N-linoleoylamino acids as chiral probes of substrate binding by soybean lipoxygenase-1.

Lipoxygenases catalyze the oxygenation of polyunsaturated fatty acids and their derivatives to produce conjugated diene hydroperoxides. Soybean lipoxygenase-1 (SBLO-1) has been the subject of intensive structural and mechanistic study, but the manner in which this enzyme binds substrates is uncertain. Previous studies suggest that the fatty acyl group of the substrate binds in an internal cavity near the catalytic iron with the polar end at the surface of the protein or perhaps external to the protein. To test this model, we have investigated two pairs of enantiomeric N-linoleoylamino acids as substrates for SBLO-1. If the amino acid moiety binds external to the protein, the kinetics and product distribution should show little or no sensitivity to the stereochemical configuration of the amino acid moiety. Consistent with this expectation, N-linoleoyl-l-valine (LLV) and N-linoleoyl-d-valine (LDV) are both good substrates with kcat/Km values that are equal within error and about 40% higher than kcat/Km for linoleic acid. Experiments with N-linoleoyl-l-tryptophan (LLT) and N-linoleoyl-d-tryptophan (LDT) were complicated by the low critical micelle concentrations (CMC = 6-8 µM) of these substances. Below the CMC, LDT is a better substrate by a factor of 2.7. The rates of oxygenation of LDT and LLT continue to rise above the CMC, with modest stereoselectivity in favor of the d enantiomer. With all of the substrates tested, the major product is the 13(S)-hydroperoxide, and the distribution of minor products is not appreciably affected by the configuration of the amino acid moiety. The absence of stereoselectivity with LLV and LDV, the modest magnitude of the stereoselectivity with LLT and LDT, and the ability micellar forms of LLT and LDT to increase the concentration of available substrate are all consistent with the hypothesis that the amino acid moiety binds largely external to SBLO-1 and interacts with it only weakly.

Spontaneous mutation of Dock7 results in lower trabecular bone mass and impaired periosteal expansion in aged female Misty mice.

Misty mice (m/m) have a loss of function mutation in Dock7 gene, a guanine nucleotide exchange factor, resulting in low bone mineral density, uncoupled bone remodeling and reduced bone formation. Dock7 has been identified as a modulator of osteoblast number and in vitro osteogenic differentiation in calvarial osteoblast culture. In addition, m/m exhibit reduced preformed brown adipose tissue innervation and temperature as well as compensatory increase in beige adipocyte markers. While the low bone mineral density phenotype is in part due to higher sympathetic nervous system (SNS) drive in young mice, it is unclear what effect aging would have in mice homozygous for the mutation in the Dock7 gene. We hypothesized that age-related trabecular bone loss and periosteal envelope expansion would be altered in m/m. To test this hypothesis, we comprehensively characterized the skeletal phenotype of m/m at 16, 32, 52, and 78wks of age. When compared to age-matched wild-type control mice (+/+), m/m had lower areal bone mineral density (aBMD) and areal bone mineral content (aBMC). Similarly, both femoral and vertebral BV/TV, Tb.N, and Conn.D were decreased in m/m while there was also an increase in Tb.Sp. As low bone mineral density and decreased trabecular bone were already present at 16wks of age in m/m and persisted throughout life, changes in age-related trabecular bone loss were not observed highlighting the role of Dock7 in controlling trabecular bone acquisition or bone loss prior to 16wks of age. Cortical thickness was also lower in the m/m across all ages. Periosteal and endosteal circumferences were higher in m/m compared to +/+ at 16wks. However, endosteal and periosteal expansion were attenuated in m/m, resulting in m/m having lower periosteal and endosteal circumferences by 78wks of age compared to +/+, highlighting the critical role of Dock7 in appositional bone expansion. Histomorphometry revealed that osteoblasts were nearly undetectable in m/m and marrow adipocytes were elevated 3.5 fold over +/+ (p=0.014). Consistent with reduced bone formation, osteoblast gene expression of Alp, Col1a1, Runx-2, Sp7, and Bglap was significantly decreased in m/m whole bone. Furthermore, markers of osteoclasts were either unchanged or suppressed. Bone marrow stromal cell migration and motility were inhibited in culture and changes in senescence markers suggest that osteoblast function may also be inhibited with loss of Dock7 expression in m/m. Finally, increased Oil Red O staining in m/m ear mesenchymal stem cells during adipogenesis highlights a potential shift of cells from the osteogenic to adipogenic lineages. In summary, loss of Dock7 in the aging m/m resulted in an impairment of periosteal and endocortical envelope expansion, but did not alter age-related trabecular bone loss. These studies establish Dock7 as a critical regulator of both cortical and trabecular bone mass, and demonstrate for the first time a novel role of Dock7 in modulating compensatory changes in the periosteum with aging.

CRISPR/Cas9-Mediated Insertion of loxP Sites in the Mouse Dock7 Gene Provides an Effective Alternative to Use of Targeted Embryonic Stem Cells.

Targeted gene mutation in the mouse is a primary strategy to understand gene function and relation to phenotype. The Knockout Mouse Project (KOMP) had an initial goal to develop a public resource of mouse embryonic stem (ES) cell clones that carry null mutations in all genes. Indeed, many useful novel mouse models have been generated from publically accessible targeted mouse ES cell lines. However, there are limitations, including incorrect targeting or cassette structure, and difficulties with germline transmission of the allele from chimeric mice. In our experience, using a small sample of targeted ES cell clones, we were successful ∼50% of the time in generating germline transmission of a correctly targeted allele. With the advent of CRISPR/Cas9 as a mouse genome modification tool, we assessed the efficiency of creating a conditional targeted allele in one gene, dedicator of cytokinesis 7 (Dock7), for which we were unsuccessful in generating a null allele using a KOMP targeted ES cell clone. The strategy was to insert loxP sites to flank either exons 3 and 4, or exons 3 through 7. By coinjecting Cas9 mRNA, validated sgRNAs, and oligonucleotide donors into fertilized eggs from C57BL/6J mice, we obtained a variety of alleles, including mice homozygous for the null alleles mediated by nonhomologous end joining, alleles with one of the two desired loxP sites, and correctly targeted alleles with both loxP sites. We also found frequent mutations in the inserted loxP sequence, which is partly attributable to the heterogeneity in the original oligonucleotide preparation.

Transcriptional regulation of the human TNFSF11 gene in T cells via a cell type-selective set of distal enhancers.

In addition to osteoblast lineage cells, the TNF-like factor receptor activator of NF-κB ligand (RANKL) is expressed in both B and T cells and may play a role in bone resorption. Rankl gene (Tnfsf11) expression in mouse T cells is mediated through multiple distal elements marked by increased transcription factor occupancy, histone tail acetylation, and RNA polymerase II recruitment. Little is known, however, of the regulation of human TNFSF11 in T cells. Accordingly, we examined the consequence of T cell activation on the expression of this factor both in Jurkat cells and in primary human T cells. We then explored the mechanism of this regulation by scanning over 400 kb of DNA surrounding the TNFSF11 locus for regulatory enhancers using ChIP-chip analysis. Histone H3/H4 acetylation enrichment identified putative regulatory regions located between -170 and -220 kb upstream of the human TNFSF11 TSS that we designated the human T cell control region (hTCCR). This region showed high sequence conservation with the mouse TCCR. Inhibition of MEK1/2 by U0126 resulted in decreased RANKL expression suggesting that stimulation through MEK1/2 was a prerequisite. ChIP-chip analysis also revealed that c-FOS was recruited to the hTCCR as well. Importantly, both the human TNFSF11 D5a/b (RLD5a/b) enhancer and segments of the hTCCR mediated robust inducible reporter activity following TCR activation. Finally, SNPs implicated in diseases characterized by dysregulated BMD co-localized to the hTCCR region. We conclude that the hTCCR region contains a cell-selective set of enhancers that plays an integral role in the transcriptional regulation of the TNFSF11 gene in human T cells.

A DNA segment spanning the mouse Tnfsf11 transcription unit and its upstream regulatory domain rescues the pleiotropic biologic phenotype of the RANKL null mouse.

Receptor activator of NF-κB ligand (RANKL) is a TNFα-like cytokine that is produced by a diverse set of lineage-specific cells and is involved in a wide variety of physiological processes that include skeletal remodeling, lymph node organogenesis, mammary gland development, and thermal regulation. Consistent with these diverse functions, control of RANKL expression is accomplished in a cell-specific fashion via a set of at least 10 regulatory enhancers that are located up to 170 kb upstream of the gene's transcriptional start site. Here we examined the in vivo consequence of introducing a contiguous DNA segment containing these components into a genetically deleted RANKL null mouse strain. In contrast to RANKL null littermates, null mice containing the transgene exhibited normalized body size, skeletal development, and bone mass as well as normal bone marrow cavities, normalized spleen weights, and the presence of developed lymph nodes. These mice also manifested normalized reproductive capacity, including the ability to lactate and to produce normal healthy litters. Consistent with this, the transgene restored endogenous-like RANKL transcript levels in several RANKL-expressing tissues. Most importantly, restoration of RANKL expression from this segment of DNA was fully capable of rescuing the complex aberrant skeletal and immune phenotype of the RANKL null mouse. RANKL also restored appropriate levels of B220+ IgM+ and B220+ IgD+ B cells in spleen. Finally, we found that RANKL expression from this transgene was regulated by exogenously administered 1,25(OH)2 D3 , parathyroid hormone (PTH), and lipopolysaccharide (LPS), thus recapitulating the ability of these same factors to regulate the endogenous gene. These findings fully highlight the properties of the Tnfsf11 gene locus predicted through previous in vitro dissection. We conclude that the mouse Tnfsf11 gene locus identified originally through unbiased chromatin immunoprecipitation with DNA microarray (ChIP-chip) analysis contains the necessary genetic information to direct appropriate tissue-specific and factor-regulated RANKL expression in vivo.

The osteoblast to osteocyte transition: epigenetic changes and response to the vitamin D3 hormone.

Osteocytes are derived from osteoblast lineage cells that become progressively embedded in mineralized bone. Development of the osteocytogenic cell line IDG-SW3 has enabled a temporal and mechanistic investigation of this process. Through RNA-sequencing analyses, we show that although substantial changes in gene expression occur during the osteoblast to osteocyte transition, the majority of the transcriptome remains qualitatively osteoblast like. Genes either up-regulated or expressed uniquely in the osteocyte include local and systemic factors such as Sost and Fgf23 as well as genes implicated in neuronal, muscle, vascular, or regulatory function. As assessed by chromatin immunoprecipitation coupled to high-throughput sequencing, numerous changes in epigenetic histone modifications also occur during osteocytogenesis; these are largely qualitative rather than quantitative. Specific epigenetic changes correlate with altered gene expression patterns that are observed during the transition. These genomic changes likely influence the highly restricted transcriptomic response to 1,25(OH)(2)D(3) that occurs during differentiation. VDR binding in osteocytes revealed an extensive cistrome co-occupied by retinoid X receptor and located predominantly at sites distal to regulated genes. Although sites of VDR binding were apparent near many 1,25(OH)(2)D(3)-regulated genes, the expression of others adjacent to VDR-binding sites were unaffected; lack of VDR binding was particularly prevalent at down-regulated genes. Interestingly, 1,25(OH)(2)D(3) was found to induce the Boc and Cdon coreceptors that are active in hedgehog signaling in osteocytes. We conclude that osteocytogenesis is accompanied by changes in gene expression that may be driven by both genetic and epigenetic components. These changes are likely responsible for the osteocyte phenotype and may contribute to reduced sensitivity to 1,25(OH)(2)D(3).

Mouse and human BAC transgenes recapitulate tissue-specific expression of the vitamin D receptor in mice and rescue the VDR-null phenotype.

The biological actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) are mediated by the vitamin D receptor (VDR), which is expressed in numerous target tissues in a cell type-selective manner. Recent studies using genomic analyses and recombineered bacterial artificial chromosomes (BACs) have defined the specific features of mouse and human VDR gene loci in vitro. In the current study, we introduced recombineered mouse and human VDR BACs as transgenes into mice and explored their expression capabilities in vivo. Individual transgenic mouse strains selectively expressed BAC-derived mouse or human VDR proteins in appropriate vitamin D target tissues, thereby recapitulating the tissue-specific expression of endogenous mouse VDR. The mouse VDR transgene was also regulated by 1,25(OH)2D3 and dibutyryl-cAMP. When crossed into a VDR-null mouse background, both transgenes restored wild-type basal as well as 1,25(OH)2D3-inducible gene expression patterns in the appropriate tissues. This maneuver resulted in the complete rescue of the aberrant phenotype noted in the VDR-null mouse, including systemic features associated with altered calcium and phosphorus homeostasis and disrupted production of parathyroid hormone and fibroblast growth factor 23, and abnormalities associated with the skeleton, kidney, parathyroid gland, and the skin. This study suggests that both mouse and human VDR transgenes are capable of recapitulating basal and regulated expression of the VDR in the appropriate mouse tissues and restore 1,25(OH)2D3 function. These results provide a baseline for further dissection of mechanisms integral to mouse and human VDR gene expression and offer the potential to explore the consequence of selective mutations in VDR proteins in vivo.

Progesterone receptor and Stat5 signaling cross talk through RANKL in mammary epithelial cells.

Progesterone (P4) stimulates proliferation of the mammary epithelium by a mechanism that involves paracrine signaling mediated from progesterone receptor (PR)-positive to neighboring PR-negative cells. Here we used a primary mouse mammary epithelial cell (MEC) culture system to define the molecular mechanism by which P4 regulates the expression of target gene effectors of proliferation including the paracrine factor receptor and activator of nuclear factor κB ligand (RANKL). MECs from adult virgin mice grown and embedded in three-dimensional basement-membrane medium resemble mammary ducts in vivo structurally and with respect to other properties including a heterogeneous pattern of PR expression, P4 induction of RANKL and other target genes in a PR-dependent manner, and a proliferative response to progestin. RANKL was demonstrated to have multiple functional P4-responsive enhancers that bind PR in a hormone-dependent manner as detected by chromatin immunoprecipitation assay. P4 also stimulated recruitment of signal transducer and activator of transcription (Stat)5a to RANKL enhancers through an apparent tethering with PR. Analysis of primary MECs from Stat5a knockout mice revealed that P4 induction of RANKL and a broad range of other PR target genes required Stat5a, as did P4-stimulated cell proliferation. In the absence of Stat5a, PR binding was lost at selective RANKL enhancers but was retained with others, suggesting that Stat5a acts to facilitate PR DNA binding at selective sites and to function as a coactivator with DNA-bound PR at others. These results show that RANKL is a direct PR target gene and that Stat5a has a novel role as a cofactor in PR-mediated transcriptional signaling in the mammary gland.

Altered thermogenesis and impaired bone remodeling in Misty mice.

Fat mass may be modulated by the number of brown-like adipocytes in white adipose tissue (WAT) in humans and rodents. Bone remodeling is dependent on systemic energy metabolism and, with age, bone remodeling becomes uncoupled and brown adipose tissue (BAT) function declines. To test the interaction between BAT and bone, we employed Misty (m/m) mice, which were reported be deficient in BAT. We found that Misty mice have accelerated age-related trabecular bone loss and impaired brown fat function (including reduced temperature, lower expression of Pgc1a, and less sympathetic innervation compared to wild-type (+/ +)). Despite reduced BAT function, Misty mice had normal core body temperature, suggesting heat is produced from other sources. Indeed, upon acute cold exposure (4°C for 6 hours), inguinal WAT from Misty mice compensated for BAT dysfunction by increasing expression of Acadl, Pgc1a, Dio2, and other thermogenic genes. Interestingly, acute cold exposure also decreased Runx2 and increased Rankl expression in Misty bone, but only Runx2 was decreased in wild-type. Browning of WAT is under the control of the sympathetic nervous system (SNS) and, if present at room temperature, could impact bone metabolism. To test whether SNS activity could be responsible for accelerated trabecular bone loss, we treated wild-type and Misty mice with the ß-blocker, propranolol. As predicted, propranolol slowed trabecular bone volume/total volume (BV/TV) loss in the distal femur of Misty mice without affecting wild-type. Finally, the Misty mutation (a truncation of DOCK7) also has a significant cell-autonomous role. We found DOCK7 expression in whole bone and osteoblasts. Primary osteoblast differentiation from Misty calvaria was impaired, demonstrating a novel role for DOCK7 in bone remodeling. Despite the multifaceted effects of the Misty mutation, we have shown that impaired brown fat function leads to altered SNS activity and bone loss, and for the first time that cold exposure negatively affects bone remodeling.

Human invariant natural killer T cells acquire transient innate responsiveness via histone H4 acetylation induced by weak TCR stimulation.

Invariant NKT cells (iNKT cells) are innate T lymphocytes that are thought to play an important role in producing an early burst of IFN-γ that promotes successful tumor immunosurveillance and antimicrobial immunity. The cellular activation processes underlying innate IFN-γ production remain poorly understood. We show here that weak T cell receptor (TCR) stimulation that does not directly activate iNKT cell IFN-γ messenger RNA transcription nevertheless induces histone H4 acetylation at specific regions near the IFNG gene locus. This renders the iNKT cells able to produce IFN-γ in an innate manner (i.e., not requiring concurrent TCR stimulation) upon exposure to IL-12 and IL-18. The iNKT cells retain the capacity for innate activation for hours to days after the initial weak TCR stimulation, although their innate responsiveness gradually declines as a function of histone deacetylation. These results explain how iNKT cells are able to mediate rapid innate IFN-γ secretion in a manner that does not require them to undergo permanent T(H1) differentiation. Moreover, our results also indicate that iNKT cell motility is maintained during activation by IL-12 and IL-18. Therefore, iNKT cells activated through this pathway can continue to migrate and may thus disseminate the IFN-γ that they produce, which may amplify its impact.

Regulation of target gene expression by the vitamin D receptor - an update on mechanisms.

Virtually all of the known biological actions of the hormonal ligand 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) are mediated by the vitamin D receptor (VDR). Following binding and activation by the ligand, the VDR localizes in the nucleus to the regulatory regions of target genes and recruits chromatin-active coregulatory complexes which, in turn, modulate transcriptional output. The failure of the VDR to function due to crippling mutations results in total hereditary resistance to 1,25(OH)(2)D(3) in both mice and humans. In this review, we summarize the structural and functional properties of the VDR and the role of 1,25(OH)(2)D(3) in receptor activation, and then describe the results of recent studies using genome-wide analyses that define the overarching principles through which the VDR modulates genes expression. We also focus on the recent analysis of a specific 1,25(OH)(2)D(3) regulated gene that provides confirmation of the principles identified through these genome-wide methodologies. Taken together, these studies suggest an unanticipated increase in the complexity of the molecular processes that govern gene regulation by hormones and other factors.

Mouse Rankl expression is regulated in T cells by c-Fos through a cluster of distal regulatory enhancers designated the T cell control region.

Receptor activator of NF-κB ligand (Rankl) is a TNF-like factor that induces the formation of osteoclasts responsible for bone resorption. Although T cell activation up-regulates this gene, the molecular mechanism of its transcriptional control remains unknown. We used ChIP-chip analysis in mouse primary T cells and a T cell hybridoma to define the regulatory enhancers responsible for this up-regulation and to characterize their properties. Elevated H3/H4 acetylation and increased RNA polymerase II density were evident at mRL-D5, a known enhancer located 76 kb upstream of the TSS, as well as at a cluster of regulatory sites located even further upstream between -123 to -156 kb, termed the T cell control region (TCCR). Based upon the ability of calcium signaling and MAPK inhibitors to block Rankl expression, we conducted further ChIP-chip analysis of the transcriptional mediators c-Fos, NF-κB, and Nfat. T cell activation induced c-Fos binding at the mRL-D5 enhancer and within the TCCR. The interaction of NF-κB was observed at the transcriptional start site and at mRL-D5. Both mRL-D5 and segments of the TCCR exhibited robust transcriptional activity in reporter assays, and site-specific mutagenesis of c-Fos and Nfat elements abrogated reporter activity, suggesting a role for both factors in the control of enhancer-mediated Rankl transcription. Finally, chromosome conformation capture analysis confirmed that mRL-D5 and segments of the TCCR were located in proximity to the Rankl gene promoter and thus potentially able to influence directly Rankl gene promoter activity. We conclude that both mRL-D5 and the TCCR represent control segments that play an integral role in Rankl expression in T cells.

Cationic substrates of soybean lipoxygenase-1.

Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of 1,4-dienes to produce conjugated diene hydroperoxides. The best substrates are anions of fatty acids; for example, linoleate is converted to 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoate. The manner in which SBLO-1 binds substrates is uncertain. In the present work, it was found that SBLO-1 will oxygenate linoleyltrimethylammonium ion (LTMA) to give primarily13(S)-hydroperoxy-9(Z),11(E)-octadecadienyltrimethylammonium ion. The rate of this process is about the same at pH 7 and pH 9 and is about 30% of the rate observed with linoleate at pH 9. At pH 7, SBLO-1 oxygenates linoleyldimethylamine (LDMA) to give primarily 13(S)-hydroperoxy-9(Z),11(E)-octadecadienyldimethylamine. The oxygenation of LDMA occurs at about the same rate as LTMA at pH 7, but more slowly at pH 9. The results demonstrate that SBLO-1 will readily oxygenate substrates in which the carboxylate of linoleate is replaced with a cationic group, and the products of these reactions have the same stereo- and regiochemistry as the products obtained from fatty acid substrates.

Emerging regulatory paradigms for control of gene expression by 1,25-dihydroxyvitamin D3.

1,25-dihydroxyvitamin D3 (1,25(OH)2D3) functions as a steroid hormone to modulate the expression of genes. Its actions are mediated by the vitamin D receptor (VDR) which binds to target genes and functions to recruit coregulatory complexes that are essential for transcriptional modulation. ChIP analysis coupled to tiled DNA microarray hybridization (ChIP-chip) or massively parallel DNA sequencing (ChIP-seq) is now providing critical new insight into how genes are regulated. In studies herein, we utilized these techniques as well as gene expression analysis to explore the actions of 1,25(OH)2D3 at the genome-wide and individual target gene levels in cells. We identify a series of overarching principles that likely define the actions of 1,25(OH)2D3 at most target genes. We discover that while VDR binding to target sites is ligand-dependent, RXR binding is ligand-independent. We also show that while VDR/RXR binding can localize to promoters, it occurs more frequently at multiple sites many kilobases from target gene promoters. We then describe a new method whereby the regulatory regions of complex genes can be evaluated using large recombineered bacterial artificial chromosomes. We conclude that these new approaches are likely to replace many of the traditional methods used to explore the regulation of transcription.

A novel distal enhancer mediates cytokine induction of mouse RANKl gene expression.

Chronic inflammatory states are associated with increased bone loss. This increase is often linked to an elevation in receptor activator of nuclear factor-kappaB ligand (RANKL), a TNFalpha-like factor essential to osteoclast formation. In this study, we document the ability of IL-6 in combination with IL-6 soluble receptor (IL-6/IL-6sR) and oncostatin M to induce Rankl expression in stromal cells via signal transducer and activator of transcription 3 (STAT3). We used chromatin immunoprecipitation-tiled DNA microarray analysis to determine sites of action of STAT3 at the Rankl locus and to assess the consequences of binding on histone H4 acetylation and RNA polymerase II recruitment. Both IL-6/IL-6 soluble receptor and oncostatin M stimulated STAT3 binding upstream of the Rankl transcriptional start site. Although previously identified enhancers bound STAT3, a more distal enhancer termed mRLD6 was a particular focus of STAT3 binding. When fused to a heterologous promoter, this enhancer was highly active, containing two functionally active STAT response elements. Importantly, small interfering RNA knockdown of Stat3 mRNA and protein, but not that of Stat1 or Stat5a, was effective in limiting Rankl mRNA up-regulation. Interestingly, although RNA polymerase II and histone H4 acetylation marked many of the enhancers under basal conditions, the levels of both were strongly increased after cytokine treatment, particularly at mRLD6. Finally, mRLD6 was also a target for forskolin-induced cellular response element-binding protein (CREB) recruitment, which potentiated cytokine activity. Our studies provide new insight into mechanisms by which glycoprotein 130 activating cytokines induce RANKL expression.