Regulation of CCN2/Connective tissue growth factor expression in the nucleus pulposus of the intervertebral disc: Role of Smad and activator protein 1 signaling

Abstract

Objective. To investigate transforming growth factor ␤ (TGF␤) regulation of connective tissue growth factor (CTGF) expression in cells of the nucleus pulposus of rats, mice, and humans.Methods. Real-time reverse transcriptionpolymerase chain reaction and Western blot analyses were used to measure CTGF expression in the nucleus pulposus. Transfections were used to measure the effects of Smads 2, 3, and 7 and activator protein 1 (AP-1) on TGF␤-mediated CTGF promoter activity.Results. CTGF expression was lower in neonatal rat discs than in skeletally mature rat discs. An increase in CTGF expression and promoter activity was observed in rat nucleus pulposus cells after TGF␤ treatment. Deletion analysis indicated that promoter constructs lacking Smad and AP-1 motifs were unresponsive to treatment. Analysis showed that full-length Smad3 and the Smad3 MH-2 domain alone increased CTGF activity. Further evidence of Smad3 and AP-1 involvement was seen when DN-Smad3, SiRNA-Smad3, Smad7, and DN-AP-1 suppressed TGF␤-mediated activation of the CTGF promoter. When either Smad3 or AP-1 sites were mutated, CTGF promoter induction by TGF␤ was suppressed. We also observed a decrease in the expression of CTGF in discs from Smad3-null mice as compared with those from wild-type mice. Analysis of human nucleus pulposus samples indicated a trend toward increasing CTGF and TGF␤ expression in the degenerated state.Conclusion. TGF␤, through Smad3 and AP-1, serves as a positive regulator of CTGF expression in the nucleus pulposus. We propose that CTGF is a part of the limited reparative response of the degenerated disc.

Runx2/Smad3 Complex Negatively Regulates TGF-β-Induced Connective Tissue Growth Factor gene Expression in Vascular Smooth Muscle Cells

“…Given that Smad3 and Smad4 form a complex in the nucleus even in the absence of TGF- 39,40) , it is likely that Runx2 acts as corepressor of the Smad3/Smad4 complex to inhibit basal activity of the CTGF promoter under basal conditions. Consistent with this assumption, previous studies showed that CTGF expression is reduced in cells derived from Smad3-null mice 41,42) , suggesting that the Smad3/Smad4 complex supports basal CTGF gene expression and is subjected to Runx2-mediated repression, irrespective of TGF- …”

CCN2 Suppresses Catabolic Effects of Interleukin-1β through α5β1 and αVβ3 Integrins in Nucleus Pulposus Cells

“…Further evidence of altered expression came from our own studies that showed a trend of increasing expression of ␤1, ␣5, and ␣V integrin subunits in severely degenerated human disc samples (grade 4 and 5) compared with moderately degenerated discs (grade 2 and 3). These trends failed to reach statistical significance because of large patient to patient variations as observed in previous studies of human disc tissues (3,11). Nonetheless, a strong positive correlation was observed between the expressions of these integrin subunits.…”

“…Le Maitre et al (47) showed that mechanotransduction through ␣5␤1 integrin was impaired in degenerate human NP cells, whereas Xia and Zhu (48) found that the fibronectin fragments that accumulate in the degenerative disc cause an increase in MMP-9 and -13 through ␣5␤1. Moreover, it is important to note that along with altered integrin expression and function, CCN2 levels are also elevated during disc degeneration (11,22). These findings beg the questions: Is CCN2 able to exert anti-catabolic effects in degenerate state?…”

“…It is worth noting that TGF-␤ treatment can override CCN2 suppression by either inflammatory cytokine (Fig. 1, F and G), perhaps explaining the elevated expression of both CCN2 and inflammatory cytokines during disc degeneration (5,11,22).…”

“…Interestingly, several groups have reported increased levels of CCN2 in degenerate and painful human discs (11,22). However, its relationship with inflammatory cytokines in the pathogenesis of disc disease remains unclear.…”