ABSTRACT: The transcription factor Kruppel-like factor 2 (KLF2) displays anticarcinogenic activities but the mechanism that underlies this activity is unknown. We show here that KLF2 is markedly downregulated in human breast cancers and that its expression positively correlates with breast cancer patient survival. We show further that KLF2 suppresses tumor development by controlling the transcriptional activity of the vitamin A metabolite retinoic acid (RA). RA regulates gene transcription by activating two types of nuclear receptors: RA receptors (RARs), which inhibit tumor development, and peroxisome proliferator-activated receptor ?/? (PPAR?/?), which promotes tumorigenesis. The partitioning of RA between these receptors is regulated by two carrier proteins: cellular retinoic acid-binding protein 2 (CRABP2), which delivers RA to RARs, and fatty acid-binding protein 5 (FABP5), which shuttles ligands to PPAR?/?. We show that KLF2 induces the expression of CRABP2 and RAR? and inhibits the expression FABP5 and PPAR?/? thereby shifting RA signaling from the pro-carcinogenic FABP5/PPAR?/? to the growth-suppressing CRABP2/RAR path. The data thus reveal that KLF2 suppresses tumor growth by controlling the transcriptional activities of RA.
Project description:Nuclear receptor-mediated signaling via RARs and PPAR? is involved in the regulation of skin homeostasis. Moreover, activation of both RAR and PPAR? was shown to alter skin inflammation. Endogenous all-trans retinoic acid (ATRA) can activate both receptors depending on specific transport proteins: Fabp5 initiates PPAR? signaling whereas Crabp2 promotes RAR signaling. Repetitive topical applications of ovalbumin (OVA) in combination with intraperitoneal injections of OVA or only intraperitoneal OVA applications were used to induce allergic dermatitis. In our mouse model, expression of IL-4, and Hbegf increased whereas expression of involucrin, Abca12 and Spink5 decreased in inflamed skin, demonstrating altered immune response and epidermal barrier homeostasis. Comprehensive gene expression analysis showed alterations of the cutaneous retinoid metabolism and retinoid-mediated signaling in allergic skin immune response. Notably, ATRA synthesis was increased as indicated by the elevated expression of retinaldehyde dehydrogenases and increased levels of ATRA. Consequently, the expression pattern of genes downstream to RAR was altered. Furthermore, the increased ratio of Fabp5 vs. Crabp2 may indicate an up-regulation of the PPAR? pathway in allergen-induced dermatitis in addition to the altered RAR signaling. Thus, our findings suggest that ATRA levels, RAR-mediated signaling and signaling involved in PPAR? pathways are mainly increased in allergen-induced dermatitis and may contribute to the development and/or maintenance of allergic skin diseases.
Project description:Upon breaching of the endometrial surface epithelium, the implanting embryo embeds in the decidualizing stroma. Retinoic acid (RA), a metabolite of vitamin A, is an important morphogen during embryonic and fetal development, although the role of the RA pathway in the surrounding decidual cells is not understood. Here we show that decidual transformation of human endometrial stromal cells (HESCs) results in profound reprogramming of the RA signaling and metabolism pathways. Differentiating HESCs downregulate the intracellular carrier proteins CRABP2 and FABP5, responsible for transfer and binding of RA to the nuclear receptors RAR and PPAR?/?, respectively. Furthermore, the expression of RAR, the receptor that mediates the pro-apoptotic effects of RA, was also inhibited. By contrast, PPAR?/?, which transduces the differentiation responses of RA, was upregulated. Decidualization was also associated with increased expression of retinol-binding protein 4 (RBP4) and various enzymes involved in the metabolism of RA and its precursor, retinaldehyde (Rald), including CYP26A1, DHRS3, and RDH12. Exposure of differentiating HESCs to RA or Rald reversed the inhibition of the CRABP2-RAR pathway, perturbed the expression of decidual marker genes and triggered cell death. Taken together, the data demonstrate that decidualizing HESCs silence RA signaling by downregulating key cytoplasmic binding proteins and by increasing retinoid metabolism. However, excessive RA exposure is toxic for decidual cells and triggers a response that may lead to pregnancy failure.
Project description:Retinoic acid (RA) protects mice from diet-induced obesity. The activity is mediated in part through activation of the nuclear receptors RA receptors (RARs) and peroxisome proliferator-activated receptor ?/? and their associated binding proteins cellular RA binding protein type II (CRABP-II) and fatty acid binding protein type 5 in adipocytes and skeletal muscle, leading to enhanced lipid oxidation and energy dissipation. It was also reported that RA inhibits differentiation of cultured preadipocytes. However, whether the hormone suppresses adipogenesis in vivo and how the activity is propagated remained unknown. In this study, we show that RA inhibits adipocyte differentiation by activating the CRABP-II/RAR? path in preadipose cells, thereby upregulating the expression of the adipogenesis inhibitors Pref-1, Sox9, and Kruppel-like factor 2 (KLF2). In turn, KLF2 induces the expression of CRABP-II and RAR?, further potentiating inhibition of adipocyte differentiation by RA. The data also indicate that RA suppresses adipogenesis in vivo and that the activity significantly contributes to the ability of the hormone to counteract diet-induced obesity.
Project description:Retinoic acid (RA), a metabolite of vitamin A, is required for the regulation of growth and development. Aberrant expression of molecules involved in RA signaling has been reported in various cancer types including glioblastoma multiforme (GBM). Cellular retinoic acid-binding protein 2 (CRABP2) has previously been shown to play a key role in the transport of RA to retinoic acid receptors (RARs) to activate their transcription regulatory activity. Here, we demonstrate that CRABP2 is predominantly located in the cytoplasm of GBM tumors. Cytoplasmic, but not nuclear, CRABP2 levels in GBM tumors are associated with poor patient survival. Treatment of malignant glioma cell lines with RA results in a dose-dependent increase in accumulation of CRABP2 in the cytoplasm. CRABP2 knockdown reduces proliferation rates of malignant glioma cells, and enhances RA-induced RAR activation. Levels of CRYAB, a small heat shock protein with anti-apoptotic activity, and GFAP, an astrocyte-specific intermediate filament protein, are greatly reduced in CRABP2-depleted cells. Restoration of CRYAB expression partially but significantly reversed the effect of CRABP2 depletion on RAR activation. Our combined in vivo and in vitro data indicate that: (i) CRABP2 is an important determinant of clinical outcome in GBM patients, and (ii) the mechanism of action of CRABP2 in GBM involves sequestration of RA in the cytoplasm and activation of an anti-apoptotic pathway, thereby enhancing proliferation and preventing RA-mediated cell death and differentiation. We propose that reducing CRABP2 levels may enhance the therapeutic index of RA in GBM patients.
Project description: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.
Project description:Long chain fatty acids (LCFA) serve as energy sources, components of cell membranes and precursors for signalling molecules. Here we show that these biological compounds also regulate gene expression and that they do so by controlling the transcriptional activities of the retinoic acid (RA)-activated nuclear receptors RAR and PPAR?/?. The data indicate that these activities of LCFA are mediated by FABP5, which delivers ligands from the cytosol to nuclear PPAR?/?. Both saturated and unsaturated LCFA (SLCFA, ULCFA) bind to FABP5, thereby displacing RA and diverting it to RAR. However, while SLCFA inhibit, ULCFA activate the FABP5/PPAR?/? pathway. We show further that, by concomitantly promoting the activation of RAR and inhibiting the activation of PPAR?/?, SLCFA suppress the oncogenic properties of FABP5-expressing carcinoma cells in cultured cells and in vivo. The observations suggest that compounds that inhibit FABP5 may constitute a new class of drugs for therapy of certain types of cancer.
Project description:All-trans-retinoic acid (RA), a potent inducer of cellular differentiation, functions as a ligand for retinoic acid receptors (RAR?, ?, and ?). RARs are activated by ligand binding, which induces transcription of direct genomic targets. However, whether embryonic stem cells respond to RA through routes that do not involve RARs is unknown. Here, we used CRISPR technology to introduce biallelic frameshift mutations in RAR?, RAR?, and RAR?, thereby abrogating all RAR functions in murine embryonic stem cells. We then evaluated RA-responsiveness of the RAR-null cells using RNA-Seq transcriptome analysis. We found that the RAR-null cells display no changes in transcripts in response to RA, demonstrating that the RARs are essential for the regulation of all transcripts in murine embryonic stem cells in response to RA. Our key finding, that in embryonic stem cells the transcriptional effects of RA all depend on RARs, addresses a long-standing topic of discussion in the field of retinoic acid signaling.
Project description:The pleiotropic activities of retinoids are mediated by two types of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). All-trans-retinoic acid (RA) transcriptionally activates RARs, but not RXRs, whereas its natural stereoisomer, 9-cis-RA, is the ligand for RXRs. Here, we demonstrate that 9-cis-RA did not transcriptionally activate RARs, whereas in the presence of all-trans-RA the transactivation of RARs was inhibited in a dose-dependent manner by 9-cis-RA. RAR homodimer complexes were destabilized in vitro in the presence of 9-cis-RA. This suggests that 9-cis-RA may be a natural antagonist of all-trans-RA for binding to RAR complexes. The levels of 9-cis-RA may determine by which pathway the transcription of retinoid-responsive genes is modulated.
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.
Project description:Transcriptional activation of the nuclear receptor RAR by retinoic acid (RA) often leads to inhibition of cell growth. However, in some tissues, RA promotes cell survival and hyperplasia, activities that are unlikely to be mediated by RAR. Here, we show that, in addition to functioning through RAR, RA activates the "orphan" nuclear receptor PPARbeta/delta, which, in turn, induces the expression of prosurvival genes. Partitioning of RA between the two receptors is regulated by the intracellular lipid binding proteins CRABP-II and FABP5. These proteins specifically deliver RA from the cytosol to nuclear RAR and PPARbeta/delta, respectively, thereby selectively enhancing the transcriptional activity of their cognate receptors. Consequently, RA functions through RAR and is a proapoptotic agent in cells with high CRABP-II/FABP5 ratio, but it signals through PPARbeta/delta and promotes survival in cells that highly express FABP5. Opposing effects of RA on cell growth thus emanate from alternate activation of two different nuclear receptors.