FRS2 via Fibroblast Growth Factor Receptor 1 Is Required for Platelet-derived Growth Factor Receptor ��-mediated Regulation of Vascular Smooth Muscle Marker Gene Expression

Vascular smooth muscle cells (VSMC) exhibit phenotypic plasticity and change from a quiescent contractile phenotype to a proliferative synthetic phenotype during physiological arteriogenesis and pathological conditions such as atherosclerosis and restenosis. Platelet-derived growth factor (PDGF)-BB is a potent inducer of the VSMC synthetic phenotype; however, much less is known about the role of fibroblast growth factor-2 (FGF2) in this process. Here, we show using signal transduction mutants of FGF receptor 1 (FGFR1) expressed in rat VSMC that the adaptor protein FRS2 is essential for FGFR1-mediated phenotypic modulation and down-regulation of VSMC smooth muscle α-actin (SMA) gene expression. In addition, we show that PDGF-BB and FGF2 act synergistically to induce cell proliferation and down-regulate SMA and SM22α in VSMC. Furthermore, we show that PDGF-BB induces tyrosine phosphorylation of FGFR1 and that this phosphorylation is mediated by PDGF receptor-β (PDGFRβ), but not c-Src. We demonstrate that FRS2 co-immunoprecipitates with PDGFRβ in a complex that requires FGFR1 and that both the extracellular and the intracellular domains of FGFR1 are required for association with PDGFRβ, whereas the cytoplasmic domain of FGFR1 is required for FRS2 association with the FGFR1-PDGFRβ complex. Knockdown of FRS2 in VSMC by RNA interference inhibited PDGF-BB-mediated down-regulation of SMA and SM22α without affecting PDGF-BB mediated cell proliferation or ERK activation. Together, these data support the notion that PDGFRβ down-regulates SMA and SM22α through formation of a complex that requires FGFR1 and FRS2 and prove novel insight into VSMC phenotypic plasticity.Phenotypic modulation of vascular smooth muscle cells (VSMC)3 is an important step in the development of several pathophysiological processes including atherosclerosis, restenosis, and vascular remodeling (1, 2). During these processes VSMC change from a contractile phenotype to a synthetic phenotype characterized by increased proliferation, migration, increased extracellular matrix production, and decreased expression of contractile proteins, including smooth muscle α-actin (SMA), SM22α, calponin, and myosin heavy chain. Several growth factors including platelet-derived growth factor-BB (PDGF-BB), fibroblast growth factor 2 (FGF2), and thrombin have been implicated in the induction of the synthetic phenotype (3). These growth factors bind cell surface receptors and activate intracellular signaling pathways that result in changes in gene expression and cellular phenotype. Understanding the interactions between these pathways may provide insights into mechanisms of phenotypic modulation of VSMC and provide new targets for therapeutic intervention in vascular disease.Experimental evidence using various in vitro and in vivo models points to a role for FGF-FGFR in the phenotypic modulation of VSMC. FGFs and FGFRs are expressed in VSMC and are up-regulated during vascular injury and in atherosclerotic plaque formation (4–6). Balloon injury of rat arteries led to an increase in FGFR expression in VSMC. The up-regulation of FGF and FGFR suggests that they contribute to the pathogenesis of vascular disease. In support of this hypothesis, administration of anti-FGF2 antibodies and FGFR tyrosine kinase inhibitors results in decreased VSMC proliferation, migration, and attenuated neointimal thickening (7).PDGF-BB binds to PDGFRβ and activates several intracellular signaling pathways including ERK, phosphatidylinositol 3-kinase/Akt, and mammalian target of rapamycin (mTOR) (8). Studies have indicated that PDGF-BB induces the release of FGF2 and activation FGFR1, resulting in sustained ERK activation and proliferation of human VSMC (9). When FGFR1 expression was inhibited by RNA interference, PDGF-BB induced transient but not sustained ERK activation.Binding of FGF2 to FGFR1 activates the ERK and phosphatidylinositol 3-kinase/Akt pathways via the adaptor protein FRS2 (10, 11). Upon FGF2 binding, FGFR1 phosphorylates FRS2 on six tyrosine residues that function as docking sites for the SH2 domain-containing proteins Grb2 and SHP2 (12, 13). Grb2 binds Gab1 leading to activation of phosphatidylinositol 3-kinase/Akt, whereas SHP2 activates the Ras-Raf-ERK pathway. FRS2 binds to FGFR1 via a Val-Thr dipeptide in the juxtamembrane region of FGFR1 (14, 15). Deletion of these two amino acids abrogates binding of FRS2 to FGFR1. To determine the role of FRS2 in FGFR1-mediated VSMC phenotypic modulation and to determine the interaction of PDGFRβ with the FGFR1 signaling pathway, we developed a set of FGFR1 signaling pathway deficient mutants and stably expressed them in rat VSMC. In this study we report that PDGFRβ, FGFR1, and FRS2 form a multi-protein complex that is essential for VSMC phenotypic modulation and that stable knockdown of FRS2 inhibits PDGF-BB-mediated down-regulation of VSMC marker gene expression but not PDGF-BB-mediated VSMC proliferation.     

Fibroblast Growth Factor–21 Ameliorates Rheumatoid Arthritis by Maintaining Articular Integrity

International Journal of Peptide Research and Therapeutics - Rheumatoid arthritis, a chronic degenerative autoimmune disease is hallmarked by tenacious inflammation of synovial membranes causing...     

Metabolic Regulator βKlotho Interacts with Fibroblast Growth Factor Receptor 4 (FGFR4) to Induce Apoptosis and Inhibit Tumor Cell Proliferation

In organs involved in metabolic homeostasis, transmembrane α and βklothos direct FGFR signaling to control of metabolic pathways. Coordinate expression of βklotho and FGFR4 is a property of mature hepatocytes. Genetic deletion of FGFR4 or βklotho in mice disrupts hepatic cholesterol/bile acid and lipid metabolism. The deletion of FGFR4 has no effect on the proliferative response of hepatocytes after liver injury. However, its absence results in accelerated progression of dimethynitrosamine-initiated hepatocellular carcinomas, indicating that FGFR4 suppresses hepatoma proliferation. The mechanism underlying the FGFR4-mediated hepatoma suppression has not been addressed. Here we show that βklotho expression is more consistently down-regulated in human and mouse hepatomas than FGFR4. Co-expression and activation by either endocrine FGF19 or cellular FGF1 of the FGFR4 kinase in a complex with βklotho restricts cell population growth through induction of apoptotic cell death in both hepatic and nonhepatic cells. The βklotho-FGFR4 partnership caused a depression of activated AKT and mammalian target of rapamycin while activating ERK1/2 that may underlie the pro-apoptotic effect. Our results show that βklotho not only interacts with heparan sulfate-FGFR4 to form a complex with high affinity for endocrine FGF19 but also impacts the quality of downstream signaling and biological end points activated by either FGF19 or canonical FGF1. Thus the same βklotho-heparan sulfate-FGFR4 partnership that mediates endocrine control of hepatic metabolism plays a role in cellular homeostasis and hepatoma suppression through negative control of cell population growth mediated by pro-apoptotic signaling.     

Effects of 3-Methylgibberellin Analogs on Gibberellin 3β-Hydroxylases and Plant Growth

Six 3-methylgibberellin analogs were synthesized, and their effects on the GA 3β-hydroxylases from immature seeds of Phaseolus vulgaris and Cucurbita maxima, and/or on the growth of dwarf rice (Oryza sativa L. cv. Tan-ginbozu) and cucumber (Cucumis sativus L. cv. Spacemaster) were investigated. 3-Methyl-GA₅ and 2, 3-didehydro-3-methyl-GA₉· inhibited the conversion of [2, 3-³H2]GA₉ to [2-³H]GA₄ by GA 3β-hydroxylases from both P. vulgaris and C. maxima at 3 μM and higher. Their C/D-ring-rearranged isomers, 2, 3-didehydro-3-methyl-DGC and 16-deoxo-2, 3-didehydro-3-methyl-DGC, inhibited 3β-hydroxylation by the enzyme from P. vulgaris threefold more strongly than the non-C/D-ring-rearranged compounds, but exhibited no effect on 3β-hydroxylation by the enzyme from C. maxima. In a dwarf rice seedling assay, 3-methyl-GA₅ and 2, 3-didehydro-3-methyl-GA₉ promoted shoot elongation at doses of 300 ng/plant and higher, and 3α-methyl-GA₁ and 3α-methyl-GA₄ at doses of 30 ng/plant and higher. In contrast 2, 3-didehydro-3-methyl-DGC inhibited shoot growth to half that of the control at a dose of 300 ng/plant, and 16-deoxo-2, 3-didehydro-3-methyl-DGC showed no effect on growth. In a cucumber seedling assay, 3α-methyl-GA₄ promoted hypocotyl elongation at doses of 300 ng/plant and higher. The other C-3 methyl compounds showed no effect on the hypocotyl elongation of cucumber seedlings.     

Platelet-Derived Growth Factor Receptor β Is Critical for Zebrafish Intersegmental Vessel Formation

Background

Platelet-derived growth factor receptor β (PDGFRβ) is a tyrosine kinase receptor known to affect vascular development. The zebrafish is an excellent model to study specific regulators of vascular development, yet the role of PDGF signaling has not been determined in early zebrafish embryos. Furthermore, vascular mural cells, in which PDGFRβ functions cell autonomously in other systems, have not been identified in zebrafish embryos younger than 72 hours post fertilization.

Methodology/Principal Findings

In order to investigate the role of PDGFRβ in zebrafish vascular development, we cloned the highly conserved zebrafish homolog of PDGFRβ. We found that pdgfrβ is expressed in the hypochord, a developmental structure that is immediately dorsal to the dorsal aorta and potentially regulates blood vessel development in the zebrafish. Using a PDGFR tyrosine kinase inhibitor, a morpholino oligonucleotide specific to PDGFRβ, and a dominant negative PDGFRβ transgenic line, we found that PDGFRβ is necessary for angiogenesis of the intersegmental vessels.

Significance/Conclusion

Our data provide the first evidence that PDGFRβ signaling is required for zebrafish angiogenesis. We propose a novel mechanism for zebrafish PDGFRβ signaling that regulates vascular angiogenesis in the absence of mural cells.     

Enhanced Expression of Integrin αvβ3 Induced by TGF-β Is Required for the Enhancing Effect of Fibroblast Growth Factor 1 (FGF1) in TGF-β-Induced Epithelial-Mesenchymal Transition (EMT) in Mammary Epithelial Cells

Epithelial-to-mesenchymal transition (EMT) plays a critical role in cancer metastasis, and is regulated by growth factors such as transforming growth factor β (TGF-β) and fibroblast growth factors (FGF) secreted from the stromal and tumor cells. However, the role of growth factors in EMT has not been fully established. Several integrins are upregulated by TGF-β1 during EMT. Integrins are involved in growth factor signaling through integrin-growth factor receptor crosstalk. We previously reported that FGF1 directly binds to integrin αvβ3 and the interaction was required for FGF1 functions such as cell proliferation and migration. We studied the role of αvβ3 induced by TGF-β on TGF-β-induced EMT. Here, we describe that FGF1 augmented EMT induced by TGF-β1 in MCF10A and MCF12A mammary epithelial cells. TGF-β1 markedly amplified integrin αvβ3 and FGFR1 (but not FGFR2). We studied if the enhancing effect of FGF1 on TGF-β1-induced EMT requires enhanced levels of both integrin αvβ3 expression and FGFR1. Knockdown of β3 suppressed the enhancement by FGF1 of TGF-β1-induced EMT in MCF10A cells. Antagonists to FGFR suppressed the enhancing effect of FGF1 on EMT. Integrin-binding defective FGF1 mutant did not augment TGF-β1-induced EMT in MCF10A cells. These findings suggest that enhanced integrin αvβ3 expression in addition to enhanced FGFR1 expression is critical for FGF1 to augment TGF-β1-induced EMT in mammary epithelial cells.     

Gαi2 Signaling Is Required for Skeletal Muscle Growth,Regeneration, and Satellite Cell Proliferation and Differentiation

We have previously shown that activation of Gαi2, an α subunit of the heterotrimeric G protein complex, induces skeletal muscle hypertrophy and myoblast differentiation. To determine whether Gαi2 is required for skeletal muscle growth or regeneration, Gαi2-null mice were analyzed. Gαi2 knockout mice display decreased lean body mass, reduced muscle size, and impaired skeletal muscle regeneration after cardiotoxin-induced injury. Short hairpin RNA (shRNA)-mediated knockdown of Gαi2 in satellite cells (SCs) leads to defective satellite cell proliferation, fusion, and differentiation ex vivo. The impaired differentiation is consistent with the observation that the myogenic regulatory factors MyoD and Myf5 are downregulated upon knockdown of Gαi2. Interestingly, the expression of microRNA 1 (miR-1), miR-27b, and miR-206, three microRNAs that have been shown to regulate SC proliferation and differentiation, is increased by a constitutively active mutant of Gαi2 [Gαi2(Q205L)] and counterregulated by Gαi2 knockdown. As for the mechanism, this study demonstrates that Gαi2(Q205L) regulates satellite cell differentiation into myotubes in a protein kinase C (PKC)- and histone deacetylase (HDAC)-dependent manner.     

Transforming Growth Factor: β Signaling Is Essential for Limb Regeneration in Axolotls

Axolotls (urodele amphibians) have the unique ability, among vertebrates, to perfectly regenerate many parts of their body including limbs, tail, jaw and spinal cord following injury or amputation. The axolotl limb is the most widely used structure as an experimental model to study tissue regeneration. The process is well characterized, requiring multiple cellular and molecular mechanisms. The preparation phase represents the first part of the regeneration process which includes wound healing, cellular migration, dedifferentiation and proliferation. The redevelopment phase represents the second part when dedifferentiated cells stop proliferating and redifferentiate to give rise to all missing structures. In the axolotl, when a limb is amputated, the missing or wounded part is regenerated perfectly without scar formation between the stump and the regenerated structure. Multiple authors have recently highlighted the similarities between the early phases of mammalian wound healing and urodele limb regeneration. In mammals, one very important family of growth factors implicated in the control of almost all aspects of wound healing is the transforming growth factor-beta family (TGF-β). In the present study, the full length sequence of the axolotl TGF-β1 cDNA was isolated. The spatio-temporal expression pattern of TGF-β1 in regenerating limbs shows that this gene is up-regulated during the preparation phase of regeneration. Our results also demonstrate the presence of multiple components of the TGF-β signaling machinery in axolotl cells. By using a specific pharmacological inhibitor of TGF-β type I receptor, SB-431542, we show that TGF-β signaling is required for axolotl limb regeneration. Treatment of regenerating limbs with SB-431542 reveals that cellular proliferation during limb regeneration as well as the expression of genes directly dependent on TGF-β signaling are down-regulated. These data directly implicate TGF-β signaling in the initiation and control of the regeneration process in axolotls.     

β-Catenin Is Required for Prostate Development and Cooperates with Pten Loss to Drive Invasive Carcinoma

Prostate cancer is a major cause of male death in the Western world, but few frequent genetic alterations that drive prostate cancer initiation and progression have been identified. β-Catenin is essential for many developmental processes and has been implicated in tumorigenesis in many tissues, including prostate cancer. However, expression studies on human prostate cancer samples are unclear on the role this protein plays in this disease. We have used in vivo genetic studies in the embryo and adult to extend our understanding of the role of β-Catenin in the normal and neoplastic prostate. Our gene deletion analysis revealed that prostate epithelial β-Catenin is required for embryonic prostate growth and branching but is dispensable in the normal adult organ. During development, β-Catenin controls the number of progenitors in the epithelial buds and regulates a discrete network of genes, including c-Myc and Nkx3.1. Deletion of β-Catenin in a Pten deleted model of castration-resistant prostate cancer demonstrated it is dispensable for disease progression in this setting. Complementary overexpression experiments, through in vivo protein stabilization, showed that β-Catenin promotes the formation of squamous epithelia during prostate development, even in the absence of androgens. β-Catenin overexpression in combination with Pten loss was able to drive progression to invasive carcinoma together with squamous metaplasia. These studies demonstrate that β-Catenin is essential for prostate development and that an inherent property of high levels of this protein in prostate epithelia is to drive squamous fate differentiation. In addition, they show that β-Catenin overexpression can promote invasive prostate cancer in a clinically relevant model of this disease. These data provide novel information on cancer progression pathways that give rise to lethal prostate disease in humans.     

ε-N-Acetylation in the Production of Recombinant Human Basic Fibroblast Growth Factor Mutein

Abstract

During large scale preparation of recombinant human basic fibroblast growth factor(hbFGF) mutein CS23(CS23) produced in Escherichia coli (E. Coli), three species, named Mf-1, Mf-2 and Mf-3 in the order of their elution, were isolated from the early fraction of Sulfated Cellulofine chromatography by high performance liquid chromatography (HPLC) on heparin-5pw. Structural analysis through HPLC peptide mapping, mass spectrometry, sequencing and amino acid composition revealed that ε-N-acetylation occurred at positions Lys135, Lys26 and Lys119 of CS23. Together with this observation, the results on large scale preparation of CS23 were also presented.     

Transforming Growth Factor-β1 (TGF-β1)-stimulated Fibroblast to Myofibroblast Differentiation Is Mediated by Hyaluronan (HA)-facilitated Epidermal Growth Factor Receptor (EGFR) and CD44 Co-localization in Lipid Rafts

Fibroblast to myofibroblast differentiation drives effective wound healing and is largely regulated by the cytokine transforming growth factor-β1 (TGF-β1). Myofibroblasts express α-smooth muscle actin and are present in granulation tissue, where they are responsible for wound contraction. Our previous studies show that fibroblast differentiation in response to TGF-β1 is dependent on and mediated by the linear polysaccharide hyaluronan (HA). Both the HA receptor, CD44, and the epidermal growth factor receptor (EGFR) are involved in this differentiation response. The aim of this study was to understand the mechanisms linking HA-, CD44-, and EGFR-regulated TGF-β1-dependent differentiation. CD44 and EGFR co-localization within membrane-bound lipid rafts was necessary for differentiation, and this triggered downstream mitogen-activated protein kinase (MAPK/ERK) and Ca²⁺/calmodulin kinase II (CaMKII) activation. We also found that ERK phosphorylation was upstream of CaMKII phosphorylation, that ERK activation was necessary for CaMKII signaling, and that both kinases were essential for differentiation. In addition, HA synthase-2 (HAS2) siRNA attenuated both ERK and CaMKII signaling and sequestration of CD44 into lipid rafts, preventing differentiation. In summary, the data suggest that HAS2-dependent production of HA facilitates TGF-β1-dependent fibroblast differentiation through promoting CD44 interaction with EGFR held within membrane-bound lipid rafts. This induces MAPK/ERK, followed by CaMKII activation, leading to differentiation. This pathway is synergistic with the classical TGF-β1-dependent SMAD-signaling pathway and may provide a novel opportunity for intervention in wound healing.     

p130Cas Is Required for Mammary Tumor Growth and Transforming Growth Factor-��-mediated Metastasis through Regulation of Smad2/3 Activity

During breast cancer progression, transforming growth factor-β (TGF-β) switches from a tumor suppressor to a pro-metastatic molecule. Several recent studies suggest that this conversion in TGF-β function depends upon fundamental changes in the TGF-β signaling system. We show here that these changes in TGF-β signaling are concomitant with aberrant expression of the focal adhesion protein, p130Cas. Indeed, elevating expression of either the full-length (FL) or just the carboxyl terminus (CT) of p130Cas in mammary epithelial cells (MECs) diminished the ability of TGF-β1 to activate Smad2/3, but increased its coupling to p38 MAPK. This shift in TGF-β signaling evoked (i) resistance to TGF-β-induced growth arrest, and (ii) acinar filling upon three-dimensional organotypic cultures of p130Cas-FL or -CT expressing MECs. Furthermore, rendering metastatic MECs deficient in p130Cas enhanced TGF-β-stimulated Smad2/3 activity, which restored TGF-β-induced growth inhibition both in vitro and in mammary tumors produced in mice. Additionally, whereas elevating TβR-II expression in metastatic MECs had no affect on their phosphorylation of Smad2/3, this event markedly enhanced their activation of p38 MAPK, leading to increased MEC invasion and metastasis. Importantly, depleting p130Cas expression in TβR-II-expressing metastatic MECs significantly increased their activation of Smad2/3, which (i) reestablished the physiologic balance between canonical and noncanonical TGF-β signaling, and (ii) reversed cellular invasion and early mammary tumor cell dissemination stimulated by TGF-β. Collectively, our findings identify p130Cas as a molecular rheostat that regulates the delicate balance between canonical and noncanonical TGF-β signaling, a balance that is critical to maintaining the tumor suppressor function of TGF-β during breast cancer progression.