PDGF-B Promoter Research Articles

Increasing Endoglin Deletion in Endothelial Cells Exacerbates the Severity of Brain Arteriovenous Malformation in Mouse.

Endoglin (ENG) mutation causes type 1 hereditary hemorrhagic telangiectasia (HHT1). HHT1 patients have arteriovenous malformations (AVMs) in multiple organs, including the brain. In mice, Eng deletion induced by R26RCreER or SM22αCre leads to AVM development in the brain and other organs. We hypothesized that an increase in Eng- negative ECs will enhance AVM severity. To increase EC Eng deletion, we used a codon-improved cre (icre), which is more potent in recombination of the floxed alleles than the wild-type (WT) cre. R26RCreER;Engf/f mice that have a Rosa promoter driving and tamoxifen (TM)-inducible WT cre expression globally, and PdgfbiCreER;Engf/f mice that have a Pdgfb promoter driving and TM-inducible icre expression in ECs were treated with three intra-peritoneal injections of TM (2.5 mg/25 g of body weight) to delete Eng globally or in the ECs. AAV-VEGF was stereotactically injected into the brain to induce brain focal angiogenesis and brain AVM. We found that icre caused more Eng deletion in the brain, indicated by a lower level of Eng proteins (p < 0.001) and fewer Eng-positive ECs (p = 0.01) than mice with WT cre. Mice with icre-mediated Eng deletion have more abnormal vessels (p = 0.02), CD68+ macrophages (p = 0.002), and hemorrhage (p = 0.04) and less vascular pericyte and smooth muscle coverage than mice with WT cre. In addition, arteriovenous shunts were detected in the intestines of icre mice, a phenotype that has not been detected in WT cre mice before. RNA-seq analysis showed that 8 out of the 10 top upregulated pathways identified by gene ontology (GO) analysis are related to inflammation. Therefore, the increase in Eng deletion in ECs exacerbates AVM severity, which is associated with enhanced inflammation. Strategies that can reduce Eng-negative ECs could be used to develop new therapies to reduce AVM severity for HHT1 patients.

Long non-coding RNA ACTA2-AS1 promotes ductular reaction by interacting with the p300/ELK1 complex

Biliary disease is associated with a proliferative/fibrogenic ductular reaction (DR). p300 is an epigenetic regulator that acetylates lysine 27 on histone 3 (H3K27ac) and is activated during fibrosis. Long non-coding RNAs (lncRNAs) are aberrantly expressed in cholangiopathies, but little is known about how they recruit epigenetic complexes and regulate DR. We investigated epigenetic complexes, including transcription factors (TFs) and lncRNAs, contributing to p300-mediated transcription during fibrosis. We evaluated p300 invivo using tamoxifen-inducible, cholangiocyte-selective, p300 knockout (KO) coupled with bile duct ligation (BDL) and Mdr KO mice treated with SGC-CBP30. Primary cholangiocytes and liver tissue were analyzed for expression of Acta2-as1 lncRNA by qPCR and RNA in situ hybridization. Invitro, we performed RNA-sequencing in human cholangiocytes with a p300 inhibitor. Cholangiocytes were exposed to lipopolysaccharide (LPS) as an injury model. We confirmed formation of a p300/ELK1 complex by immunoprecipitation (IP). RNA IP was used to examine interactions between ACTA2-AS1 and p300. Chromatin IP assays were used to evaluate p300/ELK1 occupancy and p300-mediated H3K27ac. Organoids were generated from ACTA2-AS1-depleted cholangiocytes. BDL-induced DR and fibrosis were reduced in Krt19-CreERT/p300fl/fl mice. Similarly, Mdr KO mice were protected from DR and fibrosis after SGC-CBP30 treatment. Invitro, depletion of ACTA2-AS1 reduced expression of proliferative/fibrogenic markers, reduced LPS-induced cholangiocyte proliferation, and impaired organoid formation. ACTA2-AS1 regulated transcription by facilitating p300/ELK1 binding to the PDGFB promoter after LPS exposure. Correspondingly, LPS-induced H3K27ac was mediated by p300/ELK1 and was reduced in ACTA2-AS1-depleted cholangiocytes. Cholangiocyte-selective p300 KO or p300 inhibition attenuate DR/fibrosis in mice. ACTA2-AS1 influences recruitment of p300/ELK1 to specific promoters to drive H3K27ac and epigenetic activation of proliferative/fibrogenic genes. This suggests that cooperation between epigenetic co-activators and lncRNAs facilitates DR/fibrosis in biliary diseases. We identified a three-part complex containing an RNA molecule, a transcription factor, and an epigenetic enzyme. The complex is active in injured bile duct cells and contributes to activation of genes involved in proliferation and fibrosis.

Anoikis resistant gastric cancer cells promote angiogenesis and peritoneal metastasis through C/EBPβ-mediated PDGFB autocrine and paracrine signaling

Anoikis is a type of programmed cell death induced by loss of anchorage to the extracellular matrix (ECM). Anoikis resistance (AR) is crucial for the survival of metastatic cancer cells in blood, lymphatic circulation and distant organs. Compared to ordinary cancer cells, anoikis resistant cancer cells undergo various cellular and molecular alterations, probably characterizing the cells with unique features not limited to anoikis resistance. However, the molecular mechanisms connecting anoikis resistance to other metastatic properties are still poorly understood. Here, the biological interaction between anoikis resistance and angiogenesis as well as their involvement into peritoneal metastasis of gastric cancer (GC) were investigated in vitro and in vivo. The prognostic value of key components involved in this interaction was evaluated in the GC cohort. Compared to ordinary GC cells, GCAR cells exhibited stronger metastatic and pro-angiogenic traits corresponding to elevated PDGFB secretion. Mechanistically, transcription factor C/EBPβ facilitated PDGFB transcription by directly binding to and interacting with PDGFB promoter elements, subsequently increasing PDGFB secretion. Secreted PDGFB promoted the survival of detached GC cells through a C/EBPβ-dependent self-feedback loop. Moreover, secreted PDGFB promoted angiogenesis in metastases via activation of the MAPK/ERK signaling pathway in vascular endothelial cells. Both C/EBPβ activation level and PDGFB expression were significantly elevated in GC and correlated with metastatic progression and poor prognosis of patients with GC. Overall, interaction between GCAR cells and vascular endothelial cells promotes angiogenesis and peritoneal metastasis of GC based on C/EBPβ-mediated PDGFB autocrine and paracrine signaling. C/EBPβ-PDGFB-PDGFRβ-MAPK axis promises to be potential prognostic biomarkers and therapeutic targets for peritoneal metastasis of GC.

Glucocorticoids Disrupt Skeletal Angiogenesis Through Transrepression of NF-κB-Mediated Preosteoclast Pdgfb Transcription in Young Mice.

In the growing skeleton, angiogenesis is intimately coupled with osteogenesis. Chronic, high doses of glucocorticoids (GCs) are associated with decreased bone vasculature and induce osteoporosis and growth failure. The mechanism of GC-suppression of angiogenesis and relationship to osteoporosis and growth retardation remains largely unknown. Type H vessels, which are regulated by preosteoclast (POC) platelet-derived growth factor-BB (PDGF-BB), are specifically coupled with bone formation and development. We determined the effect of GCs on POC synthesis of PDGF-BB in relation to type H vessel formation, bone mass, and bone growth in the distal femur of 2-week-old young mice receiving prednisolone or vehicle for 2, 4, or 6 weeks. After 2 weeks of prednisolone, the number of POCs were unchanged while POC synthesis of PDGF-BB was reduced. Longer treatment with prednisolone reduced POCs numbers and PDGF-BB. These changes were associated with a reduction in type H vessels, bone formation rate, bone mass, and bone length at each time point. In vitro, excessive concentrations of prednisolone (10-6 M) resulted in decreased PDGF-BB concentration and POC numbers. Conditioned medium from POC cultures treated with control concentration of prednisolone (10-7 M) or recombinant PDGF-BB stimulated endothelial tube formation, whereas conditioned medium from control concentration of prednisolone-treated POC cultures neutralized by PDGF-BB antibody or excessive prednisolone inhibited endothelial tube formation. Administration of excessive prednisolone attenuated the P65 subunit of nuclear factor kappa B (NF-κB) binding to the Pdgfb promoter, resulting in lower Pdgfb transcription. Co-treatment with excessive prednisolone and the glucocorticoid receptor (GR) antagonist (RU486), GR siRNA, or TNFα rescued NF-κB binding to the Pdgfb promoter and endothelial tube formation. These results indicate that PDGF-BB synthesis in POCs is suppressed by GCs through transrepression of GR/NF-κB, thus inhibiting type H vessel formation and associated osteoporosis and growth failure. © 2020 American Society for Bone and Mineral Research.

Commentary: The unliganded glucocorticoid receptor positively regulates the tumor suppressor gene BRCA1 through GABP beta.

Commentary: The unliganded glucocorticoid receptor positively regulates the tumor suppressor gene BRCA1 through GABP beta.

SDF-1α Induces PDGF-B Expression and the Differentiation of Bone Marrow Cells into Pericytes

Platelet-derived growth factor B (PDGF-B) and its receptor, PDGFR-β, play a critical role in pericyte maturation; however, the mechanisms by which PDGF-B is upregulated in the tumor microenvironment remain unclear. We previously showed that upregulating stromal-derived factor, SDF-1α, in VEGF(165)-inhibited Ewing's sarcoma tumors (TC/siVEGF(7-1)) induced PDGF-B mRNA expression, increased infiltration and differentiation of bone marrow cells (BMC) into pericytes and, rescued tumor growth. The purpose of this study was to investigate the mechanism by which SDF-1α increased PDGF-B expression and the role of this pathway in BM-derived pericyte differentiation. We showed that SDF-1α induced expression of PDGF-B mRNA and protein both in vitro and in vivo. In contrast, inhibiting SDF-1α downregulated PDGF-B. We cloned the 2-kb pdgf-b promoter fragment and showed that SDF-1α activates PDGF-B via a transcriptional mechanism. Chromatin immunoprecipitation showed that the ELK-1 transcription factor binds to the pdgf-b promoter in response to SDF-1α. We confirmed the correlation between the SDF-1α/PDGF-B pathway and the differentiation of PDGFR-β+ BMCs into mature pericytes using an in vitro assay. These findings show that SDF-1α regulates PDGF-B expression and that this regulation plays a critical role in the differentiation of PDGFR-β+ BMCs into mature pericytes.

Highlights of This Issue

Pericytes are critical in remodeling and stabilizing developing tumor vessels. PDGF-B induces pericyte migration and differentiation and is upregulated in several tumor types. Hamdan and colleagues show that increased PDGF-B in the tumor microenvironment is mediated by the production of SDF-1α. SDF-1α upregulated PDGF-B mRNA and protein in vitro and in vivo, whereas a specific shRNA downregulated PDGF-B. Luciferase and ChIP assays showed that PDGF-B induction by SDF-1α was transcriptionally mediated, involving binding of ELK-1 to the pdgf-b promoter. This SDF-1α/PDGF-B pathway induced bone marrow cells to differentiate into pericytes and may therefore become a new therapeutic target to inhibit vasculogenesis.Class 3 semaphorins have been shown to have tumor suppressive activities. In the current study, Nguyen and colleagues explored the role and regulation of SEMA3B and SEMA3F in endometrial cancer. Data showed a progressive decrease in SEMA3B and SEMA3F expression in human endometrial cancers. Treatment of endometrial cancer cell lines with progesterone or vitamin D restored the expression of SEMA3B and SEMA3F in cancer cells and inhibited growth and angiogenesis, suggesting that malignant behavior of cells can be reverted to their normal state by restoration of SEMA3 expression.MicroRNAs regulate gene expression by repression or degradation of mRNA of multiple target genes and, therefore, play an important role in angiogenesis, cell migration and invasion, especially in cancer. Changes in cell morphology and mobility are tightly regulated by the heterodimeric adhesion receptors, the integrins, whose bidirectional signaling function is crucial for both physiological and pathological conditions. Here, Augoff and coworkers show that miR-31, a key regulator of the cancer invasion-metastasis cascade, targets several α and β integrin subunits to suppress cell spreading in a ligand-specific manner in cancer cells.Melanoma secretes high levels of proinflammatory cytokines, including IL-1. To date, the expression of IL-1α and IL-1β in human melanoma and their functions and role(s) associated with tumor progression have yet to be elucidated. Here, Qin and colleagues examined IL-1α and IL-1β expression profiles in large-scale melanoma tissue samples and identified several important downstream molecules and kinase responders (SPAK/JNK) for endogenous IL-1 in human melanoma cells. This study suggests that IL-1α and IL-1β increase significantly from nevus to primary melanoma and interrupting IL-1 signaling can affect autophagy and inhibit growth of IL-1-positive melanoma cells.

HIV-1 Tat-Mediated Induction of Platelet-Derived Growth Factor in Astrocytes: Role of Early Growth Response Gene 1

HIV-associated neurologic disorders (HAND) are estimated to affect almost 60% of HIV-infected individuals. HIV encephalitis, the pathologic correlate of the most severe form of HAND, is often characterized by glial activation, cytokine-chemokine dysregulation, and neuronal damage and loss. However, the severity of HIV encephalitis correlates better with glial activation rather than viral load. Using the macaque model, it has been demonstrated that SIV encephalitis correlates with increased expression of the mitogen platelet-derived growth factor (PDGF) B chain in the brain. The goal of this study was to explore the role of PDGF-B chain in HIV-associated activation and proliferation of astrocytes. Specifically, the data demonstrate that exposure of rat and human astrocytes to the HIV-1 protein Tat resulted in the induction of PDGF at both the mRNA and protein levels. Furthermore, PDGF-BB induction was regulated by activation of ERK1/2 and JNK signaling pathways and the downstream transcription factor early growth response 1. Chromatin immunoprecipitation assays demonstrated binding of Egr-1 to the PDGF-B promoter. Exposure of astrocytes to PDGF-BB in turn led to increased proliferation and the release of proinflammatory cytokines MCP-1 and IL-1β. Because astrogliosis is linked to disease severity, understanding its regulation by PDGF-BB could aid in the development of therapeutic intervention strategies for HAND.

Platelet-Derived Growth Factors and Receptors in the Rat Corpus Luteum: Localization and Identification of an Effect on Luteogenesis1

Platelet-derived growth factors (PDGFs) and their receptors (PDGFRs) play a vital role in regulating cell growth and angiogenesis. In this study, the expression of the family of PDGFs and PDGFRs in the ovarian corpus luteum were identified and characterized, and an effect of their activity on development of the corpus luteum revealed. Gonadotropin-stimulated immature rats were utilized as a model of induced ovulation, luteogenesis, and pseudopregnancy. Levels of ovarian mRNA for Pdgfb and Pdgfd, and their receptor, Pdgfrb, increased significantly as early as 4 h after human chorionic gonadotropin (hCG) injection in immature rats primed with equine chorionic gonadotropin (eCG). Gonadotropin regulation of Pdgfb expression was confirmed by in vitro promoter-reporter assays, which showed a 2- to 3-fold increase in Pdgfb promoter activity in response to luteinizing hormone (LH). Inhibition studies implicated protein kinase A, phosphatidylinositol 3-kinase and mitogen activated protein kinase signaling pathways in the LH-induced upregulation. In the corpus luteum, PDGFA, PDGFB, PDGFC, and PDGFRA were localized to a population of luteal parenchymal/steroidogenic cells. PDGFRB was expressed primarily in what appeared to be cells of the luteal microvasculature. Intraovarian injection of an inhibitor of PDGF receptor activity, the tyrphostin AG1295, prior to injection of hCG in eCG-primed immature rats resulted in a significant 21.86%+/-11.15% decrease in corpora lutea per treated ovary in comparison to the contralateral vehicle-injected control ovary. In addition, the treated ovary of 3 of 16 rats showed widespread hemorrhage throughout the entire ovary, indicating a possible role for PDGF receptor activity in maintenance of the ovarian vasculature.

Dopaminergic neuronal loss in transgenic mice expressing the Parkinson's disease-associated UCH-L1 I93M mutant

The I93M mutation in ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) was reported in one German family with autosomal dominant Parkinson's disease (PD). The causative role of the mutation has, however, been questioned. We generated transgenic (Tg) mice carrying human UCHL1 under control of the PDGF-B promoter; two independent lines were generated with the I93M mutation (a high- and low-expressing line) and one line with wild-type human UCH-L1. We found a significant reduction in the dopaminergic neurons in the substantia nigra and the dopamine content in the striatum in the high-expressing I93M Tg mice as compared with non-Tg mice at 20 weeks of age. Although these changes were absent in the low-expressing I93M Tg mice, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment profoundly reduced dopaminergic neurons in this line as compared with wild-type Tg or non-Tg mice. Abnormal neuropathologies were also observed, such as silver staining-positive argyrophilic grains in the perikarya of degenerating dopaminergic neurons, in I93M Tg mice. The midbrains of I93M Tg mice contained increased amounts of insoluble UCH-L1 as compared with those of non-Tg mice, perhaps resulting in a toxic gain of function. Collectively, our data represent in vivo evidence that expression of UCHL1 I93M leads to the degeneration of dopaminergic neurons.

Distribution of galanin in the brain of a galanin-overexpressing transgenic mouse

The distribution of galanin mRNA-expressing cells and galanin-immunoreactive (IR) cell bodies and processes was studied in the brain of mice overexpressing galanin under the PDGF-B promoter (GalOE mice) and of wild type (WT) mice, both in colchicine-treated and non-treated animals. A widespread ectopic expression of galanin (both mRNA and peptide) was found, that is when neither transcript nor peptide could be seen in WT mice, not even after colchicine treatment. However, in some regions, such as claustrum, basolateral amygdala, thalamus, CA1 pyramidal cells, and Purkinje cells only galanin mRNA could be detected. The highest levels of galanin expression were observed in the forebrain structures (the mitral cells of the olfactory bulb, throughout the cortex, granular and pyramidal cell layers of the hippocampus), in the mesencephalon (nucleus ruber), in the cerebellum (lateral cerebellar nucleus), in the pons (sensory and motor nuclei of the trigeminal nerve), within the medulla oblongata (facial, prepositus and spinal trigeminal nuclei). High densities of galanin-IR fibers were found in the axonal terminals of the lateral olfactory tract, hippocampal and presumably cerebellar mossy fiber system, in several thalamic and hypothalamic regions and the lower brain stem.

127. Platelet derived growth factors and receptors contribute toward development of the corpus luteum

In this study the expression of the family of platelet derived growth factors (PDGF) and receptors in the ovarian corpus luteum was identified and characterized, and an effect of their activity on development of the corpus luteum revealed. Gonadotropin-stimulated immature rats were utilized as a model of induced ovulation, luteogenesis and pseudopregnancy, and levels of mRNA for platelet derived growth factors (PDGF-A, PDGF-B, PDGF-C and PDGF-D) and receptors (PDGF-Rα and PDGF-Rβ) in response to gonadotropins were investigated. Intraperitoneal injection of immature rats with pregnant mare’s serum gonadotropin (PMSG) followed 54 h later with human chorionic gonadotropin (hCG) resulted in a significant increase in ovarian mRNA levels for PDGF-Rβ and its ligands, PDGF-B and PDGF-D, as early as 4 h after hCG injection. Gonadotropin regulation of PDGF-B was confirmed by in vitro promoter–reporter assays, which showed a 2–3-fold increase in PDGF-B promoter activity in response to luteinising hormone (LH), and inhibition studies implicated protein kinase A, phosphatidylinositol 3-kinase and mitogen activated protein kinase signaling pathways in the LH-induced upregulation. In the corpus luteum, PDGF-Rα was localized to a subset of luteal steroidogenic cells, and PDGF-Rβ to cells of the luteal microvasculature. PDGF-A, PDGF-B and PDGF-C were also identified in a population of luteal steroidogenic cells. Intraovarian injection of an inhibitor of PDGF receptor activity, the tyrphostin AG1295, prior to injection of hCG in PMSG-primed immature rats resulted in a significant 22.85 ± 10.7% decrease in corpora lutea per treated ovary in comparison to the contralateral vehicle-injected control ovary. In addition, the treated ovary of 3 of 12 rats showed widespread hemorrhage throughout the entire ovary, indicating a possible role for PDGF receptor activity in maintenance of the ovarian vasculature. In summary, these data identify expression of members of the family of platelet derived growth factors and receptors in cells within the corpus luteum and reveal a role during development of the corpus luteum.

Distribution of galanin and galanin transcript in the brain of a galanin-overexpressing transgenic mouse

The distribution of galanin mRNA-expressing cells and galanin-immunoreactive (IR) cell bodies and processes was studied in the brain of mice overexpressing galanin under the PDGF-B promoter (GalOE mice) and of wild type (WT) mice, both in colchicine-treated and non-treated animals. In this abstract, we only describe the results in GalOE mouse. A widespread ectopic expression of galanin (both mRNA and peptide) was found, that is a situation when neither transcript nor peptide could be seen in WT mice, not even after colchicine treatment. However, in some regions, such as claustrum, basolateral amygdala, thalamus, CA1 pyramidal cells, and Purkinje cells only galanin mRNA could be detected. In the forebrain galanin was seen in the mitral cells of the olfactory bulb, throughout the cortex, in the basolateral amygdaloid nucleus, claustrum, granular and pyramidal cell layers of the hippocampus, subiculum and presubiculum. In the thalamus, the anterodorsal, mediodorsal, intermediodorsal and mediodorsal lateral nuclei, the reuniens and reticular nuclei showed ectopic expression of galanin. Within the hypothalamus, neurons of the suprachiasmatic nucleus contained galanin. In the mesencephalon, the geniculate nucleus, nucleus ruber, the mesencephalic trigeminal and reticulotegmental nuclei ectopically expressed galanin. In the cerebellum, galanin was observed in the Purkinje cells and in the lateral and interposed cerebellar nuclei. In the pons, sensory and motor nuclei of the trigeminal nerve, the laterodorsal and dorsal tegmental nuclei, the pontine, reticulotegmental and gigantocellular reticular nuclei expressed galanin. Within the medulla oblongata, labeled cells were detected in the facial, ambiguus, prepositus, lateral paragigantocellular and lateral reticular nuclei, and spinal trigeminal nucleus. High densities of galanin-IR fibers were found in the axonal terminals of the lateral olfactory tract, the hippocampal and presumably the cerebellar mossy fibers system, in several thalamic and hypothalamic regions and the lower brain stem. Possible functional consequences of galanin overexpression are discussed.

Blood Lipid Mediator Sphingosine 1-Phosphate Potently Stimulates Platelet-derived Growth Factor-A and -B Chain Expression through S1P1-Gi-Ras-MAPK-dependent Induction of Krüppel-like Factor 5

Platelet-derived growth factors (PDGFs), potent mitogens and chemoattractants for mesenchymal cell types, play essential roles in development of several organs including blood vessels, kidney, and lung, and are also implicated in the pathogenesis of atherosclerosis and malignancies. Blood lipid mediator sphingosine 1-phosphate (S1P) regulates migration, proliferation, and apoptosis in a variety of cell types through multiple G protein-coupled receptors of the Edg family, and is necessary for vascular formation at the developmental stage. We found in the present study that S1P induced severalfold increases in the mRNA and protein levels of PDGF-A and -B chains in vascular smooth muscle cells and neointimal cells. S1P stimulation of PDGF mRNA and protein expression was abolished by the small interfering RNA duplexes targeting S1P(1)/Edg1 receptor subtype. S1P stimulated the small GTPase Ras in a G(i)-dependent manner, and activated ERK and p38 MAPK in G(i)- and Ras-dependent manners. Pertussis toxin pretreatment, adenovirus-mediated Asn(17)Ras expression, the MEK inhibitor PD98059, or the p38 MAPK inhibitor SB203580 markedly suppressed PDGF mRNA and protein up-regulation, indicating the involvement of G(i)-Ras-ERK/p38 MAPK in S1P stimulation of PDGF expression. S1P stimulated expression of the transcription factor KLF5 in manners dependent on G(i), Ras, and ERK/p38 MAPK. Down-regulation of KLF5 by small interfering RNA duplexes abolished S1P-induced PDGF-A and -B chain expression. On the other hand, overexpression of KLF5 stimulated basal and S1P-induced PDGF expression. Either S1P stimulation or KLF5 overexpression increased the PDGF-B promoter activity in a cis-element-dependent manner. These results reveal the S1P(1)-triggered, G(i)-Ras-ERK/p38 MAPK-KLF5-dependent, stimulatory regulation of PDGF gene transcription in vascular smooth muscle cells.

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

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-zeta blocked nogalamycin-inducible Sp1 phosphorylation and PDGF-B promoter-dependent expression. Nogalamycin stimulated the phosphorylation of PKC-zeta (on residue Thr(410)). These findings demonstrate for the first time that PKC-zeta and Sp1 phosphorylation mediate the inducible expression of this growth factor.

Triplex targeting of human PDGF-B (c-sis, proto-oncogene) promoter specifically inhibits factors binding and PDGF-B transcription.

Human c-sis/PDGF-B proto-oncogene has been shown to be overexpressed in a large percentage of human tumor cells establishing a growth-promoting, autocrine growth circuit. Triplex forming oligonucleotides (TFOs) can recognize and bind sequences in duplex DNA, and have received considerable attention because of their potential for targeting specific genomic sites. The c-sis/PDGF-B promoter contains a unique homopurine/homopyrimidine sequence (SIS proximal element, SPE), which is crucial for binding nuclear factors that provoke transcription. In order to develop specific transcriptional inhibitors of the human c-sis/PDGF-B proto-oncogene, 20 potential TFOs targeting part or all of the SPE were screened by gel mobility analysis. DNase I footprinting shows that the TFOs we designed can form a sequence-specific triplex with the target. Protein binding assays demonstrate that triplex formation inhibits nuclear factors binding the c-sis/PDGF-B promoter. Both transient and stable transfection experiments demonstrate that the transcriptional activity of the promoter is considerably inhibited by the TFOs. We propose that TFOs represent a therapeutic potential to specifically diminish the expression of c-sis/PDGF-B proto-oncogene in various pathologic settings where constitutive expression of this gene has been observed.

Induction of Platelet-derived Growth Factor B-chain Expression by Transforming Growth Factor-β Involves Transactivation by Smads

Transforming growth factor-beta (TGF-beta) regulates a diverse array of biological processes, such as proliferation, differentiation, extracellular matrix production, and apoptosis. In cultured vascular endothelial cells, TGF-beta induces the expression of platelet-derived growth factor (PDGF) B-chain, a mitogen and chemoattractant, at the level of transcription. The molecular mechanism(s) underlying this process are not presently understood. In this study, we performed serial 5' deletion and transient transfection analysis to define a region in the PDGF-B promoter mediating inducible responsiveness to TGF-beta. This region contains an atypical nucleotide recognition element for the Smad family of transcriptional regulators. Electrophoretic mobility shift analysis revealed that nuclear proteins bound to this site in a transient and specific manner. Supershift studies demonstrated the physical association of Smad4 with the promoter. Overexpression of Smad4 activated the PDGF-B promoter and superinduced PDGF-B promoter-dependent expression in cells exposed to TGF-beta. Moreover, simultaneous cotransfection of Smad3 and Smad4 activated the PDGF-B promoter. This effect was attenuated when Smad4 was substituted with its dominant negative counterpart. Mutation of the (-81)CAGA(-78) motif in the PDGF-B promoter abrogated Smad-inducible promoter-dependent expression. Overexpression of Smad2 and Smad3 transactivated the PDGF-B promoter in a synergistic manner. These findings demonstrate the existence of a novel, functional binding element in the proximal region of the PDGF-B promoter mediating responsiveness to TGF-beta.

Novel Negative Regulatory Element in the Platelet-derived Growth Factor B Chain Promoter That Mediates ERK-dependent Transcriptional Repression

Platelet-derived growth factor (PDGF), which consists of an A and/or B chain, stimulates migration and proliferation in vascular smooth muscle cells as well as a large number of other cell types. Investigations over recent years have defined roles for several positive regulatory transcription factors in the PDGF-B promoter. However, little is known about the transcriptional mechanisms that negatively regulate this gene. Here, we used transient transfection and 5' deletion analysis to define a specific region in the PDGF-B promoter-mediating repression in vascular smooth muscle cells. Gel retardation assays revealed this region is bound by nuclear protein(s) in a specific manner. Supershift assays excluded the direct association of Sp1, Sp3, and Egr-1. Mutation of the negative regulatory element no longer supported nucleoprotein complex formation and, when introduced into the PDGF-B promoter, rescued the promoter from repression. Promoter activity was also restored by transfection of oligonucleotide decoys bearing the repressor binding site. The MEK1/2 inhibitor, PD98059, and a dominant negative construct generating inactive ERK1 increased reporter expression driven by the PDGF-B promoter. In contrast, the MEK inhibitor had no effect on the activity of the mutant PDGF-B promoter. These effects were cell type-specific, since neither suppression of the PDGF-B promoter nor nucleoprotein complex formation was observed in vascular endothelial cells. These findings define a distinct negative regulatory element in the PDGF-B promoter that interacts with nuclear protein(s) and inhibits PDGF-B promoter-dependent gene expression in an ERK-dependent manner.

Mechanical Strain Increases PDGF-B and PDGF β Receptor Expression in Vascular Smooth Muscle Cells

Cyclic mechanical strain causes proliferation of vascular smooth muscle cells, mediated in part by platelet-derived growth factor (PDGF). We examined the effect of cyclic strain on expression of PDGF-B and the PDGF β receptor. Neonatal rat vascular smooth muscle cells were exposed to 1 hertz cyclic strain on silicone elastomer plates. PDGF-B mRNA increased after 6 h of strain. In cells transfected with a PDGF-B promoter chloramphenicol acetyl transferase construct (psisCAT 6A), activity increased by 12-fold following 12 h of strain. Two neutralizing antibodies to the PDGF β receptor both reduced strain-induced [3H]thymidine incorporation by 50%. Expression of the PDGF β receptor protein increased 1.8-fold following 24 h of strain. During strain, PDGF β receptor expression was not significantly altered by neutralizing antibodies to PDGF-B. Thus, both PDGF-B and the PDGF β receptor are induced by cyclic mechanical strain and both contribute to cell proliferation in response to strain.

Zinc Finger Transcription Factors Mediate High Constitutive Platelet-derived Growth Factor-B Expression in Smooth Muscle Cells Derived from Aortae of Newborn Rats

Platelet-derived growth factor (PDGF) B-chain gene is differentially expressed in smooth muscle cells (SMCs) derived from the rat aortic wall. SMCs cultured from two week-old rats (pups) express high levels of PDGF-B mRNA, whereas cells isolated from three month-old rats (adults) express low levels of PDGF-B. Nuclear run-off experiments indicate that increased PDGF-B gene expression in pups is mediated, at least in part, at the transcriptional level. We used electrophoretic mobility shift assays and Western blot analysis to demonstrate that levels of Sp1 and Sp3, two zinc finger transcription factors which mediate basal expression of the PDGF-B gene, are elevated in pup nuclei compared with adult nuclei. The immediate-early transcription factor, Egr-1, which footprints the PDGF-B promoter, is also constitutively expressed in these cells. Transient transfection and binding studies show that these factors interact with a region in the proximal PDGF-B promoter key for basal expression in pup cells. Mutation of this proximal element in transfected pup cells attenuates reporter gene expression to levels observed in adult cells. Conversely, overexpression of Sp1 in adult cells augments PDGF-B promoter-dependent expression. Elevated PDGF-B expression in cultured newborn rat SMCs may therefore require high constitutive expression of a number of zinc finger transcription factors and their specific interactions with the proximal PDGF-B promoter.