14-3-3ζ Suppresses RANKL Signaling by Destabilizing TRAF6.

Macrophages are essential regulators of inflammation and bone loss. RANKL, a pro-inflammatory cytokine, is responsible for macrophage differentiation to osteoclasts and bone loss. We recently showed that 14-3-3ζ-knockout (YwhazKO) rats exhibit increased bone loss in the inflammatory arthritis model. 14-3-3ζ is a cytosolic adaptor protein that actively participates in many signaling transductions. However, the role of 14-3-3ζ in RANKL signaling or bone remodeling is unknown. We investigated how 14-3-3ζ affects osteoclast activity by evaluating its role in RANKL signaling. We utilized 14-3-3ζ-deficient primary bone marrow-derived macrophages (BMDMs) obtained from wildtype (Wt) and YwhazKO animals, and RAW cells generated using CRISPR-Cas9. Our results showed that 14-3-3ζ-deficient macrophages, upon RANKL stimulation, have bigger and stronger TRAP-positive multinucleated cells and increased bone resorption activity. The presence of 14-3-3ζ suppressed RANKL-induced MAPK and AKT phosphorylation, transcription factors (NFATC1 and p65) nuclear translocation, and subsequently, gene induction (Rank, Acp5, and Ctsk). Mechanistically, 14-3-3ζ interacts with TRAF6, an essential component of the RANKL receptor complex. Upon RANKL stimulation, 14-3-3ζ-TRAF6 interaction was increased, while RANK-TRAF6 interaction was decreased. Importantly, 14-3-3ζ supported TRAF6 ubiquitination and degradation by the proteasomal pathway, thus dampening the downstream RANKL signaling. Together, we show that 14-3-3ζ regulates TRAF6 levels to suppress inflammatory RANKL signaling and osteoclast activity. To the best of our knowledge, this is the first report on 14-3-3ζ regulation of RANKL signaling and osteoclast activation.

PPARG Post-translational Modifications Regulate Bone Formation and Bone Resorption.

The peroxisome proliferator-activated receptor gamma (PPARγ) regulates osteoblast and osteoclast differentiation, and is the molecular target of thiazolidinediones (TZDs), insulin sensitizers that enhance glucose utilization and adipocyte differentiation. However, clinical use of TZDs has been limited by side effects including a higher risk of fractures and bone loss. Here we demonstrate that the same post-translational modifications at S112 and S273, which influence PPARγ pro-adipocytic and insulin sensitizing activities, also determine PPARγ osteoblastic (pS112) and osteoclastic (pS273) activities. Treatment of either hyperglycemic or normoglycemic animals with SR10171, an inverse agonist that blocks pS273 but not pS112, increased trabecular and cortical bone while normalizing metabolic parameters. Additionally, SR10171 treatment modulated osteocyte, osteoblast, and osteoclast activities, and decreased marrow adiposity. These data demonstrate that regulation of bone mass and energy metabolism shares similar mechanisms suggesting that one pharmacologic agent could be developed to treat both diabetes and metabolic bone disease.

High bone mass in adult mice with diet-induced obesity results from a combination of initial increase in bone mass followed by attenuation in bone formation; implications for high bone mass and decreased bone quality in obesity.

Obesity is generally recognized as a condition which positively influences bone mass and bone mineral density (BMD). Positive effect of high body mass index (BMI) on bone has been recognized as a result of increased mechanical loading exerted on the skeleton. However, epidemiologic studies indicate that obesity is associated with increased incidence of fractures. The results presented here offer a new perspective regarding the mechanisms which may be responsible for the increase of bone mass and concurrent decrease in bone quality. Two groups of 12 week old C57BL/6 males were fed either high fat diet (HFD) or regular diet (RD) for 11 weeks. Metabolic profile, bone parameters and gene expression were assessed in these groups at the end of the experiment. Additionally, bone status was evaluated in a third group of 12 week old animals corresponding to animals at the start of the feeding period. Administration of HFD resulted in development of a diet-induced obesity (DIO), glucose intolerance, alteration in energy metabolism, and impairment in WAT function, as compared to the age-matched control animals fed RD. The expression of adiponectin, FABP4/aP2, DIO2 and FoxC2 were decreased in WAT of DIO animals, as well as transcript levels for IGFBP2, the cytokine regulating both energy metabolism and bone mass. At the end of experiment, DIO mice had higher bone mass than both control groups on RD, however they had decreased bone formation, as assessed by calcein labeling, and increased marrow adipocyte content. This study suggests that the bone mass acquired in obesity is a result of a two-phase process. First phase would consist of either beneficial effect of fat expansion to increase bone mass by increased mechanical loading and/or increased production of bone anabolic adipokines and/or nutritional effect of fatty acids. This is followed by a second phase characterized by decreased bone formation and bone turnover resulting from development of metabolic impairment.

Bone marrow fat has brown adipose tissue characteristics, which are attenuated with aging and diabetes.

Fat occupies a significant portion of bone cavity however its function is largely unknown. Marrow fat expands during aging and in conditions which affect energy metabolism, indicating that fat in bone is under similar regulatory mechanisms as other fat depots. On the other hand, its location may determine specific functions in the maintenance of the environment for bone remodeling and hematopoiesis. We have demonstrated that marrow fat has a distinctive phenotype, which resembles both, white and brown adipose tissue (WAT and BAT, respectively). Marrow adipocytes express gene markers of brown adipocytes at levels characteristic for the BAT, including transcription factor Prdm16, and regulators of thermogenesis such as deiodinase 2 (Dio2) and PGC1α. The levels of expression of BAT-specific gene markers are decreased in bone of 24 mo old C57BL/6 and in diabetic yellow agouti A(vy)/a mice implicating functional changes of marrow fat occurring with aging and diabetes. Administration of antidiabetic TZD rosiglitazone, which sensitizes cells to insulin and increases adipocyte metabolic functions, significantly increased both, BAT (UCP1, PGC1α, Dio2, ß3AR, Prdm16, and FoxC2) and WAT (adiponectin and leptin) gene expression in marrow of normoglycemic C57BL/6 mice, but failed to increase the expression of BAT, but not WAT, gene markers in diabetic mice. In conclusion, the metabolic phenotype of marrow fat combines both BAT and WAT characteristics. Decrease in BAT-like characteristics with aging and diabetes may contribute to the negative changes in the marrow environment supporting bone remodeling and hematopoiesis.

Human UDP-glucuronosyltransferase 2B7.

UDP-Glucuronosyltransferases (UGTs) are glycoproteins, localized in endoplasmic reticulum (ER) and nuclear membranes, which catalyze the confugation of a broad variety of lipophilic aglycon substrates with glucuronic acid using UDP-glucuronic acid (UDP-GlcUA) as the sugar donor. The major function of glucuronidation is to change hydrophobic compounds into hydrophilic derivatives, a process which facilitates their detoxification and excretion. However, it is also widely recognized that glucuronidation can result in compounds which are biologically active or demonstrate increased toxicity. UGTs, like other drug-metabolizing enzymes, have been postulated to be involved in controlling the steady state concentrations of nuclear receptor ligands for interactions with nuclear receptors [1,2]. One of the isoforms from the UGT2B subfamily, UGT2B7, has been found to be a major human UGT2B isoform, involved in the glucuronidation of a variety of endogenous compounds and xenobiotics. In this review, we included all available information from our studies and those of other investigators on a) the history of the identification and expression of UGT2B7 in human tissues, b) the substrate specificity of UGT2B7, c) the extrahepatic localization of UGT2B7 d) the nuclear localization of UGT2B7 and e) characterization of the UGT2B7 gene and promoter.

Glucuronidation of linoleic acid diols by human microsomal and recombinant UDP-glucuronosyltransferases: identification of UGT2B7 as the major isoform involved.

Recent reports suggest that linoleic acid (LA) epoxides and diols are associated with important physiological, pharmacological, and pathological events in vivo. We have shown recently that LA-diols are excellent substrates for human liver microsomal UDP-glucuronosyltransferases (UGTs); however, it is not known if other human tissues glucuronidate LA-diols or which UGT isozyme(s) is involved. The present studies with human intestinal microsomes indicate that glucuronidation of LA-diols occurs throughout the gastrointestinal tract, with the highest activity in the small intestine. LA-diols yielded exclusively hydroxyl-linked glucuronides, whereas LA yielded the carboxyl-linked glucuronide. Studies with human recombinant UGTs demonstrated that only UGT2B7 glucuronidated LA and LA-diols. Kinetic analysis with UGT2B7 yielded apparent K(m) values in the range of 40-70 microM and V(max) values from 4.5 to 5.4 nmol/mg x min. These studies indicate that LA and LA-diols are excellent substrates for intestinal UGTs and provide the first evidence for UGT2B7 being the major isoform involved.

Application of photoaffinity labeling with [(3)H] all trans- and 9-cis-retinoic acids for characterization of cellular retinoic acid--binding proteins I and II.

Cellular retinoic acid-binding proteins (CRABPs) are carrier proteins thought to play a crucial role in the transport and metabolism of all-trans-retinoic acid (atRA) and its derivatives within the cell. This report describes a novel photoaffinity-based binding assay involving competition between potential ligands of CRABP and [(3)H]atRA or [(3)H]-9-cis-RA for binding to the atRA-binding sites of CRABP I and II. Photoaffinity labeling of purified CRABPs with [(3)H]atRA was light- and concentration-dependent, saturable, and protected by several retinoids in a concentration-dependent manner, indicating that binding occurred in the CRABP atRA-binding site. Structure-function relationship studies demonstrated that oxidative changes to the atRA beta-ionone ring did not affect ligand potency. However, derivatives lacking a terminal carboxyl group and some cis isomers did not bind to CRABPs. These studies also identified two novel ligands for CRABPs: 5,6-epoxy-RA and retinoyl-beta-D-glucuronide (RAG). The labeling of both CRABPs with 9-cis-RA occurred with much lower affinity. Experimental evidence excluded nonspecific binding of RAG to CRABPs and UDP-glucuronosyltransferases, the enzymes responsible for RAG synthesis. These results established that RAG is an effective ligand of CRABPs. Therefore, photoaffinity labeling with [(3)H]atRA can be used to identify new ligands for CRABP and retinoid nuclear receptors and also provide information concerning the identity of amino acid(s) localized in the atRA-binding site of these proteins.

Glucuronidation of estrogens and retinoic acid and expression of UDP-glucuronosyltransferase 2B7 in human intestinal mucosa.

We have recently shown that, in human intestine, glucuronidation of androsterone and testosterone was on the nanomolar level and increased from proximal to distal intestine. In the present study, we have characterized estrogen UDP-glucuronosyltransferase activity in microsomes from intestine of seven human subjects. Intestinal microsomes from all segments of intestine from both males and females (except for one male) glucuronidated estrone (0.2-2.6 nmol/mg x min) and estradiol (0.5-3.1 nmol/mg x min) at levels 2 to 15 times higher than found with human liver microsomes (0.04-0.1 and 0.16-0.25 nmol/mg x min, for estrone and estradiol, respectively). Only with estriol were there significant hepatic glucuronidation (2. 2-4.5 nmol/mg x min) and intestinal glucuronidation activities (0.2-2.2 nmol/mg x min) that were lower than those in liver. All-trans-retinoic acid was glucuronidated by all segments of intestine from both sexes at levels 50 to 80% of those found with human liver but quite low compared with estrogen glucuronidation. In the two subjects for whom stomach was available, there was no measurable activity in stomach microsomes toward any of the substrates. UGT2B RNA expression was examined in mucosa from stomach to colon from two subjects. There was significant expression of UGT2B7, but not of UGT2B4 or UGT2B15, in all segments of intestine. To our knowledge, this is the first direct demonstration of glucuronidation of estrogens by human intestinal microsomes. Thus, in humans, the intestine may be considered as part of the overall mechanism of detoxification via glucuronidation.

Direct interaction of all-trans-retinoic acid with protein kinase C (PKC). Implications for PKC signaling and cancer therapy.

Protein kinase C (PKC) regulates fundamental cellular functions including proliferation, differentiation, tumorigenesis, and apoptosis. All-trans-retinoic acid (atRA) modulates PKC activity, but the mechanism of this regulation is unknown. Amino acid alignments and crystal structure analysis of retinoic acid (RA)-binding proteins revealed a putative atRA-binding motif in PKC, suggesting existence of an atRA binding site on the PKC molecule. This was supported by photolabeling studies showing concentration- and UV-dependent photoincorporation of [(3)H]atRA into PKCalpha, which was effectively protected by 4-OH-atRA, 9-cis-RA, and atRA glucuronide, but not by retinol. Photoaffinity labeling demonstrated strong competition between atRA and phosphatidylserine (PS) for binding to PKCalpha, a slight competition with phorbol-12-myristate-13-acetate, and none with diacylglycerol, fatty acids, or Ca(2+). At pharmacological concentrations (10 micrometer), atRA decreased PKCalpha activity through the competition with PS but not phorbol-12-myristate-13-acetate, diacylglycerol, or Ca(2+). These results let us hypothesize that in vivo, pharmacological concentrations of atRA may hamper binding of PS to PKCalpha and prevent PKCalpha activation. Thus, this study provides the first evidence for direct binding of atRA to PKC isozymes and suggests the existence of a general mechanism for regulation of PKC activity during exposure to retinoids, as in retinoid-based cancer therapy.

Structural and functional studies of UDP-glucuronosyltransferases.

UDP-Glucuronosyltransferases (UGTs) are glycoproteins localized in the endoplasmic reticulum (ER) which catalyze the conjugation of a broad variety of lipophilic aglycon substrates with glucuronic acid using UDP-glucuronic acid (UDP-GIcUA) as the sugar donor. Glucuronidation is a major factor in the elimination of lipophilic compounds from the body. In this review, current information on the substrate specificities of UGT1A and 2B family isoforms is discussed. Recent findings with regard to UGT structure and topology are presented, including a dynamic topological model of UGTs in the ER. Evidence from experiments on UGT interactions with inhibitors directed at specific amino acids, photoaffinity labeling, and analysis of amino acid alignments suggest that UDP-GIcUA interacts with residues in both the N- and C-terminal domains, whereas aglycon binding sites are localized in the N-terminal domain. The amino acids identified so far as crucial for substrate binding and catalysis are arginine, lysine, histidine, proline, and residues containing carboxylic acid. Site-directed mutagenesis experiments are critical for unambiguous identification of the active-site architecture.

Preferential binding of MyoD-E12 versus myogenin-E12 to the murine sarcoma virus enhancer in vitro.

The MyoD family of transcription factors regulates muscle-specific gene expression in vertebrates. In the adult rat, MyoD mRNA accumulates predominately in fast-twitch muscle, in particular type IIb and/or IIx fibers, whereas Myogenin mRNA is restricted to slow-twitch type I muscle fibers. Transgenic mice expressing the avian v-ski oncogene from the murine sarcoma virus (MSV) promoter-enhancer display preferential hypertrophy of type IIb fast-twitch muscle apparently because of the restricted expression of the transgene. We tested the hypothesis that preferential interactions of MyoD, as a heterodimer with E12, with the MSV enhancer, which has six E-box targets for MyoD family proteins, could contribute to v-ski gene expression in IIb muscle fibers. A series of quantitative binding studies was performed using an electrophoretic mobility shift assay to test MyoD-E12 versus Myogenin-E12 binding to the MSV enhancer. Our results indicate that MyoD-E12 binds the MSV enhancer with higher affinity and higher cooperativity than Myogenin-E12. Interestingly, MyoD-E12 bound all of the individual E-boxes tested with positive cooperativity indicating DNA-mediated dimerization of the protein subunits.

Functional selection and characterization of DNA binding sites for trp repressor of Escherichia coli.

trp repressor of Escherichia coli controls transcription initiation in operons involved in tryptophan biosynthesis by binding to operator sequences within the regulated promoters. Naturally occurring operators are homologous over an 18-base pair region and display dyad symmetry. We have examined the sequence determinants of a repressor binding site using a functional selection/polymerase chain reaction (PCR) amplification strategy. A trp repressor affinity column was generated and used to select binding-competent DNAs from a randomized pool of synthetic double-stranded DNA. DNAs that showed tryptophan-dependent high-affinity binding were eluted by addition of the tryptophan analog beta-indole acrylic acid and amplified by PCR. Following iterative cycles of affinity chromatography and PCR, the selected DNAs were cloned and sequenced. The CTAG tetranucleotide, present in the consensus sequence of all natural operators, was found in all selected DNAs. Mapping experiments utilizing the repressor affinity column showed the CTAG motif to be a critical determinant for repressor binding. Quantitative electrophoretic mobility shift assays with purified trp repressor revealed that although some of the DNAs were bound by one repressor dimer, others were bound by two repressor dimers with cooperativity. Measured binding constants ranged from 0.035 to 0.5 nM for the selected DNAs, compared with 0.1 nM for the trp operator.

Isolation and characterization of Streptomyces erythreus plasmids.

Streptomyces erythreus strains were found to carry several plasmids of molecular weights ranging from about 2 X 10(6) Mr to 40 X 10(6) Mr. Restriction enzyme maps for the streptomycete plasmids pPC7 and pPC8 were constructed for the enzymes Bg/II, EcoRI, XbaI, HindIII, BamHI and SalI. The smaller, pPC8, plasmid appears to be a naturally occurring deletion variant of pPC7. These plasmids belong to the group of conjugative streptomycete plasmids.