Targeting nuclear receptor corepressors for reversible male contraception.

Despite numerous female contraceptive options, nearly half of all pregnancies are unintended. Family planning choices for men are currently limited to unreliable condoms and invasive vasectomies with questionable reversibility. Here, we report the development of an oral contraceptive approach based on transcriptional disruption of cyclical gene expression patterns during spermatogenesis. Spermatogenesis involves a continuous series of self-renewal and differentiation programs of spermatogonial stem cells (SSCs) that is regulated by retinoic acid (RA)-dependent activation of receptors (RARs), which control target gene expression through association with corepressor proteins. We have found that the interaction between RAR and the corepressor silencing mediator of retinoid and thyroid hormone receptors (SMRT) is essential for spermatogenesis. In a genetically engineered mouse model that negates SMRT-RAR binding (SMRTmRID mice), the synchronized, cyclic expression of RAR-dependent genes along the seminiferous tubules is disrupted. Notably, the presence of an RA-resistant SSC population that survives RAR de-repression suggests that the infertility attributed to the loss of SMRT-mediated repression is reversible. Supporting this notion, we show that inhibiting the action of the SMRT complex with chronic, low-dose oral administration of a histone deacetylase inhibitor reversibly blocks spermatogenesis and fertility without affecting libido. This demonstration validates pharmacologic targeting of the SMRT repressor complex for non-hormonal male contraception.

Normalizing Microbiota-Induced Retinoic Acid Deficiency Stimulates Protective CD8(+) T Cell-Mediated Immunity in Colorectal Cancer.

Although all-trans-retinoic acid (atRA) is a key regulator of intestinal immunity, its role in colorectal cancer (CRC) is unknown. We found that mice with colitis-associated CRC had a marked deficiency in colonic atRA due to alterations in atRA metabolism mediated by microbiota-induced intestinal inflammation. Human ulcerative colitis (UC), UC-associated CRC, and sporadic CRC specimens have similar alterations in atRA metabolic enzymes, consistent with reduced colonic atRA. Inhibition of atRA signaling promoted tumorigenesis, whereas atRA supplementation reduced tumor burden. The benefit of atRA treatment was mediated by cytotoxic CD8(+) T cells, which were activated due to MHCI upregulation on tumor cells. Consistent with these findings, increased colonic expression of the atRA-catabolizing enzyme, CYP26A1, correlated with reduced frequencies of tumoral cytotoxic CD8(+) T cells and with worse disease prognosis in human CRC. These results reveal a mechanism by which microbiota drive colon carcinogenesis and highlight atRA metabolism as a therapeutic target for CRC.

Cyp26a1 supports postnatal retinoic acid homeostasis and glucoregulatory control.

Considerable evidence confirms the importance of Cyp26a1 to all-trans-retinoic acid (RA) homeostasis during embryogenesis. In contrast, despite its presence in postnatal liver as a potential major RA catabolizing enzyme and its acute sensitivity to induction by RA, some data suggested that Cyp26a1 contributes only marginally to endogenous RA homeostasis postnatally. We report reevaluation of a conditional Cyp26a1 knockdown in the postnatal mouse. The current results show that Cyp26a1 mRNA in WT mouse liver increases 16-fold upon refeeding after a fast, accompanied by an increased rate of RA elimination and a 41% decrease in the RA concentration. In contrast, Cyp26a1 mRNA in the refed homozygotic knockdown reached only 2% of its extent in WT during refeeding, accompanied by a slower rate of RA catabolism and no decrease in liver RA, relative to fasting. Refed homozygous knockdown mice also had decreased Akt1 and 2 phosphorylation and pyruvate dehydrogenase kinase 4 (Pdk4) mRNA and increased glucokinase (Gck) mRNA, glycogen phosphorylase (Pygl) phosphorylation, and serum glucose, relative to WT. Fasted homozygous knockdown mice had increased glucagon/insulin relative to WT. These data indicate that Cyp26a1 participates prominently in moderating the postnatal liver concentration of endogenous RA and contributes essentially to glucoregulatory control.

Energy status regulates levels of the RAR/RXR ligand 9-cis-retinoic acid in mammalian tissues: Glucose reduces its synthesis in ß-cells.

9-cis-retinoic acid (9cRA) binds retinoic acid receptors (RAR) and retinoid X receptors (RXR) with nanomolar affinities, in contrast to all-trans-retinoic acid (atRA), which binds only RAR with nanomolar affinities. RXR heterodimerize with type II nuclear receptors, including RAR, to regulate a vast gene array. Despite much effort, 9cRA has not been identified as an endogenous retinoid, other than in pancreas. By revising tissue analysis methods, 9cRA quantification by liquid chromatography-tandem mass spectrometry becomes possible in all mouse tissues analyzed. 9cRA occurs in concentrations similar to or greater than atRA. Fasting increases 9cRA in white and brown adipose, brain and pancreas, while increasing atRA in white adipose, liver and pancreas. 9cRA supports FoxO1 actions in pancreas ß-cells and counteracts glucose actions that lead to glucotoxicity; in part by inducing Atg7 mRNA, which encodes the key enzyme essential for autophagy. Glucose suppresses 9cRA biosynthesis in the ß-cell lines 832/13 and MIN6. Glucose reduces 9cRA biosynthesis in 832/13 cells by inhibiting Rdh5 transcription, unconnected to insulin, through cAMP and Akt, and inhibiting FoxO1. Through adapting tissue specifically to fasting, 9cRA would act independent of atRA. Widespread occurrence of 9cRA in vivo, and its self-sufficient adaptation to energy status, provides new perspectives into regulation of energy balance, attenuation of insulin and glucose actions, regulation of type II nuclear receptors, and retinoid biology.

Retinoic acid from the meninges regulates cortical neuron generation.

Extrinsic signals controlling generation of neocortical neurons during embryonic life have been difficult to identify. In this study we demonstrate that the dorsal forebrain meninges communicate with the adjacent radial glial endfeet and influence cortical development. We took advantage of Foxc1 mutant mice with defects in forebrain meningeal formation. Foxc1 dosage and loss of meninges correlated with a dramatic reduction in both neuron and intermediate progenitor production and elongation of the neuroepithelium. Several types of experiments demonstrate that retinoic acid (RA) is the key component of this secreted activity. In addition, Rdh10- and Raldh2-expressing cells in the dorsal meninges were either reduced or absent in the Foxc1 mutants, and Rdh10 mutants had a cortical phenotype similar to the Foxc1 null mutants. Lastly, in utero RA treatment rescued the cortical phenotype in Foxc1 mutants. These results establish RA as a potent, meningeal-derived cue required for successful corticogenesis.

Retinoic acid exerts sexually dimorphic effects on muscle energy metabolism and function.

The retinol dehydrogenase Rdh10 catalyzes the rate-limiting reaction that converts retinol into retinoic acid (RA), an autacoid that regulates energy balance and reduces adiposity. Skeletal muscle contributes to preventing adiposity, by consuming nearly half the energy of a typical human. We report sexually dimorphic differences in energy metabolism and muscle function in Rdh10+/- mice. Relative to wild-type (WT) controls, Rdh10+/- males fed a high-fat diet decrease reliance on fatty-acid oxidation and experience glucose intolerance and insulin resistance. Running endurance decreases 40%. Rdh10+/- females fed this diet increase fatty acid oxidation and experience neither glucose intolerance nor insulin resistance. Running endurance increases 220%. We therefore assessed RA function in the mixed-fiber type gastrocnemius muscles (GM), which contribute to running, rather than standing, and are similar to human GM. RA levels in Rdh10+/- male GM decrease 38% relative to WT. Rdh10+/- male GM increase expression of Myog and reduce Eif6 mRNAs, which reduce and enhance running endurance, respectively. Cox5A, complex IV activity, and ATP decrease. Increased centralized nuclei reveal existence of muscle malady and/or repair in GM fibers. Comparatively, RA in Rdh10+/- female GM decreases by less than half the male decrease, from a more modest decrease in Rdh10 and an increase in the estrogen-induced retinol dehydrogenase Dhrs9. Myog mRNA decreases. Cox5A, complex IV activity, and ATP increase. Centralized GM nuclei do not increase. We conclude that Rdh10/RA affects whole body energy use and insulin resistance partially through sexual dimorphic effects on skeletal muscle gene expression, structure, and mitochondria activity.

DGAT1 inhibits retinol-dependent regulatory T cell formation and mediates autoimmune encephalomyelitis.

The balance of effector versus regulatory T cells (Tregs) controls inflammation in numerous settings, including multiple sclerosis (MS). Here we show that memory phenotype CD4+ T cells infiltrating the central nervous system during experimental autoimmune encephalomyelitis (EAE), a widely studied animal model of MS, expressed high levels of mRNA for Dgat1 encoding diacylglycerol-O-acyltransferase-1 (DGAT1), an enzyme that catalyzes triglyceride synthesis and retinyl ester formation. DGAT1 inhibition or deficiency attenuated EAE, with associated enhanced Treg frequency; and encephalitogenic, DGAT1-/- in vitro-polarized Th17 cells were poor inducers of EAE in adoptive recipients. DGAT1 acyltransferase activity sequesters retinol in ester form, preventing synthesis of retinoic acid, a cofactor for Treg generation. In cultures with T cell-depleted lymphoid tissues, retinol enhanced Treg induction from DGAT1-/- but not from WT T cells. The WT Treg induction defect was reversed by DGAT1 inhibition. These results demonstrate that DGAT1 suppresses retinol-dependent Treg formation and suggest its potential as a therapeutic target for autoimmune inflammation.

RDH1 suppresses adiposity by promoting brown adipose adaptation to fasting and re-feeding.

RDH1 is one of the several enzymes that catalyze the first of the two reactions to convert retinol into all-trans-retinoic acid (atRA). Here, we show that Rdh1-null mice fed a low-fat diet gain more weight as adiposity (17% males, 13% females) than wild-type mice by 20 weeks old, despite neither consuming more calories nor decreasing activity. Glucose intolerance and insulin resistance develop following increased adiposity. Despite the increase in white fat pads, epididymal white adipose does not express Rdh1, nor does muscle. Brown adipose tissue (BAT) and liver express Rdh1 at relatively high levels compared to other tissues. Rdh1 ablation lowered body temperatures during ambient conditions. Given the decreased body temperature, we focused on BAT. A lack of differences in BAT adipogenic gene expression between Rdh1-null mice and wild-type mice, including Pparg, Prdm16, Zfp516 and Zfp521, indicated that the phenotype was not driven by brown adipose hyperplasia. Rather, Rdh1 ablation eliminated the increase in BAT atRA that occurs after re-feeding. This disruption of atRA homeostasis increased fatty acid uptake, but attenuated lipolysis in primary brown adipocytes, resulting in increased lipid content and larger lipid droplets. Rdh1 ablation also decreased mitochondrial proteins, including CYCS and UCP1, the mitochondria oxygen consumption rate, and disrupted the mitochondria membrane potential, further reflecting impaired BAT function, resulting in both BAT and white adipose hypertrophy. RNAseq revealed dysregulation of 424 BAT genes in null mice, which segregated predominantly into differences after fasting vs after re-feeding. Exceptions were Rbp4 and Gbp2b, which increased during both dietary conditions. Rbp4 encodes the serum retinol-binding protein-an insulin desensitizer. Gbp2b encodes a GTPase. Because Gbp2b increased several hundred-fold, we overexpressed it in brown adipocytes. This caused a shift to larger lipid droplets, suggesting that GBP2b affects signaling downstream of the ß-adrenergic receptor during basal thermogenesis. Thus, Rdh1-generated atRA in BAT regulates multiple genes that promote BAT adaptation to whole-body energy status, such as fasting and re-feeding. These gene expression changes promote optimum mitochondria function and thermogenesis, limiting adiposity. Attenuation of adiposity and insulin resistance suggests that RDH1 mitigates metabolic syndrome.

Examination of Fluconazole-Induced Alopecia in an Animal Model and Human Cohort.

Fluconazole-induced alopecia is a significant problem for patients receiving long-term therapy. We evaluated the hair cycle changes of fluconazole in a rat model and investigated potential molecular mechanisms. Plasma and tissue levels of retinoic acid were not found to be causal. Human patients with alopecia attributed to fluconazole also underwent detailed assessment and in both our murine model and human cohort fluconazole induced telogen effluvium. Future work further examining the mechanism of fluconazole-induced alopecia should be undertaken.

Quantification of Dehydroepiandrosterone, 17ß-Estradiol, Testosterone, and Their Sulfates in Mouse Tissues by LC-MS/MS.

We report a high-performance, liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) assay to quantify without derivatizaton dehyroepiandrosterone (DHEA), 17ß-estradiol (E2), testosterone (T), and their sulfates in serum and tissues. This assay functions well with multiple adipose depots, a previously unattained analysis. To delipidate and facilitate recovery, tissues were homogenized in acetonitrile, and the homogenate was frozen. The supernatant was evaporated, resuspended in an aqueous acetate buffer, and extracted with hexane to separate free (unconjugated) from sulfated steroids. Sulfated steroids in the aqueous medium were then hydrolyzed with sulfatase and extracted with hexane. Each extract was analyzed separately. HPLC resolution combined with the sensitivity and specificity of MS/MS allowed quantification of DHEA, E2, and T with 10, 10, and 5 fmol lower limits of quantification and linear ranges to 1 pmol. Application of the method to mouse serum and tissues reveals ranges of DHEA, E2, and T and their sulfates, and tissue-specific differences in steroid profile, especially white versus brown adipose. In addition, marginal decreases of T in all tissues and considerable increases in DHEA in male iWAT and eWAT in response to a high-fat diet further strengthen the inference regarding the role of steroid metabolism in adipogenesis. This assay permits detailed studies of interactions between adiposity and sex steroids in serum and tissues, including adipose.

Serine Threonine Kinase Receptor-Associated Protein Deficiency Impairs Mouse Embryonic Stem Cells Lineage Commitment Through CYP26A1-Mediated Retinoic Acid Homeostasis.

Retinoic acid (RA) signaling is essential for the differentiation of embryonic stem cells (ESCs) and vertebrate development. RA biosynthesis and metabolism are controlled by a series of enzymes, but the molecular regulators of these enzymes remain largely obscure. In this study, we investigated the functional role of the WD-domain protein STRAP (serine threonine kinase receptor-associated protein) in the pluripotency and lineage commitment of murine ESCs. We generated Strap knockout (KO) mouse ESCs and subjected them to spontaneous differentiation. We observed that, despite the unchanged characteristics of ESCs, Strap KO ESCs exhibited defects for lineage differentiation. Signature gene expression analyses revealed that Strap deletion attenuated intracellular RA signaling in embryoid bodies (EBs), and exogenous RA significantly rescued this deficiency. Moreover, loss of Strap selectively induced Cyp26A1 expression in mouse EBs, suggesting a potential role of STRAP in RA signaling. Mechanistically, we identified putative Krüppel-like factor 9 (KLF9) binding motifs to be critical in the enhancement of non-canonical RA-induced transactivation of Cyp26A1. Increased KLF9 expression in the absence of STRAP is partially responsible for Cyp26A1 induction. Interestingly, STRAP knockdown in Xenopus embryos influenced anterior-posterior neural patterning and impaired the body axis and eye development during early Xenopus embryogenesis. Taken together, our study reveals an intrinsic role for STRAP in the regulation of RA signaling and provides new molecular insights for ESC fate determination. Stem Cells 2018;36:1368-1379.

Immunotherapy Added to Antibiotic Treatment Reduces Relapse of Disease in a Mouse Model of Tuberculosis.

Immune-modulating drugs that target myeloid-derived suppressor cells or stimulate natural killer T cells have been shown to reduce mycobacterial loads in tuberculosis (TB). We aimed to determine if a combination of these drugs as adjunct immunotherapy to conventional antibiotic treatment could also increase therapeutic efficacy against TB. In our model of pulmonary TB in mice, we applied treatment with isoniazid, rifampicin, and pyrazinamide for 13 weeks alone or combined with immunotherapy consisting of all-trans retinoic acid, 1,25(OH)2-vitamin D3, and α-galactosylceramide. Outcome parameters were mycobacterial load during treatment (therapeutic activity) and 13 weeks after termination of treatment (therapeutic efficacy). Moreover, cellular changes were analyzed using flow cytometry and cytokine expression was assessed at the mRNA and protein levels. Addition of immunotherapy was associated with lower mycobacterial loads after 5 weeks of treatment and significantly reduced relapse of disease after a shortened 13-week treatment course compared with antibiotic treatment alone. This was accompanied by reduced accumulation of immature myeloid cells in the lungs at the end of treatment and increased TNF-α protein levels throughout the treatment period. We demonstrate, in a mouse model of pulmonary TB, that immunotherapy consisting of three clinically approved drugs can improve the therapeutic efficacy of standard antibiotic treatment.

Functions of Intracellular Retinoid Binding-Proteins.

Multiple binding and transport proteins facilitate many aspects of retinoid biology through effects on retinoid transport, cellular uptake, metabolism, and nuclear delivery. These include the serum retinol binding protein sRBP (aka Rbp4), the plasma membrane sRBP receptor Stra6, and the intracellular retinoid binding-proteins such as cellular retinol-binding proteins (CRBP) and cellular retinoic acid binding-proteins (CRABP). sRBP transports the highly lipophilic retinol through an aqueous medium. The major intracellular retinol-binding protein, CRBP1, likely enhances efficient retinoid use by providing a sink to facilitate retinol uptake from sRBP through the plasma membrane or via Stra6, delivering retinol or retinal to select enzymes that generate retinyl esters or retinoic acid, and protecting retinol/retinal from excess catabolism or opportunistic metabolism. Intracellular retinoic acid binding-proteins (CRABP1 and 2, and FABP5) seem to have more diverse functions distinctive to each, such as directing retinoic acid to catabolism, delivering retinoic acid to specific nuclear receptors, and generating non-canonical actions. Gene ablation of intracellular retinoid binding-proteins does not cause embryonic lethality or gross morphological defects. Metabolic and functional defects manifested in knockouts of CRBP1, CRBP2 and CRBP3, however, illustrate their essentiality to health, and in the case of CRBP2, to survival during limited dietary vitamin A. Future studies should continue to address the specific molecular interactions that occur between retinoid binding-proteins and their targets and their precise physiologic contributions to retinoid homeostasis and function.

Insulin regulates retinol dehydrogenase expression and all-trans-retinoic acid biosynthesis through FoxO1.

All-trans-retinoic acid (atRA), an autacoid derived from retinol (vitamin A), regulates energy balance and reduces adiposity. We show that energy status regulates atRA biosynthesis at the rate-limiting step, catalyzed by retinol dehydrogenases (RDH). Six h after re-feeding, Rdh1 expression decreased 80-90% in liver and brown adipose tissue and Rdh10 expression was decreased 45-63% in liver, pancreas, and kidney, all relative to mice fasted 16 h. atRA in the liver was decreased 44% 3 h after reduced Rdh expression. Oral gavage with glucose or injection with insulin decreased Rdh1 and Rdh10 mRNA 50% or greater in mouse liver. Removing serum from the medium of the human hepatoma cell line HepG2 increased Rdh10 and Rdh16 (human Rdh1 ortholog) mRNA expression 2-3-fold by 4 h, by increasing transcription and stabilizing mRNA. Insulin decreased Rdh10 and Rdh16 mRNA in HepG2 cells incubated in serum-free medium by inhibiting transcription and destabilizing mRNA. Insulin action required PI3K and Akt, which suppress FoxO1. Serum removal increased atRA biosynthesis 4-fold from retinol in HepG2 cells, whereas dominant-negative FoxO1 prevented the increase. Thus, energy status via insulin and FoxO1 regulate Rdh expression and atRA biosynthesis. These results reveal mechanisms for regulating atRA biosynthesis and the opposing effects of atRA and insulin on gluconeogenesis, and also suggest an interaction between atRA and insulin signaling related diseases, such as type II diabetes and cancer.

Vitamin A metabolism and mucosal immune function are distinct between BALB/c and C57BL/6 mice.

The vitamin A metabolite retinoic acid (RA) has been reported to suppress Th1 responses and enhance Th2 responses. Here, we investigated whether differences in vitamin A metabolism could underlie the differences between C57BL/6 and BALB/c mice, which are reportedly seen as Th1 and Th2 responders, respectively. BALB/c mice were shown to have higher intestinal epithelial expression of RALDH1 (where RALDH is retinaldehyde dehydrogenase), and, consequently, higher RALDH activity in MLN-DCs, leading to an increased ability to induce IgA class switching in B cells. Furthermore, within BALB/c mice, induction of IgA secretion as well as increased accumulation of regulatory T cells (Treg) in the intestinal lamina propria was observed. Additionally, as BALB/c mice are more resistant to dextran sulphate sodium (DSS) induced colitis, mice that lacked vitamin A in their diet had a more severe form of DSS-induced colitis compared to control mice. Therefore, the level of RA production and consequently the degree of RA-mediated signaling is crucial for the efficiency of the mucosal immune system.

Quantitation of retinaldehyde in small biological samples using ultrahigh-performance liquid chromatography tandem mass spectrometry.

We report an ultrahigh-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method to quantify all-trans-retinal in biological samples of limited size (15-35mg), which is especially advantageous for use with adipose. To facilitate recovery, retinal and the internal standard 3,4-didehydroretinal were derivatized in situ into their O-ethyloximes. UHPLC resolution combined with high sensitivity and specificity of MS/MS allowed quantification of retinal-O-ethyloximes with a 5-fmol lower limit of detection and a linear range from 5fmol to 1pmol. This assay revealed that extraocular concentrations of retinal range from approximately 2 to 40pmol/g in multiple tissues-the same range as all-trans-retinoic acid. All-trans-retinoic acid has high affinity (kd⩽0.4nM) for its nuclear receptors (RARα, -ß, and -γ), whereas retinal has low (if any) affinity for these receptors, making it unlikely that these retinal concentrations would activate RAR. We also show that the copious amount of vitamin A used in chow diets increases retinal in adipose depots 2- to 5-fold relative to levels in adipose of mice fed a vitamin A-sufficient diet, as recommended for laboratory rodents. This assay also is proficient for quantifying conversion of retinol into retinal in vitro and, therefore, provides an efficient method to study metabolism of retinol in vivo and in vitro.

The retinol dehydrogenase Rdh10 localizes to lipid droplets during acyl ester biosynthesis.

Rdh10 catalyzes the first step of all-trans-retinoic acid biogenesis physiologically, conversion of retinol into retinal. We show that Rdh10 associates predominantly with mitochondria/mitochondrial-associated membrane (MAM) in the absence of lipid droplet biosynthesis, but also locates with lipid droplets during acyl ester biosynthesis. Targeting to lipid droplets requires the 32 N-terminal residues, which include a hydrophobic region followed by a net positive charge. Targeting to mitochondria/MAM and/or the stability of Rdh10 require both the N-terminal and the 48 C-terminal hydrophobic residues. Rdh10 behaves similarly to cellular retinol-binding protein, type 1, which also localizes to mitochondria/MAM before lipid droplet synthesis, and associates with lipid droplets during acyl ester synthesis (Jiang, W., and Napoli, J. L. (2012) Biochem. Biophys. Acta 1820, 859-8692). LRAT, an ER protein, also associates with lipid droplets upon acyl ester biosynthesis. Colocalization of Rdh10, Crbp1, and LRAT on lipid droplets suggests a metabolon that mediates retinol homeostasis.

Retinoic acid biosynthesis is impaired in human and murine endometriosis.

Endometriosis is characterized by the presence of endometrial glands and stroma in extrauterine sites. Our objective was to determine whether endometriotic lesions (ELs) from women with endometriosis have altered retinoid levels compared with their eutopic endometrium, and to test the hypothesis that defects in all-trans retinoic acid (ATRA) biosynthesis in EL is related to reduced expression of cellular retinol-binding protein type 1 (RBP1). Retinoids were evaluated by liquid chromatography-tandem mass spectrometry and high-performance liquid chromatography in eutopic endometrial biopsies (EBs) and ELs from 42 patients with pathologically confirmed endometriosis. The ATRA levels were reduced, whereas the retinol and retinyl ester concentrations were elevated in EL compared with EB tissue. Similar results were found in a mouse model of endometriosis that used green fluorescent protein-positive endometrial tissue injected into the peritoneum of syngeneic hosts to mimic retrograde menses. The ATRA biosynthesis in vitro in retinol-treated primary human endometrial stromal cell (ESC) cultures derived from ELs was reduced compared with that of ESCs derived from patient-matched EBs. Correspondingly, RBP1 expression was reduced in tissue and ESCs derived from EL versus EB. Rbp1(-/-) mice showed reduced endometrial ATRA concentrations compared with wild type, associated with loss of tissue organization and hypercellularity. These findings provide the first quantitative measurements of ATRA in human endometrium and endometriosis, demonstrating reduced ATRA in ectopic tissue and corresponding ESC cultures. Quantitation of retinoids in murine endometriosis and in Rbp1(-/-) mice supports the contention that impaired ATRA synthesis caused by reduced RBP1 promotes an "endometriosis phenotype" that enables cells to implant and grow at ectopic sites.

Physiological insights into all-trans-retinoic acid biosynthesis.

All-trans-retinoic acid (atRA) provides essential support to diverse biological systems and physiological processes. Epithelial differentiation and its relationship to cancer, and embryogenesis have typified intense areas of interest into atRA function. Recently, however, interest in atRA action in the nervous system, the immune system, energy balance and obesity has increased considerably, especially concerning postnatal function. atRA action depends on atRA biosynthesis: defects in retinoid-dependent processes increasingly relate to defects in atRA biogenesis. Considerable evidence indicates that physiological atRA biosynthesis occurs via a regulated process, consisting of a complex interaction of retinoid binding-proteins and retinoid recognizing enzymes. An accrual of biochemical, physiological and genetic data have identified specific functional outcomes for the retinol dehydrogenases, RDH1, RDH10, and DHRS9, as physiological catalysts of the first step in atRA biosynthesis, and for the retinal dehydrogenases RALDH1, RALDH2, and RALDH3, as catalysts of the second and irreversible step. Each of these enzymes associates with explicit biological processes mediated by atRA. Redundancy occurs, but seems limited. Cumulative data support a model of interactions among these enzymes with retinoid binding-proteins, with feedback regulation and/or control by atRA via modulating gene expression of multiple participants. The ratio apo-CRBP1/holo-CRBP1 participates by influencing retinol flux into and out of storage as retinyl esters, thereby modulating substrate to support atRA biosynthesis. atRA biosynthesis requires the presence of both an RDH and an RALDH: conversely, absence of one isozyme of either step does not indicate lack of atRA biosynthesis at the site. This article is part of a Special Issue entitled: Retinoid and Lipid Metabolism.

Reorganization of cellular retinol-binding protein type 1 and lecithin:retinol acyltransferase during retinyl ester biosynthesis.

BACKGROUND: Cellular retinol-binding protein, type 1 (Crbp1), chaperones retinyl ester (RE) biosynthesis catalyzed by lecithin:retinol acyltransferase (LRAT). METHODS: We monitored the subcellular loci of LRAT and Crbp1 before and during RE biosynthesis, and compared the results to diacylglycerol:acyltransferase type 2 (DGAT2) during triacylglycerol biosynthesis in three cell lines: COS7, CHO and HepG2. RESULTS: Before initiation of RE biosynthesis, LRAT distributed throughout the endoplasmic reticulum (ER), similar to DGAT2, and Crpb1 localized with mitochondria associated membranes (MAM), surrounded by LRAT. Upon initiating RE biosynthesis in cells transfected with low amounts of vector to simulate physiological expression levels, Crpb1 remained with MAM, and both Crbp1 and MAM re-localized with LRAT. LRAT formed rings around the growing lipid droplets. LRAT activity was higher in these rings relative to the general ER. LRAT-containing rings colocalized with the lipid-droplet surface proteins, desnutrin/adipose triglyceride lipase and perilipin 2. Colocalization with lipid droplets required the 38 N-terminal amino acid residues of LRAT, and specifically K36 and R38. Formation of rings around the growing lipid droplets did not require functional microtubules. GENERAL SIGNIFICANCE: These data indicate a relationship between LRAT and Crbp1 during RE biosynthesis in which MAM-associated Crpb1 and LRAT colocalize, and both surround the growing RE-containing lipid droplet. The N-terminus of LRAT, especially K36 and R38, is essential to colocalization with the lipid droplet.