RBP7 Regulated by EBF1 Affects Th2 Cells and the Oocyte Meiosis Pathway in Bone Metastases of Bladder Urothelial Carcinoma.

BACKGROUND: Bladder urothelial carcinoma (BLCA) is a malignancy with a high incidence worldwide. One-third of patients may experience aggressive progression later on, and 70% of patients who have undergone surgical intervention will still suffer from metastasis. MATERIALS AND METHODS: RNA sequencing profiles of BLCA samples were obtained from The Cancer Genome Atlas (TCGA) database. Differential expression and univariate Cox regression analyses were performed to identify prognosis-related differentially expressed immune genes (DEIGs). Subsequently, a proportional hazards model of DEIGs was then constructed by univariate regression analysis. Differential expression and correlation analyses, CIBERSORT, Single Sample Gene Set Enrichment Analysis (ssGSEA), GSVA were conducted on transcription factors (TFs), immune cells/pathways and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The regulation network was then constructed. Eventually, ATAC-seq, ChIP-seq, scRNA-seq, and multiple online databases were employed for further validation. RESULTS: A proportional hazards model of 31 DEIGs was constructed and risk score was calculated and proven to be a independent prognostic factor. Then 5 immune genes were characterized to be significantly correlated with bone metastasis, stage and TF expression simultaneously. 4 TFs were identified to be significantly correlated with prognosis and RBP7 expression. 5 immune cells/pathways were revealed to be significantly correlated with RBP7 expression. Only 1 KEGG pathway was identified to be significant in Gene Set Enrichment Analysis (GSEA) and Gene Set Variation Analysis (GSVA) analyses. The regulatory relationship was then constructed, in which the correlation between EBF1 and RBP7 (R = 0.677, p < 0.001), Th2 cells and RBP7 (R = 0.23, p < 0.001), the oocyte meiosis pathway and RBP7 (R = 0.14, p = 0.042) were the most statistically significant. The results were further confirmed by Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq), Chromatin Immunoprecipitation sequencing (ChIP-seq), single-cell RNA sequencing (scRNA-seq), and multiple online databases validation. CONCLUSIONS: This study revealed that the EBF1-RBP7 regulatory relationship had potential importance in the bone metastasis in BLCA through Th2 cells and the oocyte meiosis pathway.

Discovery of Nonretinoid Inhibitors of CRBP1: Structural and Dynamic Insights for Ligand-Binding Mechanisms.

The dysregulation of retinoid metabolism has been linked to prevalent ocular diseases including age-related macular degeneration and Stargardt disease. Modulating retinoid metabolism through pharmacological approaches holds promise for the treatment of these eye diseases. Cellular retinol-binding protein 1 (CRBP1) is the primary transporter of all-trans-retinol (atROL) in the eye, and its inhibition has recently been shown to protect mouse retinas from light-induced retinal damage. In this report, we employed high-throughput screening to identify new chemical scaffolds for competitive, nonretinoid inhibitors of CRBP1. To understand the mechanisms of interaction between CRBP1 and these inhibitors, we solved high-resolution X-ray crystal structures of the protein in complex with six selected compounds. By combining protein crystallography with hydrogen/deuterium exchange mass spectrometry, we quantified the conformational changes in CRBP1 caused by different inhibitors and correlated their magnitude with apparent binding affinities. Furthermore, using molecular dynamic simulations, we provided evidence for the functional significance of the "closed" conformation of CRBP1 in retaining ligands within the binding pocket. Collectively, our study outlines the molecular foundations for understanding the mechanism of high-affinity interactions between small molecules and CRBPs, offering a framework for the rational design of improved inhibitors for this class of lipid-binding proteins.

Comparative molecular dynamics study on the features of binding and non-binding modes of retinoic acid in cellular retinol-binding protein (I).

Retinoids play crucial roles in various biological processes by interacting with their carrier proteins such as cellular retinol-binding protein (CRBP). Understanding the molecular interactions between retinoids and CRBP enables their pharmacological and biomedical applications. Experimentally, CRBP(I) does not bind to retinoic acid, but when arginine is introduced into 108th residue instead of glutamine (Q108R), it binds to retinoic acid. Here, molecular dynamics simulations were performed to understand the differences in the microscopic and dynamic behaviors of the non-binding wild-type CRBP(I)-retinoic acid and binding Q108R variant-retinoic acid complexes. The ligand RMSD and RMSF, the binding poses of binding motif amino acids, and the number of hydrogen bonds and salt-bridges revealed the relative instability of the non-binding complex. In particular, the ligand's terminal group showed very different dynamics and interactions. So far, most studies have focused on the binding characteristics of retinoids, but the features of their non-binding modes have not been studied well. This study provides some structural insights into the non-binding modes of a retinoid in CRBP, which may be applicable in retinoid-based drug discovery and protein engineering through computational modeling.

Cellular retinol-binding protein 1: a therapeutic and diagnostic tumor marker.

Cellular Retinol Binding Protein 1 (CRBP1) gene is a protein coding gene located on human chromosome 3q21, which codifies a protein named CRBP1. CRBP1 is widely expressed in many tissues as a chaperone protein to regulate the uptake, subsequent esterification and bioavailability of retinol. CRBP1 combines retinol and retinaldehyde with high affinity to protect retinoids from non-specific oxidation, and transports retinoids to specific enzymes to promote the biosynthesis of retinoic acid. The vital role of CRBP1 in retinoids metabolism has been gradually discovered, which has been implicated in tumorigenesis. However, the precise functions of CRBP1 in different diseases are still poorly understood. The purpose of this review is to provide an overview of the role of CRBP1 in various diseases, especially in both the promotion and inhibition of cancers, which may also offer a novel biomarker and potential therapeutic target for human diseases.

Intestinal Enteroendocrine Cell Signaling: Retinol-binding Protein 2 and Retinoid Actions.

Retinol-binding protein 2-deficient (Rbp2-/-) mice are more prone to obesity, glucose intolerance, and hepatic steatosis than matched controls. Glucose-dependent insulinotropic polypeptide (GIP) blood levels are dysregulated in these mice. The present studies provide new insights into these observations. Single cell transcriptomic and immunohistochemical studies establish that RBP2 is highly expressed in enteroendocrine cells (EECs) that produce incretins, either GIP or glucagon-like peptide-1. EECs also express an enzyme needed for all-trans-retinoic acid (ATRA) synthesis, aldehyde dehydrogenase 1 family member A1, and retinoic acid receptor-alpha, which mediates ATRA-dependent transcription. Total and GIP-positive EECs are significantly lower in Rbp2-/- mice. The plasma transport protein for retinol, retinol-binding protein 4 (RBP4) is also expressed in EECs and is cosecreted with GIP upon stimulation. Collectively, our data support direct roles for RBP2 and ATRA in cellular processes that give rise to GIP-producing EECs and roles for RBP2 and RBP4 within EECs that facilitate hormone storage and secretion.

Retinol-binding protein 2 (RBP2): More than just dietary retinoid uptake.

Retinol-binding protein 2 (RBP2, also known as cellular retinol-binding protein 2 (CRBP2)) is a member of the fatty acid-binding protein family and has been extensively studied for its role in facilitating dietary vitamin A (retinol) uptake and metabolism within enterocytes of the small intestine. RBP2 is present in highest concentrations in the proximal small intestine where it constitutes approximately 0.1-0.5% of soluble protein. Recent reports have established that RBP2 binds monoacylglycerols (MAGs) with high affinity, including the canonical endocannabinoid 2-arachidonoylglycerol (2-AG). Crystallographic studies reveal that retinol, 2-AG, or other long-chain MAGs alternatively can bind in the retinol-binding pocket of RBP2. It also has been demonstrated recently that Rbp2-deficient mice are more susceptible to developing obesity and associated metabolic phenotypes when exposed to a high fat diet, or as they age when fed a conventional chow diet. When subjected to an oral fat challenge, the Rbp2-deficient mice release into the circulation significantly more, compared to littermate controls, of the intestinal hormone glucose-dependent insulinotropic polypeptide (GIP). These new findings regarding RBP2 structure and actions within the intestine are the focus of this review.

Role of cellular retinol-binding protein, type 1 and retinoid homeostasis in the adult mouse heart: A multi-omic approach.

The main active metabolite of Vitamin A, all-trans retinoic acid (RA), is required for proper cellular function and tissue organization. Heart development has a well-defined requirement for RA, but there is limited research on the role of RA in the adult heart. Homeostasis of RA includes regulation of membrane receptors, chaperones, enzymes, and nuclear receptors. Cellular retinol-binding protein, type 1 (CRBP1), encoded by retinol-binding protein, type 1 (Rbp1), regulates RA homeostasis by delivering vitamin A to enzymes for RA synthesis and protecting it from non-specific oxidation. In this work, a multi-omics approach was used to characterize the effect of CRBP1 loss using the Rbp1-/- mouse. Retinoid homeostasis was disrupted in Rbp1-/- mouse heart tissue, as seen by a 33% and 24% decrease in RA levels in the left and right ventricles, respectively, compared to wild-type mice (WT). To further inform on the effect of disrupted RA homeostasis, we conducted high-throughput targeted metabolomics. A total of 222 metabolite and metabolite combinations were analyzed, with 33 having differential abundance between Rbp1-/- and WT hearts. Additionally, we performed global proteome profiling to further characterize the impact of CRBP1 loss in adult mouse hearts. More than 2606 unique proteins were identified, with 340 proteins having differential expression between Rbp1-/- and WT hearts. Pathway analysis performed on metabolomic and proteomic data revealed pathways related to cellular metabolism and cardiac metabolism were the most disrupted in Rbp1-/- mice. Together, these studies characterize the effect of CRBP1 loss and reduced RA in the adult heart.

Altered RBP1 Gene Expression Impacts Epithelial Cell Retinoic Acid, Proliferation, and Microenvironment.

Vitamin A is an essential diet-derived nutrient that has biological activity affected through an active metabolite, all-trans retinoic acid (atRA). Retinol-binding protein type 1 (RBP1) is an intracellular chaperone that binds retinol and retinal with high affinity, protects retinoids from non-specific oxidation, and delivers retinoids to specific enzymes to facilitate biosynthesis of RA. RBP1 expression is reduced in many of the most prevalent cancers, including breast cancer. Here, we sought to understand the relationship between RBP1 expression and atRA biosynthesis in mammary epithelial cells, as well as RBP1 expression and atRA levels in human mammary tissue. We additionally aimed to investigate the impact of RBP1 expression and atRA on the microenvironment as well as the potential for therapeutic restoration of RBP1 expression and endogenous atRA production. Using human mammary ductal carcinoma samples and a series of mammary epithelial cell lines representing different stages of tumorigenesis, we investigated the relationship between RBP1 expression as determined by QPCR and atRA via direct liquid chromatography-multistage-tandem mass spectrometry-based quantification. The functional effect of RBP1 expression and atRA in epithelial cells was investigated via the expression of direct atRA targets using QPCR, proliferation using Ki-67 staining, and collagen deposition via picrosirius red staining. We also investigated the atRA content of stromal cells co-cultured with normal and tumorigenic epithelial cells. Results show that RBP1 and atRA are reduced in mammary tumor tissue and tumorigenic epithelial cell lines. Knock down of RBP1 expression using shRNA or overexpression of RBP1 supported a direct relationship between RBP1 expression with atRA. Increases in cellular atRA were able to activate atRA direct targets, inhibit proliferation and inhibit collagen deposition in epithelial cell lines. Conditions encountered in tumor microenvironments, including low glucose and hypoxia, were able to reduce RBP1 expression and atRA. Treatment with either RARα agonist AM580 or demethylating agent Decitabine were able to increase RBP1 expression and atRA. Cellular content of neighboring fibroblasts correlated with the RA producing capacity of epithelial cells in co-culture. This work establishes a direct relationship between RBP1 expression and atRA, which is maintained when RBP1 expression is restored therapeutically. The results demonstrate diseases with reduced RBP1 could potentially benefit from therapeutics that restore RBP1 expression and endogenous atRA.

[Effect of RBP2 gene silencing on proliferation, migration and invasion of ovarian epithelial cancer SKOV3/DDP cells and its mechanism].

Objective: To explore the effect of down-regulation of retinol binding protein 2 (RBP2) expression on the biological characteristics of ovarian cancer cells and its mechanism. Methods: Knockdown of RBP2 and cisplatin (DDP)-resistant ovarian cancer cell line SKOV3/DDP-RBP2i was established, the negative control group and blank control group were also set. Cell counting kit 8 (CCK-8) was used to detect the cell proliferation ability, flow cytometry was used to detect cell apoptosis, scratch test and Transwell invasion test were used to detect cell migration and invasion ability, real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) and western blot were used to detect the expressions of molecular markers related to epithelial-mesenchymal transition (EMT). The effect of RBP2 on the growth of ovarian cancer was verified through experiment of transplanted tumors in nude mice, and the relationships between RBP2 expression and tumor metastasis and patient prognosis were analyzed using the clinical data of ovarian cancer in TCGA database. Results: After down-regulating the expression of RBP2, the proliferation ability of SKOV3/DDP cell was significantly reduced. On the fifth day, the proliferation activities of SKOV3/DDP-RBP2i group, negative control group and blank control group were (56.67±4.16)%, (84.67±3.51) and (87.00±4.00)% respectively, with statistically significant difference (P<0.001). The apoptosis rate of SKOV3/DDP-RBP2i group was (14.19±1.50)%, higher than (8.77±0.75)% of the negative control group and (7.48±0.52)% of the blank control group (P<0.001). The number of invasive cells of SKOV3/DDP-RBP2i group was (55.20±2.39), lower than (82.60±5.18) and (80.80±7.26) of the negative control group and the blank control group, respectively (P<0.001). The scratch healing rate of SKOV3/DDP-RBP2i group was (28.47±2.72)%, lower than (50.58±4.06)% and (48.92±4.63)% of the negative control group and the blank control group, respectively (P<0.001). The mRNA and protein expressions of E-cadherin in the SKOV3/DDP-RBP2i group were higher than those in the negative control group (P=0.015, P<0.001) and the blank control group (P=0.006, P<0.001). The mRNA and protein expression of N-cadherin in SKOV3/DDP-RBP2i group were lower than those in the negative control group (P=0.012, P<0.001) and the blank control group (P=0.005, P<0.001). The mRNA and protein expressions of vimentin in SKOV3/DDP-RBP2i group were also lower than those in the negative control group (P=0.016, P=0.001) and the blank control group (P=0.011, P=0.001). Five weeks after the cells inoculated into the nude mice, the tumor volume of SKOV3/DDP-RBP2i group, negative control group and blank control group were statistically significant different. The tumor volume of SKOV3/DDP-RBP2i group was smaller than those of negative control group and blank control group (P=0.001). Bioinformatics analysis showed that the expression of RBP2 in patients with metastatic ovarian cancer was higher than that without metastasis (P=0.043), and the median overall survival of ovarian cancer patients with high RBP2 expression was 41 months, shorter than 69 months of low RBP2 expression patients (P<0.001). Conclusion: Downregulation of the expression of RBP2 in SKOV3/DDP cells can inhibit cell migration and invasion, and the mechanism may be related to the inhibition of EMT.

MicroRNA-20a-5p inhibits the autophagy and cisplatin resistance in ovarian cancer via regulating DNMT3B-mediated DNA methylation of RBP1.

Ovarian cancer (OvCa) is the third most common female malignancy worldwide and poses great threats on women health. Chemotherapy is the most recommended post-surgery treatment for OvCa patients; but, cisplatin resistance is a main cause of chemotherapy failure. In addition, autophagy modulates the sensitivity of tumor cells to chemotherapeutic agents. Hence, it is significant to explore the molecular mechanism concerning the autophagy and cisplatin resistance in OvCa. In this study, quantitative real-time PCR (qRT-PCR) was used to detect miR-20a-5p expression and western blot to measure RBP1 expression. A series of assays were conducted to explore the gain-of-function effects of miR-20a-5p. Luciferase reporter assay was applied to determine the downstream target of miR-20a-5p. The results proved that miR-20a-5p represses malignant phenotypes and autophagy in cisplatin-resistant OvCa cells. In addition, DNMT3B mediates DNA methylation of RBP1 to impair the promoting effects of RBP1 on carcinogenesis and autophagy in OvCa. Through rescue experiments, we certified that miR-20a-5p inhibits the autophagy and cisplatin resistance in OvCa via DNMT3B-mediated DNA methylation of RBP1. Collectively, we demonstrated that miR-20a-5p plays a crucial role in the modulation of autophagy and cisplatin resistance in OvCa, which might offer novel insights into developing effective treatment strategies for OvCa.

Retinol binding protein 1-dependent activation of NF- κB signaling enhances the malignancy of non-glioblastomatous diffuse gliomas.

Nonglioblastomatous diffuse glioma (non-GDG) is a heterogeneous neuroepithelial tumor that exhibits a varied survival range from 4 to 13 years based on the diverse subtypes. Recent studies demonstrated novel molecular markers can predict prognosis for non-GDG patients; however, these findings as well as pathological classification strategies show obvious limitations on malignant transition due to the heterogeneity among non-GDGs. Therefore, developing reliable prognostic biomarkers and therapeutic targets have become an urgent need for precisely distinguishing non-GDG subtypes, illuminating the underlying mechanism. Nuclear factor κß (NF-κB) has been proved to be a significant nuclear transcriptional regulator with specific DNA-binding sequences to participate in multiple pathophysiological processes. However, the underlying mechanism of NF-κB activation still needs to be further investigated. Herein, our results indicated retinol-binding protein 1 (RBP1) was significantly upregulated in the IDHWT and 1p19qNon co-del non-GDG subtypes and enriched RBP1 expression was markedly correlated with more severe outcomes. Additionally, malignant signatures of the non-GDG cells including proliferation, migration, invasion, and self-renewal were significantly suppressed by lentiviral knockdown of RBP1. To further explore the underlying molecular mechanism, bioinformatics analysis was performed using databases, and the results demonstrated RBP1 was strongly correlated with tumor necrosis factor α (TNFα)-NF-κB signaling. Moreover, exogenous silencing of RBP1 reduced phosphorylation of IkB-kinase α (IKKα) and thus decreased NF-κB expression via decreasing the degradation of the IκBα protein. Altogether, these data suggested RBP1-dependent activation of NF-κB signaling promoted malignancy of non-GDG, indicating that RBP1 could be a reliable prognostic biomarker and potential therapeutic target for non-GDG.

Effect of RBP2 gene silencing on proliferation, migration and invasion of ovarian epithelial cancer SKOV3/DDP cells and its mechanism / 中华肿瘤杂志

Objective: To explore the effect of down-regulation of retinol binding protein 2 (RBP2) expression on the biological characteristics of ovarian cancer cells and its mechanism. Methods: Knockdown of RBP2 and cisplatin (DDP)-resistant ovarian cancer cell line SKOV3/DDP-RBP2i was established, the negative control group and blank control group were also set. Cell counting kit 8 (CCK-8) was used to detect the cell proliferation ability, flow cytometry was used to detect cell apoptosis, scratch test and Transwell invasion test were used to detect cell migration and invasion ability, real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) and western blot were used to detect the expressions of molecular markers related to epithelial-mesenchymal transition (EMT). The effect of RBP2 on the growth of ovarian cancer was verified through experiment of transplanted tumors in nude mice, and the relationships between RBP2 expression and tumor metastasis and patient prognosis were analyzed using the clinical data of ovarian cancer in TCGA database. Results: After down-regulating the expression of RBP2, the proliferation ability of SKOV3/DDP cell was significantly reduced. On the fifth day, the proliferation activities of SKOV3/DDP-RBP2i group, negative control group and blank control group were (56.67±4.16)%, (84.67±3.51) and (87.00±4.00)% respectively, with statistically significant difference (P<0.001). The apoptosis rate of SKOV3/DDP-RBP2i group was (14.19±1.50)%, higher than (8.77±0.75)% of the negative control group and (7.48±0.52)% of the blank control group (P<0.001). The number of invasive cells of SKOV3/DDP-RBP2i group was (55.20±2.39), lower than (82.60±5.18) and (80.80±7.26) of the negative control group and the blank control group, respectively (P<0.001). The scratch healing rate of SKOV3/DDP-RBP2i group was (28.47±2.72)%, lower than (50.58±4.06)% and (48.92±4.63)% of the negative control group and the blank control group, respectively (P<0.001). The mRNA and protein expressions of E-cadherin in the SKOV3/DDP-RBP2i group were higher than those in the negative control group (P=0.015, P<0.001) and the blank control group (P=0.006, P<0.001). The mRNA and protein expression of N-cadherin in SKOV3/DDP-RBP2i group were lower than those in the negative control group (P=0.012, P<0.001) and the blank control group (P=0.005, P<0.001). The mRNA and protein expressions of vimentin in SKOV3/DDP-RBP2i group were also lower than those in the negative control group (P=0.016, P=0.001) and the blank control group (P=0.011, P=0.001). Five weeks after the cells inoculated into the nude mice, the tumor volume of SKOV3/DDP-RBP2i group, negative control group and blank control group were statistically significant different. The tumor volume of SKOV3/DDP-RBP2i group was smaller than those of negative control group and blank control group (P=0.001). Bioinformatics analysis showed that the expression of RBP2 in patients with metastatic ovarian cancer was higher than that without metastasis (P=0.043), and the median overall survival of ovarian cancer patients with high RBP2 expression was 41 months, shorter than 69 months of low RBP2 expression patients (P<0.001). Conclusion: Downregulation of the expression of RBP2 in SKOV3/DDP cells can inhibit cell migration and invasion, and the mechanism may be related to the inhibition of EMT.

Cellular retinol binding protein-1 inhibits cancer stemness via upregulating WIF1 to suppress Wnt/ß-catenin pathway in hepatocellular carcinoma.

BACKGROUND: CRBP-1, a cytosolic chaperone of vitamin A, is identified in a serious number of cancers; however, its biological role in hepatocellular carcinoma (HCC) needs to be further explored. The aim of our present study is to explore the roles and mechanisms of CRBP-1 in regulating liver cancer by using in vitro and in vivo biology approaches. METHODS: The expression level of CRBP-1 was detected using immunohistochemistry in HCC and matching adjacent non-tumorous liver tissues. Following established stable CRBP-1 overexpressed HCC cell lines, the cell growth and tumorigenicity were investigated both in vitro and in vivo. Intracellular retinoic acid was quantified by ELISA. The relationship between CRBP-1 and WIF1 was validated by using dual luciferase and ChIP analyses. RESULTS: The low expression of CRBP-1 was observed in HCC tissues compared to the normal liver tissues, while high CRBP-1 expression correlated with clinicopathological characteristics and increased overall survival in HCC patients. Overexpression of CRBP-1 significantly inhibited cell growth and tumorigenicity both in vitro and in vivo. Moreover, overexpression of CRBP-1 suppressed tumorsphere formation and cancer stemness related genes expression in HCC. Mechanically, CRBP-1 inhibited Wnt/ß-catenin signaling pathway to suppress cancer cell stemness of HCC. Furthermore, our results revealed that CRBP-1 could increase the intracellular levels of retinoic acid, which induced the activation of RARs/RXRs leading to the transcriptional expression of WIF1, a secreted antagonist of the Wnt/ß-catenin signaling pathway, by physically interacting with the region on WIF1 promoter. CONCLUSION: Our findings reveal that CRBP-1 is a crucial player in the initiation and progression of HCC, which provide a novel independent prognostic biomarker and therapeutic target for the diagnosis and treatment of HCC.

Cellular retinoid-binding proteins transfer retinoids to human cytochrome P450 27C1 for desaturation.

Cytochrome P450 27C1 (P450 27C1) is a retinoid desaturase expressed in the skin that catalyzes the formation of 3,4-dehydroretinoids from all-trans retinoids. Within the skin, retinoids are important regulators of proliferation and differentiation. In vivo, retinoids are bound to cellular retinol-binding proteins (CRBPs) and cellular retinoic acid-binding proteins (CRABPs). Interaction with these binding proteins is a defining characteristic of physiologically relevant enzymes in retinoid metabolism. Previous studies that characterized the catalytic activity of human P450 27C1 utilized a reconstituted in vitro system with free retinoids. However, it was unknown whether P450 27C1 could directly interact with holo-retinoid-binding proteins to receive all-trans retinoid substrates. To assess this, steady-state kinetic assays were conducted with free all-trans retinoids and holo-CRBP-1, holo-CRABP-1, and holo-CRABP-2. For holo-CRBP-1 and holo-CRABP-2, the kcat/Km values either decreased 5-fold or were equal to the respective free retinoid values. The kcat/Km value for holo-CRABP-1, however, decreased ∼65-fold in comparison with reactions with free all-trans retinoic acid. These results suggest that P450 27C1 directly accepts all-trans retinol and retinaldehyde from CRBP-1 and all-trans retinoic acid from CRABP-2, but not from CRABP-1. A difference in substrate channeling between CRABP-1 and CRABP-2 was also supported by isotope dilution experiments. Analysis of retinoid transfer from holo-CRABPs to P450 27C1 suggests that the decrease in kcat observed in steady-state kinetic assays is due to retinoid transfer becoming rate-limiting in the P450 27C1 catalytic cycle. Overall, these results illustrate that, like the CYP26 enzymes involved in retinoic acid metabolism, P450 27C1 interacts with cellular retinoid-binding proteins.

Molecular basis for the interaction of cellular retinol binding protein 2 (CRBP2) with nonretinoid ligands.

Present in the small intestine, cellular retinol binding protein 2 (CRBP2) plays an important role in the uptake, transport, and metabolism of dietary retinoids. However, the recent discovery of the interactions of CRBP2 with 2-arachidonoylglycerol and other monoacylglycerols (MAGs) suggests the broader involvement of this protein in lipid metabolism and signaling. To better understand the physiological role of CRBP2, we determined its protein-lipid interactome using a fluorescence-based retinol replacement assay adapted for a high-throughput screening format. By examining chemical libraries of bioactive lipids, we provided evidence for the selective interaction of CRBP2 with a subset of nonretinoid ligands with the highest affinity for sn-1 and sn-2 MAGs that contain polyunsaturated C18-C20 acyl chains. We also elucidated the structure-affinity relationship for nonretinoid ligands of this protein. We further dissect the molecular basis for this ligand's specificity by analyzing high-resolution crystal structures of CRBP2 in complex with selected derivatives of MAGs. Finally, we identify T51 and V62 as key amino acids that enable the broadening of ligand selectivity to MAGs in CRBP2 as compared with retinoid-specific CRBP1. Thus, our study provides the molecular framework for understanding the lipid selectivity and diverse functions of CRBPs in controlling lipid homeostasis.

Retinol binding protein 1 affects Xenopus anterior neural development via all-trans retinoic acid signaling.

BACKGROUND: Retinol binding protein 1 (Rbp1) acts as an intracellular regulator of vitamin A metabolism and retinoid transport. In mice, Rbp1 deficiency decreases the capacity of hepatic stellate cells to take up all-trans retinol and sustain retinyl ester stores. Furthermore, Rbp1 is crucial for visual capacity. Although the function of Rbp1 has been studied in the mature eye, its role during early anterior neural development has not yet been investigated in detail. RESULTS: We showed that rbp1 is expressed in the eye, anterior neural crest cells (NCCs) and prosencephalon of the South African clawed frog Xenopus laevis. Rbp1 knockdown led to defects in eye formation, including microphthalmia and disorganized retinal lamination, and to disturbed induction and differentiation of the eye field, as shown by decreased rax and pax6 expression. Furthermore, it resulted in reduced rax expression in the prosencephalon and affected cranial cartilage. Rbp1 inhibition also interfered with neural crest induction and migration, as shown by twist and slug. Moreover, it led to a significant reduction of the all-trans retinoic acid target gene pitx2 in NCC-derived periocular mesenchyme. The Rbp1 knockdown phenotypes were rescued by pitx2 RNA co-injection. CONCLUSION: Rbp1 is crucial for the development of the anterior neural tissue.

Model establishment of prognostic-related immune genes in laryngeal squamous cell carcinoma.

BACKGROUND: Laryngeal squamous cell carcinoma (LSCC) is one of the most common malignant tumors of the head and neck in the world. At present, the treatment methods include surgery, radiotherapy, and chemotherapy, but the 5-year survival rate is still not ideal and the quality of life of the patients is low. Due to the relative lack of immunotherapy methods, this study aims to build a risk prediction model of related immune genes, which can be used to effectively predict the prognosis of laryngeal cancer patients, and provide targets for subsequent immunotherapy. METHODS: We collected the 111 cases of laryngeal squamous cell carcinoma and 12 matched normal samples in the The Cancer Genome Atlas Database (TCGA) gene expression quantification database. The differentially expressed related immune genes were screened by R software version 3.5.2. The COX regression model of immune related genes was constructed, and the sensitivity and specificity of the model were evaluated. The risk value was calculated according to the model, and the risk curve was drawn to verify the correlation between related immune genes, risk score, and clinical traits. RESULTS: We selected 8 immune-related genes that can predict the prognosis of LSCC in a COX regression model and plotted the Kaplan-Meier survival curve. The 5-year survival rate of the high-risk group was 16.5% (95% CI: 0.059-0.459), and that of the low-risk group was 72.9% (95% CI: 0.555-0.956). The area under the receiver operating characteristic (ROC) curve was used to confirm the accuracy of the model (AUG = 0.887). After univariate and multivariate regression analysis, the risk score can be used as an independent risk factor for predicting prognosis. The risk score (P = .021) was positively correlated with the clinical Stage classification. CONCLUSION: We screened out 8 immune genes related to prognosis: RBP1, TLR2, AQP9, BTC, EPO, STC2, ZAP70, and PLCG1 to construct risk value models, which can be used to speculate the prognosis of the disease and provide new targets for future immunotherapy.

Absence of CD36 alters systemic vitamin A homeostasis.

Fatty acid translocase (CD36) is a scavenger receptor with multiple ligands and diverse physiological actions. We recently reported that alcohol-induced hepatic retinoid mobilization is impaired in Cd36-/- mice, leading us to hypothesize that CD36 has a novel role in hepatic vitamin A mobilization. Given the central role of the liver in systemic vitamin A homeostasis we also postulated that absence of CD36 would affect whole-body vitamin A homeostasis. We tested this hypothesis in aging wild type and Cd36-/- mice, as well as mice fed a vitamin A-deficient diet. In agreement with our hypothesis, Cd36-/- mice accumulated hepatic retinyl ester stores with age to a greater extent than wild type mice. However, contrary to expectations, Cd36-/- mice consuming a vitamin A-deficient diet mobilized hepatic retinoid similar to wild type mice. Interestingly, we observed that Cd36-/- mice had significantly reduced white adipose tissue retinoid levels compared to wild type mice. In conclusion, we demonstrate that the absence of CD36 alters whole-body vitamin A homeostasis and suggest that this phenotype is secondary to the impaired chylomicron metabolism previously reported in these mice.

Disruption of retinoid homeostasis induces RBP4 overproduction in diabetes: O-GlcNAcylation involved.

BACKGROUND: Retinol-binding protein 4 (RBP4) is elevated and associated with inflammation in metabolic diseases. Disruption of the retinol cascade and O-GlcNAcylation of the RBP4 receptor (STRA6) are found in diabetic kidneys. OBJECTIVES: We investigated whether the disruption of the retinol cascade induces RBP4 overproduction and if O-linked GlcNAc modification targets RBPR2 and contributes to the disruption of retinol cascades in diabetic livers. METHODS: Western blot or immunohistochemistry for RBPR2, CRBP1, LRAT, RALDH, RARα, RARγ, RXRα, RBP4, GFAT, OGT, OGA and inflammatory markers, as well as ELISA for RBP4, were performed in livers of db/db and ob/ob mice and high glucose-cultured hepatocytes. Immunoprecipitation and dual fluorescence staining were used to explore O-GlcNAc-modified RBPR2 and RBP4 binding activity on RBPR2. Transfection of the CRBP1 gene was done to verify whether a disrupted retinol cascade induces RBP4 overproduction. OGT silencing was done to investigate the association of O-GlcNAcylation with the disruption of retinol cascade. RESULTS: Disruption of retinol cascade, RBP4 overproduction, O-GlcNAcylation of RBPR2, decreased RBP4 binding activity on RBPR2 and inflammation were found in livers of db/db and ob/ob mice and high glucose-cultured hepatocytes. CRBP1 gene transfection reversed the suppression of the cellular retinol cascade and simultaneously attenuated the RBP4 overproduction and inflammation in high glucose-treated hepatocytes. The silencing of OGT reversed the disruption of the cellular retinol cascade, RBP4 overproduction and inflammation induced by high glucose in hepatocytes. CONCLUSIONS: This study indicates that the disruption of cellular retinol cascade is strongly associated with RBP4 overproduction and inflammation in diabetic livers. RBPR2 is one target for high glucose-mediated O-linked GlcNAc modification, which causes liver retinol dyshomeostasis.

Cellular retinol binding protein 1 transfection reduces proliferation and AKT-related gene expression in H460 non-small lung cancer cells.

In recent years, new treatments with novel action mechanisms have been explored for advanced non-small cell lung cancer (NSCLC). Retinoids promote cancer cell differentiation and death and their trafficking and action is mediated from specific cytoplasmic and nuclear receptors, respectively. The purpose of this study was to investigate the effect of Cellular retinol binding protein-1 (CRBP-1) transfection in H460 human NSCLC cell line, normally not expressing CRBP-1. H460 cells were transfected by using a vector pTargeT Mammalian expression system carrying the whole sequence of CRBP-1 gene. For proliferation and apoptosis studies, cells were treated with different concentrations of all-trans Retinoic Acid (atRA) and retinol. AKT-related gene expression was analyzed by using western blot and Signosis array and results analysed by one-way analysis of variance (ANOVA) or by t-student test. CRBP-1+ showed reduced proliferation and viability in basal condition and after atRA treatment when compared to empty-transfected H460 cells. Reduced proliferation in CRBP-1+ H460 cells associated to the down-regulation of pAKT/pERK/pEGFR-related genes. In particular, gene array documented the down-regulation of AKT and Stat-3-related genes, including M-Tor, Akt1, Akt2, Akt3, Foxo1, p27, Jun. Restoration of CRBP-1 expression in H460 cells reduced proliferation and viability in both basal condition and after atRA treatment, likely by down-regulating AKT-related gene level. Further studies are needed to better clarify how those CRBP-1-related intracellular pathways contribute to counteract NSCLC progression in order to suggest a potential tool to improve efficacy of retinoid anti lung cancer adjuvant therapy.