CCN2 suppresses catabolic effects of interleukin-1ß through α5ß1 and αVß3 integrins in nucleus pulposus cells: implications in intervertebral disc degeneration.

The objective of the study was to examine the regulation of CCN2 by inflammatory cytokines, IL-1ß, and TNF-α and to determine whether CCN2 modulates IL-1ß-dependent catabolic gene expression in nucleus pulposus (NP) cells. IL-1ß and TNF-α suppress CCN2 mRNA and protein expression in an NF-κB-dependent but MAPK-independent manner. The conserved κB sites located at -93/-86 and -546/-537 bp in the CCN2 promoter mediated this suppression. On the other hand, treatment of NP cells with IL-1ß in combination with CCN2 suppressed the inductive effect of IL-1ß on catabolic genes, including MMP-3, ADAMTS-5, syndecan 4, and prolyl hydroxylase 3. Likewise, silencing of CCN2 in human NP cells resulted in elevated basal expression of several catabolic genes and inflammatory cytokines like IL-6, IL-4, and IL-12 as measured by gene expression and cytokine protein array, respectively. Interestingly, the suppressive effect of CCN2 on IL-1ß was independent of modulation of NF-κB signaling. Using disintegrins, echistatin, and VLO4, peptide inhibitors to αvß3 and α5ß1 integrins, we showed that CCN2 binding to both integrins was required for the inhibition of IL-1ß-induced catabolic gene expression. It is noteworthy that analysis of human tissues showed a trend of altered expression of these integrins during degeneration. Taken together, 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.

HIF-1-PHD2 axis controls expression of syndecan 4 in nucleus pulposus cells.

Intervertebral disc degeneration is the leading cause of chronic back pain. Recent studies show that raised level of SDC4, a cell-surface heparan sulfate (HS) proteoglycan, plays a role in pathogenesis of disc degeneration. However, in nucleus pulposus (NP) cells of the healthy intervertebral disc, the mechanisms that control expression of SDC4 and its physiological function are unknown. Hypoxia induced SDC4 mRNA and protein expression by ~2.4- and 4.4-fold (P<0.05), respectively, in NP cells. While the activity of the SDC4 promoter containing hypoxia response element (HRE) was induced 2-fold (P<0.05), the HRE mutation decreased the activity by 40% in hypoxia. Transfections with plasmids coding prolyl-4-hydroxylase domain protein 2 (PHD2) and ShPHD2 show that hypoxic expression of SDC4 mRNA and protein is regulated by PHD2 through controlling hypoxia-inducible factor 1α (HIF-1α) levels. Although overexpression of HIF-1α significantly increased SDC4 protein levels, stable suppression of HIF-1α and HIF-1ß decreased SDC4 expression by 50% in human NP cells. Finally, suppression of SDC4 expression, as well as HS function, resulted in an ~2-fold increase in sex-determining region Y (SRY)-box 9 (Sox9) mRNA, and protein (P<0.05) and simultaneous increase in Sox9 transcriptional activity and target gene expression. Taken together, our findings suggest that in healthy discs, SDC4, through its HS side chains, contributes to maintenance of the hypoxic tissue niche by controlling baseline expression of Sox9.

Hypoxia-inducible factor (HIF)-1α and CCN2 form a regulatory circuit in hypoxic nucleus pulposus cells: CCN2 suppresses HIF-1α level and transcriptional activity.

The objective of the study was to investigate if hypoxia-inducible factor (HIF)-1α and connective tissue growth factor (CCN2) form a regulatory network in hypoxic nucleus pulposus (NP) cells. A decrease in CCN2 expression and proximal promoter activity was observed in NP cells after hypoxic culture. Analysis of both human and mouse CCN2 promoters using the JASPAR core database revealed the presence of putative hypoxia response elements. Transfection experiments showed that both promoter activities and CCN2 expression decreases in hypoxia in a HIF-1α-dependent fashion. Interestingly, deletion analysis and mutation of the hypoxia responsive elements individually or in combination resulted in no change in promoter activity in response to hypoxia or in response to HIF-1α, suggesting an indirect mode of regulation. Notably, silencing of endogenous CCN2 increased HIF-1α levels and its target gene expression, suggesting a role for CCN2 in controlling basal HIF-1α levels. On the other hand, treatment of cells with rCCN2 resulted in a decrease in the ability of HIF-1α transactivating domain to recruit co-activators and diminished target gene expression. Last, knockdown of CCN2 in NP cells results in a significant decrease in GAG synthesis and expression of AGGRECAN and COLLAGEN II. Immunohistochemical staining of intervertebral discs of Ccn2 null embryos shows a decrease in aggrecan. These findings reveal a negative feedback loop between CCN2 and HIF-1α in NP cells and demonstrate a role for CCN2 in maintaining matrix homeostasis in this tissue.

Transforming growth factor ß controls CCN3 expression in nucleus pulposus cells of the intervertebral disc.

OBJECTIVE: To investigate transforming growth factor ß (TGFß) regulation of CCN3 expression in cells of the nucleus pulposus. METHODS: Real-time reverse transcription-polymerase 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 TGFß-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. TGFß 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 TGFß suppression of CCN3 promoter activity. However, p38 and ERK inhibitors blocked suppression of CCN3 by TGFß, suggesting involvement of these signaling pathways in the regulation of CCN3. Interestingly, overexpression of Smad3 in the absence of TGFß 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 TGFß 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.

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. CONCLUSION: 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.

mTORC1 restrains adipocyte lipolysis to prevent systemic hyperlipidemia.

OBJECTIVE: Pharmacological agents targeting the mTOR complexes are used clinically as immunosuppressants and anticancer agents and can extend the lifespan of model organisms. An undesirable side effect of these drugs is hyperlipidemia. Although multiple roles have been described for mTOR complex 1 (mTORC1) in lipid metabolism, the etiology of hyperlipidemia remains incompletely understood. The objective of this study was to determine the influence of adipocyte mTORC1 signaling in systemic lipid homeostasis in vivo. METHODS: We characterized systemic lipid metabolism in mice lacking the mTORC1 subunit Raptor (RaptoraKO), the key lipolytic enzyme ATGL (ATGLaKO), or both (ATGL-RaptoraKO) in their adipocytes. RESULTS: Mice lacking mTORC1 activity in their adipocytes failed to completely suppress lipolysis in the fed state and displayed prominent hypertriglyceridemia and hypercholesterolemia. Blocking lipolysis in their adipose tissue restored normal levels of triglycerides and cholesterol in the fed state as well as the ability to clear triglycerides in an oral fat tolerance test. CONCLUSIONS: Unsuppressed adipose lipolysis in the fed state interferes with triglyceride clearance and contributes to hyperlipidemia. Adipose tissue mTORC1 activity is necessary for appropriate suppression of lipolysis and for the maintenance of systemic lipid homeostasis.

Rapamycin Blocks Induction of the Thermogenic Program in White Adipose Tissue.

Rapamycin extends life span in mice, yet paradoxically causes lipid dysregulation and glucose intolerance through mechanisms that remain incompletely understood. Whole-body energy balance can be influenced by beige/brite adipocytes, which are inducible by cold and other stimuli via ß-adrenergic signaling in white adipose depots. Induction of beige adipocytes is considered a promising strategy to combat obesity because of their ability to metabolize glucose and lipids, dissipating the resulting energy as heat through uncoupling protein 1. Here, we report that rapamycin blocks the ability of ß-adrenergic signaling to induce beige adipocytes and expression of thermogenic genes in white adipose depots. Rapamycin enhanced transcriptional negative feedback on the ß3-adrenergic receptor. However, thermogenic gene expression remained impaired even when the receptor was bypassed with a cell-permeable cAMP analog, revealing the existence of a second inhibitory mechanism. Accordingly, rapamycin-treated mice are cold intolerant, failing to maintain body temperature and weight when shifted to 4°C. Adipocyte-specific deletion of the mTORC1 subunit Raptor recapitulated the block in ß-adrenergic signaling. Our findings demonstrate a positive role for mTORC1 in the recruitment of beige adipocytes and suggest that inhibition of ß-adrenergic signaling by rapamycin may contribute to its physiological effects.

Molecular regulation of CCN2 in the intervertebral disc: lessons learned from other connective tissues.

Connective tissue growth factor (CCN2/CTGF) plays an important role in extracellular matrix synthesis, especially in skeletal tissues such as cartilage, bone, and the intervertebral disc. As a result there is a growing interest in examining the function and regulation of this important molecule in the disc. This review discusses the regulation of CCN2 by TGF-ß and hypoxia, two critical determinants that characterize the disc microenvironment, and discusses known functions of CCN2 in the disc. The almost ubiquitous regulation of CCN2 by TGF-ß, including that seen in the disc, emphasizes the importance of the TGF-ß-CCN2 relationship, especially in terms of extracellular matrix synthesis. Likewise, the unique cross-talk between CCN2 and HIF-1 in the disc highlights the tissue and niche specific mode of regulation. Taken together the current literature supports an anabolic role for CCN2 in the disc and its involvement in the maintenance of tissue homeostasis during both health and disease. Further studies of CCN2 in this tissue may reveal valuable targets for the biological therapy of disc degeneration.