Critical role for NF-kappaB-induced JunB in VEGF regulation and tumor angiogenesis.
ABSTRACT: Regulation of vascular endothelial growth factor (VEGF) expression is a complex process involving a plethora of transcriptional regulators. The AP-1 transcription factor is considered as facilitator of hypoxia-induced VEGF expression through interaction with hypoxia-inducible factor (HIF) which plays a major role in mediating the cellular hypoxia response. As yet, both the decisive AP-1 subunit leading to VEGF induction and the molecular mechanism by which this subunit is activated have not been deciphered. Here, we demonstrate that the AP-1 subunit junB is a target gene of hypoxia-induced signaling via NF-kappaB. Loss of JunB in various cell types results in severely impaired hypoxia-induced VEGF expression, although HIF is present and becomes stabilized. Thus, we identify JunB as a critical independent regulator of VEGF transcription and provide a mechanistic explanation for the inherent vascular phenotypes seen in JunB-deficient embryos, ex vivo allantois explants and in vitro differentiated embryoid bodies. In support of these findings, tumor angiogenesis was impaired in junB(-/-) teratocarcinomas because of severely impaired paracrine-acting VEGF and the subsequent inability to efficiently recruit host-derived vessels.
Project description:Bone marrow (BM) angiogenesis significantly influences disease progression in multiple myeloma (MM) patients and correlates with adverse prognosis. The present study shows a statistically significant correlation of the AP-1 family member JunB with VEGF, VEGFB, and IGF1 expression levels in MM. In contrast to the angiogenic master regulator Hif-1α, JunB protein levels were independent of hypoxia. Results in tumor-cell models that allow the induction of JunB knockdown or JunB activation, respectively, corroborated the functional role of JunB in the production and secretion of these angiogenic factors (AFs). Consequently, conditioned media derived from MM cells after JunB knockdown or JunB activation either inhibited or stimulated in vitro angiogenesis. The impact of JunB on MM BM angiogenesis was finally confirmed in a dynamic 3D model of the BM microenvironment, a xenograft mouse model as well as in patient-derived BM sections. In summary, in continuation of our previous study (Fan et al., 2017), the present report reveals for the first time that JunB is not only a mediator of MM cell survival, proliferation, and drug resistance, but also a promoter of AF transcription and consequently of MM BM angiogenesis. Our results thereby underscore worldwide efforts to target AP-1 transcription factors such as JunB as a promising strategy in MM therapy.
Project description:Activating transcription factor 4 (ATF4) is a critical transcription factor for bone remodeling; however, its role in bone angiogenesis has not been established. Here we show that ablation of the Atf4 gene expression in mice severely impaired skeletal vasculature and reduced microvascular density of the bone associated with dramatically decreased expression of hypoxia-inducible factor 1? (HIF-1?) and vascular endothelial growth factor (VEGF) in osteoblasts located on bone surfaces. Results from in vivo studies revealed that hypoxia/reoxygenation induction of HIF-1? and VEGF expression leading to bone angiogenesis, a key adaptive response to hypoxic conditions, was severely compromised in mice lacking the Atf4 gene. Loss of ATF4 completely prevented endothelial sprouting from embryonic metatarsals, which was restored by addition of recombinant human VEGF protein. In vitro studies revealed that ATF4 promotion of HIF-1? and VEGF expression in osteoblasts was highly dependent upon the presence of hypoxia. ATF4 interacted with HIF-1? in hypoxic osteoblasts, and loss of ATF4 increased HIF-1? ubiquitination and reduced its protein stability without affecting HIF-1? mRNA stability and protein translation. Loss of ATF4 increased the binding of HIF-1? to prolyl hydroxylases, the enzymes that hydroxylate HIF-1a protein and promote its proteasomal degradation via the pVHL pathway. Furthermore, parathyroid hormone-related protein (PTHrP) and receptor activator of NF-?B ligand (RANKL), both well-known activators of osteoclasts, increased release of VEGF from the bone matrix and promoted angiogenesis through the protein kinase C- and ATF4-dependent activation of osteoclast differentiation and bone resorption. Thus, ATF4 is a new key regulator of the HIF/VEGF axis in osteoblasts in response to hypoxia and of VEGF release from bone matrix, two critical steps for bone angiogenesis.
Project description:Interstitial adenosine stimulates neovascularization in part through A2B adenosine receptor-dependent upregulation of vascular endothelial growth factor (VEGF). In the current study, we tested the hypothesis that A2B receptors upregulate JunB, which can contribute to stimulation of VEGF production. Using the human microvascular endothelial cell line, human mast cell line, mouse cardiac Sca1-positive stromal cells, and mouse Lewis lung carcinoma (LLC) cells, we found that adenosine receptor-dependent upregulation of VEGF production was associated with an increase in VEGF transcription, activator protein-1 (AP-1) activity, and JunB accumulation in all cells investigated. Furthermore, the expression of JunB, but not the expression of other genes encoding transcription factors from the Jun family, was specifically upregulated. In LLC cells expressing A2A and A2B receptor transcripts, only the nonselective adenosine agonist NECA (5'-N-ethylcarboxamidoadenosine), but not the selective A2A receptor agonist CGS21680 [2-p-(2-carboxyethyl) phenylethylamino-5'-N-ethylcarboxamidoadenosine], significantly increased JunB reporter activity and JunB nuclear accumulation, which were inhibited by the A2B receptor antagonist PSB603 [(8-[4-[4-((4-chlorophenzyl)piperazide-1-sulfonyl)phenyl]]-1-propylxanthine]. Using activators and inhibitors of intracellular signaling, we demonstrated that A2B receptor-dependent accumulation of JunB protein and VEGF secretion share common intracellular pathways. NECA enhanced JunB binding to the murine VEGF promoter, whereas mutation of the high-affinity AP-1 site (-1093 to -1086) resulted in a loss of NECA-dependent VEGF reporter activity. Finally, NECA-dependent VEGF secretion and reporter activity were inhibited by the expression of a dominant negative JunB or by JunB knockdown. Thus, our data suggest an important role of the A2B receptor-dependent upregulation of JunB in VEGF production and possibly other AP-1-regulated events.
Project description:Diabetes is associated with poor outcomes following acute vascular occlusive events. This results in part from a failure to form adequate compensatory microvasculature in response to ischemia. Since vascular endothelial growth factor (VEGF) is an essential mediator of neovascularization, we examined whether hypoxic up-regulation of VEGF was impaired in diabetes. Both fibroblasts isolated from type 2 diabetic patients, and normal fibroblasts exposed chronically to high glucose, were defective in their capacity to up-regulate VEGF in response to hypoxia. In vivo, diabetic animals demonstrated an impaired ability to increase VEGF production in response to soft tissue ischemia. This resulted from a high glucose-induced decrease in transactivation by the transcription factor hypoxia-inducible factor-1alpha (HIF-1alpha), which mediates hypoxia-stimulated VEGF expression. Decreased HIF-1alpha functional activity was specifically caused by impaired HIF-1alpha binding to the coactivator p300. We identify covalent modification of p300 by the dicarbonyl metabolite methylglyoxal as being responsible for this decreased association. Administration of deferoxamine abrogated methylglyoxal conjugation, normalizing both HIF-1alpha/p300 interaction and transactivation by HIF-1alpha. In diabetic mice, deferoxamine promoted neovascularization and enhanced wound healing. These findings define molecular defects that underlie impaired VEGF production in diabetic tissues and offer a promising direction for therapeutic intervention.
Project description:Chronic alcohol consumption leads to liver inflammation and cirrhosis. Alcoholic liver disease patients have increased levels of hepatic RANTES/CCL5. However, less is known about the molecular mechanisms for ethanol-induced RANTES up-regulation. In this study, we observed that liver sinusoidal endothelial cells derived from ethanol-fed rats (E-rLSECs) showed severalfold increases in RANTES and hypoxia-inducible factor 1alpha (HIF-1alpha) mRNAs compared with control rLSECs (C-rLSECs). Similar effects were seen in acute ethanol treatment of isolated rLSECs and human dermal microvascular endothelial cells. Ethanol-induced RANTES mRNA expression required ethanol metabolism, p38 MAPK, HIF-1alpha, and JNK-2, but not JNK-1. EMSA experiments showed increased HIF-1alpha binding to wild-type hypoxia response elements (HREs; -31 to -9 bp) within the RANTES promoter in response to ethanol. RANTES promoter analysis showed that cis elements proximal to the transcription start site, HRE-1 (nt -22 to -19), HRE-2 (nt -32 to -29), and AP-1 (nt -250 to -244) were required for ethanol-mediated RANTES expression. These results were corroborated by chromatin immunoprecipitation assays showing augmented HIF-1alpha binding to HRE-1. Additionally, promoter analysis revealed c-Jun, c-Jun/c-Fos, and JunD, but not JunB, bound to the AP-1 site of the RANTES promoter. Ethanol-mediated activation of NF-kappaB led to HIF-1alpha activation and concomitant RANTES expression. Plasma of ethanol-fed c-Jun(flox/flox)-Mx-1-Cre mice showed attenuated levels of RANTES compared with ethanol-fed control mice, supporting the role of c-Jun in ethanol-induced RANTES expression. Our studies showed that ethanol-mediated RANTES/CCL5 expression occurs via HIF-1alpha activation independently of hypoxia. The identification of HIF-1alpha and AP-1 in ethanol-induced RANTES expression provides new strategies to ameliorate ethanol-induced inflammatory responses.
Project description:FGF2 (fibroblast growth factor 2), but not vascular endothelial growth factor (VEGF), stimulates sustained activation of ERK2/1 for endothelial NOS3 (nitric-oxide synthase 3) protein expression in ovine fetoplacental artery endothelial cells (oFPAEC). We deciphered herein the downstream signaling of ERK2/1 responsible for NOS3 expression by FGF2 in oFPAEC. FGF2, but not VEGF, increased NOS3 mRNA levels without altering its degradation. FGF2, but not VEGF, trans-activated sheep NOS3 promoter, and this was dependent on ERK2/1 activation. FGF2 did not trans-activate NOS3 promoters with deletions upstream of the consensus AP-1 site (TGAGTC A, -678 to -685). Trans-activation of wild-type NOS3 promoter by FGF2 was significantly inhibited when either the AP-1 or the cAMP-response element (CRE)-like sequence (TGCGTCA, -752 to -758) was mutated and was completely blocked when both were mutated. EMSA analyses showed that FGF2, but not VEGF, stimulated AP-1 and CRE DNA-protein complexes primarily composed of JunB and Fra1. Chromatin immunoprecipitation assays confirmed JunB/Fra1 binding to NOS3 promoter AP-1 and CRE elements in intact cells. FGF2, but not VEGF, stimulated JunB and Fra1 expressions; all preceded NOS3 up-regulation and were inhibited by PD98059. Down-regulation of JunB or Fra-1, but not c-Jun, blocked FGF2 stimulation of NOS3 expression and NO production. AP-1 inhibition suppressed FGF2 stimulation of NOS3 expression in human umbilical vein EC and uterine artery endothelial cells. Thus, FGF2 induction of NOS3 expression is mainly mediated by AP-1-dependent transcription involving JunB and Fra1 up-regulation via sustained ERK2/1 activation in endothelial cells.
Project description:Hypoxia-dependent accumulation of vascular endothelial growth factor (VEGF) plays a major role in retinal diseases characterized by neovessel formation. In this study, we investigated whether the glial water channel Aquaporin-4 (AQP4) is involved in the hypoxia-dependent VEGF upregulation in the retina of a mouse model of oxygen-induced retinopathy (OIR). The expression levels of VEGF, the hypoxia-inducible factor-1? (HIF-1?) and the inducible form of nitric oxide synthase (iNOS), the production of nitric oxide (NO), the methylation status of the HIF-1 binding site (HBS) in the VEGF gene promoter, the binding of HIF-1? to the HBS, the retinal vascularization and function have been determined in the retina of wild-type (WT) and AQP4 knock out (KO) mice under hypoxic (OIR) or normoxic conditions. In response to 5 days of hypoxia, WT mice were characterized by (i) AQP4 upregulation, (ii) increased levels of VEGF, HIF-1?, iNOS and NO, (iii) pathological angiogenesis as determined by engorged retinal tufts and (iv) dysfunctional electroretinogram (ERG). AQP4 deletion prevents VEGF, iNOS and NO upregulation in response to hypoxia thus leading to reduced retinal damage although in the presence of high levels of HIF-1?. In AQP4 KO mice, HBS demethylation in response to the beginning of hypoxia is lower than in WT mice reducing the binding of HIF-1? to the VEGF gene promoter. We conclude that in the absence of AQP4, an impaired HBS demethylation prevents HIF-1 binding to the VEGF gene promoter and the relative VEGF transactivation, reducing the VEGF-induced retinal damage in response to hypoxia.
Project description:Hypoxia inducible factor (HIF)-1 plays an important role in cellular adaptation to hypoxia by activating oxygen-regulated genes such as vascular endothelial growth factor (VEGF) and erythropoietin. Sputum VEGF levels are reported to be decreased in COPD, despite hypoxia. Here we show that patients with COPD fail to induce HIF-1? and VEGF under hypoxic condition because of a reduction in histone deacetylase (HDAC) 7.Peripheral blood mononuclear cells (PBMCs) were obtained from patients with moderate to severe COPD (n = 21), smokers without COPD (n = 12), and nonsmokers (n = 15). PBMCs were exposed to hypoxia (1% oxygen, 5% CO(2), and 94% N(2)) for 24 h, and HIF-1? and HDAC7 protein expression in nuclear extracts were determined by sodium dodecyl sulfate poly acrylamide gel electrophoresis (SDS-PAGE)/Western blotting.HIF-1? was significantly induced by hypoxia in each group when compared with the normoxic condition (12-fold induction in nonsmokers, 24-fold induction in smokers without COPD, fourfold induction in COPD), but induction of HIF-1? under hypoxia was significantly lower in patients with COPD than in nonsmokers and smokers without COPD (P < .05 and P < .01, respectively). VEGF messenger RNA detected by quantitative real-time polymerase chain reaction was correlated with HIF-1? protein in nuclei (r = 0.79, P < .05), and HDAC7 protein expression was correlated with HIF-1? protein in nuclei (r = 0.46, P < .05). HDAC7 knockdown inhibited hypoxia-induced HIF-1? activity in U937 cells, and HIF-1? nuclear translocation and HIF-1? binding to the VEGF promoter in A549 cells.HDAC7 reduction in COPD causes a defect of HIF-1? induction response to hypoxia with impaired VEGF gene expression. This poor cellular adaptation might play a role in the pathogenesis of COPD.
Project description:The molecular mechanism triggering the organization of endothelial cells (ECs) in multicellular tubules is mechanistically still poorly understood. We demonstrate that cell-autonomous endothelial functions of the AP-1 subunit JunB are required for proper endothelial morphogenesis both in vivo in mouse embryos with endothelial-specific ablation of JunB and in in vitro angiogenesis models. By cDNA microarray analysis, we identified core-binding factor beta (CBFbeta), which together with the Runx proteins forms the heterodimeric core-binding transcription complex CBF, as a novel JunB target gene. In line with our findings, expression of the CBF target MMP-13 was impaired in JunB-deficient ECs. Reintroduction of CBFbeta into JunB-deficient ECs rescued the tube formation defect and MMP-13 expression, indicating an important role for CBFbeta in EC morphogenesis.
Project description:Mesenchymal stromal cell (MSC) markers are expressed on brain tumor-initiating cells involved in the development of hypoxic glioblastoma. Given that MSCs can survive hypoxia and that the glucose-6-phosphate transporter (G6PT) provides metabolic control that contributes to MSC mobilization and survival, we investigated the effects of low oxygen (1.2% O(2)) exposure on G6PT gene expression. We found that MSCs significantly expressed G6PT and the glucose-6-phosphatase catalytic subunit beta, whereas expression of the glucose-6-phosphatase catalytic subunit alpha and the islet-specific glucose-6-phosphatase catalytic subunit-related protein was low to undetectable. Analysis of the G6PT promoter sequence revealed potential binding sites for hypoxia inducible factor (HIF)-1alpha and for the aryl hydrocarbon receptor (AhR) and its dimerization partner, the AhR nuclear translocator (ARNT), AhR:ARNT. In agreement with this, hypoxia and the hypoxia mimetic cobalt chloride induced the expression of G6PT, vascular endothelial growth factor (VEGF), and HIF-1alpha. Gene silencing of HIF-1alpha prevented G6PT and VEGF induction in hypoxic MSCs whereas generation of cells stably expressing HIF-1alpha resulted in increased endogenous G6PT gene expression. A semisynthetic analog of the polyketide mumbaistatin, a potent G6PT inhibitor, specifically reduced MSC-HIF-1alpha cell survival. Collectively, our data suggest that G6PT may account for the metabolic flexibility that enables MSCs to survive under conditions characterized by hypoxia and could be specifically targeted within developing tumors.