Cellular uptake of retinoic acid in vitro.
Ontology highlight
ABSTRACT:
SUBMITTER: Banerjee CK
PROVIDER: S-EPMC1162166 | biostudies-other | 1980 Sep
REPOSITORIES: biostudies-other
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Project description:Cellular retinoic acid-binding protein II (CRABP-II) undergoes nuclear translocation upon binding of retinoic acid (RA). In the nucleus, CRABP-II directly binds to the nuclear receptor RAR to form a complex through which RA is "channeled" from the binding protein to the receptor. CRABP-II thus facilitates the ligation of RAR and markedly enhances its transcriptional activity. The primary sequence of CRABP-II contains three putative SUMOylation sites, centered at K45, K87, and K102. We show here that RA induces interactions of CRABP-II with the E2 SUMO ligase Ubc9 and triggers SUMOylation of the protein both in vitro and in cultured cells. Mutagenesis analyses demonstrate that K102 is the sole CRABP-II residue to be SUMOylated in response to RA. Mutation of this residue abolishes the ability of CRABP-II to undergo nuclear translocation in response RA and thus impairs CRABP-II-mediated activation of RAR. Additional observations demonstrate that apo-CRABP-II is associated with endoplasmic reticulum (ER), and that RA triggers the dissociation of CRABP-II from this location. Furthermore, we show that RA-induced dissociation of CRABP-II from the ER requires SUMOylation of K102. Hence, SUMOylation of K102 in response to RA binding is critical for dissociation of CRABP-II from ER and, consequently, for mobilization of the protein to nucleus and for its cooperation with RAR.
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Project description:All-trans-retinoic acid (atRA) is the essential derivative of vitamin A and is of interest due to its various biological key functions. As shown in the recent literature, atRA also plays a role in the failing heart during myocardial infarction, the leading cause of death globally. To date insufficient mechanistic information has been available on related hypoxia-induced cell damage and reperfusion injuries. However, it has been demonstrated that a reduction in cellular atRA uptake abrogates hypoxia-mediated cell and tissue damage, which may offer a new route for intervention. Consequently, in this study, the effect of the novel cardio-protective compound 5-methoxyleoligin (5ML) on cellular atRA uptake was tested in human umbilical-vein endothelial cells (HUVECs). For this purpose, a high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method was developed to assess intra-cellular levels of the active substance and corresponding levels of vitamin A and its derivatives, including potential cis/trans isomers. This work also focused on light-induced isomerization and the stability of biological sample material to ensure sample integrity and avoid biased conclusions. This study provides evidence of the inhibitory effect of 5ML on cellular atRA uptake, a promising step toward a novel therapy for myocardial infarction.
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Project description:We have cloned a cDNA and gene from the tobacco hornworm, Manduca sexta, which is related to the vertebrate cellular retinoic acid binding proteins (CRABPs). CRABPs are members of the superfamily of lipid binding proteins (LBPs) and are thought to mediate the effects of retinoic acid (RA) on morphogenesis, differentiation, and homeostasis. This discovery of a Manduca sexta CRABP (msCRABP) demonstrates the presence of a CRABP in invertebrates. Compared with bovine/murine CRABP I, the deduced amino acid sequence of msCRABP is 71% homologous overall and 88% homologous for the ligand binding pocket. The genomic organization of msCRABP is conserved with other CRABP family members and the larger LBP superfamily. Importantly, the promoter region contains a motif that resembles an RA response element characteristic of the promoter region of most CRABPs analyzed. Three-dimensional molecular modeling based on postulated structural homology with bovine/murine CRABP I shows msCRABP has a ligand binding pocket that can accommodate RA. The existence of an invertebrate CRABP has significant evolutionary implications, suggesting CRABPs appeared during the evolution of the LBP superfamily well before vertebrate/invertebrate divergence, instead of much later in evolution in selected vertebrates.
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Project description:Spermatogenesis is an intricate developmental process occurring in testes by which spermatogonial stem cells (SSCs) self-renew and differentiate into mature sperm. The molecular mechanisms for SSC self-renewal and differentiation, while have been well studied in mice, may differ between mice and domestic animals including pigs. To gain knowledge about the molecular mechanisms for porcine SSC self-renewal and differentiation that have to date been poorly understood, here we isolated and enriched primitive spermatogonia from neonatal porcine testes, and exposed the cells to retinoic acid, a direct inducer for spermatogonial differentiation. We then identified that retinoic acid could induce porcine primitive spermatogonial differentiation into leptotene spermatocyte-like cells, which was accompanied by a clear transcriptomic alteration, as revealed by the RNA-sequencing analysis. We also compared retinoic acid-induced in vitro porcine spermatogonial differentiation with the in vivo process, and compared retinoic acid-induced in vitro spermatogonial differentiation between pigs and mice. Furthermore, we analyzed retinoic acid-induced differentially expressed long non-coding RNAs (lncRNAs), and demonstrated that a pig-specific lncRNA, lncRNA-106504875, positively regulated porcine spermatogonial proliferation by targeting the core transcription factor ZBTB16. Taken together, these results would help to elucidate the roles of retinoic acid in porcine spermatogonial differentiation, thereby contributing to further knowledge about the molecular mechanisms underlying porcine SSC development and, in the long run, to optimization of both long-term culture and induced differentiation systems for porcine SSCs.
2023-01-09 | GSE219031 | GEO
Project description:Vitamin A deficiency in the mouse results in an arrest in the progression of undifferentiated spermatogonia to differentiating spermatogonia. The supplement of retinol to vitamin-A-deficient mice reinitiates spermatogenesis in a synchronous manner throughout the testes. It is unclear whether the effects of retinoids are the result of a direct action on germ cells or are indirectly mediated through Sertoli cells. The expression of Stimulated by retinoic acid gene 8 (Stra8), which is required for spermatogenesis, is directly related to the availability of retinoic acid (RA). Analysis of gene expression by microarrays revealed moderate levels of Stra8 transcript in gonocytes and high levels in A and B spermatogonia. Stra8 mRNA levels were greatly reduced or absent in germ cells once they entered meiosis. This study examined the effect of retinoic acid on cultured neonatal testes and isolated gonocytes/spermatogonia in vitro. THY1(+) and KIT(+) germ cells were isolated by magnetic-activated cell sorting from the testes of mice of different ages. Isolated germ cells were cultured and treated with either vehicle (ethanol) or RA without feeder cells. We found that 1) Stra8 is predominantly expressed in premeiotic germ cells, 2) RA stimulates gonocyte DNA replication and differentiation in cultured neonatal testes, 3) in the absence of feeder cells, RA directly induces the transition of undifferentiated spermatogonia to differentiating spermatogonia by stimulating Stra8 and Kit gene expression, 4) RA dramatically stimulates Stra8 expression in undifferentiated spermatogonia but has a lesser impact in differentiating spermatogonia, 5) endogenous Stra8 gene expression is higher in differentiating spermatogonia than in undifferentiated spermatogonia and could mediate the RA effects on spermatogonial maturation, and 6) RA stimulates a group of genes involved in the metabolism, storage, transport, and signaling of retinoids.
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Project description:Cyclooxygenases (COX) metabolize arachidonic acid (AA) to hydroxyeicosatetraenoic acids (HETE), which can then be oxidized by dehydrogenases, such as 15-hydroxyprostaglandin dehydrogenase (15-PGDH), to oxo-eicosatetraenoic acids (ETE). We have previously established that 11-oxo-eicosatetraenoic acid (oxo-ETE) and 15-oxo-ETE are COX-2/15-PGDH-derived metabolites. Stable isotope dilution (SID) chiral liquid chromatography coupled with electron capture atmospheric pressure chemical ionization (ECAPCI) single reaction monitoring (SRM) MS has been used to quantify uptake of 11-oxo-ETE and 15-oxo-ETE in both LoVo cells and human umbilical vein endothelial cells (HUVEC). Intracellular 11-oxo- and 15-oxo-ETE concentrations reached maximum levels within 1 h and declined rapidly, with significant quantitative differences in uptake between the LoVo cells and the HUVECs. Maximal intracellular concentrations of 11-oxo-ETE were 0.02 ng/4 × 10? cells in the LoVo cells and 0.58 ng/4 × 10? cells in the HUVECs. Conversely, maximal levels of 15-oxo-ETE were 0.21 ng/4 × 10? in the LoVo cells and 0.01 ng/4 × 10? in the HUVECs. The methyl esters of both 11-oxo- and 15-oxo-ETE increased the intracellular concentrations of the corresponding free oxo-ETEs by 3- to 8-fold. 11-oxo-ETE, 15-oxo-ETE, and their methyl esters inhibited proliferation in both HUVECs and LoVo cells at concentrations of 2-10 ?M, with 11-oxo-ETE methyl ester being the most potent inhibitor. Cotreatment with probenecid, an inhibitor of multiple drug resistance transporters (MRP)1 and 4, increased the antiproliferative effect of 11-oxo-ETE methyl ester in LoVo cells and increased the intracellular concentration of 11-oxo-ETE from 0.05 ng/4 × 10? cells to 0.18 ng/4 × 10? cells. Therefore, this study has established that the COX-2/15-PGDH-derived eicosanoids 11-oxo- and 15-oxo-ETE enter target cells, that they inhibit cellular proliferation, and that their inhibitory effects are modulated by MRP exporters.
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Project description:Meiosis is the key step in gametogenesis. However, the mechanism of mammalian meiosis remains poorly understood due to the lack of an in vitro model. Here, we report that retinoic acid (RA) is sufficient for inducing leptotene/zygotene spermatocytes from cultured mouse spermatogonial stem cells. Multiple genes regulated by RA were identified by RNA sequencing. RA in combination with pup Sertoli cell co-culture resulted in a higher induction efficiency of 28%. Comparisons in the transcriptomic profiles of the induced spermatogenic cells and the isolated ones revealed the progressive induction of the germ cells. Using this model, we showed that Stra8, Agpat3, Fam57a, Wdr91, and Sox30 contributed to the proliferation and meiosis initiation differentially. In conclusion, we have efficiently generated spermatocytes using an RA/pup Sertoli cell-based in vitro model and provided proof-of-concept evidence for its application in identifying genes involved in mammalian meiosis.
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Project description:Cellular retinoic acid-binding protein 2 (CRABP2) potently suppresses the growth of various carcinomas, but the mechanism(s) that underlies this activity remains incompletely understood. CRABP2 displays two distinct functions. The classical function of this protein is to directly deliver retinoic acid (RA) to RA receptor (RAR), a nuclear receptor activated by this hormone, in turn inducing the expression of multiple antiproliferative genes. The other function of the protein is exerted in the absence of RA and mediated by the RNA-binding and stabilizing protein HuR. CRABP2 directly binds to HuR, markedly strengthens its interactions with target mRNAs, and thus increases their stability and up-regulates their expression. Here we show that the anticarcinogenic activities of CRABP2 are mediated by both of its functions. Transcriptome analyses revealed that, in the absence of RA, a large cohort of transcripts is regulated in common by CRABP2 and HuR, and many of these are involved in regulation of oncogenic properties. Furthermore, both in cultured cells and in vivo, CRABP2 or a CRABP2 mutant defective in its ability to cooperate with RAR but competent in interactions with HuR suppressed carcinoma growth and did so in the absence of RA. Hence, transcript stabilization by the CRABP2-HuR complex significantly contributes to the ability of CRABP2 to inhibit tumorigenesis. Surprisingly, the observations also revealed that HuR regulates the expression of multiple genes involved in nuclear pore formation and is required for nuclear import of CRABP2 and for transcriptional activation by RAR. The data thus point at a novel function for this important protein.
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Project description:The treatment of solid cancers with pharmacological all-trans retinoic acid (ATRA) concentrations, even if it is a gold standard therapy for the acute promyelocytic leukaemia (APL), is not always effective due to some resistance mechanisms. Here the resistance to ATRA treatment of T24 cell line, bladder cancer, was investigated. T24 was not only resistant to cell death when treated at concentrations up to 20 µM of ATRA, but it was also able to stimulate the cellular proliferation. An over-expression of the fatty acid binding protein 5 (FABP5) in conjunction with the cellular retinol-binding protein-II (CRABP-II) down-expression was found. However, the direct inhibition of the peroxisome proliferator-activated receptor β/δ (PPARβ/δ) did not abolish T24 proliferation, but rather potentiated it. Moreover, considering the ability of the long-chain fatty acids (LCFAs) to displace ATRA from FABP5, the actions of the saturated palmitic acid (PA), unsaturated omega-6 linoleic acid (LA) and omega-3 docosahexaenoic acid (DHA) were evaluated to counteract ATRA-related proliferation. ATRA-PA co-treatment induces cellular growth inhibition, while ATRA-LA co-treatment induces cellular growth enhancement. However, even if DHA is unsaturated LCFA as LA, it was able to reverse the ATRA-induced cellular proliferation of T24, bringing the viability percentages at the levels of the control.
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Project description:Skin aging is an intricate biological process consisting of intrinsic and extrinsic alterations of epidermal and dermal structures. Retinoids play an important role in epidermal cell growth and differentiation and are beneficial to counteract skin aging. Cellular retinoic acid binding protein-II (CRABP-II) selectively binds all trans-retinoic acid, the most active retinoid metabolite, contributing to regulate intracytoplasmic retinoid trafficking and keratinocyte differentiation. Immunohistochemistry revealed a reduced epidermal and dermal CRABP-II expression in aged human and mouse skin. To better clarify the role of CRABP-II, we investigated age-related skin changes in CRABP-II knock-out mice. We documented an early reduction of keratinocyte layers, proliferation and differentiation rate, dermal and hypodermal thickness, pilosebaceous units and dermal vascularity in CRABP-II knock-out compared with wild-type mice. Ultrastructural investigation documented reduced number and secretion of epidermal lamellar bodies in CRABP-II knock-out compared with wild-type mice. Cultured CRABP-II knock-out-derived dermal fibroblasts proliferated less and showed reduced levels of TGF-β signal-related genes, Col1A1, Col1A2, and increased MMP2 transcripts compared with those from wild-type. Our data strongly support the hypothesis that a reduction of CRABP-II expression accelerates and promotes skin aging, and suggest CRABP-II as a novel target to improve the efficacy of retinoid-mediated anti-aging therapies.
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