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At least 595 records · Page 33Linked to original sources
PubMed1998

Autocrine regulation of macrophage differentiation and 92-kDa gelatinase production by tumor necrosis factor-alpha via alpha5 beta1 integrin in HL-60 cells.

Tumor necrosis factor-alpha (TNF-alpha) gene is one of the early response genes induced by phorbol 12-myristate 13-acetate (PMA) in human HL-60 myeloid leukemia cells. In the present study, we examined the role of the TNF-alpha autocrine loop in PMA-induced macrophage differentiation and gene expression of 92- and 72-kDa gelatinases (MMP-9 and MMP-2). In HL-60 cells, PMA inhibited cell proliferation and induced cell adhesion and spreading, expression of surface maturation marker OKM1 and phagocytic activity, as well as the expression of both gelatinases, which all characterize the macrophage phenotype. In contrast, TNF-alpha alone was only effective in inhibiting cell proliferation. Blocking the endogenous TNF-alpha activity with neutralizing anti-TNF-alpha antibodies abolished all these PMA-induced events with the exception of MMP-2 gene expression. Since fibronectin (FN)-mediated cell adhesion and spreading are prerequisite for both macrophage differentiation and MMP-9 gene expression in HL-60 cells, we hypothesized that TNF-alpha might be involved in modulating the expression of either the FN or its integrin receptor genes. Whereas PMA substantially enhanced the steady state mRNA and protein levels of both FN and alpha5 beta1 integrins, TNF-alpha alone had little effect on the expression of these genes. However, anti-TNF-alpha antibodies blocked PMA-induced augmentation of both alpha5 and beta1 integrin gene expression without affecting the expression of the FN gene. Our results suggest that TNF-alpha may regulate macrophage differentiation and critical matrix-degrading activities of myeloid progenitor cells in an autocrine manner by augmenting surface levels of the alpha5 beta1 integrin, thus promoting interactions with the extracellular matrix, a key event for maturation and migration of these cells during inflammation.

Autocrine Communication↗
PubMed1998

Autocrine/paracrine determinants of strain-activated brain natriuretic peptide gene expression in cultured cardiac myocytes.

The application of mechanical strain leads to activation of human brain natriuretic peptide gene promoter activity, a marker of hypertrophy, in cultured neonatal rat ventricular myocytes. We have used a combination of transient transfection analysis and reverse transcriptase-polymerase chain reaction to examine the role of locally produced factors in contributing to this activation. Conditioned media from strained, but not static, cultures led to a dose-dependent increase in human brain natriuretic peptide gene promoter activity. This increase was completely blocked by losartan or BQ-123, implying a role for angiotensin and endothelin as autocrine/paracrine mediators of the response to strain. Inclusion of the same antagonists in the cultures themselves led to only partial inhibition (approximately 60%), whereas inclusion of exogenous endothelin or angiotensin II resulted in amplification of the strain response. Angiotensin II and endothelin appear to be arrayed in series in the regulatory circuitry; the angiotensin response was blocked by BQ-123, whereas the endothelin response was unaffected by losartan. Mechanical strain was also shown to stimulate expression of the endogenous angiotensinogen, angiotensin-converting enzyme, and endothelin genes in this system. Collectively, these data indicate that locally generated angiotensin II and endothelin, acting in series, play an important autocrine/paracrine role in mediating strain-dependent activation of cardiac-specific gene expression.

Angiotensin II↗
PubMed1998

ATP activates cAMP production via multiple purinergic receptors in MDCK-D1 epithelial cells. Blockade of an autocrine/paracrine pathway to define receptor preference of an agonist.

Extracellular nucleotides regulate function in many cell types via activation of multiple P2-purinergic receptor subtypes. However, it has been difficult to define which individual subtypes mediate responses to the physiological agonist ATP. We report a novel means to determine this by exploiting the differential activation of an autocrine/paracrine signaling pathway. We used Madin-Darby canine kidney epithelial cells (MDCK-D1) and assessed the regulation of cAMP formation by nucleotides. We found that ATP, 2-methylthio-ATP (MT-ATP) and UTP increase cAMP production. The cyclooxygenase inhibitor indomethacin completely inhibited UTP-stimulated, did not inhibit MT-ATP-stimulated, and only partially blocked ATP-stimulated cAMP formation. In parallel studies, ATP and UTP but not MT-ATP stimulated prostaglandin production. By pretreating cells with indomethacin to eliminate the P2Y2/prostaglandin component of cAMP formation, we could assess the indomethacin-insensitive P2 receptor component. Under these conditions, ATP displayed a ten-fold lower potency for stimulation of cAMP formation compared with untreated cells. These data indicate that ATP preferentially activates P2Y2 relative to other P2 receptors in MDCK-D1 cells (P2Y1 and P2Y11, as shown by reverse transcriptase polymerase chain reaction) and that P2Y2 receptor activation is the principal means by which ATP increases cAMP formation in these cells. Blockade of autocrine/paracrine signaling can aid in dissecting the contribution of multiple receptor subtypes activated by an agonist.

Adenosine Triphosphate↗
PubMed1998

Tyrosine phosphorylation regulates H2O2 production in lung fibroblasts stimulated by transforming growth factor beta1.

Transforming growth factor beta1 (TGF-beta1) is a multifunctional, profibrotic cytokine involved in cellular growth and differentiation. We have previously described a cell surface-associated H2O2-generating NADH:flavin:O2 oxidoreductase (referred to as NADH oxidase) activity in human lung fibroblasts induced by TGF-beta1 (Thannickal, V. J., and Fanburg, B. L. (1995) J. Biol. Chem. 270, 30334-30338). In this study, the potential for regulation of this novel TGF-beta1-activated oxidase in fibroblasts by protein tyrosine phosphorylation was examined. Immunoblots using anti-phosphotyrosine antibody demonstrated a time-dependent but delayed phosphorylation of two proteins of 115 and 103 kDa in cells stimulated with TGF-beta1 (2 ng/ml). Similar to the effect on TGF-beta1-induced H2O2 production, phosphorylation of these proteins was blocked by the addition of actinomycin D. The protein-tyrosine kinase inhibitors genistein and herbimycin A inhibited TGF-beta1-induced protein tyrosine phosphorylation, NADH oxidase activation, and H2O2 production in a dose-dependent manner. Catalase, diphenyliodonium (an inhibitor of flavoenzymes), and suramin (an inhibitor of receptor activation, added 4 h after TGF-beta1) had no effect on the induction of protein tyrosine phosphorylation. Phosphorylation of the 115- and 103-kDa proteins preceded the generation of H2O2 production and returned to control levels when H2O2 was undetectable at 48 h after TGF-beta1 exposure. These results suggest that protein tyrosine phosphorylation by a nonreceptor protein-tyrosine kinase(s) regulates the activity of the TGF-beta1-responsive H2O2-generating NADH oxidase in human lung fibroblasts. Additionally, this study demonstrates that TGF-beta1, which binds to a serine-threonine kinase receptor, is able to induce protein tyrosine phosphorylation in a delayed manner via a signaling pathway that requires transcriptional activation.

Autocrine Communication↗
PubMed1998

Oncogenic Ha-Ras-dependent mitogen-activated protein kinase activity requires signaling through the epidermal growth factor receptor.

C3H10T1/2 fibroblasts transformed by the minimal expression of oncogenic Ha-Ras (V12H10 cells) or N-Ras (K61N10 cells) have constitutive mitogen-activated protein kinase (MAPK) activity and proliferate in serum-free medium. The constitutive MAPK activity and serum-independent proliferation of V12H10 cells are sensitive to the growth factor antagonist, suramin (Hamilton, M., and Wolfman, A. (1998) Oncogene 16, 1417-1428), suggesting that Ha-Ras-mediated regulation of the MAPK cascade is dependent upon the action of an autocrine factor. Serum-free medium conditioned by V12H10 cells contains an activity that stimulates MAPK activity in quiescent fibroblasts. This MAPK stimulatory activity could be specifically blocked by the epidermal growth factor receptor (EGFR) inhibitors, PD153035 and PD158780. These inhibitors also blocked the serum-independent proliferation of V12H10 cells. Immunodepletion of conditioned medium with antibodies to transforming growth factor alpha and EGF significantly inhibited its ability to stimulate MAPK activity. Stable transfection of EGFR-negative NR6 and EGFR-positive Swiss3T3 cells with oncogenic (G12V)Ha-Ras demonstrated that only the Ha-Ras-transfected Swiss 3T3 cells possessed constitutive MAPK activity, and this activity was sensitive to PD153035. These data suggest that autocrine activation of the EGFR is required for the regulation of the MAPK cascade in cells minimally expressing oncogenic Ha-Ras.

Animals↗
PubMed1998

Autocrine transforming growth factor alpha provides a growth advantage to malignant cells by facilitating re-entry into the cell cycle from suboptimal growth states.

CBS human colon carcinoma cells are poorly tumorigenic in athymic nude mice, whereas FET colon carcinoma cells are non-tumorigenic. Both cell lines have well differentiated properties in tissue culture. Transforming growth factor alpha (TGF-alpha) was ectopically expressed by stable transfection of a TGF-alpha cDNA under repressible tetracycline control. The TGF-alpha-transfected cells showed enhanced clonal initiation and shortened lag phase growth in tissue culture without an alteration in doubling time in exponential phase relative to untransfected cells. Furthermore, the TGF-alpha transfectants showed increased independence from exogenous growth factors in clonal growth assays and induction of DNA synthesis after release from quiescence. Growth factor independence was associated with sustained epidermal growth factor receptor activation in quiescent TGF-alpha-transfected cells and the requirement of exogenous insulin for stimulation of quiescent cells to re-enter the cell cycle. Higher cloning, reduced lag time in tissue, and the acquisition of growth factor independence for DNA synthesis without a change in doubling time of TGF-alpha-transfected cells indicate that autocrine TGF-alpha functions by facilitating re-entry into the cell cycle from sub-optimal growth states rather than promoting or controlling the proliferation of actively cycling cells. The modulation of growth regulation by autocrine TGF-alpha was associated with increased malignant properties as TGF-alpha transfectants showed increased tumorigenicity in athymic nude mice. The administration of tetracycline reversed the effects of TGF-alpha expression in these cells both in vivo and in vitro, indicating that the alterations of the biological properties were due to the expression of TGF-alpha. Since these cells are continuously grown in a completely chemically defined medium without serum supplementation, it was possible to assign the mechanism underlying the generation of growth factor independence to the replacement of a requirement for exogenous insulin in parental cells by autocrine TGF-alpha.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Low environmental pH is responsible for the induction of nitric-oxide synthase in macrophages. Evidence for involvement of nuclear factor-kappaB activation.

Stimulation of macrophages with endotoxin and/or cytokines is responsible for the expression of the inducible isoform of nitric oxide synthase (iNOS). Because macrophages are exposed to low pH within the microenvironment of inflammatory lesions, the potential role of acidic pH as an additional regulator of iNOS was investigated. Substitution of the culture medium of rat peritoneal macrophages at pH 7.4 with medium at pH 7.0 up-regulated iNOS activity, as reflected by a 2.5-fold increase in nitrite accumulation. The increase in iNOS activity was associated with a similar increase in iNOS mRNA expression that reflected an increase in iNOS mRNA synthesis rather than stability. Low environmental pH-induced iNOS gene transcription involved the activation of nuclear factor-kappaB (NF-kappaB) transcription factor since exposure of macrophages to low environmental pH both increased NF-kappaB binding activity in the nucleus and enhanced NF-kappaB-driven reporter gene expression. In addition, treatment of macrophages with pyrrolidine dithiocarbamate or n-acetyl-leucinyl-leucinyl-norleucinal, two drugs preventing NF-kappaB translocation to the nucleus, canceled low pH-induced nitrite accumulation. The overall mechanism required the synthesis of tumor necrosis factor alpha (TNFalpha). Indeed, 1) elevated TNFalpha bioactivity was observed in the medium of macrophages exposed to pH 7.0, and 2) incubation of macrophages with a neutralizing anti-TNFalpha antibody impaired both NF-kappaB activation and nitrite accumulation in response to acid challenge. In summary, exposure of macrophages to acidic microenvironment in inflammatory lesions leads to the up-regulation of iNOS activity through the activation of NF-kappaB.

Acids↗
PubMed1999

Neuregulin stimulates myogenic differentiation in an autocrine manner.

During myogenesis, mononucleated myoblasts form multinucleated myotubes by membrane fusion. Efficiency of this intercellular process can be maximized by a simultaneous progress, with a time window, of other neighboring myoblasts in the differentiation program. This phenomenon has been described as the community effect. It proposes the existence of a molecule that acts as a differentiation-inducing signal to a group of identical cells. Here, we show that neuregulin is a strong candidate for this molecule in myoblast differentiation. The expression of neuregulin increased rapidly but transiently at early stage of differentiation of rat L6 cells. Neuregulin showed a potent differentiation-promoting activity in membrane fusion and expression of myosin heavy chain. The antibodies raised against neuregulin and its cognate receptor ErbB3, which were capable of neutralizing the signal pathway, inhibited myotube formation and expression of myosin heavy chain in both L6 cells and primary rat myoblasts. The progress of differentiation was mostly halted after the expression of myogenin and cell cycle arrest. These results suggest that the activation of an autocrine signaling of neuregulin may provide a basic mechanism for the community effect observed in the differentiation of the embryonic muscle cells.

Animals↗
PubMed2000

Purification and identification of secreted oxidative stress-induced factors from vascular smooth muscle cells.

Reactive oxygen species have been implicated in the pathogenesis of atherosclerosis and hypertension, in part by promoting vascular smooth muscle cell (VSMC) growth. We have previously shown that LY83583, a generator of O-(2), activated extracellular signal-regulated kinases (ERK1/2) with early (10 min) and late (2 h) peaks and stimulated VSMC growth. To investigate whether secreted oxidative stress-induced factors (termed SOXF) from VSMC were responsible for late ERK1/2 activation in response to LY83583, we purified putative SOXF proteins from conditioned medium (2 h of LY83583 exposure) by sequential chromatography based on activation of ERK1/2. Proteins identified by capillary chromatography, electrospray ionization tandem mass spectrometry, and data base searching included heat shock protein 90-alpha (HSP90-alpha) and cyclophilin B. Western blot analysis of conditioned medium showed specific secretion of HSP90-alpha but not HSP90-beta. Immunodepletion of HSP90-alpha from conditioned medium significantly inhibited conditioned medium-induced ERK1/2 activation. Human recombinant HSP90-alpha reproduced the effect of conditioned medium on ERK1/2 activation. These results show that brief oxidative stress causes sustained release of protein factors from VSMC that can stimulate ERK1/2. These factors may be important mediators for the effects of reactive oxygen species on vascular function.

1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium ↗
PubMed2000

Tumor necrosis factor alpha up-regulates in an autocrine manner the synthesis of plasminogen activator inhibitor type-1 during induction of monocytic differentiation of human HL-60 leukemia cells.

Tumor necrosis factor-alpha (TNFalpha) critically regulates several cellular functions during monocyte/macrophage differentiation. We therefore investigated during the phorbol ester (phorbol 12-myristate 13-acetate (PMA))-induced monocyte/macrophage differentiation of the human HL-60 leukemia cells, if TNFalpha contributed to plasminogen activator inhibitor type-1 (PAI-1) synthesis that is initiated by a protein kinase Cbeta-extracellular signal-regulated kinase 2-dependent pathway (Lopez, S., Peiretti, F., Morange, P., Laouar, A., Fossat, C., Bonardo, B., Huberman, E., Juhan-Vague, I., and Nalbone, G. (1999) Thromb. Haemostasis 81, 415-422). Following PMA treatment, the level of TNFalpha mRNA strongly increased and appeared earlier than PAI-1 mRNA. An anti-TNFalpha antibody significantly inhibited the PMA-induced PAI-1 mRNA and protein levels. The recombinant human TNFalpha, which is inactive on native HL-60 cells in terms of PAI-1 synthesis, optimally potentiates it once HL-60 cells are committed into the differentiation process. The use of 1) the HL-525 cell line, a clone issued from HL-60 cells rendered resistant to PMA-induced differentiation, and 2) the transforming growth factorbeta-1/vitamin D3 differentiative mixture confirmed the relationships between the induction of differentiation and the potency of TNFalpha to up-regulate PAI-1 synthesis. In conclusion, we showed that during the induction of monocyte/macrophage differentiation, TNFalpha and PAI-1 gene expressions are activated and that synthesized TNFalpha up-regulates and prolongs, in an autocrine manner, the synthesis of PAI-1.

Autocrine Communication↗
PubMed2000

Epiregulin, a novel member of the epidermal growth factor family, is an autocrine growth factor in normal human keratinocytes.

Epiregulin is a new member of the epidermal growth factor (EGF) family purified from conditioned medium of NIH-3T3 clone T7. Some EGF family growth factors play essential roles in human keratinocytes in an autocrine manner. We show here that epiregulin is another autocrine growth factor for human keratinocytes. Epiregulin stimulated human keratinocyte proliferation under both subconfluent and confluent culture conditions in the absence of exogenous EGF family growth factors. Immunoprecipitation of [(35)S]methionine-labeled conditioned medium revealed a 5-kDa band corresponding to epiregulin. Northern blot analysis detected a 4. 8-kilobase transcript of epiregulin, and the addition of epiregulin up-regulated epiregulin mRNA synthesis. Furthermore, an anti-epiregulin blocking antibody reduced DNA synthesis by 25%. Epiregulin up-regulated the mRNA levels of heparin-binding EGF-like growth factor (HB-EGF), amphiregulin, and TGF-alpha. In turn, the addition of EGF, HB-EGF, amphiregulin, and TGF-alpha increased epiregulin mRNA levels. These results demonstrate that epiregulin acts as an autocrine growth factor in human epidermal keratinocytes and is part of auto- and cross-induction mechanisms involving HB-EGF, amphiregulin, and TGF-alpha. The mRNA expression profile resulting from induction of differentiation with high calcium and fetal calf serum revealed the differential expression of epiregulin, HB-EGF, amphiregulin, and TGF-alpha in keratinocytes. This indicates that these four growth factors have distinct, non-redundant biological functions.

Amphiregulin↗
PubMed2000

Characterization and functional significance of calcium transients in the 2-cell mouse embryo induced by an autocrine growth factor.

Growth of preimplantation embryos is influenced by autocrine trophic factors that need to act by the 2-cell stage, but their mode of action is not yet described. This report shows that late zygote and 2-cell stage mouse embryos responded to embryo-derived platelet-activating factor (PAF) with transient increases in intracellular calcium concentration ([Ca(2+)](i)). [Ca(2+)](i) transients were single global events and were specifically induced by embryo-derived PAF. They were blocked by inhibition of phospholipase C (U 73122) and an inositol trisphosphate (IP(3)) receptor antagonist (xestospongin C), indicating the release of calcium from IP(3)-sensitive intracellular stores. Transients were also inhibited by the absence of calcium from extracellular medium and partially inhibited by treatment with dihydropyridine (nifedipine, 10 micrometer), but not pimozide (an inhibitor of an embryonic T-type calcium channel). (+/-)BAY K8644 (an L-type channel agonist) induced [Ca(2+)](i) transients, yet these were completely inhibited by nifedipine (10 micrometer). The complete inhibition of BAY K8644, but only partial inhibition of PAF by nifedipine shows that L-type channels were only partly responsible for the calcium influx. Depolarization of 2-cell embryos by 50 mm K(+) did not inhibit PAF-induced calcium transients, showing that the influx channels were not voltage-dependent. Depletion of intracellular calcium stores by thapsigargin revealed the presence of store-operated channels. The interdependent requirement for IP(3)-sensitive internal calcium stores and extracellular calcium in the generation of PAF-induced transients may be explained by a requirement for capacitative calcium entry via store-operated channels. A functionally important role for the PAF-induced transients is supported by the observation that inhibition of [Ca(2+)](i) transients by a PAF-antagonist (WEB 2086) or an intracellular calcium chelator (1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetrakis-acetoxymethyl ester; BAPTA-AM) caused marked inhibition of early embryo development. Growth inhibition by BAPTA-AM was relieved by addition of exogenous PAF.

Animals↗
PubMed2001

Herpesvirus saimiri replaces ZAP-70 for CD3- and CD2-mediated T cell activation.

The protein tyrosine kinase ZAP-70 plays a pivotal role involved in signal transduction through the T cell receptor and CD2. Defects in ZAP-70 result in severe combined immunodeficiency. We report that Herpesvirus saimiri, which does not code for a ZAP-70 homologue, can replace this tyrosine kinase. H. saimiri is an oncogenic virus that transforms human T cells to stable growth based on mutual CD2-mediated activation. Although CD2-mediated proliferation of ZAP-70-deficient uninfected T cells was absent, we could establish H. saimiri-transformed T cell lines from two unrelated patients presenting with ZAP-70 deficiencies. In these cell lines, CD2 and CD3 activation were restored in terms of [Ca(2+)](i), MAPK activation, cytokine production, and proliferation. Activation-induced tyrosine phosphorylation of zeta remained defective. The transformed cells expressed very high levels of the ZAP-70-related kinase Syk. This increased expression was not observed in the primary T cells from the patients and was not due to the transformation by the virus because transformed cell lines established from control T cells did not present this particularity. In conclusion, wild type H. saimiri can restore CD2- and CD3-mediated activation in signaling-deficient human T cells. It extends our understanding of interactions between the oncogenic H. saimiri and the infected host cells.

Autocrine Communication↗
PubMed2002

Sex hormone-binding globulin in the human prostate is locally synthesized and may act as an autocrine/paracrine effector.

Sex hormone-binding globulin (SHBG) is a plasma protein synthesized and secreted by the liver. Its initial description stemmed from its ability to bind estrogens and androgens and its capacity to regulate the free concentration of the steroids that bind to it. Additionally, it participates in signal transduction for certain steroid hormones at the cell membrane. It binds with high affinity to a specific membrane receptor (R(SHBG)) in prostate stromal and epithelial cells, wherein the SHBG.R(SHBG) complex forms. An appropriate steroid binds to this complex and results in increases of intracellular cAMP. These two disparate functions of SHBG, regulation of the concentration of free steroids in plasma and signal transduction in selected tissues, raise the question of how its synthesis and secretion might be regulated so as to best perform these two disparate functions. In this paper we demonstrate that SHBG is produced in human prostate cancer cell lines (LNCaP, DU 145, and PC 3) as well as in cultured human prostate epithelial and stromal cells. In addition, in tissue sections of human prostate, we demonstrate the presence of SHBG (immunocytochemistry) and SHBG mRNA (in situ hybridization). These observations are consistent with the hypothesis that SHBG, destined to participate in signaling at the cell membrane, is locally regulated and produced.

Alternative Splicing↗
PubMed2002

Extracellular ATP is an autocrine/paracrine regulator of hypoxia-induced adventitial fibroblast growth. Signaling through extracellular signal-regulated kinase-1/2 and the Egr-1 transcription factor.

Important autocrine/paracrine functions for the adenine nucleotides have been proposed in several tissues. We addressed the possibility that extracellular ATP would modulate/mediate hypoxia-induced adventitial fibroblast growth. Acute hypoxia (3% O(2), 10-60 min) increased extracellular ATP concentrations in adventitial fibroblasts and in lung microvascular endothelial cells, and chronic hypoxia (3% O(2), 14-30 days) markedly attenuated the rate of extracellular ATP hydrolysis by ecto-nucleotidase(s). Exogenous ATP stimulated [(3)H]thymidine incorporation in fibroblasts as did UTP, ADPbeta, 2-methylthioadenosine triphosphate, adenosine 5'-(alpha,beta-methylene)triphosphate, and benzoylbenzoyl-ATP (2'-3'-O-(4-benzoylbenzoyl)-ATP), indicating that both P2Y and P2X purinoceptors can mediate mitogenic responses. Suramin (100 microm), Cibacron blue 3GA (100 microm), and pyridoxalphosphate-6-azophenyl-2',-4'-disulfonic acid (100 microm) as well as apyrase (5 units/ml) attenuated hypoxia- and ATP-induced and DNA synthesis, indicating activation and a functional role of purinoceptors under hypoxic conditions. ATP-induced DNA synthesis was augmented by hypoxia in an additive fashion, whereas ATP and hypoxia synergistically increased growth factor-induced DNA synthesis, again suggesting that ATP and hypoxia utilize similar signaling pathways to induce proliferation. Indeed, we found that ATP (100 microm) and hypoxia (3% O(2)) induced expression and activation of Egr-1 transcription factor, and both stimuli acted, in part, through a G(alpha)(i)/ERK1/2-dependent signaling pathway. Suramin, Cibacron blue 3GA, and apyrase attenuated hypoxia-induced ERK1/2 activation and Egr-1 expression. We conclude that hypoxia induces ATP release from endothelial cells and fibroblasts and that the activation of P2 purinoceptors is involved in the regulation of DNA synthesis by fibroblasts under hypoxic conditions.

Adenosine Triphosphatases↗
PubMed2002

Role for 18:1 lysophosphatidic acid as an autocrine mediator in prostate cancer cells.

Lysophosphatidic acid (LPA) is a lipid mediator that may play an important role in growth and survival of carcinomas. In this study, LPA production and response were characterized in two human prostate cancer (CaP) cell lines: PC-3 and Du145. Bombesin, a neuroendocrine peptide that is mitogenic for CaP cells, stimulated focal adhesion kinase phosphorylation and activated the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. Similar responses were elicited by 18:1 LPA (oleoyl-LPA). Studies using radioisotopic labeling revealed that both PC-3 and Du145 generate LPA and that LPA production is increased by bombesin. The kinetics of bombesin-induced phospholipase D activation and LPA production were similar. Using electrospray ionization mass spectrometry, 18:1 LPA was found to be an abundant LPA species in CaP cell medium. Structure activity studies of acyl-LPAs revealed that 18:1 LPA is most efficacious for activation of extracellular signal-regulated kinase and phospholipase D in CaP cells. Incubation with 18:1 LPA caused homologous desensitization of LPA response, whereas bombesin caused heterologous desensitization. LPA was present at nanomolar levels in medium from bombesin-treated cells. LPA extracted from the medium induced calcium mobilization in CaP cells. These results demonstrate that bioactive LPA is generated by CaP cells in response to a mitogen and suggest that 18:1 LPA can act as an autocrine mediator.

Autocrine Communication↗
PubMed2002

Transforming growth factor-beta -Smad signaling pathway cooperates with NF-kappa B to mediate nontypeable Haemophilus influenzae-induced MUC2 mucin transcription.

Transforming growth factor-beta (TGF-beta) and related factors are multifunctional cytokines that regulate diverse cellular processes, including proliferation, differentiation, apoptosis, and immune response. The involvement of TGF-beta receptor-mediated signaling in bacteria-induced up-regulation of mucin, a primary innate defensive response for mammalian airways, however, still remains unknown. Here, we report that the bacterium nontypeable Haemophilus influenzae (NTHi), an important human respiratory pathogen, utilizes the TGF-beta-Smad signaling pathway together with the TLR2-MyD88-TAK1-NIK-IKKbeta/gamma-IkappaBalpha pathway to mediate NF-kappaB-dependent MUC2 mucin transcription. The NTHi-induced TGF-beta receptor Type II phosphorylation occurred at as early as 5 min. Pretreatment of NTHi with TGF-beta neutralization antibody reduced up-regulation of MUC2 transcription. Moreover, functional cooperation of NF-kappaB p65/p50 with Smad3/4 appears to positively mediate NF-kappaB-dependent MUC2 transcription. These data are the first to demonstrate the involvement of TGF-beta receptor-mediated signaling in bacteria-induced up-regulation of mucin transcription, bring insights into the novel role of TGF-beta signaling in bacterial pathogenesis, and may lead to new therapeutic intervention of NTHi infections.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Regulation of Hex gene expression by a Smads-dependent signaling pathway.

The homeobox gene Hex is expressed in multiple cell types during embryogenesis and is required for liver and monocyte development. Hex is expressed in the foregut region of late gastrula avian and mammalian embryos in a pattern that overlaps with expression of bone morphogenetic proteins (BMPs). Here we investigate the relationship between BMP signaling and Hex gene expression. We find that Hex expression in avian anterior lateral endoderm is regulated by autocrine BMP signaling. Characterization of the mouse Hex gene promoter identified a 71-nucleotide BMP-responsive element (BRE) that is required for up-regulation of Hex by an activated BMP signaling pathway. The Hex BRE binds Smad4 and Smad1-Smad4 complexes in vitro, and in transfection assays, it is responsive to Smad1 and Smad4 but not to Smad2 and Smad4 or Smad3 and Smad4. The BRE contains two copies of a GCCGnCGC-like motif that in Drosophila is the binding site for Mad and Madea followed by two CAGAG boxes that are similar to sequences required for transforming growth factor-beta/activin responsiveness of several vertebrate genes. Mutation of the GC elements, but not the two CAGAG boxes, abolishes Smads responsiveness in the intact Hex promoter, whereas mutations in both the GC elements and CAGAG boxes show that they act cooperatively to confer Smads responsiveness to the Hex promoter. The Hex BRE can confer Smads responsiveness to a heterologous promoter, and in this context, both the GC-rich elements and the CAGAG boxes are required for Smads-dependent promoter activity. An element almost identical to the Hex BRE is present within the BMP-responsive Nkx2-5 gene promoter, suggesting that the Hex BRE represents a common response element for genes regulated by BMP signaling in the foregut region of the embryo.

Animals↗