Biochimica et Biophysica Acta 1693 (2004) 193–204 www.bba-direct.com Induction of tenascin-C by cyclic tensile strain versus growth factors: distinct contributions by Rho/ROCK and MAPK signaling pathways Matthias Chiquet*, Ana Sarasa-Renedo, Vildan Tunc¸-Civelek ITI-Research Institute for Dental and Skeletal Biology, University of Bern, Murtenstrasse 35, P.O. Box 54, CH-3010 Bern, Switzerland Received 15 April 2004; received in revised form 2 July 2004; accepted 2 August 2004 Available online 20 August 2004 Abstract 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-h1 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-h1 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. D 2004 Elsevier B.V. All rights reserved. Keywords: Extracellular matrix; Tenascin-C; Mechanical stress; TGF-h; PDGF; Gene regulation; Signaling 1. Introduction to induce genes for extracelluar matrix components indi- rectly. In various cell types, mechanical stimulation has been For homeostasis, connective tissues depend on a constant shown to trigger the release of certain auto-or paracrine input of mechanical stress in a certain physiological range growth factors which then change the transcription rate of and pattern; without such stimulation, these tissues suffer specific bmechanoresponsiveQ genes [3,4]. Transforming atrophy [1]. Thus, it is an important question how growth factor-beta (TGF-h), notorious for stimulating the mechanical signals regulate extracellular matrix (ECM) production of ECM proteins in fibroblasts, is an obvious synthesis and turnover [2]. Different mechanisms have been candidate for mediating such indirect responses to mechan- suggested. First, in many cases mechanical stress is thought ical stress [5,6]; another is connective tissue growth factor (CTGF) whose mRNA level is itself regulated by mechan- ical signals [7]. Platelet derived growth factor (PDGF) is Abbreviations: ECM, extracellular matrix; MAPK, mitogen activated released upon mechanical stimulation of smooth muscle protein kinase; ROCK, Rho-dependent protein kinase; FCS, fetal calf cells, inducing their proliferation [8]. An autocrine feedback serum; DMEM, Dulbecco’s minimal essential medium; BSA, bovine serum loop involving interleukin-4 (IL-4) was shown to be albumin; PBS, phosphate buffered saline; TGF-h, transforming growth factor-beta; PDGF, platelet derived growth factor; IL-4, interleukin-4 involved in the response of chondrocytes to mechanical * Corresponding author. Tel.: +41 31 632 8684; fax: +41 31 632 4999. signals [9], and reactive oxygen species (ROS) were E-mail address: [email protected] (M. Chiquet). implicated in mechanotransduction in heart myocytes [10]. 0167-4889/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2004.08.001 194 M. Chiquet et al. / Biochimica et Biophysica Acta 1693 (2004) 193–204 Alternatively, mechanical stimulation can trigger intra- 2. Materials and methods cellular signals directly, since physical stress is transmitted from the ECM via integrins across the cell membrane to the 2.1. The equi-biaxial strain device cytoskeleton [11]. Integrins, together with the entire machi- nery at focal contacts involved in cytoskeletal assembly and The cyclic strain device with special culture dishes was dynamics, are likely to take part in mechanotransduction designed by us and manufactured in our institute’s [12]. Pulling on ligand-engaged integrins from the outside of workshop. Its is similar in principle to the device the cell leads to the activation of intracellular protein kinases developed by Schaffer et al. [25] who also provided the [13] as well as to a stiffening of the actin cytoskeleton theoretical background. The advantage of our machine lies mediated by the small GTPase RhoA and its downstream in its simplicity and its multiwell design (Fig. 1). It allows effector Rho-dependent kinase (ROCK I/II) [14]. By such to apply sinusoidal, equi-biaxial and isotropic strain of integrin- and Rho-dependent signaling cascades, certain desired amplitude, frequency and duration to cells cultured mechanosensitive genes could be activated directly, i.e., on (ECM-coated) silicone elastomer membranes. The without an intermediate step of growth factor or cytokine scaffolds of its six-well culture dishes are moulds from release by the stimulated cells [11]. Along these lines, it has blocks of high density polyethylene (UHMW-PE, Kundert, been shown recently that Rho-dependent actin dynamics Jona, Switzerland) on a computer-controlled lathe (Pico- regulate nuclear translocation of megakaryocytic acute max 51-CNC, Fehlmann, Seon, Switzerland). Scaffolds leukemia (MAL) protein, a coactivator of transcription have the exact dimensions of commercial six-well dishes factor, serum response factor (SRF) [15]. (35-mm wells), but wells are open at the bottom. Steel In practice, it is by no means trivial to distinguish thread cases are inserted into the lower surface of the between an indirect and a direct mechanism of gene scaffolds between the wells. These allow to screw on a 2- regulation by mechanical signals. Tenascin-C (myotendi- mm-thick stainless steel base plate (16.2Â8.7 cm) with six nous antigen), for example, is an ECM protein prominently circular 35-mm diameter openings concentric with the expressed in tissues bearing high tensile stress (especially wells. A sheet of silicone membrane (0.05U, gloss/gloss; tendons and ligaments), suggesting a mechanical component Specialty Manufacturing Inc.) is sandwiched between the in its regulation [16]. Indeed, we have found that scaffold and the base plate of the culture dish, forming the immobilization of chick embryos in ovo strongly reduces bottom surface of the wells. After seeding cells on the tenascin-C expression in developing leg tendons [17], and silicone membrane (see below), two assembled culture that tenascin-C mRNA and protein are rapidly induced in dishes can be mounted onto the strain device. The device the endomysium of chick ALD muscle after applying load itself consists of a aluminum bottom platform (12Â24Â1.2 to the wing [18]. In cultured fibroblasts and heart myocytes, cm) and a rectangular top frame (18Â24Â1.2 cm) held this ECM protein was shown to be regulated by static together at their corners by four vertical steel rods (17-cm [19,20] as well as dynamic tensile stress [10],and length, 1-cm diameter). An aluminum stage (12Â24Â1.2 mechanoresponsive regulatory sequences have been cm) between lower platform and upper frame can be described in the promoter of its gene [10,19]. On the other hand, tenascin-C is strongly expressed de novo during tissue remodeling [21], wound healing [22], and in inflammation [23]. Accordingly, serum and several isolated growth factors (among them TGF-h and PDGF) are known to induce tenascin-C mRNA (for review, see Ref. [24]). Thus, any direct effects of mechanical stress on tenascin-C expression might be confounded with the concomitant release of a growth factor. In order to approach this dilemma, we compared the magnitude and time course of tenascin-C induction by serum, purified growth factors, and cyclic tensile strain in cultured chick embryo fibroblasts. We also asked whether cyclic strain and growth factors acted on tenascin-C mRNA Fig. 1. Multiwell device used to generate equi-biaxial strain on cultured fibroblasts. (1) Six-well culture dish with fibronectin-coated silicone levels in a sequential or rather in an additive or synergistic membrane bottoms onto which the cells are plated. (2) Fixed aluminum manner. Moreover, we evaluated the responses to growth frame onto which two dishes are mounted with screws. (3) Teflon rings factors and mechanical stress, respectively, for their which push into the silicone bottomed culture wells from below (the second sensitivity to antagonists of various intracellular signaling culture dish is not mounted for better view). (4) Platform which holds the pathways. The aim of these experiments was to gain Teflon rings, and which can be moved vertically along four steel rods at the corners. (5) Piston fixed to the movable platform from below (a second on information whether growth factors and cyclic tensile