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Integration of microbiota in a host begins at birth and progresses during adolescence, forming a multidirectional system of physiological interactions. Here, we present an instantaneous effect of natural, bacterial gut colonization on the acceleration of longitudinal and radial bone growth in germ-free born, 7-wk-old male rats. Changes in bone mass and structure were analyzed after 10 days following the onset of colonization through cohousing with conventional rats and revealed unprecedented acceleration of bone accrual in cortical and trabecular compartments, increased bone tissue mineral density, improved proliferation and hypertrophy of growth plate chondrocytes, bone lengthening, and preferential deposition of periosteal bone in the tibia diaphysis. In addition, the number of small in size adipocytes increased, whereas the number of megakaryocytes decreased, in the bone marrow of conventionalized germ-free rats indicating that not only bone mass but also bone marrow environment is under control of gut microbiota signaling. The changes in bone status paralleled with a positive shift in microbiota composition toward short-chain fatty acids (SCFA)-producing microbes and a considerable increase in cecal SCFA concentrations, specifically butyr-ate. Furthermore, reconstitution of the host holobiont increased hepatic expression of IGF-1 and its circulating levels. Elevated serum levels of 25-hydroxy vitamin D and alkaline phosphatase pointed toward an active process of bone formation. The acute stimulatory effect on bone growth occurred independently of body mass increase. Overall, the presented model of conventionalized germ-free rats could be used to study microbiota-based therapeutics for combatting dysbiosis-related bone disorders. [ABSTRACT FROM AUTHOR]

Lineage allocation of the marrow mesenchymal stem cells (MSCs) to osteoblasts and adipocytes is dependent on both Wnt signaling and PPARγ2 activity. Activation of PPARγ2, an essential regulator of energy metabolism and insulin sensitivity, stimulates adipocyte and suppresses osteoblast differentiation and bone formation, and correlates with decreased bone mass and increased fracture rate. In contrast, activation of Wnt signaling promotes osteoblast differentiation, augments bone accrual and reduces total body fat. This study examined the cross-talk between PPARγ2 and β-catenin, a key mediator of canonical Wnt signaling, on MSC lineage determination. Rosiglitazone-activated PPARγ2 induced rapid proteolytic degradation of β-catenin, which was prevented by either inhibiting glycogen synthase kinase 3 beta (GSK3b) activity, or blocking pro-adipocytic activity of PPARγ2 using selective antagonist GW9662 or mutation within PPARγ2 protein. Stabilization of β-catenin suppressed PPARγ2 pro-adipocytic but not anti-osteoblastic activity. Moreover, β-catenin stabilization decreased PPARγ2-mediated insulin signaling as measured by insulin receptor and FoxO1 gene expression, and protein levels of phosphorylated Akt (pAkt). Cellular knockdown of β-catenin with siRNA increased expression of adipocyte but did not affect osteoblast gene markers. Interestingly, the expression of Wnt10b was suppressed by anti-osteoblastic, but not by pro-adipocytic activity of PPARγ2. Moreover, β-catenin stabilization in the presence of activated PPARγ2 did not restore Wnt10b expression indicating a dominant role of PPARγ2 in negative regulation of pro-osteoblastic activity of Wnt signaling. In conclusion, β-catenin and PPARγ2 are in cross-talk which results in sequestration of pro-adipocytic and insulin sensitizing activity. The anti-osteoblastic activity of PPARγ2 is independent of this interaction. [ABSTRACT FROM AUTHOR]

The peroxisome proliferator activated receptor gamma (PPARG) nuclear receptor regulates energy metabolism and insulin sensitivity. In this study, we present novel evidence for an essential role of PPARG in the regulation of osteocyte function, and support for the emerging concept of the conjunction between regulation of energy metabolism and bone mass. We report that PPARG is essential for sclerostin production, a recently approved target to treat osteoporosis. Our mouse model of osteocyte-specific PPARG deletion (Dmp1CrePparγflfl or γOTKO) is characterized with increased bone mass and reduced bone marrow adiposity, which is consistent with upregulation of WNT signaling and increased bone forming activity of endosteal osteoblasts. An analysis of osteocytes derived from γOTKO and control mice showed an excellent correlation between PPARG and SOST/sclerostin at the transcript and protein levels. The 8 kb sequence upstream of Sost gene transcription start site possesses multiple PPARG binding elements (PPREs) with at least two of them binding PPARG with dynamics reflecting its activation with full agonist rosiglitazone and correlating with increased levels of Sost transcript and sclerostin protein expression (Pearson's r = 0.991, p = 0.001). Older γOTKO female mice are largely protected from TZD-induced bone loss providing proof of concept that PPARG in osteocytes can be pharmacologically targeted. These findings demonstrate that transcriptional activities of PPARG are essential for sclerostin expression in osteocytes and support consideration of targeting PPARG activities with selective modulators to treat osteoporosis. [Display omitted] • PPARG nuclear receptor in osteocytes is essential for sclerostin expression. • Osteocytic PPARG regulates bone mass and marrow adiposity. • Mice deficient in PPARG in osteocytes are protected from rosiglitazone-induced bone loss. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The lipid peroxidation product 4-hydroxynon-2-enal (4-HNE) is a strong electrophile that forms covalent adducts with proteins and, to a lesser extent, nucleic acids and phospholipids. The generation of 4-HNE appears to be an inevitable consequence of aerobic metabolism. The metabolism of 4-HNE is mainly, although not entirely, conjugative, and proceeds via Michael addition of glutathione to the double bond of 4-HNE. This reaction is catalyzed by specialized glutathione S-transferases (GSTs) exemplified by the murine mGSTA4-4. To study the (patho)physiological effects of 4-HNE in an intact organism, we disrupted the mGsta4 gene in the mouse. The resulting mGsta4 null mouse expressed no mGsta4 mRNA and no corresponding protein, had a reduced ability to conjugate 4-HNE, and had an increased steady-state level of this aldehyde in tissues. The residual conjugating activity for 4-HNE (23–64% depending on the tissue) is probably attributable to isoforms of glutathione S-transferases which have low catalytic efficiency for 4-HNE but are more abundant than mGSTA4-4, or are upregulated upon mGsta4 gene disruption. Mice homozygous for the disrupted mGsta4 allele were viable and appeared normal except for lower litter size, higher fat content in bones, and greater susceptibility to bacterial infection. The null mice had a significantly lower survival time than wild-type controls when chronically treated with relatively low doses of paraquat, a finding consistent with a role of mGSTA4-4 in the defense against oxidative stress. The mouse model should be useful for the study of degenerative conditions in which 4-HNE is postulated to be a contributing factor. [Copyright &y& Elsevier]

Inflammatory arthritis (IA) is a common disease that affects millions of individuals worldwide. Proinflammatory events during IA pathogenesis are well studied; however, loss of protective immunity remains underexplored. Earlier, we reported that 14-3-3zeta (ζ) has a role in T-cell polarization and interleukin (IL)-17A signal transduction. Here, we demonstrate that 14-3-3ζ knockout (KO) rats develop early-onset severe arthritis in two independent models of IA, pristane-induced arthritis and collagen-induced arthritis. Arthritic 14-3-3ζ KO animals showed an increase in bone loss and immune cell infiltration in synovial joints. Induction of arthritis coincided with the loss of anti-14-3-3ζ antibodies; however, rescue experiments to supplement the 14-3-3ζ antibody by passive immunization did not suppress arthritis. Instead, 14-3-3ζ immunization during the presymptomatic phase resulted in significant suppression of arthritis in both wild-type and 14-3-3ζ KO animals. Mechanistically, 14-3-3ζ KO rats exhibited elevated inflammatory gene signatures at the messenger RNA and protein levels, particularly for IL-1β. Furthermore, the immunization with recombinant 14-3-3ζ protein suppressed IL-1β levels, significantly increased anti-14-3-3ζ antibody levels and collagen production, and preserved bone quality. The 14-3-3ζ protein increased collagen expression in primary rat mesenchymal cells. Together, our findings indicate that 14-3-3ζ causes immune suppression and extracellular remodeling, which lead to a previously unrecognized IA-suppressive function. [ABSTRACT FROM AUTHOR]

Through linkage analysis of the Dahl salt-sensitive (S) rat and the spontaneously hypertensive rat (SHR), a blood pressure (BP) quantitative trait locus (QTL) was previously located on rat chromosome 9. Subsequent substitution mapping studies of this QTL revealed multiple BP QTLs within the originally identified logarithm of odds plot by linkage analysis. The focus of this study was on a 14.39 Mb region, the distal portion of which remained unmapped in our previous studies. High-resolution substitution mapping for a BP QTL in the setting of a high-salt diet indicated that an SHR-derived congenic segment of 787.9 kb containing the gene secreted phosphoprotein-2 (Spp2) lowered BP and urinary protein excretion. A nonsynonymous G/T polymorphism in the Spp2 gene was detected between the S and S.SHR congenic rats. A survey of 45 strains showed that the T allele was rare, being detected only in some substrains of SHR and WKY. Protein modeling prediction through SWISSPROT indicated that the predicted protein product of this variant was significantly altered. Importantly, in addition to improved cardiovascular and renal function, high salt-fed congenic animals carrying the SHR T variant of Spp2 had significantly lower bone mass and altered bone microarchitecture. Total bone volume and volume of trabecular bone, cortical thickness, and degree of mineralization of cortical bone were all significantly reduced in congenic rats. Our study points to opposing effects of a congenic segment containing the prioritized candidate gene Spp2 on BP and bone mass. [ABSTRACT FROM AUTHOR]

Peroxisome proliferator activated receptor gamma (PPARγ) controls both glucose metabolism and an allocation of marrow mesenchymal stem cells (MSCs) toward osteoblast and adipocyte lineages. Its activity is determined by interaction with a ligand which directs posttranscriptional modifications of PPARγ protein including dephosphorylation of Ser112 and Ser273, which results in acquiring of pro-adipocytic and insulin-sensitizing activities, respectively. PPARγ full agonist TZD rosiglitazone (ROSI) decreases phosphorylation of both Ser112 and Ser273 and its prolonged use causes bone loss in part due to diversion of MSCs differentiation from osteoblastic toward adipocytic lineage. Telmisartan (TEL), an anti-hypertensive drug from the class of angiotensin receptor blockers, also acts as a partial PPARγ agonist with insulin-sensitizing and a weak pro-adipocytic activity. TEL decreased S273pPPARγ and did not affect S112pPPARγ levels in a model of marrow MSC differentiation, U-33/γ2 cells. In contrast to ROSI, TEL did not affect osteoblast phenotype and actively blocked ROSI-induced anti-osteoblastic activity and dephosphorylation of S112pPPARγ. The effect of TEL on bone was tested side-by-side with ROSI. In contrast to ROSI, TEL administration did not affect bone mass and bone biomechanical properties measured by micro-indentation method and did not induce fat accumulation in bone, and it partially protected from ROSI-induced bone loss. In addition, TEL induced “browning” of epididymal white adipose tissue marked by increased expression of UCP1, FoxC2, Wnt10b and IGFBP2 and increased overall energy expenditure. These studies point to the complexity of mechanisms by which PPARγ acquires anti-osteoblastic and pro-adipocytic activities and suggest an importance of Ser112 phosphorylation status as being a part of the mechanism regulating this process. These studies showed that TEL acts as a full PPARγ agonist for insulin-sensitizing activity and as a partial agonist/partial antagonist for pro-adipocytic and anti-osteoblastic activities. They also suggest a relationship between PPARγ fat “browning” activity and a lack of anti-osteoblastic activity. [ABSTRACT FROM AUTHOR]

We have demonstrated the subcellular localization of the human UDP-glucuronosyltransferases (UGTs), UGT2B7 and UGT1A6, in endoplasmic reticulum (ER) and nuclear membrane from human hepatocytes and cell lines, by in situ immunostaining and Western blot. Double immunostaining for UGT2B7 and calnexin, an ER resident protein, showed that UGT2B7 was equally present in ER and nuclear membrane whereas calnexin was present almost exclusively in ER. Immunogold labeling of HK293 cells expressing UGT2B7 established the presence of UGT2B7 in both nuclear membranes. Enzymatic assays with UGT2B7 substrates confirmed the presence of functional UGT2B7 protein in ER, whole nuclei, and both outer and inner nuclear membranes. This study has identified, for the first time, the presence of UGT2B7 and UGT1A6 in the nucleus and of UGT2B7 in the inner and outer nuclear membranes. This localization may play an important functional role within nuclei: protection from toxic compounds and/or control of steady-state concentrations of nuclear receptor ligands. [Copyright &y& Elsevier]