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A novel mechanism by which thiazolidinediones facilitate the proteasomal degradation of cyclin D1 in cancer cells.

ABSTRACT: This study identifies a novel mechanism by which thiazolidinediones mediate cyclin D1 repression in prostate cancer cells. Based on the finding that the thiazolidinedione family of peroxisome proliferator-activated receptor gamma (PPARgamma) agonists mediated PPARgamma-independent cyclin D1 degradation, we developed a novel PPARgamma-inactive troglitazone derivative, STG28, with high potency in cyclin D1 ablation. STG28-mediated cyclin D1 degradation was preceded by Thr-286 phosphorylation and nuclear export, which however, were independent of glycogen synthase kinase 3beta. Mutational analysis further confirmed the pivotal role of Thr-286 phosphorylation in STG28-induced nuclear export and proteolysis. Of several kinases examined, inhibition of IkappaB kinase alpha blocked STG28-mediated cytoplasmic sequestration and degradation of cyclin D1. Pulldown of ectopically expressed Cul1, the scaffold protein of the Skp-Cullin-F-box E3 ligase, in STG28-treated cells revealed an increased association of cyclin D1 with beta-TrCP, whereas no specific binding was noted with other F-box proteins examined, including Skp2, Fbw7, Fbx4, and Fbxw8. This finding represents the first evidence that cyclin D1 is targeted by beta-TrCP. Moreover, beta-TrCP expression was up-regulated in response to STG28, and ectopic expression and small interfering RNA-mediated knock-down of beta-TrCP enhanced and protected against STG28-facilitated cyclin D1 degradation, respectively. Because cyclin D1 lacks the DSG destruction motif, mutational and modeling analyses indicate that cyclin D1 was targeted by beta-TrCP through an unconventional recognition site, (279)EEVDLACpT(286), reminiscent to that of Wee1. Moreover, we obtained evidence that this beta-TrCP-dependent degradation takes part in controlling cyclin D1 turnover when cancer cells undergo glucose starvation, which endows physiological relevance to this novel mechanism.

SUBMITTER: Wei S

PROVIDER: S-EPMC2546561 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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A novel mechanism by which thiazolidinediones facilitate the proteasomal degradation of cyclin D1 in cancer cells.

Wei Shuo S Yang Hsiao-Ching HC Chuang Hsiao-Ching HC Yang Jian J Kulp Samuel K SK Lu Pei-Jung PJ Lai Ming-Derg MD Chen Ching-Shih CS

The Journal of biological chemistry 20080723 39

This study identifies a novel mechanism by which thiazolidinediones mediate cyclin D1 repression in prostate cancer cells. Based on the finding that the thiazolidinedione family of peroxisome proliferator-activated receptor gamma (PPARgamma) agonists mediated PPARgamma-independent cyclin D1 degradation, we developed a novel PPARgamma-inactive troglitazone derivative, STG28, with high potency in cyclin D1 ablation. STG28-mediated cyclin D1 degradation was preceded by Thr-286 phosphorylation and n ...[more]

PMID: 18650423

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Project description:In chondrocytes, PTHrP maintains them in a proliferative state and prevents premature hypertrophy. The mechanism by which PTHrP does this is not fully understood. Both Runx2 and Runx3 are required for chondrocyte maturation. We recently demonstrated that cyclin D1 induces Runx2 protein phosphorylation and degradation. In the present studies, we tested the hypothesis that PTHrP regulates both Runx2 and Runx3 protein stability through cyclin D1. We analyzed the effects of cyclin D1 on Runx3 protein stability and function using COS cells, osteoprogenitor C3H10T1/2 cells and chondrogenic RCJ3.1C5.18 cells. We found that cyclin D1 induced Runx3 degradation in a dose-dependent manner and that both Myc-tagged Runx3 and endogenous Runx3 interact directly with CDK4 in COS and RCJ3.1C5.18 cells. A conserved CDK recognition site was identified in the C-terminal region of Runx3 by sequence analysis (residues 356-359). Pulse-chase experiments showed that the mutation of Runx3 at Ser356 to alanine (SA-Runx3) increased the half-life of Runx3. By contrast, the mutation at the same serine residue to glutamic acid (SE-Runx3) accelerated Runx3 degradation. In addition, SA-Runx3 was resistant to cyclin D1-induced degradation. GST-Runx3 was strongly phosphorylated by CDK4 in vitro. By contrast, CDK4 had no effect on the phosphorylation of SA-Runx3. Although both wild-type and SE-Runx3 were ubiquitylated, this was not the case for SA-Runx3. Runx3 degradation by cyclin D1 was completely blocked by the proteasome inhibitor PS1. In C3H10T1/2 cells, SA-Runx3 had a greater effect on reporter activity than SE-Runx3. The same was true for ALP activity in these cells. To investigate the role of cyclin D1 in chondrocyte proliferation and hypertrophy, we analyzed the growth plate morphology and expression of chondrocyte differentiation marker genes in Ccnd1-knockout mice. The proliferating and hypertrophic zones were significantly reduced and expression of chondrocyte differentiation marker genes and ALP activity were enhanced in 2-week-old Ccnd1-knockout mice. PTHrP significantly suppressed protein levels of both Runx2 and Runx3 in primary chondrocytes derived from wild-type mice. By contrast, the suppressive effect of PTHrP on Runx2 and Runx3 protein levels was completely abolished in primary chondrocytes derived from Ccnd1-knockout mice. Our findings demonstrate that the cell cycle proteins cyclin D1 and CDK4 induce Runx2 and Runx3 phosphorylation, ubiquitylation and proteasomal degradation. PTHrP suppresses Runx2 and Runx3 protein levels in chondrocytes through cyclin D1. These results suggest that PTHrP might prevent premature hypertrophy in chondrocytes, at least in part by inducing degradation of Runx2 and Runx3 in a cyclin-D1-dependent manner.

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A novel mechanism by which thiazolidinediones facilitate the proteasomal degradation of cyclin D1 in cancer cells.

Publications

A novel mechanism by which thiazolidinediones facilitate the proteasomal degradation of cyclin D1 in cancer cells.

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