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.     

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.     

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.     

The Nuclear Factor NF-��B Pathway in Inflammation

The nuclear factor NF-κB pathway has long been considered a prototypical proinflammatory signaling pathway, largely based on the role of NF-κB in the expression of proinflammatory genes including cytokines, chemokines, and adhesion molecules. In this article, we describe how genetic evidence in mice has revealed complex roles for the NF-κB in inflammation that suggest both pro- and anti-inflammatory roles for this pathway. NF-κB has long been considered the “holy grail” as a target for new anti-inflammatory drugs; however, these recent studies suggest this pathway may prove a difficult target in the treatment of chronic disease. In this article, we discuss the role of NF-κB in inflammation in light of these recent studies.NF-κB has long been considered a prototypical proinflammatory signaling pathway, largely based on the activation of NF-κB by proinflammatory cytokines such as interleukin 1 (IL-1) and tumor necrosis factor α (TNFα), and the role of NF-κB in the expression of other proinflammatory genes including cytokines, chemokines, and adhesion molecules, which has been extensively reviewed elsewhere. But inflammation is a complex physiological process and the role of NF-κB in the inflammatory response cannot be extrapolated from in vitro studies. In this article, we describe how genetic evidence in mice has revealed complex roles for the NF-κB pathway in inflammation.     

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.     

Silencing of Reversion-Inducing Cysteine-Rich Protein with Kazal Motifs Stimulates Hyperplastic Phenotypes through Activation of Epidermal Growth Factor Receptor and Hypoxia-Inducible Factor-2α

Reversion-inducing cysteine-rich protein with Kazal motifs (RECK, a tumor suppressor) is down-regulated by the oncogenic signals and hypoxia, but the biological function of RECK in early tumorigenic hyperplastic phenotypes is largely unknown. Knockdown of RECK by small interfering RNA (siRECK) or hypoxia significantly promoted cell proliferation in various normal epithelial cells. Hypoxia as well as knockdown of RECK by siRNA increased the cell cycle progression, the levels of cyclin D1 and c-Myc, and the phosphorylation of Rb protein (p-pRb), but decreased the expression of p21^cip1, p27^kip1, and p16^ink4A. HIF-2α was upregulated by knockdown of RECK, indicating HIF-2α is a downstream target of RECK. As knockdown of RECK induced the activation of epidermal growth factor receptor (EGFR) and treatment of an EGFR kinase inhibitor, gefitinib, suppressed HIF-2α expression induced by the silencing of RECK, we can suggest that the RECK silenicng-EGFR-HIF-2α axis might be a key molecular mechanism to induce hyperplastic phenotype of epithelial cells. It was also found that shRNA of RECK induced larger and more numerous colonies than control cells in an anchorage-independent colony formation assay. Using a xenograft assay, epithelial cells with stably transfected with shRNA of RECK formed a solid mass earlier and larger than those with control cells in nude mice. In conclusion, the suppression of RECK may promote the development of early tumorigenic hyperplastic characteristics in hypoxic stress.     

Lysine 63-linked Polyubiquitination of TAK1 at Lysine 158 Is Required for Tumor Necrosis Factor ��- and Interleukin-1��-induced IKK/NF-��B and JNK/AP-1 Activation

Transforming growth factor-β-activated kinase 1 (TAK1) plays an essential role in the tumor necrosis factor α (TNFα)- and interleukin-1β (IL-1β)-induced IκB kinase (IKK)/nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK)/activator protein 1 (AP-1) activation. Here we report that TNFα and IL-1β induce Lys⁶³-linked TAK1 polyubiquitination at the Lys¹⁵⁸ residue within the kinase domain. Tumor necrosis factor receptor-associated factors 2 and 6 (TRAF2 and -6) act as the ubiquitin E3 ligases to mediate Lys⁶³-linked TAK1 polyubiquitination at the Lys¹⁵⁸ residue in vivo and in vitro. Lys⁶³-linked TAK1 polyubiquitination at the Lys¹⁵⁸ residue is required for TAK1-mediated IKK complex recruitment. Reconstitution of TAK1-deficient mouse embryo fibroblast cells with TAK1 wild type or a TAK1 mutant containing a K158R mutation revealed the importance of this site in TNFα and IL-1β-mediated IKK/NF-κB and JNK/AP-1 activation as well as IL-6 gene expression. Our findings demonstrate that Lys⁶³-linked polyubiquitination of TAK1 at Lys¹⁵⁸ is essential for its own kinase activation and its ability to mediate its downstream signal transduction pathways in response to TNFα and IL-1β stimulation.     

Regulation of Epidermal Growth Factor Receptor Ubiquitination and Trafficking by the USP8·STAM Complex

Reversible ubiquitination of activated receptor complexes signals their sorting between recycling and degradation and thereby dictates receptor fate. The deubiquitinating enzyme ubiquitin-specific protease 8 (USP8/UBPy) has been previously implicated in the regulation of the epidermal growth factor receptor (EGFR); however, the molecular mechanisms governing its recruitment and activity in this context remain unclear. Herein, we investigate the role of USP8 in countering ligand-induced ubiquitination and down-regulation of EGFR and characterize a subset of protein-protein interaction determinants critical for this function. USP8 depletion accelerates receptor turnover, whereas loss of hepatocyte growth factor-regulated substrate (Hrs) rescues this phenotype, indicating that USP8 protects EGFR from degradation via an Hrs-dependent pathway. Catalytic inactivation of USP8 incurs EGFR hyperubiquitination and promotes receptor localization to endosomes marked by high ubiquitin content. These phenotypes require the central region of USP8, containing three extended Arg-X-X-Lys (RXXK) motifs that specify direct low affinity interactions with the SH3 domain(s) of ESCRT-0 proteins, STAM1/2. The USP8·STAM complex critically impinges on receptor ubiquitination status and modulates ubiquitin dynamics on EGFR-positive endosomes. Consequently, USP8-mediated deubiquitination slows progression of EGFR past the early-to-recycling endosome circuit in a manner dependent upon the RXXK motifs. Collectively, these findings demonstrate a role for the USP8·STAM complex as a protective mechanism regulating early endosomal sorting of EGFR between pathways destined for lysosomal degradation and recycling.     

Phosphorylation of Thr-516 and Ser-520 in the Kinase Activation Loop of MEKK3 Is Required for Lysophosphatidic Acid-mediated Optimal I��B Kinase �� (IKK��)/Nuclear Factor-��B (NF-��B) Activation

MEKK3 serves as a critical intermediate signaling molecule in lysophosphatidic acid-mediated nuclear factor-κB (NF-κB) activation. However, the precise regulation for MEKK3 activation at the molecular level is still not fully understood. Here we report the identification of two regulatory phosphorylation sites at Thr-516 and Ser-520 within the kinase activation loop that is essential for MEKK3-mediated IκB kinase β (IKKβ)/NF-κB activation. Substitution of these two residues with alanine abolished the ability of MEKK3 to activate IKKβ/NF-κB, whereas replacement with acidic residues rendered MEKK3 constitutively active. Furthermore, substitution of these two residues with alanine abolished the ability of MEKK3 to mediate lysophosphatidic acid-induced optimal IKKβ/NF-κB activation.