Distinct Mechanosensitive Ca 2þ Influx Mechanisms in Human Primary Synovial Fibroblasts Yuko Sakamoto, 1 Muneaki Ishijima, 1 Haruka Kaneko, 1 Nagomi Kurebayashi, 2 Naoki Ichikawa, 3 Ippei Futami, 1 Hisashi Kurosawa, 1 Eri Arikawa-Hirasawa 3,4 1 Department of Orthopaedics, Juntendo University School of Medicine, Tokyo, Japan, 2 Department of Pharmacology, Juntendo University School of Medicine, Tokyo, Japan, 3 Research Institute for Diseases of Old Age, Juntendo University School of Medicine, 2-1-1, Hongo, Bunkyo-ku, Tokyo, 113- 8421, Japan, 4 Department of Neurology, Juntendo University School of Medicine, Tokyo, Japan Received 3 June 2009; accepted 16 November 2009 Published online 27 January 2010 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jor.21080 ABSTRACT: Synovial cells are exposed to continually changing dynamic forces and are implicated in the maintenance of joint homeostasis. However, the mechanisms of synovial cell responses to mechanical stress are unclear. In this study, we investigated the difference between the mechanosensitive channels of human primary synovial fibroblasts (SFBs) and human primary dermal fibroblasts (DFBs) in response to mechanical stretch by uniaxial cyclic stretching and mechanical cell membrane deformation in vitro. Cyclic stretching induced orientation of SFBs and DFBs perpendicularly to the stretching direction. Furthermore, uniaxial stretching increased intracellular Ca 2þ levels in both cell types. The perpendicular orientation of DFBs was blocked by gadolinium (III) chloride (Gd 3þ , a mechanosensitive Ca 2þ channel blocker) or ruthenium red (RR, a nonselective Ca 2þ channel blocker). However, Gd 3þ did not block the stretch-induced perpendicular orientation in SFBs, while RR inhibited this orientation. Similarly, Ca 2þ influx in DFBs induced by uniaxial stretching and membrane deformation was inhibited by Gd 3þ , RR, and GsMTx-4 (another mechanosensitive Ca 2þ channel blocker), while only RR inhibited Ca 2þ influx in SFBs. Our results suggest that SFBs respond to mechanical stretch through mechanosensitive channels that are distinct from those of DFBs. ß 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:859– 864, 2010 Keywords: synovial fibroblasts; mechanical stretch; cell orientation; mechanosensitive channel; transient receptor potential (TRP) channel Cells in the synovial space are exposed to various types of dynamic forces. 1 By responding to mechanical forces, these cells play an important role in maintaining the homeostasis of tissues. Synovial fibroblasts (SFBs) are exposed to continually changing dynamic forces (e.g., >10% stretching in situ) 2 and are known to play a crucial role in the maintenance of joint homeostasis. Under physiological conditions in a variety of tissues, cyclic stretching may affect the regulation of cell morphol- ogy, proliferation, and protein synthesis. In many types of cultured cells, including fibroblasts, cyclic stretching causes cells to orient perpendicularly to the stretch axis. 3,4 It is also known that Ca 2þ influx is essential for the initial step of various types of mechanotransduction. Recently, it was reported that members of the transient receptor potential (TRP) family of nonselective Ca 2þ channels constitute some types of mechanosensitive channels. 5–9 The orientation of the cells induced by cyclic stretching is blocked by gadolinium (III) chloride (Gd 3þ , a mechano- sensitive Ca 2þ channel blocker). 3,10,11 Previous studies indicated that deformation of the cell membrane induces Ca 2þ influx in the HIG-82 rabbit synoviocyte cell line. 12 In addition, mechanical stretch induces Ca 2þ influx and Ca 2þ -dependent protein kinase C activation, which stimulates secretion of hyaluronan from synoviocytes of rabbits. 13 However, the mechanism of Ca 2þ influx in SFBs remains largely unknown. Furthermore, the effect of mechanical stretch on the cell morphology of primary human SFBs is unclear. In this study, we compare the effects of mechanical stress on SFBs with those on human dermal fibroblasts (DFBs) and demonstrate that human joint SFBs regulate their responses to mechanical stress through mechano- sensitive channels distinct from typical mechanosensi- tive channels. MATERIALS AND METHODS Cell Culture The experimental protocol was approved by the Ethics Committee of our university, and signed informed consent was obtained from each participant. Synovial tissues were obtained from five patients with anterior cruciate ligament injuries (age, 18–25; three men and two women). SFBs were cultured as described previously. 14 DFBs (Cambrex, Walkers- ville, MD) were grown as described in the manufacturer’s instructions. SFBs were cultured for three to five passages, at which time they were a homogenous population of fibroblasts. SFBs and DFBs cultured over two passages were then used in the experiments. For mechanical stretch and stimulation experiments, the culture medium was replaced with a stretching culture medium (145 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 2 mM MgCl 2 , 10 mM glucose, 10 mM HEPES, pH 7.4) or Ca 2þ -free medium (145 mM NaCl, 5 mM KCl, 2 mM MgCl 2 , 10 mM glucose, 20 mM HEPES, pH 7.4). For inhibition analyses, Gd 3þ (Sigma-Aldrich, St. Louis, MO), GsMTx-4 (Peptide Institute, Inc., Osaka, Japan), or ruthenium red (RR, Sigma-Aldrich), was added to the stretch culture medium before stimulation. Gd 3þ was prepared as described previously. 3 Cyclic Stretching of the Cells SFBs and DFBs were transferred into a silicone chamber (Strex, Osaka, Japan) at a density of 5  10 4 cells/cm 2 . The silicone chamber was attached to a stretching apparatus (Strex). The silicone chambers were cyclically stretched (uniaxial strain, 10%, 0.1 Hz) at 378C, 5% CO 2 . JOURNAL OF ORTHOPAEDIC RESEARCH JULY 2010 859 Additional Supporting Information may be found in the online version of this article. Correspondence to: Eri Arikawa-Hirasawa (T: þ81-3-3813-3111, ext. 3797; F: þ81-3-3814-3016; E-mail: ehirasaw@juntendo.ac.jp) ß 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.