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Nuclear receptors (NRs) regulate gene transcription by recruiting coregulators, involved in chromatin remodeling and assembly of the basal transcription machinery. The NR-associated protein ligand-dependent corepressor (LCoR) has previously been shown to suppress hepatic lipogenesis by decreasing the binding of steroid receptor coactivators to thyroid hormone receptor. However, the role of LCoR in adipogenesis has not been established. Here, we show that LCoR expression is reduced in the early stage of adipogenesis in vitro LCoR overexpression inhibited 3T3-L1 adipocyte differentiation, whereas LCoR knockdown promoted it. Using an unbiased affinity purification approach, we identified CCAAT/enhancer-binding protein β (C/EBPβ), a key transcriptional regulator in early adipogenesis, and corepressor C-terminal binding proteins as potential components of an LCoR-containing complex in 3T3-L1 adipocytes. We found that LCoR directly interacts with C/EBPβ through its C-terminal helix-turn-helix domain, required for LCoR's inhibitory effects on adipogenesis. LCoR overexpression also inhibited C/EBPβ transcriptional activity, leading to inhibition of mitotic clonal expansion and transcriptional repression of C / EBP α and peroxisome proliferator-activated receptor γ2 ( PPAR γ 2 ). However, LCoR overexpression did not affect the recruitment of C/EBPβ to the promoters of C / EBP α and PPAR γ 2 in 3T3-L1 adipocytes. Of note, restoration of PPARγ2 or C/EBPα expression attenuated the inhibitory effect of LCoR on adipogenesis. Mechanistically, LCoR suppressed C/EBPβ-mediated transcription by recruiting C-terminal binding proteins to the C / EBP α and PPAR γ 2 promoters and by modulating histone modifications. Taken together, our results indicate that LCoR negatively regulates early adipogenesis by repressing C/EBPβ transcriptional activity and add LCoR to the growing list of transcriptional corepressors of adipogenesis., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

The expression of 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), which acts as a placental glucocorticoid barrier, is silenced in cytotrophoblasts but substantially up-regulated during syncytialization. However, the repressive mechanism of 11β-HSD2 expression before syncytialization and how this repression is lifted during syncytialization remain mostly unresolved. Here we found that enhancer of zeste homolog 2 (EZH2) accounts for the silence of 11β-HSD2 expression via trimethylation of histone H3 lysine 27 at the promoter of the 11β-HSD2 gene. Further studies revealed that, upon syncytialization, human chorionic gonadotropin reduced the phosphorylation of retinoblastoma protein (pRB) via activation of the cAMP/PKA pathway, which sequesters E2F transcription factor 1 (E2F1), the transcription factor for EZH2 expression. As a result of inactivation of the pRB-E2F1-EZH2 pathway, the repressive marker trimethylation of histone H3 lysine 27 at the 11β-HSD2 promoter is removed, which leads to the robust expression of 11β-HSD2 during syncytialization., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Liver X receptors (LXRs) are nuclear receptors that function to modulate lipid metabolism as well as immune and inflammatory responses. Upon activation by their ligands, LXRs up-regulate a spectrum of gene transcription programs involved in cholesterol and fatty acid homeostasis. However, the mechanisms by which LXR-mediated transcriptional activation is regulated remain incompletely understood. Here, we show that PIAS1, a member of the protein inhibitor of the activated STAT family of proteins with small ubiquitin-like modifier (SUMO) E3 ligase activity, acts to suppress LXR ligand-dependent transcriptional activation of the lipogenic program in hepatocytes. We found that liver mRNA expression levels of Pias1 and Pias3 were inversely associated with those of genes involved in lipogenesis in mouse models with diet-induced or genetic obesity. Overexpression of PIAS1 in primary hepatocytes resulted in a reduction of LXR ligand-induced fatty acid synthesis and suppression of the expression of lipogenic genes, including Srebp1c and Fas. Moreover, PIAS1 was able to interact with LXRβ and repress its transcriptional activity upon ligand stimulation, which did not require PIAS1-promoted SUMO modification of LXRβ. In addition, PIAS1 could also interact with PGC-1β and attenuate its association with LXRβ, blunting the ability of PGC-1β to co-activate LXRβ. Importantly, PIAS1 impaired LXRβ binding to its target DNA sequence. Taken together, our results suggest that PIAS1 may serve as a lipogenic regulator by negatively modulating LXRs in a SUMOylation-independent manner.

Optineurin is a gene linked to amyotrophic lateral sclerosis, Paget disease of bone, and glaucoma, a major blinding disease. Mutations such as E50K were identified in glaucoma patients. We investigated herein the involvement of ubiquitin-proteasome pathway (UPP) and autophagy, two major routes for protein clearance, in processing of optineurin in a retinal ganglion cell model line RGC5 and neuronal PC12 cells. It was found that the endogenous optineurin level in neuronal cells was increased by treatment of proteasomal inhibitor but not by autophagic and lysosomal inhibitors. Multiple bands immunoreactive to anti-ubiquitin were seen in the optineurin pulldown, indicating that optineurin was ubiquitinated. In cells overexpressing wild type and E50K optineurin, the level of the proteasome regulatory β5 subunit (PSMB5, indicative of proteasome activity) was reduced, whereas that for autophagy marker microtubule-associated protein 1 light chain 3 was enhanced compared with controls. Autophagosome formation was detected by electron microscopy. The foci formed after optineurin transfection were increased upon treatment of an autophagic inhibitor but were decreased by treatment of an inducer, rapamycin. Moreover, the level of optineurin-triggered apoptosis was reduced by rapamycin. This study thus provides compelling evidence that in a normal homeostatic situation, the turnover of endogenous optineurin involves mainly UPP. When optineurin is up-regulated or mutated, the UPP function is compromised, and autophagy comes into play. A decreased PSMB5 level and an induced autophagy were also demonstrated in vivo in retinal ganglion cells of E50K transgenic mice, validating and making relevant the in vitro findings.

The human colorectal epithelium is maintained by multipotent stem cells that give rise to absorptive, mucous, and endocrine lineages. Recent evidence suggests that human colorectal cancers are likewise maintained by a minority population of so-called cancer stem cells. We have previously established a human colorectal cancer cell line with multipotent characteristics (HRA-19) and developed a serum-free medium that induces endocrine, mucous and absorptive lineage commitment by HRA-19 cells in vitro. In this study, we investigate the role of the beta1 integrin family of cell surface extracellular matrix receptors in multilineage differentiation by these multipotent human colorectal cancer cells. We show that endocrine and mucous lineage commitment is blocked in the presence of function-blocking antibodies to beta1 integrin. Function-blocking antibodies to alpha2 integrin also blocked both HRA-19 endocrine lineage commitment and enterocytic differentiation by Caco-2 human colon cancer cells; both effects being abrogated by the MEK inhibitor, PD98059, suggesting a role for ERK signaling in alpha2-mediated regulation of colorectal cancer cell differentiation. To further explore the role of alpha2 integrin in multilineage differentiation, we established multipotent cells expressing high levels of wild-type alpha2 integrin or a non-signaling chimeric alpha2 integrin. Overexpression of wild-type alpha2 integrin in HRA-19 cells significantly enhanced endocrine and mucous lineage commitment, while cells expressing the non-signaling chimeric alpha2 integrin had negligible ability for either endocrine or mucous lineage commitment. This study indicates that the collagen receptor alpha2beta1 integrin is a regulator of cell fate in human multipotent colorectal cancer cells.

Retinoblastoma (Rb) protein is a paradigm of tumor suppressors. Inactivation of Rb plays a critical role in the development of human malignancies. MDM2, an oncogene frequently found amplified and overexpressed in a variety of human tumors and cancers, directly interacts and inhibits the p53 tumor suppressor protein. In addition, MDM2 has been shown to stimulate E2F transactivation activity and promote S-phase entry independent of p53, yet the mechanism of which is still not fully understood. In this study, we demonstrate that MDM2 specifically binds to Rb C-pocket and that the central acidic domain of MDM2 is essential for Rb interaction. In addition, we show that overexpression of MDM2 reduces Rb-E2F complexes in vivo. Moreover, the ectopic expression of the wild type MDM2, but not mutant MDM2 defective in Rb interaction, stimulates E2F transactivation activity and inhibits Rb growth suppression function. Taken together, these results suggest that MDM2-mediated inhibition of Rb likely contributes to MDM2 oncogenic activity.