Mechanical induction of gene expression in connective tissue cells.

The extracellular matrices of mammals undergo coordinated synthesis and degradation, dynamic remodeling processes that enable tissue adaptations to a broad range of environmental factors, including applied mechanical forces. The soft and mineralized connective tissues of mammals also exhibit a wide repertoire of mechanical properties, which enable their tissue-specific functions and modulate cellular responses to forces. The expression of genes in response to applied forces are important for maintaining the support, attachment, and function of various organs including kidney, heart, liver, lung, joint, and periodontium. Several high-prevalence diseases of extracellular matrices including arthritis, heart failure, and periodontal diseases involve pathological levels of mechanical forces that impact the gene expression repertoires and function of bone, cartilage, and soft connective tissues. Recent work on the application of mechanical forces to cultured connective tissue cells and various in vivo force models have enabled study of the regulatory networks that control mechanically induced gene expression in connective tissue cells. In addition to the influence of mechanical forces on the expression of type 1 collagen, which is the most abundant protein of mammals, new work has shown that the expression of a wide range of matrix, signaling, and cytoskeletal proteins are regulated by exogenous mechanical forces and by the forces generated by cells themselves. In this chapter, we first discuss the fundamental nature of the extracellular matrix in health and the impact of mechanical forces. Next we consider the utilization of several, widely employed model systems for mechanical stimulation of cells. Finally, we consider in detail how application of tensile forces to cultured cardiac fibroblasts can be used for the characterization of the signaling systems by which mechanical forces regulate myofibroblast differentiation that is seen in cardiac pressure overload.

Force-induced myofibroblast differentiation through collagen receptors is dependent on mammalian diaphanous (mDia).

The development of fibrosis promotes the differentiation of myofibroblasts, pro-fibrotic cells, which contribute to tissue dysfunction. Myofibroblast differentiation is dependent on actin assembly, which in response to force, is mediated by various actin-binding proteins including the mammalian Diaphanous-related formins (mDia). We examined the role of mDia in the mechano-sensing pathway that leads to force-induced expression of alpha-smooth muscle actin (SMA), a marker and critical determinant of myofibroblast differentiation. In cells treated with siRNA to knockdown mDia and then subjected to tensile force using collagen-coated magnetite beads attached to beta1 integrins, actin assembly was inhibited at bead contact sites. Force-induced nuclear translocation of MRTF-A, a transcriptional co-activator of SMA, was reduced 50% by mDia knockdown. The expression of the transcriptional co-activator of SMA, serum response factor, was reduced by 50% after siRNA knockdown of mDia or by 100% in cells transfected with catalytically inactive mDia. Force-induced activation of the SMA promoter and SMA expression were blocked by knockdown of siRNA of mDia. In anchored collagen gel assays to measure myofibroblast-mediated contraction, knockdown of mDia reduced contraction by 50%. We conclude that mDia plays an important role in the development of force-induced transcriptional activation of SMA and myofibroblast differentiation.

FAK, PIP5KIgamma and gelsolin cooperatively mediate force-induced expression of alpha-smooth muscle actin.

During the development of pressure-induced cardiac hypertrophy, fibroblasts are activated to become myofibroblasts, which exhibit actin-cytoskeletal remodeling and express alpha-smooth muscle actin (SMA; encoded by ACTA2). Currently, the mechanosensing signaling pathways that regulate SMA expression are not defined. Because focal-adhesion complexes are putative mechanosensing organelles, we examined the role of focal adhesion kinase (FAK) and its interaction with gelsolin in the regulation of SMA expression. We subjected NIH3T3 cells to tensile forces (0.65 pN/mum(2)) by using collagen-coated magnetite beads attached to integrins. After stimulation by mechanical force, FAK and gelsolin were recruited to magnetite beads and there was increased phosphorylation of Tyr397FAK. Mechanical force enhanced SMA promoter activity by twofold; this increased activity was blocked by FAK knockdown using siRNA and by deletion of gelsolin. Force-induced nuclear translocation of MRTF-A, a transcriptional co-activator of SMA that is regulated by actin filaments, was also reduced by FAK knockdown. Phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P(2)], which uncaps gelsolin from actin filaments, was enriched at sites of force application. Type-I phosphatidylinositol 4-phosphate 5 kinase-gamma (PIP5KIgamma), which generates PtdIns(4,5)P(2), associated with FAK and was required for force-mediated SMA-promoter activity and actin assembly. Catalytically inactive PIP5KIgamma inhibited force-induced phosphorylation of FAK at Tyr397. These data suggest a novel pathway in which mechanosensing by FAK regulates actin assembly via gelsolin and the activity of PIP5KIgamma; actin assembly in turn controls SMA expression via MRTF-A.

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. Because gelsolin is a Ca(2+)-dependent actin-severing protein that mediates collagen phagocytosis, we determined whether gelsolin is a CsA target. Compared with vehicle controls, CsA treatment of wild-type mice increased collagen accumulation by 60% in periodontal tissues; equivalent increases were seen in vehicle-treated gelsolin-null mice. Collagen degradation by phagocytosis in cultured gelsolin wild-type fibroblasts was blocked by CsA, comparable to levels of vehicle-treated gelsolin-null fibroblasts. In wild-type cells treated with CsA, collagen binding was similar to that of gelsolin-null fibroblasts transfected with a gelsolin-severing mutant and treated with vehicle. CsA blocked collagen-induced Ca(2+) fluxes subjacent to bound collagen beads, gelsolin recruitment, and actin assembly at bead sites. CsA reduced gelsolin-dependent severing of actin in wild-type cells to levels similar to those in gelsolin-null fibroblasts. We conclude that CsA-induced accumulation of collagen in the extracellular matrix involves disruption of the actin-severing properties of gelsolin, thereby inhibiting the binding step of collagen phagocytosis.

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. Fibroblasts from gelsolin-null mice exhibited marked reductions in kinetics and strengthening of N-cadherin-dependent junctions when compared with wild-type cells. Experiments with lanthanum chloride (250 microm) showed that adhesion strength was dependent on entry of calcium ions subsequent to N-cadherin ligation. Cadherin-associated gelsolin severing activity was required for localized actin assembly as determined by rhodamine actin monomer incorporation onto actin barbed ends at intercellular adhesion sites. Scanning electron microscopy showed that gelsolin was an important determinant of actin filament architecture of adherens junctions at nascent N-cadherin-mediated contacts. These data indicate that increased actin barbed end generation by the severing activity of gelsolin associated with N-cadherin regulates intercellular adhesion strength.

Soluble fibrinogen-like protein 2/fibroleukin exhibits immunosuppressive properties: suppressing T cell proliferation and inhibiting maturation of bone marrow-derived dendritic cells.

Fibrinogen-like protein 2 (fgl2)/fibroleukin is a member of the fibrinogen-related protein superfamily. In addition to its established role in triggering thrombosis, it is known to be secreted by T cells. The soluble fgl2 ((s)fgl2) protein generated in a baculovirus expression system bound to both T cells and bone marrow-derived dendritic cells (DC) in a specific manner. (s)fgl2 exhibited immunomodulatory properties capable of inhibiting T cell proliferation stimulated by alloantigens, anti-CD3/anti-CD28 mAbs, and Con A in a dose-dependent manner; however, it had no inhibitory effects on CTL activity. The time- and dose-dependent inhibitory effect of (s)fgl2 on alloreactive T cell proliferation could be neutralized by a mAb against mouse fgl2. Polarization toward a Th2 cytokine profile with decreased production of IL-2 and IFN-gamma and increased production of IL-4 and IL-10 was observed in (s)fgl2-treated allogeneic cultures. Exposure of immature DC to (s)fgl2 abrogated the expression of CD80(high) and MHC class II(high) molecules and markedly inhibited NF-kappaB nuclear translocation, thus inhibiting their maturation. (s)Fgl2-treated DC had an impaired ability to stimulate allogeneic T cell proliferation. Maximal inhibition of proliferation was observed when allogeneic T cells were cultured with (s)fgl2-treated DC and (s)fgl2 protein was added in the culture. These data provide the first evidence to demonstrate that (s)fgl2 exerts immunosuppressive effects on T cell proliferation and DC maturation.

Kinetic analysis of a unique direct prothrombinase, fgl2, and identification of a serine residue critical for the prothrombinase activity.

fgl2 prothrombinase, by its ability to generate thrombin, has been shown to be pivotal to the pathogenesis of viral-induced hepatitis, cytokine-induced fetal loss syndrome, and xeno- and allograft rejection. In this study, the molecular basis of fgl2 prothrombinase activity was examined in detail. Purified fgl2 protein generated in a baculovirus expression system had no measurable prothrombinase activity, whereas the activity was restored when the purified protein was reconstituted into phosphatidyl-L-serine-containing vesicles. Reconstituted fgl2 catalyzed the cleavage of human prothrombin to thrombin with kinetics consistent with a first order reaction, with an apparent V(max) value of 6 mol/min/mol fgl2 and an apparent K(m) value for prothrombin of 8.3 microM. The catalytic activity was totally dependent on calcium, and factor Va (500 nM) enhanced the catalytic efficiency of fgl2 by increasing the apparent V(max) value to 3670 mol/min/mol fgl2 and decreasing the apparent K(m) value for prothrombin to 7.2 microM. By a combination of site-directed mutagenesis and production of truncated proteins, it was clearly shown that residue Ser(89) was critical for the prothrombinase activity of fgl2. Furthermore, fgl2 prothrombinase activity was not inhibited by antithrombin III, soybean trypsin inhibitor, 4-aminobenzamidine, aprotinin, or phenylmethylsulfonyl fluoride, whereas diisopropylfluorophosphate completely abrogated the activity. In this work we provide direct evidence that fgl2 cleaves prothrombin to thrombin consistent with serine protease activity and requires calcium, phospholipids, and factor Va for its full activity.