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PubMed2002

Adhesion-dependent signaling by macrophage migration inhibitory factor (MIF).

Proper stimulation of cell cycle progression and DNA synthesis requires cooperating signals from integrin and growth factor receptors. We previously found that the proinflammatory peptide, macrophage migration inhibitory factor (MIF), functions as an autocrine mediator of growth factor-dependent ERK MAP kinase activation and cell cycle progression. We now report that MIF secretion is induced by cell adhesion to fibronectin in quiescent mouse fibroblasts. Adhesion-mediated release of MIF subsequently promotes integrin-dependent activation of MAP kinase, cyclin D1 expression, and DNA synthesis. Secretion of MIF requires protein kinase C activity, and recombinant MIF reconstitutes the activation of MAP kinases in the presence of protein kinase C inhibition. Finally, we show that cells deficient in MIF have significantly higher retinoblastoma tumor suppressor and lower E2F transcriptional activities. These results suggest that MIF is an important autocrine mediator of adhesion-dependent signaling events and may provide mechanistic insight into how MIF regulates proliferative and oncogenic processes.

Animals↗
PubMed2003

Stimulation of airway mucin gene expression by interleukin (IL)-17 through IL-6 paracrine/autocrine loop.

Mucus hypersecretion and persistent airway inflammation are common features of various airway diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis. One key question is: does the associated airway inflammation in these diseases affect mucus production? If so, what is the underlying mechanism? It appears that increased mucus secretion results from increased mucin gene expression and is also frequently accompanied by an increased number of mucous cells (goblet cell hyperplasia/metaplasia) in the airway epithelium. Many studies on mucin gene expression have been directed toward Th2 cytokines such as interleukin (IL)-4, IL-9, and IL-13 because of their known pathophysiological role in allergic airway diseases such as asthma. However, the effect of these cytokines has not been definitely linked to their direct interaction with airway epithelial cells. In our study, we treated highly differentiated cultures of primary human tracheobronchial epithelial (TBE) cells with a panel of cytokines (interleukin-1alpha, 1beta, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, and tumor necrosis factor alpha). We found that IL-6 and IL-17 could stimulate the mucin genes, MUC5B and MUC5AC. The Th2 cytokines IL-4, IL-9, and IL-13 did not stimulate MUC5AC or MUC5B in our experiments. A similar stimulation of MUC5B/Muc5b expression by IL-6 and IL-17 was demonstrated in primary monkey and mouse TBE cells. Further investigation of MUC5B expression demonstrated that IL-17's effect is at least partly mediated through IL-6 by a JAK2-dependent autocrine/paracrine loop. Finally, evidence is presented to show that both IL-6 and IL-17 mediate MUC5B expression through the ERK signaling pathway.

Animals↗
PubMed2003

Autocrine growth factor regulation of lysyl oxidase expression in transformed fibroblasts.

Lysyl oxidase catalyzes oxidative deamination of peptidyl-lysine and hydroxylysine residues in collagens and lysine residues in elastin to form peptidyl aldehydes that are required for the formation of covalent cross-links in normal extracellular matrix biosynthesis. Lysyl oxidase in addition has tumor suppressor activity, and phenotypic reversion of transformed cell lines is accompanied by increased lysyl oxidase expression. The mechanism of low expression of lysyl oxidase in tumor cells is unknown. The present study investigates the hypothesis that autocrine growth factor pathways maintain low lysyl oxidase expression levels in c-H-ras-transformed fibroblasts (RS485 cell line). Autocrine pathways were blocked with suramin, a general inhibitor of growth factor receptor binding, and resulted in more than a 10-fold increase in lysyl oxidase expression and proenzyme production. This regulation was found to be reversible and occurred at the transcriptional level determined using lysyl oxidase promoter/reporter gene assays. Function blocking anti-fibroblast growth factor-2 (FGF-2) antibody enhanced lysyl oxidase expression in the absence of suramin. Finally, the addition of FGF-2 to suramin-treated cells completely reversed suramin stimulation of lysyl oxidase mRNA levels. Data support that an FGF-2 autocrine pathway inhibits lysyl oxidase transcription in the tumorigenic-transformed RS485 cell line. This finding may be of therapeutic significance and, in addition, provides a new experimental approach to investigate the mechanism of the tumor suppressor activity of lysyl oxidase.

3T3 Cells↗
PubMed2004

Hepatoma-derived growth factor is a neurotrophic factor harbored in the nucleus.

Hepatoma-derived growth factor (HDGF) is a heparin-binding proliferating factor originally isolated from conditioned medium of the hepatoma-derived cell line HuH-7. HDGF has greatest homology in an amino acid sequence with high mobility group 1 (HMG1), which has been characterized as a DNA-binding, inflammatory, and potent neurite outgrowth molecule. HDGF is reported to be widely expressed and act as a growth factor in many kinds of cells. However, it has not been investigated in the nervous system. Here, we show by Western blot analysis that HDGF is present in the mouse brain from the embryonic period until adulthood. In situ hybridization and immunohistochemical analyses revealed that HDGF was expressed mainly in neurons, and HDGF protein was localized to the nucleus. HDGF and high mobility group 1 were secreted under physiological conditions and released extracellularly in necrotic conditions. Furthermore, we showed that exogenously supplied HDGF had a neurotrophic effect and was able to partially prevent the cell death of neurons in which endogenous HDGF was suppressed. Therefore, we propose that HDGF is a novel type of neurotrophic factor, on account of its localization in the nucleus and its potential to function in an autocrine manner under both physiological and pathological conditions throughout life.

Animals↗
PubMed2003

Soluble urokinase-type plasminogen activator receptor inhibits cancer cell growth and invasion by direct urokinase-independent effects on cell signaling.

The urokinase-type plasminogen activator receptor (uPAR) is released from human cancers and is readily detected in blood. In animal models, soluble uPAR (SuPAR) antagonizes cancer progression; however, the mechanism by which SuPAR functions in vivo remains unclear. It is generally thought that SuPAR scavenges uPA and prevents its interaction with membrane-anchored uPAR. In this study, we demonstrate a novel molecular mechanism by which SuPAR may inhibit cancer progression. We show that SuPAR has the potential to directly and in a uPA-independent manner block the signaling activity of membrane-anchored uPAR. Whether SuPAR inhibits signaling is cell type-specific, depending on the state of the endogenous uPA-uPAR signaling system. In uPAR-deficient cells that lack endogenous uPAR signaling, including uPAR-/-murine embryonic fibroblasts and human embryonal kidney 293 cells, SuPAR functions as a partial signaling agonist that activates ERK/mitogen-activated protein kinase. By contrast, in cells with potent autocrine uPA-uPAR signaling systems, including MDA-MB 231 breast cancer cells and low density lipoprotein receptor-related protein-1-deficient murine embryonic fibroblasts, SuPAR substantially decreases ERK activation. The mechanism probably involves competitive displacement of membrane-anchored uPAR-uPA complex from signaling adaptor proteins. As a result of its effects on cell signaling, SuPAR blocks cell growth and inhibits cellular invasion of Matrigel. Cleavage of SuPAR by proteinases increases its signaling agonist activity and reverses its inhibitory effects on growth and invasion. Thus, proteolytic cleavage represents a molecular switch that neutralizes the anticancer activity of SuPAR.

Animals↗
PubMed2004

Autocrine/paracrine secreted Frizzled-related protein 2 induces cellular resistance to apoptosis: a possible mechanism of mammary tumorigenesis.

Abnormal regulation of apoptosis and cell proliferation is thought to be involved in tumor formation. The secreted Frizzled-related protein 2 (SFRP2) was detected in primary culture of canine mammary gland tumors but not in normal mammary tissues. Thus, to elucidate the role of SFRP2 in mammary tumorigenesis, we overexpressed SFRP2 in mammary gland tumor and MCF7 cells. The results indicated that SFRP2 is secreted and incorporated into the extracellular matrix (ECM) of the tumor and normal cells. In an attempt to understand the molecular basis underlying the interaction between SFRP2 and ECM, co-immunoprecipitation and cell adhesion assays were carried out. SFRP2 was found to be associated with the fibronectin-integrin protein complex and could promote cell adhesion. DNA fragmentation and caspase 3 activity analyses showed that the susceptibility of the cells to UV-induced apoptosis decreased in the context of SFRP2 overexpression. Upon disruption of the fibronectin-integrin connection, the antiapoptosis activity of SFRP2 was decreased. Moreover, SFRP2 was found to induce tumorous transformation in normal mammary epithelial cells and to inhibit apoptosis in a modified paracrine model. Collectively, our results emphasize the relevance of SFRP2 and ECM in mammary tumorigenesis and provide further insight into the mechanism of SFRP2 action.

Animals↗
PubMed2004

Induced autocrine signaling through the epidermal growth factor receptor contributes to the response of mammary epithelial cells to tumor necrosis factor alpha.

In contrast to the well known cytotoxic effects of tumor necrosis factor (TNF) alpha in many mammary cancer cells, we have found that TNF stimulates the proliferation and motility of human mammary epithelial cells (HMECs). Since the response of HMECs to TNF is similar to effects mediated by epidermal growth factor receptor (EGFR) activation, we explored the potential role of cross-talk through the EGFR signaling pathways in mediating cellular responses to TNF. Using a microarray enzyme-linked immunoassay, we found that exposure to TNF stimulated the dose-dependent shedding of the EGFR ligand transforming growth factor alpha (TGFalpha). Both proliferation and motility of HMECs induced by TNF was prevented either by inhibiting membrane protein shedding with a metalloprotease inhibitor, by blocking epidermal growth factor receptor (EGFR) kinase activity, or by limiting ligand-receptor interactions with an antagonistic anti-EGFR antibody. EGFR activity was also necessary for TNF-induced release of matrix metalloprotease-9, thought to be an essential regulator of mammary cell migration. The cellular response to TNF was associated with a biphasic temporal pattern of extracellular signal-regulated kinase (ERK) phosphorylation, which was EGFR-dependent and modulated by inhibition of metalloprotease-mediated shedding. Significantly, the late phase of ERK phosphorylation, detectable within 4 h after exposure, was blocked by the metalloprotease inhibitor batimastat, indicating that autocrine signaling through ligand shedding was responsible for this secondary wave of ERK activity. Our results indicate a novel and important role for metalloprotease activation and EGFR transmodulation in mediating the cellular response to TNF.

Autocrine Communication↗
PubMed2004

Autocrine and exogenous transforming growth factor beta control cell cycle inhibition through pathways with different sensitivity.

Human colon carcinoma cells HCT116 that lack transforming growth factor beta (TGF-beta) type II receptor (RII) demonstrated restoration of autocrine TGF-beta activity upon reexpression of RII without restoring inhibitory responses to exogenous TGF-beta treatment. RII transfectants (designated RII Cl 37) had a longer lag phase relative to NEO-transfected control cells (designated NEO pool) before entering exponential growth in tissue culture. The prolonged growth arrest of RII Cl 37 cells was associated with markedly reduced cyclin-dependent kinase (CDK)2 activity. Our results demonstrate that p21 induction by autocrine TGF-beta is responsible for reduced CDK2 activity, which at least partially contributes to prolonged growth arrest and reduced cell proliferation in RII Cl 37 cells. In contrast to RII transfectants, HCT116 cells transfected with chromosome 3 (designated HCT116Ch3), which bears the RII gene, restored the response to exogenous TGF-beta as well as autocrine TGF-beta activity. Autocrine TGF-beta activity in HCT116Ch3 cells induced p21 expression as seen in RII Cl 37 cells; however, in addition to autocrine activity, HCT116Ch3 cells responded to exogenous TGF-beta as decreased CDK4 expression and reduced pRb phosphorylation mediated a TGF-beta inhibitory response in these cells. These results indicate that autocrine TGF-beta regulates the cell cycle through a pathway different from exogenous TGF-beta in the sense that p21 is a more sensitive effector of the TGF-beta signaling pathway, which can be induced and saturated by autocrine TGF-beta, whereas CDK4 inhibition is a less sensitive effector, which can only be activated by high levels of exogenous TGF-beta

Autocrine Communication↗
PubMed2004

Autocrine transforming growth factor alpha regulates cell adhesion by multiple signaling via specific phosphorylation sites of p70S6 kinase in colon cancer cells.

Recently, we showed that autocrine transforming growth factor alpha (TGFalpha) controls the epidermal growth factor receptor (EGFR)-mediated basal expression of integrin alpha2, cell adhesion and motility in highly progressed HCT116 colon cancer cells. We also reported that the expression of basal integrin alpha2 and its biological effects are critically controlled by the constitutive activation of the ERK/MAPK pathway (Sawhney, R. S., Sharma, B., Humphrey, L. E., and Brattain, M. G. (2003) J. Biol. Chem. 278, 19861-19869). In the present report, we further examine the downstream signaling mechanisms underlying EGFR/ERK signaling and integrin alpha2 function in HCT116 cells. Selective MEK inhibitors attenuated TGFalpha-mediated basal activation of p70S6K (S6K) specifically at Thr-389, indicating that this S6K site is downstream of ERK/MAPK signaling. Cells were treated with the selective protein kinase C (PKC) inhibitor bisindolylmaleimide to determine the role of PKC in S6K activation. The Thr-421 and Ser-424 phosphorylation sites of S6K were specifically inhibited by bisindolylmaleimide, which also blocked integrin alpha2 expression, cell adhesion, and motility. These data establish a novel cell motility function of S6K via PKC activation in a cancer cell. In addition, we examined whether mammalian target of rapamycin signaling controls S6K activation. Rapamycin inhibited constitutive S6K phosphorylation specifically at Thr-389, Thr-421, and Ser-424 sites. The assignment of these phosphorylation sites on S6K to biological functions was unequivocally confirmed by transfection of cells with specific single phosphorylation site dominant negative mutants. These experiments show for the first time that autocrine TGFalpha regulates cell adhesion function by multiple signaling pathways via specific phosphorylation sites of S6K in cancer cells.

Autocrine Communication↗
PubMed2004

STAT signaling underlies difference between flagellin-induced and tumor necrosis factor-alpha-induced epithelial gene expression.

Both bacterial flagellin and the cytokine tumor necrosis factor-alpha (TNFalpha) are potent activators of intestinal epithelial cell pro-inflammatory gene expression in general; nonetheless, there seem to be distinct differences in the specific patterns of gene expression induced by these agonists. The goal of this study was to define one such difference and elucidate the signaling mechanism responsible for such differential gene induction by these agonists. We observed that expression of inducible nitric-oxide synthase is substantially induced by flagellin but only minimally expressed in response to TNFalpha. This difference seemed to be underlain by differential induction of signal transducers and activators of transcription (STAT) activation in that, whereas flagellin and TNFalpha seemed to be equipotent activators of p38 mitogen-activated protein kinase and nuclear factor-kappaB, flagellin induced substantially higher levels of STAT-1 and -3 tyrosine phosphorylation. Such flagellin-induced STAT activation exhibited delayed kinetics and was ablated by treatment with cycloheximide. Flagellin-induced activation of STAT-3 was abolished via neutralizing antibodies to interleukin (IL)-6, but not interferon (IFN)beta nor IFNgamma; none of these neutralizing antibodies had any effect on flagellin-induced STAT-1 tyrosine phosphorylation. Flagellin induced substantially more IL-6 expression than did TNFalpha, but neither agonist elicited detectable levels of IFN expression. Flagellin-induced expression of inducible nitric-oxide synthase but not IL-6, was abolished by blocking STAT activation with AG490, and was reduced by blocking STAT-3 activation with anti-IL-6. Together, these results indicate that epithelial cell induction of flagellin-specific gene expression is mediated, in part, by STAT activation that results from autocrine activation via IL-6.

Autocrine Communication↗
PubMed2004

A novel role for vascular endothelial growth factor as an autocrine survival factor for embryonic stem cells during hypoxia.

Vascular endothelial growth factor (VEGF) is best known for its angiogenic activity on endothelial cells, but it also affects neurons, pneumocytes, and other mature cell types as well as endothelial, neural, and hematopoietic progenitors. Here, we examined its effect on pluripotential embryonic stem (ES) cells under hypoxic stress. ES cells were found to produce VEGF and to express VEGF receptor-2 and neuropilin-1 (Nrp-1), a VEGF165 isoform-specific receptor. During hypoxia, expression levels of VEGF, Flk-1, and Nrp-1 were elevated. Inhibition or targeted gene inactivation of VEGF increased ES cell apoptosis during prolonged hypoxia (48 h) by about 10-fold. The survival activity of VEGF was specific since inhibition of other growth factors (including basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor, platelet-derived growth factor, and placental growth factor) had no effect. Neuropilin-1 was involved in the VEGF-survival activity since overexpression of Nrp-1 decreased hypoxia-induced apoptosis about 3-fold. The hypoxia-response element, via which hypoxia-inducible transcription factors up-regulate VEGF expression under hypoxic conditions, was critical since targeted deletion of this element in the VEGF promoter enhanced hypoxia-induced ES cell apoptosis to the same extent as VEGF inhibition or gene inactivation. Thus, VEGF plays a critical role in survival of ES cells during prolonged hypoxia.

Animals↗
PubMed2004

Kaposi's sarcoma-associated herpesvirus-encoded viral interleukin-6 is secreted and modified differently than human interleukin-6: evidence for a unique autocrine signaling mechanism.

Viral interleukin-6 (vIL-6) is a homolog of cellular IL-6 that is encoded by the Kaposi's sarcoma-associated herpesvirus (KSHV) genome. vIL-6 binds to the IL-6 signal transducer gp130 without the cooperation of the IL-6 high affinity receptor to induce STAT3 DNA binding and cell proliferation. Although vIL-6 is believed to be important in the pathogenesis of KSHV-induced diseases, its secretion and post-translational modifications have not previously been characterized. Pulse-chase analysis revealed that the half-time of vIL-6 secretion is approximately 8-fold longer than that of human IL-6. Yet, the vIL-6 signal sequence targets human IL-6 secretion to nearly wild-type levels. Surprisingly, vIL-6 was not secreted from a cell line that does not express gp130 but expression of human gp130 in these cells enabled the secretion of vIL-6. Consistent with this observation, complete maturation of gp130 N-glycans is inhibited by vIL-6 coexpression, suggesting that the binding of the receptor to vIL-6 occurs intracellularly in early or pre-Golgi compartments. Furthermore, a vIL-6 mutant containing an endoplasmic reticulum retention signal is not secreted but does still induce receptor activation and signaling. Secreted vIL-6 is completely glycosylated at both possible N-glycosylaton sites and contains a large proportion of immature high-mannose glycans that is not typical of cytokines. These findings suggest that vIL-6 may induce gp130 signaling by an exclusively autocrine mechanism that relies on intracellular binding to its receptor. During KSHV infection, vIL-6 may only induce signaling in KSHV-infected cells to benefit the viral life cycle and promote oncogenic transformation.

Amino Acid Sequence↗
PubMed2005

Alpha-tocopheryl succinate inhibits malignant mesothelioma by disrupting the fibroblast growth factor autocrine loop: mechanism and the role of oxidative stress.

We have studied the potential effect against human malignant mesotheliomas (MM) of alpha-tocopheryl succinate (alpha-TOS), a redox-silent vitamin E analog with strong pro-apoptotic and anti-cancer activity. alpha-TOS at sub-apoptotic levels inhibited proliferation of MM cell lines, while being nontoxic to nonmalignant mesothelial cells. Because MM cells are typified by a highly metastatic phenotype, we investigated the effect of alpha-TOS on genes playing a major role in MM progression. Of these, alpha-TOS down regulated fibroblast growth factor (FGF)-1 and, in particular, FGF-2 on the transcriptional level in MM cells, and this was not observed in their nonmalignant counterparts. FGF-2 short interfering RNA suppressed proliferation of MM cells. Down-regulation of FGF-2 was likely because of inhibition of the egr-1 transcription activity that was decreased in MM cells via oxidative stress induced by alpha-TOS, as evidenced by EPR spectroscopy, whereas nonmalignant cells did not show this response. Treatment of MM cells with egr-1 short interfering RNA suppressed proliferation, which was overridden by exogenously added recombinant FGF-1 and, in particular, FGF-2. An analog of coenzyme Q targeted to mitochondria and superoxide dismutase overrode inhibition of MM cell proliferation by alpha-TOS as well as alpha-TOS-induced inhibition of egr-1-dependent transactivation. Finally, alpha-TOS significantly suppressed experimental MM in immunocompromised mice. Our data suggest that alpha-TOS suppresses MM cell proliferation by disrupting the FGF-FGF receptor autocrine signaling loop by generating oxidative stress and point to the agent as a selective drug against thus far fatal mesotheliomas.

Antineoplastic Agents↗
PubMed2005

Tumor cells enhance their own CD44 cleavage and motility by generating hyaluronan fragments.

Hyaluronan (HA) is an extracellular matrix glycosaminoglycan that interacts with cell-surface receptors, including CD44. Although HA usually exists as a high molecular mass polymer, HA of a much lower molecular mass that shows a variety of biological activities can be detected under certain pathological conditions, particularly in tumors. We previously reported that low molecular weight HAs (LMW-HAs) of a certain size range induce the proteolytic cleavage of CD44 from the surface of tumor cells and promote tumor cell migration in a CD44-dependent manner. Here, we show that MIA PaCa-2, a human pancreatic carcinoma cell line, secreted hyaluronidases abundantly and generated readily detectable levels of LMW-HAs ranging from approximately 10- to 40-mers. This occurred in the absence of any exogenous stimulation. The tumor-derived HA oligosaccharides were able to enhance CD44 cleavage and tumor cell motility. Inhibition of the CD44-HA interaction resulted in the complete abrogation of these cellular events. These results are consistent with the concept that tumor cells generate HA oligosaccha-rides that bind to tumor cell CD44 through the expression of their own constitutive hyaluronidases. This enhances their own CD44 cleavage and cell motility, which would subsequently promote tumor progression. Such an autocrine/paracrine-like process may represent a novel activation mechanism that would facilitate and promote the malignant potential of tumor cells.

Animals↗
PubMed2005

Interleukin-8 secretion by fibroblasts induced by low density lipoproteins is p38 MAPK-dependent and leads to cell spreading and wound closure.

We have previously reported (Dobreva, I., Waeber, G., Mooser, V., James, R. W., and Widmann, C. (2003) J. Lipid Res. 44, 2382-2390) that low density lipoproteins (LDLs) induce activation of the p38 MAPK pathway, resulting in fibroblast spreading and lamellipodia formation. Here, we show that LDL-stimulated fibroblast spreading and wound sealing are due to secretion of a soluble factor. Using an antibody-based human protein array, interleukin-8 (IL-8) was identified as the main cytokine whose concentration was increased in supernatants from LDL-stimulated cells. Incubation of supernatants from LDL-treated cells with an anti-IL-8 blocking antibody completely abolished their ability to induce cell spreading and mediate wound closure. In addition, fibroblasts treated with recombinant IL-8 spread to the same extent as cells incubated with LDL or supernatants from LDL-treated cells. The ability of LDL and IL-8 to induce fibroblast spreading was mediated by the IL-8 receptor type II (CXCR-2). Furthermore, LDL-induced IL-8 production and subsequent wound closure required the activation of the p38 MAPK pathway, because both processes were abrogated by a specific p38 inhibitor. Therefore, the capacity of LDLs to induce fibroblast spreading and accelerate wound closure relies on their ability to stimulate IL-8 secretion in a p38 MAPK-dependent manner. Regulation of fibroblast shape and migration by lipoproteins may be relevant to atherosclerosis that is characterized by increased LDL cholesterol levels, IL-8 production, and extensive remodeling of the vessel wall.

Atherosclerosis↗
PubMed2006

Control of MyoD function during initiation of muscle differentiation by an autocrine signaling pathway activated by insulin-like growth factor-II.

The insulin-like growth factors (IGFs) play key roles in muscle development, maintenance, and repair, but their mechanisms of action are incompletely defined. We previously identified an autocrine pathway involving production of IGF-II and activation of the IGF-I receptor, phosphatidylinositol 3-kinase, and Akt in myoblast differentiation induced by MyoD in 10T1/2 mesenchymal stem cells and found that blocking this pathway prevented differentiation (Wilson, E. M., Hsieh, M. M., and Rotwein, P. (2003) J. Biol. Chem. 278, 41109-41113). We now have analyzed regulation of MyoD function in this model system. Inhibition of IGF-II production impaired the transcriptional actions of MyoD, as seen by a 70-80% decline in activity of transfected reporter genes, including the myogenin and creatine kinase promoters, and by complete inhibition of transcription of the endogenous myogenin gene but had no effect on MyoD protein levels, post-translational modifications, or nuclear localization, and neither blocked the rapid disappearance of the inhibitory molecule Id1 nor altered the nuclear expression or abundance of the MyoD binding partner E12/E47. Impaired signaling through the IGF-I receptor also did not decrease the ability of MyoD or E12/E47 to bind to target DNA sites at the proximal myogenin promoter, as assessed by chromatin immunoprecipitation assay but, rather, blocked chromatin remodeling at this site, as indicated by reduced recruitment of co-activators p300 and P/CAF and diminished acetylation of histones H3 and H4. Taken together, these results show that IGF-II-initiated signaling through the insulin-like growth factor-I receptor targets transcriptional co-regulators that are essential co-factors for MyoD and suggests that the phosphatidylinositol 3-kinase-Akt pathway plays a key role in establishing an amplification cascade that is essential for sustaining the earliest events in muscle differentiation.

Animals↗
PubMed2006

Autocrine and juxtacrine effects of amphiregulin on the proliferative, invasive, and migratory properties of normal and neoplastic human mammary epithelial cells.

Amphiregulin (AR) autocrine loops have been associated with several types of cancer. We demonstrate that SUM149 breast cancer cells have a self-sustaining AR autocrine loop. SUM149 cells are epidermal growth factor (EGF)-independent for growth, and they overexpress AR mRNA, AR membrane precursor protein, and secreted AR relative to the EGF-dependent human mammary epithelial cell line MCF10A. MCF10A cells made to overexpress AR (MCF10A AR) are also EGF-independent for growth. Treatment with the pan-ErbB inhibitor CI1033 and the anti-EGF receptor (EGFR) antibody C225 demonstrated that ligand-mediated activation of EGFR is required for SUM149 cell proliferation. AR-neutralizing antibody significantly reduced both SUM149 EGFR activity and cell proliferation, confirming that an AR autocrine loop is required for mitogenesis in SUM149 cells. EGFR tyrosine phosphorylation was dramatically decreased in both SUM149 and MCF10A AR cells after inhibition of AR cleavage with the broad spectrum metalloprotease inhibitor GM6001, indicating that an AR autocrine loop is strictly dependent on AR cleavage in culture. However, a juxtacrine assay where fixed SUM149 cells and MCF10A AR cells were overlaid on top of EGF-deprived MCF10A cells showed that the AR membrane precursor can activate EGFR. SUM149 cells, MCF10A AR cells, and MCF10A cells growing in exogenous AR were all considerably more invasive and motile than MCF10A cells grown in EGF. Moreover, AR up-regulates a number of genes involved in cell motility and invasion in MCF10A cells, suggesting that an AR autocrine loop contributes to the aggressive breast cancer phenotype.

Amphiregulin↗
PubMed2006

Smad4-dependent regulation of urokinase plasminogen activator secretion and RNA stability associated with invasiveness by autocrine and paracrine transforming growth factor-beta.

Metastasis is a primary cause of mortality due to cancer. Early metastatic growth involves both a remodeling of the extracellular matrix surrounding tumors and invasion of tumors across the basement membrane. Up-regulation of extracellular matrix degrading proteases such as urokinase plasminogen activator (uPA) and matrix metalloproteinases has been reported to facilitate tumor cell invasion. Autocrine transforming growth factor-beta (TGF-beta) signaling may play an important role in cancer cell invasion and metastasis; however, the underlying mechanisms remain unclear. In the present study, we report that autocrine TGF-beta supports cancer cell invasion by maintaining uPA levels through protein secretion. Interestingly, treatment of paracrine/exogenous TGF-beta at higher concentrations than autocrine TGF-beta further enhanced uPA expression and cell invasion. The enhanced uPA expression by exogenous TGF-beta is a result of increased uPA mRNA expression due to RNA stabilization. We observed that both autocrine and paracrine TGF-beta-mediated regulation of uPA levels was lost upon depletion of Smad4 protein by RNA interference. Thus, through the Smad pathway, autocrine TGF-beta maintains uPA expression through facilitated protein secretion, thereby supporting tumor cell invasiveness, whereas exogenous TGF-beta further enhances uPA expression through mRNA stabilization leading to even greater invasiveness of the cancer cells.

Autocrine Communication↗