JOURNAL OF BONE AND MINERAL RESEARCH Volume 10, Number 9, 1995 Blackwell Science, Inc. zyxwvutsrqpon Chondrocytes Isolated from Mature Articular Cartilage Retain the Capacity to Form Functional Gap Junctions HENRY J. DONAHUE,' FARSHID GUILAK,2 MONIQUE A. VANDER MOLEN,' KENNETH J. MCLEOD,' CLINTON T. RUBIN,' DANIEL A. GRANDE,4 and PETER R. BRINK' ABSTRACT The distribution, expression, and functionality of gap junctions was examined in bovine chondrocytes (BCs) isolated from mature articular cartilage. BC cells displayed irnmunoreactivity for connexin 43 (Cx43), a specific gap junction protein. Cx43 protein expression was confirmed by Western blot analysis, and Cx43 mRNA was detected by nuclease protection assay. Additionally, BCs were shown to be functionally coupled, as revealed by dye transfer studies, and octanol, a gap junction uncoupler, greatly attenuated coupling. Furthermore, confocal microscopy of flu03 loaded BC cells revealed that deformation-induced cytosolic CaZ+ ion (Ca2+) signals propagated from cell-to-cell via gap junctions. To our knowledge, this is the first evidence suggesting that chondrocytes isolated from adult articular cartilage express functional gap junctions. (J Bone Miner Res 1995; 101359-1364) INTRODUCTION AP JUNCTIONS ARE MEMBRANE SPANNING CHANNELS which G allow small molecules (<1 kD) to pass from cell to cell and thus facilitate intercellular communication.(') Previous studies suggest that extensive cell-to-cell communication occurs during prechondrocyte condensation prior to the onset of chondrogenesis.(2*') Furthermore, specific gap junction proteins are expressed by neonatal rat calvarial chondrocytes in sit^.(^) These findings suggest that cell-to- cell communication via gap junctions contributes to chon- drogenesis and cartilage differentiation. However, it is unclear what role, if any, gap junctional intercellular com- munication plays in the function of mature differentiated This work was supported by NIH grant AG10199 and AG13087 (HJD) and a grant from the Whitaker Foundation (FG). Portions of this work were presented at the 40th Annual Meeting of the Orthopaedic Research Society, New Orleans, LA 1994. chondrocytes. Indeed, it is unclear whether chondrocytes within mature cartilage have the capacity to express func- tional gap junctions. To address this issue we examined the distribution and expression of connexin 43 (Cx43), a specific gap junction protein, in primary cultures of mature bovine chondrocytes. We also examined the degree to which chon- drocytes communicate via gap junctions, i.e., are function- ally coupled, and to what extent intercellular signals prop- agate from chondrocyte to chondrocyte via gap junctions. MATERIALS AND METHODS zyx Cell culture Bovine chondrocytes (BCs) were isolated, following thc method of Burmeister et aI,(') from mature articular carti- lage of the hock joint of skeletally mature animals. Freshly isolated cells were plated on 25 cm2 flasks with RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 IU/ml of penicillin, and 100 Fl.g/nil 'Musculoskeletal Research Laboratory, Department of Orthopaedics, The Pennsylvania State University College of Medicine, Hershey, *Department of Orthopaedics, Duke University Medical Center, Durham, North Carolina. 3Department of Orthopaedics. State University of New York, Stony Brook, New York. 4North Shore University Hospital, Manhasset, New York. 5Department of Physiology and Biophysics, State University of New York, Stony Brook, New York. Pennsylvania. 1359