Mycobacterium smegmatis expressing a chimeric protein MPT64-proteolipid protein (PLP) 139-151 reorganizes the PLP-specific T cell repertoire favoring a CD8-mediated response and induces a relapsing experimental autoimmune encephalomyelitis.

We infected SJL mice with a recombinant Mycobacterium smegmatis expressing a chimeric protein containing the self-epitope of proteolipid protein 139-151 (p139) fused to MPT64, a secreted protein of Mycobacterium tuberculosis (rMS(p139)). Infected mice developed a relapsing experimental autoimmune encephalomyelitis (EAE), showing a prevailing demyelination of the CNS, and disease severity was significantly lower in comparison with the one that follows immunization with p139. rMS(p139) was not detected in lymph node or spleen in the course of clinical disease development or in the CNS during relapse. Infection with rMS(p139) modified the p139-specific T cell repertoire, recruiting the spontaneous p139-specific repertoire and activating CD4(+) T cells carrying the BV4 semiprivate rearrangement. T cells carrying the public BV10 rearrangement that are consistently found in the CNS during flares of disease were not activated by infection with rMS(p139) because lymph node APCs infected with rMS(p139) selectively fail to present the epitope for which BV10 cells are specific. Simultaneously, rMS(p139) expanded p139-specific CD8(+) cells more efficiently than immunization with peptide in adjuvant. SJL mice vaccinated against the CDR3 sequence of the BV10 public rearrangement reduced usage of the BV10 cells and displayed reduced symptoms during bouts of EAE. Thus, transient peripheral infection with a CNS-cross-reactive nonpathogenic Mycobacterium induces a relapsing EAE that continues long after clearance of the infectious agent. The composition of the self-reactive repertoire activated determines severity and histology of the resulting disease.

The extracellular domain of CD11d regulates its cell surface expression.

A mAb targeting the CD11d subunit of the leukocyte integrin CD11d/CD18 decreases intraspinal inflammation and oxidative damage leading to improved neurological outcomes in rodent models of SCI. CD11d/CD18 is the fourth member of the beta2-integrin family. Current evidence indicates that CD11d/CD18 is regulated differently than other beta2-integrins, suggesting that CD11d(+) leukocytes play a distinct role in inflammation. Although the transcriptional control of CD11d expression has been evaluated, control of the intracellular distribution of CD11d has not been addressed. For this reason and as a result of the potential of CD11d as a therapeutic target for SCI and possibly other CNS injuries, we investigated the intracellular localization and surface expression of CD11d in cultured cells. CD11d and CD18 were fused at their C-termini with YFP and mRFP, respectively. Flow cytometry and confocal microscopy demonstrated that rCD11d-YFP is expressed on the cell surface of leukocyte cell lines expressing CD18. In contrast, in heterologous cell lines, CD11d-YFP is retained intracellularly in the TGN. Coexpression of CD11d-YFP and CD18-mRFP relieves this intracellular restriction and allows the CD11d/CD18 heterodimer to be surface-expressed. Based on domain-swapping experiments with CD25, the extracellular domain of CD11d is required and sufficient for the observed intracellular retention in heterologous cells. Furthermore, the transmembrane and C-terminus are also required for proper heterodimerization with CD18 and localization to the plasma membrane. These findings suggest that multiple CD11d domains play a role in controlling intracellular location and association with CD18.

Src and FAK mediate cell-matrix adhesion-dependent activation of Met during transformation of breast epithelial cells.

Cell-matrix adhesion has been shown to promote activation of the hepatocyte growth factor receptor, Met, in a ligand-independent manner. This process has been linked to transformation and tumorigenesis in a variety of cancer types. In the present report, we describe a key role of integrin signaling via the Src/FAK axis in the activation of Met in breast epithelial and carcinoma cells. Expression of an activated Src mutant in non-neoplastic breast epithelial cells or in carcinoma cells was found to increase phosphorylation of Met at regulatory tyrosines in the auto-activation loop domain, correlating with increased cell spreading and filopodia extensions. Furthermore, phosphorylated Met is complexed with beta1 integrins and is co-localized with vinculin and FAK at focal adhesions in epithelial cells expressing activated Src. Conversely, genetic or pharmacological inhibition of Src abrogates constitutive Met phosphorylation in carcinoma cells or epithelial cells expressing activated Src, and inhibits filopodia formation. Interestingly, Src-dependent phosphorylation of Met requires cell-matrix adhesion, as well as actin stress fiber assembly. Phosphorylation of FAK by Src is also required for Src-induced Met phosphorylation, emphasizing the importance of the Src/FAK signaling pathway. However, stimulation of Met phosphorylation by addition of exogenous HGF in epithelial cells is refractory to inhibition of Src family kinases, indicating that HGF-dependent and Src/integrin-dependent Met activation occur via distinct mechanisms. Together these findings demonstrate a novel mechanism by which the Src/FAK axis links signals from the integrin adhesion complex to promote Met activation in breast epithelial cells.

Coordinated integrin and growth factor regulation of primary keratinocyte migration mediated through extracellular signal regulated kinase and phosphoinositide 3-kinase.

We have examined coordinated integrin and growth factor regulation of primary keratinocyte migration mediated by phosphoinositide 3-kinase (PI3K) and mitogen-activated extracellular-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK). On collagen I and fibronectin substrates, both epidermal growth factor (EGF) and hepatocyte growth factor (HGF) stimulated chemokinetic (random) and chemotactic (directional) migration. On provisional matrix, a combination of fibronectin and fibrin found in the early phase of wound healing, EGF and HGF-stimulated significant chemotactic but little or no chemokinetic cell movement. Blocking mAbs to integrin alpha2beta1 and alpha5beta1 effectively inhibited EGF- and HGF-stimulated chemokinetic and chemotactic cell movement on collagen I and fibronectin, respectively; however, HGF-stimulated chemotactic migration on collagen I was only partially inhibited by alpha2beta1 blocking mAb. Differentiated keratinocytes underwent reduced chemokinetic and chemotactic migration compared with undifferentiated keratinocytes; however, EGF-stimulated migration was reduced more than HGF-stimulated migration. When the migratory response on collagen I and fibronectin was assessed in the presence of the MEK-specific inhibitor PD98059, EGF- and HGF-stimulated chemotaxis was significantly reduced, whereas PD98059 had little effect on the stimulated chemokinesis. PI3K-specific inhibitor LY294002 reduced EGF- and HGF-stimulated chemokinesis and chemotaxis on collagen I and fibronectin. Thus beta1 integrins acted in concert with EGF and HGF to regulate migration of primary keratinocytes on extracellular matrix components via PI3K and MEK/ERK.

Lipid incorporation inhibits Src-dependent assembly of fibronectin and type I collagen by vascular smooth muscle cells.

A vital role of vascular smooth muscle cells (SMCs) is to stabilize the artery wall by elaborating fibrils of type I collagen. This is especially important in atherosclerotic lesions. However, SMCs in these lesions can be laden with lipids and the impact of this modification on collagen fibril formation is unknown. To address this, we converted human vascular SMCs to a foam cell state by incubating them with either LDL or VLDL. Biochemical markers of a SMC phenotype were preserved. However, microscopic tracking revealed a profound perturbation in the ability of the cells to assemble collagen fibrils, reducing assembly by up to 79%. This dysfunction was mirrored by an inability of smooth muscle foam cells to assemble fibronectin. Lipid-loaded SMCs did not display a generalized defect in the actin cytoskeleton and the formation of vinculin-containing focal adhesion complexes was preserved. However, lipid-loaded SMCs were unable to assemble fibrillar adhesion complexes and clustering of tensin and alpha5beta1 integrin was disordered. Moreover, phosphorylation of tensin, required for fibrillar adhesion complex formation, was suppressed by up to 57%, with a concomitant decrease in activation of Src and FAK and restriction of activated Src to the cell edges. Forced activation of Src-FAK signaling in lipid-engorged SMCs rescued both fibrillar adhesion formation and fibrillogenesis. We conclude that lipid accumulation by SMCs disables the machinery for collagen and fibronectin assembly. This previously unknown relationship between atherogenic lipids and integrin-based signaling could underlie plaque vulnerability.

Cathepsin B is involved in the trafficking of TNF-alpha-containing vesicles to the plasma membrane in macrophages.

TNF-alpha is a potent proinflammatory cytokine, essential for initiating innate immune responses against invading microbes and a key mediator involved in the pathogenesis of acute and chronic inflammatory diseases. To identify molecules involved in the production of TNF-alpha, we used a functional gene identification method using retroviral integration-mediated mutagenesis, followed by LPS-stimulated TNF-alpha production analysis in macrophages. We found that cathepsin B, a lysosomal cysteine proteinase, was required for optimal posttranslational processing of TNF-alpha in response to the bacterial cell wall component LPS. Mouse bone marrow-derived macrophages from cathepsin B-deficient mice and macrophages treated with the cathepsin B-specific chemical inhibitor CA074 methyl ester or small interfering RNA against cathepsin B secreted significantly less TNF-alpha than wild-type or nontreated macrophages. We further showed that the inhibition of cathepsin B caused accumulation of 26-kDa pro-TNF-containing vesicles. Ectopic expression of GFP-conjugated pro-TNF further suggests that pro-TNF failed to reach the plasma membrane without intracellular cathepsin B activity. Altogether, these data suggest that intracellular cathepsin B activity is involved in the TNF-alpha-containing vesicle trafficking to the plasma membrane.

Interaction of epidermal growth factor, Ca2+, and matrix metalloproteinase-9 in primary keratinocyte migration.

Elevations of epidermal growth factor (EGF) and Ca(2+) concentrations in the wound site are associated with reepithelialization during wound healing. In addition, Ca(2+) and EGF can both induce increases in matrix metalloproteinase-9 (MMP-9) synthesis. However, little is known about the interplay of these events in regulating the migration properties of primary keratinocytes on collagen I, the most abundant extracellular matrix component in the skin. We found that EGF stimulated both chemokinetic and chemotactic migration of primary keratinocytes on collagen I; however, MMP-9 was required for EGF-stimulated chemotaxis but not EGF-stimulated chemokinesis. Calcium at 0.5 mM stimulated chemokinetic migration of keratinocytes. Together, Ca(2+) and EGF stimulated higher levels of chemokinesis than either stimulus alone. Furthermore, Ca(2+) could restore the ability of keratinocytes from MMP-9 null mice to undergo EGF-stimulated chemotaxis. The phosphatidylinositol-3 kinase inhibitor LY294002 inhibited both EGF- and Ca(2+)-stimulated chemokinetic migration. In contrast, the MEK inhibitor PD98059 blocked Ca(2+)- but not EGF-stimulated chemokinetic migration of keratinocytes. A combination of PD98059 and LY294002 was required to inhibit Ca(2+) enhancement of EGF-stimulated migration completely. Calcium-stimulated chemokinesis was completely blocked by either the protein kinase C-alpha inhibitor Gö6976 or the src/fyn inhibitor PP2. Using primary keratinocytes, our results showed how the combined action of Ca(2+), EGF, and MMP-9 regulated the contributions of extracellular-regulated kinase and phosphatidylinositol-3 kinase toward chemokinetic and chemotactic migration of keratinocytes.

Chemokine receptor CXCR4-beta1 integrin axis mediates tumorigenesis of osteosarcoma HOS cells.

It is known that beta1 integrins mediate the migratory response of cells to chemokine stimulation. Also, both beta1 integrins and chemokines have roles in tumor development. In the present study, the beta1 integrin-chemokine axis is assessed using human osteosarcoma (HOS) transfectant cells expressing the CXCR4 receptor for chemokine SDF-1 (CXCL12). We first identified in vitro the specific beta1 integrins that mediated the migratory response to SDF-1 stimulation. Results showed that on collagen type I and laminin, the chemotactic response to SDF-1 was predominantly mediated by alpha2beta1 integrin. On fibronectin, SDF-1-stimulated chemotaxis involved both alpha4beta1 and alpha5beta1 integrins. A comparison of the transfectant clones expressing CXCR4 at low, intermediate, and high levels and the control transfectant revealed that the transfectant clones migratory response in vitro and their ability to form tumors in vivo was related to their levels of CXCR4 expression. In addition, treatment by injection with mAbs to CXCR4, integrin alpha2beta1, or integrin alpha5beta1 effectively inhibited the growth of HOS-CXCR4 transfectant cells in vivo. Therefore, our results show that the beta1 integrins that mediated the migratory response were also functionally linked to the enhanced tumor growth of CXCR4-expressing HOS transfectant cells.

The zebrafish band 4.1 member Mir is involved in cell movements associated with gastrulation.

Cellular processes rely on dynamic events occurring between the cortical cytoskeleton and plasma membrane. Members of the Band 4.1 superfamily, which are best known for their ability to tether the cytoskeleton to the plasma membrane, play prominent structural and regulatory roles that influence cell-cell and cell-substrate interactions, endo- and exocytosis, cell polarity, migration, proliferation, and differentiation. We have identified a new member of the zebrafish Band 4.1 superfamily, which is the homolog of human myosin regulatory light chain interacting protein (MIR), and have examined its role in embryonic development. Zebrafish Mir contains the conserved amino-terminal plasma membrane-binding FERM (Band 4.1/ezrin/radixin/moesin) domain as well as other putative protein-protein interacting domains, including a RING finger. Overall, zebrafish Mir is 71% identical to human MIR located at chromosome 6p23-p22.3, and maps on linkage group 19 to a region of synteny with human chromosome 6. In situ hybridization and RT-PCR revealed that mir is expressed maternally and ubiquitously throughout development. Blocking Mir translation using a mir-specific, morpholino-based, knock-down strategy or expressing Mir constructs lacking the RING finger domain disrupts gastrulation and leads to subsequent trunk and tail defects. In severe cases, morphants exogastrulate. The synergistic effect seen when two mir-specific morpholinos are used in conjunction reflects the specific knock-down of mir. In addition, morphant phenotypes induced by mir-specific morpholinos are rescued by overexpression of the full-length Mir. In situ hybridization analysis with mesodermal- and neural-specific markers shows that morphants exhibit a delay in cell movements associated with gastrulation, epiboly, convergence, and extension. A yeast two-hybrid analysis was performed to identify binding partners that may participate with Mir during gastrulation, and Annexin V, a calcium channel protein, was isolated. At early developmental stages, annexin V transcripts colocalize with mir, but after gastrulation, annexin V mRNA becomes localized to the distal tail region and an area in the olfactory placode. At the protein level, Mir colocalizes with Annexin V when expressed in COS cells. Together, these results indicate that Mir is essential for embryonic development and that its role in early embryonic development likely involves calcium-dependent mechanisms essential during the extensive cell movements associated with gastrulation.

Beta1 integrins are distributed in adhesion structures with fibronectin and caveolin and in coated pits.

Integrins are found in adhesion structures, which link the extracellular matrix to cytoskeletal proteins. Here, we attempt to further define the distribution of beta1 integrins in the context of their association with matrix proteins and other cell surface molecules relevant to the endocytic process. We find that beta1 integrins colocalize with fibronectin in fibrillar adhesion structures. A fraction of caveolin is also organized along these adhesion structures. The extracellular matrix protein laminin is not concentrated in these structures. The alpha4beta1 integrin exhibits a distinct distribution from other beta1 integrins after cells have adhered for 1 h to extracellular matrix proteins but is localized in adhesion structures after 24 h of adhesion. There are differences between the fibronectin receptors: alpha5beta1 integrins colocalize with adaptor protein-2 in coated pits, while alpha4beta1 integrins do not. This parallels our earlier observation that of the two laminin receptors, alpha1beta1 and alpha6beta1, only alpha1beta1 integrins colocalize with adaptor protein-2 in coated pits. Calcium chelation or inhibition of mitogen-activated protein kinase kinase, protein kinase C, or src did not affect localization of alpha1beta1 and alpha5beta1 integrins in coated pits. Likewise, the integrity of coated-pit structures or adhesion structures is not required for integrin and adaptor protein-2 colocalization. This suggests a robust and possibly constitutive interaction between these integrins and coated pits.

Vascular smooth muscle cells orchestrate the assembly of type I collagen via alpha2beta1 integrin, RhoA, and fibronectin polymerization.

Assembly of collagen into fibrils is widely studied as a spontaneous and entropy-driven process. To determine whether vascular smooth muscle cells (SMCs) impact the formation of collagen fibrils, we microscopically tracked the conversion of soluble to insoluble collagen in human SMC cultures, using fluorescent type I collagen at concentrations less than that which supported self-assembly. Collagen microaggregates were found to form on the cell surface, initially as punctate collections and then as an increasingly intricate network of fibrils. These fibrils displayed 67-nm periodicity and were found in membrane-delimited cellular invaginations. Fibril assembly was inhibited by an anti-alpha2beta1 integrin antibody and accelerated by an alpha2beta1 integrin antibody that stimulates a high-affinity binding state. Newly assembled collagen fibrils were also found to co-localize with newly assembled fibronectin fibrils. Moreover, inhibition of fibronectin assembly with an anti-alpha5beta1 integrin antibody completely inhibited collagen assembly. Collagen fibril formation was also linked to the cytoskeleton. Fibrils formed on the stretched tails of SMCs, ran parallel to actin microfilament bundles, and formed poorly on SMCs transduced with retrovirus containing cDNA for dominant-negative RhoA and robustly on SMCs expressing constitutively active RhoA. Lysophosphatidic acid, which activates RhoA and stimulates fibronectin assembly, stimulated collagen fibril formation, establishing for the first time that collagen polymerization can be regulated by soluble agonists of cell function. Thus, collagen fibril formation is under close cellular control and is dynamically integrated with fibronectin assembly, opening new possibilities for modifying collagen deposition.

Integrin alpha2beta1 on rat myeloma cells modulates interaction of alpha4beta1 integrin with vascular cell adhesion molecule-1 but not fibronectin.

It is well established that alpha2beta1 integrin functions as a receptor for collagen and laminin; whereas alpha4beta1 integrin binds fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In the present study, we showed that rat myeloma YB2/0 cells constitutively expressed alpha4beta1 but not alpha2beta1 integrin. Transfection of cDNA of mouse a2 integrin subunit resulted in the expression of heterologous alpha2beta1 integrin on YB2/0 cells (YBmalpha2). The expression of alpha2beta1 conferred YBmalpha2 cells the ability to interact with collagen and laminin. In comparison with mock transfected YB2/0 cells (YBpF), YBmalpha2 cells exhibited increases in the binding and migration on VCAM-1; in contrast, both YBpF and YBmalpha2 were similar in their interactions with fibronectin or fibronectin fragment FN-40 that contains the binding site for alpha4beta1 integrin. The interaction of alpha4beta1 with VCAM-1 was further stimulated upon ligation with alpha2beta1-specific mAb. The use of specific inhibitory mAb demonstrated the role of alpha4beta1 in mediating the observed interactions with fibronectin and VCAM-1. Therefore, results show that expression of alpha2beta1 differentially regulated alpha4alpha1 integrin function by stimulating its interactions with VCAM-1 but not fibronectin. The in vivo significance of alpha2beta1 integrin expression was demonstrated by intravital videomicroscopy showing that ligation of alpha2beta1 enhanced alpha4beta1-mediated extravasation of YBmalpha2 cells in the liver.

Adaptor protein-2 exhibits alpha 1 beta 1 or alpha 6 beta 1 integrin-dependent redistribution in rhabdomyosarcoma cells.

Downregulation of several signaling pathways, such as those stimulated by growth factor receptors, occurs by internalization of signaling receptors through clathrin-coated pits. The first step in internalization or endocytosis is interaction with AP-2, which results in coated pit formation by assembly of clathrin to AP-2. Changes in endocytosis are reflected in the distribution of AP-2 molecules at the cell surface. Integrins are receptors which mediate attachment to the extracellular matrix and also stimulate numerous intracellular signaling pathways; however, it is not known how signaling through integrins is terminated or downregulated. Endocytosis through clathrin-coated pits offers an attractive mechanism for this. This work explores the relationship between AP-2 and beta(1) integrins. RD cells grown for 24 h on collagen or laminin exhibit a redistribution of AP-2 to the cell periphery relative to those grown on fibronectin or polylysine. The total AP-2 protein levels in the cells are unaffected. Blocking alpha(1)beta(1) integrin ligand binding on collagen prevents this redistribution fully. On laminin where alpha(1)beta(1) and alpha(6)beta(1) integrins are engaged, both receptors must be simultaneously blocked to prevent AP-2 redistribution, confirming that the redistribution depends on the specific engagement of the receptors. Immunofluorescence reveals that the majority of alpha(1)beta(1) integrins colocalize with alpha(6)beta(1) integrins in linear structures identified as focal adhesions. A separate fraction of alpha(1)beta(1) integrins colocalize with AP-2 in coated pits. Interestingly, alpha(6)beta(1) integrins are not located in coated pits, demonstrating that integrin colocalization with AP-2 is not necessary to induce redistribution of AP-2.

Integrin VLA-6 (α6ß1) is transiently expressed during the development of mouse bone marrow-derived mast cells.

In the present study the involvement of VLA-6 (α6ß1) integrin, a laminin receptor, was characterized during the course of mouse bone marrow-derived mast cell (BMMC) development. Flow cytometry and immunoprecipitation revealed increases in α6 integrin expression during the first 3 weeks, followed by a decline, such that α6ß1 was no longer detectable by week 13. Using RT-PCR, transcripts for α6A but not the α6B isoform were detected. Results from immunoprecipitation and costaining with ß1-or ß4-specific mAb showed the expression of VLA-6 (α6ß1) and not α6ß4 heterodimers. Moreover, the ability of BMMC to interact with laminin correlated with the time period of VLA-6 expression. However, only 40% of adhesion to laminin was inhibited by blocking mAb for α6, indicating the involvement of additional laminin receptor(s). This is supported by the immunoprecipitation of VLA-2 integrin, also known to have laminin binding properties. Heterogeneity of VLA-6 expression was also found in connective tissue-type mast cells; thus, VLA-6hi and VLA-6lo subpopulations of peritoneal mast cells were observed. The heterogeneity of VLA-6 integrin expression in BMMC and CTMC may be relevant to the concept of mast cell heterogeneity as well as to the ability of mast cell precursors to migrate and complete their course of maturation within tissues.