Objective. To investigate transforming growth factor  (TGF) regulation of CCN3 expression in cells of the nucleus pulposus.
Methods. Real-time reverse transcriptionpolymerase chain reaction and Western blot analyses were used to measure CCN3 expression in the nucleus pulposus. Transfections were used to measure the effect of Smad3, MAPKs, and activator protein 1 (AP-1) on TGF-mediated CCN3 promoter activity. Lentiviral knockdown of Smad3 was performed to assess the role of Smad3 in CCN3 expression.
Results. CCN3 was expressed in embryonic and adult intervertebral discs. TGF decreased the expression of CCN3 and suppressed its promoter activity in nucleus pulposus cells. DN-Smad3, Smad3 small interfering RNA, or DN-AP-1 had little effect on TGF suppression of CCN3 promoter activity. However, p38 and ERK inhibitors blocked suppression of CCN3 by TGF, suggesting involvement of these signaling pathways in the regulation of CCN3. Interestingly, overexpression of Smad3 in the absence of TGF increased CCN3 promoter activity. We validated the role of Smad3 in controlling CCN3 expression in Smad3-null mice and in nucleus pulposus cells transduced with lentiviral short hairpin Smad3. In terms of function, treatment with recombinant CCN3 showed a dose-dependent decrease in the proliferation of nucleus pulposus cells. Moreover, CCN3-treated cells showed a decrease in aggrecan, versican, CCN2, and type I collagen expression.
Conclusion. The opposing effect of TGF on CCN2 and CCN3 expression and the suppression of CCN2 by CCN3 in nucleus pulposus cells further the paradigm that these CCN proteins form an interacting triad, which is possibly important in maintaining extracellular matrix homeostasis and cell numbers.
The intervertebral disc is a unique structure that permits rotation as well as flexion and extension of the human spine. It is comprised of a gel-like nucleus pulposus surrounded circumferentially by a ligamentous anulus fibrosus. Although cells comprise only ϳ1% of the tissue volume, they are critically important in maintaining a healthy disc (1). Nucleus pulposus cells secrete and organize a complex extracellular matrix that mainly contains the proteoglycan aggrecan and a small proportion of fibrillar collagens. This macromolecular assembly provides a robust hydrodynamic system that accommodates applied biomechanical forces to the spine (2-4). During degenerative disc disease, loss of disc cells, limited proteoglycan synthesis, and a shift toward synthesis of a fibrotic matrix decrease the water-binding capacity of the disc and result in a failure of the tissue to resist compressive loads (5).
Growth factors, in particular transforming growth factor  (TGF), are known to up-regulate aggrecan synthesis in the disc, and there is evidence that this molecule influences the activities of members of the CCN family of proteins (6,7). CCN proteins signal through heparan sulfate proteoglycans and integrins,