Tensin1 Requires Protein Phosphatase-1�� in Addition to RhoGAP DLC-1 to Control Cell Polarization, Migration, and Invasion

Tensin is a family of multidomain scaffold proteins that bind the cytoplasmic tail of β-integrins and localize to adhesions that anchor stress fibers in cells. Tensin expression is suppressed in cancer, especially metastatic cancer. The N-terminal domain of tensin1 associates with protein phosphatase-1α (PP1α) and mediates PP1α localization to adhesions. Here, we show F302A mutation in a KVXF motif of tensin1 abrogates binding to PP1α. The SH2 domain in tensin family member c-ten requires R474 to bind a RhoGAP called DLC-1 (deleted in liver cancer). We mutated the corresponding residue in tensin1, R1488A, and showed this reduces association with DLC-1. Unexpectedly, tensin1 F302A also had reduced association with DLC-1. Expression of tensin1 F302A or R1488A showed similar dominant phenotypes, with reduced cell polarization, lowered MLC20 phosphorylation and reduced levels of RhoA(GTP) compared with cells expressing tensin1 WT. However, migration and invasion of metastatic MDA MB 231 breast cancer cells were differentially affected by tensin1 mutated at F302A or R1488A. Cancer cells stably expressing F302A tensin1 showed increased migration and invasion compared with cells stably expressing either R1488A tensin1 or WT tensin1. This suggests that PP1α bound to tensin1 has additional effects in reducing migration and invasion that are not mediated through DLC-1. Our results show the importance of PP1α binding to tensin1 for the regulation of cell polarization, migration, and invasion.     

Comprehensive Analysis of Phosphorylation Sites in Tensin1 Reveals Regulation by p38MAPK

Tensin1 is the archetype of a family of focal adhesion proteins. Tensin1 has a phosphotyrosine binding domain that binds the cytoplasmic tail of β-integrin, a Src homology 2 domain that binds focal adhesion kinase, p130Cas, and the RhoGAP called deleted in liver cancer-1, a phosphatase and tensin homology domain that binds protein phosphatase-1α and other regions that bind F-actin. The association between tensin1 and these partners affects cell polarization, migration, and invasion. In this study we analyzed the phosphorylation of human S-tag-tensin1 expressed in HEK293 cells by mass spectrometry. Peptides covering >90% of the sequence initially revealed 50 phosphorylated serine/phosphorylated threonine (pSer/pThr) but no phosphorylated tyrosine (pTyr) sites. Addition of peroxyvanadate to cells to inhibit protein tyrosine phosphatases exposed 10 pTyr sites and addition of calyculin A to cells to inhibit protein phosphatases type 1 and 2A gave a total of 62 pSer/pThr sites. We also characterized two sites modified by O-linked N-acetylglucosamine. Tensin1 F302A, which does not bind protein phosphatase-1, showed > twofold enhanced phosphorylation of seven sites. The majority of pSer/pThr have adjacent proline (Pro) residues and we show endogenous p38 mitogen activated protein kinase (MAPK) associated with and phosphorylated tensin1 in an in vitro kinase assay. Recombinant p38α MAPK also phosphorylated S-tag-tensin1, resulting in decreased binding with deleted in liver cancer-1. Activation of p38 MAPK in cells by sorbitol-induced hyperosmotic stress increased phosphorylation of S-tag-tensin1, which reduced binding to deleted in liver cancer-1 and increased binding to endogenous pTyr proteins, including p130Cas and focal adhesion kinase. These data demonstrate that tensin1 is extensively phosphorylated on Ser/Thr residues in cells and phosphorylation by p38 MAPK regulates the specificity of the tensin1 Src homology 2 domain for binding to different proteins. Tensin1 provides a hub for connecting signaling pathways involving p38 MAP kinase, tyrosine kinases and RhoGTPases.Tensin1 is a protein localized at focal adhesions that acts as a scaffold for signaling (1). The tensin1 phosphotyrosine binding (PTB)1 domain binds the cytoplasmic tail of β-integrin (2), presumed to be the basis for focal adhesion localization. Human tensin1 interacts with actin by capping the barbed ends and cross-linking actin filaments through two different actin binding regions (3). Actin binding regions were identified in chicken tensin1 at residues 1–263, 263–463, and 889–1143 (4). The C terminus region of tensin1, as well as family members tensin2, tensin3, and c-ten, has adjacent Src homology 2 (SH2) and PTB domains that interact with the tyrosine phosphorylated proteins Dok2 and PDK1 (5) as well as PI3 kinase, p130Cas, and focal adhesion kinase (FAK) (6), thereby posing a role for tensin1 in multiple signal transduction pathways. The N-terminal region of tensin1 contains a domain that is related in sequence to the tumor suppressor protein and PIP3 phosphatase called phosphatase and tensin homologue (PTEN) (3). This domain of tensin1 binds the alpha isoform of protein phosphatase 1 (PP1) (7), the major protein Ser/Thr phosphatase in cells that regulates a variety of signaling pathways. The SH2 domain of tensin1 also associates with a RhoGAP protein called deleted in liver cancer-1 (DLC-1) but does not require Tyr phosphorylation of DLC-1 (8). DLC-1 has a role in cell migration and is a negative regulator of tumor formation (8–10). Human breast carcinoma, prostate carcinoma, head and neck squamous cell carcinoma, and melanoma all exhibit reduced expression of tensin1, suggesting a tumor suppressor action (11). In addition, various cancer cell lines do not express detectable levels of tensin1 protein relative to normal fibroblasts that have abundant expression (1, 7). Re-expression of tensin1 in cancer cells promoted formation of focal adhesions (4) and decreased migration and invasion of MDA MB 231 human breast cancer cells (12). Taken together, these studies support a model for tensin1 as a tumor suppressor that acts as a scaffold protein for various signaling enzymes.Tensin1 was first shown to be tyrosine phosphorylated following concentration by immunoprecipitation and immunoblotting with a pTyr antibody (6). Tyrosine phosphorylation of tensin1 was only detected if fibroblasts were plated on fibronectin, laminin, or vitronectin (13), suggesting that tensin1 tyrosine phosphorylation depends on integrin-mediated signaling. Jiang et al. (14) showed increased tyrosine phosphorylation of tensin1 when cells were treated with platelet-derived growth factor. In addition, epidermal growth factor treatment of human gastric epithelial cells stimulated tyrosine phosphorylation of tensin1 and this stimulation was inhibited with the nonsteroidal anti-inflammatory drug indomethacin (15). Cells transformed by the oncogene p210BCR/ABL contained tyrosine phosphorylated tensin1 (16). Treatment of rat aortic smooth muscle cells with angiotensin or thrombin also showed an increase in tensin1 tyrosine phosphorylation (17). Rapid turnover of pTyr by phosphatases presumably keeps tensin1 pTyr levels low in cells following stimulation. Different publications report tensin1 is phosphorylated on Ser and Thr residues, but data supporting these claims was not shown (1, 3, 18, 19). Phosphoproteomics implementing shotgun mass spectrometry techniques have turned up as many as 20 pTyr, 30 pSer, and 8 pThr peptides from human tensin (www.phosphosite.org). However, to date no comprehensive analysis of tensin1 phosphorylation has been reported.We previously identified residue F302 in the KVEF motif in tensin1 as necessary for PP1α binding (12). Tensin1 F302A showed a reduced electrophoretic mobility in SDS-PAGE compared with tensin1 wild type, suggesting an increase in tensin1 phosphorylation because of absence of bound PP1. We also observed less DLC-1 binding to tensin1 F302A, but it is not known whether this was because of an increase in tensin1 phosphorylation (12). The tensin1 F302A did not suppress cancer cell invasion like tensin1 wild type (12), and this could be because of loss of PP1 binding, or less DLC-1 binding, or changes in phosphorylation.In the present study we comprehensively analyze the phosphorylation of human S-tag-tensin1. Addition of phosphatase inhibitors to cells is shown to enhance phosphorylation to yield a total of 62 Ser/Thr phosphorylation sites and expose 10 Tyr sites not otherwise seen. The majority of Ser/Thr sites have adjacent proline residues and we identify p38α MAPK activity associated with tensin1. The p38MAPK phosphorylation of tensin1 alters binding of DLC-1, p130Cas and FAK. Our results demonstrate that tensin1 is extensively phosphorylated on Ser/Thr residues in addition to Tyr residues and this phosphorylation alters association with its SH2 domain binding partners.     

Independent interactions of phosphorylated β-catenin with E-cadherin at cell-cell contacts and APC at cell protrusions

Background

The APC tumour suppressor functions in several cellular processes including the regulation of β-catenin in Wnt signalling and in cell adhesion and migration.

Findings

In this study, we establish that in epithelial cells N-terminally phosphorylated β-catenin specifically localises to several subcellular sites including cell-cell contacts and the ends of cell protrusions. N-terminally phosphorylated β-catenin associates with E-cadherin at adherens junctions and with APC in cell protrusions. We isolated APC-rich protrusions from stimulated cells and detected β-catenin, GSK3β and CK1α, but not axin. The APC/phospho-β-catenin complex in cell protrusions appears to be distinct from the APC/axin/β-catenin destruction complex. GSK3β phosphorylates the APC-associated population of β-catenin, but not the cell junction population. β-catenin associated with APC is rapidly phosphorylated and dephosphorylated. HGF and wound-induced cell migration promote the localised accumulation of APC and phosphorylated β-catenin at the leading edge of migrating cells. APC siRNA and analysis of colon cancer cell lines show that functional APC is required for localised phospho-β-catenin accumulation in cell protrusions.

Conclusions

We conclude that N-terminal phosphorylation of β-catenin does not necessarily lead to its degradation but instead marks distinct functions, such as cell migration and/or adhesion processes. Localised regulation of APC-phospho-β-catenin complexes may contribute to the tumour suppressor activity of APC.     

Structural basis and kinetics of force-induced conformational changes of an αA domain-containing integrin

Background

Integrin α_Lβ₂ (lymphocyte function-associated antigen, LFA-1) bears force upon binding to its ligand intercellular adhesion molecule 1 (ICAM-1) when a leukocyte adheres to vascular endothelium or an antigen presenting cell (APC) during immune responses. The ligand binding propensity of LFA-1 is related to its conformations, which can be regulated by force. Three conformations of the LFA-1 αA domain, determined by the position of its α₇-helix, have been suggested to correspond to three different affinity states for ligand binding.

Methodology/Principal Findings

The kinetics of the force-driven transitions between these conformations has not been defined and dynamically coupled to the force-dependent dissociation from ligand. Here we show, by steered molecular dynamics (SMD) simulations, that the αA domain was successively transitioned through three distinct conformations upon pulling the C-terminus of its α₇-helix. Based on these sequential transitions, we have constructed a mathematical model to describe the coupling between the αA domain conformational changes of LFA-1 and its dissociation from ICAM-1 under force. Using this model to analyze the published data on the force-induced dissociation of single LFA-1/ICAM-1 bonds, we estimated the force-dependent kinetic rates of interstate transition from the short-lived to intermediate-lived and from intermediate-lived to long-lived states. Interestingly, force increased these transition rates; hence activation of LFA-1 was accelerated by pulling it via an engaged ICAM-1.

Conclusions/Significance

Our study defines the structural basis for mechanical regulation of the kinetics of LFA-1 αA domain conformational changes and relates these simulation results to experimental data of force-induced dissociation of single LFA-1/ICAM-1 bonds by a new mathematical model, thus provided detailed structural and kinetic characterizations for force-stabilization of LFA-1/ICAM-1 interaction.     

Mechanism of cigarette smoke condensate-induced acute inflammatory response in human bronchial epithelial cells

Background

To demonstrate the involvement of tobacco smoking in the pathophysiology of lung disease, the responses of pulmonary epithelial cells to cigarette smoke condensate (CSC) — the particulate fraction of tobacco smoke — were examined.

Methods

The human alveolar epithelial cell line A549 and normal human bronchial epithelial cells (NHBEs) were exposed to 0.4 μg/ml CSC, a concentration that resulted in >90% cell survival and <5% apoptosis. Changes in gene expression and signaling responses were determined by RT-PCR, western blotting and immunocytofluorescence.

Results

NHBEs exposed to CSC showed increased expression of the inflammatory mediators sICAM-1, IL-1β, IL-8 and GM-CSF, as determined by RT-PCR. CSC-induced IL-1β expression was reduced by PD98059, a blocker of mitogen-actived protein kinase (MAPK) kinase (MEK), and by PDTC, a NFκB inhibitor. Analysis of intracellular signaling pathways, using antibodies specific for phosphorylated MAPKs (extracellular signal-regulated kinase [ERK]-1/2), demonstrated an increased level of phosphorylated ERK1/2 with increasing CSC concentration. Nuclear localization of phosphorylated ERK1/2 was seen within 30 min of CSC exposure and was inhibited by PD98059. Increased phosphorylation and nuclear translocation of IκB was also seen after CSC exposure. A549 cells transfected with a luciferase reporter plasmid containing a NFκB-inducible promoter sequence and exposed to CSC (0.4 μg/ml) or TNF-α (50 ng/ml) had an increased reporter activity of approximately 2-fold for CSC and 3.5-fold for TNF-α relative to untreated controls.

Conclusion

The acute phase response of NHBEs to cigarette smoke involves activation of both MAPK and NFκB.     

Lack of renal 11 beta-hydroxysteroid dehydrogenase type 2 at birth, a targeted temporal window for neonatal glucocorticoid action in human and mice

Background

Glucocorticoid hormones play a major role in fetal organ maturation. Yet, excessive glucocorticoid exposure in utero can result in a variety of detrimental effects, such as growth retardation and increased susceptibility to the development of hypertension. To protect the fetus, maternal glucocorticoids are metabolized into inactive compounds by placental 11beta-hydroxysteroid dehydrogenase type2 (11βHSD2). This enzyme is also expressed in the kidney, where it prevents illicit occupation of the mineralocorticoid receptor by glucocorticoids. We investigated the role of renal 11βHSD2 in the control of neonatal glucocorticoid metabolism in the human and mouse.

Methods

Cortisol (F) and cortisone (E) concentrations were measured in maternal plasma, umbilical cord blood and human newborn urine using HPLC. 11βHSD2 activity was indirectly assessed by comparing the F/E ratio between maternal and neonatal plasma (placental activity) and between plasma and urine in newborns (renal activity). Direct measurement of renal 11βHSD2 activity was subsequently evaluated in mice at various developmental stages. Renal 11βHSD2 mRNA and protein expression were analyzed by quantitative RT-PCR and immunohistochemistry during the perinatal period in both species.

Results

We demonstrate that, at variance with placental 11βHSD2 activity, renal 11βHSD2 activity is weak in newborn human and mouse and correlates with low renal mRNA levels and absence of detectable 11βHSD2 protein.

Conclusions

We provide evidence for a weak or absent expression of neonatal renal 11βHSD2 that is conserved among species. This temporal and tissue-specific 11βHSD2 expression could represent a physiological window for glucocorticoid action yet may constitute an important predictive factor for adverse outcomes of glucocorticoid excess through fetal programming.     

A novel dimeric inhibitor targeting Beta2GPI in Beta2GPI/antibody complexes implicated in antiphospholipid syndrome

Background

β2GPI is a major antigen for autoantibodies associated with antiphospholipid syndrome (APS), an autoimmune disease characterized by thrombosis and recurrent pregnancy loss. Only the dimeric form of β2GPI generated by anti-β2GPI antibodies is pathologically important, in contrast to monomeric β2GPI which is abundant in plasma.

Principal Findings

We created a dimeric inhibitor, A1-A1, to selectively target β2GPI in β2GPI/antibody complexes. To make this inhibitor, we isolated the first ligand-binding module from ApoER2 (A1) and connected two A1 modules with a flexible linker. A1-A1 interferes with two pathologically important interactions in APS, the binding of β2GPI/antibody complexes with anionic phospholipids and ApoER2. We compared the efficiency of A1-A1 to monomeric A1 for inhibition of the binding of β2GPI/antibody complexes to anionic phospholipids. We tested the inhibition of β2GPI present in human serum, β2GPI purified from human plasma and the individual domain V of β2GPI. We demonstrated that when β2GPI/antibody complexes are formed, A1-A1 is much more effective than A1 in inhibition of the binding of β2GPI to cardiolipin, regardless of the source of β2GPI. Similarly, A1-A1 strongly inhibits the binding of dimerized domain V of β2GPI to cardiolipin compared to the monomeric A1 inhibitor. In the absence of anti-β2GPI antibodies, both A1-A1 and A1 only weakly inhibit the binding of pathologically inactive monomeric β2GPI to cardiolipin.

Conclusions

Our results suggest that the approach of using a dimeric inhibitor to block β2GPI in the pathological multivalent β2GPI/antibody complexes holds significant promise. The novel inhibitor A1-A1 may be a starting point in the development of an effective therapeutic for antiphospholipid syndrome.     

B-cell depletion therapy in patients with diffuse systemic sclerosis associates with a significant decrease in PDGFR expression and activation in spindle-like cells in the skin

Introduction

Recently, several studies assessing the clinical efficacy of rituximab (RTX) in systemic sclerosis (SSc) have reported encouraging results. We aimed at exploring whether RTX exerts its beneficial effects on fibrosis through attenuation of platelet-derived growth factor receptor (PDGFR) pathway activation.

Methods

We immunohistochemically assessed skin biopsies obtained from eight patients with SSc prior to and 6 months following RTX treatment, three control SSc patients (at the same time points) and three healthy subjects. We assessed the expression of platelet-derived growth factor, PDGFR and phosphorylated (activated) PDGFR.

Results

We found a strong correlation of PDGFRα and PDGFRβ expression on spindle-like cells and collagen deposition in SSc biopsies (r = 0.97 and r = 0.96 for PDGFRα and PDGFRβ, respectively; P < 0.0001 for both), indicating a strong link between PDGFR expression and fibrosis. Expression of PDGFRα and PDGFRβ in the papillary dermis significantly decreased following RTX administration (mean ± standard error of the mean at baseline vs. 6 months, respectively: PDGFRα, 42.05 ± 5.03 vs. 26.85 ± 3.00, P = 0.004; and PDGFRβ, 37.14 ± 4.94 vs. 24.01 ± 3.27, P = 0.012). Similarly, expression of phosphorylated PDGFRα and PDGFRβ in the papillary dermis significantly decreased following RTX administration (P = 0.006 and P = 0.013 for phospho-PDGFRα and phospho-PDGFRβ, respectively). No changes in platelet-derived growth factor tissue expression or serum levels were found following RTX treatment.

Conclusion

RTX may favorably affect skin fibrosis through attenuation of PDGFR expression and activation, a finding that supports a disease-modifying role of RTX in SSc. Large-scale, multicenter studies are needed to further explore the efficacy of RTX in SSc.     

Zona pellucida domain-containing protein β-tectorin is crucial for zebrafish proper inner ear development

Background

The zona pellucida (ZP) domain is part of many extracellular proteins with diverse functions from structural components to receptors. The mammalian β-tectorin is a protein of 336 amino acid residues containing a single ZP domain and a putative signal peptide at the N-terminus of the protein. It is 1 component of a gel-like structure called the tectorial membrane which is involved in transforming sound waves into neuronal signals and is important for normal auditory function. β-Tectorin is specifically expressed in the mammalian and avian inner ear.

Methodology/Principal Findings

We identified and cloned the gene encoding zebrafish β-tectorin. Through whole-mount in situ hybridization, we demonstrated that β-tectorin messenger RNA was expressed in the otic placode and specialized sensory patch of the inner ear during zebrafish embryonic stages. Morpholino knockdown of zebrafish β-tectorin affected the position and number of otoliths in the ears of morphants. Finally, swimming behaviors of β-tectorin morphants were abnormal since the development of the inner ear was compromised.

Conclusions/Significance

Our results reveal that zebrafish β-tectorin is specifically expressed in the zebrafish inner ear, and is important for regulating the development of the zebrafish inner ear. Lack of zebrafish β-tectorin caused severe defects in inner ear formation of otoliths and function.     

βB1-crystallin: thermodynamic profiles of molecular interactions

Background

β-Crystallins are structural proteins maintaining eye lens transparency and opacification. Previous work demonstrated that dimerization of both βA3 and βB2 crystallins (βA3 and βB2) involves endothermic enthalpy of association (∼8 kcal/mol) mediated by hydrophobic interactions.

Methodology/Principal Findings

Thermodynamic profiles of the associations of dimeric βA3 and βB1 and tetrameric βB1/βA3 were measured using sedimentation equilibrium. The homo- and heteromolecular associations of βB1 crystallin are dominated by exothermic enthalpy (−13.3 and −24.5 kcal/mol, respectively).

Conclusions/Significance

Global thermodynamics of βB1 interactions suggest a role in the formation of stable protein complexes in the lens via specific van der Waals contacts, hydrogen bonds and salt bridges whereas those β-crystallins which associate by predominately hydrophobic forces participate in a weaker protein associations.     

The effects of sleep hypoxia on coagulant factors and hepatic inflammation in emphysematous rats

Objectives

To develop a sleep hypoxia (SH) in emphysema (SHE) rat model and to explore whether SHE results in more severe hepatic inflammation than emphysema alone and whether the inflammation changes levels of coagulant/anticoagulant factors synthesized in the liver.

Methods

Seventy-five rats were put into 5 groups: SH control (SHCtrl), treated with sham smoke exposure (16 weeks) and SH exposure (12.5% O₂, 3 h/d, latter 8 weeks); emphysema control (ECtrl), smoke exposure and sham SH exposure (21% O₂); short SHE (SHEShort), smoke exposure and short SH exposure (1.5 h/d); mild SHE (SHEMild), smoke exposure and mild SH exposure (15% O₂); standard SHE (SHEStand), smoke exposure and SH exposure. Therefore, ECtrl, SHEShort, SHEMild and SHEStand group were among emphysematous groups. Arterial blood gas (ABG) data was obtained during preliminary tests. After exposure, hepatic inflammation (interleukin -6 [IL-6] mRNA and protein, tumor necrosis factor α [TNFα] mRNA and protein) and liver coagulant/anticoagulant factors (antithrombin [AT], fibrinogen [FIB] and Factor VIII [F VIII]) were evaluated. SPSS 11.5 software was used for statistical analysis.

Results

Characteristics of emphysema were obvious in emphysematous groups and ABGs reached SH criteria on hypoxia exposure. Hepatic inflammation parameters and coagulant factors are the lowest in SHCtrl and the highest in SHEStand while AT is the highest in SHCtrl and the lowest in SHEStand. Inflammatory cytokines of liver correlate well with coagulant factors positively and with AT negatively.

Conclusions

When SH is combined with emphysema, hepatic inflammation and coagulability enhance each other synergistically and produce a more significant liver-derivative inflammatory and prothrombotic status.     

Characterization of the functional domain of β2-microglobulin from the Asian seabass, Lates calcarifer

Background

β2-Microglobulin (β₂M) is the light chain of major histocompatibility class I (MHC I) that binds non-covalently with the α heavy chain. Both proteins attach to the antigen peptide, presenting a complex to the T cell to be destroyed via the immune mechanism.

Methodology/Principal Findings

In this study, a cDNA sequence encoding β₂M in the Asian seabass (Lates calcarifer) was identified and analyzed using in silico approaches to predict and characterize its functional domain. The β₂M cDNA contains an open reading frame (ORF) of 351 bases with a coding capacity of 116 amino acids. A large portion of the protein consists of the IG constant domain (IGc1), similar to β₂M sequences from other species studied thus far. Alignment of the IGc1 domains of β₂M from L. calcarifer and other species shows a high degree of overall conservation. Seven amino acids were found to be conserved across taxa whereas conservation between L. calcarifer and other fish species was restricted to 14 amino acids at identical conserved positions.

Conclusion/Significance

As the L. calcarifer β₂M protein analyzed in this study contains a functional domain similar to that of β₂M proteins in other species, it can be postulated that the β₂M proteins from L. calcarifer and other organisms are derived from a common ancestor and thus have a similar immune function. Interestingly, fish β₂M genes could also be classified according to the ecological habitat of the species, i.e. whether it is from a freshwater, marine or euryhaline environment.     

The LDL Receptor-Related Protein 1 (LRP1) Regulates the PDGF Signaling Pathway by Binding the Protein Phosphatase SHP-2 and Modulating SHP-2- Mediated PDGF Signaling Events

Background

The PDGF signaling pathway plays a major role in several biological systems, including vascular remodeling that occurs following percutaneous transluminal coronary angioplasty. Recent studies have shown that the LDL receptor-related protein 1 (LRP1) is a physiological regulator of the PDGF signaling pathway. The underlying mechanistic details of how this regulation occurs have yet to be resolved. Activation of the PDGF receptor β (PDGFRβ) leads to tyrosine phosphorylation of the LRP1 cytoplasmic domain within endosomes and generates an LRP1 molecule with increased affinity for adaptor proteins such as SHP-2 that are involved in signaling pathways. SHP-2 is a protein tyrosine phosphatase that positively regulates the PDGFRβ pathway, and is required for PDGF-mediated chemotaxis. We investigated the possibility that LRP1 may regulate the PDGFRβ signaling pathway by binding SHP-2 and competing with the PDGFRβ for this molecule.

Methodology/Principal Findings

To quantify the interaction between SHP-2 and phosphorylated forms of the LRP1 intracellular domain, we utilized an ELISA with purified recombinant proteins. These studies revealed high affinity binding of SHP-2 to phosphorylated forms of both LRP1 intracellular domain and the PDGFRβ kinase domain. By employing the well characterized dynamin inhibitor, dynasore, we established that PDGF-induced SHP-2 phosphorylation primarily occurs within endosomal compartments, the same compartments in which LRP1 is tyrosine phosphorylated by activated PDGFRβ. Immunofluorescence studies revealed colocalization of LRP1 and phospho-SHP-2 following PDGF stimulation of fibroblasts. To define the contribution of LRP1 to SHP-2-mediated PDGF chemotaxis, we employed fibroblasts expressing LRP1 and deficient in LRP1 and a specific SHP-2 inhibitor, NSC-87877. Our results reveal that LRP1 modulates SHP-2-mediated PDGF-mediated chemotaxis.

Conclusions/Significance

Our data demonstrate that phosphorylated forms of LRP1 and PDGFRβ compete for SHP-2 binding, and that expression of LRP1 attenuates SHP-2-mediated PDGF signaling events.     

Dystroglycan, Tks5 and Src mediated assembly of podosomes in myoblasts

Background

Dystroglycan is a ubiquitously expressed cell adhesion receptor best understood in its role as part of the dystrophin glycoprotein complex of mature skeletal muscle. Less is known of the role of dystroglycan in more fundamental aspects of cell adhesion in other cell types, nor of its role in myoblast cell adhesion.

Principal Findings

We have examined the role of dystroglycan in the early stages of myoblast adhesion and spreading and found that dystroglycan initially associates with other adhesion proteins in large puncta morphologically similar to podosomes. Using a human SH3 domain phage display library we identified Tks5, a key regulator of podosomes, as interacting with β-dystroglycan. We verified the interaction by immunoprecipitation, GST-pulldown and immunfluorescence localisation. Both proteins localise to puncta during early phases of spreading, but importantly following stimulation with phorbol ester, also localise to structures indistinguishable from podosomes. Dystroglycan overexpression inhibited podosome formation by sequestering Tks5 and Src. Mutation of dystroglycan tyrosine 890, previously identified as a Src substrate, restored podosome formation.

Conclusions

We propose therefore, that Src-dependent phosphorylation of β-dystroglycan results in the formation of a Src/dystroglycan complex that drives the SH3-mediated association between dystroglycan and Tks5 which together regulate podosome formation in myoblasts.     

Delayed re-epithelialization in periostin-deficient mice during cutaneous wound healing

Background

Matricellular proteins, including periostin, are important for tissue regeneration.

Methods and Findings

Presently we investigated the function of periostin in cutaneous wound healing by using periostin-deficient (−/−) mice. Periostin mRNA was expressed in both the epidermis and hair follicles, and periostin protein was located at the basement membrane in the hair follicles together with fibronectin and laminin γ2. Periostin was associated with laminin γ2, and this association enhanced the proteolytic cleavage of the laminin γ2 long form to produce its short form. To address the role of periostin in wound healing, we employed a wound healing model using WT and periostin−/− mice and the scratch wound assay in vitro. We found that the wound closure was delayed in the periostin−/− mice coupled with a delay in re-epithelialization and with reduced proliferation of keratinocytes. Furthermore, keratinocyte proliferation was enhanced in periostin-overexpressing HaCaT cells along with up-regulation of phosphorylated NF-κB.

Conclusion

These results indicate that periostin was essential for keratinocyte proliferation for re-epithelialization during cutaneous wound healing.