Regulation and Function of CCN2 in the Intervertebral Disc: A Critical Anabolic Factor in Nucleus Pulposus Cells
Lower back pain resulting from intervertebral disc degeneration is one of the most common musculoskeletal conditions. The intervertebral disc comprises a central, proteoglycan-rich nucleus pulposus (NP) that is surrounded circumferentially by a fibrocartilagenous annulus fibrosus (AF). Together these structures form a unique hydrodynamic system that accommodates biomechanical forces on the spine. Cells of the NP reside within an avascular microenvironment and, unlike other tissues, have adapted to low oxygen tension by stabilizing Hypoxia Inducible Factor 1-alpha (HIF-1alpha). NP cells must maintain homeostasis of the extracellular matrix in response to stresses, growth factors and cytokines present in this unique microenvironment. Due to increasing catabolic activity and the change in matrix synthesis to a more fibrotic composition, disc degeneration is characterized by the loss of proteoglycans. The resulting dehydration causes a reduction in disc height and altered mechanical loading that leads to progressive changes in the tissue microenvironment and cell function. Inflammatory cytokines, such as IL-1beta and TNF-alpha, and growth factors such as TGF-beta, increase in the degenerate disc and are thought to mediate the degenerative process. Although some contributing factors of disc degeneration have been identified, the underlying molecular mechanisms of disc degeneration and of tissue homeostasis in the healthy state are largely unknown. Connective tissue growth factor (CCN2/CTGF) is a matricellular protein that has emerged as an important regulator of extracellular matrix homeostasis in skeletal tissues such as cartilage and bone. For this reason, we were interested in examining the regulation and function of this protein in the intervertebral disc. The aims of this work were twofold: The first aim was to determine the regulation of CCN2 by TGF-beta and hypoxia, factors central to the unique NP microenvironment, as well as the regulation of CCN2 by inflammatory cytokines induced during disc degeneration. The second aim was to determine the function of CCN2 in the healthy and degenerate NP. Results of our study showed that TGF-beta through Smad3 and AP1, increased CCN2 expression in the NP. Analysis of human NP indicated a trend of increasing CCN2 and TGF-beta expression with degeneration. Meanwhile, TGF-beta was shown to decrease the expression of CCN3, a CCN protein with anti-proliferative effects on NP cells and reciprocal functions to CCN2 in terms of matrix homeostasis. We propose that the TGF-beta - CCN2 axis is a part of a limited reparative response in the degenerate disc. In a separate study, we showed that hypoxia, through a HIF-1alpha, suppressed CCN2 expression and, conversely, that CCN2 also serves to suppress HIF-1alpha transcription and activity. It is possible that hypoxic suppression serves to tightly regulate CCN2 production in the NP and that this negative feedback loop is important in the maintenance of tissue homeostasis. In this study, we also showed that the NP of CCN2-null mice contain reduced levels of proteoglycans, specifically aggrecan, compared to controls confirming an anabolic role for CCN2 in the NP. Lastly, we found that inflammatory cytokines, IL-1beta and TNF-alpha, suppressed CCN2 through NF-kappaB signaling. Moreover, CCN2 interaction with both alphavbeta3 and alpha5beta1 integrins is required for its suppressive effects on IL-1beta-induced catabolic gene expression and its anabolic effect on aggrecan expression. These results suggest that CCN2 and inflammatory cytokines form a functional negative feedback loop in NP cells that may be important in the pathogenesis of disc disease. Taken together, it is clear that CCN2 plays an important anabolic role in matrix homeostasis in the healthy NP; whether it functions similarly in degenerate NP cells remains to be discovered.
Biology|Molecular biology|Cellular biology
Tran, Cassie M, "Regulation and Function of CCN2 in the Intervertebral Disc: A Critical Anabolic Factor in Nucleus Pulposus Cells" (2013). ETD Collection for Thomas Jefferson University. AAI3600913.