Activation of RXR and RAR signaling promotes myogenic differentiation of myoblastic C2C12 cells

Differentiation of embryonic and adult myogenic progenitors undergoes a complex series of cell rearrangements and specification events which are controlled by distinct gene regulatory networks. Delineation of the molecular mechanisms that regulate skeletal muscle specification and formation should be important for understanding congenital myopathies and muscular degenerative diseases. Retinoic acid (RA) signaling plays an important role in development. However, the role of RA signaling in adult myogenic progenitors is poorly understood. Here, we investigate the role of RA signaling in regulating myogenic differentiation of myoblastic progenitor cells. Using the mouse myoblast progenitor C2C12 line as a model, we have found that the endogenous expression of most RAR and RXR isotypes is readily detected. While the nuclear receptor co-repressors are highly expressed, two of the three nuclear receptor co-activators and the enzymes involved in RA synthesis are expressed at low level or undetectable, suggesting that the RA signaling pathway may be repressed in myogenic progenitors. Using the α-myosin heavy chain promoter-driven reporter (MyHC-GLuc), we have demonstrated that either ATRA or 9CRA is able to effectively induce myogenic differentiation, which can be synergistically enhanced when both ATRA and 9CRA are used. Upon ATRA and 9CRA treatment of C2C12 cells the expression of late myogenic markers significantly increases. We have further shown that adenovirus-mediated exogenous expression of RARα and/or RXRα is able to effectively induce myogenic differentiation in a ligand-independent fashion. Morphologically, ATRA- and 9CRA-treated C2C12 cells exhibit elongated cell body and become multi-nucleated myoblasts, and even form myoblast fusion. Ultrastructural analysis under transmission electron microscope reveals that RA-treated myogenic progenitor cells exhibit an abundant presence of muscle fibers. Therefore, our results strongly suggest that RA signaling may play an important role in regulating myogenic differentiation.     

Retinoic acid activates human inducible nitric oxide synthase gene through binding of RARalpha/RXRalpha heterodimer to a novel retinoic acid response element in the promoter

Human inducible nitric oxide synthase (hiNOS) catalyzes nitric oxide (NO) which has a significant effect on tumor suppression and cancer therapy. Here we revealed the detailed molecular mechanism involved in the regulation of hiNOS expression induced by retinoic acid (RA). We showed that RARalpha/RXRalpha heterodimer was important in hiNOS promoter activation, hiNOS protein expression, and NO production. Serial deletion and site-directed mutation analysis revealed two half-sites of retinoic acid response element (RARE) spaced by 5bp located at -172 to -156 in the hiNOS promoter. EMSA and ChIP assays demonstrated that RARalpha/RXRalpha directly bound to this RARE of hiNOS promoter. Our results suggested the identification of a novel RARE in the hiNOS promoter and the roles of the nuclear receptors (RARalpha/RXRalpha) in the induction of hiNOS by RA.     

Structure basis of bigelovin as a selective RXR agonist with a distinct binding mode

The nuclear receptor retinoid X receptor (RXR) functions potently in the regulation of homeostasis and cell development, while rexinoids as RXR agonists have proved their therapeutic potential in the treatment of metabolic diseases and cancer. Here, the natural product bigelovin was identified as a selective RXRα agonist. Interestingly, this compound could not transactivate RXRα:RXRα homodimer but could enhance the transactivation of RXRα:peroxisome proliferator-activated receptor γ heterodimer and repress that of RXRα:liver X receptor (LXR) α heterodimer, while it had no effects on RXRα:farnesoid X receptor heterodimer. Considering that the effective role of LXR response element involved transactivation of sterol regulatory element-binding protein-1c mediated by RXRα:LXRα in triglyceride elevation, such LXR response element repressing by bigelovin has obviously addressed its potency for further research. Moreover, our determined crystal structure of the bigelovin-activated RXRα ligand-binding domain with the coactivator human steroid receptor coactivator-1 peptide revealed that bigelovin adopted a distinct binding mode. Compared with the known RXR ligands, bigelovin lacks the acidic moiety in structure, which indicated that the acidic moiety rendered little effects on RXR activation. Our results have thereby provided new insights into the structure-based selective rexinoids design with bigelovin as a potential lead compound.     

Molecular determinants of the interactions between SRC-1 and LXR/RXR heterodimers

Liver X receptor (LXR)/retinoid X receptor (RXR) heterodimers have been shown to perform critical functions in cholesterol and lipid metabolism. Here, we have conducted a comparative analysis of the contributions of LXR and RXR binding to steroid receptor coactivator-1 (SRC-1), which contains three copies of the NR box. We demonstrated that the coactivator-binding surface of LXR, but not that of RXR, is critically important for physical and functional interactions with SRC-1, thereby confirming that RXR functions as an allosteric activator of SRC-1-LXR interaction. Notably, we identified NR box-2 and -3 as the essential binding targets for the SRC-1-induced stimulation of LXR transactivity, and observed the competitive in vitro binding of NR box-2 and -3 to LXR.

Structured summary

MINT-7986678, MINT-7986639, MINT-7986700, MINT-7986720, MINT-7986736, MINT-7986760, MINT-7986787: LXR (uniprotkb:Q13133) physically interacts (MI:0915) with SRC1 (uniprotkb:Q15788) and RXR (uniprotkb:P19793) by pull down (MI:0096)MINT-7986596, MINT-7986621: SRC1 (uniprotkb:Q15788) physically interacts (MI:0915) with LXR (uniprotkb:Q13133) by pull down (MI:0096)MINT-7986555, MINT-7986575: LXR (uniprotkb:Q13133) physically interacts (MI:0915) with SRC1 (uniprotkb:Q15788) by two hybrid (MI:0018)MINT-7986808, MINT-7986907, MINT-7986890: SRC1 (uniprotkb:Q15788) binds (MI:0407) to LXR (uniprotkb:Q13133) by pull down (MI:0096)MINT-7986822, MINT-7986848, MINT-7986865: SRC1 (uniprotkb:Q15788) binds (MI:0407) to RXR (uniprotkb:P19793) by pull down (MI:0096)     

Retinoids and their targets in vertebrate development

The elaboration of the effect of retinoic acid on limb morphogenesis has prompted renewed investigation into the teratology of retinoic acid treatment, with the hope that such analysis might give insight into mechanisms of vertebrate patterning. Retinoids, their nuclear receptors and their cytoplasmic binding proteins are now known to be deployed throughout development, but the extent to which they are natural agents of morphogenesis remains obscure. The study of retinoic acid receptors may offer molecular insight into gene regulation underlying vertebrate pattern formation.     

Molecular Determinants Required for Selective Interactions between the Thyroid Hormone Receptor Homodimer and the Nuclear Receptor Corepressor N-CoR

The unliganded nuclear receptor (NR) generally recruits the NR corepressor (N-CoR) and the silencing mediator of retinoid and thyroid hormone receptor via its direct binding to the extended helical motif within dual NR-interaction domains (IDs) of corepressors. Interestingly, N-CoR has a third ID (ID3) upstream of two IDs (ID1 and ID2) and its core motif (IDVII), rather than an extended helical motif, is known to be involved directly in the exclusive interaction of ID3 with the thyroid hormone receptor (TR). Here, we investigated the molecular determinants of the TR interaction with ID3 to understand the molecular basis of the N-CoR preference shown by the TR homodimer. Using a one- plus two-hybrid system, we identified the specific residues of N-CoR-ID2 and N-CoR-ID3 that are required for stable association of N-CoR with the TR homodimer. By swapping experiments and mutagenesis studies, we found that the C-terminally flanked residues of the core motif of ID3 contribute to the TR preference for N-CoR-ID3, suggesting that an extended three-turn helix might form within the ID3 via a C-terminal extension (IDVIITRQI) and participate directly in the TR-specific interaction. Structural modeling of the ID3 motif on TR-LBD is consistent with this conclusion. Notably, we identified a novel interaction between N-CoR-ID3 and orphan NR RevErb that is mediated by the residues crucial also in TR binding. These observations raise the intriguing possibility that NR homodimers such as TR and RevErb display preferential binding to the N-CoR corepressor via their specific interactions with ID3, which is normally absent from the silencing mediator of retinoid and thyroid hormone receptor.     

X-Ray Structures of the LXRα LBD in Its Homodimeric Form and Implications for Heterodimer Signaling

Liver X receptors (LXRs) are nuclear receptors that are central regulators of cholesterol homeostasis, and synthetic LXR agonists have shown promise as promoters of reverse cholesterol transport and anti-inflammatory agents. Here, we present three X-ray structures of three different agonists bound to the ligand binding domain of LXRα. These compounds are GW3965, F₃methylAA, and a benzisoxazole urea, and we show that these diverse chemical scaffolds address common structural themes, leading to high binding affinity for LXR. Our structures show the LXRα ligand binding domain in its homodimeric form, an arrangement previously thought to be stereochemically difficult. A comparison with existing structures of the LXRβ homodimer and LXRα:RXR (retinoid X receptor) heterodimers explains differences in dimer affinity and leads us to propose a model for allosteric activation in nuclear receptor dimers, in which an unactivated RXR partner provides an inhibitory tail wrap to the cofactor binding pocket of LXR.     

Retinoids synergized with insulin to induce Srebp-1c expression and activated its promoter via the two liver X receptor binding sites that mediate insulin action

We have reported that the rat liver lipophilic extract (LE) synergized with insulin to induce Gck and Srebp-1c in primary rat hepatocytes. After identification of retinol and retinal in LE, only their effects in the absence or presence of insulin on Gck, but not that on Srebp-1c, were investigated subsequently. The retinoid effects on the Srebp-1c expression and the activation of its promoter were examined with real-time PCR and reporter gene assays, respectively. In primary hepatocytes, retinal and retinoic acid (RA) synergized with insulin to induce Srebp-1c expression. This induction was followed by the elevation of its target gene, fatty acid synthase. Activation of retinoid X receptor, but not retinoic acid receptor, was responsible for the induction of Srebp-1c expression. RA, but not retinal, also induced Srebp-1c expression in a dose dependent manner in INS-1 cells. The RA responsive elements in Srebp-1c promoter were determined as previously identified two liver X receptor elements responsible for mediating insulin action. We conclude that retinoids regulate hepatic Srebp-1c expression through activation of retinoid X receptor. The RA- and insulin-induced Srebp-1c expression converged at the same sites in its promoter, indicating the roles of vitamin A in regulation of hepatic gene expression.     

Characterization of stress sensitivity and chaperone activity of Hsp105 in mammalian cells

Nuclear receptor and apoptosis inducer NGFI-B translocates out of the nucleus as a heterodimer with RXR in response to different apoptosis stimuli, and therefore represents a potential pharmacological target. We found that the cytosolic levels of NGFI-B and RXRα were increased in cultures of cerebellar granule neurons 2 h after treatment with glutamate (excitatory neurotransmitter in the brain, involved in stroke). To find a time-window for potential intervention the neurons were transfected with gfp-tagged expressor plasmids for NGFI-B and RXR. The default localization of NGFI-Bgfp and RXRgfp was nuclear, however, translocation out of the nucleus was observed 2–3 h after glutamate treatment. We therefore hypothesized that the time-window between treatment and translocation would allow late protection against neuronal death. The RXR ligand 9-cis retinoic acid was used to arrest NGFI-B and RXR in the nucleus. Addition of 9-cis retinoic acid 1 h after treatment with glutamate reduced the cytosolic translocation of NGFI-B and RXRα, the cytosolic translocation of NGFI-Bgfp observed in live neurons, as well as the neuronal death. However, the reduced translocation and the reduced cell death were not observed when 9-cis retinoic acid was added after 3 h. Thus, late protection from glutamate induced death by addition of 9-cis retinoic acid is possible in a time-window after apoptosis induction.     

Retinoid regulated association of transcriptional co-regulators and the polycomb group protein SUZ12 with the retinoic acid response elements of Hoxa1, RARbeta(2), and Cyp26A1 in F9 embryonal carcinoma cells

Hox gene expression is activated by all-trans retinoic acid (RA), through binding to retinoic acid receptor-retinoid X receptor (RAR-RXR) heterodimers bound at RA response elements (RAREs) of target genes. The RARs and RXRs each have three isotypes (alpha, beta, and gamma), which are encoded by distinct genes. Hox genes are also repressed by polycomb group proteins (PcG), though how these proteins are targeted is unclear. We used chromatin immunoprecipitation assays to investigate the association of RXRalpha, RARgamma, cofactors, and the PcG protein SUZ12 with the Hoxa1, RARbeta2, and Cyp26A1 RAREs in F9 embryonal carcinoma cells (teratocarcinoma stem cells) during RA treatment. We demonstrate that RARgamma and RXRalpha are associated with RAREs prior to and during RA treatment. pCIP, p300, and RNA polymerase II levels increased at target RAREs upon exposure to RA. Conversely, SUZ12 was found associated with all RAREs studied and these associations were attenuated by treatment with RA. Upon RA removal, SUZ12 re-associated with RAREs. H3ac, H3K4me2, and H3K27me3 marks were simultaneously detected at target loci, indicative of a bivalent domain chromatin structure. During RA mediated differentiation, H3K27me3 levels decreased at target RAREs whereas H3ac and H3K4me2 levels remained constant. These studies provide insight into the dynamics of association of co-regulators with RAREs and demonstrate a novel link between RA signaling and PcG repression.     

A new regulatory mechanism of NF-kappaB activation by I-kappaBbeta in cancer cells

Transglutaminase 2 (TGase 2) catalyzes covalent isopeptide bond formation between glutamine and lysine residues. Recently, we reported that TGase 2 activates nuclear factor-kappa B (NF-κB) by depleting inhibitor of NF-κBα (I-κBα) levels via polymer formation. Furthermore, TGase 2 expression synergistically increases NF-κB activity with canonical pathway. The major I-κB proteins such as I-κBα and I-κBβ resemble each other in both primary sequence and tertiary structure. However, I-κBβ does not degrade fully, while I-κBα degrades immediately in response to most stimuli. We found that I-κBβ does not contain any of the previously identified TGase 2 target sites. In this study, both an in vitro cross-linking assay and a TGase 2 transfection assay revealed that I-κBβ is independent from TGase 2-mediated polymerization. Furthermore, increased I-κBβ expression reversed NF-κB activation in cancer cells, compensating for the loss of I-κBα via TGase 2 polymerization.     

Transcriptional suppression of nephrin in podocytes by macrophages: roles of inflammatory cytokines and involvement of the PI3K/Akt pathway

Expression of nephrin, a crucial component of the glomerular slit diaphragm, is downregulated in patients with proteinuric glomerular diseases. Using conditionally immortalized reporter podocytes, we found that bystander macrophages as well as macrophage-derived cytokines IL-1beta and TNF-alpha markedly suppressed activity of the nephrin gene promoter in podocytes. The cytokine-initiated repression was reversible, observed on both basal and inducible expression, independent of Wilms' tumor suppressor WT1, and caused in part via activation of the phosphatidylinositol-3-kinase/Akt pathway. These results indicated a novel mechanism by which activated macrophages participate in the induction of proteinuria in glomerular diseases.