Emergent Coordination of the CHKB and CPT1B Genes in Eutherian Mammals: Implications for the Origin of Brown Adipose Tissue.

Mitochondrial fatty acid oxidation (FAO) contributes to the proton motive force that drives ATP synthesis in many mammalian tissues. In eutherian (placental) mammals, brown adipose tissue (BAT) can also dissipate this proton gradient through uncoupling protein 1 (UCP1) to generate heat, but the evolutionary events underlying the emergence of BAT are unknown. An essential step in FAO is the transport of cytoplasmic long chain acyl-coenzyme A (acyl-CoA) into the mitochondrial matrix, which requires the action of carnitine palmitoyltransferase 1B (CPT1B) in striated muscle and BAT. In eutherians, the CPT1B gene is closely linked to the choline kinase beta (CHKB) gene, which is transcribed from the same DNA strand and terminates just upstream of CPT1B. CHKB is a rate-limiting enzyme in the synthesis of phosphatidylcholine (PC), a predominant mitochondrial membrane phospholipid, suggesting that the coordinated expression of CHKB and CPT1B may cooperatively enhance mitochondrial FAO. The present findings show that transcription of the eutherian CHKB and CPT1B genes is linked within a unitary epigenetic domain targeted to the CHKB gene, and that that this regulatory linkage appears to have resulted from an intergenic deletion in eutherians that significantly altered the distribution of CHKB and CPT1B expression. Informed by the timing of this event relative to the emergence of BAT, the phylogeny of CHKB-CPT1B synteny, and the insufficiency of UCP1 to account for eutherian BAT, these data support a mechanism for the emergence of BAT based on the acquisition of a novel capacity for adipocyte FAO in a background of extant UCP1.

High Fat Diet Dysregulates Hypothalamic-Pituitary Axis Gene Expression Levels which are Differentially Rescued by EPA and DHA Ethyl Esters.

SCOPE: Dietary fat composition can modulate gene expression in peripheral tissues in obesity. Observations of the dysregulation of growth hormone (GH) in obesity indicate that these effects extend to the hypothalamic-pituitary (H-P) axis. The authors thus determine whether specific high fat (HF) diets influence the levels of Gh and other key gene transcripts in the H-P axis. METHODS AND RESULTS: C57BL/6 mice are fed a lean control diet or a HF diet in the absence or presence of OA, EPA, or DHA ethyl esters. Comparative studies are conducted with menhaden fish oil. The HF diet lowered pituitary Gh mRNA and protein levels, and cell culture studies reveal that elevated insulin and glucose can reduce Gh transcripts. Supplementation of the HF diet with OA, EPA, DHA, or menhaden fish oil do not improve pituitary Gh levels. The HF diet also impaired the levels of additional genes in the pituitary and hypothalamus, which are selectively rescued with EPA or DHA ethyl esters. The effects of EPA and DHA are more robust relative to fish oil. CONCLUSION: A HF diet can affect H-P axis transcription, which can be mitigated in some genes by EPA and DHA, but not fish oil in most cases.

RARα/RXR synergism potentiates retinoid responsiveness in cutaneous T-cell lymphoma cell lines.

Retinoids, natural and synthetic derivatives of vitamin A, induce cellular changes by activating nuclear retinoic acid receptors (RAR) and retinoid X receptors (RXR). Although the ability of retinoids to govern gene expression is exploited clinically for cancer therapeutics, the full benefit of retinoid-based strategies is unrealized due to detrimental side effects. Delineating the receptors that prompt cellular outcomes is critical to advancing retinoid-based approaches. Here, we identify the receptors that evoke multiple responses in cutaneous T-cell lymphoma (CTCL). The data demonstrate that RARα drives integrin ß7-dependent adhesion and CCR9-mediated chemotaxis in CTCL cells. Of note, concomitant activation of RARα and RXR nuclear receptors yielded synergistic increases in adhesion and migration at concentrations where single agents were ineffective. As the established paradigm of retinoid action in CTCL is apoptosis and growth arrest, the role of RARα/RXR in these events was studied. As with adhesion and migration, RARα/RXR synergism prompted apoptosis and dampened CTCL cell proliferation. Strikingly, RARα/RXR synergism induced responses from CTCL cell lines previously reported to be unresponsive to retinoids. These data provide a novel framework that may further refine a proven CTCL therapy.

Soluble guanylyl cyclase-activated cyclic GMP-dependent protein kinase inhibits arterial smooth muscle cell migration independent of VASP-serine 239 phosphorylation.

Coronary artery disease (CAD) accounts for over half of all cardiovascular disease-related deaths. Uncontrolled arterial smooth muscle (ASM) cell migration is a major component of CAD pathogenesis and efforts aimed at attenuating its progression are clinically essential. Cyclic nucleotide signaling has long been studied for its growth-mitigating properties in the setting of CAD and other vascular disorders. Heme-containing soluble guanylyl cyclase (sGC) synthesizes cyclic guanosine monophosphate (cGMP) and maintains vascular homeostasis predominantly through cGMP-dependent protein kinase (PKG) signaling. Considering that reactive oxygen species (ROS) can interfere with appropriate sGC signaling by oxidizing the cyclase heme moiety and so are associated with several CVD pathologies, the current study was designed to test the hypothesis that heme-independent sGC activation by BAY 60-2770 (BAY60) maintains cGMP levels despite heme oxidation and inhibits ASM cell migration through phosphorylation of the PKG target and actin-binding vasodilator-stimulated phosphoprotein (VASP). First, using the heme oxidant ODQ, cGMP content was potentiated in the presence of BAY60. Using a rat model of arterial growth, BAY60 significantly reduced neointima formation and luminal narrowing compared to vehicle (VEH)-treated controls. In rat ASM cells BAY60 significantly attenuated cell migration, reduced G:F actin, and increased PKG activity and VASP Ser239 phosphorylation (pVASP·S239) compared to VEH controls. Site-directed mutagenesis was then used to generate overexpressing full-length wild type VASP (FL-VASP/WT), VASP Ser239 phosphorylation-mimetic (FL-VASP/239D) and VASP Ser239 phosphorylation-resistant (FL-VASP/239A) ASM cell mutants. Surprisingly, FL-VASP/239D negated the inhibitory effects of FL-VASP/WT and FL-VASP/239A cells on migration. Furthermore, when FL-VASP mutants were treated with BAY60, only the FL-VASP/239D group showed reduced migration compared to its VEH controls. Intriguingly, FL-VASP/239D abrogated the stimulatory effects of FL-VASP/WT and FL-VASP/239A cells on PKG activity. In turn, pharmacologic blockade of PKG in the presence of BAY60 reversed the inhibitory effect of BAY60 on naïve ASM cell migration. Taken together, we demonstrate for the first time that BAY60 inhibits ASM cell migration through cGMP/PKG/VASP signaling yet through mechanisms independent of pVASP·S239 and that FL-VASP overexpression regulates PKG activity in rat ASM cells. These findings implicate BAY60 as a potential pharmacotherapeutic agent against aberrant ASM growth disorders such as CAD and also establish a unique mechanism through which VASP controls PKG activity.

Differential epigenetic and transcriptional response of the skeletal muscle carnitine palmitoyltransferase 1B (CPT1B) gene to lipid exposure with obesity.

The ability to increase fatty acid oxidation (FAO) in response to dietary lipid is impaired in the skeletal muscle of obese individuals, which is associated with a failure to coordinately upregulate genes involved with FAO. While the molecular mechanisms contributing to this metabolic inflexibility are not evident, a possible candidate is carnitine palmitoyltransferase-1B (CPT1B), which is a rate-limiting step in FAO. The present study was undertaken to determine if the differential response of skeletal muscle CPT1B gene transcription to lipid between lean and severely obese subjects is linked to epigenetic modifications (DNA methylation and histone acetylation) that impact transcriptional activation. In primary human skeletal muscle cultures the expression of CPT1B was blunted in severely obese women compared with their lean counterparts in response to lipid, which was accompanied by changes in CpG methylation, H3/H4 histone acetylation, and peroxisome proliferator-activated receptor-δ and hepatocyte nuclear factor 4α transcription factor occupancy at the CPT1B promoter. Methylation of specific CpG sites in the CPT1B promoter that correlated with CPT1B transcript level blocked the binding of the transcription factor upstream stimulatory factor, suggesting a potential causal mechanism. These findings indicate that epigenetic modifications may play important roles in the regulation of CPT1B in response to a physiologically relevant lipid mixture in human skeletal muscle, a major site of fatty acid catabolism, and that differential DNA methylation may underlie the depressed expression of CPT1B in response to lipid, contributing to the metabolic inflexibility associated with severe obesity.

Lipid exposure elicits differential responses in gene expression and DNA methylation in primary human skeletal muscle cells from severely obese women.

The skeletal muscle of obese individuals exhibits an impaired ability to increase the expression of genes linked with fatty acid oxidation (FAO) upon lipid exposure. The present study determined if this response could be attributed to differential DNA methylation signatures. RNA and DNA were isolated from primary human skeletal muscle cells (HSkMC) from lean and severely obese women following lipid incubation. mRNA expression and DNA methylation were quantified for genes that globally regulate FAO [PPARγ coactivator (PGC-1α), peroxisome proliferator-activated receptors (PPARs), nuclear respiratory factors (NRFs)]. With lipid oversupply, increases in NRF-1, NRF-2, PPARα, and PPARδ expression were dampened in skeletal muscle from severely obese compared with lean women. The expression of genes downstream of the PPARs and NRFs also exhibited a pattern of not increasing as robustly upon lipid exposure with obesity. Increases in CpG methylation near the transcription start site with lipid oversupply were positively related to PPARδ expression; increases in methylation with lipid were depressed in HSkMC from severely obese women. With severe obesity, there is an impaired ability to upregulate global transcriptional regulators of FAO in response to lipid exposure. Transient changes in DNA methylation patterns and differences in the methylation signature with severe obesity may play a role in the transcriptional regulation of PPARδ in response to lipid. The persistence of differential responses to lipid in HSkMC derived from lean and obese subjects supports the possibility of stable epigenetic programming of skeletal muscle cells by the respective environments.

Growth hormone enhances LNCaP prostate cancer cell motility.

PURPOSE: Prostate cancer cells are responsive to multiple hormones and growth factors that can affect cell function. These effects may include modulating cell proliferation and apoptosis, but the ability to impinge on the metastatic potential of prostate cancer cells by affecting cell motility should also be considered, as prostate tumor metastasis correlates with limited therapeutic options and poor prognosis. Human growth hormone (hGH) can affect the growth and survival of prostate cancer cells, but the effect of hGH on prostate cancer cell motility is unknown. In the present study, the potential for exogenous and autocrine hGH to directly affect prostate cancer cell motility was addressed. MATERIALS AND METHODS: The effects of exogenous and autocrine hGH on the chemokinesis and chemotaxis of LNCaP prostate cancer cells were tested using cell monolayer wound healing and Boyden chamber invasion assays. The signaling pathways underlying these effects were resolved with chemical inhibitors and the correlation with cytoskeletal actin reorganization evaluated microscopically by staining cells with fluor-conjugated phalloidin. RESULTS: Both exogenous and autocrine hGH augmented the migration and invasion of LNCaP cells, and hGH itself acted as a chemoattractant. This activity was dependent upon the STAT5, MEK1/2 and PI3K signaling pathways, and was accompanied by an alteration in cellular actin organization. CONCLUSIONS: hGH may enhance the metastatic potential of prostate cancer cells, both as a stimulant of cellular motility and invasiveness and as a chemoattractant.

Prostaglandins and n-3 polyunsaturated fatty acids in the regulation of the hypothalamic-pituitary axis.

The hypothalamic-pituitary (H-P) axis integrates complex physiological and environmental signals and responds to these cues by modulating the synthesis and secretion of multiple pituitary hormones to regulate peripheral tissues. Prostaglandins are a component of this regulatory system, affecting multiple hormone synthesis and secretion pathways in the H-P axis. The implications of these actions are that physiological processes or disease states that alter prostaglandin levels in the hypothalamus or pituitary can impinge on H-P axis function. Considering the role of prostaglandins in mediating inflammation, the potential for neuroinflammation to affect H-P axis function in this manner may be significant. In addition, the mitigating effects of n-3 polyunsaturated fatty acids (n-3 PUFA) on the inflammation-associated synthesis of prostaglandins and their role as substrates for pro-resolving lipid mediators may also include effects in the H-P axis. One context in which neuroinflammation may play a role is in the etiology of diet-induced obesity, which also correlates with altered pituitary hormone levels. This review will survey evidence for the actions of prostaglandins and other lipid mediators in the H-P axis, and will address the potential for obesity-associated inflammation and n-3 PUFA to impinge on these mechanisms.

Eicosapentaenoic and docosahexaenoic acid ethyl esters differentially enhance B-cell activity in murine obesity.

EPA and DHA are not biologically equivalent; however, their individual activity on B cells is unknown. We previously reported fish oil enhanced murine B-cell activity in obesity. To distinguish between the effects of EPA and DHA, we studied the ethyl esters of EPA and DHA on murine B-cell function as a function of time. We first demonstrate that EPA and DHA maintained the obese phenotype, with no improvements in fat mass, adipose inflammatory cytokines, fasting insulin, or glucose clearance. We then tested the hypothesis that EPA and DHA would increase the frequency of splenic B cells. EPA and DHA differentially enhanced the frequency and/or percentage of select B-cell subsets, correlating with increased natural serum IgM and cecal IgA. We next determined the activities of EPA and DHA on ex vivo production of cytokines upon lipopolysaccharide stimulation of B cells. EPA and DHA, in a time-dependent manner, enhanced B-cell cytokines with DHA notably increasing IL-10. At the molecular level, EPA and DHA differentially enhanced the formation of ordered microdomains but had no effect on Toll-like receptor 4 mobility. Overall, the results establish differential effects of EPA and DHA in a time-dependent manner on B-cell activity in obesity, which has implications for future clinical studies.

Distinct chromatin configurations regulate the initiation and the maintenance of hGH gene expression.

For many mammalian genes, initiation of transcription during embryonic development must be subsequently sustained over extensive periods of adult life. It remains unclear whether maintenance of gene expression reflects the same set of pathways as are involved in initial gene activation. The human pituitary growth hormone (hGH-N) locus is activated in the differentiating somatotrope midway through embryogenesis by a multicomponent locus control region (LCR). DNase I-hypersensitive site I (HSI) of the LCR is essential to full developmental activation of the hGH-N locus. Here we demonstrate that conditional deletion of HSI from the active hGH locus in the adult pituitary effectively silences hGH-N expression. Analyses of chromatin structure and locus positioning demonstrate that a specific subset of the HSI functions active in the embryo retain their HSI dependence in the adult pituitary. These functions sustain engagement of the hGH locus with polymerase II (Pol II) factories, histone acetylation at the hGH-N promoter, and looping of the LCR to its target promoter. These data reveal that HSI is essential to both the maintenance and the initiation phases of gene expression. These observations contribute to our mechanistic understanding of how stable patterns of mammalian gene expression are established in a terminally differentiated cell.

Differential effects of exogenous and autocrine growth hormone on LNCaP prostate cancer cell proliferation and survival.

The prostate gland is regulated by multiple hormones and growth factors that may also affect prostate tumorigenesis. Growth hormone (GH) contributes to prostate development and function, but the direct effects of GH on prostate cancer cells are not well understood. The expression of endogenous GH in prostate cancer cell lines has also been observed, suggesting the potential for an effect of autocrine GH. In the present study, we measure the levels of GH and GH receptor (GHR) mRNA in multiple prostate cancer and normal prostate-derived cell lines, and compare the effects of exogenous and autocrine GH on LNCaP prostate cancer cell proliferation and apoptosis, and the associated signal transduction pathways. We found that GHR and GH expression were higher in the prostate cancer cell lines, and that exogenous GH increased LNCaP cell proliferation, but had no effect on apoptosis. In contrast, autocrine GH overexpression reduced LNCaP cell proliferation and increased apoptosis. The distinct actions of exogenous and autocrine GH were accompanied by differences in the involvement of GHR-associated signal transduction pathways, and were paralleled by an alteration in the subcellular localization of GHR, in which autocrine GH appeared to sequester GHR in the Golgi and endoplasmic reticulum. This alteration of GHR trafficking may underlie a distinct mode of GH-mediated signaling associated with the effect of autocrine GH. These findings clarify the potential effects of GH on prostate cancer cell function, and indicate that the activity of autocrine GH may be distinct from that of endocrine GH in prostate cancer cells.

POU1F1-mediated activation of hGH-N by deoxyribonuclease I hypersensitive site II of the human growth hormone locus control region.

The human growth hormone gene (hGH-N) is regulated by a distal locus control region (LCR) composed of five deoxyribonuclease I hypersensitive sites (HSs). The region encompassing HSI and HSII contains the predominant pituitary somatotrope-specific hGH-N activation function of the LCR. This activity was attributed primarily to POU1F1 (Pit-1) elements at HSI, as linkage to HSI was sufficient for properly regulated hGH-N expression in transgenic mice, while HSII alone had no activity. However, the presence of HSII in conjunction with HSI further enhanced hGH-N transgene expression, indicating additional determinants of pituitary hGH-N activation in the HSII region, but limitations of transgenic models and previous ex vivo systems have prevented the characterization of HSII. In the present study, we employ a novel minichromosome model of the hGH-N regulatory domain and show that HSII confers robust POU1F1-dependent activation of hGH-N in this system. This effect was accompanied by POU1F1-dependent histone acetylation and methylation throughout the minichromosome LCR/hGH-N domain. A series of in vitro DNA binding experiments revealed that POU1F1 binds to multiple sites at HSII, consistent with a direct role in HSII function. Remarkably, POU1F1 binding was localized in part to the 3' untranslated region of a primate-specific LINE-1 (long interspersed nuclear element 1) retrotransposon, suggesting that its insertion during primate evolution may have conferred function to the HSII region in the context of pituitary GH gene regulation. These observations clarify the function of HSII, expanding the role of POU1F1 in hGH LCR activity, and provide insight on the molecular evolution of the LCR.

Expression of Pit-1 in nonsomatotrope cell lines induces human growth hormone locus control region histone modification and hGH-N transcription.

The POU domain transcription factor Pit-1 is expressed in somatotropes, lactotropes, and thyrotropes of the anterior pituitary. Pit-1 is essential for the establishment of these lineages during development and regulates the expression of genes encoding the peptide hormones secreted by each cell type, including the growth hormone gene expressed in somatotropes. In contrast to rodent growth hormone loci, the human growth hormone (hGH) locus is regulated by a distal locus control region (LCR), which is required in cis for the proper expression of the hGH gene cluster in transgenic mice. The hGH LCR mediates a domain of histone acetylation targeted to the hGH locus that is associated with distal hGH-N activation, and the discrete determinants of this activity coincide with DNaseI hypersensitive site (HS) I of the LCR. The identification of three in vitro Pit-1 binding sites within the HS-I region suggested a model in which Pit-1 binding at HS-I initiates the chromatin modification mechanism associated with hGH LCR activity. To test this hypothesis directly and to determine whether Pit-1 expression is sufficient to confer hGH locus histone acetylation and activate hGH-N transcription from an inactive locus, we expressed Pit-1 in nonpituitary cell types. We show that Pit-1 expression established a domain of histone hyperacetylation at the LCR and hGH-N promoter in these cells similar to that observed in pituitary chromatin. This was accompanied by the activation of hGH-N transcription and an increase in intergenic and CD79b transcripts proximal to HS-I. These effects were coincident with Pit-1 occupancy at HS-I and the hGH-N promoter and were observed irrespective of the basal histone modification status of HS-I in the heterologous cell line. These findings are consistent with a role for Pit-1 as an initiating factor in hGH locus activation during somatotrope ontogeny, acting through binding sites at HS-I of the hGH LCR.

Notch-1 signaling is lost in prostate adenocarcinoma and promotes PTEN gene expression.

Prostate tumorigenesis is associated with loss of PTEN gene expression. We and others have recently reported that PTEN is regulated by Notch-1 signaling. Herein, we tested the hypothesis that alterations of the Notch-1 signaling pathway are present in human prostate adenocarcinoma and that Notch-1 signaling regulates PTEN gene expression in prostate cells. Prostate adenocarcinoma cases were examined by immunohistochemistry for ligand cleaved (activated) Notch-1 protein. Tumor foci exhibited little cleaved Notch-1 protein, but expression was observed in benign tissue. Both tumor and benign tissue expressed total (uncleaved) Notch-1. Reduced Hey-1 expression was seen in tumor foci but not in benign tissue, confirming loss of Notch-1 signaling in prostate adenocarcinoma. Retroviral expression of constitutively active Notch-1 in human prostate tumor cell lines resulted in increased PTEN gene expression. Incubation of prostate cell lines with the Notch-1 ligand, Delta, resulted in increased PTEN expression indicating that endogenous Notch-1 regulates PTEN gene expression. Chromatin immunoprecipitation demonstrated that CBF-1 was bound to the PTEN promoter. These data collectively indicate that defects in Notch-1 signaling may play a role in human prostate tumor formation in part via a mechanism that involves regulation of the PTEN tumor suppressor gene.

A single base difference between Pit-1 binding sites at the hGH promoter and locus control region specifies distinct Pit-1 conformations and functions.

Activation of the human growth hormone (hGH-N) gene in pituitary somatotropes is mediated by a locus control region (LCR). This LCR is composed of DNase I-hypersensitive sites (HS) located -14.5 kb to -32 kb relative to the hGH-N promoter. HSI, at -14.5 kb, is the dominant determinant of hGH-N expression and is essential for establishment of a 32-kb domain of histone acetylation that encompasses the active hGH locus. This activity is conferred by three binding sites for the POU domain transcription factor Pit-1. These Pit-1 elements are sufficient to activate hGH-N expression in the mouse pituitary. In contrast, Pit-1 sites at the hGH-N promoter are consistently unable to mediate similar activity. In the present study, we demonstrate that the functional difference between the promoter-proximal and the HSI Pit-1 binding sites can be attributed in part to a single base difference. This base affects the conformation of the Pit-1/DNA complex, and reciprocal exchange of the divergent bases between the two sets of Pit-1 elements results in a partial reversal of their transgenic activities. These data support a model in which the Pit-1 binding sites in the hGH LCR allosterically program the bound Pit-1 complex for chromatin activating functions.

Specification of unique Pit-1 activity in the hGH locus control region.

The human GH (hGH) gene cluster is regulated by a remote 5' locus control region (LCR). HSI, an LCR component located 14.5 kb 5' to the hGH-N promoter, constitutes the primary determinant of high-level hGH-N activation in pituitary somatotropes. HSI encompasses an array of three binding sites for the pituitary-specific POU homeodomain factor Pit-1. In the present report we demonstrate that all three Pit-1 sites in the HSI array contribute to LCR activity in vivo. Furthermore, these three sites as a unit are fully sufficient for position-independent and somatotrope-restricted hGH-N transgene activation. In contrast, the hGH-N transgene is not activated by Pit-1 sites native to either the hGH-N or rat (r)GH gene promoters. These findings suggest that the structures of the Pit-1 binding sites at HSI specify distinct chromatin-dependent activities essential for LCR-mediated activation of hGH in the developing pituitary.