Activation of Rac and Cdc42 by Integrins Mediates Cell Spreading

Adhesion to ECM is required for many cell functions including cytoskeletal organization, migration, and proliferation. We observed that when cells first adhere to extracellular matrix, they spread rapidly by extending filopodia-like projections and lamellipodia. These structures are similar to the Rac- and Cdc42-dependent structures observed in growth factor-stimulated cells. We therefore investigated the involvement of Rac and Cdc42 in adhesion and spreading on the ECM protein fibronectin. We found that integrin-dependent adhesion led to the rapid activation of p21-activated kinase, a downstream effector of Cdc42 and Rac, suggesting that integrins activate at least one of these GTPases. Dominant negative mutants of Rac and Cdc42 inhibit cell spreading in such a way as to suggest that integrins activate Cdc42, which leads to the subsequent activation of Rac; both GTPases then contribute to cell spreading. These results demonstrate that initial integrin-dependent activation of Rac and Cdc42 mediates cell spreading.     

Regulation of Rho family GTPases by cell-cell and cell-matrix adhesion

Integrins and cadherins are transmembrane adhesion receptors that are necessary for cells to interact with the extracellular matrix or adjacent cells, respectively. Integrins and cadherins initiate signaling pathways that modulate the activity of Rho family GTPases. The Rho proteins Cdc42, Rac1, and RhoA regulate the actin cytoskeleton. Cdc42 and Rac1 are primarily involved in the formation of protrusive structures, while RhoA generates myosin-based contractility. Here we examine the differential regulation of RhoA, Cdc42, and Rac1 by integrin and cadherin signaling. Integrin and cadherin signaling leads to a decrease in RhoA activity and activation of Cdc42 and Rac1. When the normal RhoA suppression is antagonized or RhoA signaling is increased, cells exhibited impaired spreading on the matrix protein fibronectin and decreased cell-cell adhesion. Spreading on fibronectin and the formation of cell-cell adhesions is decreased in cells expressing dominant negative forms of Cdc42 or Rac1. These data demonstrate that integrins and cadherins regulate Rho proteins in a comparable manner and lead us to speculate that these changes in Rho protein activity participate in a feedback mechanism that promotes further cell-matrix or cell-cell interaction, respectively.     

Fibronectin-dependent collagen I deposition modulates the cell response to fibronectin

Communication between cells and the extracellular matrix (ECM) is critical for regulation of cell growth, survival, migration, and differentiation. Remodeling of the ECM can occur under normal physiological conditions, as a result of tissue injury, and in certain pathological conditions. ECM remodeling leads to alterations in ECM composition and organization that can alter many aspects of cell behavior, including cell migration. The cell migratory response varies depending on the type, amount, and organization of ECM molecules present, as well as the integrin and proteoglycan repertoire of the cells. We and others have shown that the deposition of several ECM molecules, including collagen types I and III, depends on the presence and stability of ECM fibronectin. Hence, the effect of fibronectin and fibronectin matrix on cell function may partially depend on its ability to direct the deposition of collagen in the ECM. In this study, we used collagen-binding fibronectin mutants and recombinant peptides that interfere with fibronectin-collagen binding to show that fibronectin-dependent collagen I deposition regulates the cell migratory response to fibronectin. These data show that the ability of fibronectin to organize other proteins in the ECM is an important aspect of fibronectin function and highlight the importance of understanding how interactions between ECM proteins influence cell behavior.     

Matrix rigidity regulates spatiotemporal dynamics of Cdc42 activity and vacuole formation kinetics of endothelial colony forming cells

Recent evidence has shown that endothelial colony forming cells (ECFCs) may serve as a cell therapy for improving blood vessel formation in subjects with vascular injury, largely due to their robust vasculogenic potential. The Rho family GTPase Cdc42 is known to play a primary role in this vasculogenesis process, but little is known about how extracellular matrix (ECM) rigidity affects Cdc42 activity during the process. In this study, we addressed two questions: Does matrix rigidity affect Cdc42 activity in ECFC undergoing early vacuole formation? How is the spatiotemporal activation of Cdc42 related to ECFC vacuole formation? A fluorescence resonance energy transfer (FRET)-based Cdc42 biosensor was used to examine the effects of the rigidity of three-dimensional (3D) collagen matrices on spatiotemporal activity of Cdc42 in ECFCs. Collagen matrix stiffness was modulated by varying the collagen concentration and therefore fibril density. The results showed that soft (150 Pa) matrices induced an increased level of Cdc42 activity compared to stiff (1 kPa) matrices. Time-course imaging and colocalization analysis of Cdc42 activity and vacuole formation revealed that Cdc42 activity was colocalized to the periphery of cytoplasmic vacuoles. Moreover, soft matrices generated faster and larger vacuoles than stiff matrices. The matrix-driven vacuole formation was enhanced by a constitutively active Cdc42 mutant, but significantly inhibited by a dominant-negative Cdc42 mutant. Collectively, the results suggest that matrix rigidity is a strong regulator of Cdc42 activity and vacuole formation kinetics, and that enhanced activity of Cdc42 is an important step in early vacuole formation in ECFCs.     

The interaction of human papillary and reticular fibroblasts and human keratinocytes in the contraction of three-dimensional floating collagen lattices

Fibroblasts derived from the papillary and reticular dermis of human skin and human keratinocytes show differences in their abilities to contract floating three-dimensional gels constructed from type I collagen. Reticular fibroblasts produce greater gel contraction than papillary fibroblasts. When equal numbers of papillary and reticular fibroblasts are mixed in the gels, papillary fibroblasts consistently inhibit gel contraction by reticular fibroblasts indicating interaction between these cell types in the contraction process. Surprisingly, keratinocytes alone produce greater gel contraction than that produced by either fibroblast type. Cooperativity in the gel contraction process is observed when fibroblasts are incorporated into the collagen matrix and keratinocytes are seeded onto the gel surface. Keratinocytes and dermal fibroblasts adhere to the collagen fibril to induce gel contraction by different mechanisms. Fibroblast contraction of collagen gels does not require fibronectin but is a serum-dependent reaction. In contrast, keratinocyte contraction of collagen gels occurs in a serum-free environment. Polyclonal, affinity-purified antibodies to human plasma fibronectin at high concentrations do not inhibit gel contraction by keratinocytes, making unlikely the possibility that fibronectin synthesized by the keratinocyte is a significant factor in the gel contraction process. We are currently examining the possibilities either that keratinocytes are synthesizing other adhesion proteins or that receptors on the cell surface can interact directly with the collagen fiber.     

Microarray-based identification of aminopeptidase N target genes in keratinocyte conditioned medium-stimulated dermal fibroblasts

Of the many processes that affect the outcome of wound repair, epidermal-dermal interactions are essential to extracellular matrix (ECM) remodeling and in particular, soluble factors released by keratinocytes are known to have a direct impact on the production of ECM by dermal fibroblasts. Aminopeptidase N (APN) has recently been proposed as a cell-surface receptor for stratifin and is responsible for the stratifin-mediated matrix metalloproteinase-1 (MMP-1) upregulation in fibroblasts. The present study examines whether modulation of APN gene expression has any impact on the fibroblast ECM gene expression profile. The result reveals that in the presence of keratinocyte-derived soluble factors, transient knockdown of APN in dermal fibroblasts affects the expression of key ECM components such as fibronectin, tenascin-C, MMP-1, MMP-3, and MMP-12. The regulatory effects of APN on fibronectin and selective MMPs appear to be associated with receptor-mediated signal transduction independently of its peptidase activity. On the contrary, inhibition of the APN enzymatic activity by bestatin significantly reduces the tenascin-C expression and enhances the contraction of fibroblast-populated collagen gel, suggesting an activity-dependent regulation of fibroblast contractility by APN. The overall effects of APN on the expression of fibronectin, tenascin-C, and MMPs in fibroblasts propose an important role for APN in the regulation of keratinocyte-mediated ECM remodeling and fibroblast contractile activity.     

Spike formation by fibroblasts adhering to fibrillar collagen I gel

The fibrillar collagen I gel induced the formation of numerous dendritic cell-like protrusions (cell spikes) from the cell body, whereas monomeric collagen I induced typical cell spreading with filopodia and lamellipodia in skin fibroblasts. Peripheral, not central stress fibers appeared upon adhesion to fibrillar collagen gel, whereas both types of fibers were evident upon adhesion to monomeric collagen. Microtubules and vimentin filaments were elongated inside stress fibers along the terminal tip of cell spikes. Spike formation was totally inhibited by nocodazole and severely delayed by cytochalasin D. This suggests that cell spike formation is dependent on microtubules rather than on F-actin. We then investigated the intracellular signaling responsible for cytoskeleton organization to identify the key factor that induces cell spike morphology. During cell spike formation, FAK and CAS were activated. More CAS was activated in cells on fibrillar collagen gel than on the monomeric form, whereas FAK was activated to the same level on either. At 90 min of culture, Rac1 was activated in cells on monomeric collagen I, whereas Cdc42, Rac1 and RhoA were activated in cells on fibrillar collagen gel. These results suggest that microtubule organization via CAS and small GTPases is important for the cell spike formation that is involved in collagen gel contraction and in wound retraction in skin.     

Spatial organization of extracellular matrix and fibroblast activity: effects of serum, transforming growth factor beta, and fibronectin

The goal of our research is to understand reciprocal relationships between cell function and tissue organization. We studied the regulation of fibroblast activity in an in vitro culture model that recapitulates in continuous fashion the cycle of events occurring during connective tissue repair. We present evidence that concomitant with spatial reorganization of the extracellular matrix, there was a dramatic decline in extracellular matrix synthesis and cell proliferation. Therefore, spatial reorganization was a crucial turning point for fibroblast activity. Factors that regulated the timing of spatial reorganization included serum, transforming growth factor beta, and fibronectin. By accelerating spatial reorganization of the cultures, transforming growth factor beta led to a relative decrease in cell proliferation and extracellular matrix synthesis. By retarding spatial reorganization of the cultures, fibronectin led to a relative increase in cell proliferation and extracellular matrix synthesis. The results indicate that spatial information in the three-dimensional cell-matrix interaction permits higher order, tissue-level regulation of fibroblast function.     

Fibronectin polymerization regulates the composition and stability of extracellular matrix fibrils and cell-matrix adhesions

Remodeling of extracellular matrices occurs during development, wound healing, and in a variety of pathological processes including atherosclerosis, ischemic injury, and angiogenesis. Thus, identifying factors that control the balance between matrix deposition and degradation during tissue remodeling is essential for understanding mechanisms that regulate a variety of normal and pathological processes. Using fibronectin-null cells, we found that fibronectin polymerization into the extracellular matrix is required for the deposition of collagen-I and thrombospondin-1 and that the maintenance of extracellular matrix fibronectin fibrils requires the continual polymerization of a fibronectin matrix. Further, integrin ligation alone is not sufficient to maintain extracellular matrix fibronectin in the absence of fibronectin deposition. Our data also demonstrate that the retention of thrombospondin-1 and collagen I into fibrillar structures within the extracellular matrix depends on an intact fibronectin matrix. An intact fibronectin matrix is also critical for maintaining the composition of cell-matrix adhesion sites; in the absence of fibronectin and fibronectin polymerization, neither alpha5beta1 integrin nor tensin localize to fibrillar cell-matrix adhesion sites. These data indicate that fibronectin polymerization is a critical regulator of extracellular matrix organization and stability. The ability of fibronectin polymerization to act as a switch that controls the organization and composition of the extracellular matrix and cell-matrix adhesion sites provides cells with a means of precisely controlling cell-extracellular matrix signaling events that regulate many aspects of cell behavior including cell proliferation, migration, and differentiation.     

Fibronectin production by cultured human lung fibroblasts in three-dimensional collagen gel culture

Summary In vivo, fibroblasts are distributed in a three-dimensional (3-D) connective tissue matrix. Fibronectin is a major product of fibroblasts in routine cell culture and is thought to regulate many aspects of fibroblast biology. In this context, we sought to determine if the interaction of fibroblasts with a 3-D matrix might affect fibronectin production. To examine this hypothesis, fibronectin production by fibroblasts cultured in a 3-D collagen gel or on plastic dishes was measured by ELISA. Fibroblasts in 3-D gel culture produced more fibronectin than those in monolayer culture. Fibroblasts in 3-D culture produced increasing amounts of fibronectin when the collagen concentration of the gel was increased. The 3-D nature of the matrix appeared to be crucial because plating the fibroblasts on the surface of a plastic dish underneath a collagen gel was not different from plating them on a plastic dish in the absence of collagen. In addition to increased fibronectin production, the distribution of the fibronectin produced in 3-D culture was different from that of monolayer culture. In monolayer culture, more than half of the fibronectin was released into the culture medium. In 3-D culture, however, approximately two-thirds remained in the collagen gel. In summary, the presence of a 3-D collagen matrix increases fibroblast fibronectin production and results in greater retention of fibronectin in the vicinity of the producing cells.     

The role of the arginine-glycine-aspartic acid-directed cellular binding to type I collagen and rat mesenchymal cells in colorectal tumour differentiation

The relationship between the adhesion of five human colorectal carcinoma cell lines to extracellular matrix (ECM) proteins, namely type I collagen, type IV collagen, fibronectin, laminin and basement membrane extract (Matrigel), and the ability of these cells to express morphological differentiation when grown in a basement membrane extract (Matrigel) or on normal rat mesenchymal cells has been examined. Two cell lines, SW1222 and HRA-19, organised into glandular structures, with well-defined polarity when cultured on both substrata as well as in three-dimensional (3D) collagen gel culture as previously shown. The remaining three cell lines (SW620, SW480 and HT29) grew as loose aggregates or as they would normally grow on tissue culture plastic. Addition to the culture medium of a hexapeptide, containing the cell-matrix recognition sequence arginine-glycine-aspartic acid (RGD), inhibited attachment and glandular formation of SW1222 and HRA-19 when these cells were grown on living mesenchymal cells, but not in Matrigel. The morphological differentiation of HRA-19 cells in 3D-collagen was also inhibited by the same RGD-containing peptide, as previously shown for SW1222 cells. Attachment of the remaining three cell lines was inhibited on mesenchyme but not in Matrigel, further supporting the specificity of the peptide effect on epithelial-mesenchymal binding. In conclusion we have shown that colorectal tumour cells are able to bind ECM proteins and that the cellular binding is an essential step in the induction of the morphological differentiation seen on living mesenchymal cells, in basement membrane extracts and in type I collagen gel.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of fibroblast morphology and adhesion during collagen matrix remodeling

When fibroblasts are placed within a three-dimensional collagen matrix, cell locomotion results in translocation of the flexible collagen fibrils of the matrix, a remodeling process that has been implicated in matrix morphogenesis during development and wound repair. In the current experiments, we studied formation and maturation of cell-matrix interactions under conditions in which we could distinguish local from global matrix remodeling. Local remodeling was measured by the movement of collagen-embedded beads towards the cells. Global remodeling was measured by matrix contraction. Our observations show that no direct relationship occurs between protrusion and retraction of cell extensions and collagen matrix remodeling. As fibroblasts globally remodel the collagen matrix, however, their overall morphology changes from dendritic to stellate/bipolar, and cell-matrix interactions mature from punctate to focal adhesion organization. The less well organized sites of cell-matrix interaction are sufficient for translocating collagen fibrils, and focal adhesions only form after a high degree of global remodeling occurs in the presence of growth factors. Rho kinase activity is required for maturation of fibroblast morphology and formation of focal adhesions but not for translocation of collagen fibrils.     

N-cadherin cell-cell adhesion complexes are regulated by fibronectin matrix assembly

Fibronectin is a principal component of the extracellular matrix. Soluble fibronectin molecules are assembled into the extracellular matrix as insoluble, fibrillar strands via a cell-dependent process. In turn, the interaction of cells with the extracellular matrix form of fibronectin stimulates cell functions critical for tissue repair. Cross-talk between cell-cell and cell-extracellular matrix adhesion complexes is essential for the organization of cells into complex, functional tissue during embryonic development and tissue remodeling. Here, we demonstrate that fibronectin matrix assembly affects the organization, composition, and function of N-cadherin-based adherens junctions. Using fibronectin-null mouse embryonic myofibroblasts, we identified a novel quaternary complex composed of N-cadherin, β-catenin, tensin, and actin that exists in the absence of a fibronectin matrix. In the absence of fibronectin, homophilic N-cadherin ligation recruited both tensin and α5β1 integrins into nascent cell-cell adhesions. Initiation of fibronectin matrix assembly disrupted the association of tensin and actin with N-cadherin, released α5β1 integrins and tensin from cell-cell contacts, stimulated N-cadherin reorganization into thin cellular protrusions, and decreased N-cadherin adhesion. Fibronectin matrix assembly has been shown to recruit α5β1 integrins and tensin into fibrillar adhesions. Taken together, these studies suggest that tensin serves as a common cytoskeletal link for integrin- and cadherin-based adhesions and that the translocation of α5β1 integrins from cell-cell contacts into fibrillar adhesions during fibronectin matrix assembly is a novel mechanism by which cell-cell and cell-matrix adhesions are coordinated.     

Collagen gel contraction assays: From modelling wound healing to quantifying cellular interactions with three-dimensional extracellular matrices

Cells respond to and actively remodel the extracellular matrix (ECM). The dynamic and bidirectional interaction between cells and ECM, especially their mechanical interactions, has been found to play an essential role in triggering a series of complex biochemical and biomechanical signal pathways and in regulating cellular functions and behaviours. The collagen gel contraction assay (CGCA) is a widely used method to investigate cell–ECM interactions in 3D environments and provides a mechanically associated readout reflecting 3D cellular contractility. In this review, we summarize various versions of CGCA, with an emphasis on recent high-throughput and low-consumption CGCA techniques. More importantly, we focus on the technique of force monitoring during the contraction of collagen gel, which provides a quantitative characterization of the overall forces generated by all the resident cells in the collagen hydrogel. Accordingly, we present recent biological applications of the CGCA, which have expanded from the initial wound healing model to other studies concerning cell–ECM interactions, including fibrosis, cancer, tissue repair and the preparation of biomimetic microtissues.     

Ultrastructural analysis of mineralized matrix from human osteoblastic cells: Effect of tumor necrosis factor-alpha

Recent work by a number of investigators has demonstrated that the process of bone matrix formation and mineralization is under the influence of growth factors and cytokines present in the local environment. Utilizing primary and established osteoblast cell culture systems, these studies have examined the regulation of bone matrix protein synthesis and deposition into the extracellular matrix (ECM) and subsequent mineralization. In previous studies, we have utilized the human osteoblastic cell line, HOS TE85, to study the effects of Tumor Necrosis Factor - alpha (TNF-) on the regulation of matrix proteins and proteolytic function in monolayer cultures as well as during the development and calcification of ECM formed by HOS TE85 cells during extended culture. Our studies demonstrate that TNF- inhibited formation and mineralization of nodules. In the study reported here, we evaluated the ultrastructural morphology of the cell-matrix complex formed by HOS TE85 cells in the presence and absence of TNF- at selected time points during the matrix development process utilizing both transmission electron microscopy and light microscopy. In the presence of TNF-, the cell-matrix complex does not develop normally, with a lack of organization and mineralization, when compared to untreated cells. The lack of mineralization appears to result from the lack of normal collagen fibril deposition and formation of an appropriate ECM essential for the mineralization process. These results support our previous observations that TNF- inhibits HOS TE85 cells from forming a mineralizing ECM by inhibiting incorporation of collagen into the ECM and inducing the synthesis of proteolytic enzymes capable of degrading collagen in the ECM.     

Hyaluronan Controls the Deposition of Fibronectin and Collagen and Modulates TGF-β1 Induction of Lung Myofibroblasts

The contribution of hyaluronan-dependent pericellular matrix to TGF-β1-driven induction and maintenance of myofibroblasts is not understood. Hyaluronan is an extracellular matrix (ECM) glycosaminoglycan important in cell adhesion, proliferation and migration, and is implicated in myofibroblast formation and maintenance. Reduced turnover of hyaluronan has been linked to differentiation of myofibroblasts and potentiation of lung fibrosis. Fibronectin is a fibril forming adhesive glycoprotein that is also upregulated following induction with TGF-β1. Although they are known to bind each other, the interplay between hyaluronan and fibronectin in the pericellular matrix during myofibroblast induction and matrix assembly is not clear. This study addresses the role of hyaluronan and its interaction with fibrillar matrix components during myofibroblast formation. Hyaluronan and fibronectin were increased and co-localized in the ECM following myofibroblast induction by TGF-β1. Inhibition of hyaluronan synthesis in TGF-β1-induced lung myofibroblasts over a 4 day period with 4-methyl umbelliferone (4-MU) further enhanced myofibroblast morphology, caused increased deposition of fibronectin and type I collagen in the ECM, and increased expression of alpha-smooth muscle actin and hyaluronan synthase 2 (HAS2) mRNA. Hyaluronan oligosaccharides or hyaluronidase treatment, which more effectively disrupted the pericellular matrix, had similar effects. CD44 and β1 integrins co-localized in the cell membrane and along some stress fibers. However, CD44 and hyaluronan were specifically excluded from focal adhesions, and associated primarily with cortical actin. Time-lapse imaging of the immediate effects of hyaluronidase digestion showed that hyaluronan matrix primarily mediates attachment of membrane and cortical actin between focal contacts, suggesting that surface adhesion through hyaluronan and CD44 is distinct from focal adhesion through β1 integrins and fibronectin. Fluorescein-labeled hyaluronan bound regularly along fibronectin fibers and co-localized more with β1 integrin and less with CD44. Therefore, the hyaluronan matrix can interfere with the assembly of fibrillar ECM components, and this interplay regulates the degree of myofibroblast formation. These data also suggest that adhesion through hyaluronan matrix impacts cytoskeletal organization, and is potentially part of a clutch mechanism that regulates stick and slip of myofibroblasts by affecting the adhesion to and organization of fibronectin and collagen.     

Extracellular matrix production and regulation in micropatterned endothelial cells

Production and maintenance of extracellular matrix (ECM) is an essential aspect of endothelial cell (EC) function. ECM surfaces composed of collagen type IV and laminin support an atheroprotective endothelium, while fibronectin may encourage an atheroprone endothelium through inflammation or wound repair signaling. ECs maintain this underlying structure through regulation of protein production and degradation, yet the role of cytoskeletal alignment on this regulation is unknown. To examine the regulation and production of ECM by ECs with an atheroprotective phenotype, ECs were micropatterned onto lanes, which created an elongated EC morphology similar to that seen with unidirectional fluid shear stress application. Collagen IV and fibronectin protein production were measured as were gene expression of collagen IV, fibronectin, laminin, MMP2, MMP9, TIMP1, TIMP2, and TGF-β1. ECs were also treated with TNF to simulate an injury model. Micropattern-induced elongation led to significant increases in collagen IV and fibronectin protein production, and collagen IV, laminin, and TGF-β1 gene expression, but no significant changes in the MMP or TIMP genes. TNF treatment significantly increased collagen IV gene and protein production. These results suggest that the increase in ECM synthesis in micropattern-elongated ECs is likely regulated with TGF-β1, and this increase in ECM could be relevant to the atheroprotection needed for maintenance of a healthy endothelium in vivo.     

Extracellular matrix organization modulates fibroblast growth and growth factor responsiveness

To learn more about the relationship between extracellular matrix organization, cell shape, and cell growth control, we studied DNA synthesis by fibroblasts in collagen gels that were either attached to culture dishes or floating in culture medium during gel contraction. After 4 days of contraction, the collagen density (initially 1.5 mg/ml) reached 22 mg/ml in attached gels and 55 mg/ml in floating gels. After contraction, attached collagen gels were well organized; collagen fibrils were aligned in the plane of cell spreading; and fibroblasts had an elongated, bipolar morphology. Floating collagen gels, however, were unorganized; collagen fibrils were arranged randomly; and fibroblasts had a stellate morphology. DNA synthesis by fibroblasts in contracted collagen gels was suppressed if the gels were floating in medium but not if the gels were attached, and inhibition was independent of the extent of gel contraction. Therefore, growth of fibroblasts in contracted collagen gels could be regulated by differences in extracellular matrix organization and cell shape independently of extracellular matrix density. We also compared the responses of fibroblasts in contracted collagen gels and monolayer culture to peptide growth factors including fibroblast growth factor, platelet-derived growth factor, transforming growth factor-beta, and interleukin 1. Cells in floating collagen gels were generally unresponsive to any of the growth factors. Cells in attached collagen gels and monolayer culture were affected similarly by fibroblast growth factor but not by the others. Our results indicate that extracellular matrix organization influenced not only cell growth, but also fibroblast responsiveness to peptide growth factors.