ABSTRACT
It has long been appreciated that insulin action is closely tied to circadian rhythms. However, the mechanisms that dictate diurnal insulin sensitivity in metabolic tissues are not well understood. Retinol-binding protein 4 (RBP4) has been implicated as a driver of insulin resistance in rodents and humans, and it has become an attractive drug target in type II diabetes. RBP4 is synthesized primarily in the liver where it binds retinol and transports it to tissues throughout the body. The retinol-RBP4 complex (holo-RBP) can be recognized by a cell-surface receptor known as stimulated by retinoic acid 6 (STRA6), which transports retinol into cells. Coupled to retinol transport, holo-RBP can activate STRA6-driven Janus kinase (JAK) signaling and downstream induction of signal transducer and activator of transcription (STAT) target genes. STRA6 signaling in white adipose tissue has been shown to inhibit insulin receptor responses. Here, we examined diurnal rhythmicity of the RBP4/STRA6 signaling axis and investigated whether STRA6 is necessary for diurnal variations in insulin sensitivity. We show that adipose tissue STRA6 undergoes circadian patterning driven in part by the nuclear transcription factor REV-ERBα. Furthermore, STRA6 is necessary for diurnal rhythmicity of insulin action and JAK/STAT signaling in adipose tissue. These findings establish that holo-RBP and its receptor STRA6 are potent regulators of diurnal insulin responses and suggest that the holo-RBP/STRA6 signaling axis may represent a novel therapeutic target in type II diabetes.
Subject(s)
Circadian Rhythm , Insulin/metabolism , Membrane Proteins/metabolism , Adipose Tissue, White/metabolism , Animals , Diabetes Mellitus, Type 2/metabolism , Membrane Proteins/deficiency , Mice , Mice, Knockout , Signal TransductionABSTRACT
The RNA-binding protein HuR binds to elements rich in adenylate and uridylate (AU-rich elements) in target mRNAs and stabilizes them against degradation. The complete spectrum of genes whose expression is regulated by HuR and are the basis for the broad range of cellular functions of the protein is incompletely understood. We show that HuR controls the expression of multiple components of the nuclear import machinery. Consequently, HuR is crucial for the nuclear import of cellular retinoic acid-binding protein 2 (CRABP2), which delivers RA to the nuclear retinoic acid receptor (RAR) and whose mobilization to the nucleus is mediated by a 'classical-like' nuclear localization signal (NLS). HuR is also required for heregulin-induced nuclear translocation of the NFκB subunit p65, which contains both classical and non-canonical NLSs. HuR thus regulates the transcriptional activities of both RAR and NFκB. The observations reveal that HuR plays a central role in regulating nuclear import of proteins.
Subject(s)
Cell Nucleus/metabolism , ELAV-Like Protein 1/metabolism , Active Transport, Cell Nucleus/drug effects , Animals , Apoproteins/metabolism , Cell Nucleus/drug effects , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/metabolism , HEK293 Cells , Humans , Karyopherins/metabolism , MCF-7 Cells , Mice , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Quinazolines/pharmacology , RNA Stability/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Retinoic Acid/metabolism , Tretinoin/pharmacology , beta Karyopherins/metabolismABSTRACT
Vitamin A, retinol, circulates in blood bound to retinol binding protein (RBP). In some tissues, the retinol-RBP complex (holo-RBP) is recognized by a membrane receptor, termed STRA6, which mediates uptake of retinol into cells. Recent studies have revealed that, in addition to serving as a retinol transporter, STRA6 is a ligand-activated cell surface signaling receptor that, upon binding of holo-RBP activates JAK/STAT signaling, culminating in the induction of STAT target genes. It has further been shown that retinol transport and cell signaling by STRA6 are critically interdependent and that both are coupled to intracellular vitamin A metabolism. The molecular mechanism of action of STRA6 and its associated machinery is beginning to be revealed, but further work is needed to identify and characterize the complete range of genes and associated signaling cascades that are regulated by STRA6 in different tissues. An understanding of STRA6 is clinically relevant, as for example, it has been shown to be hyper- activated in obese animals, leading to insulin resistance. A potential role for STRA6 in other pathologies, including cancer, awaits further investigation.
Subject(s)
Membrane Proteins/physiology , Vitamin A/metabolism , Animals , Anophthalmos/genetics , Anophthalmos/metabolism , Biological Transport , Gene Expression Regulation , Humans , Insulin Resistance , Janus Kinases/physiology , Lung Diseases/genetics , Lung Diseases/metabolism , Membrane Proteins/deficiency , Membrane Proteins/genetics , Mice , Mice, Knockout , Microphthalmos/genetics , Microphthalmos/metabolism , Obesity/metabolism , Organ Specificity , Prealbumin/physiology , Retinol-Binding Proteins, Plasma/metabolism , STAT Transcription Factors/physiology , Signal Transduction/physiology , Transcription, GeneticABSTRACT
It has long been established that the transcriptional activity of retinoic acid (RA) is mediated by members of the nuclear receptor family of ligand-activated transcription factors termed RA receptors (RARs). More recent observations have established that RA also activates an additional nuclear receptor, PPARß/δ. Partitioning RA between RARs and PPARß/δ is governed by different intracellular lipid-binding proteins: cellular RA binding protein 2 (CRABP2) delivers RA to nuclear RARs and a fatty acid binding protein (FABP5) delivers the hormone from the cytosol to nuclear PPARß/δ. Consequently, RA signals through RARs in CRABP2-expressing cells, but activates PPARß/δ in cells that express a high level of FABP5. RA elicits different and sometimes opposing responses in cells that express different FABP5/CRABP2 ratios because PPARß/δ and RARs regulate the expression of distinct sets of genes. An overview of the observations that led to the discovery of this non-classical activity of RA are presented here, along with a discussion of evidence demonstrating the involvement of the dual transcriptional activities of RA in regulating energy homeostasis, insulin responses, and adipocyte and neuron differentiation.
Subject(s)
Gene Expression Regulation/drug effects , PPAR delta/physiology , PPAR-beta/physiology , Transcription, Genetic/drug effects , Tretinoin/pharmacology , Adipogenesis/drug effects , Adipogenesis/genetics , Adipose Tissue/metabolism , Animals , Biological Transport , Fatty Acid-Binding Proteins/physiology , Forecasting , Gene Expression Regulation/genetics , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/genetics , Humans , Models, Molecular , Neoplasm Proteins/physiology , Neurogenesis/drug effects , Neurogenesis/genetics , Obesity/metabolism , PPAR delta/drug effects , PPAR-beta/drug effects , Protein Conformation , Receptors, Retinoic Acid/physiologyABSTRACT
Endocannabinoids modulate multiple behaviors, including learning and memory. We show that the endocannabinoid anandamide (AEA) can alter neuronal cell function both through its established role in activation of the G-protein-coupled receptor CB1, and by serving as a precursor for a potent agonist of the nuclear receptor PPARß/δ, in turn up-regulating multiple cognition-associated genes. We show further that the fatty acid-binding protein FABP5 controls both of these functions in vivo. FABP5 both promotes the hydrolysis of AEA into arachidonic acid and thus reduces brain endocannabinoid levels, and directly shuttles arachidonic acid to the nucleus where it delivers it to PPARß/δ, enabling its activation. In accordance, ablation of FABP5 in mice results in excess accumulation of AEA, abolishes PPARß/δ activation in the brain, and markedly impairs hippocampus-based learning and memory. The data indicate that, by controlling anandamide disposition and activities, FABP5 plays a key role in regulating hippocampal cognitive function.
Subject(s)
Arachidonic Acids/metabolism , Brain/physiology , Cognition/physiology , Endocannabinoids/metabolism , Fatty Acid-Binding Proteins/metabolism , Neoplasm Proteins/metabolism , PPAR delta/metabolism , PPAR-beta/metabolism , Polyunsaturated Alkamides/metabolism , Active Transport, Cell Nucleus , Animals , Blotting, Western , Brain/metabolism , Cell Line, Tumor , Cell Nucleus/metabolism , Fatty Acid-Binding Proteins/genetics , Gene Regulatory Networks , Maze Learning/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Microscopy, Confocal , Neoplasm Proteins/genetics , Oligonucleotide Array Sequence Analysis , PPAR delta/agonists , PPAR delta/genetics , PPAR-beta/agonists , PPAR-beta/genetics , Protein Binding , Reverse Transcriptase Polymerase Chain Reaction , Thiazoles/pharmacology , Transcriptome/drug effectsABSTRACT
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.
Subject(s)
ELAV Proteins/genetics , Gene Expression Regulation, Neoplastic , RNA, Messenger/genetics , Receptors, Retinoic Acid/genetics , Transcriptional Activation , Active Transport, Cell Nucleus , Animals , Carcinogenesis/genetics , Cell Line, Tumor , Cell Nucleus/metabolism , Cell Proliferation , ELAV Proteins/metabolism , ELAV-Like Protein 1 , Genes, Reporter , Humans , Luciferases/genetics , Luciferases/metabolism , Mice , RNA Stability , RNA, Messenger/metabolism , Receptors, Retinoic Acid/metabolism , Signal Transduction , Tretinoin/metabolism , Tretinoin/pharmacologyABSTRACT
Fatty acid-binding proteins (FABPs) are a widely expressed group of calycins that play a well established role in solubilizing cellular fatty acids. Recent studies, however, have recast FABPs as active participants in vital lipid-signaling pathways. FABP5, like its family members, displays a promiscuous ligand binding profile, capable of interacting with numerous long chain fatty acids of varying degrees of saturation. Certain "activating" fatty acids induce the protein's cytoplasmic to nuclear translocation, stimulating PPARß/δ transactivation; however, the rules that govern this process remain unknown. Using a range of structural and biochemical techniques, we show that both linoleic and arachidonic acid elicit FABP5's translocation by permitting allosteric communication between the ligand-sensing ß2 loop and a tertiary nuclear localization signal within the α-helical cap of the protein. Furthermore, we show that more saturated, nonactivating fatty acids inhibit nuclear localization signal formation by destabilizing this activation loop, thus implicating FABP5 specifically in cis-bonded, polyunsaturated fatty acid signaling.
Subject(s)
Fatty Acid-Binding Proteins/metabolism , Fatty Acids/metabolism , PPAR gamma/metabolism , PPAR-beta/metabolism , Animals , Arachidonic Acid/chemistry , Arachidonic Acid/metabolism , Arachidonic Acid/pharmacology , Binding Sites , COS Cells , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Chlorocebus aethiops , Cytoplasm/drug effects , Cytoplasm/metabolism , Fatty Acid-Binding Proteins/chemistry , Fatty Acid-Binding Proteins/genetics , Fatty Acids/chemistry , Fatty Acids/pharmacology , Humans , Ligands , Linoleic Acid/chemistry , Linoleic Acid/metabolism , Linoleic Acid/pharmacology , MCF-7 Cells , Models, Molecular , Molecular Structure , Mutation , Nuclear Localization Signals/chemistry , Nuclear Localization Signals/metabolism , PPAR gamma/genetics , PPAR-beta/genetics , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Protein Transport/drug effects , Transcriptional Activation/drug effectsABSTRACT
Vitamin A, retinol, circulates in blood bound to retinol-binding protein (RBP). At some tissues, holo-RBP is recognized by a plasma membrane receptor termed STRA6, which serves a dual role: it mediates transport of retinol from RBP into cells, and it functions as a cytokine receptor that, on binding holo-RBP, activates JAK2/STAT5 signaling. As STAT target genes include SOCS3, an inhibitor of insulin receptor, holo-RBP suppresses insulin responses in STRA6-expressing cells. We have shown previously that the two functions of STRA6 are interdependent. These observations suggest factors that regulate STRA6-mediated retinol transport may also control STRA6-mediated cell signaling. One such factor is retinol metabolism, which enables cellular uptake of retinol by maintaining an inward-directed concentration gradient. We show here that lecithin:retinol acyl transferase (LRAT), which catalyzes esterification of retinol to its storage species retinyl esters, is necessary for activation of the STRA6/JAK2/STAT5 cascade by holo-RBP. In accordance, LRAT-null mice are protected from holo-RBP-induced suppression of insulin responses. Hence, STRA6 signaling, which requires STRA6-mediated retinol transport, is supported by LRAT-catalyzed retinol metabolism. The observations demonstrate that STRA6 regulates key cellular processes by coupling circulating holo-RBP levels and intracellular retinol metabolism to cell signaling.
Subject(s)
Acyltransferases/metabolism , Membrane Proteins/metabolism , Retinol-Binding Proteins/metabolism , Acyltransferases/genetics , Animals , Eating/physiology , Glucose Tolerance Test , Hep G2 Cells , Humans , Membrane Proteins/genetics , Mice , NIH 3T3 Cells , Real-Time Polymerase Chain Reaction , Retinol-Binding Proteins/genetics , Suppressor of Cytokine Signaling 3 Protein , Suppressor of Cytokine Signaling Proteins/genetics , Suppressor of Cytokine Signaling Proteins/metabolism , Vitamin A/metabolismABSTRACT
The plasma membrane protein STRA6 is thought to mediate uptake of retinol from its blood carrier retinol-binding protein (RBP) into cells and to function as a surface receptor that, upon binding of holo-RBP, activates a JAK/STAT cascade. It was suggested that STRA6 signaling underlies insulin resistance induced by elevated serum levels of RBP in obese animals. To investigate these activities in vivo, we generated and analyzed Stra6-null mice. We show that the contribution of STRA6 to retinol uptake by tissues in vivo is small and that, with the exception of the eye, ablation of Stra6 has only a modest effect on retinoid homeostasis and does not impair physiological functions that critically depend on retinoic acid in the embryo or in the adult. However, ablation of Stra6 effectively protects mice from RBP-induced suppression of insulin signaling. Thus one biological function of STRA6 in tissues other than the eye appears to be the coupling of circulating holo-RBP levels to cell signaling, in turn regulating key processes such as insulin response.
Subject(s)
Insulin Resistance , Insulin/metabolism , Membrane Proteins/metabolism , Obesity/metabolism , Retinol-Binding Proteins, Plasma/metabolism , Vitamin A/metabolism , 3T3-L1 Cells , Animals , Eye , Insulin/genetics , Membrane Proteins/genetics , Mice , Mice, Knockout , Obesity/genetics , Obesity/pathology , Retinol-Binding Proteins, Plasma/genetics , Signal Transduction/geneticsABSTRACT
It currently is believed that vitamin A, retinol, functions through active metabolites: the visual chromophore 11-cis-retinal, and retinoic acids, which regulate gene transcription. Retinol circulates in blood bound to retinol-binding protein (RBP) and is transported into cells by a membrane protein termed "stimulated by retinoic acid 6" (STRA6). We show here that STRA6 not only is a vitamin A transporter but also is a cell-surface signaling receptor activated by the RBP-retinol complex. Association of RBP-retinol with STRA6 triggers tyrosine phosphorylation, resulting in recruitment and activation of JAK2 and the transcription factor STAT5. The RBP-retinol/STRA6/JAK2/STAT5 signaling cascade induces the expression of STAT target genes, including suppressor of cytokine signaling 3 (SOCS3), which inhibits insulin signaling, and peroxisome proliferator-activated receptor gamma (PPARγ), which enhances lipid accumulation. These observations establish that the parental vitamin A molecule is a transcriptional regulator in its own right, reveal that the scope of biological functions of the vitamin is broader than previously suspected, and provide a rationale for understanding how RBP and retinol regulate energy homeostasis and insulin responses.
Subject(s)
Gene Expression Regulation , Insulin/metabolism , Retinol-Binding Proteins/metabolism , Signal Transduction , Vitamin A/metabolism , Animals , Enzyme Activation/drug effects , Gene Expression Regulation/drug effects , Hep G2 Cells , Humans , Janus Kinase 2/metabolism , Membrane Proteins/metabolism , Mice , Models, Biological , Phosphorylation/drug effects , Protein Binding/drug effects , STAT5 Transcription Factor/metabolism , Signal Transduction/drug effects , Transcriptional Activation/drug effects , Triglycerides/metabolismABSTRACT
Retinoic acid (RA) regulates gene transcription by activating the nuclear receptors retinoic acid receptor (RAR) and peroxisome proliferator-activated receptor (PPAR) ß/δ and their respective cognate lipid-binding proteins CRABP-II and FABP5. RA induces neuronal differentiation, but the contributions of the two transcriptional pathways of the hormone to the process are unknown. Here, we show that the RA-induced commitment of P19 stem cells to neuronal progenitors is mediated by the CRABP-II/RAR path and that the FABP5/PPARß/δ path can inhibit the process through induction of the RAR repressors SIRT1 and Ajuba. In contrast with its inhibitory activity in the early steps of neurogenesis, the FABP5/PPARß/δ path promotes differentiation of neuronal progenitors to mature neurons, an activity mediated in part by the PPARß/δ target gene PDK1. Hence, RA-induced neuronal differentiation is mediated through RAR in the early stages and through PPARß/δ in the late stages of the process. The switch in RA signaling is accomplished by a transient up-regulation of RARß concomitantly with a transient increase in the CRABP-II/FABP5 ratio at early stages of differentiation. In accordance with these conclusions, hippocampi of FABP5-null mice display excess accumulation of neuronal progenitor cells and a deficit in mature neurons versus wild-type animals.
Subject(s)
Antineoplastic Agents/pharmacology , Hippocampus/metabolism , Nerve Tissue Proteins/metabolism , Neural Stem Cells/metabolism , Neurogenesis/drug effects , Neurons/metabolism , PPAR-beta/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Retinoic Acid/metabolism , Signal Transduction/drug effects , Tretinoin/pharmacology , Animals , Cell Line , Fatty Acid-Binding Proteins/genetics , Fatty Acid-Binding Proteins/metabolism , Hippocampus/cytology , LIM Domain Proteins/genetics , LIM Domain Proteins/metabolism , Mice , Mice, Mutant Strains , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Nerve Tissue Proteins/genetics , Neural Stem Cells/cytology , Neurogenesis/physiology , Neurons/cytology , PPAR-beta/genetics , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Pyruvate Dehydrogenase Acetyl-Transferring Kinase , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Retinoic Acid/genetics , Signal Transduction/physiology , Sirtuin 1/genetics , Sirtuin 1/metabolismABSTRACT
Vitamin A, retinol, circulates in blood bound to serum retinol binding protein (RBP) and is transported into cells by a membrane protein termed stimulated by retinoic acid 6 (STRA6). It was reported that serum levels of RBP are elevated in obese rodents and humans, and that increased level of RBP in blood causes insulin resistance. A molecular mechanism by which RBP can exert such an effect is suggested by the recent discovery that STRA6 is not only a vitamin A transporter but also functions as a surface signaling receptor. Binding of RBP-ROH to STRA6 induces the phosphorylation of a tyrosine residue in the receptor C-terminus, thereby activating a JAK/STAT signaling cascade. Consequently, in STRA6-expressing cells such as adipocytes, RBP-ROH induces the expression of STAT target genes, including SOCS3, which suppresses insulin signaling, and PPARγ, which enhances lipid accumulation. RBP-retinol thus joins the myriad of cytokines, growth factors and hormones which regulate gene transcription by activating cell surface receptors that signal through activation of Janus kinases and their associated transcription factors STATs. This article is part of a Special Issue entitled Retinoid and Lipid Metabolism.
Subject(s)
Insulin Resistance , Insulin/metabolism , Lipid Metabolism , Membrane Proteins/metabolism , Retinol-Binding Proteins/metabolism , Vitamin A/metabolism , Animals , Homeostasis , Humans , Janus Kinases/metabolism , Lipids , PPAR gamma/biosynthesis , PPAR gamma/metabolism , Retinol-Binding Proteins/genetics , STAT Transcription Factors/biosynthesis , STAT Transcription Factors/metabolism , Signal Transduction , Suppressor of Cytokine Signaling 3 Protein , Suppressor of Cytokine Signaling Proteins/biosynthesis , Suppressor of Cytokine Signaling Proteins/metabolism , Vitamin A/bloodABSTRACT
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.
Subject(s)
Active Transport, Cell Nucleus , Receptors, Retinoic Acid/metabolism , Sumoylation , Tretinoin/metabolism , Animals , COS Cells , Cell Line, Tumor , Cell Nucleus/metabolism , Chlorocebus aethiops , Endoplasmic Reticulum/metabolism , HEK293 Cells , Humans , Transcription, Genetic , Ubiquitin-Conjugating Enzymes/metabolismABSTRACT
In preadipocytes, retinoic acid (RA) regulates gene expression by activating the nuclear RA receptor (RAR) and its cognate intracellular lipid-binding protein CRABP-II. It was previously reported that RA inhibits adipocyte differentiation but only when administered early during the differentiation program. The data presented here indicate that the diminished ability of RA to activate RAR following induction of differentiation stems from down-regulation of CRABP-II. The observations show that expression of CRABP-II in preadipocytes is repressed by all three components of the classical hormonal mixture that induces adipocyte differentiation, i.e. isobutylmethylxanthine, insulin, and dexamethasone. Isobutylmethylxanthine-dependent activation of protein kinase A triggered the phosphorylation of the transcription factor cAMP-response element-binding protein, which induced the expression of the cAMP-response element-binding protein family repressor cAMP-response element modulator. In turn, cAMP-response element modulator was found to associate with a cognate response element in the CRABP-II promoter and to repress CRABP-II expression. The data further show that CRABP-II is a direct target gene for the glucocorticoid receptor and that it is subjected to dexamethasone-induced glucocorticoid receptor-mediated repression during adipogenesis. Finally, the observations demonstrate that permanent repression of CRABP-II in mature adipocytes is exerted by the master regulator of adipocyte differentiation CCAAT/enhancer-binding protein alpha and is directly mediated through CCAAT/enhancer-binding protein alpha-response elements in the CRABP-II promoter. Taken together, the observations emphasize the important role of CRABP-II in regulating the transcriptional activity of RA through RAR, and they demonstrate that repression of this gene is critical for allowing adipogenesis to proceed.
Subject(s)
Adipocytes/metabolism , Cell Differentiation , Receptors, Retinoic Acid/metabolism , 3T3-L1 Cells , Adipocytes/cytology , Animals , Base Sequence , Chromatin Immunoprecipitation , Cyclic AMP-Dependent Protein Kinases/metabolism , DNA Primers , Mice , Polymerase Chain ReactionABSTRACT
Epidermal growth factors and their receptors (EGFRs) promote breast cancer cell proliferation and can drive tumorigenesis. However, the molecular mechanisms that mediate these effects are incompletely understood. We previously showed that mammary tumor development in the mouse model of breast cancer MMTV-neu, a model characterized by amplification of the EGFR ErbB2 in mammary tissue, correlates with a marked up-regulation of fatty acid-binding protein 5 (FABP5). FABP5 functions to deliver ligands to and enhance the transcriptional activity of the nuclear receptor peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta), a receptor whose target genes include genes involved in cell growth and survival. We show here that in MCF-7 mammary carcinoma cells, EGFR signaling directly up-regulates the expression of FABP5. The data demonstrate that treatment of these cells with the EGFR ligand heregulin-beta1 signals through the ERK and the phophatidylinositol-3-kinase cascades, resulting in activation of the transcription factor NF-kappaB. In turn, NF-kappaB induces the expression of FABP5 through two cognate response elements in the promoter of this gene. The observations further demonstrate that FABP5 and PPARbeta/delta are critical mediators of the ability of EGFR to enhance cell proliferation, indicating that this transcriptional pathway plays a key role in EGFR-induced tumorigenesis. Additional observations indicate that the expression of FABP5 is down-regulated by the Krüppel-like factor KLF2, suggesting a tumor suppressor activity for this factor.
Subject(s)
Carcinoma/metabolism , Fatty Acid-Binding Proteins/physiology , Gene Expression Regulation, Neoplastic , PPAR delta/metabolism , PPAR-beta/metabolism , Cell Line, Tumor , Cell Nucleus/metabolism , Cell Proliferation , Humans , Intercellular Signaling Peptides and Proteins , Ligands , Lipids/chemistry , Models, Biological , NF-kappa B/metabolism , Neuregulin-1/metabolismABSTRACT
The vitamin A metabolite all-trans-retinoic acid (RA) regulates multiple biological processes by virtue of its ability to regulate gene expression. It thus plays critical roles in embryonic development and is involved in regulating growth, remodeling, and metabolic responses in adult tissues. RA can also suppress carcinoma cell growth and is currently used in treatment of some cancers. Growth inhibition by RA may be exerted by induction of differentiation, cell cycle arrest, or apoptosis, or by a combination of these activities. Paradoxically, in the context of some cells, RA not only fails to inhibit growth but, instead, enhances proliferation and survival. This review focuses on the involvement of RA in regulating apoptotic responses. It includes brief overviews of transcriptional signaling by RA and of apoptotic pathways, and then addresses available information on the mechanisms by which RA induces apoptosis or, conversely, inhibits cell death and enhances survival.
Subject(s)
Apoptosis/physiology , Gene Expression Regulation/physiology , Receptors, Retinoic Acid/physiology , Tretinoin/physiology , Animals , Antineoplastic Agents , Apoptosis/drug effects , Humans , Signal Transduction/drug effects , Signal Transduction/physiologyABSTRACT
The uptake of dietary lipids from the small intestine is a complex process that depends on the activities of specific membrane receptors with yet unknown regulatory mechanisms. Using both mouse models and human cell lines, we show here that intestinal lipid absorption by the scavenger receptor class B type 1 (SR-BI) is subject to control by retinoid signaling. Retinoic acid via retinoic acid receptors induced expression of the intestinal transcription factor ISX. ISX then repressed the expression of SR-B1 and the carotenoid-15,15'-oxygenase Bcmo1. BCMO1 acts downstream of SR-BI and converts absorbed beta,beta-carotene to the retinoic acid precursor, retinaldehyde. Using BCMO1-knockout mice, we demonstrated increased intestinal SR-BI expression and systemic beta,beta-carotene accumulation. SR-BI-dependent accumulation of beta,beta-carotene was prevented by dietary retinoids that induced ISX expression. Thus, our study revealed a diet-responsive regulatory network that controls beta,beta-carotene absorption and vitamin A production by negative feedback regulation. The role of SR-BI in the intestinal absorption of other dietary lipids, including cholesterol, fatty acids, and tocopherols, implicates retinoid signaling in the regulation of lipid absorption more generally and has clinical implications for diseases associated with dyslipidemia.
Subject(s)
Intestinal Mucosa/metabolism , Scavenger Receptors, Class B/metabolism , Transcription Factors/metabolism , Vitamin A/metabolism , beta Carotene/metabolism , beta-Carotene 15,15'-Monooxygenase/physiology , Animals , Blotting, Western , Cells, Cultured , Chromatin Immunoprecipitation , Colon/cytology , Colon/metabolism , Fluorescent Antibody Technique , Humans , Immunoenzyme Techniques , Intestinal Absorption , Intestines/cytology , Mice , Mice, Inbred C57BL , Mice, Knockout , Promoter Regions, Genetic , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Retinoic Acid/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Scavenger Receptors, Class B/genetics , Transcription Factors/geneticsABSTRACT
Retinoic acid (RA) displays potent anticarcinogenic activities that are mediated by the nuclear retinoic acid receptors (RARs). However, use of RA in oncology is limited by RA resistance acquired during carcinogenesis. Moreover, in some cancers, RA facilitates rather than inhibits growth. A clue to this paradoxical behavior was recently suggested by the findings that RA also activates PPARbeta/delta, a receptor involved in mitogenic and anti-apoptotic activities. The observations that partitioning of RA between its two receptors is regulated by two intracellular lipid-binding proteins-CRABP-II, which targets RA to RAR, and FABP5, which delivers it to PPARbeta/delta-further suggest that RA resistance may stem from the deregulation of the binding proteins, resulting in activation of PPARbeta/delta rather than RAR. Here, we show that, in the RA-resistant mouse model of breast cancer MMTV-neu, RA indeed activates the nonclassical RA receptor PPARbeta/delta. This behavior was traced to an aberrantly high intratumor FABP5/CRABP-II ratio. Decreasing this ratio in mammary tissue diverted RA from PPARbeta/delta to RAR and suppressed tumor growth. The data demonstrate the existence of a mechanism that underlies RA resistance in tumors, indicate that CRABP-II functions as a tumor suppressor, and suggest that the inhibition of FABP5 may comprise a therapeutic strategy for overcoming RA resistance in some tumors.
Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma/metabolism , Drug Resistance, Neoplasm , Fatty Acid-Binding Proteins/metabolism , Mammary Neoplasms, Animal/metabolism , Neoplasm Proteins/metabolism , Receptors, Retinoic Acid/agonists , Tretinoin/pharmacology , Animals , Cell Line, Tumor , Disease Models, Animal , Fatty Acid-Binding Proteins/antagonists & inhibitors , Mice , Mice, Transgenic , Neoplasm Proteins/antagonists & inhibitors , PPAR delta/agonists , PPAR-beta/agonists , Receptors, Retinoic Acid/genetics , Receptors, Retinoic Acid/metabolism , Transcription, Genetic/drug effectsABSTRACT
The anticarcinogenic activities of retinoic acid (RA) are believed to be mediated by the nuclear RA receptor (RAR) and by the RA-binding protein cellular RA-binding protein-II (CRABP-II). In MCF-7 mammary carcinoma cells, growth inhibition by RA entails an early cell cycle arrest followed by induction of apoptosis. Here, we aimed to obtain insights into the initial cell cycle response. We show that a 3- to 5-h RA pulse is sufficient for inducing a robust growth arrest 2 to 4 days later, demonstrating inhibition of the G1-S transition by RA is triggered by immediate-early RAR targets and does not require the continuous presence of the hormone throughout the arrest program. Expression array analyses revealed that RA induces the expression of several genes involved in cell cycle regulation, including the p53-controlled antiproliferative gene B-cell translocation gene, member 2 (Btg2) and the BTG family member Tob1. We show that induction of Btg2 by RA does not require de novo protein synthesis and is augmented by overexpression of CRABP-II. Additionally, we identify a RA response element in the Btg2 promoter and show that the element binds retinoid X receptor/RAR heterodimers in vitro, is occupied by the heterodimers in cells, and can drive RA-induced activation of a reporter gene. Hence, Btg2 is a novel direct target for RA signaling. In concert with the reports that Btg2 inhibits cell cycle progression by down-regulating cyclin D1, induction of Btg2 by RA was accompanied by a marked decrease in cyclin D1 expression. The observations thus show that the antiproliferative activity of RA in MCF-7 cells is mediated, at least in part, by Btg2.
Subject(s)
Breast Neoplasms/drug therapy , Genes, cdc/drug effects , Immediate-Early Proteins/genetics , Receptors, Retinoic Acid/metabolism , Tretinoin/pharmacology , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Growth Processes/genetics , Cell Line, Tumor , G1 Phase/drug effects , Genes, Tumor Suppressor , Humans , Receptors, Retinoic Acid/biosynthesis , Receptors, Retinoic Acid/genetics , Signal Transduction/drug effects , Transcriptional Activation , Tretinoin/pharmacokinetics , Tumor Suppressor Proteins , Up-Regulation/drug effectsABSTRACT
Fatty acid-binding protein 4 (FABP4) delivers ligands from the cytosol to the nuclear receptor PPARgamma in the nucleus, thereby enhancing the transcriptional activity of the receptor. Notably, FABP4 binds multiple ligands with a similar affinity but its nuclear translocation is activated only by specific compounds. To gain insight into the structural features that underlie the ligand-specificity in activation of the nuclear import of FABP4, we solved the crystal structures of the protein complexed with two compounds that induce its nuclear translocation, and compared these to the apo-protein and to FABP4 structures bound to non-activating ligands. Examination of these structures indicates that activation coincides with closure of a portal loop phenylalanine side-chain, contraction of the binding pocket, a subtle shift in a helical domain containing the nuclear localization signal of the protein, and a resultant change in oligomeric state that exposes the nuclear localization signal to the solution. Comparisons of backbone displacements induced by activating ligands with a measure of mobility derived from translation, libration, screw (TLS) refinement, and with a composite of slowest normal modes of the apo state suggest that the helical motion associated with the activation of the protein is part of the repertoire of the equilibrium motions of the apo-protein, i.e. that ligand binding does not induce the activated configuration but serves to stabilize it. Nuclear import of FABP4 can thus be understood in terms of the pre-existing equilibrium hypothesis of ligand binding.