GPS2-dependent corepressor/SUMO pathways govern anti-inflammatory actions of LRH-1 and LXRbeta in the hepatic acute phase response.

The orphan receptor LRH-1 and the oxysterol receptors LXRalpha and LXRbeta are established transcriptional regulators of lipid metabolism that appear to control inflammatory processes. Here, we investigate the anti-inflammatory actions of these nuclear receptors in the hepatic acute phase response (APR). We report that selective synthetic agonists induce SUMOylation-dependent recruitment of either LRH-1 or LXR to hepatic APR promoters and prevent the clearance of the N-CoR corepressor complex upon cytokine stimulation. Investigations of the APR in vivo, using LXR knockout mice, indicate that the anti-inflammatory actions of LXR agonists are triggered selectively by the LXRbeta subtype. We further find that hepatic APR responses in small ubiquitin-like modifier-1 (SUMO-1) knockout mice are increased, which is due in part to diminished LRH-1 action at APR promoters. Finally, we provide evidence that the metabolically important coregulator GPS2 functions as a hitherto unrecognized transrepression mediator of interactions between SUMOylated nuclear receptors and the N-CoR corepressor complex. Our study extends the knowledge of anti-inflammatory mechanisms and pathways directed by metabolic nuclear receptor-corepressor networks to the control of the hepatic APR, and implies alternative pharmacological strategies for the treatment of human metabolic diseases associated with inflammation.

SUMO-1 regulates body weight and adipogenesis via PPARγ in male and female mice.

Properly functioning adipose tissue is essential for normal insulin sensitivity of the body. When mice are kept on high-fat diet (HFD), adipose tissue expands, adipocytes increase in size and number, and the mice become obese. Many of these changes are mediated by the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ), the activity of which is regulated by multiple posttranslational modifications, including SUMOylation. To address the role of small ubiquitin-like modifier-1 (SUMO-1) in PPARγ function in vivo, particularly in fat cell biology, we subjected Sumo1-knockout mice to HFD. Sumo1-null mice gained less weight and had smaller and fewer adipocytes in their gonadal fat tissue on HFD, but their glucose tolerance was similar to that of wild-type littermates. Adipogenesis was impaired in Sumo1-null cells, and expression of PPARγ target genes was attenuated. In addition, both Sumo1-null cells and Sumo1-null mice responded less efficiently to rosiglitazone, a PPARγ agonist. These findings indicate that SUMO-1 is important also for transcriptional activation by the PPARγ signaling pathway and not only for trans-repressive functions of PPARγ as previously reported.

The phytoestrogen genistein is a tissue-specific androgen receptor modulator.

To enable studies of androgen signaling in different tissues in vivo, we generated an androgen receptor (AR) reporter mouse line by inserting a luciferase gene construct into the murine genome. The construct is driven by four copies of androgen-responsive elements from the mouse sex-limited protein gene (slp-HRE2) and a minimal thymidine kinase promoter. Luciferase activity was readily measurable in a number of murine tissues, including prostate, lung, testis, brain, and skeletal muscle, and testosterone administration elicited a significant increase in reporter gene activity in these tissues. Consumption of isoflavonoid genistein is linked to reduced risk of prostate cancer, but direct effects of genistein on the AR pathway are not well understood. To examine androgen-modulating activity of genistein in vivo, male mice received daily doses of genistein (10 mg/kg) for 5 d. In intact males, genistein was antiandrogenic in testis, prostate, and brain, and it attenuated reporter gene activity by 50-80%. In castrated males, genistein exhibited significant androgen agonistic activity in prostate and brain by increasing reporter gene activity over 2-fold in both tissues. No antiandrogenic action was seen in lung or skeletal muscle of intact males. Gene expression profiling of the murine prostate under the same experimental conditions revealed that genistein modulates androgen-dependent transcription program in prostate in a fashion similar to that observed in reporter mice by luciferase expression. In conclusion, genistein is a partial androgen agonist/antagonist in some but not in all mouse tissues and should be considered as a tissue-specific AR modulator.

Androgen receptor and androgen-dependent gene expression in lung.

The androgen receptor (AR) mediates the effects of male sex steroids. There are major sex differences in lung development and pathologies, including lung cancer. In this report, we show that Ar is mainly expressed in type II pneumocytes and the bronchial epithelium of murine lung and that androgen treatment increases AR protein levels in lung cells. Androgen administration altered significantly murine lung gene expression profiles; for example, by up-regulating transcripts involved in oxygen transport and down-regulating those in DNA repair and DNA recombination. Androgen exposure also affected the gene expression profile in a human lung adenocarcinoma-derived cell line, A549, by up- or down-regulating significantly some 200 transcripts, including down-regulation of genes involved in cell respiration. Dexamethasone treatment of A549 cells augmented expression of transcript sets that overlapped in part with those up-regulated by androgen in these cells. Moreover, a human lung cancer tissue array revealed that different lung cancer types are all AR-positive. Our results indicate that adult lung is an AR target tissue and suggest that AR plays a role in lung cancer biology.

Sumo-1 function is dispensable in normal mouse development.

To elucidate SUMO-1 functions in vivo, we targeted by homologous recombination the last three exons of the murine Sumo-1 gene. Sumo-1 mRNA abundance was reduced to one-half in heterozygotes and was undetectable in Sumo-1(-/-) mice, and SUMO-1-conjugated RanGAP1 was detectable in wild-type mouse embryo fibroblasts (MEFs) but not in Sumo-1(-/-) MEFs, indicating that gene targeting yielded Sumo-1-null mice. Sumo-1 mRNA is expressed in all tissues of wild-type mice, and its abundance is highest in the testis, brain, lungs, and spleen. Sumo-2 and Sumo-3 mRNAs are also expressed in all tissues, but their abundance was not upregulated in Sumo-1-null mice. The development and function of testis are normal in the absence of Sumo-1, and Sumo-1(-)(/)(-) mice of both sexes are viable and fertile. In contrast to a previous report (F. S. Alkuraya et al., Science 313:1751, 2006), we did not observe embryonic or early postnatal demise of Sumo-1-targeted mice; genotypes of embryos and 21-day-old mice were of predicted Mendelian ratios, and there was no defect in lip and palate development in Sumo-1(+/-) or Sumo-1(-/-) embryos. The ability of Sumo-1(-/-) MEFs to differentiate into adipocyte was not different from that of wild-type MEFs. Collectively, our results support the notion that most, if not all, SUMO-1 functions are compensated for in vivo by SUMO-2 and SUMO-3.

Identification of a short PIASx gene promoter that directs male germ cell-specific transcription in vivo.

PIASx gene encodes two SUMO E3 ligases that are highly expressed in the testis. We have isolated and analyzed the promoter of the murine PIASx gene. Electrophoretic mobility shift assays with testicular nuclear extracts showed that the proximal promoter forms a major DNA-protein complex containing Sp1, Sp2, and Sp3 transcription factors. Reporter gene assays in cultured cells indicated that a fragment comprising nucleotides from -168 to +76 relative to transcription start site is sufficient for basal promoter activity in cultured cells, but these in vitro assays failed to reveal clear differences in promoter activity between testis- and non-testis-derived cell lines. Interestingly, however, the proximal promoter encompasses the elements necessary for a testis-specific transcription in vivo, as it directed beta-galactosidase expression exclusively to male germ cells in transgenic mice. In conclusion, we have characterized the minimal PIASx promoter that can be used for highly specific targeting of transgene expression to male germ cells.