Regulation of CCN2/connective tissue growth factor expression in the nucleus pulposus of the intervertebral disc: role of Smad and activator protein 1 signaling.
OBJECTIVE
To investigate transforming growth factor beta (TGFbeta) regulation of connective tissue growth factor (CTGF) expression in cells of the nucleus pulposus of rats, mice, and humans.


METHODS
Real-time reverse transcription-polymerase 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 TGFbeta-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 TGFbeta 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 TGFbeta-mediated activation of the CTGF promoter. When either Smad3 or AP-1 sites were mutated, CTGF promoter induction by TGFbeta 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 TGFbeta expression in the degenerated state.


CONCLUSIONS
TGFbeta, 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.
Tran
Cassie M
CM
Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Markova
Dessislava
D
Smith
Harvey E
HE
Susarla
Bala
B
Ponnappan
Ravi Kumar
RK
Anderson
D Greg
DG
Symes
Aviva
A
Shapiro
Irving M
IM
Risbud
Makarand V
MV
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D055513:Connective Tissue Growth Factor / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D007403:Intervertebral Disc / Q000378:metabolism* / Q000473:pathology; D008159:Lumbar Vertebrae / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D018345:Mice, Knockout; D008875:Middle Aged; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction / Q000502:physiology; D051785:Smad Proteins / Q000235:genetics / Q000378:metabolism*; D013045:Species Specificity; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000494:pharmacology
C526317:CCN2 protein, human; C526318:CCN2 protein, mouse; C526316:CCN2 protein, rat; D034741:RNA, Small Interfering; D051785:Smad Proteins; D018808:Transcription Factor AP-1; D016212:Transforming Growth Factor beta; D055513:Connective Tissue Growth Factor