Modulation of connexin43 alters expression of osteoblastic differentiation markers

Division of Musculoskeletal Sciences, Department of Orthopaedics and Rehabilitation, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania Submitted 23 August 2005 ; accepted in final form 22 November 2005 Gap junctional channels between cells provide a pathway for exchange of regulatory ions and small molecules. We previously demonstrated that expression of connexins and cell-to-cell communication parallel osteoblastic differentiation and that nonspecific pharmacological inhibitors of gap junctional communication inhibit alkaline phosphatase activity. In this study, we stably transfected connexin (Cx)43 antisense cDNA into the immortalized human fetal osteoblastic cell line hFOB 1.19 (hFOB/Cx43 – ). hFOB/Cx43 – cells express lower levels of Cx43 protein and mRNA and display a 50% decrease in gap junctional intercellular communication relative to control [hFOB/plasmid vector control (pvc)]. This suggests that other connexins, such as Cx45, which is expressed to a similar degree in hFOB/Cx43 – cells and hFOB/pvc cells, contribute to cell-to-cell communication in hFOB 1.19 cells. We observed almost total inhibition of alkaline phosphatase activity in hFOB/Cx43 – cells despite only a 50% decrease in cell-to-cell communication. This suggests the intriguing possibility that Cx43 expression per se, independent of cell-to-cell communication, influences alkaline phosphatase activity and perhaps bone cell differentiation. Quantitative real-time RT-PCR revealed that mRNA levels for osteocalcin and core binding factor 1 (Cbfa1) increased as a function of time in hFOB/pvc but were inhibited in hFOB/Cx43 – . Osteopontin mRNA levels were increased in hFOB/Cx43 – relative to hFOB/pvc and decreased as a function of time in both hFOB/Cx43 – and hFOB/pvc. Transfection with Cx43 antisense did not affect expression of type I collagen in hFOB 1.19 cells. These results suggest that gap junctional intercellular communication and expression of Cx43 contribute to alkaline phosphatase activity, as well as osteocalcin, osteopontin, and Cbfa1 expression in osteoblastic cells. gap junction communication; alkaline phosphatase activity; osteopontin; osteocalcin; hFOB 1.19 Address for reprint requests and other correspondence: Z. Li, Division of Musculoskeletal Sciences, Dept. of Orthopaedics & Rehabilitation, Pennsylvania State Univ. College of Medicine 500 University Dr., Hershey, PA 17033 ( zhongyongli{at}psu.edu )