MicroRNAs (miRNAs) play crucial roles in bone metabolism. In the present study, we found that miR-148a is dramatically upregulated during osteoclastic differentiation of circulating CD14þ peripheral blood mononuclear cells (PBMCs) induced by macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-kB ligand (RANKL). Overexpression of miR-148a in CD14þ PBMCs promoted osteoclastogenesis, whereas inhibition of miR-148a attenuated osteoclastogenesis. V-maf musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB) is a transcription factor negatively regulating RANKL-induced osteoclastogenesis. miR-148a directly targeted MAFB mRNA by binding to the 3 0 untranslated region (3 0 UTR) and repressed MAFB protein expression. In vivo, our study showed that silencing of miR-148a using a specific antagomir-inhibited bone resorption and increased bone mass in mice receiving ovariectomy (OVX) and in sham-operated control mice. Furthermore, our results showed that miR-148a levels significantly increased in CD14þ PBMCs from lupus patients and resulted in enhanced osteoclastogenesis, which contributed to the lower bone mineral density (BMD) in lupus patients compared with normal controls. Thus, our study provides a new insight into the roles of miRNAs in osteoclastogenesis, and contributes to a new therapeutic pathway for osteoporosis. ß
In many cases, CYP3A4 exhibits unusual kinetic characteristics that result from the metabolism of multiple substrates that coexist at the active site. In the present study, we observed that ␣-naphthoflavone (␣-NF) exhibited a differential effect on CYP3A4-mediated product formation as shown by an increase and decrease, respectively, of the carboxylic acid (P 2 ) and -3-hydroxylated (P 1 ) metabolites of losartan, while losartan was found to be an inhibitor of the formation of the 5,6-epoxide of ␣-NF. Thus, to address this problem, a kinetic model was developed on the assumption that CYP3A4 can accommodate two distinct and independent binding domains for the substrates within the active site, and the resulting velocity equations were employed to predict the kinetic parameters for all possible enzyme-substrate species. Our results indicate that the predicted values had a good fit with the experimental observations. Therefore, the kinetic constants can be used to adequately describe the nature of the metabolic interaction between the two substrates. Applications of the model provide some new insights into the mechanism of drug-drug interactions at the level of CYP3A4.
As the mechanical competence of trabecular bone is a function of its apparent density and 3-D distribution, assessment of 3-D trabecular structural characteristics may improve our ability to understand the pathophysiology of osteoporosis, to test the efficacy of pharmaceutical intervention, and to estimate bone biomechanical properties. We have studied ovariectomy-induced osteopenia in rats and its treatment with agents such as estrogen and sodium fluoride. We have demonstrated that 3-D micro-computed tomography (microCT) can directly quantify mouse trabecular and cortical bone structure with an isotropic resolution of 6 microm(3). MicroCT is also useful for studying osteoporosis in mice and phenotypes of mice with gene manipulation, such as SHIP-knockout mice, which are severely osteoporotic due to increased numbers of hyperresorptive osteoclasts, PTHrP heterozygous-null mice, and mice with Zmpste24 deficiency. MicroCT can quantify osteogenesis in mouse Ilizarov leg-lengthening procedures, osteoconduction in a rat cranial defect model, and structural changes in arthritic rabbits, rats, and mice. In clinical studies, we evaluated longitudinal changes in the iliac crests. Paired bone biopsies from the same premenopausal and postmenopausal women showed the changes in 3-D trabecular structure, such as decreased trabecular thickness, shifting of trabecular model from platelike structure to rodlike structure, and decreased degree of anisotropy were remarkable. Treatment with PTH in postmenopausal women with osteoporosis significantly improved trabecular morphology with a shift toward a more platelike structure, increased trabecular connectivity density, and increased cortical thickness. Paired bone biopsy specimens from the iliac crest in postmenopausal women with osteoporosis before and an average of 2 years after beginning of estrogen replacement therapy demonstrated that posttreatment biopsies showed a significant change in the ratio of plates to rods and statistically insignificant changes in other 3-D trabecular parameters. Thus, microCT can characterize 3-D structure of various animal models, and the longitudinal changes in 3-D bone microarchitectural integrity that deteriorates in the transmenopausal period, is preserved with HRT, and is improved with PTH treatment in postmenopausal women.
1. The selectivity of eight chemical inhibitors has been extensively evaluated with 10 cDNA-expressed human cytochrome P450 isoforms (CYP). The results indicate that sulphaphenazole, quinidine and alpha-naphthoflavone are selective inhibitors of CYP2C9 (IC50 = 0.5-0.7 microM), CYP2D6 (0.3-0.4 microM) and CYP1A (0.05-5 microM) respectively on the basis of the IC50, which are much lower than those of other P450 isoforms (> 10-fold). 2. Ketoconazole exhibited potent inhibition of both CYP3A4-catalysed metabolism of phenanthrene, testosterone, diazepam (IC50 = 0.03-0.5 microM) and CYP1A1-catalysed deethylation of 7-ethoxycoumarin (0.33 microM). The selectivity of ketoconazole for other P450s was highly related to the concentration used. 3. Diethyldithiocarbamate, orphenadrine and furafylline were shown separately to be less selective inhibitors of CYP2E1, CYP2B6 and CYP1A isoforms by a broad range of IC50 that overlap those observed with other P450 isoforms. 4. Furafylline, quinidine and alpha-naphthoflavone activated CYP3A4-catalysed phenanthrene metabolism by 1.7-, 2- and 15-fold respectively. 5. The selectivity of orphenadrine and ketoconazole was further examined by using inhibitory monoclonal antibodies (MAb). Inhibitory MAb specific for the individual P450 isoforms may be of greater value than chemical inhibitors.
Cytochrome P450 3A4 (CYP3A4) plays a prominent role in the metabolism of a vast array of drugs and xenobiotics and exhibits broad substrate specificities. Most cytochrome P450-mediated reactions follow simple Michaelis-Menten kinetics. These parameters are widely accepted to predict pharmacokinetic and pharmacodynamic consequences in vivo caused by exposure to one or multiple drugs. However, CYP3A4 in many cases exhibits allosteric (sigmoidal) characteristics that make the Michaelis constants difficult to estimate. In the present study, diazepam, temazepam and nordiazepam were employed as substrates of CYP3A4 to propose a kinetic model. The model hypothesized that CYP3A4 contains two substrate-binding sites in a single active site that are both distinct and co-operative, and the resulting velocity equation had a good fit with the sigmoidal kinetic observations. Therefore, four pairs of the kinetic estimates (KS1, kalpha, KS2, kbeta, KS3, kdelta, KS4 and kgamma) were resolved to interpret the features of binding affinity and catalytic ability of CYP3A4. Dissociation constants KS1 and KS2 for two single-substrate-bound enzyme molecules (SE and ES) were 3-50-fold greater than KS3 and KS4 for a two-substrate-bound enzyme (SES), while respective rate constants kdelta and kgamma were 3-218-fold greater than kalpha and kbeta, implying that access and binding of the first molecule to either site in an active pocket of CYP3A4 can enhance the binding affinity and reaction rate of the vacant site for the second substrate. Thus our results provide some new insights into the co-operative binding of two substrates in the inner portions of an allosteric CYP3A4 active site.
Early-life stress is a potent risk factor for development of psychiatric conditions such as depression. The underlying mechanisms remain poorly understood. Here, we used the early-life social isolation (ESI) model of early-life stress in rats to characterize development of depressive-like behavior, the role of microglia, levels of histone methylation, as well as expression of glutamate receptor subunits in the hippocampus. We found that depressive-like behavior was induced after ESI as determined by sucrose preference and forced swimming tests. Increased expression of microglial activation marker, Iba1, was observed in the hippocampus of the ESI group, while expression of the microglial CD200 receptor, which promotes microglial quiescence, significantly decreased. In addition, increased levels of proinflammatory cytokines, interleukin 1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor-alpha (TNF-α) were observed in the hippocampus of the ESI group. Moreover, ESI increased levels of neuronal H3K9me2 (a repressive marker of transcription) and its associated "writer" enzymes, G9a and G9a-like protein, in the hippocampus. ESI also decreased expression of hippocampal NMDA receptor subunits, NR1, and AMPA receptor subunits, GluR1 and GluR2, which are involved in synaptic plasticity, but it did not affect expression of PSD95 and NR2B. Interestingly, treatment with minocycline to block microglial activation induced by ESI inhibited increases in hippocampal microglia and prevented ESI-induced depressive-like behavior as well as increases in IL-1β, IL-6, and TNF-α. Notably, minocycline also triggered downregulation of H3K9me2 expression and restored expression of NR1, GluR1, and GluR2. These results suggest that ESI induces depressive-like behavior, which may be mediated by microglial signaling.
The precursor of brain derived neurotrophic factor (proBDNF), the unprocessed BDNF gene product, binds to its receptors and exerts the opposing biologic functions of mature BDNF. proBDNF is expressed in the peripheral tissues but the functions of peripheral proBDNF remain elusive. Here we showed that proBDNF and its predominant receptor, p75 pan-neurotrophin receptor were upregulated in the nerve fibers and inflammatory cells in the local tissue in inflammatory pain. Neutralization of proBDNF by polyclonal antibody attenuated pain in different models of inflammatory pain. Unilateral intra-plantar supplementation of proBDNF by injecting exogenous proBDNF or ectopic overexpression resulted in pain hypersensitivity and induced spinal phosphorylated extracellular signal-regulated kinase activation. Exogenous proBDNF injection induced the infiltration of inflammatory cells and the activation of proinflammatory cytokines, suggesting that inflammatory reaction contributed to the pro-algesic effect of proBDNF. Finally, we generated monoclonal anti-proBDNF antibody that could biologically block proBDNF. Administration of monoclonal Ab-proBDNF attenuated various types of inflammatory pain and surgical pain. Thus, peripheral proBDNF is a potential pain mediator and anti-proBDNF pretreatment may alleviate the development of inflammatory pain.
Eukaryotic cells sense extracellular glucose concentrations via diverse mechanisms to regulate the expression of genes involved in metabolic control. One such example is the tight correlation between the expression of thioredoxin-interacting protein (Txnip) and extracellular glucose levels. In this report, we show that the transcription of the Txnip gene is induced by adenosine-containing molecules, of which an intact adenosine moiety is necessary and sufficient. Txnip promoter contains a carbohydrate response element, which mediates the induction of Txnip expression by these molecules in a glucose-dependent manner. Max-like protein X and MondoA are transcription factors previously shown to stimulate glucose-dependent Txnip expression and are shown here to convey stimulatory signals from extracellular adenosine-containing molecules to the Txnip promoter. The regulatory role of these molecules may be exerted via amplifying glucose signaling. Hence, this revelation may pave the way for interventions aimed toward metabolic disorders resulting from abnormal glucose homeostasis.
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