Effect of a peroxisome proliferator-activated receptor gamma sumoylation mutant on neointimal formation after balloon injury in rats.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor regulating inflammation, atherosclerosis, insulin sensitivity and adipogenesis. Recently, it has been discovered that modification by the small ubiquitin-like modifier (SUMO) plays an important role in PPARgamma activity. In the present study, we investigated the effect of sumoylation on the antiatherogenic property of PPARgamma. PPARgamma-K107R sumoylation mutant, PPARgamma-wild type (WT) and control genes were transfected on vascular smooth muscle cells (VSMCs) to compare their effect on the proliferation and migration. Adenoviral vectors expressing the PPARgamma-K107R, PPARgamma-WT or control gene were delivered into the carotid arteries of rats after balloon injury. The PPARgamma-K107R increased the transcriptional activity of peroxisome proliferator response element (PPRE) and had a more potent transcriptional repression activity on the inducible nitric oxide synthase (iNOS) promoter as compared to the other sumoylation mutants or WT. PPARgamma-K107R or WT gene transfer inhibited VSMCs proliferation and migration to a greater extent than the control. The PPARgamma-K107R had more potent activity than PPARgamma-WT in this regard. PPARgamma-K107R or WT transfer showed a significantly lower intima-media ratio (IMR) than the control after balloon injury in rats. Again, the delivery of the PPARgamma-K107R decreased IMR further compared to PPARgamma-WT. In addition, the PPARgamma-K107R transfer showed a lower proliferation index and a higher apoptotic index than PPARgamma-WT. In conclusion, the PPARgamma sumoylation mutant K107R strongly inhibited VSMCs proliferation and migration, sustained apoptosis, and reduced neointimal formation after balloon injury. These results indicate that desumoylation at K107 in PPARgamma might play an important role against atherosclerosis.

SUMO-specific protease SUSP4 positively regulates p53 by promoting Mdm2 self-ubiquitination.

The p53 tumour suppressor has a key role in the control of cell growth and differentiation, and in the maintenance of genome integrity. p53 is kept labile under normal conditions, but in response to stresses, such as DNA damage, it accumulates in the nucleus for induction of cell-cycle arrest, DNA repair or apoptosis. Mdm2 is an ubiquitin ligase that promotes p53 ubiquitination and degradation. Mdm2 is also self-ubiquitinated and degraded. Here, we identified a novel cascade for the increase in p53 level in response to DNA damage. A new SUMO-specific protease, SUSP4, removed SUMO-1 from Mdm2 and this desumoylation led to promotion of Mdm2 self-ubiquitination, resulting in p53 stabilization. Moreover, SUSP4 competed with p53 for binding to Mdm2, also resulting in p53 stabilization. Overexpression of SUSP4 inhibited cell growth, whereas knockdown of susp4 by RNA interference (RNAi) promoted of cell growth. UV damage induced SUSP4 expression, leading to an increase in p53 levels in parallel with a decrease in Mdm2 levels. These findings establish a new mechanism for the elevation of cellular p53 levels in response to UV damage.

Negative modulation of RXRalpha transcriptional activity by small ubiquitin-related modifier (SUMO) modification and its reversal by SUMO-specific protease SUSP1.

Retinoid X receptor alpha (RXRalpha) belongs to a family of ligand-activated transcription factors that regulate many aspects of metazoan life. Here we demonstrate that RXRalpha is a target substrate of a small ubiquitin-related modifier (SUMO)-specific protease, SUSP1, which is capable of controlling the transcriptional activity of RXRalpha. RXRalpha was modified by SUMO-1 in vivo as well as in vitro, and the Lys-108 residue within the IKPP sequence of RXRalpha AF-1 domain was identified as the major SUMO-1 acceptor site. Prevention of SUMO modification by Lys-to-Arg mutation led to an increase not only in the transcriptional activity of RXRalpha but also in the activity of its heterodimeric complex with retinoic acid receptor-alpha or peroxisome proliferator-activated receptor-gamma (PPARgamma). SUSP1 co-localized with RXRalpha in the nucleus and removed SUMO-1 from RXRalpha but not from androgen receptor or PPARgamma. Moreover, overexpression of SUSP1 caused an increase in the transcriptional activity of RXRalpha, whereas small hairpin RNA-mediated knockdown of endogenous SUSP1 led to a decrease in RXRalpha activity. These results suggest that SUSP1 plays an important role in the control of the transcriptional activity of RXRalpha and thus in the RXRalpha-mediated cellular processes.

Roles of sumoylation of a reptin chromatin-remodelling complex in cancer metastasis.

Defining the functional modules within transcriptional regulatory factors that govern switching between repression and activation events is a central issue in biology. Recently, we have reported the dynamic role of a beta-catenin-reptin chromatin remodelling complex in regulating a metastasis suppressor gene KAI1 (ref.1), which is capable of inhibiting the progression of tumour metastasis. Here, we identify signalling factors that confer repressive function on reptin and hence repress the expression of KAI1. Biochemical purification of a reptin-containing complex has revealed the presence of specific desumoylating enzymes that reverse the sumoylation of reptin that underlies its function as a repressor. Desumoylation of reptin alters the repressive function of reptin and its association with HDAC1. Furthermore, the sumoylation status of reptin modulates the invasive activity of cancer cells with metastatic potential. These data clearly define a functional model and provide a novel link for SUMO modification in cancer metastasis.

Sumoylation increases HIF-1alpha stability and its transcriptional activity.

HIF-1 is closely involved in various biological processes, including angiogenesis, energy metabolism, and cell survival. HIF-1 consists of an oxygen-sensitive HIF-1alpha and oxygen-insensitive HIF-1beta. Oxygen-sensitive HIF-1alpha is subjected to post-translational modifications such as hydroxylation, ubiquitination, and acetylation, which are related to the regulation of its stability. In this present study, we found that the ectopic expression of SUMO-1 increased HIF-1alpha stability by the co-transfection study with HIF-1alpha and SUMO-1. Furthermore, the ectopic expression of SUMO-1 enhanced the transcriptional activity of HIF-1alpha. In the subsequent immunoprecipitation assay, SUMO-1 was co-immunoprecipitated with HIF-1alpha, implying that HIF-1alpha is covalently modified by SUMO-1. Thereafter, using a series of lysine mutants in the ODD domain, we found that HIF-1alpha was sumoylated at Lys(391) and Lys(477), suggesting that sumoylation at these two lysine residues enhances HIF-1alpha stability by possibly modulating other post-translational modifications. Altogether, we demonstrate that HIF-1alpha is upregulated through SUMO-1 modification at Lys(391)/Lys(477) residues, which may stabilize HIF-1alpha and enhance its transcriptional activity.