No abstract

Fibrosis is a major cause of end-stage renal disease, and although initiation factors have been elucidated, uncertainty concerning the downstream pathways has hampered the development of anti-fibrotic therapies. CCN2 (CTGF) functions downstream of transforming growth factor (TGF)-beta, driving increased extracellular matrix (ECM) accumulation and fibrosis. We examined the possibility that CCN3 (NOV), another CCN family member with reported biological activities that differ from CCN2, might act as an endogenous negative regulator of ECM and fibrosis. We show that cultured rat mesangial cells express CCN3 mRNA and protein, and that TGF-beta treatment reduced CCN3 expression levels while increasing CCN2 and collagen type I activities. Conversely, either the addition of CCN3 or CCN3 overexpression produced a marked down-regulation of CCN2 followed by virtual blockade of both collagen type I transcription and its accumulation. This finding occurred in both growth-arrested and CCN3-transfected cells under normal growth conditions after TGF-beta treatment. These effects were not attributable to altered cellular proliferation as determined by cell cycle analysis, nor were they attributable to interference of Smad signaling as shown by analysis of phosphorylated Smad3 levels. In conclusion, both CCN2 and CCN3 appear to act in a yin/yang manner to regulate ECM metabolism. CCN3, acting downstream of TGF-beta to block CCN2 and the up-regulation of ECM, may therefore serve to naturally limit fibrosis in vivo and provide opportunities for novel, endogenous-based therapeutic treatments.

p21 is a potent inhibitor of cyclin-dependent kinases capable of arresting cell cycle progression. p21 is primarily regulated at the transcriptional level by several transcription factors, including p53. Previously, we reported that certain members of the E2F family of transcription factors may activate p21 transcription via a p53-independent mechanism. To further elucidate the consequences of E2F-1-regulated induction of p21, we developed cell lines with a tamoxifen-dependent form of E2F-1. We confirmed direct interaction of E2F-1 with the proximal region of the p21 promoter. Interestingly, elevated E2F-1 activity was sufficient to arrest a substantial subset of cells in S phase and this effect was correlated to and dependent on the induction of p21 protein. Since E2F proteins control genes required for cell cycle progression and are activated by various oncogenic events, we believe that the p21-dependent arrest described in this report represents an additional mechanism that guards against unrestricted cell proliferation.

Prior work in the CCN field, including our own, suggested to us that there might be co-regulatory activity and function as part of the actions of this family of cysteine rich cytokines. CCN2 is now regarded as a major pro-fibrotic molecule acting both down-stream and independent of TGF-beta1, and appears causal in the disease afflicting multiple organs. Since diabetic renal fibrosis is a common complication of diabetes, and a major cause of end stage renal disease (ESRD), we examined the possibility that CCN3 (NOV), might act as an endogenous negative regulator of CCN2 with the capacity to limit the overproduction of extracellular matrix (ECM), and thus prevent, or ameliorate fibrosis. We demonstrate, using an in vitro model of diabetic renal fibrosis, that both exogenous treatment with CCN3 and transfection with the over-expression of the CCN3 gene in mesangial cells markedly down-regulates CCN2 activity and blocks ECM over-accumulation stimulated by TGF-beta1. Conversely, TGF-beta1 treatment reduces endogenous CCN3 expression and increases CCN2 activity and matrix accumulation, indicating an important, novel yin/yang effect. Using the db/db mouse model of diabetic nephropathy, we confirm the expression of CCN3 in the kidney, with temporal localization that supports these in vitro findings. In summary, the results corroborate our hypothesis that one function of CCN3 is to regulate CCN2 activity and at the concentrations and conditions used down-regulates the effects of TGF-beta1, acting to limit ECM turnover and fibrosis in vivo. The findings suggest opportunities for novel endogenous-based therapy either by the administration, or the upregulation of CCN3.

The CDK inhibitor p21 WAF1/CIP1 is a negative regulator of the cell cycle, and its expression is induced during terminal dierentiation in vitro and in vivo. Expression of p21 is controlled at the transcriptional level by both p53-dependent and -independent mechanisms. Our previous studies established that p21 is expressed in the Caco-2 adenocarcinoma cell line, and its expression is induced by a p53-independent mechanism during dierentiation of these cells. Here we have found that transcription of p21 in Caco-2 cells is controlled primarily by the transcription factors Sp1 and Sp3 through two Sp1 binding sites, Sp1-1 and Sp1-2, located between 7119 and 7114 bp and between 7109 and 7104 bp of the p21 promoter, respectively. Sp1 and Sp3 binding to the p21 promoter increased during Caco-2 cell dierentiation, while the absolute level of Sp1 did not change and the absolute level of Sp3 increased approximately twofold. Transfection experiments in the SL2 Drosophila cell line that lacks endogenous Sp3 activity demonstrated that Sp1 transactivates the p21 promoter primarily through the Sp1-2 site, while Sp3 acts through the Sp1-1 site. In these cells Sp3 is a stronger transactivator of the p21 promoter than Sp1. Our data suggest that induction of p21 transcription during Caco-2 dierentiation is modulated by Sp1/Sp3 interactions with the p21 promoter. Oncogene (2000) 19, 5182 ± 5188.

Fibrosis is at the core of the high morbidity and mortality rates associated with the complications of diabetes and obesity, including diabetic nephropathy (DN), without any US Food and Drug Administration-approved drugs with this specific target. We recently provided the first evidence that the matricellular protein CCN3 (official symbol NOV) functions in a reciprocal manner, acting on the profibrotic family member CCN2 to inhibit fibrosis in a mesangial cell model of DN. Herein, we used the BT/BR ob/ob mouse as a best model of human obesity and DN progression to determine whether recombinant human CCN3 could be used therapeutically, and the mechanisms involved. Eight weeks of thrice-weekly i.p. injections (0.604 and 6.04 μg/kg of recombinant human CCN3) beginning in early-stage DN completely blocked and/or reversed the up-regulation of mRNA expression of kidney cortex fibrosis genes (CCN2, Col1a2, TGF-β1, and PAI-1) seen in placebo-treated diabetic mice. The treatment completely blocked glomerular fibrosis, as determined by altered mesangial expansion and deposition of laminin. Furthermore, it protected against, or reversed, podocyte loss and kidney function reduction (rise in plasma creatinine concentration); albuminuria was also greatly reduced. This study demonstrates the potential efficacy of recombinant human CCN3 treatment in DN and points to mechanisms operating at multiple levels or pathways, upstream (eg, protecting against cell injury) and downstream (eg, regulating CCN2 activity and extracellular matrix metabolism).

We recently reported that E2F1 could transactivate the p21 promoter via cis-acting elements between 7119 to +16 bp of the p21 gene. Here we show that activated V12-H-Ras can induce the p21 promoter through the same region of the p21 promoter by a p53-independent mechanism in NIH3T3 cells. In contrast, activated Ras was not able to induce the p21 promoter in E2F1 7/7 ®broblasts, suggesting that E2F1 is required for induction of the p21 promoter by activated Ras. Cotransfection of increasing concentrations of dominant negative E2F1 alone, or with dominant negative DP1 into NIH3T3 cells suppressed induction of the p21 promoter by activated Ras. These data suggest that p53-independent induction of the p21 promoter by activated Ras is mediated at least in part by E2F1. Oncogene (2000) 19, 961 ± 964.

Cooker LA, Peterson D, Rambow J, Riser ML, Riser RE, Najmabadi F, Brigstock D, Riser BL. TNF-␣, but not IFN-␥, regulates CCN2 (CTGF), collagen type I, and proliferation in mesangial cells: possible roles in the progression of renal fibrosis.