Involvement of protein kinase A in fibroblast growth factor-2-activated transcription

Polypeptide growth factors activate common signal transduction pathways, yet they can induce transcription of different target genes. The mechanisms that control this specificity are not completely understood. Recently, we have described a fibroblast growth factor (FGF)-inducible response element, FiRE, on the syndecan-1 gene. In NIH 3T3 cells, the FiRE is activated by FGF-2 but not by several other growth factors, such as platelet-derived growth factor or epidermal growth factor, suggesting that FGF-2 activates signaling pathways that diverge from pathways activated by other growth factors. In this paper, we report that the activation of FiRE by FGF-2 requires protein kinase A (PKA) in NIH 3T3 cells. The PKA-specific inhibitor H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide) blocked the FGF-2-induced activation of FiRE, the transcription of the syndecan-1 gene, and cell proliferation. Also, expression of a dominant-negative form of PKA inhibited the FGF-2-induced FiRE activation and the transcription of the syndecan-1 gene. The binding of activator protein-1 transcription-factor complexes, required for the activation of FiRE, was blocked by inhibition of PKA activity before FGF-2 treatment. In accordance with the growth factor specificity of FiRE, the activity of PKA was stimulated by FGF-2 but not by platelet-derived growth factor or epidermal growth factor. Furthermore, a portion of the PKA catalytic subunit pool was translocated to the nucleus by FGF-2. Noticeably, the total cellular cAMP concentration was not affected by FGF-2 stimulus. We propose that the FGF-2-selective transcriptional activation through FiRE is caused by the ability of FGF-2 to control PKA activity.

Growth factor-mediated Fyn signaling regulates α-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor expression in rodent neocortical neurons

Src-family protein tyrosine kinases (PTKs) transduce signals to regulate neuronal development and synaptic plasticity. However, the nature of their activators and molecular mechanisms underlying these neural processes are unknown. Here, we show that brain-derived neurotrophic factor (BDNF) and platelet-derived growth factor enhance expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor 1 and 2/3 proteins in rodent neocortical neurons via the Src-family PTK(s). The increase in AMPA receptor levels was blocked in cultured neocortical neurons by addition of a Src-family-selective PTK inhibitor. Accordingly, neocortical cultures from Fyn-knockout mice failed to respond to BDNF whereas those from wild-type mice responded. Moreover, the neocortex of young Fyn mutants exhibited a significant in vivo reduction in these AMPA receptor proteins but not in their mRNA levels. In vitro kinase assay revealed that BDNF can indeed activate the Fyn kinase: It enhanced tyrosine phosphorylation of Fyn as well as that of enolase supplemented exogenously. All of these results suggest that the Src-family kinase Fyn, activated by the growth factors, plays a crucial role in modulating AMPA receptor expression during brain development.

Signaling through fibroblast growth factor receptor 2b plays a key role in the development of the exocrine pancreas

The development of the pancreas depends on epithelial-mesenchymal interactions. Fibroblast growth factors (FGFs) and their receptors (FGFRs 1–4) have been identified as mediators of epithelial-mesenchymal interactions in different organs. We show here that FGFR-2 IIIb and its ligands FGF-1, FGF-7, and FGF-10 are expressed throughout pancreatic development. We also show that in mesenchyme-free cultures of embryonic pancreatic epithelium FGF-1, FGF-7, and FGF-10 stimulate the growth, morphogenesis, and cytodifferentiation of the exocrine cells of the pancreas. The role of FGFs signaling through FGFR-2 IIIb was further investigated by inhibiting FGFR-2 IIIb signaling in organocultures of pancreatic explants (epithelium + mesenchyme) by using either antisense FGFR-2 IIIb oligonucleotides or a soluble recombinant FGFR-2 IIIb protein. Abrogation of FGFR-2 IIIb signaling resulted in a considerable reduction in the size of the explants and in a 2-fold reduction of the development of the exocrine cells. These results demonstrate that FGFs signaling through FGFR-2 IIIb play an important role in the development of the exocrine pancreas.

Functional interaction between c-Jun and promoter factor Sp1 in epidermal growth factor-induced gene expression of human 12(S)-lipoxygenase

The functional role of the interaction between c-Jun and simian virus 40 promoter factor 1 (Sp1) in epidermal growth factor (EGF)-induced expression of 12(S)-lipoxygenase gene in human epidermoid carcinoma A431 cells was studied. Coimmunoprecipitation experiments indicated that EGF stimulated interaction between c-Jun and Sp1 in a time-dependent manner. Overexpression of Ha-ras and c-Jun also enhanced the amount of c-Jun binding to Sp1. In addition, the c-Jun dominant negative mutant TAM-67 not only inhibited the coimmunoprecipitated c-Jun binding to Sp1 in a dose-dependent manner in cells overexpressing c-Jun but also reduced promoter activity of the 12(S)-lipoxygenase gene induced by c-Jun overexpression. Treatment of cells with EGF increased the interaction between the Sp1 oligonucleotide and nuclear c-Jun/Sp1 in a time-dependent manner. Furthermore, EGF activated the chimeric promoter consisting of 10 tandem GAL4-binding sites, which replaced the three Sp1-binding sites in the 12(S)lipoxygenase promoter only when coexpressed with GAL4-c-Jun () fusion proteins. These results indicate that the direct interaction between c-Jun and Sp1 induced by EGF cooperatively activated expression of the 12(S)-lipoxygenase gene, and that Sp1 may serve at least in part as a carrier bringing c-Jun to the promoter, thus transactivating the transcriptional activity of 12(S)-lipoxygenase gene.

Regulation of vascular endothelial growth factor (VEGF) gene transcription by estrogen receptors α and β

Vascular endothelial growth factor (VEGF) mediates angiogenic activity in a variety of estrogen target tissues. To determine whether estrogen has a direct transcriptional effect on VEGF gene expression, we developed a model system by transiently transfecting human VEGF promoter-luciferase reporter constructs into primary human endometrial cells and into Ishikawa cells, derived from a well-differentiated human endometrial adenocarcinoma. In primary endometrial epithelial cells, treatment with 17β-estradiol (E2) resulted in a 3.8-fold increase in luciferase activity, whereas a 3.2-fold induction was demonstrated for stromal cells. Our Ishikawa cells had less than 100 functional estrogen receptors (ER)/cell and were therefore cotransfected with expression vectors encoding either the α- or the β-form of the human ER. In cells cotransfected with ERα, E2 induced 3.2-fold induction in VEGF-promoter luciferase activity. A 2.3-fold increase was observed in cells cotransfected with ERβ. Through specific deletions, the E2 response was restricted to a single 385-bp PvuII-SstI fragment in the 5′ flanking DNA. Cotransfection of this upstream region with a DNA binding domain ER mutant, or site-directed mutagenesis of a variant ERE within this fragment, resulted in the loss of the E2 response. Electromobility shift assays demonstrated that this same ERE sequence specifically binds estradiol-ER complexes. These studies demonstrate that E2-regulated VEGF gene transcription requires a variant ERE located 1.5 kb upstream from the transcriptional start site. Site-directed mutagenesis of this ERE abrogated E2-induced VEGF gene expression.

CYR61, a product of a growth factor-inducible immediate early gene, promotes angiogenesis and tumor growth

CYR61 is a secreted, cysteine-rich, heparin-binding protein encoded by a growth factor-inducible immediate–early gene. Acting as an extracellular, matrix-associated signaling molecule, CYR61 promotes the adhesion of endothelial cells through interaction with the integrin αVβ3 and augments growth factor-induced DNA synthesis in the same cell type. In this study, we show that purified CYR61 stimulates directed migration of human microvascular endothelial cells in culture through an αVβ3-dependent pathway and induces neovascularization in rat corneas. Both the chemotactic and angiogenic activities of CYR61 can be blocked by specific anti-CYR61 antibodies. Whereas most human tumor-derived cell lines tested express CYR61, the gastric adenocarcinoma cell line RF-1 does not. Expression of the CYR61 cDNA under the regulation of a constitutive promoter in RF-1 cells significantly enhances the tumorigenicity of these cells as measured by growth in immunodeficient mice, resulting in tumors that are larger and more vascularized than those produced by control RF-1 cells. Taken together, these results identify CYR61 as an angiogenic inducer that can promote tumor growth and vascularization; the results also suggest potential roles for CYR61 in physiologic and pathologic neovascularization.

Vascular endothelial growth factor: Direct neuroprotective effect in in vitro ischemia

Vascular endothelial growth factor (VEGF) is a hypoxia-inducible angiogenic peptide with recently identified neurotrophic effects. Because some neurotrophic factors can protect neurons from hypoxic or ischemic injury, we investigated the possibility that VEGF has similar neuroprotective properties. In HN33, an immortalized hippocampal neuronal cell line, VEGF reduced cell death associated with an in vitro model of cerebral ischemia: at a maximally effective concentration of 50 ng/ml, VEGF approximately doubled the number of cells surviving after 24 h of hypoxia and glucose deprivation. To investigate the mechanism of neuroprotection by VEGF, the expression of known target receptors for VEGF was measured by Western blotting, which showed that HN33 cells expressed VEGFR-2 receptors and neuropilin-1, but not VEGFR-1 receptors. The neuropilin-1 ligand placenta growth factor-2 failed to reproduce the protective effect of VEGF, pointing to VEGFR-2 as the site of VEGF's neuroprotective action. Two phosphatidylinositol 3′-kinase inhibitors, wortmannin and LY294002, reversed the neuroprotective effect of VEGF, implicating the phosphatidylinositol 3′-kinase/Akt signal transduction system in VEGF-mediated neuroprotection. VEGF also protected primary cultures of rat cerebral cortical neurons from hypoxia and glucose deprivation. We conclude that in addition to its known role as an angiogenic factor, VEGF may exert a direct neuroprotective effect in hypoxic-ischemic injury.

β-Adrenergic receptor-induced activation of nerve growth factor gene transcription in rat cerebral cortex involves CCAAT/enhancer-binding protein δ

Stimulation of β-adrenergic receptors (BAR) by clenbuterol (CLE) increases nerve growth factor (NGF) biosynthesis in the rat cerebral cortex but not in other regions of the brain. We have explored the transcription mechanisms that may account for the cortex-specific activation of the NGF gene. Although the NGF promoter contains an AP-1 element, AP-1-binding activity in the cerebral cortex was not induced by CLE, suggesting that other transcription factors govern the brain area-specific induction of NGF. Because BAR activation increases cAMP levels, we examined the role of CCAAT/enhancer-binding proteins (C/EBP), some of which are known to be cAMP-inducible. In C6–2B glioma cells, whose NGF expression is induced by BAR agonists, (i) CLE increased C/EBPδ-binding activity, (ii) NGF mRNA levels were increased by overexpressing C/EBPδ, and (iii) C/EBPδ increased the activity of an NGF promoter–reporter construct. Moreover, DNase footprinting and deletion analyses identified a C/EBPδ site in the proximal region of the NGF promoter. C/EBPδ appears to be responsible for the BAR-mediated activation of the NGF gene in vivo, since CLE elicited a time-dependent increase in C/EBPδ-binding activity in the cerebral cortex only. Our data suggest that, while AP-1 may regulate basal levels of NGF expression, C/EBPδ is a critical component determining the area-specific expression of NGF in response to BAR stimulation.

Systemic hypoxia changes the organ-specific distribution of vascular endothelial growth factor and its receptors

Vascular endothelial growth factor (VEGF) plays a key role in physiological blood vessel formation and pathological angiogenesis such as tumor growth and ischemic diseases. Hypoxia is a potent inducer of VEGF in vitro. Here we demonstrate that VEGF is induced in vivo by exposing mice to systemic hypoxia. VEGF induction was highest in brain, but also occurred in kidney, testis, lung, heart, and liver. In situ hybridization analysis revealed that a distinct subset of cells within a given organ, such as glial cells and neurons in brain, tubular cells in kidney, and Sertoli cells in testis, responded to the hypoxic stimulus with an increase in VEGF expression. Surprisingly, however, other cells at sites of constitutive VEGF expression in normal adult tissues, such as epithelial cells in the choroid plexus and kidney glomeruli, decreased VEGF expression in response to the hypoxic stimulus. Furthermore, in addition to VEGF itself, expression of VEGF receptor-1 (VEGFR-1), but not VEGFR-2, was induced by hypoxia in endothelial cells of lung, heart, brain, kidney, and liver. VEGF itself was never found to be up-regulated in endothelial cells under hypoxic conditions, consistent with its paracrine action during normoxia. Our results show that the response to hypoxia in vivo is differentially regulated at the level of specific cell types or layers in certain organs. In these tissues, up- or down-regulation of VEGF and VEGFR-1 during hypoxia may influence their oxygenation after angiogenesis or modulate vascular permeability.

Conservation of fibroblast growth factor function in lens regeneration

In urodele amphibians, lens induction during development and regeneration occurs through different pathways. During development, the lens is induced from the mutual interaction of the ectoderm and the optic vesicle, whereas after lentectomy the lens is regenerated through the transdifferentiation of the iris-pigmented epithelial cells. Given the known role of fibroblast growth factors (FGFs) during lens development, we examined whether or not the expression and the effects of exogenous FGF during urodele lens regeneration were conserved. In this paper, we describe expression of FGF-1 and its receptors, FGFR-2 (KGFR and bek variants) and FGFR-3, in newts during lens regeneration. Expression of these genes was readily observed in the dedifferentiating pigmented epithelial cells, and the levels of expression were high in the lens epithelium and the differentiating fibers and lower in the retina. These patterns of expression implied involvement of FGFs in lens regeneration. To further elucidate this function, we examined the effects of exogenous FGF-1 and FGF-4 during lens regeneration. FGF-1 or FGF-4 treatment in lentectomized eyes resulted in the induction of abnormalities reminiscent to the ones induced during lens development in transgenic mice. Effects included transformation of epithelial cells to fiber cells, double lens regeneration, and lenses with abnormal polarity. These results establish that FGF molecules are key factors in fiber differentiation, polarity, and morphogenesis of the lens during regeneration even though the regenerating lens is induced by a different mechanism than in lens development. In this sense, FGF function in lens regeneration and development should be regarded as conserved. Such conservation should help elucidate the mechanisms of lens regeneration in urodeles and its absence in higher vertebrates.

Role of transforming growth factor-α in von Hippel– Lindau (VHL)−/− clear cell renal carcinoma cell proliferation: A possible mechanism coupling VHL tumor suppressor inactivation and tumorigenesis

Mutations of the VHL tumor suppressor gene occur in patients with VHL disease and in the majority of sporadic clear cell renal carcinomas (VHL−/− RCC). Loss of VHL protein function is associated with constitutive expression of mRNAs encoding hypoxia-inducible proteins, such as vascular endothelial growth factor. Overproduction of angiogenic factors might explain why VHL−/− RCC tumors are so highly vascularized, but whether this overproduction is sufficient for oncogenesis still remains unknown. In this report, we examined the activity of transforming growth factor-α (TGF-α), another VHL-regulated growth factor. We show that TGF-α mRNA and protein are hypoxia-inducible in VHL−/− RCC cells expressing reintroduced VHL. In addition to its overexpression by VHL−/− RCC cells, TGF-α can also act as a specific growth-stimulatory factor for VHL−/− RCC cells expressing reintroduced wild-type VHL, as well as primary renal proximal tubule epithelial cells, the likely site of origin of RCC. This role is in contrast to those of other growth factors overexpressed by VHL−/− RCC cells, such as vascular endothelial growth factor and TGF-β1, which do not stimulate RCC cell proliferation. A TGF-α-specific antisense oligodeoxynucleotide blocked TGF-α production in VHL−/− RCC cells, which led to the dependence of those cells on exogenous growth factors to sustain growth in culture. Growth of VHL−/− RCC cells was also significantly reduced by a drug that specifically inhibits the epidermal growth factor receptor, the receptor through which TGF-α stimulates proliferation. These results suggest that the generation of a TGF-α autocrine loop as a consequence of VHL inactivation in renal proximal tubule epithelial cells may provide the uncontrolled growth stimulus necessary for the initiation of tumorigenesis.

Sp1 phosphorylation regulates inducible expression of platelet-derived growth factor B-chain gene via atypical protein kinase C-ζ

Platelet-derived growth factor (PDGF) is a broadly expressed mitogenic and chemotactic factor with diverse roles in a number of physiologic and pathologic settings. The zinc finger transcription factors Sp1, Sp3 and Egr-1 bind to overlapping elements in the proximal PDGF B-chain promoter and activate transcription of this gene. The anthracycline nogalamycin has previously been reported to inhibit the capacity of Egr-1 to bind DNA in vitro. Here we used electrophoretic mobility shift assays to show that nogalamycin added to cells in culture did not alter the interaction of Egr-1 with the PDGF-B promoter. Instead, it enhanced the capacity of Sp1 to bind DNA. Nogalamycin increased PDGF-B mRNA expression at the level of transcription, which was abrogated by mutation of the Sp1 binding site in the PDGF-B promoter or overexpression of mutant Sp1. Rather than increasing total levels of Sp1, nogalamycin altered the phosphorylation state of the transcription factor. Overexpression of dominant-negative PKC-ζ blocked nogalamycin-inducible Sp1 phosphorylation and PDGF-B promoter-dependent expression. Nogalamycin stimulated the phosphorylation of PKC-ζ (on residue Thr410). These findings demonstrate for the first time that PKC-ζ and Sp1 phosphorylation mediate the inducible expression of this growth factor.

Lack of a role for transforming growth factor-β in cytotoxic T lymphocyte antigen-4-mediated inhibition of T cell activation

Similarities in the phenotypes of mice deficient for cytotoxic T lymphocyte antigen-4 (CTLA-4) or transforming growth factor-β1 (TGF-β1) and other observations have led to speculation that CTLA-4 mediates its inhibitory effect on T cell activation via costimulation of TGF-β production. Here, we examine the role of TGF-β in CTLA-4-mediated inhibition of T cell activation and of CTLA-4 in the regulation of TGF-β production. Activation of AND TCR transgenic mouse T cells with costimulatory receptor-specific antigen presenting cells results in efficient costimulation of proliferation by CD28 ligation and inhibition by CTLA-4 ligation. Neutralizing antibody to TGF-β does not reverse CTLA-4-mediated inhibition. Also, CTLA-4 ligation equally inhibits proliferation of wild-type, TGF-β1−/−, and Smad3−/− T cells. Further, CTLA-4 engagement does not result in the increased production of either latent or active TGF-β by CD4+ T cells. These results indicate that CTLA-4 ligation does not regulate TGF-β production and that CTLA-4-mediated inhibition can occur independently of TGF-β. Collectively, these data demonstrate that CTLA-4 and TGF-β represent distinct mechanisms for regulation of T cell responses.

Predominant role of endothelial nitric oxide synthase in vascular endothelial growth factor-induced angiogenesis and vascular permeability

Nitric oxide (NO) plays a critical role in vascular endothelial growth factor (VEGF)-induced angiogenesis and vascular hyperpermeability. However, the relative contribution of different NO synthase (NOS) isoforms to these processes is not known. Here, we evaluated the relative contributions of endothelial and inducible NOS (eNOS and iNOS, respectively) to angiogenesis and permeability of VEGF-induced angiogenic vessels. The contribution of eNOS was assessed by using an eNOS-deficient mouse, and iNOS contribution was assessed by using a selective inhibitor [l-N6-(1-iminoethyl) lysine, l-NIL] and an iNOS-deficient mouse. Angiogenesis was induced by VEGF in type I collagen gels placed in the mouse cranial window. Angiogenesis, vessel diameter, blood flow rate, and vascular permeability were proportional to NO levels measured with microelectrodes: Wild-type (WT) ≥ WT with l-NIL or iNOS−/− > eNOS−/− ≥ eNOS−/− with l-NIL. The role of NOS in VEGF-induced acute vascular permeability increase in quiescent vessels also was determined by using eNOS- and iNOS-deficient mice. VEGF superfusion significantly increased permeability in both WT and iNOS−/− mice but not in eNOS−/− mice. These findings suggest that eNOS plays a predominant role in VEGF-induced angiogenesis and vascular permeability. Thus, selective modulation of eNOS activity is a promising strategy for altering angiogenesis and vascular permeability in vivo.

From transforming growth factor-β signaling to androgen action: Identification of Smad3 as an androgen receptor coregulator in prostate cancer cells

Although transforming growth factor-β (TGF-β) has been identified to mainly inhibit cell growth, the correlation of elevated TGF-β with increasing serum prostate-specific antigen (PSA) levels in metastatic stages of prostate cancer has also been well documented. The molecular mechanism for these two contrasting effects of TGF-β, however, remains unclear. Here we report that Smad3, a downstream mediator of the TGF-β signaling pathway, functions as a coregulator to enhance androgen receptor (AR)-mediated transactivation. Compared with the wild-type AR, Smad3 acts as a strong coregulator in the presence of 1 nM 5α-dihydrotestosterone, 10 nM 17β-estradiol, or 1 μM hydroxyflutamide for the LNCaP mutant AR (mtAR T877A), found in many prostate tumor patients. We further showed that endogenous PSA expression in LNCaP cells can be induced by 5α-dihydrotestosterone, and the addition of the Smad3 further induces PSA expression. Together, our findings establish Smad3 as an important coregulator for the androgen-signaling pathway and provide a possible explanation for the positive role of TGF-β in androgen-promoted prostate cancer growth.