Teet Velling

cDNA cloning and chromosomal localization of human alpha(11) integrin. A collagen-binding, I domain-containing, beta(1)-associated integrin alpha-chain present in muscle tissues.

We previously identified a novel integrin α-chain in human fetal muscle cells (Gullberg, D., Velling, T., Sjöberg, G., and Sejersen, T. (1995) Dev. Dyn. 204, 57–65). We have now isolated the full-length cDNA for this integrin subunit, α11. The open reading frame of the cDNA encodes a precursor of 1188 amino acids. The predicted mature protein of 1166 amino acids contains seven conserved FG-GAP repeats, an I domain with a metal ion-dependent adhesion site motif, a short transmembrane region, and a unique cytoplasmic domain of 24 amino acids containing the sequence GFFRS. α11, like other I domain integrins, lacks a dibasic cleavage site for generation of a heavy chain and a light chain, and it contains three potential divalent cation binding sites in repeats 5–7. The presence of 22 inserted amino acids in the extracellular stalk portion (amino acids 804–826) distinguishes the α11 integrin sequence from other integrin α-chains. Amino acid sequence comparisons reveal the highest identity of 42% with the α10 integrin chain. Immunoprecipitation with antibodies to α11 integrin captures a 145-kDa protein distinctly larger than the 140-kDa α2 integrin chain when analyzed by SDS-polyacrylamide gel electrophoresis under nonreducing conditions. Fluorescence in situ hybridization maps the integrin α11 gene to chromosome 15q23, in the vicinity of an identified locus for Bardet-Biedl syndrome. Based on Northern blotting, integrin α11 mRNA levels are high in the adult human uterus and in the heart and intermediate in skeletal muscle and some other tissues tested. During in vitro myogenic differentiation, α11 mRNA and protein are up-regulated. Studies of ligand binding properties show that α11β1binds collagen type I-Sepharose, and cultured muscle cells localize α11β1 into focal contacts on collagen type I. Future studies will reveal the importance of α11β1 for muscle development and integrity in adult muscle and other tissues.

Laminins during muscle development and in muscular dystrophies.

Abstract. Cellular interactions with the extracellular matrix during muscle formation and in muscular dystrophy have received increased interest during the past years. Laminins constitute a growing family of proteins with complex expression patterns in forming basement membranes during muscle development. In skeletal muscle, laminins constitute major ligands for cell surface receptors involved in the transmission of force from the cell interior, but laminins might also influence signal transmission events during muscle formation and in muscle regeneration. During myogenesis the laminin α1 chain is present around the epithelial somite; but later, in forming muscle, the laminin α1 chain is restricted to the myotendinous junction. The laminin α2,α4 and α5 chains are major laminin chains in the muscle basement membrane during muscle formation, but laminin α4 and α5 chains are absent in adult muscle. The importance of laminins for muscle integrity is manifested in congenital muscular dystrophies with defects in the laminin α2 chain. There is no good evidence for the presence of laminin α1 chain in dystrophic muscle, but some other fetal muscle laminins can be detected in dystrophic muscle. Characterization of laminin expression patterns in muscular dystrophies might be of diagnostic and therapeutic value. In this paper, we review the recent publications on the biological functions of muscle laminins and discuss their roles in skeletal muscle.

Integrins During Muscle Development and in Muscular Dystrophies

Cellular interactions with the extracellular matrix (ECM) have been shown to be important for a number of developmental events from the time of fertilization up till the maturation of the organism. In the following review we will discuss what is currently known about these interactions with special emphasis on the role of integrins during the formation of skeletal muscle. The importance of cell-ECM interactions will also be illustrated by a discussion of what happens when these interactions go awry, as happens in muscular dystrophies.

Distinct α7Aβ1 and α7Bβ1 integrin expression patterns during mouse development: α7A is restricted to skeletal muscle but α7B is expressed in striated muscle, vasculature, and nervous system

The laminin binding α7β1 integrin has been described as a major integrin in skeletal muscle. The RNA coding for the cytoplasmic domain of α7 integrin undergoes alternative splicing to generate two major forms, denoted α7A and α7B. In the current paper, we have examined the developmental expression patterns of the α7A and α7B splice variants in the mouse. The α7 integrin expression is compared to that of the nonintegrin laminin receptor dystroglycan and to that of laminin‐α1 and laminin‐α2 chains. α7A integrin was found by in situ hybridization to be specific to skeletal muscle. Antibodies specific for α7B integrin and in situ hybridization revealed the presence of α7 mRNA and α7B protein in the E10 myotome and later in primary and secondary myotubes. In the heart, α7B integrin was not detectable in the endocardium or myocardium during embryonic and fetal heart development. Northern blot analysis and immunohistochemistry revealed a postnatal induction of α7B in the myocardium. In addition to striated muscle, α7B integrin was localized to previously unreported nonmuscle locations such as a subset of vascular endothelia and restricted sites in the nervous system. Comparison of the α7 integrin expression pattern with that of different laminin isoforms and dystroglycan revealed a coordinated temporal expression of dystroglycan, α7 integrin, and laminin‐α2, but not laminin‐α1, in the forming skeletal muscle. We conclude that the α7A and α7B integrin variants are expressed in a developmentally regulated, tissue‐specific pattern suggesting different functions for the two splice forms.

β1-Integrins induce phosphorylation of Akt on serine 473 independently of focal adhesion kinase and Src family kinases

Adhesion by means of β1‐integrins induces the phosphorylation of Akt, an event strictly dependent on the activity of the phosphatidylinositol 3‐kinase (PI3K). Binding of the p85 regulatory subunit of PI3K to phosphorylated tyrosine 397 in focal adhesion kinase (FAK) is considered to be the mechanism of cell adhesion‐induced activation of class Ia PI3K. Here we show that PI3K‐dependent phosphorylation of Akt in response to ligation of β1‐integrins occurs efficiently in the absence of FAK tyrosine phosphorylation. Akt S473 phosphorylation was strongly promoted both in cells expressing the integrin subunit splice variant β1B, which is unable to activate FAK, and in FAK knockout cells. In addition, we found this phosphorylation to be independent of the Src family kinases Src, Fyn and Yes. These results indicate that a major pathway for adhesion‐dependent activation of PI3K/Akt is triggered by the membrane proximal part of the β1 subunit in a FAK and Src‐independent manner.

PI3-kinase p110α mediates β1 integrin-induced Akt activation and membrane protrusion during cell attachment and initial spreading.

Integrin-mediated cell adhesion activates several signaling effectors, including phosphatidylinositol 3-kinase (PI3K), a central mediator of cell motility and survival. To elucidate the molecular mechanisms of this important pathway the specific members of the PI3K family activated by different integrins have to be identified. Here, we studied the role of PI3K catalytic isoforms in β1 integrin-induced lamellipodium protrusion and activation of Akt in fibroblasts. Real-time total internal reflection fluorescence imaging of the membrane-substrate interface demonstrated that β1 integrin-mediated attachment induced rapid membrane spreading reaching essentially maximal contact area within 5-10 min. This process required actin polymerization and involved activation of PI3K. Isoform-selective pharmacological inhibition identified p110α as the PI3K catalytic isoform mediating both β1 integrin-induced cell spreading and Akt phosphorylation. A K756L mutation in the membrane-proximal part of the β1 integrin subunit, known to cause impaired Akt phosphorylation after integrin stimulation, induced slower cell spreading. The initial β1 integrin-regulated cell spreading as well as Akt phosphorylation were sensitive to the tyrosine kinase inhibitor PP2, but were not dependent on Src family kinases, FAK or EGF/PDGF receptor transactivation. Notably, cells expressing a Ras binding-deficient p110α mutant were severely defective in integrin-induced Akt phosphorylation, but exhibited identical membrane spreading kinetics as wild-type p110α cells. We conclude that p110α mediates β1 integrin-regulated activation of Akt and actin polymerization important for survival and lamellipodia dynamics. This could contribute to the tumorigenic properties of cells expressing constitutively active p110α.

TF/FVIIa Transactivate PDGFRβ to Regulate PDGF-BB–Induced Chemotaxis in Different Cell Types: Involvement of Src And PLC

Background—We have previously reported the potentiation of PDGF-BB–induced chemotaxis of fibroblasts, vascular smooth muscle cells, and endothelial cells by FVIIa. Here we studied the role of TF/FVIIa and the induced signaling pathways in regulation of chemotaxis of human monocytes, fibroblasts, and porcine aorta endothelial cells. Methods and Results—Human monocytes were obtained by using Ficoll-Paque gradient and the MACS system (for highly purified population), fibroblasts and PAE cells have been characterized previously. Inhibitors of selected signaling intermediates were used, and the effect of TF/FVIIa on the migratory response of all cells to chemotactic agents was analyzed. The induced signaling was studied by immunoprecipitation and Western blotting. TF/FVIIa complex selectively enhanced PDGF-BB–induced chemotaxis in a Src-family, PLC, and PAR-2–dependent manner. Using PAE cells we identified c-Src and c-Yes as the Src-family members activated by TF/FVIIa. We report for the first time the PAR-2 and Src family-dependent transactivation of PDGFR&bgr; by TF/FVIIa involving phosphorylation of a subset of PDGFR&bgr; tyrosines. Conclusions—The described transactivation is a likely mechanism of TF/FVIIa-mediated regulation of PDGF-BB–induced chemotaxis. Similar behavior of 3 principally different cell types in our experimental setup may reflect a general function of TF in regulation of cell migration.

Tenascin-C expression correlates with macrophage invasion in Duchenne muscular dystrophy and in myositis

Tenascin-C (TN-C) is an extracellular matrix protein expressed during development in several tissues, but restricted to only a few areas in normal adult tissues. By immunizing mice with human fetal myoblasts we generated a monoclonal antibody to TN-C and mapped the epitope to the aminoterminal end containing EGF-like repeats. Using this antibody we detected by immunohistochemistry TN-C in the epimysium and perimysium of human fetal muscles, as well as in nonfibrillar deposits in myoblast cultures. In situ hybridization did not reveal any signal within human fetal muscle groups, suggesting that non-muscle cells synthesize the majority of the tenascin that localizes in and around human fetal muscle. Immunohistochemical analysis of muscle biopsies from Duchenne/Becker muscular dystrophy and myositis patients revealed that TN-C is expressed in skeletal muscle. Although the patterns of TN-C immunoreactivity were quite different in the two disease entities, the endomysial TN-C reactivity in both DMD/BMD and in myositis invariably correlated with the presence of macrophages.

Analysis of the human integrin α11 gene (ITGA11) and its promoter

Abstract Integrin α11β1 is a collagen receptor which is expressed in a subset of mesenchymally-derived tissues during embryogenesis. Based on available human chromosome 15-derived sequences and genomic PCR, the complete exon structure of ITGA11, including the proximal promoter, was assembled into 30 exons. The inserted region (encoding amino acids 804–826) distinguishing α11 from other integrin α chains, was placed in the very beginning of exon 20. PCR data failed to show alternative splicing of RNA transcribed from this region. Using the oligo-capping technique a major transcription start site was mapped 30 nucleotides upstream of the translation start and identified as an abbreviated initiator sequence. Promoter sequence analysis in silico suggested the presence of multiple binding sites for transcription factors in the region upstream of the transcription start. 3 kb of the 5′ flanking sequence was isolated and used to generate luciferase promoter constructs. In the fibrosarcoma cell line HT1080 a core promoter [nt (−)127–(+)25], a potential silencer region [nt (−)400–(−)127] and a potential enhancer region [nt (−)1519–(−)400], were identified as being important for α11 transcription in mesenchymal cells. Furthermore, studies of the promoter region will provide valuable information regarding the molecular mechanisms underlying the cell- and tissue- specific expression pattern of ITGA11.