Insulin-Like Growth Factor I (IGF-1) Ec/Mechano Growth Factor – A Splice Variant of IGF-1 within the Growth Plate

Human insulin-like growth factor 1 Ec (IGF-1Ec), also called mechano growth factor (MGF), is a splice variant of insulin-like growth factor 1 (IGF-1), which has been shown in vitro as well as in vivo to induce growth and hypertrophy in mechanically stimulated or damaged muscle. Growth, hypertrophy and responses to mechanical stimulation are important reactions of cartilaginous tissues, especially those in growth plates. Therefore, we wanted to ascertain if MGF is expressed in growth plate cartilage and if it influences proliferation of chondrocytes, as it does in musculoskeletal tissues. MGF expression was analyzed in growth plate and control tissue samples from piglets aged 3 to 6 weeks. Furthermore, growth plate chondrocyte cell culture was used to evaluate the effects of the MGF peptide on proliferation. We showed that MGF is expressed in considerable amounts in the tissues evaluated. We found the MGF peptide to be primarily located in the cytoplasm, and in some instances, it was also found in the nucleus of the cells. Addition of MGF peptides was not associated with growth plate chondrocyte proliferation.     

Transforming Growth Factor β Regulates P-Body Formation through Induction of the mRNA Decay Factor Tristetraprolin

Transforming growth factor β (TGF-β) is a potent growth regulator and tumor suppressor in normal intestinal epithelium. Likewise, epithelial cell growth is controlled by rapid decay of growth-related mRNAs mediated through 3′ untranslated region (UTR) AU-rich element (ARE) motifs. We demonstrate that treatment of nontransformed intestinal epithelial cells with TGF-β inhibited ARE-mRNA expression. This effect of TGF-β was promoted through increased assembly of cytoplasmic RNA processing (P) bodies where ARE-mRNA localization was observed. P-body formation was dependent on TGF-β/Smad signaling, as Smad3 deletion abrogated P-body formation. In concert with increased P-body formation, TGF-β induced expression of the ARE-binding protein tristetraprolin (TTP), which colocalized to P bodies. TTP expression was necessary for TGF-β-dependent P-body formation and promoted growth inhibition by TGF-β. The significance of this was observed in vivo, where colonic epithelium deficient in TGF-β/Smad signaling or TTP expression showed attenuated P-body levels. These results provide new insight into TGF-β''s antiproliferative properties and identify TGF-β as a novel mRNA stability regulator in intestinal epithelium through its ability to promote TTP expression and subsequent P-body formation.     

Structural Properties of HIV Integrase��Lens Epithelium-derived Growth Factor Oligomers

Integrase (IN) is the catalytic component of the preintegration complex, a large nucleoprotein assembly critical for the integration of the retroviral genome into a host chromosome. Although partial crystal structures of human immunodeficiency virus IN alone and its complex with the integrase binding domain of the host factor PSIP1/lens epithelium-derived growth factor (LEDGF)/p75 are available, many questions remain regarding the properties and structures of LEDGF-bound IN oligomers. Using analytical ultracentrifugation, multiangle light scattering, and small angle x-ray scattering, we have established the oligomeric state, stoichiometry, and molecular shapes of IN·LEDGF complexes in solution. Analyses of intact IN tetramers bound to two different LEDGF truncations allow for placement of the integrase binding domain by difference analysis. Modeling of the small angle x-ray scattering envelopes using existing structural data suggests domain arrangements in the IN oligomers that support and extend existing biochemical data for IN·LEDGF complexes and lend new insights into the quaternary structure of LEDGF-bound IN tetramers. These IN oligomers may be involved in stages of the viral life cycle other than integration, including assembly, budding, and early replication.     

A portrait of Transforming Growth Factor β superfamily signalling: Background matters

Ligands of the Transforming Growth Factor β superfamily like Transforming Growth Factor β and Bone Morphogenetic Proteins govern developmental processes and regulate adult homeostasis by controlling cellular proliferation, survival, differentiation and migration. Aberrant signalling activity is associated with human disorders such as cancer, cardiovascular, musculoskeletal, or fibrotic disease. Upon binding to specific sets of cognate cell surface receptors, family members induce highly similar pathways which include canonical SMAD dependent signalling as well as pathways without direct involvement of SMAD proteins, which activate signalling molecules like mitogen-activated protein kinases or small GTPases. The diverse ligand functionalities are achieved through regulation and modulation of the pathways at all levels, resulting in a highly quantitative and context sensitive signal integration reflecting the cellular state and background. Strategies to target Transforming Growth Factor β or Bone Morphogenetic Protein pathways have been developed on the basis of our current understanding and have proven a highly beneficial potential.     

Patients with Encapsulating Peritoneal Sclerosis Have Increased Peritoneal Expression of Connective Tissue Growth Factor (CCN2), Transforming Growth Factor-β1, and Vascular Endothelial Growth Factor

Introduction

Encapsulating peritoneal sclerosis (EPS) is a devastating complication of peritoneal dialysis (PD). The pathogenesis is not exactly known and no preventive strategy or targeted medical therapy is available. CCN2 has both pro-fibrotic and pro-angiogenic actions and appears an attractive target. Therefore, we studied peritoneal expression of CCN2, as well as TGFβ1 and VEGF, in different stages of peritoneal fibrosis.

Materials and methods

Sixteen PD patients were investigated and compared to 12 hemodialysis patients and four pre-emptively transplanted patients. Furthermore, expression was investigated in 12 EPS patients in comparison with 13 PD and 12 non-PD patients without EPS. Peritoneal tissue was taken during kidney transplantation procedure or during EPS surgery. In a subset of patients, CCN2 protein levels in peritoneal effluent and plasma were determined. Samples were examined by qPCR, histology, immunohistochemistry, and ELISA.

Results

Peritoneal CCN2 expression was 5-fold higher in PD patients compared to pre-emptively transplanted patients (P<0.05), but did not differ from hemodialysis patients. Peritoneal expression of TGFβ1 and VEGF were not different between the three groups; neither was peritoneal thickness. Peritoneum of EPS patients exhibited increased expression of CCN2 (35-fold, P<0.001), TGFβ1 (24-fold, P<0.05), and VEGF (77-fold, P<0.001) compared to PD patients without EPS. In EPS patients, CCN2 protein was mainly localized in peritoneal endothelial cells and fibroblasts. CCN2 protein levels were significantly higher in peritoneal effluent of EPS patients compared to levels in dialysate of PD patients (12.0±4.5 vs. 0.91±0.92 ng/ml, P<0.01), while plasma CCN2 levels were not increased.

Conclusions

Peritoneal expression of CCN2, TGFβ1, and VEGF are significantly increased in EPS patients. In early stages of peritoneal fibrosis, only CCN2 expression is slightly increased. Peritoneal CCN2 overexpression in EPS patients is a locally driven response. The potential of CCN2 as biomarker and target for CCN2-inhibiting agents to prevent or treat EPS warrants further study.     

Hyaluronan Synthesis Mediates the Fibrotic Response of Keratocytes to Transforming Growth Factor β

TGFβ induces fibrosis in healing corneal wounds, and in vitro corneal keratocytes up-regulate expression of several fibrosis-related genes in response to TGFβ. Hyaluronan (HA) accumulates in healing corneas, and HA synthesis is induced by TGFβ by up-regulation of HA synthase 2. This study tested the hypothesis that HA acts as an extracellular messenger, enhancing specific fibrotic responses of keratocytes to TGFβ. HA synthesis inhibitor 4-methylumbelliferone (4MU) blocked TGFβ induction of HA synthesis in a concentration-dependent manner. 4MU also inhibited TGFβ-induced up-regulation of α-smooth muscle actin, collagen type III, and extra domain A-fibronectin. Chemical analogs of 4MU also inhibited fibrogenic responses in proportion to their inhibition of HA synthesis. 4MU, however, showed no effect on TGFβ induction of luciferase by the 3TP-Lux reporter plasmid. Inhibition of HA using siRNA to HA synthase 2 reduced TGFβ up-regulation of smooth muscle actin, fibronectin, and cell division. Similarly, brief treatment of keratocytes with hyaluronidase reduced TGFβ responses. These results suggest that newly synthesized cell-associated HA acts as an extracellular enhancer of wound healing and fibrosis in keratocytes by augmenting a limited subset of the cellular responses to TGFβ.     

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.     

Activation of the Connective Tissue Growth Factor (CTGF)-Transforming Growth Factor β 1 (TGF-β 1) Axis in Hepatitis C Virus-Expressing Hepatocytes

Background

The pro-fibrogenic cytokine connective tissue growth factor (CTGF) plays an important role in the development and progression of fibrosis in many organ systems, including liver. However, its role in the pathogenesis of hepatitis C virus (HCV)-induced liver fibrosis remains unclear.

Methods

In the present study, we assessed CTGF expression in HCV-infected hepatocytes using replicon cells containing full-length HCV genotype 1 and the infectious HCV clone JFH1 (HCV genotype 2) by real-time PCR, Western blot analysis and confocal microscopy. We evaluated transforming growth factor β1 (TGF-β1) as a key upstream mediator of CTGF production using neutralizing antibodies and shRNAs. We also determined the signaling molecules involved in CTGF production using various immunological techniques.

Results

We demonstrated an enhanced expression of CTGF in two independent models of HCV infection. We also demonstrated that HCV induced CTGF expression in a TGF-β1-dependent manner. Further dissection of the molecular mechanisms revealed that CTGF production was mediated through sequential activation of MAPkinase and Smad-dependent pathways. Finally, to determine whether CTGF regulates fibrosis, we showed that shRNA-mediated knock-down of CTGF resulted in reduced expression of fibrotic markers in HCV replicon cells.

Conclusion

Our studies demonstrate a central role for CTGF expression in HCV-induced liver fibrosis and highlight the potential value of developing CTGF-based anti-fibrotic therapies to counter HCV-induced liver damage.     

The Role of SnoN in Transforming Growth Factor β1-induced Expression of Metalloprotease-Disintegrin ADAM12

Increased expression of metalloprotease-disintegrin ADAM12 is a hallmark of several pathological conditions, including cancer, cardiovascular disease, and certain inflammatory diseases of the central nervous system or the muscoskeletal system. We show that transforming growth factor β1 (TGFβ1) is a potent inducer of ADAM12 mRNA and protein in mouse fibroblasts and in mouse and human mammary epithelial cells. Induction of ADAM12 is detected within 2 h of treatment with TGFβ1, is Smad2/Smad3-dependent, and is a result of derepression of the Adam12 gene. SnoN, a negative regulator of the TGFβ signaling pathway, is a master regulator of ADAM12 expression in response to TGFβ1 stimulation. Overexpression of SnoN in NIH3T3 cells reduces the magnitude of ADAM12 induction by TGFβ1 treatment. Down-regulation of SnoN expression by short hairpin RNA enhances TGFβ1-induced expression of ADAM12. In a panel of TGFβ1-responsive cancer cell lines with high expression of SnoN, induction of ADAM12 by TGFβ1 is significantly impaired, suggesting that the endogenous SnoN plays a role in regulating ADAM12 expression in response to TGFβ1. Identification of SnoN as a repressor of the ADAM12 gene should contribute to advances in the studies on the role of ADAM12 in tumor progression and in the development of other pathologies.     

Effect of Epidermal Growth Factor on Follicular Development and Steroidogenesis in Perfused Rat Ovary

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Autoactivation of Transforming Growth Factor β-activated Kinase 1 Is a Sequential Bimolecular Process

Transforming growth factor-β-activated kinase 1 (TAK1), an MAP3K, is a key player in processing a multitude of inflammatory stimuli. TAK1 autoactivation involves the interplay with TAK1-binding proteins (TAB), e.g. TAB1 and TAB2, and phosphorylation of several activation segment residues. However, the TAK1 autoactivation is not yet fully understood on the molecular level due to the static nature of available x-ray structural data and the complexity of cellular systems applied for investigation. Here, we established a bacterial expression system to generate recombinant mammalian TAK1 complexes. Co-expression of TAK1 and TAB1, but not TAB2, resulted in a functional and active TAK1-TAB1 complex capable of directly activating full-length heterotrimeric mammalian AMP-activated protein kinase (AMPK) in vitro. TAK1-dependent AMPK activation was mediated via hydrophobic residues of the AMPK kinase domain αG-helix as observed in vitro and in transfected cell culture. Co-immunoprecipitation of differently epitope-tagged TAK1 from transfected cells and mutation of hydrophobic αG-helix residues in TAK1 point to an intermolecular mechanism of TAB1-induced TAK1 autoactivation, as TAK1 autophosphorylation of the activation segment was impaired in these mutants. TAB1 phosphorylation was enhanced in a subset of these mutants, indicating a critical role of αG-helix residues in this process. Analyses of phosphorylation site mutants of the activation segment indicate that autophosphorylation of Ser-192 precedes TAB1 phosphorylation and is followed by sequential phosphorylation of Thr-178, Thr-187, and finally Thr-184. Finally, we present a model for the chronological order of events governing TAB1-induced TAK1 autoactivation.     

Modeling the Epidermal Growth Factor—Epidermal Growth Factor Receptor L2 Domain Interaction: Implications for the Ligand Binding Process

Abstract

Signaling from the epidermal growth factor (EGF) receptor is triggered by the binding of lig-ands such as EGF or transforming growth factor alpha (TGF-α) and subsequent receptor dimerization. An understanding of these processes has been hindered by the lack of structural information about the ligand-bound, dimerized EGF receptor. Using an NMR-derived structure of EGF and a homology model of the major ligand binding domain of the EGF receptor and experimental data, we modeled the binding of EGF to this EGF receptor fragment. In this low resolution model of the complex, EGF sits across the second face of the EGF receptor L2 domain and EGF residues 10–16, 36–37, 40–47 bind to this face. The structural model is largely consistent with previously published NMR data describing the residues of TGF-α which interact strongly with the EGF receptor. Other EGF residues implicated in receptor binding are accounted by our proposal that the ligand binding is a two-step process with the EGF binding to at least one other site of the receptor. This three-dimensional model is expected to be useful in the design of ligand-based antagonists of the receptor.     

Does Phospholipid Methylation Play a Role in the Primary Mechanism of Action of Nerve Growth Factor?

Nerve growth factor (NGF)-untreated (naive) and neurite-bearing NGF-treated ("primed") PC12 rat pheochromocytoma cells were used as model system to study the role of phospholipid methylation in the NGF mechanism of action. The neurite-bearing cultures were deprived of NGF for 3 h before experimentation. Under both experimental conditions, the cells were labelled with [methyl-3H]methionine and then challenged with NGF for time periods ranging from 5 s to 30 min. Methylated phospholipids were extracted and then resolved and identified by TLC as phosphatidyl mono-, di-, and trimethyl ethanolamine. Quantification of the amount of radioactivity incorporated into each of the phospholipids indicated that NGF does not significantly alter phospholipid methylation either in naive or in neurite-bearing cells. Furthermore, using a methyltransferase inhibitor, it was found that neurite outgrowth still occurs when phospholipid methylation is almost completely blocked. These results indicate that phospholipid methylation does not play a primary role in the mechanism of action of NGF.     

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.