Structural characterization of a non-heme iron active site in zeolites that hydroxylates methane.

Iron-containing zeolites exhibit unprecedented reactivity in the low-temperature hydroxylation of methane to form methanol. Reactivity occurs at a mononuclear ferrous active site, α-Fe(II), that is activated by N2O to form the reactive intermediate α-O. This has been defined as an Fe(IV)=O species. Using nuclear resonance vibrational spectroscopy coupled to X-ray absorption spectroscopy, we probe the bonding interaction between the iron center, its zeolite lattice-derived ligands, and the reactive oxygen. α-O is found to contain an unusually strong Fe(IV)=O bond resulting from a constrained coordination geometry enforced by the zeolite lattice. Density functional theory calculations clarify how the experimentally determined geometric structure of the active site leads to an electronic structure that is highly activated to perform H-atom abstraction.

Metabolic Profiling of the Novel Hypoxia-Inducible Factor 2α Inhibitor PT2385 In Vivo and In Vitro.

PT2385 is a first-in-class, selective small-molecule inhibitor of hypoxia-inducible factor-2α (HIF-2α) developed for the treatment of advanced clear cell renal cell carcinoma. Preclinical results demonstrated that PT2385 has potent antitumor efficacy in mouse xenograft models of kidney cancer. It also has activity toward metabolic disease in a mouse model. However, no metabolism data are currently publically available. It is of great importance to characterize the metabolism of PT2385 and identify its effect on systemic homeostasis in mice. High-resolution mass spectrometry-based metabolomics was performed to profile the biotransformation of PT2385 and PT2385-induced changes in endogenous metabolites. Liver microsomes and recombinant drug-metabolizing enzymes were used to determine the mechanism of PT2385 metabolism. Real-time polymerase chain reaction analysis was employed to investigate the reason for the PT2385-induced bile acid dysregulation. A total of 12 metabolites of PT2385 was characterized, generated from hydroxylation (M1, M2), dihydroxylation and desaturation (M3, M4), oxidative-defluorination (M7), glucuronidation (M8), N-acetylcysteine conjugation (M9), and secondary methylation (M5, M6) and glucuronidation (M10, M11, and M12). CYP2C19 was the major contributor to the formation of M1, M2, and M7, UGT2B17 to M8, and UGT1A1/3 to M10-M12. The bile acid metabolites taurocholic acid and tauro-ß-muricholic acid were elevated in serum and liver of mice after PT2385 treatment. Gene expression analysis further revealed that intestinal HIF-2α inhibition by PT2385 treatment upregulated the hepatic expression of CYP7A1, the rate-limiting enzyme in bile acid synthesis. This study provides metabolic data and an important reference basis for the safety evaluation and rational clinical application of PT2385.

Differential DNA Hydroxymethylation in Human Uterine Leiomyoma Cells Depending on the Phase of Menstrual Cycle and Presence of MED12 Gene Mutations.

Using immunofluorescence with specific antibodies, we analyzed DNA hydroxymethylation in uncultured cells from 25 human uterine leiomyomas considering the menstrual cycle phase during surgery and the presence of MED12 gene mutations. It was found that each tumor node had specific DNA hydroxymethylation level that did not depend on the presence of mutations in MED12 gene, but depended on the phase of menstrual cycle. The degree of DNA hydroxymethylation was significantly lower in cells of leiomyomas excised during the luteal phase compared to the follicular phase (p=0.0431). Hormonal status changing at various phases of menstrual cycle is a factor affecting DNA hydroxymethylation in leiomyoma cells.

New horizons in hypoxia signaling pathways.

Investigation into the regulation of the erythropoietin gene by oxygen led to the discovery of a process of direct oxygen sensing that transduces many cellular and systemic responses to hypoxia. The oxygen-sensitive signal is generated through the catalytic action of a series of 2-oxoglutarate-dependent oxygenases that regulate the transcription factor hypoxia-inducible factor (HIF) by the post-translational hydroxylation of specific amino acid residues. Here we review the implications of the unforeseen complexity of the HIF transcriptional cascade for the physiology and pathophysiology of hypoxia, and consider the origins of post-translational hydroxylation as a signaling process.

Hypoxic behavior in cells under controlled microfluidic environment.

BACKGROUND: Depleted oxygen levels, known as hypoxia, causes considerable changes in the cellular metabolism. Hypoxia-inducible factors (HIF) act as the major protagonist in orchestrating manifold hypoxic responses by escaping cellular degradation mechanisms. These complex and dynamic intracellular responses are significantly dependent on the extracellular environment. In this study, we present a detailed model of a hypoxic cellular microenvironment in a microfluidic setting involving HIF hydroxylation. METHODS: We have modeled the induction of hypoxia in a microfluidic chip by an unsteady permeation of oxygen from the microchannel through a porous polydimethylsiloxane channel wall. Extracellular and intracellular interactions were modeled with two different mathematical descriptions. Intracellular space is directly coupled to the extracellular environment through uptake and consumption of oxygen and ascorbate similar to cells in vivo. RESULTS: Our results indicate a sharp switch in HIF hydroxylation behavior with changing prolyl hydroxylase levels from 0.1 to 4.0µM. Furthermore, we studied the effects of extracellular ascorbate concentration, using a new model, to predict its accumulation inside the cell over a relevant physiological range. In different hypoxic conditions, the cellular environment showed a significant dependence on oxygen levels in resulting intracellular response. CONCLUSIONS: Change in hydroxylation behavior and nutrient supplementation can have significant potential in designing novel therapeutic interventions in cancer and ischemia/reperfusion injuries. GENERAL SIGNIFICANCE: The hybrid mathematical model can effectively predict intracellular behavior due to external influences providing valuable directions in designing future experiments.

Enigmatic 5-hydroxymethyluracil: Oxidatively modified base, epigenetic mark or both?

The aim of this review is to describe the reactions which lead to generation of 5-hydroxymethyluracil, as well as the repair processes involved in its removal from DNA, and its level in various cells and urine. 5-hydroxymethyluracil may be formed during the course of the two processes: oxidation/hydroxylation of thymine with resultant formation of 5-hydroxymethyluracil paired with adenine (produced by reactive oxygen species), and reacting of reactive oxygen species with 5-methylcytosine forming 5-hydroxymethylcytosine, followed by its deamination to 5-hydroxymethyluracil mispaired with guanine. However, other, perhaps enzymatic, mechanism(s) may be involved in formation of 5-hydroxymethyluracil mispaired with guanine. Indeed, this mispair may be also formed as a result of deamination of 5-hydroxymethylcytosine, recently described "sixth" DNA base. It was demonstrated that 5-hydroxymethyluracil paired with adenine can be also generated by TET enzymes from thymine during mouse embryonic cell differentiation. Therefore, it is possible that 5-hydroxymethyluracil is epigenetic mark. The level of 5-hydroxymethyluracil in various somatic tissues is relatively stable and resembles that observed in lymphocytes, about 0.5/10(6) dN in human colon, colorectal cancer as well as various rat and porcine tissues. Experimental evidence suggests that SMUG1 and TDG are main enzymes involved in removal of 5-hydroxymethyluracil from DNA. 5-hydroxymethyluracil, in form of 5-hydroxymethyluridine, was also detected in rRNA, and together with SMUG1 may play a role in rRNA quality control. To summarize, 5-hydroxymethyluracil is with no doubt a product of both enzymatic and reactive oxygen species-induced reaction. This modification may probably serve as an epigenetic mark, providing additional layer of information encoded within the genome. However, the pool of 5-hydroxymethyluracil generated as a result of oxidative stress is also likely to disturb physiological epigenetic processes, and as such may be defined as a lesion. Altogether this suggests that 5-hydroxymethyluracil may be either a regulatory or erroneous compound.

Cytochrome P450 ω-Hydroxylases in Inflammation and Cancer.

Cytochrome P450-dependent ω-hydroxylation is a prototypic metabolic reaction of CYP4 family members that is important for the elimination and bioactivation of not only therapeutic drugs, but also endogenous compounds, principally fatty acids. Eicosanoids, derived from arachidonic acid, are key substrates in the latter category. Human CYP4 enzymes, mainly CYP4A11, CYP4F2, and CYP4F3B, hydroxylate arachidonic acid at the omega position to form 20-HETE, which has important effects in tumor progression and on angiogenesis and blood pressure regulation in the vasculature and kidney. CYP4F3A in myeloid tissue catalyzes the ω-hydroxylation of leukotriene B4 to 20-hydroxy leukotriene B4, an inactivation process that is critical for the regulation of the inflammatory response. Here, we review the enzymology, tissue distribution, and substrate selectivity of human CYP4 ω-hydroxylases and their roles as catalysts for the formation and termination of the biological effects of key eicosanoid metabolites in inflammation and cancer progression.

Evaluation of 89 compounds for identification of substrates for cynomolgus monkey CYP2C76, a new bupropion/nifedipine oxidase.

Cynomolgus monkeys are widely used in preclinical studies during drug development because of their evolutionary closeness to humans, including their cytochrome P450s (P450s). Most cynomolgus monkey P450s are almost identical (≥90%) to human P450s; however, CYP2C76 has low sequence identity (approximately 80%) to any human CYP2Cs. Although CYP2C76 has no ortholog in humans and is partly responsible for species differences in drug metabolism between cynomolgus monkeys and humans, a broad evaluation of potential substrates for CYP2C76 has not yet been conducted. In this study, a screening of 89 marketed compounds, including human CYP2C and non-CYP2C substrates or inhibitors, was conducted to find potential CYP2C76 substrates. Among the compounds screened, 19 chemicals were identified as substrates for CYP2C76, including substrates for human CYP1A2 (7-ethoxyresorufin), CYP2B6 (bupropion), CYP2D6 (dextromethorphan), and CYP3A4/5 (dextromethorphan and nifedipine), and inhibitors for CYP2B6 (sertraline, clopidogrel, and ticlopidine), CYP2C8 (quercetin), CYP2C19 (ticlopidine and nootkatone), and CYP3A4/5 (troleandomycin). CYP2C76 metabolized a wide variety of the compounds with diverse structures. Among them, bupropion and nifedipine showed high selectivity to CYP2C76. As for nifedipine, CYP2C76 formed methylhydroxylated nifedipine, which was not produced by monkey CYP2C9, CYP2C19, or CYP3A4, as identified by mass spectrometry and estimated by a molecular docking simulation. This unique oxidative metabolite formation of nifedipine could be one of the selective marker reactions of CYP2C76 among the major CYP2Cs and CYP3As tested. These results suggest that monkey CYP2C76 contributes to bupropion hydroxylation and formation of different nifedipine oxidative metabolites as a result of its relatively large substrate cavity.

Characterization of phase I metabolism of resibufogenin and evaluation of the metabolic effects on its antitumor activity and toxicity.

Resibufogenin (RB), one of the major active compounds of the traditional Chinese medicine Chansu, has displayed great potential as a chemotherapeutic agent in oncology. However, it is a digoxin-like compound that also exhibits extremely cardiotoxic effects. The present study aimed to characterize the metabolic behaviors of RB in humans as well as to evaluate the metabolic effects on its bioactivity and toxicity. The phase I metabolic profile in human liver microsomes was characterized systemically, and the major metabolite was identified as marinobufagenin (5ß-hydroxylresibufogenin, 5-HRB) by liquid chromatography-mass spectrometry and nuclear magnetic imaging techniques. Both cytochrome P450 (P450) reaction phenotyping and inhibition assays using P450-selective chemical inhibitors demonstrated that CYP3A4 was mainly involved in RB 5ß-hydroxylation with much higher selectivity than CYP3A5. Kinetic characterization demonstrated that RB 5ß-hydroxylation in both human liver microsomes and human recombinant CYP3A4 obeyed biphasic kinetics and displayed similar apparent kinetic parameters. Furthermore, 5-HRB could significantly induce cell growth inhibition and apoptosis in A549 and H1299 by facilitating apoptosome assembly and caspase activation. Meanwhile, 5-HRB displayed very weak cytotoxicity of human embryonic lung fibroblasts, and in mice there was a greater tolerance to acute toxicity. In summary, CYP3A4 dominantly mediated 5ß-hydroxylation and was found to be a major metabolic pathway of RB in the human liver, whereas its major metabolite (5-HRB) displayed better druglikeness than its parent compound RB. Our findings lay a solid foundation for RB metabolism studies in humans and encourage further research on the bioactive metabolite of RB.

Hypoxia reduces the efficiency of elisidepsin by inhibiting hydroxylation and altering the structure of lipid rafts.

The mechanism of action of elisidepsin (PM02734, Irvalec®) is assumed to involve membrane permeabilization via attacking lipid rafts and hydroxylated lipids. Here we investigate the role of hypoxia in the mechanism of action of elisidepsin. Culturing under hypoxic conditions increased the half-maximal inhibitory concentration and decreased the drug's binding to almost all cell lines which was reversed by incubation of cells with 2-hydroxy palmitic acid. The expression of fatty acid 2-hydroxylase was strongly correlated with the efficiency of the drug and inversely correlated with the effect of hypoxia. Number and brightness analysis and fluorescence anisotropy experiments showed that hypoxia decreased the clustering of lipid rafts and altered the structure of the plasma membrane. Although the binding of elisidepsin to the membrane is non-cooperative, its membrane permeabilizing effect is characterized by a Hill coefficient of ~3.3. The latter finding is in agreement with elisidepsin-induced clusters of lipid raft-anchored GFP visualized by confocal microscopy. We propose that the concentration of elisidepsin needs to reach a critical level in the membrane above which elisidepsin induces the disruption of the cell membrane. Testing for tumor hypoxia or the density of hydroxylated lipids could be an interesting strategy to increase the efficiency of elisidepsin.

Characterization of inhibition kinetics of (S)-warfarin hydroxylation by noscapine: implications in warfarin therapy.

Noscapine is an antitussive and potential anticancer drug. Clinically significant interactions between warfarin and noscapine have been previously reported. In this study, to provide a basis for warfarin dosage adjustment, the inhibition kinetics of noscapine against warfarin metabolism was characterized. Our enzyme kinetics data obtained from human liver microsomes and recombinant CYP2C9 proteins indicate that noscapine is a competitive inhibitor of the (S)-warfarin 7-hydroxylation reaction by CYP2C9. Interestingly, noscapine also inhibited (S)-warfarin metabolism in a NADPH- and time-dependent manner, and removal of unbound noscapine and its metabolites by ultrafiltration did not reverse inhibition of (S)-warfarin metabolism by noscapine, suggesting mechanism-based inhibition of CYP2C9 by noscapine. Spectral scanning of the reaction between CYP2C9 and noscapine revealed the formation of an absorption spectrum at 458 nm, indicating the formation of a metabolite-intermediate complex. Surprisingly, noscapine is a 2- to 3-fold more efficient inactivator of CYP2C9.2 and CYP2C9.3 variants than it is of the wild type, by unknown mechanisms. Based on the inhibitory kinetic data, (S)-warfarin exposure is predicted to increase up to 7-fold (depending on CYP2C9 genotypes) upon noscapine coadministration, mainly due to mechanism-based inactivation of CYP2C9 by noscapine. Together, these results indicate that mechanism-based inhibition of CYP2C9 by noscapine may dramatically alter pharmacokinetics of warfarin and provide a basis for warfarin dosage adjustment when noscapine is coadministered.

Metabolism and excretion of 6-chloro-9-(4-methoxy-3,5-dimethylpyridin-2-ylmethyl)-9H-purin-2-ylamine, an HSP90 inhibitor, in rats and dogs and assessment of its metabolic profile in plasma of humans.

6-Chloro-9-(4-methoxy-3,5-dimethyl-pyridin-2-ylmethyl)-9H-purin-2-ylamine (BIIB021), a synthetic HSP90 inhibitor, exhibited promising antitumor activity in preclinical models and was in development for the treatment of breast cancer. The metabolism and excretion of BIIB021 was investigated in rats and dogs after oral administration of [(14)C]BIIB021. The administered radioactive dose was quantitatively recovered in both species, and feces/bile was the major route of excretion. Metabolic profiling revealed that BIIB021 is extensively metabolized primarily via hydroxylation of the methyl group (M7), O-demethylation (M2), and to a lesser extent by glutathione conjugation (M8 and M9). M7 was further metabolized to form the carboxylic acid (M3) and glucuronide conjugate (M4). Human plasma obtained from the phase I study in cancer patients were also analyzed to assess the metabolism of BIIB021 in humans and to ensure that selected animal species were exposed to all human major metabolites. Results suggested that BIIB021 is metabolized via hydroxylation followed by carboxylation and glucuronidation in humans consistent with rat and dog; however, an additional dominant circulating metabolite, hydroxylation at the purine ring (M10), was identified in humans. Preliminary in vitro studies using liver cytosolic fractions indicated that M10 formation is primarily catalyzed by aldehyde oxidase (AO). AO catalytic activity for M10 formation was the highest in the monkey, followed by mouse, human, and rat. The apparent K(m) and V(max) values of M10 formation were 174 ± 8 µM and 14.0 ± 0.3 pmol•min(-1)•mg protein(-1) in human and 132 ± 9 µM and 131 ± 4 pmol•min(-1)•mg protein(-1) in monkey, respectively.

Ospemifene metabolism in humans in vitro and in vivo: metabolite identification, quantitation, and CYP assignment of major hydroxylations.

BACKGROUND: The metabolism of ospemifene, a novel nonsteroidal selective estrogen receptor modulator, was investigated as part of its development. METHODS: Metabolite identification, tentative quantitation, and CYP assignment of ospemifene were performed in human liver microsomes or homogenate incubations and in plasma samples from volunteer humans. The potential contributions of CYP enzymes were determined by recombinant human CYPs. Metabolite identification and tentative quantification were performed by liquid chromatography-mass spectrometry. RESULTS: The relative abundances of metabolites produced were dependent on ospemifene concentration and liver preparation, but the largest quantities of 4- and 4'-hydroxy-ospemifene (and their glucuronides in smaller quantities) were produced in human liver microsomes at low ospemifene concentrations. Other metabolites were detected in in vitro incubation with human liver including a direct glucuronide of ospemifene and some metabolites with only minor abundance. In human plasma samples, 4-hydroxy-ospemifene was the most abundant metabolite, representing about 25% of the abundance of the parent compound. All the other metabolites detected in plasma, including 4'-hydroxy-ospemifene, represented <7% of the abundance of ospemifene. Several CYP enzymes participated in 4-hydroxylation, including CYP2C9, CYP2C19, CYP2B6, and CYP3A4, whereas CYP3A enzymes were the only ones to catalyze 4'-hydroxylation. CONCLUSIONS: In vitro incubations with liver preparations provided a rather reliable starting point in the search for potential metabolites in clinical settings. The in vitro metabolite profile is informative for the in vivo metabolite profile, especially regarding the major hydroxylated metabolites. However, it is anticipated that extended in vivo exposures may result in an increased production of more distal metabolites from major metabolites.

Structure-dependent activities of hydroxylated polybrominated diphenyl ethers on human estrogen receptor.

Polybrominated diphenyl ethers (PBDEs) have been shown to affect the estrogen receptor (ER) signaling pathway, and one of the proposed disruption mechanisms is direct binding of hydroxylated PBDE (OH-PBDE) to ER. In this paper, the binding affinity of 22 OH-PBDEs with different degrees of bromination to ER was assessed quantitatively using a surface plasmon resonance biosensor technique. Seven OH-PBDEs were found to bind directly with ER with KD ranging from 1.46x10(-7) M to 7.90x10(-6) M, and the affinity is in the order of 6-OH-BDE-047>/=4'-OH-BDE-049>4'-OH-BDE-017>6'-OH-BDE-099>/=5'-OH-BDE-099>2'-OH-BDE-007>3'-OH-BDE-028. In MVLN luciferase gene reporter assays, 10 low-brominated OH-PBDEs induced luciferase activity alone, but are 10(5) to 10(7) fold less potent than E2. Their estrogenic activity is in the order of 4'-OH-BDE-049>4'-OH-BDE-017>2'-OH-BDE-007>3'-OH-BDE-028>3-OH-BDE-047>/=3'-OH-BDE-007. The good correlation between estrogenic activity and ER binding affinity of the low-brominated OH-PBDEs strongly suggest that these compounds induce ER transcriptional activity by binding directly with ER. The other 12 high-brominated OH-PBDEs inhibited luciferase activity of E2 to various degrees, demonstrating their antagonistic activity. Molecular docking analysis of the ER/OH-PBDE complexes revealed two distinctive binding modes between low- and high-brominated OH-PBDEs which provided rationale for the difference in their ER activity.

Estrogen receptor ß sustains epithelial differentiation by regulating prolyl hydroxylase 2 transcription.

Estrogen receptor ß (ERß) promotes the degradation of hypoxia inducible factor 1α (HIF-1α), which contributes to the ability of this hormone receptor to sustain the differentiation of epithelial and carcinoma cells. Although the loss of ERß and consequent HIF-1 activation occur in prostate cancer with profound consequences, the mechanism by which ERß promotes the degradation of HIF-1α is unknown. We report that ERß regulates the ligand (3ß-adiol)-dependent transcription of prolyl hydroxylase 2 (PHD2) also known as Egl nine homolog 1 (EGLN1), a 2-oxoglutarate-dependent dioxygenase that hydroxylates HIF-1α and targets it for recognition by the von Hippel-Lindau tumor suppressor and consequent degradation. ERß promotes PHD2 transcription by interacting with a unique estrogen response element in the 5' UTR of the PHD2 gene that functions as an enhancer. PHD2 itself is critical for maintaining epithelial differentiation. Loss of PHD2 expression or inhibition of its function results in dedifferentiation with characteristics of an epithelial-mesenchymal transition, and exogenous PHD2 expression in dedifferentiated cells can restore an epithelial phenotype. Moreover, expression of HIF-1α in cells that express PHD2 does not induce dedifferentiation but expression of HIF-1α containing mutations in the proline residues that are hydroxylated by PHD2 induces dedifferentiation. These data describe a unique mechanism for the regulation of HIF-1α stability that involves ERß-mediated transcriptional regulation of PHD2 and they highlight an unexpected role for PHD2 in maintaining epithelial differentiation.

Hydroxylated polybrominated diphenyl ethers exhibit different activities on thyroid hormone receptors depending on their degree of bromination.

Polybrominated diphenyl ethers (PBDEs) have been shown to disrupt thyroid hormone (TH) functions in experimental animals, and one of the proposed disruption mechanisms is direct binding of hydroxylated PBDE (OH-PBDE) to TH receptors (TRs). However, previous data on TH receptor binding and TH activity of OH-PBDEs were very limited and sometimes inconsistent. In the present paper, we examined the binding potency of ten OH-PBDEs with different degrees of bromination to TR using a fluorescence competitive binding assay. The results showed that the ten OH-PBDEs bound to TR with potency that correlated to their bromination level. We further examined their effect on TR using a coactivator binding assay and GH3 cell proliferation assay. Different TR activities of OH-PBDEs were observed depending on their degree of bromination. Four low-brominated OH-PBDEs (2'-OH-BDE-28, 3'-OH-BDE-28, 5-OH-BDE-47, 6-OH-BDE-47) were found to be TR agonists, which recruited the coactivator peptide and enhanced GH3 cell proliferation. However, three high-brominated OH-PBDEs (3-OH-BDE-100, 3'-OH-BDE-154, 4-OH-BDE-188) were tested to be antagonists. Molecular docking was employed to simulate the interactions of OH-PBDEs with TR and identify the structural determinants for TR binding and activity. According to the docking results, low-brominated OH-PBDEs, which are weak binders but TR agonists, bind with TR at the inner side of its binding pocket, whereas high-brominated compounds, which are potent binders but TR antagonists, reside at the outer region. These results indicate that OH-PBDEs have different activities on TR (agonistic or antagonistic), possibly due to their different binding geometries with the receptor.

(R)-ß-lysine-modified elongation factor P functions in translation elongation.

Post-translational modification of bacterial elongation factor P (EF-P) with (R)-ß-lysine at a conserved lysine residue activates the protein in vivo and increases puromycin reactivity of the ribosome in vitro. The additional hydroxylation of EF-P at the same lysine residue by the YfcM protein has also recently been described. The roles of modified and unmodified EF-P during different steps in translation, and how this correlates to its physiological role in the cell, have recently been linked to the synthesis of polyproline stretches in proteins. Polysome analysis indicated that EF-P functions in translation elongation, rather than initiation as proposed previously. This was further supported by the inability of EF-P to enhance the rate of formation of fMet-Lys or fMet-Phe, indicating that the role of EF-P is not to specifically stimulate formation of the first peptide bond. Investigation of hydroxyl-(ß)-lysyl-EF-P showed 30% increased puromycin reactivity but no differences in dipeptide synthesis rates when compared with the ß-lysylated form. Unlike disruption of the other genes required for EF-P modification, deletion of yfcM had no phenotypic consequences in Salmonella. Taken together, our findings indicate that EF-P functions in translation elongation, a role critically dependent on post-translational ß-lysylation but not hydroxylation.

JMJD5, a Jumonji C (JmjC) domain-containing protein, negatively regulates osteoclastogenesis by facilitating NFATc1 protein degradation.

Osteoclastogenesis is a highly regulated process governed by diverse classes of regulators. Among them, nuclear factor of activated T-cells calcineurin-dependent 1 (NFATc1) is the primary osteoclastogenic transcription factor, and its expression is transcriptionally induced during early osteoclastogenesis by receptor activation of nuclear factor κB ligand (RANKL), an osteoclastogenic cytokine. Here, we report the novel enzymatic function of JMJD5, which regulates NFATc1 protein stability. Among the tested Jumonji C (JmjC) domain-containing proteins, decreased mRNA expression levels during osteoclastogenesis were found for JMJD5 in RAW264 cells stimulated by RANKL. To examine the functional role of JMJD5 in osteoclast differentiation, we established stable JMJD5 knockdown cells, and osteoclast formation was assessed. Down-regulated expression of JMJD5 led to accelerated osteoclast formation together with induction of several osteoclast-specific genes such as Ctsk and DC-STAMP, suggesting that JMJD5 is a negative regulator in osteoclast differentiation. Although JMJD5 was recently reported as a histone demethylase for histone H3K36me2, no histone demethylase activity was detected in JMJD5 in vitro or in living cells, even for other methylated histone residues. Instead, JMJD5 co-repressed transcriptional activity by destabilizing NFATc1 protein. Protein hydroxylase activity mediated by the JmjC domain in JMJD5 was required for the observed functions of JMJD5. JMJD5 induced the association of hydroxylated NFATc1 with the E3 ubiquitin ligase Von Hippel-Lindau tumor suppressor (VHL), thereby presumably facilitating proteasomal degradation of NFATc1 via ubiquitination. Taken together, the present study demonstrated that JMJD5 is a post-translational co-repressor for NFATc1 that attenuates osteoclastogenesis.

Runx2 protein stabilizes hypoxia-inducible factor-1α through competition with von Hippel-Lindau protein (pVHL) and stimulates angiogenesis in growth plate hypertrophic chondrocytes.

The regulation of hypoxia-inducible factor-1α (HIF-1α) during endochondral bone formation is not fully understood. Here, we investigated the cross-talk between HIF-1α and Runt-related transcription factor 2 (Runx2) in the growth plate. Runx2 caused the accumulation of HIF-1α protein in ATDC5 chondrocytes and HEK293 cells under normoxic conditions. Runx2 also increased the nuclear translocation of HIF-1α when coexpressed in HEK293 cells and interacted with HIF-1α at the oxygen-dependent degradation domain (ODDD). In addition, Runx2 competed with von Hippel-Lindau tumor suppressor protein by directly binding to ODDD-HIF-1α and significantly inhibited the ubiquitination of HIF-1α, even though Runx2 did not change the hydroxylation status of HIF-1α. Furthermore, overexpression of Runx2 resulted in the significant enhancement of vascular endothelial growth factor (VEGF) promoter reporter activity and protein secretion. Runx2 significantly increased angiogenic activity in human umbilical vein endothelial cells in vitro. In wild-type mice, HIF-1α and Runx2 were colocalized in hypertrophic chondrocytes in which the cluster of differentiation 31 (CD31) protein was expressed at embryonic day 15.5 (E15.5). In contrast, the expression of HIF-1α was markedly reduced in areas of CD31 expression in Runx2(-/-) mice. These results suggest that Runx2 stabilizes HIF-1α by binding to ODDD to block the interaction between von Hippel-Lindau protein and HIF-1α. In conclusion, Runx2, HIF-1α, and VEGF may regulate vascular angiogenesis spatially and temporally in the hypertrophic zone of the growth plate during endochondral bone formation.

Hydroxylated estrogens (2-OH-E2 AND 4-OH-E2) do not activate cAMP/PKA and ERK1/2 pathways activation in a breast cancer MCF-7 cell line.

OBJECTIVE: The current study was undertaken to determine the involvement of cAMP/PKA and MAPK-mediated signalling pathways in the regulation of cell proliferation by hydroxylated metabolites of 17ß-estradiol (E2). METHODS: MCF-7, human breast cancer cells, were cultured in phenol red-free DMEM and treated with 1 nM 2-OH-E2 or 4-OH-E2. E2 was used as a positive control. Cell proliferation was measured using the BrdU incorporation assay. Cellular levels of cAMP and PKA were determined using ELISA kits. ERK1/2 protein expression was evaluated by Western Blot analysis. To determine the involvement of different intracellular pathways in the regulation of cell proliferation appropriate activators or inhibitors were used. RESULTS: Hydroxylated estrogens, as E2, exhibited no influence on cAMP accumulation and PKA activation. In concomitant treatments with forskolin, cell proliferation decreased to the amount noted under the influence of forskolin alone. A PKA inhibitor (PKI) had no statistically significant effect on proliferation stimulated by E2 and its hydroxylated metabolites. Phospho-ERK1/2 protein expression in cells stimulated with E2, 2-OH-E2 and 4-OH-E2 was not significantly different from the control. However, co-treatment with both PD98059 and E2 or its hydroxylated metabolites reversed the effect of tested compounds on cell proliferation. CONCLUSIONS: We have shown that E2 hydroxylated metabolites do not activate cAMP/PKA in breast cancer cells and confirm previously published data, which showed a lack of ERK1/2 activation in a breast cancer cell line. The observed reversible action of PD98059 on cell proliferation can be explained by the fact that hydroxylated estrogens, as E2, stimulate secretion of a number of growth factors, which affect MAPK activity, suggested by Lobenhofer et al. (2000).