely understood. Smooth muscle cells express at least 3 families of receptor tyrosine kinases that mediate cell migration: platelet-derived growth factor (PDGF) receptors, the trk family of neurotrophin receptors, and insulin-like growth factor 1 receptor. The neurotrophin, nerve growth factor (NGF), and insulin-like growth factor 1 induce the migration but not the proliferation of smooth muscle cells, whereas PDGF-BB stimulates both responses. To determine whether distinct signaling pathways downstream of receptor tyrosine kinases specifically mediate smooth muscle cell migration or proliferation, the ligand-induced activation of different signaling pathways in smooth muscle cells was examined. NGF induces prolonged activation of the Shc/MAP kinase pathway and phospholipase Cgamma compared with PDGF-BB. The activation of phosphatidylinositol-3 kinase, however, was 10-fold greater in response to PDGF-BB compared with NGF. Insulin-like growth factor 1 activates only phosphatidylinositol-3 kinase. Pharmacological inhibitors of phosphatidylinositol-3 kinase, Wortmannin and LY294002, inhibit PDGF-BB and NGF-induced migration, whereas an inhibitor of MAP kinase kinase, PD98059, has no effect. Our results suggest that (1) different receptor tyrosine kinases use similar patterns of activation of signaling pathways to mediate distinct biological outcomes of cell migration and proliferation, (2) NGF activates signaling proteins in smooth muscle cells similar to those activated during NGF-induced neuronal differentiation, and (3) the combinatorial effects of different signaling pathways are important for the regulation of smooth muscle cell migration and proliferation. Further studies using mutant trk receptors will help to define the signal transduction pathways mediating NGF-induced smooth muscle cell migration.

Animals↗

PubMed1998

Lasp-1 is a regulated phosphoprotein within the cAMP signaling pathway in the gastric parietal cell.

Activation of the cAMP signaling pathway is correlated with increased secretory-related events in a wide variety of cell types including the gastric parietal cell. Within this pathway, as well as in other intracellular signaling pathways, protein phosphorylation serves as a major downstream regulatory mechanism. However, although agonist and cAMP-dependent activation of cAMP-dependent protein kinase (PKA) has been demonstrated, little is currently known about the downstream in vivo phosphoprotein substrates of this enzyme. Here we report the isolation, microsequencing, and cloning of a LIM and SH3 domain-containing, cAMP-responsive, 40-kDa phosphoprotein (pp40) from rabbit gastric parietal cells. The deduced amino acid sequence for pp40 is 93.5%, homologous with the putative protein product of the human gene lasp-1, which was recently identified based on its overexpression in some breast carcinomas. In addition to LIM and SH3 domains, the rabbit homolog contains two highly conserved PKA consensus sequences as well as two conserved SH2 binding motifs and several other putative protein kinase phosphorylation sites, including two for tyrosine kinase(s). Combined Northern and Western blot analyses indicate that pp40/lasp-1 is widely expressed (through a single 3.3-kb message) not only in epithelial tissues but also in muscle and brain. Furthermore, stimulation of isolated parietal cells, distal colonic crypts, and pancreatic cells with the adenylyl cyclase activator forskolin leads to the appearance of a higher molecular weight form of pp40/lasp-1, a finding which is consistent with an increase in protein phosphorylation. Thus pp40/lasp-1 appears to be regulated within the cAMP signaling pathway in a wide range of epithelial cell types. Because the cAMP-dependent increase in pp40 phosphorylation is correlated with secretory responses in the parietal cell and because pp40 appears to be widely distributed among various secretory tissues, this newly defined signaling protein may play an important role in modulating ionic transport or other secretory-related activities in many different cell types.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Signal pathways and ligament cell adhesiveness.

The influence of signal pathways involved in the adhesion of fibroblasts from the anterior cruciate and medial collateral ligaments to fibronectin was investigated. Specific emphasis was paid to the cyclic adenosine monophosphate and Ca2+/phospholipid pathways to determine the signaling mediated by integrin receptors during cell binding and spreading on a fibronectin-coated glass surface and to compare the roles of these two pathways in integrin-mediated adhesion in fibroblasts from the two ligaments. Individual cell adhesion strengths were determined using a micropipette-micromanipulation system after the cells were treated with signal pathway inhibiting agents. Adhesion in fibroblasts from the medial collateral ligament was significantly reduced by inhibiting agents for Gi protein, protein kinase A, protein kinase C, protein kinase G, phospholipase C, and calmodulin, which suggests a crucial role for cyclic adenosine monophosphate and Ca2+/phospholipid signaling in integrin-mediated adhesion of these fibroblasts. Adhesion in fibroblasts from the anterior cruciate ligament, however, was reduced only by a protein kinase C inhibiting agent and was increased by inhibiting agents for protein kinase A, protein kinase G, and calmodulin, which suggests only a partial role of Ca2+/phospholipid signaling in integrin-mediated adhesion of these fibroblasts. On the basis of additional parallel studies on the role of intracellular calcium in integrin-mediated adhesion, medial collateral ligament and anterior cruciate ligament fibroblast adhesion was calcium dependent throughout the 60 minute time course of adhesion experiments. Fibroblasts from the medial collateral ligament demonstrated a 2.2-fold increase in cytosolic free calcium upon binding to fibronectin, whereas fibroblasts from the anterior cruciate ligament demonstrated no significant increase in calcium. Overall, the study of the intrinsic differences between anterior cruciate ligament and medial collateral ligament fibroblasts in their signal pathways upon binding to fibronectin may reveal information important for further explaining the lack of functional healing response in the anterior cruciate ligament after injury.

Adult↗

PubMed2001

Phosphoinositide 3-kinase regulates crosstalk between Trk A tyrosine kinase and p75(NTR)-dependent sphingolipid signaling pathways.

The mechanism of crosstalk between signaling pathways coupled to the Trk A and p75(NTR) neurotrophin receptors in PC12 cells was examined. In response to nerve growth factor (NGF), Trk A activation inhibited p75(NTR)-dependent sphingomyelin (SM) hydrolysis. The phosphoinositide 3-kinase (PI 3-kinase) inhibitor, LY294002, reversed this inhibition suggesting that Trk A activation of PI 3-kinase is necessary to inhibit sphingolipid signaling by p75(NTR). In contrast, SM hydrolysis induced by neurotrophin-3 (NT-3), which did not activate PI-3 kinase, was uneffected by LY294002. However, transient expression of a constituitively active PI 3-kinase inhibited p75(NTR)-dependent SM hydrolysis by both NGF and NT-3. Intriguingly, NGF induced an association of activated PI 3-kinase with acid sphingomyelinase (SMase). This interaction localized to caveolae-related domains and correlated with a 50% decrease in immunoprecipitated acid SMase activity. NGF-stimulated PI 3-kinase activity was necessary for inhibition of acid SMase but was not required for ligand-induced association of the p85 subunit of PI 3-kinase with the phospholipase. Finally, this interaction was specific for NGF since EGF did not induce an association of PI 3-kinase with acid SMase. In summary, our data suggest that PI 3-kinase regulates the inhibitory crosstalk between Trk A tyrosine kinase and p75(NTR)-dependent sphingolipid signaling pathways and that this interaction localizes to caveolae-related domains.

Animals↗

PubMed2000

Effects of TNF-alpha on expression of ICAM-1 in human airway epithelial cells in vitro. Signaling pathways controlling surface and gene expression.

Signaling pathways associated with tumor necrosis factor (TNF)-alpha-induced intercellular adhesion molecule 1 (ICAM-1) surface and gene expression were investigated in well differentiated normal human bronchial epithelial (NHBE) cells in air-liquid interface primary culture. Cells were exposed to human recombinant TNF-alpha (hrTNF-alpha; 0.015 to 150 ng/ml [specific activity, 2.86 x 10(7) U/mg]). TNF-alpha enhanced ICAM-1 surface expression (measured by flow cytometry) and steady-state messenger RNA (mRNA) levels (assessed by Northern hybridization) in concentration- and time-dependent manners. TNF-alpha-induced ICAM-1 surface and gene expression were both blocked by the RNA polymerase II inhibitor actinomycin D (0.1 microg/ml), and surface expression was attenuated by a neutralizing monoclonal antibody directed against the TNF-alpha receptor p55 (TNF-RI). The intracellular signaling pathway leading to enhanced expression appeared to involve activation of a phospholipase C that hydrolyzes phosphatidylcholine (PC-PLC) because D609, a specific PC-PLC inhibitor, attenuated TNF-alpha-induced increases in production of diacyl-glycerol (DAG), a hydrolysis product of PC-PLC, and also attenuated TNF-alpha enhancement of ICAM-1 surface and gene expression. Because DAG formed by action of PC-PLC can activate protein kinase C (PKC), involvement of PKC was investigated. The specific PKC inhibitor calphostin C blocked both surface and gene expression of ICAM-1 in response to TNF-alpha in a concentration-dependent manner. Finally, TNF-alpha stimulated binding of p65 and/or c-rel complexes to the nuclear factor (NF)-kappaB consensus binding site found on the ICAM-1 promoter, and binding of these complexes was inhibited by D609. The results support the following pathway, whereby TNF-alpha enhances expression of ICAM-1 in NHBE cells: TNF-alpha --> TNF-RI --> PC-PLC --> DAG --> PKC --> (NF-kappaB?) --> ICAM-1 mRNA --> ICAM-1 surface expression.

Antibodies, Monoclonal↗