Retinoic Acid Stimulation Research Articles

Maternal Vitamin A Supplementation Expands Adipose Progenitor Population through Promoting Vascular Development

Retinoic acid regulates various types of progenitor differentiation and tissue development including adipose development. However, the impact of maternal vitamin A supplementation on fetal adipose development and its long‐term consequence in adulthood remain unclear. To address, we supplemented maternal mice with 0, 15 or 30 IU/mL retinyl palmitate in water during gestation and lactation. Adipose tissue and serum of weanling offspring, and E7.5, 12.5 and 18.5 fetuses were collected. We found that retinoic acid is the main retinoid in fetuses that was elevated by maternal vitamin A supplementation. The populations of platelet‐derived growth factor receptor alpha positive (PDGFRα+) adipocyte progenitors in both fetuses and weaned offspring were increased due to vitamin A supplementation, which were correlated with heightened vascular density in adipose tissue at weaning. We further found that retinoic acid upregulated vascular endothelial growth factor α (VEGFα) which might explain the enhanced vasculogenesis in adipose tissue of fetuses supplemented with vitamin A. Using florescence tracing, we found that, under cold or retinoic acid stimulation, PDGFRα+ progenitor cells were located in neovascular system. In summary, our results reveal that maternal vitamin A supplementation increases offspring adipose progenitor populations by promoting vascular system development during the fetal stage, which is mediated by retinoic acid receptor signaling.Support or Funding InformationSupported by NIH R01HD067449 and USDA‐NIFA 2015‐67015‐23219

NPM and BRG1 Mediate Transcriptional Resistance to Retinoic Acid in Acute Promyelocytic Leukemia

Perturbation in the transcriptional control of genes driving differentiation is an established paradigm whereby oncogenic fusion proteins promote leukemia. From a retinoic acid (RA)-sensitive acute promyelocytic leukemia (APL) cell line, we derived an RA-resistant clone characterized by a block in transcription initiation, despite maintaining wild-type PML/RARA expression. We uncovered an aberrantinteraction among PML/RARA, nucleophosmin (NPM), and topoisomerase II beta (TOP2B). Surprisingly, RA stimulation in these cells results in enhanced chromatin association of the nucleosome remodeler BRG1. Inhibition of NPM or TOP2B abrogated BRG1 recruitment. Furthermore, NPM inhibition and targeting BRG1 restored differentiation when combined with RA. Here, we demonstrate a role for NPM and BRG1 in obstructing RA differentiation and implicate chromatin remodeling in mediating therapeutic resistance in malignancies. NPM mutations are the most common genetic change in patients with acute leukemia (AML); therefore, our model may be applicable to other more common leukemias driven by NPM.

Retinoic Acid Induces Embryonic Stem Cell Differentiation by Altering Both Encoding RNA and microRNA Expression.

Retinoic acid (RA) is a vitamin A metabolite that is essential for early embryonic development and promotes stem cell neural lineage specification; however, little is known regarding the impact of RA on mRNA transcription and microRNA levels on embryonic stem cell differentiation. Here, we present mRNA microarray and microRNA high-output sequencing to clarify how RA regulates gene expression. Using mRNA microarray analysis, we showed that RA repressed pluripotency-associated genes while activating ectoderm markers in mouse embryonic stem cells (mESCs). Moreover, RA modulated the DNA methylation of mESCs by altering the expression of epigenetic-associated genes such as Dnmt3b and Dnmt3l. Furthermore, H3K4me2, a pluripotent histone modification, was repressed by RA stimulation. From microRNA sequence data, we identified two downregulated microRNAs, namely, miR-200b and miR-200c, which regulated the pluripotency of stem cells. We found that miR-200b or miR-200c deficiency suppressed the expression of pluripotent genes, including Oct4 and Nanog, and activated the expression of the ectodermal marker gene Nestin. These results demonstrate that retinoid induces mESCs to differentiate by regulating miR-200b/200c. Our findings provide the landscapes of mRNA and microRNA gene networks and indicate the crucial role of miR-200b/200c in the RA-induced differentiation of mESCs.

Resveratrol ameliorates imiquimod-induced psoriasis-like skin inflammation in mice.

BackgroundThe polyphenol resveratrol has anti-inflammatory effects in various cells, tissues, animals and human settings of low-grade inflammation. Psoriasis is a disease of both localized and systemic low-grade inflammation. The Sirtuin1 enzyme thought to mediate the effects of resveratrol is present in skin and resveratrol is known to down regulate NF-κB; an important contributor in the development of psoriasis. Consequently we investigated whether resveratrol has an effect on an Imiquimod induced psoriasis-like skin inflammation in mice and sought to identify candidate genes, pathways and interleukins mediating the effects.MethodsThe study consisted of three treatment groups: A control group, an Imiquimod group and an Imiquimod+resveratrol group. Psoriasis severity was assessed using elements of the Psoriasis Area Severity Index, skin thickness measurements, and histological examination. We performed an RNA microarray from lesional skin and afterwards Ingenuity pathway analysis to identify affected signalling pathways. Our microarray was compared to a previously deposited microarray to determine if gene changes were psoriasis-like, and to a human microarray to determine if findings could be relevant in a human setting.ResultsImiquimod treatment induced a psoriasis-like skin inflammation. Resveratrol significantly diminished the severity of the psoriasis-like skin inflammation. The RNA microarray revealed a psoriasis-like gene expression-profile in the Imiquimod treated group, and highlighted several resveratrol dependent changes in relevant genes, such as increased expression of genes associated with retinoic acid stimulation and reduced expression of genes involved in IL-17 dependent pathways. Quantitative PCR confirmed a resveratrol dependent decrease in mRNA levels of IL-17A and IL-19; both central in developing psoriasis.ConclusionsResveratrol ameliorates psoriasis, and changes expression of retinoic acid stimulated genes, IL-17 signalling pathways, IL-17A and IL-19 mRNA levels in a beneficial manner, which suggests resveratrol, might have a role in the treatment of psoriasis and should be explored further in a human setting.

RA induces differentiation of multipotent P19 cells towards male germ cell.

Generating male germ cells in vitro from multipotent stem cells is still a challenge for stem cell biologists. The difficulty is caused by the lack of knowledge about spermatogenesis molecular-controlling mechanisms. In vivo, PGCs differentiate into male germ cells in a very complicated environment through many middle steps. In this study, we use the pluripotent p19 cells to test their responses to different retinoic acid (RA) concentrations by evaluating markers for stem cells (bmp4, egr3), primordial germ cells (ddx4), spermatogonia (c-kit), premeiotic cells (stra8), and male germ cells (dazl and plzf). We have found that cyp26b1, which will catalyze RA, increases dramatically in p19 cells 1 d after RA treatment. Bmp3, egr3, and stra8 are stimulated after 1 d of RA treatment and then recover to normal after 3 d of RA treatment. C-kit keeps being expressed when treated with 10 nM-4 μM RA. Dazl and plzf are gained after 3 d of stimulation. The morphology of RA (100 nM-4 μM)-treated cells changes distinctively, and cell colonies are formed. Typical neural cell-like and germ cell-like morphologies appear in the 100 nM and 4 μM RA groups, respectively. We conclude that 100-500 nM RA can cause responses in p19 cells, but a high concentration of RA (1-4 μM) can drive these pluripotent cells' differentiation towards male germ cells. However, high concentrations of RA are also toxic. Some colonies that survived from 4 μM RA begin to express ddx4 and c-kit. Selection of the c-kit(+), dazl(+), and ddx4(+) cells after RA stimulation and creating a special culture medium for their propagation might benefit successful spermatogenesis induction in vitro.

Abstract 3470: Transforming growth factor-beta (TGFβ)-mediated growth inhibition of human myeloid leukemia cells is CAK-independent but links to cdc25c-CDK1 pathway

Abstract The cell cycle progression is controlled by cyclin-dependent kinases (CDKs). CDK-Activating Kinase (CAK) is an enzyme complex that is capable of phosphorylating (activating) all CDKs and is essential for G1 and G2 CDK activities. CAK is composed of CDK7, cyclin H, and Mat 1. CDK (CDK1 and CDK2) activity is also controlled by two inhibitory phosphorylation sites (tyrosine 15 and threonine 14) on the CDK. Dephosphorylation of these two sites by cdc25c activates CDK activity. We have previously reported that TGFβ inhibits growth of human myeloid leukemia cells via downregulation of multiple CDKs and cyclins. In this study, we investigated the effect of TGFβ on activity and expression of CAK and cdc25, the two upstream molecules of cell cycle regulatory molecules in human myeloid leukemia cells. TGF-β inhibited the proliferation of TF-1 and MV4-11 accompanied by an increase in p27 level and decrease in CDK1, CDK2, CDK4, cyclin A, cyclin D3, and cylcin B. However, TGF-β had no effect on the expression of CDK7, CDK 9, CDK11, cyclin H, Mat1, and their complexes (CDK7-cyclin H and CDK7-Mat1) for the time period tested up to 72 hours, detected by Western blot and immunoprecipitation, respectively. There were no detectable changes in the phosphorylation status of CDK7, CDK9, CDK11 in the cells treated with TGF-β as compared with the cells without TGFβ. In contrast, these cells showed marked decrease in the levels of MAT1 and CDK7 in response to retinoic acid and phorbol ester (PMA) stimulation. On the other hand, TGFβ significantly downregulated expression of cdc25c starting from 3h and with a maximum inhibition being observed at 72h. We also observed a transient dephosphorylation (activation) of cdc25c followed by significant decrease in the amount of dephophorylated 25c at 48 and 72h. Taken together, our data suggest that TGFβ-induced growth inhibition of human myeloid leukemia cells is CAK-independent but is linked to inhibition of early entry into mitosis of the cells by downregulating cdc25c-CDK1 pathway. Most likely, the downregulation of multiple CDKs and cyclins by TGFβ is regulated at translational or posttranslational but not transcriptional levels, because TGFβ had no any effect on the expression and phosphorylation of the CDKs that are involved in regulation of transcription factor TFII (CDK7 and CDK11) and TAK/P-TEFb (CDK9), respectively. (Supported by Faculty Incentive Grant and NIH-NIGMS MBRS RISE: R25 GM059244-13, Barry University). Citation Format: Alejandra Toro, Daria Vasilyeva, Talia Guardia, Tamara Guardia, Jazmine Duran, Xiaotang Hu. Transforming growth factor-beta (TGFβ)-mediated growth inhibition of human myeloid leukemia cells is CAK-independent but links to cdc25c-CDK1 pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3470. doi:10.1158/1538-7445.AM2014-3470

Endothelial Cell–Derived Chemerin Promotes Dendritic Cell Transmigration

ChemR23 is a chemotactic receptor expressed by APCs, such as dendritic cells, macrophages, and NK cells. Chemerin, the ChemR23 ligand, was detected by immunohistochemistry, to be associated with inflamed endothelial cells in autoimmune diseases, such as lupus erythematosus, psoriasis, and rheumatoid arthritis. This study reports that blood and lymphatic murine endothelial cells produce chemerin following retinoic acid stimulation. Conversely, proinflammatory cytokines, such as TNF-α, IFN-γ, and LPS, or calcitriol, are not effective. Retinoic acid-stimulated endothelial cells promoted dendritic cell adhesion under shear stress conditions and transmigration in a ChemR23-dependent manner. Activated endothelial cells upregulated the expression of the atypical chemotactic receptor CCRL2/ACKR5, a nonsignaling receptor able to bind and present chemerin to ChemR23(+) dendritic cells. Accordingly, activated endothelial cells expressed chemerin on the plasma membrane and promoted in a more efficient manner chemerin-dependent transmigration of dendritic cells. Finally, chemerin stimulation of myeloid dendritic cells induced the high-affinity binding of VCAM-1/CD106 Fc chimeric protein and promoted VCAM-1-dependent arrest to immobilized ligands under shear stress conditions. In conclusion, this study reports that retinoic acid-activated endothelial cells can promote myeloid and plasmacytoid dendritic cell transmigration across endothelial cell monolayers through the endogenous production of chemerin, the upregulation of CCRL2, and the activation of dendritic cell β1 integrin affinity.

Conserved Two-Step Regulatory Mechanism of Human Epithelial Differentiation

SummaryHuman epithelia are organized in a hierarchical structure, where stem cells generate terminally differentiated cells via intermediate progenitors. This two-step differentiation process is conserved in all tissues, but it is not known whether a common gene set contributes to its regulation. Here, we show that retinoic acid (RA) regulates early human prostate epithelial differentiation by activating a tightly coexpressed set of 80 genes (e.g., TMPRSS2). Response kinetics suggested that some of these genes could be direct RA targets, whereas others are probably responding indirectly to RA stimulation. Comparative bioinformatic analyses of published tissue-specific microarrays and a large-scale transcriptomic data set revealed that these 80 genes are not only RA responsive but also significantly coexpressed in many human cell systems. The same gene set preferentially responds to androgens during terminal prostate epithelial differentiation, implying a cell-type-dependent interplay between RA and tissue-specific transcription factor-mediated signaling in regulating the two steps of epithelial differentiation.

Notch pathway regulates female germ cell meiosis progression and early oogenesis events in fetal mouse

A critical process of early oogenesis is the entry of mitotic oogonia into meiosis, a cell cycle switch regulated by a complex gene regulatory network. Although Notch pathway is involved in numerous important aspects of oogenesis in invertebrate species, whether it plays roles in early oogenesis events in mammals is unknown. Therefore, the rationale of the present study was to investigate the roles of Notch signaling in crucial processes of early oogenesis, such as meiosis entry and early oocyte growth. Notch receptors and ligands were localized in mouse embryonic female gonads and 2 Notch inhibitors, namely DAPT and L-685,458, were used to attenuate its signaling in an in vitro culture system of ovarian tissues from 12.5 days post coitum (dpc) fetus. The results demonstrated that the expression of Stra8, a master gene for germ cell meiosis, and its stimulation by retinoic acid (RA) were reduced after suppression of Notch signaling, and the other meiotic genes, Dazl, Dmc1, and Rec8, were abolished or markedly decreased. Furthermore, RNAi of Notch1 also markedly inhibited the expression of Stra8 and SCP3 in cultured female germ cells. The increased methylation status of CpG islands within the Stra8 promoter of the oocytes was observed in the presence of DAPT, indicating that Notch signaling is probably necessary for maintaining the epigenetic state of this gene in a way suitable for RA stimulation. Furthermore, in the presence of Notch inhibitors, progression of oocytes through meiosis I was markedly delayed. At later culture periods, the rate of oocyte growth was decreased, which impaired subsequent primordial follicle assembly in cultured ovarian tissues. Taken together, these results suggested new roles of the Notch signaling pathway in female germ cell meiosis progression and early oogenesis events in mammals.

C-kit expression profile and regulatory factors during spermatogonial stem cell differentiation

BackgroundIt has been proven that c-kit is crucial for proliferation, migration, survival and maturation of spermatogenic cells. A periodic expression of c-kit is observed from primordial germ cells (PGCs) to spermatogenetic stem cells (SSCs), However, the expression profile of c-kit during the entire spermatogenesis process is still unclear. This study aims to reveal and compare c-kit expression profiles in the SSCs before and after the anticipated differentiation, as well as to examine its relationship with retinoic acid (RA) stimulation.ResultsWe have found that there are more than 4 transcripts of c-kit expressed in the cell lines and in the testes. The transcripts can be divided into short and long categories. The long transcripts include the full-length canonical c-kit transcript and the 3′ end short transcript. Short transcripts include the 3.4 kb short transcript and several truncated transcripts (1.9-3.2 kb). In addition, the 3.4 kb transcript (starting from intron 9 and covering exons 10 ~ 21) is discovered to be specifically expressed in the spermatogonia. The extracellular domain of Kit is obtained in the spermatogonia stage, but the intracellular domain (50 kDa) is constantly expressed in both SSCs and spermatogonia. The c-kit expression profiles in the testis and the spermatogonial stem cell lines vary after RA stimulation. The wave-like changes of the quantitative expression pattern of c-kit (increase initially and decrease afterwards) during the induction process are similar to that of the in vivo male germ cell development process.ConclusionsThere are dynamic transcription and translation changes of c-kit before and after SSCs’ anticipated differentiation and most importantly, RA is a significant upstream regulatory factor for c-kit expression.

Acute promyelocytic leukemia mutated to radioresistance suppressed monocyte lineage differentiation by phorbol 12-myristate 13-acetate

Induction of myeloid differentiation in radioresistant HL60 cells (Res-HL60) was examined to clarify the developmental mechanism of radioresistant leukemia. Compared to wild-type HL60 cells (Wt-HL60), Res-HL60 were smaller and strongly expressed CD38. Under all-trans retinoic acid (ATRA) stimulation, Res-HL60 continued to proliferate slowly and with similar level of CD11b expression to Wt-HL60. Phorbol 12-myristate 13-acetate (PMA) strongly suppressed proliferation of Res-HL60, downregulated CD14, and affected mRNA expression. These results suggested that the specific myeloid differentiation of Res-HL60 suppressed monocyte lineage by ATRA and PMA occurred through regulation of mRNA expression.

PMA withdrawal in PMA‐treated monocytic THP‐1 cells and subsequent retinoic acid stimulation, modulate induction of apoptosis and appearance of dendritic cells

To analyse proliferation, differentiation and apoptosis in THP-1 cells after stimulation with phorbol 12-myristate 13-acetate (PMA) and retinoic acid (RA). PMA and RA were used in a three-step-procedure: (i) treatment with 6, 30, 60nm PMA, that induced initial, intermediate and advanced levels of monocyte-macrophage transition, respectively; (ii) recovery in PMA-free medium; (iii) incubation with 4μm RA. Cultures were characterized cytokinetically (flow cytometry/bromodeoxyuridine uptake) and immunocytochemically (static cytometry) for expression of CD14, CD11b (monocyte-macrophage) and DC-SIGN (dendritic cell: DCs) markers. Some treatments determined appearance of monocyte/macrophage, dendritic and apoptotic phenotypes, percentages of which were related to PMA dose used in step 1, and dependent on presence/absence of PMA and RA. PMA withdrawal induced dedifferentiation and partial restoration of proliferative activity, specially in 6 and 30nm PMA-derived cells. Recovery in the presence of serum (fundamental to DC appearance) indicated that depending on differentiation level, cell proliferation and apoptosis were inversely correlated. Treatment with 30nm PMA induced intermediate levels of monocytic-macrophagic differentiation, with expression of alternative means of differentiation and acquisition of DCs without using cytokines, after PMA withdrawal and RA stimulation. Our experimental conditions favoured differentiation, dedifferentiation and transdifferentiational pathways, in monocytic THP-1 cells, the balance of which could be related to both cell proliferation and cell death.

RALDH2, the enzyme for retinoic acid synthesis, mediates meiosis initiation in germ cells of the female embryonic chickens

Meiosis is a process unique to the differentiation of germ cells and exhibits sex-specific in timing. Previous studies showed that retinoic acid (RA) as the vitamin A metabolite is crucial for controlling Stra8 (Stimulated by retinoic acid gene 8) expression in the gonad and to initiate meiosis; however, the mechanism by which retinoid-signaling acts has remained unclear. In the present study, we investigated the role of the enzyme retinaldehyde dehydrogenase 2 (RALDH2) which catalyzes RA synthesizes by initiating meiosis in chicken ovarian germ cells. Meiotic germ cells were first detected at day 15.5 in chicken embryo ovary when the expression of synaptonemal complex protein 3 (Scp3) and disrupted meiotic cDNA 1 homologue (Dmc1) became elevated, while Stra8 expression was specifically up-regulated at day 12.5 before meiosis onset. It was observed from the increase in Raldh2 mRNA expression levels and decreases in Cyp26b1 (the enzyme for RA catabolism) expression levels during meiosis that requirement for RA accumulation is essential to sustain meiosis. This was also revealed by RA stimulation of the cultured ovaries with the initiation of meiosis response, and the knocking down of the Raldh2 expression during meiosis, leading to abolishment of RA-dependent action. Altogether, these studies indicate that RA synthesis by the enzyme RALDH2 and signaling through its receptor is crucial for meiosis initiation in chicken embryonic ovary.

Effects of vitamin A status on the responses of the hepatic genes’ expression to insulin and retinoic acid in primary hepatocytes from Zucker lean and fatty rats

Insulin‐mediated regulation of hepatic gene expression plays a key role in glucose and lipid homeostasis. This study was aimed to examine the hepatic gene expression in response to insulin and retinoic acid (RA) treatments in primary hepatocytes from Zucker rats of different vitamin A (VA) status. Primary hepatocytes were isolated from Zucker lean (ZL) or fatty (ZF) rats fed either vitamin A sufficient (VAS) or deficient diet (VAD) for 8 weeks after weaning. The mRNA levels of Gck, Pck1, Srebp‐1c and Pklr after insulin and RA stimulation for 6 hours were determined using real‐time PCR. In hepatocytes of ZL rats on either diet, insulin synergized with RA to induce the expression of Gck and Srebp‐1c. This was impaired in hepatocytes of ZF rats on VAS, but partially recovered in those on VAD diet. In lean hepatocytes, the Pck1 expression was suppressed by insulin and induced by RA respectively. The insulin‐suppressed Pck1 expression was impaired in hepatocytes from ZF rats fed VAS diet and partially restored in that fed VAD diet. The RA‐induced Pck1 expression was normal in hepatocytes from ZF rats fed VAS, and was enhanced in that fed VAD. The Pklr expression was similar between ZL and ZF and not affected by VA status. We conclude that VA status modulate the insulin‐ and RA‐regulated hepatic gene expression in hepatocytes of ZF rats. It suggests that micronutrients contribute to the hepatic insulin response.Grant Funding Source: AHA

Retinoic acid-induced upregulation of the metalloendopeptidase nardilysin is accelerated by co-expression of the brain-specific protein p42 IP4 (centaurin α1; ADAP1) in neuroblastoma cells

The mainly neuronally expressed protein p42 IP4 (centaurin α1; ADAP1), which interacts with the metalloendopeptidase nardilysin (NRD) was found to be localized in neuritic plaques in Alzheimer disease (AD) brains. NRD was shown to enhance the cleavage of the amyloid precursor protein (APP) by α-secretases, thereby increasing the release of neuroprotective sAPPα. We here investigated in vitro the biochemical interaction of p42 IP4 and NRD and studied the physiological interaction in SH-SY5Y cells. NRD is a member of the M16 family of metalloendopeptidases. Some members of this M16 family act bi-functionally, as protease and as non-enzymatic scaffold protein. Here, we show that p42 IP4 enhances the enzymatic activity of NRD 3–4 times. However, p42 IP4 is not a substrate for NRD. Furthermore, we report that differentiation of SH-SY5Y cells by stimulation with 10 μM retinoic acid (RA) results in upregulation of NRD protein levels, with a 6-fold rise after 15 days. NRD is expressed in the neurites of RA-stimulated SH-SY5Y cells, and localized in vesicular structures. Since p42 IP4 is not expressed in untreated SH-SY5Y cells, we could use this cell system as a model to find out, whether there is a functional interaction. Interestingly, SH-SY5Y cells, which we stably transfected with GFP-tagged-p42 IP4 showed an enhanced NRD protein expression already at an earlier time point after RA stimulation.

Concerted Action of Aldehyde Dehydrogenases Influences Depot-Specific Fat Formation

Vitamin A metabolite retinoic acid (RA) regulates life-sustaining differentiation processes and metabolic homeostasis. The aldehyde dehydrogenase-1 (Aldh1) family of enzymes (Aldh1a1, a2, and a3) catalyzes RA production from retinaldehyde and thereby controls concentrations of this transcriptionally active metabolite. The hierarchy of Aldh1 functions in adipose tissue has not been elucidated. We hypothesized that Aldh1 enzymes produce endogenous RA and regulate adipogenesis and fat formation in a fat depot-specific manner. We demonstrate that adipogenesis in vitro is accompanied by RA production generated primarily by Aldh1a1. In Aldh1a1-deficient adipocytes, adipogenesis is impaired compared with wild-type adipocytes due to markedly reduced expression of PPARγ regulated through zinc-finger protein 423 (ZFP423)-dependent mechanisms. These effects were recovered to some extent either by RA stimulation or overexpression of any of the Aldh1 enzymes in Aldh1a1(-/-) cells arguing that Aldh1a1 plays a dominant role in autocrine RA production. In vivo studies in C57/BL6 and Aldh1a1(-/-) mice on a regular diet revealed that multiple Aldh1 enzymes regulate differences in the formation of sc and visceral fat. In Aldh1a1(-/-) mice, visceral fat essentially lacked all Aldh1 expression. This loss of RA-producing enzymes was accompanied by 70% decreased expression of ZFP423, PPARγ, and Fabp4 in visceral fat of Aldh1a1(-/-) vs. wild-type mice and by the predominant loss of visceral fat. Subcutaneous fat of Aldh1a1(-/-) mice expressed Aldh1a3 for RA production that was sufficient to maintain expression of ZFP423 and PPARγ and sc fat mass. Our data suggest a paradigm for regulation of fat depots through the concerted action of Aldh1 enzymes that establish RA-dependent tandem regulation of transcription factors ZFP423 and PPARγ in a depot-specific manner.

Retinoic acid stimulation of VEGF secretion from human endometrial stromal cells is mediated by production of reactive oxygen species

It is widely accepted that vascular endothelial growth factor (VEGF) is involved in angiogenic functions that are necessary for successful embryonic implantation. We have shown that retinoic acid (RA), which is known to play a necessary role in early events in pregnancy, can combine with transcriptional activators of VEGF (e.g. TPA, TGF-β, IL-1β) to rapidly induce VEGF secretion from human endometrial stromal cells through a translational mechanism of action. We have now determined that this stimulation of VEGF by RA is mediated through an increased production of cellular reactive oxygen species (ROS). Results indicated that RA, but not TPA or TGF-β, directly increases ROS production in endometrial stromal cells and that the co-stimulating activity of RA on VEGF secretion can be mimicked by direct addition of H2O2. Importantly, co-treatment of RA with TPA or TGF-β further stimulated ROS production in a fashion that positively correlated with levels of VEGF secretion. The antioxidants N-acetylcysteine and glutathione monoethyl ester inhibited both RA + TPA and RA + TGF-β-stimulated secretion of VEGF, as well as RA-induced ROS production. Treatment of cells with RA resulted in a shift in the glutathione (GSH) redox potential to a more oxidative state, suggesting that the transduction pathway leading to increased VEGF secretion is at least partially mediated through the antioxidant capacity of GSH couples. The specificity of this action on GSH-sensitive signalling pathways is suggested by the determination that RA had no effect on the redox potential of thioredoxin. Together, these findings predict a redox-mediated mechanism for retinoid regulation of localized VEGF secretion in the human endometrium that may be necessary for the successful establishment of pregnancy.

Retinoic Acid-Stimulation of VEGF Secretion from Human Endometrial Stromal Cells Is Mediated by Production of Reactive Oxygen Species.

Non-technicalsummary Vascular endothelial growth factor (VEGF) is an angiogenic factor that plays a primary role in blood vessel development in uterine endometrial tissue during embryo implantation and early growth. Previously, we determined that retinoic acid (RA) can act as a co-factor to rapidly induce VEGF secretion from human endometrial stromal cells. We show here that stimulation of VEGF by RA is directly mediated by increased production of reactive oxygen species (ROS) in these cells. These findings predict a ROS-mediated mechanism for RA regulation of localized VEGF secretion in the human endometrium that may be necessary for the successful establishment of pregnancy. The results obtained may provide new targets for therapeutic intervention. Abstract It is widely accepted that vascular endothelial growth factor (VEGF) is involved in angiogenic functions that are necessary for successful embryonic implantation. We have shown that retinoic acid (RA), which is known to play a necessary role in early events in pregnancy, can combine with transcriptional activators of VEGF (e.g. TPA, TGF-β ,I L-1β) to rapidly induce VEGF secretion from human endometrial stromal cells through a translational mechanism of action. We have now determined that this stimulation of VEGF by RA is mediated through an increased production of cellular reactive oxygen species (ROS). Results indicated that RA, but not TPA or TGF-β, directly increases ROS production in endometrial stromal cells and that the co-stimulating activity of RA on VEGF secretion can be mimicked by direct addition of H2O2. Importantly, co-treatment of RA with TPA or TGF-β further stimulated ROS production in a fashionthatpositivelycorrelatedwithlevelsofVEGFsecretion.TheantioxidantsN-acetylcysteine andglutathionemonoethylesterinhibitedbothRA +TPAandRA +TGF-β-stimulatedsecretion of VEGF, as well as RA-induced ROS production. Treatment of cells with RA resulted in a shift in the glutathione (GSH) redox potential to a more oxidative state, suggesting that the transduction pathwayleadingtoincreasedVEGFsecretionisatleastpartiallymediatedthroughtheantioxidant capacity of GSH couples. The specificity of this action on GSH-sensitive signalling pathways is suggested by the determination that RA had no effect on the redox potential of thioredoxin. Together, these findings predict a redox-mediated mechanism for retinoid regulation of localized VEGFsecretioninthehumanendometriumthatmaybenecessaryforthesuccessfulestablishment of pregnancy.

Chromatin and DNA methylation dynamics during retinoic acid-induced RET gene transcriptional activation in neuroblastoma cells

Although it is well known that RET gene is strongly activated by retinoic acid (RA) in neuroblastoma cells, the mechanisms underlying such activation are still poorly understood. Here we show that a complex series of molecular events, that include modifications of both chromatin and DNA methylation state, accompany RA-mediated RET activation. Our results indicate that the primary epigenetic determinants of RA-induced RET activation differ between enhancer and promoter regions. At promoter region, the main mark of RET activation was the increase of H3K4me3 levels while no significant changes of the methylation state of H3K27 and H3K9 were observed. At RET enhancer region a bipartite chromatin domain was detected in unstimulated cells and a prompt demethylation of H3K27me3 marked RET gene activation upon RA exposure. Moreover, ChIP experiments demonstrated that EZH2 and MeCP2 repressor complexes were associated to the heavily methylated enhancer region in the absence of RA while both complexes were displaced during RA stimulation. Finally, our data show that a demethylation of a specific CpG site at the enhancer region could favor the displacement of MeCP2 from the heavily methylated RET enhancer region providing a novel potential mechanism for transcriptional regulation of methylated RA-regulated loci.

F‐spondin regulates chondrocyte terminal differentiation and endochondral bone formation

This study examines the role of F-spondin, an extracellular matrix protein of osteoarthritic cartilage, during chondrocyte maturation in embryonic growth plate cartilage. In chick tibia, F-spondin expression localized to the hypertrophic and calcified zones of the growth plate. Functional studies using tibial organ cultures indicated that F-spondin inhibited (∼35%, p = 0.02), and antibodies to F-spondin increased (∼30%, p < 0.1) longitudinal limb growth relative to untreated controls. In cell cultures, induction of chondrocyte maturation, by retinoic acid (RA) or transforming growth factor (TGF)-β treatment led to a significant upregulation of F-spondin (p < 0.05). F-spondin transfection increased mineral deposition, alkaline phosphatase (AP) and matrix metalloproteinase (MMP)-13 mRNA levels (p < 0.05), and AP activity following RA stimulation, compared to mock transfected controls. Using AP as a differentiation marker we then investigated the mechanism of F-spondin promaturation effects. Blocking endogenous F-spondin via its thrombospondin (TSR) domain inhibited RA induced AP activity 40% compared to controls (p < 0.05). The stimulatory effect of F-spondin on AP expression was also inhibited following depletion of TGF-β from culture supernatants. Our findings indicate that F-spondin is expressed in embryonic cartilage, where it has the capacity to enhance chondrocyte terminal differentiation and mineralization via interactions in its TSR domain and TGF-β dependent pathways.