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Endogenous opioids and oligodendroglial function: possible autocrine/paracrine effects on cell survival and development.

Previous work has shown that oligodendrocytes (OLs) express both micro- and kappa-opioid receptors. In developing OLs, micro receptor activation increases OL proliferation, while the kappa-antagonist nor-binaltorphimine (NorBNI) affects OL differentiation. Because exogenous opioids were not present in our defined culture medium, we hypothesized that NorBNI blocked endogenous opioids produced by the OLs themselves. To test this, intact and partially processed proenkephalin and prodynorphin-derived peptides were assessed in OLs using immunocytochemistry or Western blot analysis, or both. Immature OLs possessed large amounts of intact and partially processed proenkephalin precursors, as well as posttranslational products of prodynorphin including dynorphin A (1-17). With maturation, however, intact or partially processed proenkephalin was expressed by only about 50% of OLs, while dynorphin A (1-17) was undetectable. To assess the function of OL-derived opioids, the effect of kappa-agonists/antagonists on OL differentiation and death was explored. kappa-Agonists alone had no effect. In contrast, NorBNI significantly increased OL death. Additive OL losses were evident when NorBNI was paired with toxic levels of glutamate, suggesting that kappa-receptor blockade alone is sufficient to induce OL death. Thus, the results indicate that OLs express proenkephalin and prodynorphin peptides in a developmentally regulated manner, and further suggest that opioids produced by OLs modulate OL maturation and survival through local (i.e., autocrine and/or paracrine) mechanisms.

Aging↗

HIV-1 tat protein induces a migratory phenotype in human fetal microglia by a CCL2 (MCP-1)-dependent mechanism: possible role in NeuroAIDS.

Acquired immune deficiency syndrome (AIDS) encephalitis and dementia are characterized by neuronal loss, astrogliosis, and microglia activation and migration that contribute to the formation of multinucleated giant cells. Despite extensive evidence of pathological changes in the brain of infected individuals, the mechanisms of human immune deficiency virus type 1 (HIV-1) entry, microglia migration, and viral propagation within the brain are still not completely understood. In this study, we report that the induction of a migratory phenotype in human fetal microglia by the HIV-1 transactivator protein, tat, is mediated by the chemokine, CCL2. CCL2 or tat treatment alone induced rearrangement of actin and the formation of microglial processes. The time course of cell membrane ruffling induced by CCL2 was faster (5-30 min) than that elicited by tat treatment (2-3 h). Our previous data in human fetal microglia showed that tat induces CCL2 expression. Thus, we examined whether tat-induced microglia membrane ruffling and process formation, critical components in cell migration, are mediated by the secretion of CCL2 by these cells. To test this hypothesis, we treated microglia with tat protein in the presence of neutralizing CCL2 antibodies. Co-treatment with neutralizing CCL2 antibodies resulted in the loss of tat-induced membrane ruffling. Tat treatment of microglia induced polarization of CCR2, the receptor for CCL2, to the leading edge of processes, further suggesting a CCL2-dependent mechanism of tat-induced microglia migration. Our data indicate that tat facilitates microglia migration by inducing autocrine CCL2 release. Our results suggest that tat induced CCL2 secretion may be one of the early signals during NeuroAIDS.

AIDS Dementia Complex↗

Neuregulin-1 enhances survival of human astrocytic glioma cells.

Malignant astrocytic gliomas, referred to as astrocytomas, represent the most commonly diagnosed adult primary brain tumor. These tumors are characterized by unrelenting growth that is often resistant to chemotherapy and radiation therapy. Tumor expansion into the healthy surrounding brain tissue produces severe and often fatal consequences. In this study, we examine the potential for the neuregulin-1/erbB receptor signaling cascade to contribute to this process by modulating glioma cell growth. Using antibodies specific for the erbB receptors, we demonstrate the expression patterns for the erbB2, erbB3, and erbB4 receptors in human glioma biopsy samples. We then verify receptor expression in a panel of human glioma cell lines. Next, we investigate the status of the erbB2 and erbB3 receptors in the human glioma cell lines and find that they are constitutively tyrosine-phosphorylated and heterodimerized. Subsequently, we demonstrate that theses same cell lines express membrane bound and released forms of neuregulins, the erbB receptor ligands, suggesting a possible autocrine or paracrine signaling network. Furthermore, we show that exogenous activation of erbB2 and erbB3 receptors in U251 glioma cells by recombinant Nrg-1beta results in enhanced glioma cell growth under conditions of serum-deprivation. This enhancement is due to an increase in cell survival rather than an increase in cell proliferation and is dependent on the activation of erbB2 and phosphatidylinositol-3 kinase (PI3K). Moreover, Nrg-1beta activates an inhibitor of apoptosis, Akt, implying a possible role for this kinase in mediating Nrg-1beta effects in gliomas. This data suggests that glioma cells may use autocrine or paracrine neuregulin-1/erbB receptor signaling to enhance cell survival under conditions where growth would otherwise be limited.

Apoptosis↗

Schwann cells express erythropoietin receptor and represent a major target for Epo in peripheral nerve injury.

Erythropoietin (Epo) expresses potent neuroprotective activity in the peripheral nervous system; however, the underlying mechanism remains incompletely understood. In this study, we demonstrate that Epo is upregulated in sciatic nerve after chronic constriction injury (CCI) and crush injury in rats, largely due to local Schwann cell production. In uninjured and injured nerves, Schwann cells also express Epo receptor (EpoR), and its expression is increased during Wallerian degeneration. CCI increased the number of Schwann cells at the injury site and the number was further increased by exogenously administered recombinant human Epo (rhEpo). To explore the activity of Epo in Schwann cells, primary cultures were established. These cells expressed cell-surface Epo receptors, with masses of 71 and 62 kDa, as determined by surface protein biotinylation and affinity precipitation. The 71-kDa species was rapidly but transiently tyrosine-phosphorylated in response to rhEpo. ERK/MAP kinase was also activated in rhEpo-treated Schwann cells; this response was blocked by pharmacologic antagonism of JAK-2. RhEpo promoted Schwann cell proliferation, as determined by BrdU incorporation. Cell proliferation was ERK/MAP kinase-dependent. These results support a model in which Schwann cells are a major target for Epo in injured peripheral nerves, perhaps within the context of an autocrine signaling pathway. EpoR-induced cell signaling and Schwann cell proliferation may protect injured peripheral nerves and promote regeneration.

Animals↗

Constitutive neuregulin-1/ErbB signaling contributes to human vestibular schwannoma proliferation.

Vestibular schwannomas (VSs) are benign tumors that arise from the Schwann cells (SCs) lining the vestibular nerve. VS cells survive and proliferate far from neurons and axonally derived growth factors. We have previously shown that VSs produce the glial growth factor, neuregulin-1 (NRG1), and its receptors, ErbB2 and ErbB3. In the present work, we explore the contribution of constitutive NRG1:ErbB signaling to human VS cell proliferation. We confirm that human VSs, which express markers of immature and denervated SCs, also express endogenous NRG1 and activated ErbB2. We find that a blocking anti-NRG1 antibody and trastuzumab (Herceptin, HCN), a humanized anti-ErbB2 inhibitory monoclonal antibody, effectively inhibit NRG1 induced SC proliferation. Treatment of primary VS cultures with anti-NRG1 or HCN reduces cell proliferation in the absence of exogenous NRG1. Furthermore, conditioned medium from VS cell cultures contains NRG1 and stimulates SC proliferation in SC cultures, an effect that is inhibited by anti-NRG1 and HCN. These data suggest an autocrine pathway of VS growth stimulation involving NRG and ErbB receptors. Inhibition of constitutive NRG:ErbB signaling reduces VS cell proliferation in vitro and may have therapeutic potential for patients with VSs.

Animals↗

PACAP/PAC1 autocrine system promotes proliferation and astrogenesis in neural progenitor cells.

The Pituitary adenylate cyclase-activating peptide (PACAP) ligand/type 1 receptor (PAC1) system regulates neurogenesis and gliogenesis. It has been well established that the PACAP/PAC1 system induces differentiation of neural progenitor cells (NPCs) through the Gs-mediated cAMP-dependent signaling pathway. However, it is unknown whether this ligand/receptor system has a function in proliferation of NPCs. In this study, we identified that PACAP and PAC1 were highly expressed and co-localized in NPCs of mouse cortex at embryonic day 14.5 (E14.5) and found that the PACAP/PAC1 system potentiated growth factor-induced proliferation of mouse cortical NPCs at E14.5 via Gq-, but not Gs-, mediated PLC/IP3-dependent signaling pathway in an autocrine manner. Moreover, PAC1 activation induced elongation of cellular processes and a stellate morphology in astrocytes that had the bromodeoxyuridine (BrdU)-incorporating ability of NPCs. Consistent with this notion, we determined that the most BrdU positive NPCs differentiated to astrocytes through PAC1 signaling. These results suggest that the PACAP/PAC1 system may play a dual role in neural/glial progenitor cells not only differentiation but also proliferation in the cortical astrocyte lineage via Ca2+-dependent signaling pathways through PAC1.

Animals↗

Cellular localization of endothelin-1 and increased production in liver injury in the rat: potential for autocrine and paracrine effects on stellate cells.

Endothelin (ET) peptides have been implicated in the pathogenesis of several biological processes within the liver. ET levels are elevated in the circulation of patients with cirrhosis, and recent data suggest that ET may be overproduced in the liver itself in this condition. The aims of the current study were to elucidate the cellular source and expression of endothelin-1 (ET-1) in normal and injured liver, and to investigate its biological effects on stellate cells, the primary target of ETs in the liver. In normal hepatic cells, preproET-1 messenger RNA (mRNA) was detected in only nonparenchymal cells, predominantly in sinusoidal endothelial cells. After biliary fibrosis and early cirrhosis induced by bile duct ligation, preproET-1 mRNA and immunoreactive ET levels increased with progressive injury in whole liver extracts, as well as in isolated stellate and endothelial cell fractions. Eight days after bile duct ligation, the relative increase in preproET-1 mRNA was 1.6- and 7.6-fold above normal in sinusoidal endothelial and stellate cells, respectively. Additionally, immunoreactive ET peptide levels increased by 60% +/- 27% over basal values in sinusoidal endothelial cells and 98% +/- 40% in stellate cells. Cultured stellate cells responded dramatically to exogenous ET-1 by the spreading and up-regulation of smooth muscle alpha actin expression. Furthermore, in early culture before cellular activation, ET-1 (10 nmol/L) caused over a twofold increase in [3H]thymidine incorporation, while activated cells (i.e., those cultured for >1 week) exposed to ET-1 exhibited up to a fivefold decrease in [3H]thymidine incorporation. The data indicate that not only is ET-1 overproduced by both sinusoidal endothelial and stellate cells during liver injury, but that it also has potent effects on features of stellate cell activation. We conclude that autocrine and paracrine production of ET-1 is prominent and is likely to be important in the pathogenesis of hepatic diseases.

Actins↗

Establishment of a functional HGF/C-MET autocrine loop in spontaneous transformants of WB-F344 rat liver stem-like cells.

A model of spontaneous malignant transformation was used to evaluate the molecular changes that take place in WB-F344 rat liver epithelial cells during neoplastic transformation and tumorigenesis. A comparison of wild-type low-passage WB-F344 cells to spontaneously transformed tumor cell lines revealed that the majority of the tumor cell lines have an increased capacity for autonomous proliferation and motility when maintained in serum-free media. In the current study, we show that c-met is expressed at some level in wild-type WB-F344 cells and in all of the spontaneously transformed tumor cell lines, and that 9/16 of the tumor cell lines have acquired hepatocyte growth factor (HGF) expression. In vitro growth of HGF-expressing tumor cell lines is inhibited as much as 68% by the addition of neutralizing antibodies to HGF or antisense HGF oligonucleotides, indicating that the production of HGF by the tumor cells is partially responsible for driving autonomous proliferation in a subset of tumor cell lines. Furthermore, conditioned media collected from HGF-expressing tumor cell lines stimulates DNA synthesis in wild-type WB-F344 cells, and this effect can be abrogated by pre-incubation of the conditioned media with neutralizing antibodies to HGF. Because HGF is a motility-promoting growth factor, all cell lines were evaluated to determine if expression of HGF stimulated motogenesis. All tumor cell lines (regardless of HGF expression) were highly motile in comparison with wild-type WB-F344 cells, with a 3.5-fold to 20-fold greater number of motile cells. The high basal rate of motility characteristic of the tumor cell lines is not a result of the production of HGF, because it is also a property of the cell lines that do not express HGF messenger RNA. Furthermore, tumor cell motility is not inhibited by antisense oligonucleotides or neutralizing antibodies. Establishment of an autocrine HGF/c-met loop in a subset of spontaneously transformed WB-F344 cell lines may influence development and/or expression of the tumorigenic phenotype by driving cellular proliferation.

Animals↗

Para/autocrine regulation of estrogen receptors in hippocampal neurons.

Previous studies have shown that estrogens, originating from ovaries, have a wide variety of estrogen receptor (ER)-mediated effects in the hippocampus. In the present study, we have investigated whether estrogens, which are synthesized in the hippocampus, could induce these effects as well. As a parameter, we used ER expression in response to estrogen synthesis, because estrogen receptors are ligand-inducible transcription factors. The experiments were carried out with cultures of isolated adult rat hippocampal cells, which contained about 95% neurons and about 5% oligodendrocytes in serum-free and steroid-free medium. Hippocampal neurons express both estrogen receptor isoforms (ERalpha and ERbeta), as shown by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. The release of estrogens by hippocampal neurons was quantified by radioimmunoassay (RIA). The ER isoforms (alpha and beta) were studied by semiquantitative immunocytochemical image analysis. Hippocampal cells precultured for 4 days were found to synthesize 17beta-estradiol for the next 8 days. This synthesis was completely inhibited by letrozol, an aromatase inhibitor. Inhibition of estrogen synthesis by letrozol induced a significant decrease in ERalpha expression, but an increase in ERbeta. As a control, supplementation of the medium with 17beta-estradiol resulted in a significant increase of ERalpha expression, whereas ERbeta was downregulated. Our findings provide evidence for a de novo synthesis of estrogens in the hippocampus, differential regulation of estrogen receptor isoforms by estrogen and consequently for a para/autocrine loop of estrogen action in the hippocampus.

Animals↗

A synthetic matrix metalloproteinase inhibitor prevents squamous carcinoma cell proliferation by interfering with epidermal growth factor receptor autocrine loops.

Head and neck squamous cell carcinoma (HNSCC) is characterized by its capacity to invade adjacent tissues and to metastasize locoregionally. Evidence suggests that matrix metalloproteinases (MMPs) may play a causal role in HNSCC progression. While evaluating the role of MMPs in the invasion process, we made the surprising observation that a broad-spectrum MMP inhibitor, (marimastat, BB2516), inhibited the growth in vitro of some HNSCC cell lines. This inhibitory effect was only found in HNSCC cell lines overexpressing epidermal growth factor receptors. The effects of the MMP inhibitor could be reversed by adding exogenous c-erbB ligands, suggesting that the phenomenon may be related to autocrine ligand processing. This hypothesis was supported by the finding that the growth-inhibitory effect of marimastat was directly related to its ability to prevent the release of major c-erbB ligands including transforming growth factor-alpha, betacellulin and heregulin beta1 from HNSCC. Marimastat was also found to potentiate the cytotoxic effects of cisplatin both in vitro and in vivo. Our results indicate that the cleavage of several c-erbB ligands from membrane-anchored precursors requires MMP activity. We conclude that MMP inhibitors could prevent tumor progression not only by inhibiting invasion and angiogenesis, as previously shown, but also by their ability to inhibit autocrine signaling through the c-erbB receptors. Clinical trials to test this hypothesis in HNSCC should be considered.

Autocrine Communication↗

Malignant mesothelioma growth inhibition by agents that target the VEGF and VEGF-C autocrine loops.

Malignant mesothelioma (MM) is a locally aggressive tumor that originates from the mesothelial cells of the pleural and sometimes peritoneal surface. Conventional treatments for MM, consisting of chemotherapy or surgery give little survival benefit to patients, who generally die within 1 year of diagnosis. Hence, there is an urgent need for the development of alternative therapies. Vascular endothelial growth factor (VEGF) is an autocrine growth factor for MM. The closely related molecule, VEGF-C, is also implicated in malignant mesothelioma growth. VEGF-C and its cognate receptor VEGFR-3 are co-expressed in mesothelioma cell lines. A functional VEGF-C autocrine growth loop was demonstrated in mesothelioma cells by targeting VEGF-C expression and binding to VEGFR-3. The ability of novel agents that reduce the levels of VEGF and VEGF-C to inhibit mesothelioma cell growth in vitro was assessed. Antisense oligonucleotide (ODN) complementary to VEGF that inhibited VEGF and VEGF-C expression simultaneously specifically inhibited mesothelioma cell growth. Similarly, antibodies to VEGF receptor (VEGFR-2) and VEGF-C receptor (VEGFR-3) were synergistic in inhibiting mesothelioma cell growth. In addition, a diphtheria toxin-VEGF fusion protein (DT-VEGF), which is toxic to cells that express VEGF receptors was very effective in inhibiting mesothelioma cell growth in vitro. These results indicate that targeting VEGF and VEGF-C simultaneously may be an effective therapeutic approach for malignant mesothelioma.

Antibodies↗

Modulation of sulfur mustard induced cell death in human epidermal keratinocytes using IL-10 and TNF-alpha.

We compared the effects of overexpressing a tightly regulated anti-inflammatory cytokine, interleukin 10 (IL-10), and the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) on sulfur mustard induced cytotoxicity in human epidermal keratinocytes. Both cytokines were overexpressed when compared with the cells transfected with the empty vector as determined by quantitative ELISA. Cells overexpressing interleukin 10 suppressed the pro-inflammatory cytokines interleukin 8 and interleukin 6 following exposure to 50-300 microM sulfur mustard. These cells exhibited delayed onset of sulfur mustard induced cell death. On the other hand, cells overexpressing tumor necrosis factor alpha induced a sustained elevation in both interleukin 6 and 8 expression following exposure to 50-300 microM sulfur mustard. These cells were sensitized to the effects of sulfur mustard that resulted in an increased sulfur mustard induced cell death. Normal human epidermal keratinocytes treated with sulfur mustard exhibited elevated levels of tumor necrosis factor alpha expression and increased activity of nuclear factor kappa B. Gene array data indicated that cells overexpressing interleukin 10 induced several genes that are involved in growth promotion and cell-fate determination. We, therefore, identify IL-10 and TNF-alpha signal transduction pathways and their components as possible candidates for early therapeutic intervention against sulfur mustard induced cell injury.

Autocrine Communication↗

Tumor necrosis factor-alpha mediates RANK ligand stimulation of osteoclast differentiation by an autocrine mechanism.

Osteoblasts or bone marrow stromal cells are required as supporting cells for the in vitro differentiation of osteoclasts from their progenitor cells. Soluble receptor activator of nuclear factor-kappaB ligand (RANKL) in the presence of macrophage colony-stimulating factor (M-CSF) is capable of replacing the supporting cells in promoting osteoclastogenesis. In the present study, using Balb/c-derived cultures, osteoclast formation in both systems-osteoblast/bone-marrow cell co-cultures and in RANKL-induced osteoclastogenesis-was inhibited by antibody to tumor necrosis factor-alpha (TNF-alpha), and was enhanced by the addition of this cytokine. TNF-alpha itself promoted osteoclastogenesis in the presence of M-CSF. However, even at high concentrations of TNF-alpha the efficiency of this activity was much lower than the osteoclastogenic activity of RANKL. RANKL increased the level of TNF-alpha mRNA and induced TNF-alpha release from osteoclast progenitors. Furthermore, antibody to p55 TNF-alpha receptors (TNF receptors-1) (but not to p75 TNF-alpha receptors (TNF receptors-2) inhibited effectively RANKL- (and TNF-alpha() induced osteoclastogenesis. Anti-TNF receptors-1 antibody failed to inhibit osteoclastogenesis in C57BL/6-derived cultures. Taken together, our data support the hypothesis that in Balb/c, but not in C57BL/6 (strains known to differ in inflammatory responses and cytokine modulation), TNF-alpha is an autocrine factor in osteoclasts, promoting their differentiation, and mediates, at least in part, RANKL's induction of osteoclastogenesis.

Acid Phosphatase↗

Autocrine and paracrine nitric oxide regulate attachment of human osteoclasts.

Nitric oxide (NO) can reduce bone loss in chronic bone diseases. NO inhibits or kills osteoclasts, but the mechanism of action of NO in human bone turnover is not clear. To address this, we studied effects of NO on attachment and motility of human osteoclasts on mineralized and tissue culture substrates under defined conditions. Osteoclasts were differentiated in vitro from CD14 selected monocytes in RANKL and CSF-1, and characterized by cathepsin K expression, tartrate-resistant acid phosphatase (TRAP) activity, acid secretion, and lacunar resorption. Cell attachment was labeled with monoclonal antibody 23C6, specific for a binding domain of a key osteoclast attachment protein, the CD51/CD61 integrin dimer (alpha(v)beta(3)), with or without cell permeabilization. A ring of integrin attachment during bone degradation delimits an extracellular acid compartment, while alpha(v)beta(3) forms focal attachments on non-resorbable substrates. On resorbable substrate but not non-resorbable substrate, alpha(v)beta(3) labeling required cell permeabilization, in keeping with the membrane-matrix apposition that excludes large molecules and allows extracellular acidification. Acid secretion was labeled with the fluorescent weak base indicator lysotracker. NO donors, S-nitroso-N-acetyl penicillamine (SNAP) or sodium nitroprusside (SNP), downmodulated acid secretion simultaneously with cytoskeletal rearrangement, with alpha(v)beta(3) redistributed to a discontinuous pattern that labeled, on bone substrate, without membrane permeabilization. These effects were reversible, and an inhibitor of NO synthesis, N(G)-monomethyl-L-arginine (l-NMMA), increased acid secretion and decreased heterogeneity of attachment structures, showing that NO is an autocrine regulator of attachment. A hydrolysis-resistant activating cGMP analog 8-(4-chlorophenylthio)guanosine-3',5'-cyclic monophosphate replicated effects of NO donors, while an inhibiting analog, 8-(4-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate, Rp-isomer, opposed them. On tissue culture or mineralized substrates, NO or cGMP analogs directly regulated motility; after washout cells reattached and survived for days. We conclude that NO is produced by human osteoclasts and regulates acid secretion and cellular motility, in keeping with autocrine and paracrine NO regulation of the resorption cycle.

Acid Phosphatase↗

Vascular endothelial growth factor (VEGF-A) expression in human mesenchymal stem cells: autocrine and paracrine role on osteoblastic and endothelial differentiation.

Angiogenesis is essential in bone fracture healing for restoring blood flow to the fracture site. Vascular endothelial growth factor (VEGF) and its receptor have been implicated in this process. Despite the importance of angiogenesis for the healing processes of damaged bones, the role of VEGF signaling in modulation of osteogenic differentiation in human mesenchymal stem cells has not been investigated in great detail. We examined the expression of VEGF-A and VEGFR-1 in human adult mesenchymal stem cells derived from trabecular bone (hTBCs). VEGF-A was found to be secreted in a differentiation dependent manner during osteogenesis. Transcripts for VEGF-A were also seen to be elevated during osteogenesis. In addition, transcripts for VEGF-A and the corresponding receptor VEGFR-1 were upregulated under hypoxic conditions in undifferentiated hTBCs. To investigate the signaling of VEGF-A on osteogenesis recombinant hTBCs were generated. High expression of VEGF-A stimulated mineralization, whereas high expression of sFLT-1, an antagonist to VEGF-A, reduced mineralization suggesting that VEGF-A acts as autocrine factor for osteoblast differentiation. In addition, VEGF-A secreted by hTBCs promotes sprouting of endothelial cells (HUVE) demonstrating a paracrine role in blood vessel formation. In summary, an in vitro analysis of transgene effects on cellular behavior can be used to predict an effective ex vivo gene therapy.

Adenoviridae↗

Ligand-independent regulation of ErbB4 receptor phosphorylation by activated Ras.

The ErbB family of receptor tyrosine kinases regulates cell growth, differentiation and survival. Activation of the receptors is induced by specific growth factors in an autocrine, paracrine or juxtacrine manner. The activated ErbB receptors turn on a large variety of signaling cascades, including the prominent Ras-dependent signaling pathways. The activated Ras can induce secretion of growth factors such as EGF and neuregulin, which activate their respective receptors. In the present study, we demonstrate for the first time that activated Ras can activate ErbB4 receptor in a ligand-independent manner. Expression of constitutively active H-Ras(12V), K-Ras(12V) or N-Ras(13V) in PC12-ErbB4 cells induced ErbB4-receptor phosphorylation, indicating that each of the most abundant Ras isoforms can induce receptor activation. NRG-induced phosphorylation of ErbB4 receptor was blocked by the soluble ErbB4 receptor, which had no effect on the Ras-induced receptor phosphorylation. Moreover, conditioned medium from H-Ras(12V)-transfected PC12-ErbB4 cells had no effect on receptor phosphorylation. It thus indicates that Ras induces ErbB4 phosphorylation in a ligand-independent manner. Each of the Ras effector domain mutants, H-Ras(12V)S35, H-Ras(12V)C40, and H-Ras(12V)G37, which respectively activate Raf1, PI3K, and RalGEF, induced a small but significant receptor phosphorylation. The PI3K inhibitor LY294002 and the MEK inhibitor PD98059 caused a partial inhibition of the Ras-induced ErbB4 receptor phosphorylation. Using a mutant ErbB4 receptor, which lacks kinase activity, we demonstrated that the Ras-mediated ErbB4 phosphorylation depends on the kinase activity of the receptor and facilitates ligand-independent neurite outgrowth in PC12-ErbB4 cells. These experiments demonstrate a novel mechanism controlling ErbB receptor activation. Ras induces ErbB4 receptor phosphorylation in a non-autocrine manner and this activation depends on multiple Ras effector pathways and on ErbB4 kinase activity.

Animals↗

Regulation of bone formation by adiponectin through autocrine/paracrine and endocrine pathways.

Since interaction between bone and lipid metabolism has been suggested, this study investigated the regulation of bone metabolism by adiponectin, a representative adipokine, by analyzing deficient and overexpressing transgenic mice. We initially confirmed that adiponectin and its receptors were expressed in osteoblastic and osteoclastic cells, indicating that adiponectin can act on bone not only through an endocrine pathway as a hormone secreted from fat tissue, but also through an autocrine/paracrine pathway. There was no abnormality in bone mass or turnover of adiponectin-deficient (Ad-/-) mice, possibly due to an equivalent balance of the two pathways. In the culture of bone marrow cells from the Ad-/- mice, osteogenesis was decreased compared to the wild-type (WT) cell culture, indicating a positive effect of endogenous adiponectin through the autocrine/paracrine pathway. To examine the endocrine action of adiponectin, we analyzed transgenic mice overexpressing adiponectin in the liver, and found no abnormality in the bone. Addition of recombinant adiponectin in cultured osteoprogenitor cells suppressed osteogenesis, suggesting that the direct action of circulating adiponectin was negative for bone formation. In the presence of insulin, however, this suppression was blunted, and adiponectin enhanced the insulin-induced phosphorylations of the main downstream molecule insulin receptor substrate-1 and Akt. These lines of results suggest three distinct adiponectin actions on bone formation: a positive action through the autocrine/paracrine pathway by locally produced adiponectin, a negative action through the direct pathway by circulating adiponectin, and a positive action through the indirect pathway by circulating adiponectin via enhancement of the insulin signaling.

Adiponectin↗

Existence of autocrine loop between interleukin-6 and transforming growth factor-beta1 in activated rat pancreatic stellate cells.

Interleukin (IL)-6 is a proinflammatory cytokine assumed to participate in pancreatic fibrosis by activating pancreatic stellate cells (PSCs). Autocrine TGF-beta1 is to central in PSC functional regulation. In this study, we examined IL-6 secretion from culture-activated rat PSCs and its regulatory mechanism. Activated PSCs express and secrete IL-6. When anti-TGF-beta1 neutralizing antibody was added in the culture medium, IL-6 secretion from activated PSCs was inhibited, whereas exogenous TGF-beta1 added in the culture medium enhanced IL-6 expression and secretion by PSCs in a dose dependent manner. Infection of PSCs with an adenovirus expressing dominant-negative Smad2/3 attenuated basal and TGF-beta1-stimulated IL-6 expression and secretion of PSCs. We also demonstrated the reciprocal effect of PSCs-secreted IL-6 on autocrine TGF-beta1. Anti-IL-6 neutralizing antibody inhibited TGF-beta1 secretion from PSCs. Preincubation of cells with 10 nM PD98059, an extracellular signal-regulated kinase (ERK)-dependent pathway inhibitor, attenuated IL-6-enhanced TGF-beta1 expression and secretion of PSCs. In addition, IL-6 activated ERK in PSCs. These data indicate the existence of autocrine loop between IL-6 and TGF-beta1 through ERK- and Smad2/3-dependent pathways in activated PSCs.

Animals↗