ABSTRACT
Mechanical forces are important for connective tissue homeostasis. How do fibroblasts sense mechanical stress and how do they translate this information into an adaptive remodeling of the extracellular matrix (ECM)? Tenascin-C is rapidly induced in vivo by loading muscles and in vitro by stretching fibroblasts. Regulation of tenascin-C expression by mechanical signals occurs at the transcriptional level. Integrin receptors physically link the ECM to the cytoskeleton and act as force transducers: intracellular signals are triggered when integrins engage with ECM, and later when forces are applied. We found that cyclic strain does not induce tenascin-C messenger ribonucleic acid (mRNA) in fibroblasts lacking the beta1-integrin chain. An important link in integrin-dependent mechanotransduction is the small guanosine 5'-triphosphatase. RhoA and its target kinase, ROCK. In fibroblasts, cyclic strain activates RhoA and thereby induces ROCK-dependent actin assembly. Interestingly, tenascin-C mRNA induction by cyclic strain was suppressed by relaxing the cytoskeleton with a ROCK inhibitor or by actin depolymerization. Conversely, chemical activators of RhoA enhanced the effect of strain both on actin dynamics and on tenascin-C expression. Thus, RhoA/ROCK-controlled actin dynamics are required for the induction of specific ECM genes by mechanical stress. These findings have implications for the understanding of regeneration and for tissue engineering.
Subject(s)
Fibroblasts/metabolism , Gene Expression Regulation , Mechanotransduction, Cellular/physiology , Animals , Connective Tissue/metabolism , Exercise , HumansABSTRACT
In chick embryo fibroblasts, the mRNA for extracellular matrix protein tenascin-C is induced 2-fold by cyclic strain (10%, 0.3 Hz, 6 h). This response is attenuated by inhibiting Rho-dependent kinase (ROCK). The RhoA/ROCK signaling pathway is primarily involved in actin dynamics. Here, we demonstrate its crucial importance in regulating tenascin-C expression. Cyclic strain stimulated RhoA activation and induced fibroblast contraction. Chemical activators of RhoA synergistically enhanced the effects of cyclic strain on cell contractility. Interestingly, tenascin-C mRNA levels perfectly matched the extent of RhoA/ROCK-mediated actin contraction. First, RhoA activation by thrombin, lysophosphatidic acid, or colchicine induced tenascin-C mRNA to a similar extent as strain. Second, RhoA activating drugs in combination with cyclic strain caused a super-induction (4- to 5-fold) of tenascin-C mRNA, which was again suppressed by ROCK inhibition. Third, disruption of the actin cytoskeleton with latrunculin A abolished induction of tenascin-C mRNA by chemical RhoA activators in combination with cyclic strain. Lastly, we found that myosin II activity is required for tenascin-C induction by cyclic strain. We conclude that RhoA/ROCK-controlled actin contractility has a mechanosensory function in fibroblasts that correlates directly with tenascin-C gene expression. Previous RhoA/ROCK activation, either by chemical or mechanical signals, might render fibroblasts more sensitive to external tensile stress, e.g., during wound healing.
Subject(s)
Actins/metabolism , Connective Tissue/metabolism , Fibroblasts/metabolism , Protein Serine-Threonine Kinases/metabolism , Tenascin/metabolism , rhoA GTP-Binding Protein/metabolism , Actin Cytoskeleton/drug effects , Actin Cytoskeleton/metabolism , Animals , Cell Line, Transformed , Cells, Cultured , Chick Embryo , Connective Tissue/ultrastructure , Enzyme Activation/drug effects , Enzyme Activation/physiology , Fibroblasts/ultrastructure , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , Intracellular Signaling Peptides and Proteins , Mechanotransduction, Cellular/drug effects , Mechanotransduction, Cellular/physiology , Mice , Microtubules/drug effects , Microtubules/metabolism , Myosin Type II/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Stress, Mechanical , Tenascin/genetics , Tensile Strength/physiology , Transcriptional Activation/drug effects , Transcriptional Activation/genetics , Wound Healing/drug effects , Wound Healing/physiology , rho-Associated Kinases , rhoA GTP-Binding Protein/agonistsABSTRACT
Expression of the extracellular matrix (ECM) protein tenascin-C is induced in fibroblasts by growth factors as well as by tensile strain. Mechanical stress can act on gene regulation directly, or indirectly via the paracrine release of soluble factors by the stimulated cells. To distinguish between these possibilities for tenascin-C, we asked whether cyclic tensile strain and soluble factors, respectively, induced its mRNA via related or separate mechanisms. When cyclic strain was applied to chick embryo fibroblasts cultured on silicone membranes, tenascin-C mRNA and protein levels were increased twofold within 6 h compared to the resting control. Medium conditioned by strained cells did not stimulate tenascin-C mRNA in resting cells. Tenascin-C mRNA in resting cells was increased by serum; however, cyclic strain still caused an additional induction. Likewise, the effect of TGF-beta1 or PDGF-BB was additive to that of cyclic strain, whereas IL-4 or H2O2 (a reactive oxygen species, ROS) did not change tenascin-C mRNA levels. Antagonists for distinct mitogen-activated protein kinases (MAPK) inhibited tenascin-C induction by TGF-beta1 and PDGF-BB, but not by cyclic strain. Conversely, a specific inhibitor of Rho-dependent kinase strongly attenuated the response of tenascin-C mRNA to cyclic strain, but had limited effect on induction by growth factors. The data suggest that regulation of tenascin-C in fibroblasts by cyclic strain occurs independently from soluble mediators and MAPK pathways; however, it requires Rho/ROCK signaling.
