Fibronectin's central cell-binding domain supports focal adhesion formation and Rho signal transduction

Fibroblast adhesion to fibronectin (FN) induces formation of focal adhesions (FAs), structures that have significant effect on cell migration and signaling. FA formation requires actomyosin-based contractility that is regulated by Rho-dependent myosin light chain (MLC) phosphorylation. Previous studies indicated that the FN central cell-binding (and integrin-binding) domain (CBD) is insufficient for FA formation and that the major heparin-binding domain (HepII) facilitates FA formation in a Rho-dependent manner. We describe here conditions under which FN CBD alone is sufficient for FA formation in both human dermal fibroblasts and the FN-null murine fibroblasts. CBD-mediated FA formation is dependent on its surface adsorption and the adhesion activity of the cells. Attachment of FN-null fibroblasts to CBD elicits the same biphasic regulation of Rho activity as seen on intact FN, whereas adhesion to HepII alone does not activate Rho. Activation of Rho requires high levels of integrin occupancy. However, FN or CBD may induce FAs without increased activation of Rho (i.e. the basal level of GTP-Rho induces sufficient phospho-MLC for FA assembly under this condition). In contrast, adhesion to HepII alone does not sustain MLC phosphorylation. Pulse stimulation of cells on CBD or HepII with lysophosphatidic acid elevates Rho GTP loading to the same level, but the lysophosphatidic acid-stimulated MLC phosphorylation is significantly lower in cells on HepII than on CBD. Coating HepII with suboptimal concentrations of CBD induces FAs without increased activation of Rho. Therefore, FN CBD can support FA formation and generate contraction by activating Rho or by facilitating Rho downstream signaling.     

Involvement of microtubules and Rho pathway in TGF-beta1-induced lung vascular barrier dysfunction

Transforming growth factor-beta1 (TGF-beta1) is a cytokine critically involved in acute lung injury and endothelial cell (EC) barrier dysfunction. We have studied TGF-beta1-mediated signaling pathways and examined a role of microtubule (MT) dynamics in TGF-beta1-induced actin cytoskeletal remodeling and EC barrier dysfunction. TGF-beta1 (0.1-50 ng/ml) induced dose-dependent decrease in transendothelial electrical resistance (TER) in bovine pulmonary ECs, which was linked to increased actin stress fiber formation, myosin light chain (MLC) phosphorylation, EC retraction, and gap formation. Inhibitor of TGF-beta1 receptor kinase RI (5 microM) abolished TGF-beta1-induced TER decline, whereas inhibitor of caspase-3 zVAD (10 microM) was without effect. TGF-beta1-induced EC barrier dysfunction was linked to partial dissolution of peripheral MT meshwork and decreased levels of stable (acetylated) MT pool, whereas MT stabilization by taxol (5 microM) attenuated TGF-beta1-induced barrier dysfunction and actin remodeling. TGF-beta1 induced sustained activation of small GTPase Rho and its effector Rho-kinase; phosphorylation of myosin binding subunit of myosin specific phosphatase; MLC phosphorylation; EC contraction; and gap formation, which was abolished by inhibition of Rho and Rho-kinase, and by MT stabilization with taxol. Finally, elevation of intracellular cAMP induced by forskolin (50 microM) attenuated TGF-beta1-induced barrier dysfunction, MLC phosphorylation, and protected the MT peripheral network. These results suggest a novel role for MT dynamics in the TGF-beta1-mediated Rho regulation, EC barrier dysfunction, and actin remodeling.     

Activation of Rho signaling contributes to lysophosphatidic acid-induced contraction of intact ileal smooth muscle of guinea-pig

To elucidate the possible role of Rho A/Rho-kinase on lysophosphatidic acid (LPA)-induced contraction in intact guinea-pig ileal smooth muscle, we examined effects of pretreatment with a specific inhibitor of Rho-kinase (Y-27632) on the LPA-induced contraction and MLC20 phosphorylation. In addition, we investigated whether LPA actually elicits an activation of Rho A by studying subcellular distribution of Rho A in unstimulated and stimulated smooth muscles by LPA. LPA induced a less intense, but sustained, contraction compared with ACh, and was accompanied by significant increases in MLC20 phosphorylation. The effects of LPA on tension and MLC20 phosphorylation were inhibited by Y-27632. The ACh-induced contraction, but not increases in MLC20 phosphorylation, was partially inhibited by Y-27632. High K+-induced contraction was unaffected by the inhibitor. LPA stimulated translocation of Rho A from the cytosol to the membrane fraction of the muscle. Translocation of Rho A was also induced by ACh and high K+. These results suggest that LPA-induced contraction of intact ileal smooth muscle is dominated through activation of Rho A and Rho-kinase and subsequent increases in MLC20 phosphorylation.     

Rho-ROCK signaling differentially regulates chondrocyte spreading on fibronectin and bone sialoprotein

The mammalian growth plate is a dynamic structure rich in extracellular matrix (ECM). Interactions of growth plate chondrocytes with ECM proteins regulate cell behavior. In this study, we compared chondrocyte adhesion and spreading dynamics on fibronectin (FN) and bone sialoprotein (BSP). Chondrocyte adhesion and spreading were also compared with fibroblasts to analyze potential cell-type-specific effects. Chondrocyte adhesion to BSP is independent of posttranslational modifications but is dependent on the RGD sequence in BSP. Whereas chondrocytes and fibroblasts adhered at similar levels on FN and BSP, cells displayed more actin-dependent spread on FN despite a 16x molar excess of BSP adsorbed to plastic. To identify intracellular mediators responsible for this difference in spreading, we investigated focal adhesion kinase (FAK)-Src and Rho-Rho kinase (ROCK) signaling. Although activated FAK localized to the vertices of adhered chondrocytes, levels of FAK activation did not correlate with the extent of spreading. Furthermore, Src inhibition reduced chondrocyte spreading on both FN and BSP, suggesting that FAK-Src signaling is not responsible for less cell spreading on BSP. In contrast, inhibition of Rho and ROCK in chondrocytes increased cell spreading on BSP and membrane protrusiveness on FN but did not affect cell adhesion. In fibroblasts, Rho inhibition increased fibroblast spreading on BSP while ROCK inhibition changed membrane protrusiveness of FN and BSP. In summary, we identify a novel role for Rho-ROCK signaling in regulating chondrocyte spreading and demonstrate both cell- and matrix molecule-specific mechanisms controlling cell spreading.     

Mechanism of RhoA/Rho kinase activation in endothelin-1- induced contraction in rabbit basilar artery

This study was undertaken to demonstrate the role of the RhoA/Rho kinase pathway in endothelin-1 (ET-1)-induced contraction of the rabbit basilar artery. Isometric tension and Western blot were used to examine ET-1-induced contraction and RhoA activation. The upstream effect on ET-1-induced RhoA activity was determined by using ET(A) and ET(B) receptor antagonists, protein kinase C (PKC), tyrosine kinase, and phosphatidylinositol-3 kinase inhibitors. The downstream effect of ET-1-induced contraction and RhoA activity was studied in the presence of the Rho kinase inhibitor Y-27632. The effect of Rho kinase inhibitor on ET-1-induced myosin light chain (MLC) phosphorylation was investigated by using urea-glycerol-PAGE immunoblotting. We found 1) ET-1 increased RhoA activity (membrane binding RhoA) in a concentration-dependent manner; 2) ET(A), but not ET(B), receptor antagonist abolished the effect of ET-1 on RhoA activation; 3) phosphodylinositol-3 kinase inhibitor, but not PKC and tyrosine kinase inhibitors, reduced ET-1-induced RhoA activation; 4) Rho kinase inhibitor Y-27632 (10 microM) inhibited ET-1-induced contraction; and 5) ET-1 increased the level of MLC phosphorylation. Rho kinase inhibitor Y-27632 reduced the effect of ET-1 on MLC phosphorylation. This study demonstrated that RhoA/Rho kinase activation is involved in ET-1-induced contraction in the rabbit basilar artery. Phosphodylinositol-3 kinase and MLC might be the upstream and downstream factors of RhoA activation.     

Tenascin-C suppresses Rho activation

Cell binding to extracellular matrix (ECM) components changes cytoskeletal organization by the activation of Rho family GTPases. Tenascin-C, a developmentally regulated matrix protein, modulates cellular responses to other matrix proteins, such as fibronectin (FN). Here, we report that tenascin-C markedly altered cell phenotype on a three-dimensional fibrin matrix containing FN, resulting in suppression of actin stress fibers and induction of actin-rich filopodia. This distinct morphology was associated with complete suppression of the activation of RhoA, a small GTPase that induces actin stress fiber formation. Enforced activation of RhoA circumvented the effects of tenascin. Effects of active Rho were reversed by a Rho inhibitor C3 transferase. Suppression of GTPase activation allows tenascin-C expression to act as a regulatory switch to reverse the effects of adhesive proteins on Rho function. This represents a novel paradigm for the regulation of cytoskeletal organization by ECM.     

Biphasic role of TGF-beta1 in signal transduction and crosstalk

TGF-beta1 induces cell cycle activation in mouse embryonic fibroblasts by down regulation of p27(Kip1) but it can also induce delay of EGF-induced cell cycle activation in these cells under similar conditions. In an attempt to determine the basis for these responses, the study of early TGF-beta1-induced signal transduction pathways in the presence and absence of EGF was undertaken. It is proposed that a likely target for the inhibition by TGF-beta1 of the early EGF-induced p42/p44 MAPK is at the c-Raf locus. The finding that the catalytic subunits of PKA are associated with Raf-1 within minutes following application of TGF-beta1 but not EGF in fibroblasts arrested in early G1 is suggestive of a role of PKA-Raf-1 interaction in TGF-beta1 induced delay of EGF-induced cell cycle kinetics. A model for TGF-beta1 induced translocation to the plasma membrane-associated Raf-1 is proposed. Reports that Rho-like GTPase activity is critical for the activation of TGF-beta1 downstream pathways raises the question as to whether Rho proteins are involved in these observed TGF-beta1-induced responses. Post-receptor signaling mechanisms for TGF-beta1 and cross-talk with PKA-mediated pathways are examined in an effort to explain the modulation by TGF-beta1 of mitogen-induced cell proliferation in mesenchymal cells.     

Rho plays a central role in regulating local cell-matrix mechanical interactions in 3D culture

The purpose of this study was to assess quantitatively the role of the small GTPase Rho on cell morphology, f-actin organization, and cell-induced matrix remodeling in 3D culture. Human corneal fibroblasts (HTK) were infected with adenoviruses that express green fluorescent protein (GFP) or GFP-N19Rho (dominant negative Rho). One day later cells were plated inside collagen matrices and allowed to spread for 24 h. Cells were fixed and stained for f-actin. Fluorescent (for f-actin) and reflected light (for collagen fibrils) images were acquired using confocal microscopy. Fourier transform analysis was used to assess local collagen fibril alignment, and changes in cell morphology and collagen density were measured using MetaMorph. The decrease in matrix height was used as an indicator of global matrix contraction. HTK and HTK-GFP cells induced significant global matrix contraction; this was inhibited by N19Rho. HTK and HTK-GFP fibroblasts generally had a bipolar morphology and occasional intracellular stress fibers. Collagen fibrils were compacted and aligned parallel to stress fibers and pseudopodia. In contrast, HTK-GFPN19 cells were elongated, and had a more cortical f-actin distribution. Numerous small extensions were also observed along the cell body. In addition, both local collagen fibril density and alignment were significantly reduced. Rho plays a key role in regulating both the morphology and mechanical behavior of corneal fibroblasts in 3D culture. Overall, the data suggest that Rho-kinase dependent cell contractility contributes to global and local matrix remodeling, whereas Rho dependent activation of mDia and/or other downstream effectors regulates the structure and number of cell processes.     

Cyclic AMP blocks bacterial lipopolysaccharide-induced myosin light chain phosphorylation in endothelial cells through inhibition of Rho/Rho kinase signaling

During Gram-negative sepsis bacterial LPS induces endothelial cell contraction, actin reorganization, and loss of endothelial integrity by an unknown signal mechanism. In this study, we provide evidence that LPS-stimulation of endothelial cells (HUVEC) decreases myosin light chain (MLC) phosphatase, resulting in an increase in MLC phosphorylation followed by cell contraction. All of these LPS effects could be blocked by the Rho-GTPase inhibitor C3 transferase from Clostridium botulinum or the Rho kinase inhibitor Y-27632. These data suggest that LPS induces MLC phosphorylation via Rho/Rho kinase-mediated inhibition of MLC phosphatase in HUVEC. Furthermore, we observed that cAMP-elevating drugs, known to exert a vasoprotective function, mimicked the effects of C3 transferase and Y-27632, i.e., inhibited LPS-induced MLC phosphatase inactivation and MLC phosphorylation. cAMP elevation did not inhibit myosin phosphorylation induced by constitutively active V14Rho or the MLC phosphatase inhibitor calyculin and did not induce phosphorylation of RhoA in HUVEC, indicating inhibition of an upstream regulator of Rho/Rho kinase. Taken together, Rho/Rho kinase appears to be a central target for inflammatory mediators causing endothelial cell contraction such as bacterial toxins, but also for vasoprotective molecules elevating intracellular cAMP.     

Hyperosmotic stress activates Rho: differential involvement in Rho kinase-dependent MLC phosphorylation and NKCC activation

Hyperosmotic stress initiates adaptive responses, including phosphorylation of myosin light chain (MLC) and concomitant activation of Na+-K+-Cl- cotransporter (NKCC). Because the small GTPase Rho is a key regulator of MLC phosphorylation, we investigated 1) whether Rho is activated by hyperosmotic stress, and if so, what the triggering factors are, and 2) whether the Rho/Rho kinase (ROK) pathway is involved in MLC phosphorylation and NKCC activation. Rho activity was measured in tubular epithelial cells by affinity pulldown assay. Hyperosmolarity induced rapid (<1 min) and sustained (>20 min) Rho activation that was proportional to the osmotic concentration and reversed within minutes upon restoration of isotonicity. Both decreased cell volume at constant ionic strength and elevated total ionic strength at constant cell volume were capable of activating Rho. Changes in [Na+] and [K+] at normal total salinity failed to activate Rho, and Cl- depletion did not affect the hyperosmotic response. Thus alterations in cellular volume and ionic strength but not individual ion concentrations seem to be the critical triggering factors. Hyperosmolarity induced mono- and diphosphorylation of MLC, which was abrogated by the Rho-family blocker Clostridium toxin B. ROK inhibitor Y-27632 suppressed MLC phosphorylation under isotonic conditions and prevented its rise over isotonic levels in hypertonically stimulated cells. ML-7 had a smaller inhibitory effect. In contrast, it abolished the hypertonic activation of NKCC, whereas Y-27632 failed to inhibit this response. Thus hyperosmolarity activates Rho, and Rho/ROK pathway contributes to basal and hyperosmotic MLC phosphorylation. However, the hypertonic activation of NKCC is ROK independent, implying that the ROK-dependent component of MLC phosphorylation can be uncoupled from NKCC activation.     

Involvement of calcineurin in transforming growth factor-beta-mediated regulation of extracellular matrix accumulation

Calcineurin is a calcium-dependent, serine/threonine phosphatase that functions as a signaling intermediate. In this study, we investigated the role of calcineurin in transforming growth factor-beta (TGF-beta)-mediated cellular effects and examined the signaling pathway involved in activation of calcineurin. Calcineurin is activated by TGF-beta in a time- and dose-dependent manner. Consistent with increased phosphatase activity, the calcineurin substrate, NFATc1, is dephosphorylated and transported to the nucleus. Inhibition of calcineurin prior to the addition of TGF-beta revealed that calcineurin is required for TGF-beta-mediated accumulation of extracellular matrix (ECM) proteins but not cell hypertrophy. Conversely, overexpression of constitutively active calcineurin was sufficient to induce ECM protein expression. The mechanism of calcineurin activation by TGF-beta was found to be induction of a low, sustained increase of intracellular calcium. Chelation of extracellular calcium blocked both TGF-beta-mediated calcium influx and calcineurin activity. Finally, calcium entry was found to be dependent upon generation of reactive oxygen species (ROS) including superoxide anion and hydrogen peroxide. Accordingly, inhibition of ROS generation also blocked TGF-beta-mediated calcineurin phosphatase activity and decreased ECM accumulation. In conclusion, this study describes a new pathway for TGF-beta-mediated regulation of ECM via generation of ROS, calcium influx, and activation of calcineurin.     

Diverse signaling pathways regulate fibroblast differentiation and transformation through Rho kinase activation

This study examined the role of agonist-induced Rho kinase (ROCK) involvement in the morphological outcome of pulmonary-derived fibroblasts. Normal human lung fibroblasts (NHLF) spontaneously differentiate into network-like structures in a two-dimensional growth factor reduced Matrigel matrix-based assay. Sphingosine 1-phosphate (SPP), a bioactive phospholipid that regulates angiogenesis, inhibited fibroblast morphogenesis in a dose-dependent manner, virtually eliminating the presence of multi-cellular structures at 500 nM. Pretreatment with the Rho kinase-specific inhibitor, H1152, eradicated the high dose SPP-induced inhibition. Similarly, NHLFs transfected with Rho kinase siRNA prevented SPP-induced inhibition of the fibroblast morphogenesis. Alternatively, transforming growth factor-beta1 (TGF-beta1), a cytokine recognized as a key mediator of wound healing, terminally differentiates NHLF into myofibroblasts as evidenced by the expression of the smooth muscle cell isoform of alpha-actin (alpha-SMA). H1152 suppressed TGF-beta1-induced alpha-SMA expression in a dose-dependent manner. Similarly, treatment with Rho kinase siRNA reduced alpha-SMA expression by greater than 50%. SPP treatment had no effect on TGF-beta1-induced transformation into myofibroblasts, and TGF-beta1 treatment did not alter fibroblast morphogenesis. This study suggests a dual regulatory role for Rho kinase in cellular regulation of fibroblasts in which SPP-induced Rho kinase activation via a G-protein coupled receptor suppresses fibroblast morphogenesis while TGF-beta1-induced Rho kinase activation through a serine/threonine kinase receptor culminates in transformation into myofibroblasts.     

ALK5 and Smad4 are involved in TGF-beta1-induced pulmonary endothelial permeability

The ability of inflammatory cytokine TGF-beta1 to alter endothelial cell phenotype suggests its role in the regulation of vascular endothelial cell permeability. We demonstrate that depletion of TGF-beta1 receptor ALK5 and regulatory protein Smad4, but not ALK1 receptor attenuates TGF-beta1-induced permeability increase and significantly inhibits TGF-beta1-induced EC contraction manifested by actin stress fiber formation and increased MLC and MYPT1 phosphorylation. Consistent with these results, EC treatment with SB 431542, an inhibitor of ALK5 but not ALK1 receptor, significantly attenuates TGF-beta1-induced permeability. Thus, our data demonstrate for the first time direct link between TGF-beta1-mediated activation of ALK5/Smad and EC barrier dysfunction.     

Effects of lactoferrin on collagen gel contractile activity and myosin light chain phosphorylation in human fibroblasts.

When fibroblasts are plated on a type I collagen gel they reduce the size of the gel and the extent of collagen gel contraction reflects the motile activity of the fibroblasts. We found that both bovine and human lactoferrin (Lf) enhanced the collagen gel contractile activity of WI-38 human fibroblasts. Rho inhibitor (exoenzyme C3), Rho kinase inhibitor (Y-27632), myosin light chain kinase inhibitor (ML-7), MEK inhibitor (PD98059) and Src family tyrosine kinase inhibitor inhibited the Lf-enhanced collagen gel contraction. Treatment of fibroblasts with Lf induced the phosphorylation of myosin light chain (MLC) within 30 min. Lf-enhanced MLC phosphorylation was inhibited by Y-27632 and ML-7. These results suggest that Lf promotes the motility of fibroblasts by regulating MLC phosphorylation.     

Modulatory Roles for Integrin Activation and the Synergy Site of Fibronectin during Matrix Assembly

Initiation of fibronectin (FN) matrix assembly is dependent on specific interactions between FN and cell surface integrin receptors. Here, we show that de novo FN matrix assembly exhibits a slow phase during initiation of fibrillogenesis followed by a more rapid growth phase. Mn²⁺, which acts by enhancing integrin function, increased the rate of FN fibril growth, but only after the initial lag phase. The RGD cell-binding sequence in type III repeat 10 is an absolute requirement for initiation by α5β1 integrin. To investigate the role of the cell-binding synergy site in the adjacent repeat III₉, a full-length recombinant FN containing a synergy mutation, FN(syn⁻), was tested for its ability to form fibrils. Mutation of this site drastically reduced FN assembly by CHOα5 cells. Only sparse short fibrils were formed even after prolonged incubation, indicating that FN(syn⁻) is defective in progression of the assembly process. These results show that the synergy site is essential for α5β1-mediated accumulation of a FN matrix. However, the incorporation of FN(syn⁻) into fibrils and the deoxycholate-insoluble matrix could be stimulated by Mn²⁺. Therefore, exogenous activation of integrin receptors can overcome the requirement for FN’s synergy site as well as modulate the rate of FN matrix formation.     

Mildly oxidized low density lipoprotein induces contraction of human endothelial cells through activation of Rho/Rho kinase and inhibition of myosin light chain phosphatase.

Mildly oxidized low density lipoprotein (mox-LDL) is critically involved in the early atherogenic responses of the endothelium and increases endothelial permeability through an unknown signal pathway. Here we show that (i) exposure of confluent human endothelial cells (HUVEC) to mox-LDL but not to native LDL induces the formation of actin stress fibers and intercellular gaps within minutes, leading to an increase in endothelial permeability; (ii) mox-LDL induces a transient decrease in myosin light chain (MLC) phosphatase that is paralleled by an increase in MLC phosphorylation; (iii) phosphorylated MLC stimulated by mox-LDL is incorporated into stress fibers; (iv) cytoskeletal rearrangements and MLC phosphorylation are inhibited by C3 transferase from Clostridium botulinum, a specific Rho inhibitor, and Y-27632, an inhibitor of Rho kinase; and (v) mox-LDL does not increase intracellular Ca(2+) concentration. Our data indicate that mox-LDL induces endothelial cell contraction through activation of Rho and its effector Rho kinase which inhibits MLC phosphatase and phosphorylates MLC. We suggest that inhibition of this novel cell signaling pathway of mox-LDL could be relevant for the prevention of atherosclerosis.