Pamma D. Arora

Intracellular osteopontin is an integral component of the CD44-ERM complex involved in cell migration.

Osteopontin (OPN) is a secreted glycoprotein with mineral‐ and cell‐binding properties that can regulate cell activities through integrin receptors. Previously, we identified an intracellular form of osteopontin with a perimembranous distribution in migrating fetal fibroblasts (Zohar et al., J Cell Physiol 170:88–98, 1997). Since OPN and CD44 expression are increased in migrating cells, we analyzed the relationship of these proteins with immunofluorescence and confocal microscopy. A distinct co‐localization of perimembranous OPN and cell‐surface CD44 was observed in fetal fibroblasts, periodontal ligament cells, activated macrophages, and metastatic breast cancer cells. The co‐localization of OPN and CD44 was prominent at the leading edge of migrating fibroblasts, where OPN also co‐localized with the ezrin/radixin/moesin (ERM) protein ezrin, as well as in cell processes and at attachment sites of hyaluronan‐coated beads. The subcortical location of OPN in these cells was verified by cell‐surface biotinylation experiments in which biotinylated CD44 and non‐biotinylated OPN were isolated from complexes formed with hyaluronan‐coated beads and identified with immunoblotting. That perimembranous OPN represents secreted protein internalized by endocytosis or phagocytosis appeared to be unlikely since exogenous OPN that was added to cell cultures could not be detected inside the cells. A physical association with OPN, CD44, and ERM, but not with vinculin or α‐actin, was indicated by immunoadsorption and immunoblotting of cell proteins in complexes extracted from hyaluronan‐coated beads. The functional significance of OPN in this complex was demonstrated using OPN−/− and CD−/− mouse fibroblasts which displayed impaired migration and a reduced attachment to hyaluronan‐coated beads. These studies indicate that OPN exists as an integral component of a hyaluronan‐CD44‐ERM attachment complex that is involved in the migration of embryonic fibroblasts, activated macrophages, and metastatic cells. J. Cell. Physiol. 184:118–130, 2000.

α11 integrin stimulates myofibroblast differentiation in diabetic cardiomyopathy

AIMS Diabetic cardiomyopathy is characterized by the production of a disorganized fibrotic matrix in the absence of coronary atherosclerosis and hypertension. We examined whether adhesion of cardiac fibroblasts to glycated collagens mediates the differentiation of pro-fibrotic myofibroblasts, which may contribute to cardiac fibrosis. METHODS AND RESULTS By microarray, we found that methylglyoxal-treated collagen selectively enhanced α11 integrin expression in human cardiac fibroblasts, while levels of other collagen-binding integrins (α1, α2, and α10) were unchanged. Similar increases in α11 integrin mRNA and protein expression were observed in cardiac fibroblasts from streptozotocin (STZ)-treated Sprague-Dawley rats. In human cardiac fibroblasts plated on methyglyoxal-treated collagen and in cardiac fibroblasts from diabetic rats, transforming growth factor (TGF)-β2 but not TGF-β1 or TGF-β3 was increased compared with controls. Knock-down of α11 integrin and TGF-β receptors with small-interfering RNA blocked the increased expression of TGF-β2, α-smooth muscle actin (α-SMA), and α11 integrin that were induced in cells plated on methylglyoxal-treated collagen. Further, inhibition of Smad3 signalling blocked methylglyoxal-collagen up-regulation of α11 integrin and α-SMA expression. Rats with STZ-induced diabetes exhibited increased phosphorylation of Smad3 in cardiac tissues compared with control rats. CONCLUSION Interactions between α11 integrins and the Smad-dependent TGF-β2 signalling may contribute to the formation of pro-fibrotic myofibroblasts and the development of a fibrotic interstitium in diabetic cardiomyopathy.

FAK, PIP5KIγ and gelsolin cooperatively mediate force-induced expression of α-smooth muscle actin

During the development of pressure-induced cardiac hypertrophy, fibroblasts are activated to become myofibroblasts, which exhibit actin-cytoskeletal remodeling and express α-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/μm2) 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)P2], which uncaps gelsolin from actin filaments, was enriched at sites of force application. Type-I phosphatidylinositol 4-phosphate 5 kinase-γ (PIP5KIγ), which generates PtdIns(4,5)P2, associated with FAK and was required for force-mediated SMA-promoter activity and actin assembly. Catalytically inactive PIP5KIγ 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 PIP5KIγ; actin assembly in turn controls SMA expression via MRTF-A.

Inelastic behaviour of collagen networks in cell-matrix interactions and mechanosensation.

The mechanical properties of extracellular matrix proteins strongly influence cell-induced tension in the matrix, which in turn influences cell function. Despite progress on the impact of elastic behaviour of matrix proteins on cell–matrix interactions, little is known about the influence of inelastic behaviour, especially at the large and slow deformations that characterize cell-induced matrix remodelling. We found that collagen matrices exhibit deformation rate-dependent behaviour, which leads to a transition from pronounced elastic behaviour at fast deformations to substantially inelastic behaviour at slow deformations (1 μm min−1, similar to cell-mediated deformation). With slow deformations, the inelastic behaviour of floating gels was sensitive to collagen concentration, whereas attached gels exhibited similar inelastic behaviour independent of collagen concentration. The presence of an underlying rigid support had a similar effect on cell–matrix interactions: cell-induced deformation and remodelling were similar on 1 or 3 mg ml−1 attached collagen gels while deformations were two- to fourfold smaller in floating gels of high compared with low collagen concentration. In cross-linked collagen matrices, which did not exhibit inelastic behaviour, cells did not respond to the presence of the underlying rigid foundation. These data indicate that at the slow rates of collagen compaction generated by fibroblasts, the inelastic responses of collagen gels, which are influenced by collagen concentration and the presence of an underlying rigid foundation, are important determinants of cell–matrix interactions and mechanosensation.

Actin polymerization stabilizes α4β1 integrin anchors that mediate monocyte adhesion

Leukocytes arrested on inflamed endothelium via integrins are subjected to force imparted by flowing blood. How leukocytes respond to this force and resist detachment is poorly understood. Live-cell imaging with Lifeact-transfected U937 cells revealed that force triggers actin polymerization at upstream α4β1 integrin adhesion sites and the adjacent cortical cytoskeleton. Scanning electron microscopy revealed that this culminates in the formation of structures that anchor monocyte adhesion. Inhibition of actin polymerization resulted in cell deformation, displacement, and detachment. Transfection of dominant-negative constructs and inhibition of function or expression revealed key signaling steps required for upstream actin polymerization and adhesion stabilization. These included activation of Rap1, phosphoinositide 3-kinase γ isoform, and Rac but not Cdc42. Thus, rapid signaling and structural adaptations enable leukocytes to stabilize adhesion and resist detachment forces.

Flightless I is a focal adhesion-associated actin-capping protein that regulates cell migration

The role of adhesion‐associated actin‐binding proteins in cell migration is not well defined. In mouse fibroblasts we screened for focal adhesion‐associated proteins that were isolated with collagen‐coated beads and detected by tandem mass spectrometry. We identified flightless I (FliI) as an actin‐binding protein in focal adhesion fractions, which was verified by immunoblotting. By confocal microscopy most FliI was distributed throughout the cytosol and in focal adhesions. By sedimentation assays and in vitro binding assays, we found that FliI associates with actin filaments and actin monomers. Assays using purified proteins showed that FliI inhibits actin polymerization and caps but does not sever actin filaments. Cells with FliI knockdown or cells overexpressing FliI migrated more or less rapidly, respectively, than wild‐type controls. Compared with controls, cells with FliI knockdown were less adherent than wild‐type cells, exhibited reduced numbers of focal adhesions containing activated β1 integrins and vinculin, and exhibited increased incorporation of actin monomers into nascent filaments at focal adhesions. These data indicate that FliI regulates cell migration through its localization to focal adhesions and its ability to cap actin filaments, which collectively affect focal adhesion maturation.—Mohammad, I., Arora, P. D., Naghibzadeh, Y., Wang, Y., Li, J., Mascarenhas, W., Janmey, P. A., Dawson, J. F., McCulloch, C. A. Flightless I is a focal adhesion‐associated actin‐capping protein that regulates cell migration. FASEB J. 26, 3260–3272 (2012). www.fasebj.org

Glycated Collagen Induces α11 Integrin Expression Through TGF-β2 and Smad3.

The adhesion of cardiac fibroblasts to the glycated collagen interstitium in diabetics is associated with de novo expression of the α11 integrin, myofibroblast formation and cardiac fibrosis. We examined how methylglyoxal‐glycated collagen regulates α11 integrin expression. In cardiac fibroblasts plated on glycated collagen but not glycated fibronectin, there was markedly increased α11 integrin and α‐smooth muscle actin expression. Compared with native collagen, binding of purified α11β1 integrin to glycated collagen was reduced by >fourfold, which was consistent with reduced fibroblast attachment to glycated collagen. Glycated collagen strongly enhanced the expression of TGF‐β2 but not TGF‐β1 or TGF‐β3. The increased expression of TGF‐β2 was inhibited by triple helical collagen peptides that mimic the α11β1 integrin binding site on type I collagen. In cardiac fibroblasts transfected with α11 integrin luciferase promoter constructs, glycated collagen activated the α11 integrin promoter. Analysis of α11 integrin promoter truncation mutants showed a novel Smad2/3 binding site located between −809 and −1300 nt that was required for promoter activation. We conclude that glycated collagen in the cardiac interstitium triggers an autocrine TGF‐β2 signaling pathway that stimulates α11 integrin expression through Smad2/3 binding elements in the α11 integrin promoter, which is important for myofibroblast formation and fibrosis. J. Cell. Physiol. 230: 327–336, 2015.

Collagen remodeling by phagocytosis is determined by collagen substrate topology and calcium-dependent interactions of gelsolin with nonmuscle myosin IIA in cell adhesions

Cell adhesion to collagen presented on beads activates Ca2+ entry and promotes the formation of phagosomes enriched with NMMIIA and gelsolin. The Ca2+-dependent interaction of gelsolin and NMMIIA in turn enables actin remodeling and enhances collagen degradation by phagocytosis.

Glycated collagen induces a11 integrin expression through TGF-ß2 and Smad3

The adhesion of cardiac fibroblasts to the glycated collagen interstitium in diabetics is associated with de novo expression of the α11 integrin, myofibroblast formation and cardiac fibrosis. We examined how methylglyoxal‐glycated collagen regulates α11 integrin expression. In cardiac fibroblasts plated on glycated collagen but not glycated fibronectin, there was markedly increased α11 integrin and α‐smooth muscle actin expression. Compared with native collagen, binding of purified α11β1 integrin to glycated collagen was reduced by >fourfold, which was consistent with reduced fibroblast attachment to glycated collagen. Glycated collagen strongly enhanced the expression of TGF‐β2 but not TGF‐β1 or TGF‐β3. The increased expression of TGF‐β2 was inhibited by triple helical collagen peptides that mimic the α11β1 integrin binding site on type I collagen. In cardiac fibroblasts transfected with α11 integrin luciferase promoter constructs, glycated collagen activated the α11 integrin promoter. Analysis of α11 integrin promoter truncation mutants showed a novel Smad2/3 binding site located between −809 and −1300 nt that was required for promoter activation. We conclude that glycated collagen in the cardiac interstitium triggers an autocrine TGF‐β2 signaling pathway that stimulates α11 integrin expression through Smad2/3 binding elements in the α11 integrin promoter, which is important for myofibroblast formation and fibrosis. J. Cell. Physiol. 230: 327–336, 2015.

Force-induced apoptosis mediated by the Rac/Pak/p38 signalling pathway is regulated by filamin A.

Cells in mechanically challenged environments cope with high-amplitude exogenous forces that can lead to cell death, but the mechanisms that mediate force-induced apoptosis and the identity of mechanoprotective cellular factors are not defined. We assessed apoptosis in NIH 3T3 and HEK (human embryonic kidney)-293 cells exposed to tensile forces applied through β1-integrins. Apoptosis was mediated by Rac-dependent activation of p38α. Depletion of Pak1 (p21-activated kinase 1), a downstream effector of Rac, prevented force-induced p38 activation and apoptosis. Rac was recruited to sites of force transfer by filamin A, which inhibited force-induced apoptosis mediated by Rac and p38α. We conclude that, in response to tensile force, filamin A regulates Rac-dependent signals, which induce apoptosis through Pak1 and p38.