Cellular Retinoic Acid Binding Protein Research Articles

The interplay between retinoic acid binding proteins and retinoic acid degrading enzymes in modulating retinoic acid concentrations.

The active metabolite of vitamin A, all-trans-retinoic acid (atRA), is critical for maintenance of many cellular processes. Although the enzymes that can synthesize and clear atRA in mammals have been identified, their tissue and cell-type specific roles are still not fully established. Based on the plasma protein binding, tissue distribution and lipophilicity of atRA, atRA partitions extensively to lipid membranes and other neutral lipids in cells. As a consequence, free atRA concentrations in cells are expected to be exceedingly low. As such mechanisms must exist that allow sufficiently high atRA concentrations to occur for binding to retinoic acid receptor (RARs) and for RAR mediated signaling. Kinetic simulations suggest that cellular retinoic acid binding proteins (CRABPs) provide a cytosolic reservoir for atRA to allow high enough cytosolic concentrations that enable RAR signaling. Yet, the different CRABP family members CRABP1 and CRABP2 may serve different functions in this context. CRABP1 may reside in the cytosol as a member of a cytosolic signalosome and CRABP2 may bind atRA in the cytosol and localize to the nucleus. Both CRABPs appear to interact with the atRA-degrading cytochrome P450 (CYP) family 26 enzymes in the endoplasmic reticulum. These interactions, together with the expression levels of the CRABPs and CYP26s, likely modulate cellular atRA concentration gradients and tissue atRA concentrations in a tightly coordinated manner. This review provides a summary of the current knowledge of atRA distribution, metabolism and protein binding and how these characteristics may alter tissue atRA concentrations.

C21ORF2 mutations point towards primary cilia dysfunction in amyotrophic lateral sclerosis.

Progressive loss of motor neurons is the hallmark of the neurodegenerative disease amyotrophic lateral sclerosis (ALS), but the underlying disease mechanisms remain incompletely understood. In this study, we investigate the effects of C21ORF2 mutations, a gene recently linked to ALS, and find that primary cilia are dysfunctional. Human patient-derived mutant C21ORF2 motor neurons have a reduced ciliary frequency and length. We report that C21ORF2 is located at the basal body of the primary cilium, and mutations associated with ALS alter this localization. Furthermore, we show that a reduction of C21ORF2 levels in cell lines and motor neurons is sufficient to cause fewer primary cilia and reduced cilial length. This ciliary dysfunction leads to defective downstream sonic hedgehog signalling and reduces the expression of cellular retinoic acid binding protein 1 (CRABP1), a protein involved in motor neuron maintenance and survival. In a compartmentalized co-culture system of motor neurons and muscle cells, these ciliary defects were associated with a reduced ability of neuromuscular junction formation. Interestingly, these cilia defects are seemingly not restricted to C21ORF2 ALS, as we also observed perturbed primary cilia in cultured motor neurons and post-mortem motor cortex from patients with the most common genetic subtype of ALS caused by repeat expansions in the C9ORF72 gene. Finally, overexpression of C21ORF2 in mutant C21ORF2 motor neurons rescued the ciliary frequency and length, CRAPBP1 expression and neuromuscular junction formation, confirming the importance of primary cilia for motor neuron function. These results point towards primary cilia dysfunction contributing to motor neuron degeneration in ALS and open new avenues for further research and interventions for this as yet untreatable disease.

Cellular retinoic acid-binding proteins regulate germ cell proliferation and sex determination in zebrafish.

Cellular retinoic acid (RA)-binding proteins (Crabps) solubilize intracellular RA and transport it to its nuclear receptors or cytoplasmic degradation enzymes. Despite their extreme conservation across chordates, genetic studies of Crabp function have revealed few essential functions. We have generated loss-of-function mutations in all four zebrafish Crabps and find essential roles for Crabp2 proteins in gonad development and sex determination. Transgenic RA reporters show strong RA responses in germ cells at the bipotential stage of gonad development. Double mutants lacking the functions of both Crabp2a and Crabp2b predominantly become male, which correlates with their smaller gonad size and reduced germ cell proliferation during gonad development at late larval and early juvenile stages. In contrast, mutants lacking the functions of both Crabp1a and Crabp1b have normal sex ratios. Exogenous RA treatments at bipotential gonad stages increase germ cell number, consistent with a direct role for RA in promoting germ cell proliferation. Our results suggest essential functions for Crabps in gonad development and sex determination.

Structural requirements for the specific binding of CRABP2 to cyclin D3

Cellular retinoic acid binding protein 2 (CRABP2) transports retinoic acid from the cytoplasm to the nucleus where it then transfers its cargo to retinoic acid receptor-containing complexes leading to activation of gene transcription. We demonstrate using purified proteins that CRABP2 is also a cyclin D3-specific binding protein and that the CRABP2 cyclin D3 binding site and the proposed CRABP2 nuclear localization sequence overlap. Both sequences are within the helix-loop-helix motif that forms a lid to the retinoic acid binding pocket. Mutations within this sequence that block both cyclin D3 and retinoic acid binding promote formation of a CRABP2 structure in which the retinoic acid binding pocket is occupied by an alternative lid conformation. Structural and functional analysis of CRABP2 and cyclin D3 mutants combined with AlphaFold models of the ternary CDK4/6-cyclin D3-CRABP2 complex supports the identification of an α-helical protein binding site on the cyclin D3 C-terminal cyclin box fold.

CRABP1 Enhances the Proliferation of the Dermal Papilla Cells of Hu Sheep through the Wnt/β-catenin Pathway

Background: The homologous proteins identified as cellular retinoic acid-binding proteins I and II (CRABP-I and CRABP-II) belong to a subset of intracellular proteins characterized by their robust affinity for retinoic acid, which plays an indispensable role in the development of hair follicle, including differentiation, proliferation, and apoptosis in keratinocytes. Previous research on Hu sheep hair follicles revealed the specific expression CRABP1 in dermal papilla cells (DPCs), suggesting that CRABP1 has a potential role in regulating the DPC population. Therefore, the main purpose of this study is to expose the performance of the CRABP1 genes in the development and proliferation of DPCs. Methods: Initially, overexpression and inhibition of CRABP1 in the DPCs were conducted through overexpression vector and siRNA. CCK-8, EDU, and RT-PCR cell cycle assays and immunostaining were performed to evaluate the proliferation and cell cycle of dermal papilla cells (DPCs). Although, the influence of CRABP1 upon β-catenin in dermal papilla cells (DPCs) was found using immunofluorescence labeling. Finally, RT-PCR was conducted to assess the impact of CRABP1 on the expression levels of CTNNB1, TCF4, and LEF1 in DPCs involved in the Wnt/β-catenin signaling pathway. Results: The results showed that CRABP1 overexpression promotes the growth rates of DPCs and significantly enhances the proportion of S-phase cells compared with the control group (p < 0.05). The results were the opposite when CRABP1 was a knockdown. In contrast, there was a significant decline in the mRNA expression levels of CTNNβ1, LEF1 (p < 0.05), and TCF4 (p < 0.01) by CRABP1 knockdown. Conclusions: This study found that CRABP1 influences the expression of important genes within the Wnt/β-catenin signaling pathway and promotes DPC proliferation. This investigation provides a theoretical framework to explain the mechanisms that control hair follicle morphogenesis and development.

Overcoming retinoic acid resistance in HER2-enriched breast cancers: role of MYC.

HER2-enriched (HER2+) breast cancers express high levels of the growth-promoting HER2 protein. Although these cancers are treated with the HER2-targeted drug, trastuzumab, resistance to treatment is common. Retinoic acid (RA) is an anti-cancer agent that has been successfully used for the treatment of leukemia and holds promise for the treatment of solid cancers, including breast cancer. The HER2 gene is frequently co-amplified with RARA, a key determinant of RA sensitivity in breast cancers. It seems surprising, therefore, that HER2+ breast cancers are refractory to RA treatment. Here, we show that MYC mediates RA resistance by suppressing the expression of cellular retinoic acid binding protein 2 (CRABP2), resulting in RARα inactivation. CRABP2 is an intracellular RA transporter that delivers RA to the nuclear receptor RARα for its activation. Our results indicate that response to RA is enhanced by MYC depletion in HER2+ breast cancer cells and that RA treatment enhances trastuzumab responsiveness. Our findings support the use of RA and trastuzumab for the treatment of subsets of patients with breast cancers that are HER2-RARα co-amplified and have low levels of MYC.

CRABP2 promotes cell migration and invasion by activating PI3K/AKT and MAPK signalling pathways via upregulating LAMB3 in prostate cancer.

Prostate cancer (PCa) has become a worldwide health burden among men. Previous studies have suggested that cellular retinoic acid binding protein 2 (CRABP2) significantly affects the regulation of cell proliferation, motility and apoptosis in multiple cancers; however, the effect of CRABP2 on PCa is poorly reported. CRABP2 expression in different PCa cell lines and its effect on different cellular functions varied. While CRABP2 promotes cell migration and invasion, it appears to inhibit cell proliferation specifically in PC-3 cells. However, the proliferation of DU145 and 22RV1 cells did not appear to be significantly affected by CRABP2. Additionally, CRABP2 had no influence on the cell cycle distribution of PCa cells. The RNA-seq assay showed that overexpressing CRABP2 upregulated laminin subunit beta-3 (LAMB3) mRNA expression, and the enrichment analyses revealed that the differentially expressed genes were enriched in the phosphoinositide 3-kinase (PI3K)/activated protein kinase B (AKT) and mitogen-activated protein kinase (MAPK) signalling pathways. The following western blot experiments also confirmed the upregulated LAMB3 protein level and the activation of the PI3K/AKT and MAPK signalling pathways. Moreover, overexpressing CRABP2 significantly inhibited tumour growth in vivo. In conclusion, CRABP2 facilitates cell migration and invasion by activating PI3K/AKT and MAPK signalling pathways through upregulating LAMB3 in PCa.

Prognostic role of CRABP2 in lung cancer: a meta-analysis

BackgroundThe prognostic value of cellular retinoic acid-binding protein 2 (CRABP2), in lung cancer patients remains to be uncertained. Therefore, our research attempted to assess the relationship between CRABP2 and survival analysis in lung cancer patients through meta-analysis.MethodRelated literature retrieved from Cochrane Library, Ovid, Embase, PubMed, the CNKI, and the Web of Science. The latest update of the search was May 1, 2023. The outcome indicators included as effective measures in the study were hazard ratio (HR), and 95% confidence interval (CI). The Stata 12.0 software was used to analyze the data.ResultsA total of4 studies were finally enrolled in our meta-analysis. The increased plasma level of CRABP2 predicted poor OS in lung cancer patient with a combined HR of 1.14 (95% CI: 1.00–1.30), and were not associated with poor PFS with combined HR: 1.15% CI: 0.63–2.09) in lung cancer patients.ConclusionsOur meta-analysis found the increased plasma level of CRABP2 was associated with poor OS independently in NSCLC patients. The plasma CRABP2 level may be an indicator of biological aggressiveness of the tumor. Our research was promising regarding the feasibility and utility of plasma CRABP2 as a novel prognostic biomarker in NSCLC, and the findings warrant further investigation.

FOXD2-AS1 promotes malignant cell behavior in oral squamous cell carcinoma via the miR-378 g/CRABP2 axis

BackgroundOral squamous cell cancer (OSCC) is a prevalent malignancy in oral cavity, accounting for nearly 90% of oral malignancies. It ranks sixth among the most common types of cancer worldwide and is responsible for approximately 145,000 deaths each year. It is widely accepted that noncoding RNAs participate cancer development in competitive regulatory interaction, knowing as competing endogenous RNA (ceRNA) network, whereby long non-coding RNA (lncRNA) function as decoys of microRNAs to regulate gene expression. LncRNA FOXD2-AS1 was reported to exert an oncogenic role in OSCC. Nevertheless, the ceRNA network mediated by FOXD2-AS1 was not investigated yet. This study aimed to explore the effect of FOXD2-AS1 on OSCC cell process and the underlying ceRNA mechanism.MethodsFOXD2-AS1 expression in OSCC cells were determined via reverse transcription and quantitative polymerase chain reaction. Short hairpin RNA targeting FOXD2-AS1 was transfected into OSCC cells to silence FOXD2-AS1 expression. Then, loss-of-function experiments (n = 3 each assay) were performed to measure cell proliferation, apoptosis, migration, and invasion using colony formation, TdT-mediated dUTP Nick-End Labeling, wound healing and Transwell assays, respectively. RNA binding relation was verified by RNA immunoprecipitation and luciferase reporter assays. Rescue experiments were designed to validate whether FOXD2-AS1 affects cell behavior via the gene cellular retinoic acid binding protein 2 (CRABP2). Statistics were processed by GraphPad Prism 6.0 Software and SPSS software.ResultsFOXD2-AS1 was significantly upregulated in Cal27 and SCC9 cells (6.8 and 6.4 folds). In response to FOXD2-AS1 knockout, OSCC cell proliferation, migration and invasion were suppressed (approximately 50% decrease) while OSCC cell apoptosis was enhanced (more than two-fold increase). FOXD2-AS1 interacted with miR-378 g to alter CRABP2 expression. CRABP2 upregulation partly rescued (*p < 0.05, **p < 0.01, ***p < 0.001) the inhibitory impact of FOXD2-AS1 depletion on malignant characteristics of OSCC cells.ConclusionFOXD2-AS1 enhances OSCC malignant cell behaviors by interacting with miR-378 g to regulate CRABP2 expression.

Identification of cellular retinoic acid binding protein 2 (CRABP2) as downstream target of nuclear factor I/X (NFIX): implications for skeletal dysplasia syndromes.

Nuclear factor I/X (NFIX) mutations are associated with 2 skeletal dysplasias, Marshall-Smith (MSS) and Malan (MAL) syndromes. NFIX encodes a transcription factor that regulates expression of genes, including Bobby sox (BBX) and glial fibrillary acidic protein (GFAP) in neural progenitor cells and astrocytes, respectively. To elucidate the role of NFIX mutations in MSS, we studied their effects in fibroblast cell lines obtained from 5 MSS unrelated patients and 3 unaffected individuals. The 5 MSS NFIX frameshift mutations in exons 6-8 comprised 3 deletions (c.819-732_1079-948del, c.819-471_1079-687del, c.819-592_1079-808del), an insertion (c.1037_1038insT), and a duplication (c.1090dupG). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analyses using MSS and unrelated control fibroblasts and in vitro expression studies in monkey kidney fibroblast (COS-7) cells showed that frameshift mutations in NFIX exons 6-8 generated mutant transcripts that were not cleared by nonsense-mediated-decay mechanisms and encoded truncated NFIX proteins. Moreover, BBX or GFAP expression was unaffected in the majority of MSS fibroblasts. To identify novel NFIX downstream target genes, RNA sequencing and proteomics analyses were performed on mouse embryonic fibroblast (MEF) cells derived from control Nfix+/+, Nfix+/Del2, Nfix+/Del24, NfixDel24/Del24, Nfix+/Del140, and NfixDel140/Del140 mice, compared with NfixDel2/Del2 mice which had developmental, skeletal, and neural abnormalities. This identified 191 transcripts and 815 proteins misregulated in NfixDel2/Del2 MEFs with ≥2-fold-change (P <0 .05). Validation studies using qRT-PCR and western blot analyses confirmed that 2 genes, cellular retinoic acid binding protein 2 (Crabp2) and vascular cell adhesion molecule 1 (Vcam1), were misregulated at the RNA and protein levels in NfixDel2/Del2 MEFs, and that CRABP2 and VCAM1 expressions were altered in 60%-100% of MSS fibroblast cells. Furthermore, in vitro luciferase reporter assays confirmed that NFIX directly regulates CRABP2 promoter activity. Thus, these altered genes and pathways may represent possible targets for drugs as potential treatments and therapies for MSS.

Retinol and retinol binding protein 4 levels and COVID-19: a Mendelian randomization study

BackgroundThe Corona Virus Disease 2019 (COVID-19) pandemic has struck globally. Whether the related proteins of retinoic acid (RA) signaling pathway are causally associated with the risk of COVID-19 remains unestablished. We conducted a two-sample Mendelian randomization (MR) study to assess the associations of retinol, retinol binding protein 4 (RBP4), retinol dehydrogenase 16 (RDH16) and cellular retinoic acid binding protein 1 (CRABP1) with COVID-19 in European population.MethodsThe outcome utilized the summary statistics of COVID-19 from the COVID-19 Host Genetics Initiative. The exposure data were obtained from public genome wide association study (GWAS) database. We extracted SNPs from exposure data and outcome data. The inverse variance weighted (IVW), MR-Egger and Wald ratio methods were employed to assess the causal relationship between exposure and outcome. Sensitivity analyses were performed to ensure the validity of the results.ResultsThe MR estimates showed that retinol was associated with lower COVID-19 susceptibility using IVW (OR: 0.69, 95% CI: 0.53–0.90, P: 0.0065), whereas the associations between retinol and COVID-19 hospitalization or severity were not significant. RBP4 was associated with lower COVID-19 susceptibility using the Wald ratio (OR: 0.83, 95% CI: 0.72–0.95, P: 0.0072). IVW analysis showed RDH16 was associated with increased COVID-19 hospitalization (OR: 1.10, 95% CI: 1.01–1.18, P: 0.0199). CRABP1 was association with lower COVID-19 susceptibility (OR: 0.95, 95% CI: 0.91–0.99, P: 0.0290) using the IVW.ConclusionsWe found evidence of possible causal association of retinol, RBP4, RDH16 and CRABP1 with the susceptibility, hospitalization and severity of COVID-19. Our study defines that retinol is significantly associated with lower COVID-19 susceptibility, which provides a reference for the prevention of COVID-19 with vitamin A supplementation.

Hypothalamic GABAergic Neurons Expressing Cellular Retinoic Acid Binding Protein 1 (CRABP1) Are Sensitive to Metabolic Status and Liraglutide in Male Mice

Introduction: Owing to their privileged anatomical location, neurons of the arcuate nucleus of the hypothalamus (ARC) play critical roles in sensing and responding to metabolic signals such as leptin and glucagon-like peptide 1 (GLP-1). In addition to the well-known proopiomelanocortin (POMC)- and agouti-related peptide (AgRP)-expressing neurons, subpopulations of GABAergic neurons are emerging as key regulators of energy balance. However, the precise identity of these metabolic neurons is still elusive. Here, we identified and characterized the molecular signature of a novel population of GABAergic neurons of the ARC expressing Cellular retinoic acid binding protein 1 (Crabp1). Methods: Using a combination of immunohistochemistry and in situ hybridization techniques, we investigated the expression of Crabp1 across the mouse brain and characterized the molecular identity of Crabp1ARC neurons. We also determined whether Crabp1ARC neurons are sensitive to fasting, leptin, and GLP1R agonism by assessing cFOS immunoreactivity as a marker of neuronal activity. Results: Crabp1ARC neurons represent a novel GABAergic neuronal population robustly enriched in the ARC and are distinct from the prototypical melanocortin neurons. Crabp1ARC neurons overlap with three subpopulations of yet uncharacterized ARC neurons expressing Htr3b, Tbx19, and Tmem215. Notably, Crabp1ARC neurons express receptors for metabolic hormones and their activity is modulated by the nutritional state and GLP1R agonism. Conclusion: Crabp1ARC neurons represent a novel heterogeneous population of GABAergic neurons sensitive to metabolic status.

4D label-free quantitative proteomic analysis identifies CRABP1 as a novel candidate gene for litter size in rabbits†.

In commercial rabbit breeding, litter size is a crucial reproductive trait. This trait directly determines the reproductive ability of female rabbits and is crucial for evaluating the production efficiency. We here compared differentially expressed proteins of in the ovary tissue from New Zealand female rabbits with high (H) and low (L) litter sizes by using 4D label-free quantitative proteomic technology and identified 92 differential proteins. The biological functions of these proteins were revealed through gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Most distributions of GO and KEGG were related to reproduction, growth development, and metabolism. Furthermore, a novel candidate gene cellular retinoic acid binding protein-1 (CRABP1), which was highly expressed in the L group, was selected for further biological function verification. The Cell Counting Kit-8 (CCK-8) assay and flow cytometry analysis revealed that CRABP1 can promote granulosa cell (GC) apoptosis and inhibit GC proliferation. Furthermore, qRT-PCR and western blotting analysis revealed that CRABP1 regulates the genes (HSD17B1, Wnt-10b, FSHR, TAF4B, BMP15, and BMP6) and protein (Wnt-10b) associated with steroid hormone synthesis and follicle development. The PCR product direct sequencing method revealed single nucleotide polymorphisms in the core promoter region of CRABP1. Luciferase activity assays revealed that the transcriptional activity of the GG genotype was significantly higher than that of the TT or TG genotype. Different genotypes are accompanied by changes in transcription factors, which indicates that T-359G polymorphism can regulate CRABP1 expression. In general, we identified litter size-related genes and revealed the mechanism underlying the effect of CRABP1 on litter size. CRABP1 serves as a key factor in the reproductive capacity of rabbits and can act as a molecular biomarker for the breeding of New Zealand rabbits.

CRABP2 regulates infiltration of cancer-associated fibroblasts and immune response in melanoma.

Finding biomarkers for immunotherapy is an urgent issue in cancer treatment. Cellular retinoic acid-binding protein 2 (CRABP2) is a controversial factor in the occurrence and development of human tumors. However, there is limited research on the relationship between CRABP2 and immunotherapy response. This study found that negative correlations of CRABP2 and immune checkpoint markers (PD-1, PD-L1, and CTLA-4) were observed in breast invasive carcinoma (BRCA), skin cutaneous melanoma (SKCM), stomach adenocarcinoma (STAD) and testicular germ cell tumors (TGCT). In particular, in SKCM patients who were treated with PD-1 inhibitors, high levels of CRABP2 predicted poor prognosis. Additionally, CRABP2 expression was elevated in cancer-associated fibroblasts (CAFs) at the single-cell level. The expression of CRABP2 was positively correlated with markers of CAFs, such as MFAP5, PDPN, ITGA11, PDGFRα/β and THY1 in SKCM. To validate the tumor-promoting effect of CRABP2 in vivo, SKCM xenograft mice models with CRABP2 overexpression have been constructed. These models showed an increase in tumor weight and volume. Enrichment analysis indicated that CRABP2 may be involved in immune-related pathways of SKCM, such as extracellular matrix (ECM) receptor interaction and epithelial-mesenchymal transition (EMT). The study suggests that CRABP2 may regulate immunotherapy in SKCM patients by influencing infiltration of CAFs. In conclusion, this study provides new insights into the role of CRABP2 in immunotherapy response. The findings suggest that CRABP2 may be a promising biomarker for PD-1 inhibitors in SKCM patients. Further research is needed to confirm these findings and to explore the clinical implications of CRABP2 in immunotherapy.

Opposing effects of CRABP1 and CRABP2 homologs on the proliferation of breast cancer cells and their sensitivity to retinoic acid

Resistance of tumor cells to retinoic acid (RA), a promising therapeutic agent, is the major factor limiting the use of RA in clinical practice. The mechanisms of RA resistance are still poorly understood. Cellular Retinoic Acid Binding Proteins, CRABP1 and CRABP2, are essential mediators of RA signaling, but the role of the two CRABP homologs in regulating cellular sensitivity to RA has not been well studied. In addition, the effects of CRABP1 and CRABP2 on cell proliferation have not been compared. Here, using a broad panel of breast cancer cell lines with different levels of RA sensitivity/resistance, we show for the first time that in RA-sensitive cells, CRABP1 expression is restricted by methylation and protein levels are highly variable. In moderately RA-resistant lines, a high level of CRABP1 is observed both at the mRNA and protein levels, unchanged by inhibition of DNA methylation. The maximally resistant cell lines are characterized by complete repression of CRABP1 implemented at transcriptional and posttranscriptional levels, and exogenous expression of each of CRABP homologs has no effect on the studied characteristics. CRABP1 and CRABP2 proteins have opposing effects on proliferation and sensitivity to RA. Specifically, in initially RA-sensitive cells CRABP1 stimulates and CRABP2 reduces proliferation and resistance to RA, while in more resistant cells the role of each homolog in both of these indications is reversed. Overall, we have shown for the first time that CRABP proteins exert different effects on the growth and sensitivity to RA of breast cancer cells (stimulation, suppression, or no effect) depending on the baseline level of RA-sensitivity, with the effects of CRABP1 and CRABP2 homologs on the studied properties always being opposite.

Opposite Effects of CRABP1 and CRABP2 Homologs on Proliferation of Breast Cancer Cells and Their Sensitivity to Retinoic Acid.

Resistance of tumor cells to retinoic acid (RA), a promising therapeutic agent, is the major factor limiting the use of RA in clinical practice. The mechanisms of resistance to RA are still poorly understood. Cellular Retinoic Acid Binding Proteins, CRABP1 and CRABP2, are essential mediators of RA signaling, but role of the two CRABP homologs in regulating cellular sensitivity to RA has not been well studied. In addition, the effects of CRABP1 and CRABP2 on cell proliferation have not been compared. Here, using a broad panel of breast cancer cell lines with different levels of RA sensitivity/resistance, we show for the first time that in the RA-sensitive cells, CRABP1 expression is restricted by methylation, and protein levels are highly variable. In the moderately-RA-resistant cell lines, high level of CRABP1 is observed both at the mRNA and protein levels, unchanged by inhibition of DNA methylation. The cell lines with maximum resistance to RA are characterized by complete repression of CRABP1 expression realized at transcriptional and posttranscriptional levels, and exogenous expression of each of the CRABP homologs has no effect on the studied characteristics. CRABP1 and CRABP2 proteins have opposing effects on proliferation and sensitivity to RA. In particular, CRABP1 stimulates and CRABP2 reduces proliferation and resistance to RA in the initially RA-sensitive cells, while in the more resistant cells the role of each homolog in both of these parameters is reversed. Overall, we have shown for the first time that CRABP proteins exert different effects on the growth and sensitivity to RA of breast cancer cells (stimulation, suppression, or no effect) depending on the baseline level of RA-sensitivity, with the effects of CRABP1 and CRABP2 homologs on the studied properties always being opposite.

Cellular Retinoic Acid Binding Protein 2 (CRABP2), Up-regulated by HPV E6/E7, Leads to Aberrant Activation of the Integrin β1/FAK/ERK Signaling Pathway and Aggravates the Malignant Phenotypes of Cervical Cancer.

The ectopic expression of cellular retinoic acid binding protein 2 (CRABP2) is associated with various tumorigenesis. However, the effects of CRABP2 on the progression of cervical cancer are still unclear. The current study aimed to investigate the role of CRABP2 in the malignant phenotypes of cervical cancer cells. CRABP2 was artificially regulated in CaSki, SiHa, and C-33A cells. CCK-8 assay and flow cytometry were used to assess the cell proliferation and apoptosis abilities, respectively. Wound healing assay and transwell assay were employed to measure the cell migration and invasion abilities, respectively. The results showed that CRABP2 was highly expressed in cervical carcinoma tissues and cell lines, and its high expression was associated with poor overall survival. Knockdown of CRABP2 promoted the cell apoptosis and inhibited cell proliferation, migration, and invasion in cervical carcinoma cells, whereas CRABP2 overexpression exhibited the opposite results. Mechanically, CRABP2 silencing suppressed the Integrin β1/FAK/ERK signaling via HuR. Treatment with siITGB1 or a FAK inhibitor PF-562271 or an ERK inhibitor FR180204 reversed the promoting effects of CRABP2 on cell proliferation, migration, and invasion. Moreover, the overexpression of CRABP2 reverted the HPV16 E6/E7 knockdown-induced inhibition of cell proliferation, migration, and invasion in cervical cancer cells. These results suggested that HPV16 E6/E7 promoted the malignant phenotypes of cervical cancer by upregulating the expression of CRABP2. In conclusion, CRABP2, upregulated by HPV E6/E7, promoted the progression of cervical cancer through activating the Integrin β1/FAK/ERK signaling pathway via HuR.

Depleting Cellular Retinoic Acid Binding Protein 1 Impairs UPRmt.

Mitochondrial dysfunction underlines neurodegenerative diseases which are mostly characterized by progressive degeneration of neurons. We previously reported that Cellular retinoic acid Binding protein 1 (Crabp1) knockout (CKO) mice spontaneously developed age-dependent motor degeneration, with defects accumulated in spinal motor neurons (MNs), the only cell type in spinal cord that expresses CRABP1. Here we uncovered that mitochondrial DNA (mtDNA) content and the expression of genes involved in respiration were significantly reduced in CKO mouse spinal cord, accompanied by significantly elevated reactive oxygen species (ROS) and unfolded protein load, indicating that CRABP1 deficiency caused mitochondrial dysfunction. Further analyses of spinal cord tissues revealed significant reduction in the expression and activity of superoxide dismutase 2 (SOD2), as well as defected mitochondrial unfolded protein response (UPRmt) pathway, specifically an increase in ATF5 mRNA but not its protein level, which suggested failure in the translational response of ATF5 in CKO. Consistently, eukaryotic initiation factor-2α, (eIF2α) phosphorylation was reduced in CKO spinal cord. In a CRABP1 knockdown MN1 model, siCrabp1-MN1, we validated the cell-autonomous function of CRABP1 in modulating the execution of UPRmt. This study reveals a new functional role for CRABP1 in the execution of mitochondrial stress response, that CRABP1 modulates eIF2α phosphorylation thereby contributing to ATF5 translational response that is needed to mitigate mitochondria stress.

Molecular basis for cellular retinoic acid-binding protein 1 in modulating CaMKII activation.

Introduction: Cellular retinoic acid (RA)-binding protein 1 (CRABP1) is a highly conserved protein comprised of an anti-parallel, beta-barrel, and a helix-turn-helix segment outside this barrel. Functionally, CRABP1 is thought to bind and sequester cytosolic RA. Recently, CRABP1 has been established as a major mediator of rapid, non-genomic activity of RA in the cytosol, referred to as "non-canonical" activity. Previously, we have reported that CRABP1 interacts with and dampens the activation of calcium-calmodulin (Ca2+-CaM)-dependent kinase 2 (CaMKII), a major effector of Ca2+ signaling. Through biophysical, molecular, and cellular assays, we, herein, elucidate the molecular and structural mechanisms underlying the action of CRABP1 in dampening CaMKII activation. Results: We identify an interaction surface on CRABP1 for CaMKII binding, located on the beta-sheet surface of the barrel, and an allosteric region within the helix segment outside the barrel, where both are important for interacting with CaMKII. Molecular studies reveal that CRABP1 preferentially associates with the inactive form of CaMKII, thereby dampening CaMKII activation. Alanine mutagenesis of residues implicated in the CaMKII interaction results in either a loss of this preference or a shift of CRABP1 from associating with the inactive CaMKII to associating with the active CaMKII, which corresponds to changes in CRABP1's effect in modulating CaMKII activation. Conclusions: This is the first study to elucidate the molecular and structural basis for CRABP1's function in modulating CaMKII activation. These results further shed insights into CRABP1's functional involvement in multiple signaling pathways, as well as its extremely high sequence conservation across species and over evolution.

Downregulation of cellular retinoic acid binding protein 1 fosters epithelial-mesenchymal transition in thyroid cancer.

Cellular retinoic acid binding protein 1 (CRABP1) participates in the regulation of retinoid signaling. Previous studies showed conflicting results regarding the role of CRABP1 in tumor biology, including protumorigenic and tumor-suppressive effects in different types of cancer. Our bioinformatics analyses suggested that CRABP1 expression was downregulated in thyroid cancer. Ectopic expression of CRABP1 in thyroid cancer cells suppressed migratory and invasive activity without affecting cell growth or cell cycle distribution. In transformed normal thyroid follicular epithelial cells, silencing of CRABP1 expression increased invasiveness. Additionally, CRABP1 overexpression was associated with downregulation of the mesenchymal phenotype. Kinase phosphorylation profiling indicated that CRABP1 overexpression was accompanied by a decrease in phosphorylation of epidermal growth factor (EGF) receptor and downstream phosphorylation of Akt, STAT3, and FAK, which were reversed by exogenous EGF treatment. Immunohistochemical analysis of our tissue microarrays revealed an inverse association between CRABP1 expression and disease stage of differentiated thyroid cancer. Taken together, our results suggest that CRABP1 expression is aberrantly lost in thyroid cancer, and this downregulation promotes the epithelial-mesenchymal transition at least partly through modulating EGF receptor signaling.