Regulating Cell Matrix Research Articles

Integrin α2 is an early marker for osteoclast differentiation that contributes to key steps in osteoclastogenesis.

Osteoclasts determine bone tissue turnover. Their increased activity causes osteoporosis, their dysfunction osteopetrosis. Murine monocytic ER-Hoxb8 cells differentiate into OCs upon treatment with M-CSF and RANKL and upregulate the collagen-binding integrin α2β1 distinctly earlier than other OC markers, such as the OC-associated receptor, OSCAR. Integrin α2β1 promotes OC differentiation at multiple levels by stimulating differentiation-relevant genes, by regulating cell matrix adhesion and the formation of adhesion-promoting protrusions, and by the upregulation of proteins involved in precursor cell fusion. The two key factors in osteoclastogenesis, RANK and NFATc1, were essentially unaffected after knocking out the ITGA2 gene encoding integrin α2 subunit. However, compared to integrin α2β1 expressing ER-Hoxb8 cells, ITGA2-deficient cells adhered differently with more branched filopodia and significantly longer tunneling nanotubes. Despite the higher number of fusion-relevant TNTs, they form fewer syncytia. They also resorb less hydroxyapatite, because integrin α2β1 regulates expression of lacuna proteins necessary for bone matrix resorption. The impaired syncytia formation of ITGA2-deficient OC precursor cells also correlated with reduced gene activation of fusion-supporting DC-STAMP and with an almost abolished transcription of tetraspanin CD9. CD9 only partially colocalized with integrin α2β1 in TNTs and filopodia of integrin α2β1-expressing OC precursors. Our findings define integrin α2β1 as an early marker of OC differentiation.

Noncanonical Wnt/Ror2 signaling regulates cell-matrix adhesion to prompt directional tumor cell invasion in breast cancer.

Cell-extracellular matrix (ECM) interactions represent fundamental exchanges during tumor progression, yet how particular signal-transduction factors prompt the conversion of tumor cells into migratory populations capable of systemic spread during metastasis remains elusive. We demonstrate that the noncanonical Wnt receptor, Ror2, regulates tumor cell-driven matrix remodeling and invasion in breast cancer. Ror2 loss-of-function (LOF) triggers the disruption of E-cadherin within tumor cells, accompanied by an increase in tumor cell invasion and collagen realignment in three-dimensional cultures. RNA sequencing of Ror2-deficient organoids further uncovered alterations in actin cytoskeleton, cell adhesion, and collagen cross-linking gene expression programs. Spatially, we pinpoint the up-regulation and redistribution of α5 and β3 integrins together with the production of fibronectin in areas of invasion downstream of Ror2 loss. Wnt/β-catenin-dependent and Wnt/Ror2 alternative Wnt signaling appear to regulate distinct functions for tumor cells regarding their ability to modify cell-ECM exchanges during invasion. Furthermore, blocking either integrin or focal adhesion kinase (FAK), a downstream mediator of integrin-mediated signal transduction, abrogates the enhanced migration observed upon Ror2 loss. These results reveal a critical function for the alternative Wnt receptor, Ror2, as a determinant of tumor cell-driven ECM exchanges during cancer invasion and metastasis.

Untangling the Extracellular Matrix of Idiopathic Epiretinal Membrane: A Path Winding among Structure, Interactomics and Translational Medicine.

Idiopathic epiretinal membranes (iERMs) are fibrocellular sheets of tissue that develop at the vitreoretinal interface. The iERMs consist of cells and an extracellular matrix (ECM) formed by a complex array of structural proteins and a large number of proteins that regulate cell–matrix interaction, matrix deposition and remodelling. Many components of the ECM tend to produce a layered pattern that can influence the tractional properties of the membranes. We applied a bioinformatics approach on a list of proteins previously identified with an MS-based proteomic analysis on samples of iERM to report the interactome of some key proteins. The performed pathway analysis highlights interactions occurring among ECM molecules, their cell receptors and intra- or extracellular proteins that may play a role in matrix biology in this special context. In particular, integrin β1, cathepsin B, epidermal growth factor receptor, protein-glutamine gamma-glutamyltransferase 2 and prolow-density lipoprotein receptor-related protein 1 are key hubs in the outlined protein–protein cross-talks. A section on the biomarkers that can be found in the vitreous humor of patients affected by iERM and that can modulate matrix deposition is also presented. Finally, translational medicine in iERM treatment has been summed up taking stock of the techniques that have been proposed for pharmacologic vitreolysis.

Endoglin and MMP14 Contribute to Ewing Sarcoma Spreading by Modulation of Cell-Matrix Interactions.

Endoglin (ENG) is a mesenchymal stem cell (MSC) marker typically expressed by active endothelium. This transmembrane glycoprotein is shed by matrix metalloproteinase 14 (MMP14). Our previous work demonstrated potent preclinical activity of first-in-class anti-ENG antibody-drug conjugates as a nascent strategy to eradicate Ewing sarcoma (ES), a devastating rare bone/soft tissue cancer with a putative MSC origin. We also defined a correlation between ENG and MMP14 expression in ES. Herein, we show that ENG expression is significantly associated with a dismal prognosis in a large cohort of ES patients. Moreover, both ENG/MMP14 are frequently expressed in primary ES tumors and metastasis. To deepen in their functional relevance in ES, we conducted transcriptomic and proteomic profiling of in vitro ES models that unveiled a key role of ENG and MMP14 in cell mechano-transduction. Migration and adhesion assays confirmed that loss of ENG disrupts actin filament assembly and filopodia formation, with a concomitant effect on cell spreading. Furthermore, we observed that ENG regulates cell–matrix interaction through activation of focal adhesion signaling and protein kinase C expression. In turn, loss of MMP14 contributed to a more adhesive phenotype of ES cells by modulating the transcriptional extracellular matrix dynamics. Overall, these results suggest that ENG and MMP14 exert a significant role in mediating correct spreading machinery of ES cells, impacting the aggressiveness of the disease.

Fermitin family homolog 2 (Kindlin-2) affects vascularization during the wound healing process by regulating the Wnt/β-catenin signaling pathway in vascular endothelial cells

ABSTRACT Kindlin-2 is a member of the FERM-containing cytoskeletal protein family that regulates cell–matrix interactions. Previous studies have shown that Kindlin-2 recruits focal adhesion proteins and regulates integration by binding to the focal adhesion region of the integrin β-segment. Although Kindlin-2 has been reported to be involved in various skin diseases and many kinds of tumors, its role in the skin wound healing process remains unclear. The aim of the present study was to investigate the role of Kindlin-2 in the regulation of wound healing. The effects of Kindlin-2 on wound healing were studied by a wound healing model, kindlin-2 (±) mice. The effects of Kindlin-2 on cell migration, cellular tube formation, and cell adhesion and spreading were evaluated in human umbilical vein endothelial cells (HUVECs) with downregulated Kindlin-2 expression. We found that the expression of kindlin-2 was elevated in wound healing tissues and that interfering with the expression of Kindlin-2 delayed the wound healing process and reduced neovascularization. We found that the wound healing of kindlin-2 (±) mice was delayed, with a decreased number of new blood vessels. Furthermore, depletion of Kindlin-2 impaired HUVEC spreading, migration and tube formation. Intriguingly, we found that kindlin-2 binds to β-catenin in the Wnt/β-catenin signaling pathway and cooperates with β-catenin to enter the nucleus from the cytoplasm, activating the downstream Wnt/β-catenin signaling pathway. Taken together, these results help to elucidate the mechanism of Kindlin-2 in the regulation of the wound healing process and provide a theoretical basis for further study of wound healing and abnormal healing.

The extracellular matrix as a multitasking player in disease.

Extracellular matrices (ECMs) are highly specialized and dynamic three-dimensional (3D) scaffolds into which cells reside in tissues. ECM is composed of a variety of fibrillar components, such as collagens, fibronectin, and elastin, and non-fibrillar molecules as proteoglycans, hyaluronan, and glycoproteins including matricellular proteins. These macromolecular components are interconnected forming complex networks that actively communicate with cells through binding to cell surface receptors and/or matrix effectors. ECMs exert diverse roles, either providing tissues with structural integrity and mechanical properties essential for tissue functions or regulating cell phenotype and functions to maintain tissue homeostasis. ECM molecular composition and structure vary among tissues, and is markedly modified during normal tissue repair as well as during the progression of various diseases. Actually, abnormal ECM remodeling occurring in pathologic circumstances drives disease progression by regulating cell-matrix interactions. The importance of matrix molecules to normal tissue functions is also highlighted by mutations in matrix genes that give rise to genetic disorders with diverse clinical phenotypes. In this review, we present critical and emerging issues related to matrix assembly in tissues and the multitasking roles for ECM in diseases such as osteoarthritis, fibrosis, cancer, and genetic diseases. The mechanisms underlying the various matrix-based diseases are also discussed. Research focused on the highly dynamic 3D ECM networks will help to discover matrix-related causative abnormalities of diseases as well as novel diagnostic tools and therapeutic targets.

Keratin 6 regulates collective keratinocyte migration by altering cell-cell and cell-matrix adhesion.

The a and b isoforms of keratin 6 (K6), a type II intermediate filament (IF) protein, are robustly induced upon injury to interfollicular epidermis. We previously showed that complete loss of K6a/K6b stimulates keratinocyte migration, correlating with enhanced Src activity. In this study, we demonstrate that this property is cell autonomous, depends on the ECM, and results from elevated speed, enhanced directionality, and an increased rate of focal adhesion disassembly. We show that myosin IIA interacts with K6a/K6b, that its levels are markedly reduced in Krt6a/Krt6b-null keratinocytes, and that inhibiting myosin ATPase activity normalizes the enhanced migration potential of Krt6a/Krt6b-null cells. Desmoplakin, which mediates attachment of IFs to desmosomes, is also expressed at reduced levels and is mislocalized to the nucleus in Krt6a/Krt6b-null cells, correlating with defects in cell adhesion. These findings reveal that K6a/K6b modulate keratinocyte migration by regulating cell-matrix and cell-cell adhesion and highlight a role for keratins in collective cell migration.

SMOC1 silencing suppresses the angiotensin II-induced myocardial fibrosis of mouse myocardial fibroblasts via affecting the BMP2/Smad pathway.

SPARC-related modular calcium binding 1 (SMOC1) represents a vital member of the SPARC matricellular protein family that regulates cell matrix interaction through binding to cell-surface receptors. The present study aimed to investigate the roles and molecular mechanisms of SMOC1 silencing on the fibrosis of myocardial fibroblasts (MFBs). Cell Counting kit-8 and flow cytometry assays were performed to determine cell viability and reactive oxygen species (ROS) content, respectively. ELISA was performed to detect the expression of associated cytokines and matrix proteins. Western blot analysis and reverse transcription-quantitative polymerase chain reaction assays were used to evaluate the expression of associated proteins and mRNAs, respectively. The results revealed that SMOC1 silencing suppressed the cell viability of angiotensin II (Ang II)-treated MFBs. SMOC1 silencing reduced the ROS content and oxidative stress in MFBs in response to Ang II. Furthermore, SMOC1 silencing downregulated the expression levels of fibrosis-associated proteins in Ang II-treated MFBs. SMOC1 silencing affected the bone morphogenetic protein 2 (BMP2)/Smad signaling pathway in Ang II-treated MFBs. In conclusion, the results of the present study suggested that SMOC1 silencing suppressed the Ang II-induced myocardial fibrosis of mouse MFBs through affecting the BMP2/Smad signaling pathway.

Engineering of endothelial cell response on biphasic polyurethane matrix

Polyurethanes (PUs) are composed of soft and hard segments, and segmental interactions induce biphasic morphologies which can influence endothelial cell (EC) organization by regulating cell–matrix and cell–cell interactions. In this study, we explored this effect using poly(caprolactone) (PCL)-based PU, where the soft segment was composed of PCL and the hard segment was composed of hexamethylene diisocyanate (HDI) and L-tyrosine-based dipeptide (DTH). The composition of the PUs was varied by altering the PCL molecular weight and correspondingly, different phase morphologies were observed. Organization and functional state of ECs on these PUs showed that composition and phase morphology of PU have a significant effect on cellular response. The ECs formed an organized network with cord-like structures which resulted in interconnected loops when soft and hard segment fractions were phase-separated. VE-cadherin (for cell–cell adherence) and vinculin (for cell–matrix focal adhesion) localized at the tip of interconnecting cells in the tube structures indicated synchronized cell–cell and cell–matrix interactions.

Control of fibrotic changes through the synergistic effects of anti-fibronectin antibody and an RGDS-tagged form of the same antibody

TGF-β and myofibroblasts play a key role in fibrosis, characterized by aberrant synthesis and deposition of extracellular matrix (ECM) proteins, such as fibronectin (Fn) and collagen type I. There are two major roles played by integrins in the fibrotic pathology: (i) Fn-integrin interaction, coupled with cytokines like TGF-β, facilitates the self-polymerization of Fn and regulates cell–matrix fibrillar adhesions, thereby promoting fibrillogenesis; (ii) Integrin interaction with an RGD (arginine-glycine–aspartic) consensus sequence in the latent TGF-β, resulting in its activation. This study describes an anti-fibrotic strategy using a combination of two antibodies: Fn52 (targeted against the N-terminal 30 kDa region of fibronectin, a major site for Fn self-association), and its engineered form, Fn52RGDS (which binds to integrins). Interestingly, a synergistic effect of the cocktail in causing a decline in fibrotic features was confirmed in the context of fibrotic posterior capsular opacification (PCO), mediated by the lens epithelial cells (left behind after cataract surgery). Inclusion of Fn52RGDS to Fn52 aids in better diffusion of the antibodies; such combination therapies could be useful in the context of pathologies involving extensive remodeling of the fibronectin matrix, where the thick ECM offers a major challenge for efficient drug delivery.

Fabrication and characterisation of biomimetic, electrospun gelatin fibre scaffolds for tunica media-equivalent, tissue engineered vascular grafts

It is increasingly recognised that biomimetic, natural polymers mimicking the extracellular matrix (ECM) have low thrombogenicity and functional motifs that regulate cell–matrix interactions, with these factors being critical for tissue engineered vascular grafts especially grafts of small diameter. Gelatin constitutes a low cost substitute of soluble collagen but gelatin scaffolds so far have shown generally low strength and suture retention strength. In this study, we have devised the fabrication of novel, electrospun, multilayer, gelatin fibre scaffolds, with controlled fibre layer orientation, and optimised gelatin crosslinking to achieve not only compliance equivalent to that of coronary artery but also for the first time strength of the wet tubular acellular scaffold (swollen with absorbed water) same as that of the tunica media of coronary artery in both circumferential and axial directions. Most importantly, for the first time for natural scaffolds and in particular gelatin, high suture retention strength was achieved in the range of 1.8–1.94N for wet acellular scaffolds, same or better than that for fresh saphenous vein. The study presents the investigations to relate the electrospinning process parameters to the microstructural parameters of the scaffold, which are further related to the mechanical performance data of wet, crosslinked, electrospun scaffolds in both circumferential and axial tubular directions. The scaffolds exhibited excellent performance in human smooth muscle cell (SMC) proliferation, with SMCs seeded on the top surface adhering, elongating and aligning along the local fibres, migrating through the scaffold thickness and populating a transverse distance of 186μm and 240μm 9days post-seeding for scaffolds of initial dry porosity of 74 and 83%, respectively.

Matrix RGD ligand density and L1CAM-mediated Schwann cell interactions synergistically enhance neurite outgrowth

The innate biological response to peripheral nerve injury involves a complex interplay of multiple molecular cues to guide neurites across the injury gap. Many current strategies to stimulate regeneration take inspiration from this biological response. However, little is known about the balance of cell–matrix and Schwann cell–neurite dynamics required for regeneration of neural architectures. We present an engineered extracellular matrix (eECM) microenvironment with tailored cell–matrix and cell–cell interactions to study their individual and combined effects on neurite outgrowth. This eECM regulates cell–matrix interactions by presenting integrin-binding RGD (Arg–Gly–Asp) ligands at specified densities. Simultaneously, the addition or exclusion of nerve growth factor (NGF) is used to modulate L1CAM-mediated Schwann cell–neurite interactions. Individually, increasing the RGD ligand density from 0.16 to 3.2mM resulted in increasing neurite lengths. In matrices presenting higher RGD ligand densities, neurite outgrowth was synergistically enhanced in the presence of soluble NGF. Analysis of Schwann cell migration and co-localization with neurites revealed that NGF enhanced cooperative outgrowth between the two cell types. Interestingly, neurites in NGF-supplemented conditions were unable to extend on the surrounding eECM without the assistance of Schwann cells. Blocking studies revealed that L1CAM is primarily responsible for these Schwann cell–neurite interactions. Without NGF supplementation, neurite outgrowth was unaffected by L1CAM blocking or the depletion of Schwann cells. These results underscore the synergistic interplay between cell–matrix and cell–cell interactions in enhancing neurite outgrowth for peripheral nerve regeneration.

Cyclin D1 localizes in the cytoplasm of keratinocytes during skin differentiation and regulates cell–matrix adhesion

The function of Cyclin D1 (CycD1) has been widely studied in the cell nucleus as a regulatory subunit of the cyclin-dependent kinases Cdk4/6 involved in the control of proliferation and development in mammals. CycD1 has been also localized in the cytoplasm, where its function nevertheless is poorly characterized. In this work we have observed that in normal skin as well as in primary cultures of human keratinocytes, cytoplasmic localization of CycD1 correlated with the degree of differentiation of the keratinocyte. In these conditions, CycD1 co-localized in cytoplasmic foci with exocyst components (Sec6) and regulators (RalA), and with β1 integrin, suggesting a role for CycD1 in the regulation of keratinocyte adhesion during differentiation. Consistent with this hypothesis, CycD1 overexpression increased β1 integrin recycling and drastically reduced the ability of keratinocytes to adhere to the extracellular matrix. We propose that localization of CycD1 in the cytoplasm during skin differentiation could be related to the changes in detachment ability of keratinocytes committed to differentiation.

Rhodocetin-αβ–induced Neuropilin-1–cMet Association Triggers Restructuring of Matrix Contacts in Endothelial Cells

The snake venom component rhodocetin-αβ (RCαβ) stimulates endothelial cell motility in an α2β1 integrin-independent manner. We aimed to elucidate its cellular and molecular mechanisms. We identified neuropilin-1 (Nrp1) as a novel target of RCαβ by protein-chemical methods. RCαβ and vascular endothelial growth factor (VEGF)-A avidly bind to Nrp1. Instead of acting as VEGF receptor 2 coreceptor, Nrp1 associates upon RCαβ treatment with cMet. Furthermore, cell-based ELISAs and kinase inhibitor studies showed that RCαβ induces phosphorylation of tyrosines 1234/1235 [corrected] and thus activation of cMet. Consequently, paxillin is phosphorylated at Y31, which is redistributed from streak-like focal adhesions to spot-like focal contacts at the cell perimeter, along with α2β1 integrin, thereby regulating cell-matrix interactions. Cortactin is abundant in the cell perimeter, where it is involved in the branching of the cortical actin network of lamellipodia, whereas tensile force-bearing actin stress fibers radiating from focal adhesions disappear together with zyxin, a focal adhesion marker, on RCαβ treatment. Our data demonstrate that (1) Nrp1 is a novel target for venom components, such as RCαβ; (2) Nrp1 coupled to cMet regulates the type of cell-matrix interactions in a manner involving paxillin phosphorylation; and (3) altered cell-matrix interactions determine endothelial cell migration and cellular force management.

Alpha-actinin interactions with syndecan-4 are integral to fibroblast–matrix adhesion and regulate cytoskeletal architecture

All cells of the musculoskeletal system possess transmembrane syndecan proteoglycans, notably syndecan-4. In fibroblasts it regulates integrin-mediated adhesion to the extracellular matrix. Syndecan-4 null mice have a complex wound repair phenotype while their fibroblasts have reduced focal adhesions and matrix contraction abilities. Signalling through syndecan-4 core protein to the actin cytoskeleton involves protein kinase Cα and Rho family G proteins but also direct interactions with α-actinin. The contribution of the latter interaction to cell–matrix adhesion is not defined but investigated here since manipulation of Rho GTPase and its downstream targets could not restore a wild type microfilament organisation to syndecan-4 null cells. Microarray and protein analysis revealed no significant alterations in mRNA or protein levels for actin- or α-actinin associated proteins when wild type and syndecan-4 knockout fibroblasts were compared. The binding site for syndecan-4 cytoplasmic domain was identified as spectrin repeat 4 of α-actinin while further experiments confirmed the importance of this interaction in stabilising cell–matrix junctions. However, α-actinin is also present in adherens junctions, these organelles not being disrupted in the absence of syndecan-4. Indeed, co-culture of wild type and knockout cells led to adherens junction-associated stress fibre formation in cells lacking syndecan-4, supporting the hypothesis that the proteoglycan regulates cell–matrix adhesion and its associated microfilament bundles at a post-translational level. These data provide an additional dimension to syndecan function related to tension at the cell–matrix interface, wound healing and potentially fibrosis.

Matrix metalloproteinases (with accent to collagenases)

Matrix metalloproteinases (MMPs) are a major group of enzymes that regulate cell–matrix composition. The MMPs are zinc-dependent endopeptidases known for their ability to cleave one or several extracellular matrix (ECM) constituents, as well as nonmatrix proteins. This review focuses on structural and functional elements of MMPs, and their roles in physiological and pathological processes, in which it is believed the MMPs play an important, or even indispensable role. According to their structural and functional characteristics, MMP family members have been classified into six different but closely related subgroups with fairly characteristic but often overlapping substrate specificities. MMP synthesis and functions are regulated by transcriptional activation, post-transcriptional processing (release of pro-domain, cell surface shedding), and control of activity by a family of endogenous inhibitors collectively known as tissue inhibitors of metalloproteinases (TIMPs). The balance of MMPs to TIMPs therefore determines matrix turnover, where either an excess of MMPs or a deficit of TIMPs may result in excess ECM degradation. Moreover, the recent development of selective and nonselective inhibitors of MMPs would also provide new insights in the knowledge of the relationship between activation of inflammatory cells and tissue remodeling and propose new therapeutic possibilities to the treatment of inflammatory disease.

Role of Kindlin-2 in Fibroblast Functions: Implications for Wound Healing

Kindlins are a protein family that regulates cell-matrix interactions. Although kindlin-2 is known to be essential for the early developmental processes, its role in the homeostasis of adult tissues has remained elusive. In this study, we used new domain-specific antibodies and small interfering RNA technology to uncover physiological functions of kindlin-2 in human dermal fibroblasts in vitro and in adult skin in situ. In primary dermal fibroblasts, kindlin-2 is essential for the integrin clustering and activation, and it regulates cell adhesion and directed migration by guiding formation and maturation of focal adhesions (FAs) and organization of the cytoskeleton. These functions are linked to the transmission of mechanical cues between cells and their microenvironment, typically in processes wherein fibroblasts differentiate to myofibroblasts under mechanical tension. In concert, kindlin-2 was shown to be required for the stabilization and maturation of FAs and stress fibers in activated fibroblasts and myofibroblasts. These findings implicated that in physiological wound closure and tissue repair, the prediction was validated by strong upregulation of kindlin-2 in contractile myofibroblasts during middle stages of wound healing in human skin. Taken together, the data reveal a physiological role for kindlin-2 in skin fibroblasts under normal steady-state conditions and during tissue regeneration.

Rap1A regulates generation of T regulatory cells via LFA-1-dependent and LFA-1-independent mechanisms

The small GTPase Rap1A has a critical role in regulating cell–matrix and cell–cell adhesion. In T lymphocytes, Rap1A mediates LFA-1 activation and LFA-1-mediated adhesion. LFA-1 reduces the threshold of TCR signals for low affinity ligands. Previously, we determined that mice expressing constitutively active Rap1A on T cells have increased frequency of CD103 + T regulatory cells (Treg). We hypothesized that Rap1A-GTP might affect the differentiation of Treg by regulating LFA-1 activation. Using Foxp3-GFP-KI, LFA-1-KO and Rap1A-GTP-Tg mice we determined that Rap1A has an active role in the development of thymic Treg but LFA-1 is not mandatory for this function. Rap1A is also involved in the generation of peripheral Treg and this effect is mediated via LFA-1-dependent and LFA-1-independent mechanisms. Identification of the signaling pathways via which Rap1-GTP contributes to the differentiation of Treg will provide new insights to the function of Rap1A and to designing targeted approaches for generation of Treg for therapeutic applications.

Coronin 1C negatively regulates cell–matrix adhesion and motility of intestinal epithelial cells

Coronins, WD-repeat actin-binding proteins, are known to regulate cell motility by coordinating actin filament turnover in lamellipodia of migrating cell. Here we report a novel mechanism of Coronin 1C-mediated cell motility that involves regulation of cell–matrix adhesion. RNAi silencing of Coronin 1C in intestinal epithelial cells enhanced cell migration and modulated lamellipodia dynamics by increasing the persistence of lamellipodial protrusion. Coronin 1C-depleted cells showed increased cell–matrix adhesions and enhanced cell spreading compared to control cells, while over-expression of Coronin 1C antagonized cell adhesion and spreading. Enhanced cell–matrix adhesion of coronin-deficient cells correlated with hyperphosphorylation of focal adhesion kinase (FAK) and paxillin, and an increase in number of focal adhesions and their redistribution at the cell periphery. siRNA depletion of FAK in coronin-deficient cells rescued the effects of Coronin 1C depletion on motility, cell–matrix adhesion, and spreading. Thus, our findings provide the first evidence that Coronin 1C negatively regulates epithelial cell migration via FAK-mediated inhibition of cell–matrix adhesion.

Bcl2 enhances c-Myc-mediated MMP-2 expression of vascular smooth muscle cells

Matrix metalloproteinases (MMPs) are a major group of enzymes that regulate cell matrix composition. In this paper, our results show that c-Myc significantly induced vascular smooth muscle cells (VSMCs) migration and invasion, compared with the results, the T58A had more effectively than WT c-Myc, which was associated with c-Myc increased MMP-2 gene expression and activity. Silenced c-Myc led to reduce MMP-2 gene expression and activity, as well as decrease VSMC migration and invasion, indicating c-Myc is required for MMP-2 mediated VSMC migration and invasion. However, S62A had no effect on VSMC migration and invasion, which was in line with S62A had no effect on the c-Myc transcriptional activity. To better understand whether Bcl2 cooperate with c-Myc on MMP-2 function, our data show that although Bcl2 had no effect on the MMP-2 activity, the coexpressing c-Myc and Bcl2 significantly increased MMP-2 gene expression and activity. Our results suggest that phosphorylation of Bcl2 (T70E and EEE) had more effectively on the MMP-2 activity, which resulted from T70E and EEE severely increased c-Myc transcriptional activity by directly binding to c-Myc. The findings show that phosphorylation of Bcl2 enhanced c-Myc-mediated MMP-2 activity.