βKlotho Is Required for Fibroblast Growth Factor 21 Effects on Growth and Metabolism

Fibroblast growth factor 21 (FGF21) is a fasting-induced hepatokine that has potent pharmacologic effects in mice, which include improving insulin sensitivity and blunting growth. The single-transmembrane protein βKlotho functions as a coreceptor for FGF21 in?vitro. To determine if βKlotho is required for FGF21 action in?vivo, we generated whole-body and adipose tissue-selective βKlotho-knockout mice. All of the effects of FGF21 on growth and metabolism were lost in whole-body βKlotho-knockout mice. Selective elimination of βKlotho in adipose tissue blocked the acute insulin-sensitizing effects of FGF21. Taken together, these data demonstrate that βKlotho is essential for FGF21 activity and that βKlotho in adipose tissue contributes to the beneficial metabolic actions of FGF21.     

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.     

Antitumor Effect of AZD4547 in a Fibroblast Growth Factor Receptor 2–Amplified Gastric Cancer Patient–Derived Cell Model

BACKGROUND: FGFR2 amplification is associated with aggressive gastric cancer (GC), and targeted drugs have been developed for treatment of GC. We evaluated the antitumor activity of an FGFR inhibitor in FGFR2-amplified GC patients with peritoneal carcinomatosis. METHODS: Two GC patients with FGFR2 amplification confirmed by fluorescence in situ hybridization showed peritoneal seeding and malignant ascites. We used the patient-derived xenograft model; patient-derived cells (PDCs) from malignant ascites were used to assess FGFR2 expression and its downstream pathway using immunofluorescence analysis and immunoblot assay in vitro. Apoptosis and cell cycle arrest after treatment of FGFR inhibitor were analyzed by Annexin V-FITC assay and cell cycle analysis. RESULTS: FGFR2 amplification was verified in both PDC lines. AZD4547 as an FGFR inhibitor decreased proliferation of PDCs, and the IC₅₀ value was estimated to be 250 nM in PDC#1 and 210 nM in PDC#2. FGFR inhibitor also significantly decreased levels of phosphorylated FGFR2 and downstream signaling molecules in FGFR2-amplified PDC lines. In cell cycle analysis, apoptosis was significantly increased in AZD4547-treated cells compared with nontreated cells. The proportion of cells in the sub-G1 stage was significantly higher in AZD4547-treated PDCs than in control cells. CONCLUSION: Our findings suggest that FGFR2 amplification is a relevant therapeutic target in GC with peritoneal carcinomatosis.     

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.     

Tyrosine Phosphorylation of β-Catenin and Plakoglobin Enhanced by Hepatocyte Growth Factor and Epidermal Growth Factor in Human Carcinoma Cells

The effect of hepatocyte growth factor /scatter factor (HGF/SF) and epidermal growth factor (EGF) on cadherin-mediated adhesion of human carcinoma cells was studied. HGF/SF induced scattering of colonic adenocarcinoma HT29 and gastric adenocarcinomas MKN7 and MKN74 cells. Likewise, EGF induced scattering of HT29 and MKN7 cells. These cells expressed E-cadherin, which was concentrated at cell-cell contact sites. When the scattering of these cells was induced by HGF/SF or EGF, the E-cadherin concentration at cell-cell boundaries tended to decrease. Irnmunoblotting analyses, however, demonstrated that these growth factor treatments did not alter the expression of E-cadherin and E-cadherin-associated proteins, α- and β-catenin and plakoglobin. β-Catenin, plakoglobin and an unidentified 115-kDa molecule associated with E-cadherin were found to be phosphorylated at tyrosine residues, and these phosphorylations were enhanced by the growth factor treatments. These results suggest that HGF/SF and EGF may modulate the function of the cadherin-catenin system via tyrosine phosphorylation of cadherin-associated proteins.     

G Protein α Subunit 14 Mediates Fibroblast Growth Factor 2-Induced Cellular Responses in Human Endothelial Cells

During pregnancy, a tremendous increase in fetoplacental angiogenesis is associated with elevated blood flow. Aberrant fetoplacental vascular function may lead to pregnancy complications including pre-eclampsia. Fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A (VEGFA) are crucial regulators of fetoplacental endothelial function. G protein α subunit 14 (GNA14), a member of Gαq/11 subfamily is involved in mediating hypertensive diseases and tumor vascularization. However, little is known about roles of GNA14 in mediating the FGF2- and VEGFA-induced fetoplacental endothelial function. Using human umbilical vein endothelial cells (HUVECs) cultured under physiological chronic low oxygen (3% O₂) as a cell model, we show that transfecting cells with adenovirus carrying GNA14 complementary DNA (cDNA; Ad-GNA14) increases (p < 0.05) protein expression of GNA14. GNA14 overexpression blocks (p < 0.05) FGF2-stimulated endothelial migration, whereas it enhances (p < 0.05) endothelial monolayer integrity (maximum increase of ~35% over the control at 24 hr) in response to FGF2. In contrast, GNA14 overexpression does not significantly alter VEGFA-stimulated cell migration, VEGFA-weakened cell monolayer integrity, and intracellular Ca⁺⁺ mobilization in response to adenosine triphosphate (ATP), FGF2, and VEGFA. GNA14 overexpression does not alter either FGF2- or VEGFA-induced phosphorylation of ERK1/2. However, GNA14 overexpression time-dependently elevates (p < 0.05) phosphorylation of phospholipase C-β3 (PLCβ3) at S1105 in response to FGF2, but not VEGFA. These data suggest that GNA14 distinctively mediates fetoplacental endothelial cell migration and permeability in response to FGF2 and VEGFA, possibly in part by altering activation of PLCβ3 under physiological chronic low oxygen.     

The Clinical Significance of Interleukin–1 Receptor Antagonist +2018 Polymorphism in Rheumatoid Arthritis

ObjectiveInterleukin-1 receptor antagonist (IL-1Ra) acts as an inhibitor of IL-1; which is one of the culprit cytokines in rheumatoid arthritis (RA). Although +2018 polymorphism of IL-1Ra has been implicated in the pathogenesis of RA, its importance remains poorly understood. Hence, the purpose of this study was to determine the clinical significance of interleukin-1 receptor antagonist (IL-1Ra) +2018 polymorphism in RA.MethodsPolymerase chain reaction (PCR) and sequencing were used to determine the genotypes of the IL-1Ra +2018 for 77 RA patients and 18 healthy controls. All RA patients were assessed for the disease activity score that includes 28 joints (DAS28) and radiographic disease damage based on Modified Sharp Score (MSS).ResultsThe frequency of the T/T and C/T genotypes did not differ significantly (p = 0.893) between the RA patients and the controls. The C/T genotype had significantly higher mean disease activity (DAS 28) and disease damage (MSS) scores with p values of 0.017 and 0.004, respectively. Additionally, the ESR (erythrocyte sedimentation rate), CRP (C-reactive protein), the number of swollen and tender joints were higher for the C/T individuals. On multivariate analysis the CRP, swollen joint count and MSS remained significant with the following p values i.e. 0.045, 0.046 and less than 0.05.ConclusionsC/T genotype of IL-1Ra +2018 prognosticates more aggressive disease in RA.     

Phosphoprotein Enriched in Astrocytes 15 kDa (PEA-15) Reprograms Growth Factor Signaling by Inhibiting Threonine Phosphorylation of Fibroblast Receptor Substrate 2α

Changes in cellular expression of phosphoprotein enriched in astrocytes of 15 kDa (PEA-15) are linked to insulin resistance, tumor cell invasion, and cellular senescence; these changes alter the activation of the extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein (MAP) kinase pathway. Here, we define the mechanism whereby increased PEA-15 expression promotes and sustains ERK1/2 activation. PEA-15 binding prevented ERK1/2 membrane recruitment and threonine phosphorylation of fibroblast receptor substrate 2α (FRS2α), a key link in fibroblast growth factor (FGF) receptor activation of ERK1/2. This reduced threonine phosphorylation led to increased FGF-induced tyrosine phosphorylation of FRS2α, thereby enhancing downstream signaling. Conversely, short hairpin RNA-mediated depletion of endogenous PEA-15 led to reduced FRS2α tyrosine phosphorylation. Thus, PEA-15 interrupts a negative feedback loop that terminates growth factor receptor signaling downstream of FRS2α. This is the dominant mechanism by which PEA-15 activates ERK1/2 because genetic deletion of FRS2α blocked the capacity of PEA-15 to activate the MAP kinase pathway. Thus, PEA-15 prevents ERK1/2 localization to the plasma membrane, thereby inhibiting ERK1/2-dependent threonine phosphorylation of FRS2α to promote activation of the ERK1/2 MAP kinase pathway.     

Cartilage–Specific Over-Expression of CCN Family Member 2/Connective Tissue Growth Factor (CCN2/CTGF) Stimulates Insulin-Like Growth Factor Expression and Bone Growth

Previously we showed that CCN family member 2/connective tissue growth factor (CCN2) promotes the proliferation, differentiation, and maturation of growth cartilage cells in vitro. To elucidate the specific role and molecular mechanism of CCN2 in cartilage development in vivo, in the present study we generated transgenic mice overexpressing CCN2 and analyzed them with respect to cartilage and bone development. Transgenic mice were generated expressing a ccn2/lacZ fusion gene in cartilage under the control of the 6 kb-Col2a1-enhancer/promoter. Changes in cartilage and bone development were analyzed histologically and immunohistologically and also by micro CT. Primary chondrocytes as well as limb bud mesenchymal cells were cultured and analyzed for changes in expression of cartilage–related genes, and non-transgenic chondrocytes were treated in culture with recombinant CCN2. Newborn transgenic mice showed extended length of their long bones, increased content of proteoglycans and collagen II accumulation. Micro-CT analysis of transgenic bones indicated increases in bone thickness and mineral density. Chondrocyte proliferation was enhanced in the transgenic cartilage. In in vitro short-term cultures of transgenic chondrocytes, the expression of col2a1, aggrecan and ccn2 genes was substantially enhanced; and in long-term cultures the expression levels of these genes were further enhanced. Also, in vitro chondrogenesis was strongly enhanced. IGF-I and IGF-II mRNA levels were elevated in transgenic chondrocytes, and treatment of non-transgenic chondrocytes with recombinant CCN2 stimulated the expression of these mRNA. The addition of CCN2 to non-transgenic chondrocytes induced the phosphorylation of IGFR, and ccn2-overexpressing chondrocytes showed enhanced phosphorylation of IGFR. Our data indicates that the observed effects of CCN2 may be mediated in part by CCN2-induced overexpression of IGF-I and IGF-II. These findings indicate that CCN2-overexpression in transgenic mice accelerated the endochondral ossification processes, resulting in increased length of their long bones. Our results also indicate the possible involvement of locally enhanced IGF-I or IGF-II in this extended bone growth.     

Phosphorylation of Serine 779 in Fibroblast Growth Factor Receptor 1 and 2 by Protein Kinase C? Regulates Ras/Mitogen-activated Protein Kinase Signaling and Neuronal Differentiation

The FGF receptors (FGFRs) control a multitude of cellular processes both during development and in the adult through the initiation of signaling cascades that regulate proliferation, survival, and differentiation. Although FGFR tyrosine phosphorylation and the recruitment of Src homology 2 domain proteins have been widely described, we have previously shown that FGFR is also phosphorylated on Ser⁷⁷⁹ in response to ligand and binds the 14-3-3 family of phosphoserine/threonine-binding adaptor/scaffold proteins. However, whether this receptor phosphoserine mode of signaling is able to regulate specific signaling pathways and biological responses is unclear. Using PC12 pheochromocytoma cells and primary mouse bone marrow stromal cells as models for growth factor-regulated neuronal differentiation, we show that Ser⁷⁷⁹ in the cytoplasmic domains of FGFR1 and FGFR2 is required for the sustained activation of Ras and ERK but not for other FGFR phosphotyrosine pathways. The regulation of Ras and ERK signaling by Ser⁷⁷⁹ was critical not only for neuronal differentiation but also for cell survival under limiting growth factor concentrations. PKCϵ can phosphorylate Ser⁷⁷⁹ in vitro, whereas overexpression of PKCϵ results in constitutive Ser⁷⁷⁹ phosphorylation and enhanced PC12 cell differentiation. Furthermore, siRNA knockdown of PKCϵ reduces both growth factor-induced Ser⁷⁷⁹ phosphorylation and neuronal differentiation. Our findings show that in addition to FGFR tyrosine phosphorylation, the phosphorylation of a conserved serine residue, Ser⁷⁷⁹, can quantitatively control Ras/MAPK signaling to promote specific cellular responses.     

RNA Interference against Platelet-Derived Growth Factor Receptor α mRNA Inhibits Fibroblast Transdifferentiation in Skin Lesions of Patients with Systemic Sclerosis

Objective

To down-regulate expression of mRNA for the platelet-derived growth factor receptor (PDGFR)-α, block the signalling pathway of PDGF and its receptor, and study their influence on fibroblast transdifferentiation to myofibroblasts in systemic sclerosis (SSc).

Methods

Fibroblasts from skin lesions of SSc patients and health adult controls were cultured in vitro, and α-smooth muscle actin (α-SMA) expression was determined by immunocytochemistry. Both groups of fibroblasts were stimulated with PDGF-AA, transforming growth factor β₁ (TGF-β₁), and costimulated with PDGF-AA and TGF-β₁, then PDGFR-α and α-SMA mRNA and protein expression were detected with RT-PCR and WB respectively. Three pairs of siRNAs targeting different PDGFR-α mRNA sequences were synthesized for RNAi. SSc and control fibroblasts were transfected with PDGFR-α siRNA; stimulated with PDGF-AA; and assessed for PDGFR-α and α-SMA mRNA and protein expression.

Results

Although the fibroblasts from both groups had similar morphology, the SSc skin lesions had significantly more myofibroblasts than control skin lesions. PDGF-AA stimulation, TGF-β₁ stimulation, and costimulation significantly up-regulated PDGFR-α and α-SMA mRNA and protein expression in SSc fibroblasts compared to control (P<0.05), and costimulation had the strongest effects (P<0.05). All three pairs of siRNAs suppressed PDGFR-α mRNA and protein expression (P<0.05), but siRNA₁₄₉₅ had the highest gene-silencing efficiency (P<0.05). PDGFR-α siRNA attenuated the effects of PDGF-AA through up-regulating PDGFR-α and α-SMA mRNA and protein expression and inhibiting fibroblast transdifferentiation to myofibroblasts in SSc (P<0.05).

Conclusions

PDGFR-α over-expression in SSc fibroblasts bound PDGF-AA more efficiently and promoted fibroblast transdifferentiation, which was enhanced by TGF-β₁. PDGFR-α siRNA down-regulated PDGFR-α expression, blocked binding to PDGF-AA, and inhibited fibroblast transdifferentiation to myofibroblasts.     

Activation of p107 by Fibroblast Growth Factor,Which Is Essential for Chondrocyte Cell Cycle Exit,Is Mediated by the Protein Phosphatase 2A/B55α Holoenzyme

The phosphorylation state of pocket proteins during the cell cycle is determined at least in part by an equilibrium between inducible cyclin-dependent kinases (CDKs) and serine/threonine protein phosphatase 2A (PP2A). Two trimeric holoenzymes consisting of the core PP2A catalytic/scaffold dimer and either the B55α or PR70 regulatory subunit have been implicated in the activation of p107/p130 and pRB, respectively. While the phosphorylation state of p107 is very sensitive to forced changes of B55α levels in human cell lines, regulation of p107 in response to physiological modulation of PP2A/B55α has not been elucidated. Here we show that fibroblast growth factor 1 (FGF1), which induces maturation and cell cycle exit in chondrocytes, triggers rapid accumulation of p107-PP2A/B55α complexes coinciding with p107 dephosphorylation. Reciprocal solution-based mass spectrometric analysis identified the PP2A/B55α complex as a major component in p107 complexes, which also contain E2F/DPs, DREAM subunits, and/or cyclin/CDK complexes. Of note, p107 is one of the preferred partners of B55α, which also associates with pRB in RCS cells. FGF1-induced dephosphorylation of p107 results in its rapid accumulation in the nucleus and formation of larger complexes containing p107 and enhances its interaction with E2F4 and other p107 partners. Consistent with a key role of B55α in the rapid activation of p107 in chondrocytes, limited ectopic expression of B55α results in marked dephosphorylation of p107 while B55α knockdown results in hyperphosphorylation. More importantly, knockdown of B55α dramatically delays FGF1-induced dephosphorylation of p107 and slows down cell cycle exit. Moreover, dephosphorylation of p107 in response to FGF1 treatment results in early recruitment of p107 to the MYC promoter, an FGF1/E2F-regulated gene. Our results suggest a model in which FGF1 mediates rapid dephosphorylation and activation of p107 independently of the CDK activities that maintain p130 and pRB hyperphosphorylation for several hours after p107 dephosphorylation in maturing chondrocytes.