Retinoic Acid Binding Research Articles

Estrogen regulates the expression of retinoic acid synthesis enzymes and binding proteins in mouse skin

Topical 17-beta-estradiol (E2) regulates the hair cycle, hair shaft differentiation, and sebum production. Vitamin A also regulates sebum production. Vitamin A metabolism proteins localized to the pilosebaceous unit (PSU; hair follicle and sebaceous gland); and were regulated by E2 in other tissues. This study tests the hypothesis that E2 also regulates vitamin A metabolism in the PSU. First, aromatase and estrogen receptors localized to similar sites as retinoid metabolism proteins during mid-anagen. Next, female and male wax stripped C57BL/6J mice were topically treated with E2, the estrogen receptor antagonist ICI 182,780 (ICI), letrozole, E2 plus letrozole, or vehicle control (acetone) during mid-anagen. E2 or one of its inhibitors regulated most of the vitamin A metabolism genes and proteins examined in a sex-dependent manner. Most components were higher in females and reduced with ICI in females. ICI reductions occurred in the premedulla, sebaceous gland, and epidermis. Reduced E2 also reduced RA receptors in the sebaceous gland and bulge in females. However, reduced E2 increased the number of retinal dehydrogenase 2 positive hair follicle associated dermal dendritic cells in males. These results suggest that estrogen regulates vitamin A metabolism in the skin. Interactions between E2 and vitamin A have implications in acne treatment, hair loss, and skin immunity.

Abstract 658: CRABP2 - a novel biomarker for high risk endometrial cancer

Abstract Introduction: The incidence of endometrial cancer (EC) is rising - a consequence of the current obesity epidemic. An improved understanding of obesity-induced EC is needed to manage this growing cohort of patients. Here-in we present Cellular Retinoic Acid Binding Protein 2 (CRABP2), which was identified as an obesity-related gene in the BDII/Han rat spontaneous EC model, as a novel biomarker in high-risk EC. Methods:BDII/Han rats were fed either a high (N=12) or low calorie (N=7) diet for 15 months. RNA-seq, - differential gene expression and gene set enrichment analysis (GSEA) were performed on endometrial tumors from both groups. The association between CRABP2 mRNA expression and standard clinico-pathological features was examined in human EC using the TCGA. Following dichotomization of patients based on their CRABP2 mRNA expression using regression tree analysis, univariate and multivariate Cox regression survival analyses were performed. Differential gene expression analysis and GSEA were performed on human tumors stratified according to CRABP2 expression levels. In-silico findings were validated using immunohistochemistry (IHC) assessing CRABP2 expression in normal endometrium, primary and metastatic endometrial carcinoma. Results:Weight gain in high fat diet (HFD) rats was double that of normal chow diet rats (50 g vs. 25 g). The incidence of cancer was similar in both groups (4/7-57% vs. 4/12-33%; p=0.37). 368 differentially expressed genes (DEGs) were identified between both groups. CRABP2 was upregulated in tumors from the HFD group. Regression tree analysis identified 60 (11%) tumors in the TCGA with increased CRABP2 mRNA expression. Increased CRABP2 expression was associated with serous EC (p<0.001), grade 3 (p<0.001) and advanced stage (p=0.015). Univariate Cox regression analysis revealed that high CRABP2 expression is associated with reduced overall survival (HR=2.67, 95% CI 1.64-4.33, p<0.001). This remained true following multivariate cox regression analysis in which age, histological type, stage and grade were confounded for (HR=2.06, 95% CI 1.24-3.41, p=0.004). 224 DEGs were identified between CRABP2 high (N=60) and low (N=468) tumors in the TCGA. GSEA analysis revealed an enrichment of pathways involved in developmental biology and embryonic stem cell-like gene expression signatures in the CRABP2-high cohort. IHC confirmed increased expression of cytoplasmic CRABP2 in EC compared to normal endometrium. CRABP2 expression was significantly increased in serous compared to endometrioid cancer (p<0.05) and a comparison between primary and metastatic serous carcinoma demonstrated increased expression in metastatic cases. Conclusion: A HFD increased CRABP2 expression in rat EC. In humans, increased expression of CRABP2 is associated with high-risk EC. Transcriptomic analysis and validation using IHC suggests that CRABP2 and retinoic acid signalling may be an important therapeutic target in advanced EC. Citation Format: Donagh Egan, Bruce Moran, Michael Wilkinson, William Gallagher, Xavier Matias-Guiu, Walter Kolch, Carel LeRoux, Donal Brennan. CRABP2 - a novel biomarker for high risk endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 658.

Abstract 1401: MYC mediates retinoic acid resistance by suppressing cellular retinoic acid-binding protein (CRABP2) transcription in HER2-enriched breast cancers

Abstract Introduction: HER2-positive (HER2+) breast cancers express high levels of the growth-promoting HER2 protein. The development of a targeted drug, trastuzumab, has greatly improved clinical outcome for HER2+ patients. However, intrinsic and acquired resistance to trastuzumab are common. Clearly, additional therapeutic interventions are required for the clinical management of HER2-enriched breast cancers. Retinoic acid (RA) is a vital signaling molecule that regulates multiple biological processes, including cell proliferation, differentiation, and death. RA has been successfully used in the treatment of acute promyelocytic leukemia. RA also holds promise for the treatment of solid cancers like breast cancer. RA action is mediated through transcription factor RARα and the RA binding protein CRABP2, which deliver RA to RARα in the nucleus. The HER2 gene (ERBB2) is frequently co-amplified with the gene encoding RARα, a key determinant of RA sensitivity. It seems surprising, therefore, that HER2+ breast cancers are refractory to RA treatment. MYC is an oncogene that inhibits RARα activity in leukemia cells. Importantly, MYC is preferentially amplified and overexpressed in HER2+ breast cancers. My research aims to elucidate the mechanism underlying RA resistance in HER2+ breast cancers, with a special focus on the role of MYC. Hypothesis: MYC attenuates RA action by inhibiting the CRABP2-RARα pathway. Results: RNA levels of MYC are negatively correlated with CRABP2 RNA levels. Depletion of MYC upregulates CRABP2 at both the RNA and protein levels. Furthermore, we found that MYC binds to the CRABP2 promoter region suggesting that MYC may directly suppress CRABP2 gene transcription activity. Our results show that ectopic expression of MYC inhibits, whereas depletion of MYC activates RAR activity. Consistently, ectopic expression of MYC increases RA resistance, whereas depletion of MYC sensitizes cells to RA treatment. When CRABP2 is depleted along with MYC-knockdown, cell proliferation is rescued, suggesting that MYC mediates RA resistance, at least partially through downregulation of CRABP2. We also found that RA treatment enhances trastuzumab responsiveness in HER2+ breast cancer cells. Significance: This study sheds light on the role and mechanism of MYC in governing RA resistance in HER2+ breast cancer cells. Our results support the use of RA and trastuzumab for the treatment of subsets of patients with HER2+/low MYC breast cancers. Citation Format: Won-Shik Choi, Rong-Zong Liu, Roseline Godbout. MYC mediates retinoic acid resistance by suppressing cellular retinoic acid-binding protein (CRABP2) transcription in HER2-enriched breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1401.

Regulation of exosome secretion by cellular retinoic acid binding protein 1 contributes to systemic anti-inflammation

BackgroundIntercellular communications are important for maintaining normal physiological processes. An important intercellular communication is mediated by the exchange of membrane-enclosed extracellular vesicles. Among various vesicles, exosomes can be detected in a wide variety of biological systems, but the regulation and biological implication of exosome secretion/uptake remains largely unclear.MethodsCellular retinoic acid (RA) binding protein 1 (Crabp1) knockout (CKO) mice were used for in vivo studies. Extracellular exosomes were monitored in CKO mice and relevant cell cultures including embryonic stem cell (CJ7), macrophage (Raw 264.7) and hippocampal cell (HT22) using Western blot and flow cytometry. Receptor Interacting Protein 140 (RIP140) was depleted by Crispr/Cas9-mediated gene editing. Anti-inflammatory maker was analyzed using qRT-PCR. Clinical relevance was accessed by mining multiple clinical datasets.ResultsThis study uncovers Crabp1 as a negative regulator of exosome secretion from neurons. Specifically, RIP140, a pro-inflammatory regulator, can be transferred from neurons, via Crabp1-regulated exosome secretion, into macrophages to promote their inflammatory polarization. Consistently, CKO mice, defected in the negative control of exosome secretion, have significantly elevated RIP140-containing exosomes in their blood and cerebrospinal fluid, and exhibit an increased vulnerability to systemic inflammation. Clinical relevance of this pathway is supported by patients’ data of multiple inflammatory diseases. Further, the action of Crabp1 in regulating exosome secretion involves its ligand and is mediated by its downstream target, the MAPK signaling pathway.ConclusionsThis study presents the first evidence for the regulation of exosome secretion, which mediates intercellular communication, by RA-Crabp1 signaling. This novel mechanism can contribute to the control of systemic inflammation by transferring an inflammatory regulator, RIP140, between cells. This represents a new mechanism of vitamin A action that can modulate the homeostasis of system-wide innate immunity without involving gene regulation.EB6KXQYbkzhu3R444VcR3RVideo

Maprotiline Suppresses Cholesterol Biosynthesis and Hepatocellular Carcinoma Progression Through Direct Targeting of CRABP1.

Hepatocellular carcinoma (HCC) remains one of the leading causes of cancer-related death and has a poor prognosis worldwide, thus, more effective drugs are urgently needed. In this article, a small molecule drug library composed of 1,056 approved medicines from the FDA was used to screen for anticancer drugs. The tetracyclic compound maprotiline, a highly selective noradrenergic reuptake blocker, has strong antidepressant efficacy. However, the anticancer effect of maprotiline remains unclear. Here, we investigated the anticancer potential of maprotiline in the HCC cell lines Huh7 and HepG2. We found that maprotiline not only significantly restrained cell proliferation, colony formation and metastasis in vitro but also exerted antitumor effects in vivo. In addition to the antitumor effect alone, maprotiline could also enhance the sensitivity of HCC cells to sorafenib. The depth studies revealed that maprotiline substantially decreased the phosphorylation of sterol regulatory element-binding protein 2 (SREBP2) through the ERK signaling pathway, which resulted in decreased cholesterol biosynthesis and eventually impeded HCC cell growth. Furthermore, we identified cellular retinoic acid binding protein 1 (CRABP1) as a direct target of maprotiline. In conclusion, our study provided the first evidence showing that maprotiline could attenuate cholesterol biosynthesis to inhibit the proliferation and metastasis of HCC cells through the ERK-SREBP2 signaling pathway by directly binding to CRABP1, which supports the strategy of repurposing maprotiline in the treatment of HCC.

Overexpression of CRABP2 inhibits dexamethasone-induced apoptosis in human osteoblast cells

BackgroundThe purpose of the current study was to explore the role and underlying mechanism of cellular retinoic acid binding protein 2 (CRABP2) in dexamethasone (DEX)-induced apoptosis in human osteoblast cells.MethodsGSE10311 was downloaded from the Gene Expression Omnibus (GEO) database to identify the differentially expressed genes (DEGs) by the limma/R package. Primary human osteoblast was isolated and treated with different concentration of DEX (0, 10-8, 10-7, 10-6, 10-5, and 10-4 mol/L), and cell viability and flow cytometry were used to detect cell proliferation and apoptosis. A CRABP2 overexpression plasmid (oe-CRABP2) was used to overexpress CRABP2, and western blotting was conducted to detect protein expression.ResultsWe found that CRABP2 was downregulated in the DEX-treated group. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses indicated that DEGs were associated with PI3K/Akt signaling pathway. DEX downregulated CRABP2 gene and protein expression, inhibited viability, and induced human osteoblast apoptosis. Overexpression of CRABP2 reversed DEX-induced apoptosis in human osteoblast. Moreover, overexpression of CRABP2 delayed the progression of DEX-induced osteonecrosis of the femoral head (ONFH) animal model.ConclusionIn conclusion, CRABP2 is effective at inhibiting DEX-induced human osteoblast apoptosis and delayed ONFH progression.

MLL5 improves ATRA driven differentiation and promotes xenotransplant engraftment in acute promyelocytic leukemia model

Although the mixed lineage leukemia 5 (MLL5) gene has prognostic implications in acute promyelocyte leukemia (APL), the underlying mechanism remains to be elucidated. Here, we demonstrate the critical role exerted by MLL5 in APL regarding cell proliferation and resistance to drug-induced apoptosis, through mtROS regulation. Additionally, MLL5 overexpression increased the responsiveness of APL leukemic cells to all-trans retinoic acid (ATRA)-induced differentiation, via regulation of the epigenetic modifiers SETD7 and LSD1. In silico analysis indicated that APL blasts with MLL5high transcript levels were associated with retinoic acid binding and downstream signaling, while MLL5low blasts displayed decreased expression of epigenetic modifiers (such as KMT2C, PHF8 and ARID4A). Finally, APL xenograft transplants demonstrated improved engraftment of MLL5-expressing cells and increased myeloid differentiation over time. Concordantly, evaluation of engrafted blasts revealed increased responsiveness of MLL5-expressing cells to ATRA-induced granulocytic differentiation. Together, we describe the epigenetic changes triggered by the interaction of MLL5 and ATRA resulting in enhanced granulocytic differentiation.

LINC01816 promotes the migration, invasion and epithelial‑mesenchymal transition of thyroid carcinoma cells by sponging miR‑34c‑5p and regulating CRABP2 expression levels.

Thyroid carcinoma (THCA) is a common type of endocrine system cancer and its current clinical treatment method is surgical resection. Long non-coding RNAs (lncRNAs) have been revealed to serve important roles in a variety of complex human diseases. Therefore, determining the association between lncRNAs and diseases may provide novel insight into disease-related lncRNAs, with the aim of improving disease treatments and diagnoses. Long intergenic non-protein coding RNA 1816 (LINC01816) was identified to be associated with the survival of patients with colorectal cancer using the IDHI-MIRW method. The present study aimed to investigate the role and molecular mechanism of LINC01816 in THCA. Analysis of datasets from The Cancer Genome Atlas database revealed that the upregulation of LINC01816 expression levels was associated with a variety of cancer types. Reverse transcription-quantitative PCR analysis demonstrated that compared with the normal thyroid tissues, the expression levels of LINC01816 were upregulated in THCA tissues. The results of wound healing and Transwell assays, and western blotting demonstrated that the overexpression of LINC01816 could strengthen the invasive and migratory abilities of THCA cells and enhance epithelial-mesenchymal transition progression. Analysis using the starBase website and dual-luciferase reporter assays identified that microRNA (miR)-34c-5p was a target of LINC01816. The overexpression of miR-34c-5p could inhibit the invasive and migratory abilities of THCA cells, in addition to inhibiting the cellular retinoic acid binding protein 2 (CRABP2) overexpression-induced effects on invasion, migration and EMT processes. In conclusion, the findings of the present study indicated that LINC01816 may be capable of sponging miR-34c-5p to upregulate CRABP2 expression levels, which subsequently promoted the invasion, migration and EMT of THCA cells. Therefore, targeting the LINC01816/miR-34c-5p/CRABP2 pathway may be an effective therapeutic approach for patients with THCA.

Polyethylene glycol perturbs the unfolding of CRABP I: A correlation between experimental and theoretical approach

The importance of macromolecules paves the way towards a detailed molecular level investigation as all most all cellular processes occurring at the interior of cells in the form of proteins, enzymes, and other biological molecules are significantly affected because of their crowding. Thus, exploring the role of crowding environment on the denaturation and renaturation kinetics of protein molecules is of great importance. Here, CRABP I (cellular retinoic acid binding protein I) is employed as a model protein along with different molecular weights of Polyethylene glycol (PEG) as molecular crowders. The experimental evaluations are done by accessing the protein secondary structure analysis using circular dichroism (CD) spectroscopy and unfolding kinetics using intrinsic fluorescence of CRABP I at 37 °C to mimic the in vivo crowding environment. The unfolding kinetics results indicated that both PEG 2000 and PEG 4000 act as stabilizers by retarding the unfolding kinetic rates. Both kinetic and stability outcomes presented the importance of crowding environment on stability and kinetics of CRABP I. The molecular dynamics (MD) studies revealed that thirteen PEG 2000 molecules assembled during the 500 ns simulation, which increases the stability and percentage of β-sheet. The experimental findings are well supported by the molecular dynamics simulation results.

Dietary Vitamin A Impacts Refractory Telogen.

Hair follicles cycle through periods of growth (anagen), regression (catagen), rest (telogen), and release (exogen). Telogen is further divided into refractory and competent telogen based on expression of bone morphogenetic protein 4 (BMP4) and wingless-related MMTV integration site 7A (WNT7A). During refractory telogen hair follicle stem cells (HFSC) are inhibited. Retinoic acid synthesis proteins localized to the hair follicle and this localization pattern changed throughout the hair cycle. In addition, excess retinyl esters arrested hair follicles in telogen. The purpose of this study was to further define these hair cycle changes. BMP4 and WNT7A expression was also used to distinguish refractory from competent telogen in C57BL/6J mice fed different levels of retinyl esters from two previous studies. These two studies produced opposite results; and differed in the amount of retinyl esters the dams consumed and the age of the mice when the different diet began. There were a greater percentage of hair follicles in refractory telogen both when mice were bred on an unpurified diet containing copious levels of retinyl esters (study 1) and consumed excess levels of retinyl esters starting at 12 weeks of age, as well as when mice were bred on a purified diet containing adequate levels of retinyl esters (study 2) and remained on this diet at 6 weeks of age. WNT7A expression was consistent with these results. Next, the localization of vitamin A metabolism proteins in the two stages of telogen was examined. Keratin 6 (KRT6) and cellular retinoic acid binding protein 2 (CRABP2) localized almost exclusively to refractory telogen hair follicles in study 1. However, KRT6 and CRABP2 localized to both competent and refractory telogen hair follicles in mice fed adequate and high levels of retinyl esters in study 2. In mice bred and fed an unpurified diet retinol dehydrogenase SDR16C5, retinal dehydrogenase 2 (ALDH1A2), and cytochrome p450 26B1 (CYP26B1), enzymes and proteins involved in RA metabolism, localized to BMP4 positive refractory telogen hair follicles. This suggests that vitamin A may contribute to the inhibition of HFSC during refractory telogen in a dose dependent manner.

Genome-Wide Association Study and Transcriptome Differential Expression Analysis of the Feather Rate in Shouguang Chickens.

The feather rate phenotype in chicks, including early-feathering and late-feathering phenotypes, are widely used as a sexing system in the poultry industry. The objective of this study was to obtain candidate genes associated with the feather rate in Shouguang chickens. In the present study, we collected 56 blood samples and 12 hair follicle samples of flight feathers from female Shouguang chickens. Then we identified the chromosome region associated with the feather rate by genome-wide association analysis (GWAS). We also performed RNA sequencing and analyzed differentially expressed genes between the early-feathering and late-feathering phenotypes using HISAT2, StringTie, and DESeq2. We identified a genomic region of 10.0–13.0 Mb of chromosome Z, which is statistically associated with the feather rate of Shouguang chickens at one-day old. After RNA sequencing analysis, 342 differentially expressed known genes between the early-feathering (EF) and late-feathering (LF) phenotypes were screened out, which were involved in epithelial cell differentiation, intermediate filament organization, protein serine kinase activity, peptidyl-serine phosphorylation, retinoic acid binding, and so on. The sperm flagellar 2 gene (SPEF2) and prolactin receptor (PRLR) gene were the only two overlapping genes between the results of GWAS and differential expression analysis, which implies that SPEF2 and PRLR are possible candidate genes for the formation of the chicken feathering phenotype in the present study. Our findings help to elucidate the molecular mechanism of the feather rate in chicks.

Single-cell transcriptomic analysis of small and large wounds reveals the distinct spatial organization of regenerative fibroblasts.

Wound‐induced hair follicle neogenesis (WIHN) has been an important model to study hair follicle regeneration during wound repair. However, the cellular and molecular components of the dermis that make large wounds more regenerative are not fully understood. Here, we compare and contrast recently published scRNA‐seq data of small scarring wounds to wounds that regenerate in hope to elucidate the role of fibroblasts lineages in WIHN. Our analysis revealed an over‐representation of the newly identified upper wound fibroblasts in regenerative wound conditions, which express the retinoic acid binding protein Crabp1. This regenerative cell type shares a similar gene signature to the murine papillary fibroblast lineage, which are necessary to support hair follicle morphogenesis and homeostasis. RNA velocity analysis comparing scarring and regenerating wounds revealed the divergent trajectories towards upper and lower wound fibroblasts and that the upper populations were closely associated with the specialized dermal papilla. We also provide analyses and explanation reconciling the inconsistency between the histological lineage tracing and the scRNA‐seq data from recent reports investigating large wounds. Finally, we performed a computational test to map the spatial location of upper wound fibroblasts in large wounds which revealed that upper peripheral fibroblasts might harbour equivalent regenerative competence as those in the centre. Overall, our scRNA‐seq reanalysis combining multiple samples suggests that upper wound fibroblasts are required for hair follicle regeneration and that papillary fibroblasts may migrate from the wound periphery to the centre during wound re‐epithelialization. Moreover, data from this publication are made available on our searchable web resource: https://skinregeneration.org/.

Abstract 6141: Role of aldehyde dehydrogenase 1B1 in colon carcinogenesis in a newly developed mouse model

Abstract Introduction: Aldehyde dehydrogenases (ALDH), particularly ALDH1B1 has been shown to have increased expression in both human and APCMin mice colorectal tumors. Studies have proposed the use of ALDH1B1 as a potential novel human colorectal tumor marker. Previous in vitro studies have shown that ALDH1B1 plays a critical role in colon carcinogenesis. The goal of this study is to understand the in vivo role of ALDH1B1 in colon carcinogenesis using a newly developed mouse model, APCMinAldh1b1−/− mice, through characterization of its phenotype in tumor growth and development, and elucidating the underlying potential molecular mechanisms. Methods: We generated APCMinAldh1b1−/-. At 12-14 weeks old, the experimental group APCMinAldh1b1−/- (n=6) and control group APCMinAldh1b1+/+ (n=7) were euthanized. Tumor counts, size and burden for each intestinal segments were recorded. H&E staining was performed and observed for tumor types and counts. Immunohistochemistry using the markers Ki-67 (proliferation), Cleave Caspase 3 (apoptosis), KRT20 (differentiation) were done in colon. IHC of retinoic acid binding proteins (anti-CRBP2 and anti-FABP5) were also performed Results: The experimental group had shown significantly higher total average intestinal tumor burden (6.8 fold) (p=0.0007) and total average tumor counts observed (3.6 fold) (p=0.02). In the colon, there was a 3.5-fold increase in average tumor burden (p=0.017) with a 2.7-fold increase in average tumor size (p=0.025)in the experimental group. We also noticed a trend of increased average tumor counts observed in the colon of the experimental group although statistically insignificant. Average tumor counts of ileum observed was 9-fold higher in experimental group with only 1 mice from control group showed observable small tumor growth which did not meet criteria for tumor size and tumor burden calculation. There were no significant differences in observable tumor counts, tumor size and tumor burden in jejunum. Histologically, all tumors were pedunculated adenocarcinomas. Many unobservable small tumors less than 1mm width and length were seen in the H&E of both ileum and jejunum in the experimental and control group. In the control group, there was less observable tumors compared to the experimental group although the tumor counts on H&E in both groups showed no major differences. This indicates that the tumor size in the experimental group was larger than the control group. On the contrary, average tumor counts of colon in H&E were lower compared to tumor counts observed in both control and experimental groups, indicating low number of small unobservable tumors in colon. Although the tumor counts of colon on H&E and observation between the two groups were statistically insignificant, we noticed a pattern of increased tumor counts in the experimental group suggesting a potential increase in tumor incidence. Immunohistochemical staining of colon is ongoing. Conclusion: ALDH1B1 reduced tumor growth rate in the ileum, and reduced tumor size in the colon suggesting its role in tumor promotion and progression. A larger number of experimental and control group is required to establish its potentially tumor-initiating role in the colon. Further characterization of ALDH1B1 in colon carcinogenesis is ongoing. We are also elucidating the role and regulation of retinoic acid signaling by ALDH1B1 in colorectal tumor growth. Citation Format: Wan Ying Tan, David. J. Orlicky, Ying Chen, Yatrik M. Shah, David C. Thompson, Vasilis Vasiliou. Role of aldehyde dehydrogenase 1B1 in colon carcinogenesis in a newly developed mouse model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6141.

Analysis of CRABP2 and FABP5 genes in primary and recurrent pterygium tissues.

The etiology of pterygium remains unclear, but ultraviolet (UV) radiation is generally considered to be major risk factor. Pterygium has similarity features with many cancers, including inflammation, invasion, cell proliferation, anti-apoptosis, angiogenesis and recurrence after resection. Retinoic acid via cellular retinoic acid binding protein 2 (CRABP2) is involved in cell cycle arrest, apoptosis and differentiation, while it via fatty acid binding protein 5 (FABP5) is involved in survival, cell proliferation and angiogenesis, which pathway gets activated depends on the CRABP2/FABP5 ratio. Alterations of retinoid signaling were found in many cancer types. The deregulated retinoid signaling may also contribute to the development and/or recurrence of pterygium. The aim of our study was to determine mRNA and protein expressions of CRABP2 and FABP5 and ratio of CRABP2/FABP5 in primer and recurrent pterygium tissues. Pterygia tissues were collected from 30 eyes of 30 patients undergoing pterygium excision. CRABP2 and FABP5 mRNA and protein expression were assessed using Real-time PCR and Western blotting through examination of excised specimens from pterygium and conjunctiva tissues. The ratio of CRABP2/FABP5 gene expression was not altered when primary pterygium tissues compared normal conjunctival tissues (1.00-fold change). Whereas the ratio of CRABP2/ FABP5 gene expression was decreased when recurrent pterygium tissues compared normal conjunctival tissues (0.81-fold change). Understanding etiopathogenesis of pterygium may aid in the find of more promising treatments to prevent pterygium in earlier stages.

Abstract B27: Cellular retinoic acid binding protein 2 (CRABP2) is a novel biomarker and potential therapeutic target for high-grade serous ovarian carcinomas

Abstract Ovarian cancer ranks fifth in cancer deaths among women in the United States, accounting for more deaths than any other gynecologic cancer. There is an unmet medical need for biomarkers that can detect high-grade serous ovarian carcinoma (HGSOC) at an early stage. Our laboratory evaluated the biomarker potential of cellular retinoic acid binding protein 2 (CRABP2). Using Western blots (WB) and immunohistochemistry (IHC), we show that CRABP2 is expressed and secreted by HGSOC cells and tissues and is absent in normal fallopian tube (FT) epithelium. CRABP2 expression was associated with poor overall survival in patients with HGSOC. Proteomic analyses quantified specific secretion of CRABP2 in conditioned media from HGSOC cell lines, primary ascites-derived tumor cells, and serum from HGSOC patients. Expression of CRABP2 was positively associated with CRABP2 copy number amplifications by mining The Cancer Genome Atlas database. In addition, DNA methylation studies identified a putative enhancer downstream of CRABP2 that is hyper-methylated in fallopian tube epithelia (FTE) tissue and hypomethylated in HGSOC cells and tissues that expressed CRABP2. Inhibition of DNA methylation with DNA methytransferase (DNMT) inhibitors resulted in robust expression of CRABP2 protein in FT cell lines. Finally, a finding supported by CRABP2 knockdown experiments showed that CRABP2 loss triggers cell death in HGSOC cell lines. In conclusion, CRABP2 may serve as a novel biomarker for HGSOC with clinical potential as a therapeutic target. Citation Format: Yi Feng, Michael Gillette, Eric Kuhn, David Klinkebiel, Marilyn A. Mitchell, Kai Doberstein, Daniele Chaves-Moreira, Sho Sato, Haineng Xu, Brett Bomwell, Michelle S. Hirsch, Carolina Reyes, Adam R. Karpf, Michael J. Birrer, Steven J. Skates, Steven A. Carr, Ronny Drapkin. Cellular retinoic acid binding protein 2 (CRABP2) is a novel biomarker and potential therapeutic target for high-grade serous ovarian carcinomas [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B27.

CRABP2 and FABP5 expression levels in diseased and normal pancreas

Recently, stromal targeting, by agents such as All trans retinoic acid (ATRA), has been regarded as a promising avenue for the treatment of pancreatic ductal adenocarcinoma (PDAC). The intra-cellular transportation of ATRA to the nuclear receptors is performed by either: fatty acid binding protein 5 (FABP5) or cellular retinoic acid binding protein 2 (CRABP2), dictating the transcription of downstream genes and, thus, eventual cell phenotype. Here, we explored the levels of each protein, in pancreatic tissues of patients presenting with a range of pancreatic diseases (pancreatic ductal adenocarcinoma (PDAC), chronic pancreatitis (CP), cholangiocarcinoma (CC)).We demonstrate that there is a significantly lower CRABP2 and FABP5 expression in activated fibroblasts or pancreatic stellate cells (PSC) in PDAC, as well as other diseased pancreas as in CC and CP, versus quiescent fibroblasts. The quiescent fibroblasts consistently show a pattern of high FABP5:CRABP2 ratio, whereas PSC in all non-PDAC tissues showed a low FABP5:CRABP2 ratio. PSC in PDAC patients had a range of FABP5:CRABP2 ratios (high, even and low).There was a lower CRABP2 expression in cancerous epithelial cells (PDAC) versus normal epithelial cells. This is also present in other disease states (CP, CC). Contrasting to the patterns seen for fibroblasts, the FABP5 expression in PDAC epithelial cells matched that of the normal epithelial cells. However, the normal epithelial cells had a high FABP5:CRABP2 ratio, compared to the PDAC epithelial cells. These ratios may have correlation with tumor progression, and overall survival. These findings could be confirmed in in vitro cell lysates. CRABP2 and FABP5 levels and ratios could serve as valuable biomarkers.

Sonic Hedgehog-Gli1 Signaling and Cellular Retinoic Acid Binding Protein 1 Gene Regulation in Motor Neuron Differentiation and Diseases.

Cellular retinoic acid-binding protein 1 (CRABP1) is highly expressed in motor neurons. Degenerated motor neuron-like MN1 cells are engineered by introducing SODG93A or AR-65Q to model degenerated amyotrophic lateral sclerosis (ALS) or spinal bulbar muscular atrophy neurons. Retinoic acid (RA)/sonic hedgehog (Shh)-induced embryonic stem cells differentiation into motor neurons are employed to study up-regulation of Crabp1 by Shh. In SODG93A or AR-65Q MN1 neurons, CRABP1 level is reduced, revealing a correlation of motor neuron degeneration with Crabp1 down-regulation. Up-regulation of Crabp1 by Shh is mediated by glioma-associated oncogene homolog 1 (Gli1) that binds the Gli target sequence in Crabp1′s neuron-specific regulatory region upstream of minimal promoter. Gli1 binding triggers chromatin juxtaposition with minimal promoter, activating transcription. Motor neuron differentiation and Crabp1 up-regulation are both inhibited by blunting Shh with Gli inhibitor GANT61. Expression data mining of ALS and spinal muscular atrophy (SMA) motor neurons shows reduced CRABP1, coincided with reduction in Shh-Gli1 signaling components. This study reports motor neuron degeneration correlated with down-regulation in Crabp1 and Shh-Gli signaling. Shh-Gli up-regulation of Crabp1 involves specific chromatin remodeling. The physiological and pathological implication of this regulatory pathway in motor neuron degeneration is supported by gene expression data of ALS and SMA patients.

Vitamin A Metabolism During Refractory Telogen

Abstract Objectives Hair follicles cycle through periods of growth (anagen), regression (catagen) and rest (telogen). Telogen is further divided into refractory and competent telogen based on the expression of bone morphogenetic protein 4 (BMP4). Previously, the expression of a complete set of proteins involved in retinoic acid (RA) synthesis and signaling localized to the hair follicle and changed throughout the hair cycle. In addition, excess dietary vitamin A arrested the hair cycle in telogen; while retinol dehydrogenases short-chain dehydrogenase/reductase family 16C members 5 and 6 (Sdr16c5−/−/Sdr16c6−/−) double null mice had an accelerated the hair cycle. The purpose of this study was to further define these changes in the hair cycle. Methods The localization of RA synthesis proteins SDR16C5, retinol dehydrogenase 10 (RDH10), retinal dehydrogenase 2 (ALDH1A2), cellular RA binding protein 2 (CRABP2), RA degradation enzyme cytochrome p450 26B1 (CYP26B1), and BMP4 was examined in telogen hair follicles in female C57BL/6 J mice by immunohistochemistry. Immunohistochemistry with an antibody against BMP4 was also used to mark refractory telogen in the previous dietary vitamin A study. Results All proteins localized to BMP4 positive refractory telogen hair follicles. SDR16C5 and ALDH1A2 were also seen in BMP4 negative competent telogen hair follicles, but at a lower level. RDH10 was expressed in both BMP4 negative and positive hair follicles at similar levels. BMP4 expression was also used to distinguish refractory from competent telogen in C57BL/6 J mice fed different levels of vitamin A. Both low and excess dietary vitamin A resulted a greater percentage of hair follicles in refractory telogen in different studies. Conclusions In conclusion, RA synthesis and signaling may be stronger in refractory telogen and contribute to the inhibition of the hair cycle. Funding Sources NIH/NIAMS, Internal funding.

Computational and Spectroscopic Characterization of the Photocycle of an Artificial Rhodopsin.

The photocycle of a reversible photoisomerizing rhodopsin mimic (M2) is investigated. This system, based on the cellular retinoic acid binding protein, is structurally different from natural rhodopsin systems, but exhibits a similar isomerization upon light irradiation. More specifically, M2 displays a 15-cis to all-trans conversion of retinal protonated Schiff base (rPSB) and all-trans to 15-cis isomerization of unprotonated Schiff base (rUSB). Here we use hybrid quantum mechanics/molecular mechanics (QM/MM) tools coupled with transient absorption and cryokinetic UV-vis spectroscopies to investigate these isomerization processes. The results suggest that primary rPSB photoisomerization of M2 occurs around the C13═C14 double bond within 2 ps following an aborted-bicycle pedal (ABP) isomerization mechanism similar to natural microbial rhodopsins. The rUSB isomerization is much slower and occurs within 48 ps around the C15═N double bond. Our findings reveal the possibility to engineer naturally occurring mechanistic features into artificial rhodopsins and also constitute a step toward understanding the photoisomerization of UV pigments. We conclude by reinforcing the idea that the presence of the retinal chromophore inside a tight protein cavity is not mandatory to exhibit ABP mechanism.

Topical Proretinal Nanoparticles: Biological Activities, Epidermal Proliferation and Differentiation, Follicular Penetration, and Skin Tolerability

Proretinal nanoparticles, the retinilidene-chitosan nanoparticles, have been developed to overcome the physicochemical instability of retinal and to lessen the dose-dependent cutaneous irritation, through sustaining the release of retinoid. Compared to conventional retinal at the same concentration, proretinal nanoparticles had no cytotoxicity and could induce a spontaneously immortalized human keratinocyte line to express more cellular retinoic acid binding protein-2. Compared to rats topically applied with conventional retinal which showed clear skin irritation and inflammation, daily topical application of proretinal nanoparticles to rats for 28 consecutive days produced neither irritation nor inflammation but significantly increased epidermal proliferation, epidermal thickness, cellular retinoic acid binding protein- 2 expression, and up-regulation of various differentiation markers including keratin 5, keratin 10, keratin 14, cellular retinoic acid binding protein-2, and proliferating cell nuclear antigen. Through the use of confocal laser scanning microscopy, we observed the in vivo follicular penetration of proretinal nanoparticles with the depth of penetration independent of postapplication time. Proretinal nanoparticles provide better biological activities of retinoids on epidermis and could eliminate the side effect of retinoid dermatitis.