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PubMed1992

The OX-44 molecule couples to signaling pathways and is associated with CD2 on rat T lymphocytes and a natural killer cell line.

The MRC OX-44 molecule, which is expressed on all peripheral leukocytes, identifies the subset of thymocytes capable of proliferating in response to alloantigens and lectins (Paterson, D.J., J.R. Green, W.A. Jefferies, M. Puklavec, and A.F. Williams. 1987. J. Exp. Med. 165:1). When we isolated monoclonal antibodies (mAbs) on the basis of their ability to activate the phosphatidylinositol signaling pathway in RNK-16 cells (a rat leukemia line with natural killer activity), three of the resulting mAbs recognized the OX-44 molecule. Addition of these mAbs to RNK-16 elicits protein tyrosine phosphorylation, generates inositol phosphates, and increases the concentration of cytoplasmic free calcium. These responses require the addition of intact mAb and are not observed with F(ab')2 fragments. One of these mAbs (7D2) is mitogenic for freshly isolated rat splenic T cells and synergizes with a mAb to the T cell antigen receptor in this activation. A 50-60-kD glycoprotein coprecipitates with the OX-44 molecule from RNK-16 cells and rat splenic T cells. Peptide mapping and reprecipitation studies indicate that the coprecipitating molecule is CD2. Thus, the OX-44 molecule can couple to multiple signaling pathways and associates with CD2 on both RNK-16 and rat T cells.

Animals↗
PubMed1994

A role for Raf-1 in the divergent signaling pathways mediating insulin-stimulated glucose transport.

Downstream mediators of insulin signaling are thought to include multiple cytoplasmic serine/threonine kinases such as the product of the cellular proto-oncogene c-raf-1. To investigate a role for the Raf-1 protein kinase in insulin-stimulated glucose transport, a gene encoding an oncogenically activated Raf-1 mutant was introduced into 3T3-L1 fibroblasts by retroviral gene transfer. Expression of activated Raf-1 in differentiated 3T3-L1 adipocytes markedly increases hexose uptake compared to control adipocytes or those infected with the retroviral vector. Basal 2-deoxyglucose uptake in adipocytes expressing activated Raf-1 is approximately 40-fold higher than in parental adipocytes, and insulin further increases uptake about 1.2-fold. As determined by the plasma membrane "sheet" assay, Raf-1-expressing adipocytes contain greatly elevated levels of the ubiquitous glucose transporter (GLUT1) on the cell surface in the absence or presence of insulin. Total cellular GLUT1 protein is increased about 5-fold. In contrast, activated Raf-1 affects neither the expression of the "insulin-responsive" glucose transporter (GLUT4) nor its cellular distribution; GLUT4 is virtually undetectable on the plasma membrane in the absence of insulin and translocates normally following the addition of hormone. These data suggest that activation of Raf-1 mediates the chronic effect of insulin on hexose uptake but is not sufficient for the rapid translocation of GLUT4. Moreover, the differential effects of activated Raf-1 expression on the two transporter isoforms define divergent signaling pathways by which insulin regulates glucose transport in cultured adipocytes.

3T3 Cells↗
PubMedDate not supplied

Two growth factor signalling pathways in fibroblasts distinguished by pertussis toxin.

The primary action of a family of mitogens including bombesin, bradykinin, vasopressin and alpha-thrombin is to activate the hydrolysis of polyphosphoinositides. Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) by phospholipase C is mediated through coupling of surface receptors to a GTP-binding protein (Gp protein) which, in some cells, is inactivated by the toxin of Bordetella pertussis. It is not known whether this signalling pathway is involved in initiating DNA replication, whereas it has been firmly established that reinitiation of DNA synthesis can be triggered without activation of PtdIns(4,5)P2 hydrolysis by, for example, EGF (epidermal growth factor), FGF (fibroblast growth factor) and insulin/IGF-I (insulin-like growth factor-I), members of a class of mitogens known to activate receptor tyrosine kinases. Taking advantage of the fact that Chinese hamster lung fibroblasts respond to either class of mitogens and that their Gp protein appears to be sensitive to pertussis toxin, we have now analysed the toxin's effect on reinitiation of DNA synthesis and find that it inhibits up to 95% of thrombin-induced mitogenicity without affecting EGF- or FGF-induced DNA synthesis and proliferation. These findings strongly suggest that activation of PtdIns(4,5)P2-phospholipase C has a determinant function in growth control, and confirm the existence of alternative growth factor-signalling pathways independent of polyphosphoinositide breakdown.

Adenosine Diphosphate Ribose↗
PubMed1989

Use of a selectable marker regulated by alpha interferon to obtain mutations in the signaling pathway.

We have selected mutations in genes encoding components of the signaling pathway for alpha interferon (IFN-alpha) by using a specially constructed cell line. The upstream region of the IFN-regulated human gene 6-16 was fused to the Escherichia coli guanine phosphoribosyltransferase (gpt) gene and transfected into hypoxanthine-guanine phosphoribosyltransferase-negative human cells. These cells express gpt only in the presence of IFN-alpha. They grow in medium containing hypoxanthine, aminopterin, and thymidine plus IFN and are killed by 6-thioguanine plus IFN. Two different types of mutants were obtained after treating the cells with mutagens. A recessive mutant, selected in 6-thioguanine plus IFN, was completely resistant to IFN-alpha but responded normally to IFN-gamma and, unexpectedly, partially to IFN-beta. A constitutive mutant, selected in hypoxanthine-aminopterin-thymidine alone, was abnormal in expressing endogenous genes in the absence of IFN. Both types revert infrequently, allowing selection for complementation of the defects by transfection.

Blotting, Northern↗
PubMed1991

Cholera toxin induces expression of the immediate-early response gene JE via a cyclic AMP-independent signaling pathway.

Cholera toxin (CT) activates expression of two immediate-early response genes (JE and TIS10) in quiescent BALB/c 3T3 cells. Increases in cyclic AMP (cAMP) levels in response to CT are likely responsible for the induction of TIS10 gene expression, since treatment with 8-Br-cAMP and increasing the intracellular levels of cAMP by treatment with forskolin induce TIS10 gene expression. In contrast, neither forskolin nor 8-Br-cAMP induces JE gene expression. 3-Isobutyl-1-methylxanthine, which stabilizes intracellular cAMP, potentiates CT-induced TIS10 gene expression but has no effect on CT-induced JE gene expression. Thus, induction of JE by CT is independent of the cAMP produced in response to CT. Induction of JE by CT does not require protein kinase C (PKC), since depleting cells of PKC activity has no effect on the induction of JE by CT. CT-induced expression of JE can be distinguished from CT-induced TIS10 gene expression by using protein kinase inhibitors and inhibitors of arachidonic acid metabolism, further suggesting distinct signaling pathways for CT-induced JE and TIS10 gene expression. Thus, induction of JE gene expression by CT results from the activation of an intracellular signaling pathway that is independent of cAMP production. This pathway is independent of PKC activity and uniquely sensitive to inhibitors of protein kinases and arachidonic acid metabolism.

1-Methyl-3-isobutylxanthine↗
PubMed1992

Interleukin-4 (IL-4) receptor expression on human T cells is affected by different intracellular signaling pathways and by IL-4 at transcriptional and posttranscriptional level.

Interleukin-4 (IL-4) modulates the survival, proliferation, and differentiation of a variety of hematopoietic cells. The effects are mediated through a single class of high-affinity receptors for IL-4. To understand the biologic effects of IL-4 on human T cells, we studied the regulation of IL-4 receptor (IL-4R) gene expression. We showed that IL-4R mRNA accumulation in human T cells is enhanced fourfold after activation of different secondary signaling pathways by concanavalin A (Con A), phorbol myristate acetate (PMA), the calcium ionophore A23187, and combinations of these factors. This could be ascribed to an increase in the IL-4R transcription rate and to stabilization of IL-4R mRNA resulting in a half-life of 80 to 90 minutes (v 35 to 40 minutes in resting T cells). IL-4 did enhance the IL-4R mRNA accumulation by a factor 10, which was caused by an increase in the IL-4R transcription rate and prolonging the half-life of IL-4R transcripts to 140 to 160 minutes. Finally, it was shown that A23187 induced IL-4R mRNA expression is a protein synthesis-dependent process. In contrast, Con A-, PMA-, Con A + PMA-, and Con A + A23187-induced expression of IL-4R mRNA is protein-synthesis independent. Cyclosporine A inhibited the A23187- and Con A + A23187-induced IL-4R mRNA accumulation, whereas Con A-, PMA-, and Con A + PMA-induced IL-4R mRNA expression was not affected by this drug. These data indicate that expression of IL-4 receptors on human T cells can be modulated by different intracellular signaling pathways at both transcriptional and posttranscriptional levels.

Calcimycin↗
PubMed1994

Monocyte deactivation by interleukin 10 via inhibition of tyrosine kinase activity and the Ras signaling pathway.

Activation of monocytes by bacterial lipopolysaccharides (LPSs) is a central component in the pathogenesis of septic shock syndrome. Interleukin 10 (IL-10) is a potent monocyte-deactivating factor and transcriptionally inhibits LPS-induced expression of proinflammatory mediators. The intracellular signaling pathways of LPS have been only partially characterized and mechanisms of IL-10 signaling remain unknown. We show that LPS activates the protein tyrosine kinase (PTK) p56lyn and that this is associated with tyrosine phosphorylation of the protooncogene product Vav. These events are completely blocked by the tyrosine kinase inhibitor herbimycin A. LPS also increases Ras activation in monocytes. LPS-triggered phosphorylation of mitogen-activated protein kinase is a downstream activation event that is also reduced by herbimycin A. Analysis of the IL-10 effects shows that it completely inhibits the p56lyn tyrosine kinase activation and all other subsequent events in this pathway including Ras activation. The IL-10 effects are selective since it reduced PTK-dependent cytokine mRNA expression but not the PTK independent induction of c-jun and c-fos mRNA in LPS-activated monocytes. These results identify the Ras signaling pathway as a component of intracellular signaling in LPS-stimulated monocytes and define early events in this response as targets of monocyte deactivation by IL-10.

Base Sequence↗
PubMed1994

Alteration in the agonist/antagonist balance of antiestrogens by activation of protein kinase A signaling pathways in breast cancer cells: antiestrogen selectivity and promoter dependence.

We find that stimulation of the protein kinase A (PKA) signaling pathway in MCF-7 human breast cancer cells changes the agonist/antagonist activity of tamoxifen and related antiestrogens; it activates or enhances their estrogen agonist activity and reduces their ability to antagonize the effects of estradiol (E2). In MCF-7 human breast cancer cells which contain high levels of endogenous estrogen receptor (ER), the antiestrogen trans-hydroxy-tamoxifen (TOT) fails to stimulate transcription of the estrogen-responsive promoter-reporter constructs estrogen response element (ERE)-TATA-chloramphenicol acetyl transferase (CAT), (ERE)2-TATA-CAT, and pS2-CAT. However, when cells are treated with isobutyl methylxanthine plus cholera toxin (which increases intracellular cAMP approximately 10-fold), or with 8-bromo-cAMP, or are transfected with expression vectors for the PKA catalytic subunits, the transcriptional activity of the antiestrogen-ER complex is now increased, to levels 20-75% that of E2, and TOT also becomes much less effective in antagonizing the stimulation of transcription by E2. Although this alteration in the agonist and antagonist activity of TOT is observed with three promoter-reporter constructs, containing a simple TATA promoter or a more complex, pS2 promoter, elevation of cAMP did not enhance the transcription by either TOT or E2 of the reporter plasmid ERE-thymidine kinase-CAT. Thus, this phenomenon is promoter specific. The maximal stimulatory effects of isobutylmethylxanthine plus cholera toxin and PKA catalytic subunits on TOT and E2 transcriptional enhancement were not additive, consistent with the hypothesis that they are both acting via stimulation of the same signal transduction pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1995

Transforming G protein-coupled receptors potently activate JNK (SAPK). Evidence for a divergence from the tyrosine kinase signaling pathway.

The expression of human muscarinic acetylcholine receptors (mAChRs) in NIH 3T3 cells has been used as a model for studying proliferative signaling through G protein-coupled receptors. In this biological system, the m1 class of mAChRs can effectively transduce mitogenic signals (Stephens, E.V., Kalinec, G., Brann, M.R., and Gutkind, J.S. (1993) Oncogene 8, 19-26) and induce malignant transformation if persistently activated (Gutkind, J.S., Novotny, E.A., Brann, M.R., and Robbins, K.C. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4703-4708). Moreover, available evidence suggests that the m1-signaling pathway converges at the level of p21ras with that emerging from tyrosine kinase receptors (Crespo, P., Xu, N., Simonds, W.F., and Gutkind, J.S. (1994) Nature 369, 418-420). To explore nuclear events involved in growth regulation by G protein-coupled receptors in this setting, we compared the effect of platelet-derived growth factor (PDGF) and the cholinergic agonist, carbachol, on the expression of mRNA for members of the jun and fos family of nuclear proto-oncogenes. We found that activation of m1 receptors by carbachol induces the expression of a distinct set of nuclear transcription factors. In particular, carbachol caused a much greater induction of c-jun mRNA and AP-1 activity. These responses did not correlate with protein kinase C stimulation nor with the activation of mitogen-activated protein (MAP) kinases. Recently, it has been shown that a novel family of kinases structurally related to MAP kinases, stress-activated protein kinases, or Jun kinases (JNKs), phosphorylate in vivo the amino-terminal transactivating domain of the c-Jun protein, thereby increasing its transcriptional activity. In view of our results, this observation prompted us to ask whether m1 and PDGF can differentially activate JNKs. Here, we show that m1 mAChRs can induce a remarkable increase in JNK activity, which was temporally distinct from that of MAP kinase and was entirely protein kinase C independent. In contrast, PDGF failed to activate JNK in these cells, although it stimulated MAP kinase to an extent even greater than that for carbachol. These findings demonstrate that G protein-coupled receptors can signal through pathways leading to the activation of JNK, thus diverging at this level with those signaling routes utilized by tyrosine kinase receptors.

3T3 Cells↗
PubMed1994

The Ras/Raf signaling pathway is required for progression of mouse embryos through the two-cell stage.

We have used microinjection of antisense oligonucleotides, monoclonal antibody, and the dominant negative Ras N-17 mutant to interfere with Ras expression and function in mouse oocytes and early embryos. Microinjection of either ras antisense oligonucleotides or anti-Ras monoclonal antibody Y13-259 did not affect normal progression of oocytes through meiosis and arrest at metaphase II. However, microinjection of fertilized eggs with constructs expressing Ras N-17 inhibited subsequent development through the two-cell stage. The inhibitory effect of Ras N-17 was overcome by simultaneous injection of a plasmid expressing an active raf oncogene, indicating that it resulted from interference with the Ras/Raf signaling pathway. In contrast to the inhibition of two-cell embryo development resulting from microinjection of pronuclear stage eggs, microinjection of late two-cell embryos with Ras N-17 expression constructs did not affect subsequent cleavages and development to morulae and blastocysts. It thus appears that the Ras/Raf signaling pathway, presumably activated by autocrine growth factor stimulation, is specifically required at the two-cell stage, which is the time of transition between maternal and embryonic gene expression in mouse embryos.

Animals↗
PubMed1994

Contribution of known mitogenic signaling pathways to induction of DNA synthesis in quiescent Chinese hamster fibroblasts.

The mitogenic pathways so far identified in mammalian cells fall into three main categories: tyrosine kinase, kinase C, and the cAMP-dependent pathways. In quiescent murine 3T3 fibroblasts, all three signaling pathways synergize with each other to restart DNA synthesis. In order to establish if the same was true in other rodent fibroblast lines we studied the effects of factors, known to modulate the above-mentioned pathways, on DNA synthesis in Chinese hamster embryo fibroblasts (CHEF/18). The factors examined were: (1) EGF and insulin representative of tyrosine kinase-activating growth factors, (2) TPA as specific activator of protein kinase C, (3) cholera toxin, dibutyryl cyclic AMP, and theophylline as compounds increasing cAMP levels. We found that EGF alone is a strong mitogen in CHEF/18 cells, probably because it can modulate by itself all three pathways. Although cAMP acts as a growth enhancer in 3T3 cells, in CHEF/18 where high levels of cAMP were found, increased concentrations of this second messenger produce strong DNA synthesis inhibition and temporal disturbance of ribosomal protein S6 phosphorylation. Possible interpretations of these findings are presented.

1-Methyl-3-isobutylxanthine↗
PubMed1995

The octadecanoic pathway: signal molecules for the regulation of secondary pathways.

Plant defense against microbial pathogens and herbivores relies heavily on the induction of defense proteins and low molecular weight antibiotics. The signals between perception of the aggression, gene activation, and the subsequent biosynthesis of secondary compounds are assumed to be pentacylic oxylipin derivatives. The rapid, but transient, synthesis of cis-jasmonic acid was demonstrated after insect attack on a food plant and by microbial elicitor addition to plant suspension cultures. This effect is highly specific and not caused by a number of environmental stresses such as light, heavy metals, or cold or heat shock. Elicitation of Eschscholtzia cell cultures also led to a rapid alkalinization of the growth medium prior to jasmonate formation. Inhibition of this alkalinization process by the protein kinase inhibitor staurosporine also inhibited jasmonate formation. The induction of specific enzymes in the benzo[c]phenanthridine alkaloid pathway leading to the antimicrobial sanguinarine was induced to a qualitatively and quantitatively similar extent by fungal elicitor, methyl jasmonate, and its linolenic acid-derived precursor 12-oxophytodienoic acid. It is herein proposed that a second oxylipid cascade may exist in plants starting from linoleic acid via 15,16-dihydro-12-oxophytodienoic acid to 9,10-dihydrojasmonate. Experiments with synthetic trihomojasmonate demonstrated that beta-oxidation is not a prerequisite for biological activity and that 12-oxophytodienoic acid and derivatives are most likely fully active as signal transducers. Octadecanoic acid-derived compounds are essential elements in modulating the synthesis of antibiotic compounds and are thus integral to plant defense.

Animals↗
PubMed1995

Cyclic AMP signaling pathways are important in IL-1 beta transcriptional regulation.

An intact cAMP response element (CRE) in the upstream regulatory sequence of IL-1 beta (-2755/-2762) has been shown to be essential for maintaining full IL-1 beta inducibility following treatment with LPS, PMA, or TNF-alpha. In the present study, using the recombinant plasmid pIL-1(4.0 kb)-chloramphenicol acetyltransferase, containing 4.0 kb of the IL-1 beta upstream regulatory sequence, we have demonstrated that dibutyryl cAMP treatment alone is capable of induction. Due to the critical nature of the CRE for the induction of IL-1 beta transcription, an effort was made to determine the importance of the cAMP signaling pathway(s) by determining whether CRE binding protein (CREB) and other CREB/activating transcription factor (ATF) family members that responded to cAMP were associated with the DNA-protein complex that forms at this site. Nuclear extracts prepared from LPS-treated THP-1 5A cells were fractionated by ammonium sulfate precipitation and heparin-Sepharose chromatography, and the resulting fractions were characterized in electrophoretic gel mobility shift assays. These purification steps resulted in an approximately 100-fold enrichment of the proteins binding to the CRE site. Western blot analysis of isolated fractions, using CREB- and ATF-1-specific Ab showed an increased level of these proteins in the enriched fractions. Tryptic digest and DNase I protection studies showed the presence of CREB protein in the complex at the CRE site. Supershift electrophoretic gel mobility shift assays and immunoprecipitation analysis provided further evidence that both CREB and ATF-1 are present in the complex. In addition, an increase in CREB phosphorylation was observed when THP-1 5A cells were treated with dibutyryl cAMP, LPS, or both. These studies confirm the importance of a cAMP signaling pathway(s) in the regulation of IL-1 beta at the transcriptional level.

Base Sequence↗
PubMed2025

miR-9-5p/HMMR regulates the tumorigenesis and progression of clear cell renal cell carcinoma through EMT and JAK1/STAT1 signaling pathway.

BACKGROUND: The most common malignant type of kidney cancer is clear cell renal cell carcinoma (ccRCC). The expression levels of hyaluronan-mediated motility receptor (HMMR) in many tumor types are significantly elevated. HMMR is closely associated with tumor-related progression, treatment resistance, and poor prognosis, and has yet to be fully investigated in terms of its expression patterns and molecular mechanisms of action in ccRCC. Further research is imperative to elucidate these aspects. METHODS: We used The Cancer Genome Atlas (TCGA) database to preliminarily investigate HMMR expression and function in ccRCC and the data for 19 samples from the NCBI GEO database (GSE207493) for single-cell analysis. We assessed the differential expression level of HMMR between ccRCC cancerous tissues and their matched non-tumor tissues. Subsequently, a series of in vivo and in vitro experiments were designed to elucidate the biological function of HMMR in ccRCC, including Transwell assays, CCK-8 assays, clone formation assays and subcutaneous xenograft experiments in nude mice. Through bioinformatics analysis, we identified potential microRNAs (miRNAs) that may regulate HMMR, as well as the possible signaling pathways involved. Finally, we conducted a series of cellular functional experiments to validate our hypotheses regarding the HMMR axis. RESULTS: HMMR expression was significantly up-regulated in tumor tissues of ccRCC patients, and elevated HMMR expression level showed a strong correlation with ccRCC progression and adverse prognoses of patients. Knocking down HMMR inhibited the proliferative and migratory abilities of ccRCC cells, while its overexpression amplified these oncogenic properties. In nude mice model, reduced HMMR expression inhibited ccRCC tumor proliferation in vivo. Furthermore, overexpression of an upstream transcriptional regulator, miR-9-5p, effectively downregulated HMMR expression and thus impeded ccRCC cells proliferation and migration. HMMR might influence ccRCC growth via the Epithelial-Mesenchymal Transition (EMT) pathway and the Janus Kinase 1/Signal Transducer and Activator of Transcription 1 (JAK1/STAT1) pathway. CONCLUSIONS: HMMR is overexpressed in ccRCC, and there is a significant link between high HMMR expression and tumor progression, as well as poor patient prognosis. Specifically, HMMR could be targeted and inhibited by miR-9-5p and might modulate the tumorigenesis and progression of ccRCC through both EMT and JAK1/STAT1 signaling pathway.

Carcinoma, Renal Cell↗
PubMed1995

Plant homologues of components of MAPK (mitogen-activated protein kinase) signal pathways in yeast and animal cells.

As they respond to numerous extracellular and intracellular stimuli, plants develop various morphological features and the capacity for a large variety of physiological processes during their growth. If we are to understand the molecular basis of such developments, we must elucidate the way in which signals generated by such stimuli can be transduced into plant cells and transmitted by cellular components to induce the appropriate terminal events. In yeast and animal systems, signal pathways that are known collectively as MAPK (mitogen-activated protein kinase) cascades have been shown to play a central role in the transmission of various signals. The components of these pathways include the MAPK family, the activator kinases of the MAPK family (the MAPKK family) and the activator kinases of the MAPKK family (the MAPKKK family). The members of each respective family are structurally conserved and signals are transmitted by similar phosphotransfer reactions at corresponding steps that are mediated by a specific member of each family in turn. Both cDNAs and genes that encode putative homologues of these components have recently been isolated from plant sources. Some of them have been shown to be related not only structurally but also functionally to members of the MAPK cascades of other organisms. These findings suggest that plants have signal pathways that are analogues to the MAPK cascades in yeast and animal cells but it remains to be proven that plant homologues do in fact constitute kinase cascades.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Mitogen-induced tyrosine phosphorylation of 41 kDa and 43 kDa proteins. Potential role in integrating multiple mitogenic signalling pathways.

We have examined the possible involvement of pertussis toxin (PT)-sensitive GTP-binding protein and protein kinase C (PKC) in mitogen-induced tyrosine phosphorylation of the 41 kDa and 43 kDa cytosol proteins using PT-pretreated (inactivation of PT-sensitive GTP-binding protein) or phorbol 12-myristate 13-acetate (PMA)-pretreated (depletion of PKC) mouse fibroblasts. The effects of the inactivation of PT-sensitive GTP-binding protein and the depletion of PKC on mitogen-stimulated tyrosine phosphorylation of the proteins were similar and varied significantly and systematically in response to growth factors. The important finding was that such inhibitory effects of PT-sensitive GTP-binding protein inactivation and PKC depletion on protein tyrosine phosphorylation induced by each mitogen always correlated well with their inhibitory effects on each mitogen-stimulated DNA synthesis. Although the extent of platelet-derived-growth-factor-induced phosphorylation of the proteins was decreased to approx. 50% in PT- and PMA-pretreated cells compared with native cells, protein phosphorylation itself was not affected and occurred at identical sites on each protein in native, PT- and PMA-pretreated cells. These results suggest that: (1) 41 kDa and 43 kDa proteins are located downstream of PT-sensitive GTP-binding protein and PKC in the mitogenic signalling pathways of growth factors, (2) protein phosphorylation occurs via a cascade of events which includes the activation of the receptor tyrosine kinases, PKC and other unidentified kinase(s) which directly participate(s) in the phosphorylation of the 41 kDa and 43 kDa proteins, and (3) their phosphorylation may play an important role in integrating multiple mitogenic signalling pathways.

3T3 Cells↗
PubMed1995

Chemokines regulate cellular polarization and adhesion receptor redistribution during lymphocyte interaction with endothelium and extracellular matrix. Involvement of cAMP signaling pathway.

Leukocyte recruitment is a key step in the inflammatory reaction. Several changes in the cell morphology take place during lymphocyte activation and migration: spheric-shaped resting T cells become polarized during activation, developing a well defined cytoplasmic projection designated as cellular uropod. We found that the chemotactic and proinflammatory chemokines RANTES, MCP-1, and, to a lower extent, MIP-1 alpha, MIP-1 beta, and IL-8, were able to induce uropod formation and ICAM-3 redistribution in T lymphoblasts adhered to ICAM-1 or VCAM-1. A similar chemokine-mediated effect was observed during T cells binding to the fibronectin fragments of 38- and 80-kD, that contain the binding sites for the integrins VLA-4 and VLA-5, respectively. The uropod structure concentrated the ICAM-3 adhesion molecule (a ligand for LFA-1), and emerged to the outer milieu from the area of contact between lymphocyte and protein ligands. In addition, we found that other adhesion molecules such as ICAM-1, CD43, and CD44, also redistributed to the lymphocyte uropod upon RANTES stimulation, whereas a wide number of other cell surface receptors did not redistribute. Chemokines displayed a selective effect among different T cell subsets; MIP-1 beta had more potent action on CD8+ T cells and tumor infiltrating lymphocytes (TIL), whereas RANTES and MIP-1 alpha targeted selectively CD4+ T cells. We have also examined the involvement of cAMP signaling pathway in uropod formation. Interestingly, several cAMP agonists were able to induce uropod formation and ICAM-3 redistribution, whereas H-89, a specific inhibitor of the cAMP-dependent protein kinase, abrogated the chemokine-mediated uropod formation, thus pointing out a role for cAMP-dependent signaling in the development of this cytoplasmic projection. Since the lymphocyte uropod induced by chemokines was completely abrogated by Bordetella pertussis toxin, the formation of this membrane projection appears to be dependent on G proteins signaling pathways. In addition, the involvement of myosin-based cytoskeleton in uropod formation and ICAM-3 redistribution in response to chemokines was suggested by the prevention of this phenomenon with the myosin-disrupting agent butanedione monoxime. Interestingly, this agent also inhibited the ICAM-3-mediated cell aggregation, but not the cell adhesion to substrata. Altogether, these results demonstrate that uropod formation and adhesion receptor redistribution is a novel function mediated by chemokines; this phenomenon may represent a mechanism that significantly contributes to the recruitment of circulating leukocytes to inflammatory foci.

Antigens, CD↗
PubMed1994

A novel role for the nitric oxide-cGMP signaling pathway: the control of epithelial function in Drosophila.

The nitric oxide (NO) signaling pathway plays major roles in the vertebrate vascular, nervous, and immune systems. Here we present evidence that all the elements in the NO pathway are present in, and act to control epithelial fluid secretion by, the Malpighian tubules of an insect, Drosophila melanogaster. This finding will allow both a physiological and a molecular genetic dissection of the NO pathway in the same tissue.

3',5'-Cyclic-GMP Phosphodiesterases↗