Evidence that PI3K, Rac, Rho, and Rho kinase are involved in basic fibroblast growth factor-stimulated fibroblast-Collagen matrix contraction

Fibroblast-collagen matrix contraction has been used as a model system to study how cells organize connective tissue. Previous work showed that lysophosphatidic acid (LPA)-stimulated floating collagen matrix contraction is independent of Rho kinase while platelet-derived growth factor (PDGF)-stimulated contraction is Rho kinase-dependent. The current studies were carried out to determine the signaling mechanisms of basic fibroblast growth factor (bFGF)-stimulated fibroblast-collagen matrix contraction. Both bFGF and LPA promoted equally collagen matrix contraction well. Three different inhibitors, LY294002 for phosphatidylinositol-3-kinase (PI3K), C3 exotransferase for Rho and Y27632 for Rho kinase, suppressed the bFGF-stimulated fibroblast-collagen matrix contraction. With bFGF stimulation, fibroblasts spread with prominent stress fiber network formation and focal adhesions. In the presence of Rho kinase inhibitor, focal adhesions and stress fibers were mostly lost. We demonstrated that bFGF stimulation for fibroblast caused transient Rac and Rho activation but did not activate Cdc42. In addition, bFGF enhanced fibroblast migration in wound healing assay. The present study implicates PI3K, Rac, Rho, and Rho kinase as being involved in bFGF-stimulated collagen matrix contraction. The elucidation of bFGF-triggered signal transduction may be an important clue to understand the roles of bFGF in wound healing.     

LPA-stimulated fibroblast contraction of floating collagen matrices does not require Rho kinase activity or retraction of fibroblast extensions

Fibroblasts synthesize, organize, and maintain connective tissues during development and in response to injury and fibrotic disease. These morphogenetic processes depend on cell-matrix remodeling, which has been investigated using cells cultured in three-dimensional collagen matrices. The current studies were carried out to test the role of Rho kinase activity and retraction of fibroblast extensions on the matrix remodeling process. We found that remodeling (contraction) of floating collagen matrices stimulated by lysophosphatidic acid (LPA) did not require Rho kinase activity or retraction of fibroblast extensions. On the other hand, LPA-stimulated contraction of restrained matrices became Rho kinase dependent after the matrices were allowed to develop mechanical loading for 2-4 h, suggesting that the remodeling process itself was able to feed back to modulate cell behavior in an iterative process. Modulation was specific for LPA since fibroblast-collagen matrix contraction stimulated by platelet-derived growth factor was Rho kinase dependent before or after mechanical loading developed.     

Different molecular motors mediate platelet-derived growth factor and lysophosphatidic acid-stimulated floating collagen matrix contraction

Fibroblast-collagen matrix contraction has been used as a model system to study how cells organize connective tissue. Previous work showed that lysophosphatidic acid (LPA)-stimulated floating collagen matrix contraction is independent of Rho kinase, whereas platelet-derived growth factor (PDGF)-stimulated contraction is Rho kinase-dependent. The current studies were carried out to learn more about the molecular motors responsible for LPA- and PDGF-stimulated contraction. We found that neither PDGF nor LPA-dependent contractile mechanisms require myosin II regulatory light chain kinase or increased phosphorylation of myosin II regulatory light chain (measured as diphosphorylation). Low concentrations of the specific myosin II inhibitor blebbistatin blocked PDGF-stimulated matrix contraction and LPA-stimulated retraction of fibroblast dendritic extensions but not LPA-stimulated matrix contraction. These data suggest that PDGF- and LPA-stimulated floating matrix contraction utilize myosin II-dependent and -independent mechanisms, respectively. LPA-dependent, Rho kinase-independent force generation also was detected during fibroblast spreading on collagen-coated coverslips.     

Increased myosin light chain phosphorylation is not required for growth factor stimulation of collagen matrix contraction.

Previous research suggested the possibility that contraction of floating collagen matrices by human fibroblasts required increased myosin light chain (MLC) phosphorylation. In the current studies, we show that increased MLC phosphorylation was neither necessary for platelet-derived growth factor (PDGF)-dependent matrix contraction nor sufficient for lysophosphatidic acid (LPA)-dependent contraction. In contrast, increased MLC phosphorylation did appear to be coupled to the formation of stress fibers by cells spreading in monolayer culture. Signal transduction pathways required for PDGF- and LPA-dependent matrix contraction involved phosphatidylinositol 3-kinase and the G(i) class of heterotrimeric G proteins, respectively. Our results indicate that PDGF- and LPA-dependent contraction of floating collagen matrices can be uncoupled from an increase in MLC phosphorylation.     

Decreased PDGF Receptor Kinase Activity in Fibroblasts Contracting Stressed Collagen Matrices

Fibroblasts cultured in mechanically stressed collagen matrices proliferate, whereas cells in floating collagen matrices become quiescent. Previous research indicated that one factor contributing to cell quiescence in floating matrices was reduced receptor autophosphorylation in response to PDGF stimulation (i.e., PDGF receptor desensitization). To learn more about the mechanism of PDGF receptor desensitization, we analyzed changes in PDGF receptor autophosphorylation and receptor kinase activity after stressed collagen matrices were switched to floating conditions, which results in rapid cell contraction and dissipation of mechanical stress. PDGF receptor desensitization occurred during contraction stimulated by serum but not in the absence of serum, and desensitization was prevented by inhibitors of contraction but not by inhibitors of the contraction-activated cyclic AMP signaling pathway. Receptor desensitization resulted from decreased receptor kinase activity rather than from elevated protein tyrosine phosphatase activity, and only receptors unoccupied at the time of contraction were affected. After contraction, radiolabeled PDGF binding to the cells was decreased, which suggested that receptor desensitization resulted from a contraction-dependent change in receptor availability or affinity.     

Multiple signaling pathways mediate compaction of collagen matrices by EGF-stimulated fibroblasts

Fibroblasts stimulated by EGF within collagen matrices generate contraction forces that are likely of importance to cell migration and matrix compaction during wound healing. We have employed an in vitro fibroblast-embedded collagen matrix compaction assay to ascertain signaling pathway components downstream of EGFR activation leading to generation and transmission of contractile force. EGF compacts this floating collagen matrix to a similar extent as PDGF. We demonstrate that compaction requires EGFR kinase activity, yet is maximal in magnitude at intermediate EGF concentrations. This suggests that transmission of EGFR-induced contractile force to the matrix can be mitigated by consequent anti-adhesive effects of EGFR signaling in a dose-dependent manner. Treatment with pharmacological inhibitors demonstrated involvement of the signaling components extracellular signal-regulated kinase (ERK), Rho kinase, and myosin light chain kinase (MLCK) in the force generation and/or transmission process. Moreover, treatment with the pan-calpain inhibitor ALLN and isoform-specific downregulation of m-calpain (CAPN2) using RNA interference determined m-calpain to be a key component of the EGF-induced force response. ALLN treatment modulated the compaction response in a biphasic manner, enhancing matrix deformation to the greatest extent at intermediate concentrations. Our findings have thus identified key signals downstream of EGFR, which integrate in a complex manner to generate and transmit contractile forces to yield matrix deformation.     

Budding yeast Cdc5 phosphorylates Net1 and assists Cdc14 release from the nucleolus

Lysophosphatidic acid (LPA) has been shown to be a potent mitogen for vascular smooth muscle cells. Src-dependent transactivation of receptor tyrosine kinases has been previously demonstrated to mediate LPA-induced activation of MAP kinase ERK1/2. Furthermore, generation of reactive oxygen species (ROS) by LPA is also known to contribute to MAP kinase activation. Rho family small G-proteins Rac and Cdc42, and their immediate downstream effector p21-activated kinase (PAK), have been demonstrated to mediate important effects on the cytoskeleton that are relevant for cell migration and proliferation. In the present report we evaluated stimulation of PAK by LPA in rat aortic vascular smooth muscle cells (VSMC) by PAK immunocomplex MBP in-gel kinase assay. LPA increased PAK activity 3-fold, peaking at 5 min and showing sustained activation up to 45 min. Inhibition of tyrosine kinases by pretreatment of VSMC with genistein or specific inhibition of Src by PP1 greatly diminished LPA-induced PAK activation, whereas specific inhibition of PDFG- and EGF receptor kinase by tyrphostin AG1296 and AG1478 had no effect. Furthermore, inhibition of Galpha(i) by pertussis toxin and inhibition of NADH/NADPH oxidase by diphenylene iodonium also diminished LPA-induced stimulation of PAK. This is the first study to demonstrate that LPA activates PAK. In VSMC, PAK activation by LPA is mediated by Galpha(i) and is dependent on Src, whereas EGF- or PDGF receptor transactivation are not involved. Furthermore, generation of ROS is required for LPA-induced activation of PAK.     

Activation of p21-activated kinase 1 is required for lysophosphatidic acid-induced focal adhesion kinase phosphorylation and cell motility in human melanoma A2058 cells.

Lysophosphatidic acid (LPA), one of the naturally occurring phospholipids, stimulates cell motility through the activation of Rho family members, but the signaling mechanisms remain to be elucidated. In the present study, we investigated the roles of p21-activated kinase 1 (PAK1) on LPA-induced focal adhesion kinase (FAK) phosphorylation and cell motility. Treatment of human melanoma cells A2058 with LPA increased phosphorylation and activation of PAK1, which was blocked by treatment with pertussis toxin and by inhibition of phosphoinositide 3-kinase (PI3K) with an inhibitor LY294002 or by overexpression of catalytically inactive mutant of PI3Kgamma, indicating that LPA-induced PAK1 activation was mediated via a Gi protein and the PI3Kgamma signaling pathway. In addition, we demonstrated that Rac1/Cdc42 signals acted as upstream effector molecules of LPA-induced PAK activation. However, Rho-associated kinase, MAP kinase kinase 1/2 or phospholipase C might not be involved in LPA-induced PAK1 activation or cell motility stimulation. Furthermore, PAK1 was necessary for FAK phosphorylation by LPA, which might cause cell migration, as transfection of the kinase deficient mutant of PAK1 or PAK auto-inhibitory domain significantly abrogated LPA-induced FAK phosphorylation. Taken together, these findings strongly indicated that PAK1 activation was necessary for LPA-induced cell motility and FAK phosphorylation that might be mediated by sequential activation of Gi protein, PI3Kgamma and Rac1/Cdc42.     

Corneal stromal cells use both high- and low-contractility migration mechanisms in 3-D collagen matrices

Corneal keratocyte migration can impact both corneal clarity and refractive outcome following injury or refractive surgery. In this study, we investigated how culture conditions, ECM properties, and Rho kinase activity regulate the mechanics of keratocyte migration, using a nested collagen matrix model. Time-lapse imaging demonstrated that both serum and PDGF stimulate keratocyte migration into the outer matrix. Although the velocity of cell migration was similar, cells in serum were bipolar and induced significant matrix deformation during migration, whereas PDGF induced extension of branching dendritic processes with smaller, more localized force generation. These differences in cell-induced matrix reorganization were verified with a global matrix contraction assay and confocal reflection imaging, using both bovine and rat tail collagen. When constructs were detached from the substrate to lower the effective stiffness, migration was significantly reduced in serum; but was unchanged in PDGF. These differences in migration mechanics were mediated, in part, by Rho kinase. Overall, corneal keratocytes can effectively migrate through collagen matrices using varying degrees of cellular force generation. Low-contractility migration may facilitate keratocyte repopulation of the stroma following surgery or injury, without altering the structural and mechanical properties that are critical to maintaining corneal transparency.     

p85 beta-PIX is required for cell motility through phosphorylations of focal adhesion kinase and p38 MAP kinase

Lysophosphatidic acid (LPA) mediates diverse biological responses, including cell migration, through the activation of G-protein-coupled receptors. Recently, we have shown that LPA stimulates p21-activated kinase (PAK) that is critical for focal adhesion kinase (FAK) phosphorylation and cell motility. Here, we provide the direct evidence that p85 beta-PIX is required for cell motility of NIH-3T3 cells by LPA through FAK and p38 MAP kinase phosphorylations. LPA induced p85 beta-PIX binding to FAK in NIH-3T3 cells that was inhibited by pretreatment of the cells with phosphoinositide 3-kinase inhibitor, LY294002. Furthermore, the similar inhibition of the complex formation was also observed, when the cells were transfected with either p85 beta-PIX mutant that cannot bind GIT or dominant negative mutants of Rac1 (N17Rac1) and PAK (PAK-PID). Transfection of the cells with specific p85 beta-PIX siRNA led to drastic inhibition of LPA-induced FAK phosphorylation, peripheral redistribution of p85 beta-PIX with FAK and GIT1, and cell motility. p85 beta-PIX was also required for p38 MAP kinase phosphorylation induced by LPA. Finally, dominant negative mutant of Rho (N19Rho)-transfected cells did not affect PAK activation, while the cells stably transfected with p85 beta-PIX siRNA or N17Rac1 showed the reduction of LPA-induced PAK activation. Taken together, the present data suggest that p85 beta-PIX, located downstream of Rac1, is a key regulator for the activations of FAK or p38 MAP kinase and plays a pivotal role in focal complex formation and cell motility induced by LPA.     

Lysophosphatidic acid enhances collagen gel contraction by hepatic stellate cells: association with rho-kinase

We studied the effect of lysophosphatidic acid (LPA) on collagen gel contraction by cultured rat hepatic stellate cells (HSCs) in association with the function of Rho-kinase, one of the target molecules of small GTPase Rho. Binding studies showed a single class-binding site of LPA on HSCs. LPA enhanced the contraction of a collagen lattice seeded with HSCs. LPA increased the number of HSCs with polygonal morphology that contained actin stress fibers, and enhanced the phosphorylation of myosin light chain and the assembly of focal adhesion kinase and RhoA around fibronectin-coated beads seeded on HSCs. The electric cell-substrate impedance sensor system showed that LPA enhanced adhesion of HSC to extracellular substrate. All the effects of LPA were suppressed by Y-27632, Rho-kinase inhibitor. These data support the notion that LPA is involved in modulating HSC morphology, its attachment to surrounding extracellular matrix and its contraction by a mechanism involving Rho-kinase.     

ROCK-generated contractility regulates breast epithelial cell differentiation in response to the physical properties of a three-dimensional collagen matrix

Breast epithelial cells differentiate into tubules when cultured in floating three-dimensional (3D) collagen gels, but not when the cells are cultured in the same collagen matrix that is attached to the culture dish. These observations suggest that the biophysical properties of collagenous matrices regulate epithelial differentiation, but the mechanism by which this occurs is unknown. Tubulogenesis required the contraction of floating collagen gels through Rho and ROCK-mediated contractility. ROCK-mediated contractility diminished Rho activity in a floating 3D collagen gel, and corresponded to a loss of FAK phosphorylated at Y397 localized to 3D matrix adhesions. Increasing the density of floating 3D collagen gels also disrupted tubulogenesis, promoted FAK phosphorylation, and sustained high Rho activity. These data demonstrate the novel finding that breast epithelial cells sense the rigidity or density of their environment via ROCK-mediated contractility and a subsequent down-regulation of Rho and FAK function, which is necessary for breast epithelial tubulogenesis to occur.     

Quantitative assessment of local collagen matrix remodeling in 3-D culture: the role of Rho kinase

The purpose of this study was to quantitatively assess the role of Rho kinase in modulating the pattern and amount of local cell-induced collagen matrix remodeling. Human corneal fibroblasts were plated inside 100-microm thick fibrillar collagen matrices and cultured for 24 h in media with or without the Rho kinase inhibitor Y-27632. Cells were then fixed and stained with phalloidin. Fluorescent (for f-actin) and reflected light (for collagen fibrils) 3-D optical section images were acquired using laser confocal microscopy. Fourier transform analysis was used to assess collagen fibril alignment, and 3-D cell morphology and local collagen density were measured using MetaMorph. Culture in serum-containing media induced significant global matrix contraction, which was inhibited by blocking Rho kinase (p<0.001). Fibroblasts generally had a bipolar morphology and intracellular stress fibers. Collagen fibrils were compacted and aligned parallel to stress fibers and pseudopodia. When Rho kinase was inhibited, cells had a more cortical f-actin distribution and dendritic morphology. Both local collagen fibril density and alignment were significantly reduced (p<0.01). Overall, the data suggests that Rho kinase-dependent contractile force generation leads to co-alignment of cells and collagen fibrils along the plane of greatest resistance, and that this process contributes to global matrix contraction.     

Cdc42 regulates extracellular matrix remodeling in three dimensions

Extracellular matrix (ECM) actively participates in normal cell regulation and in the process of tumor progression. The Rho GTPase Cdc42 has been shown to regulate cell-ECM interaction in conventional two-dimensional culture conditions by using dominant mutants of Cdc42 in immortalized cell lines that may introduce nonspecific effects. Here, we employ three-dimensional culture systems for conditional gene targeted primary mouse embryonic fibroblasts that better simulate the reciprocal and adaptive interactions between cells and surrounding matrix to define the role of Cdc42 signaling pathways in ECM organization. Cdc42 deficiency leads to a defect in global cell-matrix interactions reflected by a decrease in collagen gel contraction. The defect is associated with an altered cell-matrix interaction that is evident by morphologic changes and reduced focal adhesion complex formation. The matrix defect is also associated with a reduction in synthesis and activation of matrix metalloproteinase 9 (MMP9) and altered fibronectin deposition patterning. A Cdc42 mutant rescue experiment found that downstream of Cdc42, p21-activated kinase (PAK), but not Par6 or WASP, may be involved in regulating collagen gel contraction and fibronectin organization. Thus, in addition to the previously implicated roles in intracellular regulation of actin organization, proliferation, and vesicle trafficking, Cdc42 is essential in ECM remodeling in three dimensions.     

Synergism among lysophosphatidic acid, beta1A integrins, and epidermal growth factor or platelet-derived growth factor in mediation of cell migration.

GD25 cells lacking the beta1 integrin subunit or expressing beta1A with certain cytoplasmic mutations have poor directed cell migration to platelet-derived growth factor (PDGF) or epidermal growth factor (EGF), ligands of receptor tyrosine kinases, or to lysophosphatidic acid (LPA), a ligand of G-protein-coupled receptors (Sakai, T., Zhang, Q., F?ssler, R., and Mosher, D. F. (1998) J. Cell Biol. 141, 527-538 and Sakai, T., Peyruchaud, O., F?ssler, R., and Mosher, D. F. (1998) J. Biol. Chem. 273, 19378-19382). We demonstrate here that LPA synergizes with signals induced by beta1A integrins and ligated EGF or PDGF receptors to modulate migration. When LPA was mixed with EGF or PDGF, migration was greater than with EGF or PDGF alone. The enhancement was greater for beta1A-expressing cells than for beta1-null cells. Cells expressing beta1A with mutations of prolines or tyrosines in conserved cytoplasmic NPXY motifs had blunted migratory responses to mixtures of LPA and EGF or PDGF. The major effects on beta1A-expressing cells of LPA when combined with EGF or PDGF were to sensitize cells so that maximal responses were obtained with >10-fold lower concentrations of growth factor and increase the chemokinetic component of migration. Sensitization by LPA was lost when cells were preincubated with pertussis toxin or C3 exotransferase. There was no evidence for transactivation or sensitization of receptors for EGF or PDGF by LPA. EGF or PDGF and LPA caused activation of mitogen-activated protein kinase by pertussis toxin-insensitive and -sensitive pathways respectively, but activation was not additive. These findings indicate that signaling pathways initiated by the cytoplasmic domains of ligated beta1A integrins and tyrosine kinase receptors interact with signaling pathways initiated by LPA to facilitate directed cell migration.     

Ligand density and integrin repertoire regulate cellular response to LPA

Engagement of integrin receptors by the extracellular matrix (ECM) protein fibronectin (FN) activates intracellular signaling, cytoskeletal reorganization and cellular tension. The soluble factor lysophosphatidic acid (LPA) acts through Rho GTPase and its effector Rho kinase (ROCK) to enhance α5β1 integrin-mediated cell spreading on the Arg-Gly-Asp (RGD) cell-binding domain of FN. A second cell-binding site for α4 integrins resides in the CS1 segment of the alternatively spliced V region of FN. We show here that LPA treatment of α4β1-expressing CHOα4 cells on FN induced a significant decrease in spread cell area. LPA also decreased apoptosis induced by serum-deprivation in CHOα4 and human A375 melanoma cells in an α4β1-dependent manner. Improvement in cell viability and changes in cell morphology were dependent on ROCK and on the number of substrate binding sites for α4β1. LPA signaling combined with α4β1-mediated adhesion appears to sustain cell viability in situations where FN matrix is limiting. Such cooperation may impact dynamic cellular events such as wound healing, fibrosis, and metastasis.     

Nested collagen matrices: a new model to study migration of human fibroblast populations in three dimensions

Fibroblast-3D collagen matrix culture provides a model system to analyze cell physiology under conditions that more closely resemble tissue than conventional 2D cell culture. Previous work has focused primarily on remodeling and contraction of collagen matrices by fibroblasts, and there has been little research on migration of cell populations within the matrix. Here, we introduce a nested collagen matrix model to analyze migration of fibroblasts in 3D collagen matrices. Nested collagen matrices were prepared by embedding contracted cell-containing matrices (also called dermal equivalents) inside cell-free matrices; migration occurred from the former to the latter. Control experiments with human dermal fragments in place of dermal equivalents confirmed the reliability of the model. Human fibroblast migration in nested collagen matrices occurred after a lag phase of 8-16 h, and cells migrating out of the inner matrices were bipolar with leading dendritic extensions. Migration was myosin II, Rho kinase and metalloproteinase-dependent but did not require plasma fibronectin. Platelet-derived growth factor but not lysophosphatidic acid or serum stimulated cell migration, although all three of these physiological agonists promote matrix remodeling and contraction. The nested collagen matrix model is a relatively easy, rapid and quantitative method to measure migration of cell populations. Our studies using this model demonstrate important differences between regulation of fibroblast migration and remodeling in collagen matrices.     

Regulation of LPA-promoted myofibroblast contraction: role of Rho, myosin light chain kinase, and myosin light chain phosphatase

Myofibroblasts generate the contractile force responsible for wound healing and pathological tissue contracture. In this paper the stress-relaxed collagen lattice model was used to study lysophosphatidic acid (LPA)-promoted myofibroblast contraction and the role of the small GTPase Rho and its downstream targets Rho kinase and myosin light chain phosphatase (MLCPPase) in regulating myofibroblast contraction. In addition, the regulation of myofibroblast contraction was compared with that of smooth muscle cells. LPA-promoted myofibroblast contraction was inhibited by the myosin light chain kinase (MLCK) inhibitors KT5926 and ML-7; however, in contrast to that observed in smooth muscle cells, elevation of intracellular calcium alone was not sufficient to promote myofibroblast contraction. These results suggest that Ca(2+)-mediated activation of MLCK, while necessary, is not sufficient to promote myofibroblast contraction. The specific Rho inactivator C3-transferase and the Rho kinase inhibitor Y-27632 inhibited LPA-promoted myofibroblast contraction, suggesting that contraction depends on activation of the Rho/Rho kinase pathway. Calyculin, a type 1 phosphatase inhibitor known to inhibit MLCPPase, could promote myofibroblast contraction in the absence of LPA, as well as restore contraction in the presence of C3-transferase or Y-27632. Together these results support a model whereby Rho/Rho kinase-mediated inhibition of MLCPPase is necessary for LPA-promoted myofibroblast contraction, in contrast to smooth muscle cells in which Ca(2+) activation of MLCK alone is sufficient to promote contraction.     

Cell surface receptors activate p21-activated kinase 1 via multiple Ras and PI3-kinase-dependent pathways

p21-activated kinases (PAKs) were the first identified mammalian members of a growing family of Ste20-like serine–threonine protein kinases. In this study, we show that PAK1 can be stimulated by carbachol, lysophosphatidic acid (LPA), epidermal growth factor (EGF), and phorbol 12-myristate 13-acetate (PMA) by multiple independent and overlapping pathways. Dominant-negative Ras, Rac, and Cdc42 inhibited PAK1 activation by all of these agonists, while active Rac1 and Cdc42 were sufficient to maximally activate PAK1 in the absence of any treatment. Active Ras induced only a weak activation of PAK1 that could be potentiated by muscarinic receptor stimulation. Studies using inhibitors of the EGF receptor tyrosine kinase, phosphatidylinositol 3-kinase (PI3-kinase) and protein kinase C (PKC) revealed that all of the cell surface agonists could activate PAK1 through pathways independent of PKC, that EGF stimulated a PI3-kinase dependent pathway to stimulate PAK1, and that muscarinic receptor stimulation of PAK1 was predominantly mediated through this EGF-R-dependent mechanism. Activation of PAK1 by LPA was independent of PI3-kinase and the EGF receptor, but was inhibited by dominant-negative RhoA. These results identify multiple Ras-dependent pathways to activation of PAK1.