Roumen Pankov

Transmembrane crosstalk between the extracellular matrix--cytoskeleton crosstalk.

Integrin-mediated cell adhesions provide dynamic, bidirectional links between the extracellular matrix and the cytoskeleton. Besides having central roles in cell migration and morphogenesis, focal adhesions and related structures convey information across the cell membrane, to regulate extracellular-matrix assembly, cell proliferation, differentiation, and death. This review describes integrin functions, mechanosensors, molecular switches and signal-transduction pathways activated and integrated by adhesion, with a unifying theme being the importance of local physical forces.

Transmembrane crosstalk between the extracellular matrix and the cytoskeleton

Integrin-mediated cell adhesions provide dynamic, bidirectional links between the extracellular matrix and the cytoskeleton. Besides having central roles in cell migration and morphogenesis, focal adhesions and related structures convey information across the cell membrane, to regulate extracellular-matrix assembly, cell proliferation, differentiation, and death. This review describes integrin functions, mechanosensors, molecular switches and signal-transduction pathways activated and integrated by adhesion, with a unifying theme being the importance of local physical forces.

Fibronectin at a glance

Fibronectin (FN) mediates a wide variety of cellular interactions with the extracellular matrix (ECM) and plays important roles in cell adhesion, migration, growth and differentiation ( [Mosher, 1989][1]; [Carsons, 1989][2]; [Hynes, 1990][3]; [Yamada and Clark, 1996][4]). FN is widely expressed by

Cell interactions with three-dimensional matrices

Signaling and other cellular functions differ in three-dimensional compared with two-dimensional systems. Cell adhesion structures can evolve in vitro towards in-vivo-like adhesions with distinct biological activities. In this review, we examine recent advances in studies of interactions of fibroblasts with collagen gels and fibronectin-containing matrices that mimic in vivo three-dimensional microenvironments. These three-dimensional systems are illuminating mechanisms of cell-matrix interactions in living organisms.

A Rac switch regulates random versus directionally persistent cell migration

Directional migration moves cells rapidly between points, whereas random migration allows cells to explore their local environments. We describe a Rac1 mechanism for determining whether cell patterns of migration are intrinsically random or directionally persistent. Rac activity promoted the formation of peripheral lamellae that mediated random migration. Decreasing Rac activity suppressed peripheral lamellae and switched the cell migration patterns of fibroblasts and epithelial cells from random to directionally persistent. In three-dimensional rather than traditional two-dimensional cell culture, cells had a lower level of Rac activity that was associated with rapid, directional migration. In contrast to the directed migration of chemotaxis, this intrinsic directional persistence of migration was not mediated by phosphatidylinositol 3′-kinase lipid signaling. Total Rac1 activity can therefore provide a regulatory switch between patterns of cell migration by a mechanism distinct from chemotaxis.

A specific α5β1-integrin conformation promotes directional integrin translocation and fibronectin matrix formation

Integrin adhesion receptors are structurally dynamic proteins that adopt a number of functionally relevant conformations. We have produced a conformation-dependent anti-α5 monoclonal antibody (SNAKA51) that converts α5β1 integrin into a ligand-competent form and promotes fibronectin binding. In adherent fibroblasts, SNAKA51 preferentially bound to integrins in fibrillar adhesions. Clustering of integrins expressing this activation epitope induced directional translocation of α5β1, mimicking fibrillar adhesion formation. Priming of α5β1 integrin by SNAKA51 increased the accumulation of detergent-resistant fibronectin in the extracellular matrix, thus identifying an integrin conformation that promotes matrix assembly. The SNAKA51 epitope was mapped to the calf-1/calf-2 domains. We propose that the action of the antibody causes the legs of the integrin to change conformation and thereby primes the integrin to bind ligand. These findings identify SNAKA51 as the first anti-integrin antibody to selectively recognize a subset of adhesion contacts, and they identify an integrin conformation associated with integrin translocation and fibronectin matrix formation.

Specific β1 Integrin Site Selectively Regulates Akt/Protein Kinase B Signaling via Local Activation of Protein Phosphatase 2A

Integrin transmembrane receptors generate multiple signals, but how they mediate specific signaling is not clear. Here we test the hypothesis that particular sequences along the β1 integrin cytoplasmic domain may exist that are intimately related to specific integrin-mediated signaling pathways. Using systematic alanine mutagenesis of amino acids conserved between different β integrin cytoplasmic domains, we identified the tryptophan residue at position 775 of human β1 integrin as specific and necessary for integrin-mediated protein kinase B/Akt survival signaling. Stable expression of a β1 integrin mutated at this amino acid in GD25 β1-null cells resulted in reduction of Akt phosphorylation at both Ser473 and Thr308 activation sites. As a consequence, the cells were substantially more sensitive to serum starvation-induced apoptosis when compared with cells expressing wild type β1 integrin. This inactivation of Akt resulted from increased dephosphorylation by a localized active population of protein phosphatase 2A. Both Akt and protein phosphatase 2A were present in β1integrin-organized cytoplasmic complexes, but the activity of this phosphatase was 2.5 times higher in the complexes organized by the mutant integrin. The mutation of Trp775 specifically affected Akt signaling, without effects on other integrin-activated pathways including phosphoinositide 3-kinase, MAPK, JNK, and p38 nor did it influence activation of the integrin-responsive kinases focal adhesion kinase and Src. The identification of Trp775 as a specific site for integrin-mediated Akt signaling supports the concept of specificity of signaling along the integrin cytoplasmic domain.

AP-1, ETS, and transcriptional silencers regulate retinoic acid-dependent induction of keratin 18 in embryonic cells.

The differentiation of both embryonal carcinoma (EC) and embryonic stem (ES) cells can be triggered in culture by exposure to retinoic acid and results in the transcriptional induction of both the endogenous mouse keratin 18 (mK18) intermediate filament gene and an experimentally introduced human keratin 18 (K18) gene as well as a variety of other markers characteristic of extraembryonic endoderm. The induction of K18 in EC cells is limited, in part, by low levels of ETS and AP-1 transcription factor activities which bind to sites within a complex enhancer element located within the first intron of K18. RNA levels of ETS-2, c-Jun, and JunB increase upon the differentiation of ES cells and correlate with increased expression of K18. Occupancy of the ETS site, detected by in vivo footprinting methods, correlates with K18 induction in ES cells. In somatic cells, the ETS and AP-1 elements mediate induction by a variety of oncogenes associated with the ras signal transduction pathway. In EC cells, in addition to the induction by these limiting transcription factors, relief from negative regulation is mediated by three silencer elements located within the first intron of the K18 gene. These silencer elements function in F9 EC cells but not their differentiated derivatives, and their activity is correlated with proteins in F9 EC nuclei which bind to the silencers and are reduced in the nuclei of differentiated F9 cells. The induction of K18, associated with the differentiation of EC cells to extraembryonic endoderm, is due to a combination of relief from negative regulation and activation by members of the ETS and AP-1 transcription factor families.

β1 Integrin Cytoplasmic Domain Residues Selectively Modulate Fibronectin Matrix Assembly and Cell Spreading through Talin and Akt-1

The integrin β1 cytoplasmic domain (tail) serves as a scaffold for numerous intracellular proteins. The mechanisms by which the tail coordinates these proteins to facilitate extracellular matrix assembly and cell spreading are not clear. This study demonstrates that the β1 cytoplasmic domain can regulate cell spreading on fibronectin and fibronectin matrix assembly through Akt- and talin-dependent mechanisms, respectively. To identify these mechanisms, we characterized GD25 cells expressing the β1 integrin cytoplasmic domain mutants W775A and R760A. Although cell spreading appears normal in R760A mutant-integrin cells compared with wild type, it is inhibited in W775A mutant cells. In contrast, both mutant cell lines show defective fibronectin matrix assembly. Inhibition of cell spreading, but not matrix assembly, in the W775A mutant cells is due to a specific defect in Akt-1 activation. In addition, we find that both W775A and R760A mutant integrins have reduced surface expression of the 9EG7 epitope that correlates with reduced recruitment of talin to β1 integrin cytoplasmic complexes. Down-regulation of talin with small interfering RNA or expression of green fluorescent protein-talin head domain inhibits matrix assembly in β1 wild-type cells, mimicking the defect seen with the W775A and R760A mutant cells. These results demonstrate distinct mechanisms by which integrins regulate cell spreading and matrix assembly through the β1 integrin cytoplasmic tail.

Three‐dimensional matrix induces sustained activation of ERK1/2 via Src/Ras/Raf signaling pathway

Research in cell signaling often depends on tissue culture, but the artificial substrates used to grow cells in vitro are likely to distort the conclusions, particularly when adhesion‐mediated signaling events are investigated. Studies of signal transduction pathways operating in cells grown in three‐dimensional (3D) matrices provide a better system, giving a closer insight of the cell signaling in vivo. We compared the steady‐state levels of ERK1/2 activity in primary human fibroblasts, induced by cell‐derived 3D fibronectin matrix or fibronectin, coated on flat surfaces. 3D environment caused ERK1/2 stimulation concomitant with a 2.5‐fold increase in Ras GTP loading and Src activation. Under these conditions FAK autophosphorylation was suppressed. Treatment with Src inhibitor PP2 abolished these effects indicating that 3D fibronectin matrix activated ERK1/2 through Src/Ras/Raf pathway, bypassing FAK. These observations suggest that within in vivo‐like conditions Src may have a leading role in the induction of sustained ERK1/2 activation.