Role of the small GTPase activating protein IQGAP1 in collagen phagocytosis.

Many adult connective tissues undergo continuous remodeling to maintain matrix homeostasis. Physiological remodeling involves the degradation of collagen fibers by the intracellular cathepsin-dependent phagocytic pathway. We considered that a multidomain, small GTPase activating protein, IQGAP1, which is involved in the generation of cell extensions, is required for collagen phagocytosis, possibly arising from its interactions with cdc42 and the actin-binding protein Flightless I (FliI).

Multifunctional roles of gelsolin in health and diseases.

Gelsolin, a Ca(2+) -regulated actin filament severing, capping, and nucleating protein, is an ubiquitous, multifunctional regulator of cell structure and metabolism. More recent data show that gelsolin can act as a transcriptional cofactor in signal transduction and its own expression and function can be influenced by epigenetic changes. Here, we review the functions of the plasma and cytoplasmic forms of gelsolin, and their manifold impacts on cancer, apoptosis, infection and inflammation, cardiac injury, pulmonary diseases, and aging.

The role of FilGAP-filamin A interactions in mechanoprotection.

Cells in mechanically active environments are subjected to high-amplitude exogenous forces that can lead to cell death. Filamin A (FLNa) may protect cells from mechanically induced death by mechanisms that are not yet defined. We found that mechanical forces applied through integrins enhanced Rac-mediated lamellae formation in FLNa-null but not FLNa-expressing cells.

Rap1 activation in collagen phagocytosis is dependent on nonmuscle myosin II-A.

Rap1 enhances integrin-mediated adhesion but the link between Rap1 activation and integrin function in collagen phagocytosis is not defined. Mass spectrometry of Rap1 immunoprecipitates showed that the association of Rap1 with nonmuscle myosin heavy-chain II-A (NMHC II-A) was enhanced by cell attachment to collagen beads. Rap1 colocalized with NM II-A at collagen bead-binding sites.

Cyclosporin inhibition of collagen remodeling is mediated by gelsolin.

Cyclosporin A (CsA) inhibits collagen remodeling by interfering with the collagen-binding step of phagocytosis. In rapidly remodeling connective tissues such as human periodontium this interference manifests as marked tissue overgrowth and loss of function. Previous data have shown that CsA inhibits integrin-induced release of Ca(2+) from internal stores, which is required for the binding step of collagen phagocytosis.

Role of p38 in stress activation of Sp1.

Cell stressors such as physical forces can activate Sp1-dependent genes but the regulatory mechanisms are not defined. We determined if the stress-induced MAP kinase, p38, can phosphorylate Sp1 and thereby regulate the Sp1 target gene FLNA. We used Rat-2 cells and human gingival fibroblasts to examine stress-induced activation of an Sp1-dependent gene and SL2 cells, an Sp1-deficient model system, to facilitate interaction studies of transfected Sp1 with regulatory factors.

Phosphorylation of N-cadherin-associated cortactin by Fer kinase regulates N-cadherin mobility and intercellular adhesion strength.

Cortactin regulates the strength of nascent N-cadherin-mediated intercellular adhesions through a tyrosine phosphorylation-dependent mechanism. Currently, the functional significance of cortactin phosphorylation and the kinases responsible for the regulation of adhesion strength are not defined. We show that the nonreceptor tyrosine kinase Fer phosphorylates cadherin-associated cortactin and that this process is involved in mediating intercellular adhesion strength.

Collagen phagocytosis by fibroblasts is regulated by decorin.

Decorin is a small, leucine-rich proteoglycan that binds to collagen and regulates fibrillogenesis. We hypothesized that decorin binding to collagen inhibits phagocytosis of collagen fibrils. To determine the effects of decorin on collagen degradation, we analyzed phagocytosis of collagen and collagen/decorin-coated fluorescent beads by Rat-2 and gingival fibroblasts.

Smooth muscle actin determines mechanical force-induced p38 activation.

The mitogen-activated protein kinase p38 is activated by mechanical force, but the cellular elements that mediate force-induced p38 phosphorylation are not defined. As alpha-smooth muscle actin (SMA) is an actin isoform associated with force generation in fibroblasts, we asked if SMA participates in the activation of p38 by force. Tensile forces (0.

Cortactin associates with N-cadherin adhesions and mediates intercellular adhesion strengthening in fibroblasts.

The regulation of N-cadherin-mediated intercellular adhesion strength in fibroblasts is poorly characterized; this is due, in part, to a lack of available quantitative models. We used a recombinant N-cadherin chimeric protein and a Rat 2 fibroblast, donor-acceptor cell model, to study the importance of cortical actin filaments and cortactin in the strengthening of N-cadherin adhesions. In wash-off assays, cytochalasin D (1 microM) reduced intercellular adhesion by threefold, confirming the importance of cortical actin filaments in strengthening of N-cadherin-mediated adhesions.

Regulation of intercellular adhesion strength in fibroblasts.

The regulation of adherens junction formation in cells of mesenchymal lineage is of critical importance in tumorigenesis but is poorly characterized. As actin filaments are crucial components of adherens junction assembly, we studied the role of gelsolin, a calcium-dependent, actin severing protein, in the formation of N-cadherin-mediated intercellular adhesions. With a homotypic, donor-acceptor cell model and plates or beads coated with recombinant N-cadherin-Fc chimeric protein, we found that gelsolin spatially co-localizes to, and is transiently associated with, cadherin adhesion complexes.

Gelsolin mediates collagen phagocytosis through a rac-dependent step.

The role of gelsolin, a calcium-dependent actin-severing protein, in mediating collagen phagocytosis, is not defined. We examined alpha 2 beta 1 integrin-mediated phagocytosis in fibroblasts from wild-type (WT) and gelsolin knockout (Gsn(-)) mice. After initial contact with collagen beads, collagen binding and internalization were 60% lower in Gsn(-) than WT cells.

Regulation of tension-induced mechanotranscriptional signals by the microtubule network in fibroblasts.

Mechanical loading of connective tissues induces the expression of extracellular matrix and cytoskeletal genes that are involved in matrix remodeling. These processes depend in part on force transmission through beta1 integrins and actin filaments, but the role of microtubules in regulating mechanotranscriptional responses is not well defined. We assessed the involvement of microtubules in the mechanotranscriptional regulation of filamin A, an actin-cross-linking protein that protects cells against force-induced apoptosis by stabilizing cell membranes.

Differential binding to dorsal and ventral cell surfaces of fibroblasts: effect on collagen phagocytosis.

Matrix remodeling by phagocytic fibroblasts is essential for growth and development but the regulatory processes are undefined. We evaluated the impact of spreading on the binding step of collagen phagocytosis with a novel culture system that more closely replicates phagocytosis in vivo than previous models. 3T3 cells were plated on collagen-coated beads, thereby loading only ventral surfaces (adhesion with spreading), or were allowed to spread on collagen films and then loaded with beads on their dorsal surfaces (adhesion without spreading).

Interaction of p38 and Sp1 in a mechanical force-induced, beta 1 integrin-mediated transcriptional circuit that regulates the actin-binding protein filamin-A.

Connective tissue cells in mechanically active environments survive applied physical forces by modifying actin cytoskeletal structures that stabilize cell membranes. In fibroblasts, tensile forces induce the expression of filamin-A, a mechanoprotective actin-binding protein, but the mechanisms and protein interactions by which force activates filamin-A transcription are not defined. We found that in fibroblasts, application of tensile forces through collagen-coated magnetite beads to cell surface beta(1) integrins induced filamin-A expression.