Expression Of Cellular Retinol-binding Protein Research Articles

Massive bowel resection modulates the expression of genes involved in lipid and cholesterol metabolism in rats.

We have previously shown that vitamin A-absorptive function was enhanced in bowel-resected rats via increased expression of cellular retinol-binding protein II (CRBP II). Recently, CRBP II was shown to bind not only to retinol but also to monoacylglycerols to modulate gut endocrine signaling. We hypothesized that the increased CRBP II in bowel-resected rats had broader effects than vitamin A metabolism. Acetyl-CoA carboxylase 1 (fatty-acid biosynthesis) and sterol O-acyltransferase 1 (cholesterol esterification) expressions were down-regulated in the bowel-resected rats. Adjustment of nutritional absorption may take place in a limited area of the small intestine by the modulation of gene expression.

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.

Relative mRNA expression of CRBP IV gene and its association with layer economic traits in Rhode Island Red chicken

Cellular retinol-binding protein (CRBP) IV gene plays an important role in absorption, transport, metabolism and homeostasis of retinol (Vitamin A) and its derivatives. Vitamin A plays vital role in ovarian follicular growth, oviduct and uterine environments, embryo and its development, and oocyte maturation. Present investigation was carried out in Rhode Island Red (RIR) chicken to assess relative mRNA expression of CRBP IV gene in kidney, liver and oviduct tissues, collected from 12 birds at 40 weeks of age from four different egg production-body weight groups, viz. high egg production-high body weight (HEP-HBW), high egg production-low body weight (HEP-LBW), low egg production-high body weight (LEP-HBW) and low egg production-low body weight (LEPLBW), using qRT-PCR method. Data on body weight at 20 weeks of age (BW20) and layer traits, viz. age at sexual maturity (ASM), egg weight at 28 (EW28) and 40 (EW40) weeks and egg production up to 40 weeks of age (EP40) were analyzed by LS ANOVA taking sire as random and hatch as fixed effects. LS means of ASM, BW20, EW28, EW40 and EP40 were 135.19±1.15 days, 1347.13±15.28 g, 42.49±0.27 g, 48.19±0.42 g and 124.55±1.94 eggs, respectively. Expression data was analyzed by LS ANOVA using SAS. The basal mRNA expression of CRBP IV gene differed significantly among different egg production-body weight groups in kidney only. The 40-ΔCt values of mRNA expression levels of CRBP IV gene in the four groups were 35.81±1.60, 29.49±1.60, 32.16±1.60 and 39.02±1.60, respectively. Significantly lower expression was observed in HEP-LBW group. Similarly, tissues differed significantly for the CRBP IV gene expression. The 40-ΔCt values of mRNA expression levels of CRBP IV gene in kidney, liver and oviduct were 34.12±0.87, 31.67±0.87 and 37.97±0.87, respectively. Highest expression was observed in oviduct. The study generated significant information on basal mRNA expression of CRBP IV gene in poultry which may have great potential for use in selection for higher reproductive efficiencies.

Inhibitory effect of chitooligosaccharides on retinol metabolism and bioavailability in mice.

This study investigated the intervention effects of chitooligosaccharides (COS) on retinol metabolism and included comparisons of the retinol level, retinol binding protein 4 (RBP4) content, key genes, and protein expression between mice on a COS-enriched diet and a normal diet. The results showed that COS markedly decreased the retinol and RBP4 concentrations in the serum and liver. Furthermore, COS suppressed the mRNA and protein expression of RBP4, cellular retinol binding protein 1 (CRBP1), lecithin: retinol acyltransferase (LRAT) and cytochrome P45026A1 (CYP26A1). In addition, COS inhibited the mRNA expression of stimulated by retinoic acid 6 (STRA6). However, the protein expression of STRA6 was not significantly decreased. Thus, COS reduced the retinol concentration in the serum and disrupted the metabolism of retinol. The intervention mechanism of COS on retinol metabolism may be attributed to the modulation of RBP4, CRBP1, LRAT, STRA6, and CYP26A1 expression at the mRNA and protein levels. PRACTICAL APPLICATIONS: Chitooligosaccharides (COS), known to be the degradation products of chitosan, have been found to induce pinkeye in industrial workers who participate in the manufacturing of COS. Meanwhile, 5% population with COS dietary supplement also have similar phenomenon. The aim of this study is to explore the possible mechanism underlay of this potential risk. The results of this study showed that high exposure to COS during manufacture influences retinol metabolism and leads to a decrease in retinol content, ultimately causing pinkeye. These findings provide new evidence for understanding COS-induced retinol metabolism alteration and drawing attention toward the prevention of potential risk in high-exposure populations.

Relative mRNA expression of cellular retinol-binding protein IV gene in various tissues of Rhode Island Red chicken

Cellular retinol-binding protein (CRBP) IV plays an important role in absorption, transport, homeostasis and excretion of retinol (vitamin A) and its active derivative for embryo development. In oviparous vertebrates, vitamin A plays vital role in reproductive performance. Variation in CRBP-IV expression may have varied functional roles. Present investigation was carried out to assess relative mRNA expression of CRBP IV gene in kidney, liver and oviduct tissues of Rhode Island Red (RIR) chicken, collected from 12 birds, randomly chosen at 40 weeks of age, from four different egg production-body weight groups, viz., high egg production-high body weight (HEP-HBW), high egg production-low body weight (HEP-LBW), low egg production-high body weight (LEP-HBW) and low egg production-low body weight (LEP-LBW). CRBP-IV gene expression and its relative expression was quantified by qRT-PCR and by 2(—HCt) method, respectively. In kidney, expression of CRBP IV gene was 2029.16 folds higher in LEP-LBW, followed by HEP-HBW (79.83 folds), and LEP-HBW (6.38 folds) groups against the HEP-LBW. In liver, CRBP IV gene was expressed 10.85 folds more in HEP-LBW group, followed by 9.22 and 2.08 folds in HEP-HBW and LEP-HBW groups against LEP-LBW. In Oviduct, the CRBP IV gene was expressed 29.28 folds more in HEP-LBW group, followed by 12.39 and 4.19 folds more in HEP-HBW and LEP-HBW groups against LEP-LBW. The study generated significant information about fold changes in basal mRNA expression of CRBP IV gene in various tissues which may be useful in selection for higher productivity of chicken.

Cellular retinol-binding protein-1 expression increases with increasing clinical severity of alopecia areata.

Alopecia areata (AA) is multifactorial disease mostly autoimmune affecting anagen hair follicles. Many researchers hypothesize that adequate retinoic acid (RA) levels are important for proper hair follicle behavior. Previous animal studies revealed increase in RA synthesis proteins and decrease in RA degradation proteins in AA patients when compared with controls. To evaluate cellular retinol-binding protein-1 expression in lesional skin of alopecia areata in comparison with controls, in an attempt to know its role in the pathogenesis of alopecia areata . Immunohistochemical expression of cellular retinol-binding protein-1 CRBP1 was evaluated in skin biopsies taken from lesions of alopecia areata in 30 patients and 10 normal biopsy specimens taken from skin of healthy controls (HC) who were within the same age and sex. CRBP1 expression was significantly increased in lesional alopecia areata skin in comparison with normal skin of controls (P<0.001*). Significant positive correlation was found between expression of CRBP-1 and percentage of hair loss in the scalp (SALT score; r=0.840, P=<0.001). These results may enhance the idea of the possible role of CRBP1 in the pathogenesis of AA, and ensuring the importance of its level in AA treatment.

Expression and potential role of cellular retinol binding protein I in psoriasis.

Psoriasis is a diffuse chronic skin disorder characterized from accelerated epidermal turnover and inflammatory cell infiltrate. Retinoids influence keratinocyte proliferation and differentiation as well as inflammatory response. Cellular retinol binding protein (CRBPI) regulates intracellular vitamin A bioavailability and contributes to maintain skin homeostasis. The aim of present study was to investigate the expression of CRBPI and its role in the pathogenesis of skin psoriasis. Immunohistochemistry revealed more diffuse and increased CRBPI expression in all epidermal layers of human psoriatic lesions except in the stratum corneum. An imiquimod-induced psoriatic-like model documented the increase of skin lesional area and severity index score as well as of the severity of microscopic features as parakeratosis, papillomatosis and spongiosis in CRBPI-knockout compared to wild-type mice, associated to the increased keratinocyte CK17 and Ki-67 expression and the reduction of CK1, CRABPII and RXRα. Gene array of imiquimod-induced psoriatic skin documented the greater up-regulation of EGF/PDGF-related genes and down-regulation of EGR1 and pro-inflammatory IL-related genes in CRBPI-knockout compared to wild-type mice. Finally, CRBPI transfection in HaCaT cells increased AKT and NF-κB-related genes and proteins and down-regulated IL-2, IL-6 and IL-8 pro-inflammatory signalling. Although not recognized as a psoriatic susceptibility gene in our cohort of patients, the present data strongly supported the potential role of CRBPI to sustain keratinocyte proliferation and differentiation and to counteract pro-inflammatory genes expression in psoriatic lesions.

CRBP-1 over-expression is associated with poor prognosis in tongue squamous cell carcinoma

BackgroundTongue squamous cell carcinoma (TSCC) is one of the most common malignancies of oral squamous cell carcinomas. Cellular retinol binding protein-1 (CRBP-1) as a carrier protein transports retinol from the liver storage site to peripheral tissue. Up-regulated expression of CRBP-1 is associated with some tumor types such as prostate cancer, breast cancer and ovarian cancer as reported, but its role in TSCC remains uncertain.MethodsIn this study, an integrated bioinformatics analysis based on the multiple cancer microarray data sets available from Oncomine database was conducted to view the differential expression of CRBP-1 between TSCC and the adjacent non-tumorous tissues. Quantitative real-time polymerase chain reaction (qRT-PCR), western blotting (WB) and immunohistochemical (IHC) assays were performed to investigate CRBP-1 expression in 101 paraffin-embeded TSCC tissues and 48 pairs of freshly frozen tissues. Kaplan-Meier curve and univariate and multivariate Cox-regression analysis were used to estimate the association between CRBP-1 expression and patients’ prognosis. Then western blotting, MTT, transwell migration and invasion assays were performed in TSCC cell lines to investigate the effects of CRBP-1 on cellular proliferation and invasion.ResultsCompared with the matched adjacent non-tumorous tissues, the expression of CRBP-1 was significantly up-regulated in TSCC tissues, which correlated with the differentiation state (P = 0.003), N classification (P = 0.048), the clinical stage (P = 0.048) and death (P = 0.001). The Kaplan-Meier curve showed that TSCC patients with higher CRBP-1 expression levels had lower overall survival rates than those with lower CRBP-1 expression levels. A univariate and multivariate analysis demonstrated that CRBP-1 was an independent prognostic factor (P < 0.05). Furthermore, we knocked down CRBP-1 expression and observed that TSCC cell proliferation and invasion in vitro were significantly blocked, as determined by MTT and transwell assays.ConclusionsUp-regulated expression of CRBP-1 is associated with poor prognosis in TSCC, so it might potentially serve as an additional prognostic marker, and the inhibition of CRBP-1 might provide new therapeutic approaches for TSCC.

Comprehensive molecular exploration identified promoter DNA methylation of the CRBP1 gene as a determinant of radiation sensitivity in rectal cancer

Background:Neoadjuvant chemoradiotherapy (NCRT) for advanced rectal cancer (RC) is a well-evidenced therapy; however, some RC patients have no therapeutic response. Patient selection for NCRT so that non-responsive patients are excluded has been subjective. To date, no molecular markers indicating radiation sensitivity have been reported.Methods:We irradiated six colorectal cancer (CRC) cell lines and identified HCT116 cells as radiation-sensitive and HCT15 and DLD-1 cells as radiation resistant. Using a microarray, we selected candidate radiation sensitivity marker genes by choosing genes whose expression was consistent with a radiation-resistant or sensitive cell phenotype.Results:Among candidate genes, cellular retinol binding protein 1 (CRBP1) was of particular interest because it was not only induced in HCT116 cells by tentative 10 Gy radiation treatments, but also its expression was increased in HCT116-derived radiation-resistant cells vs parental cells. Forced expression of CRBP1 decreased the viability of both HCT15 and DLD-1 cells in response to radiation therapy. We also confirmed that CRBP1 was epigenetically silenced by hypermethylation of its promoter DNA, and that the quantitative methylation value of CRBP1 significantly correlated with histological response in RC patients with NCRT (P=0.031).Conclusions:Our study identified CRBP1 as a radiation-sensitive predictor in RC.

High expression of cellular retinol binding protein-1 in lung adenocarcinoma is associated with poor prognosis.

Adenocarcinoma, the most common non-small cell lung cancer is a leading cause of death worldwide, with a low overall survival (OS) despite increasing attempts to achieve an early diagnosis and accomplish surgical and multimodality treatment strategies. Cellular retinol binding protein-1 (CRBP-1) regulates retinol bioavailability and cell differentiation, but its role in lung cancerogenesis remains uncertain. CRBP-1 expression, clinical outcome and other prognostic factors were investigated in 167 lung adenocarcinoma patients. CRBP-1 expression was evaluated by immunohistochemistry of tissue microarray sections, gene copy number analysis and tumor methylation specific PCR. Effects of CRBP-1 expression on proliferation/apoptosis gene array, protein and transcripts were investigated in transfected A549 lung adenocarcinoma cells. CRBP-1(High) expression was observed in 62.3% of adenocarcinomas and correlated with increased tumor grade and reduced OS as an independent prognostic factor. CRBP-1 gene copy gain also associated with tumor CRBP-1(High) status and dedifferentiation. CRBP-1-transfected (CRBP-1(+)) A549 grew more than CRBP-1(-) A549 cells. At >1μM concentrations, all trans-retinoic acid and retinol reduced viability more in CRBP-1(+) than in CRBP-1(-) A549 cells. CRBP-1(+) A549 cells showed up-regulated RARα/ RXRα and proliferative and transcriptional genes including pAkt, pEGFR, pErk1/2, creb1 and c-jun, whereas RARβ and p53 were strongly down-regulated; pAkt/pErk/ pEGFR inhibitors counteracted proliferative advantage and increased RARα/RXRα, c-jun and CD44 expression in CRBP-1(+) A549 cells. CRBP-1(High) expression in lung adenocarcinoma correlated with increased tumor grade and reduced OS, likely through increased Akt/Erk/EGFR-mediated cell proliferation and differentiation. CRBP-1(High) expression can be considered an additional marker of poor prognosis in lung adenocarcinoma patients.

Mechanical force and tensile strain activated hepatic stellate cells and inhibited retinol metabolism.

Hepatic stellate cells (HSCs), the principal producers of extracellular matrix proteins, play a major role in the development of liver fibrosis which is accompanied with elevated sinusoidal pressure and portal hypertension. We have isolated primary rat HSCs and investigated the effect of mechanical pressure and tensile strain on retinol metabolism in the cells. Mechanical force and tensile strain significantly increased the expression of α-smooth muscle actin (α-SMA) and collagen I, and notably inhibited the expression of cellular retinol-binding protein I (CRBP-I), lecithin-retinol acyltransferase (LRAT), retinyl ester hydrolase (REH), retinoic acid receptor-β (RAR-β) and retinoid X receptor-α (RXR-α). Such effects were partially reversed by the retinoid X receptor antagonist, HX531, and the retinoic acid receptor antagonist, LE135. Mechanical force and tensile strain significantly activate HSCs by regulating the retinoid metabolic pathway. Activation of HSCs can therefore be manipulated through mechanical force and tensile strain in vitro.

The role of cellular retinol-binding protein-1 on pulmonary structural remodeling after rat myocardial infarction

To observe the expression of cellular retinol-binding protein-1 (CRBP-1) in pulmonary tissues after rat myocardial infarction (MI) and uncover the role of CRBP-1 on the pulmonary structural remodeling. MI was produced in male Wistar rats by left coronary ligation. Rats were sacrificed to obtain the lung at the 3(rd), 6(th), 15(th), 30(th), and 45(th) day after operation. After weighted, the rat lungs were fixed in 4% formalin and embedded in paraffin. Sections were cut and stained with hematoxylin and eosin (HE), Masson's trichrome (MT), rabbit anti-CRBP-1 antibody. Thirty-nine Wistar rats survived and developed MI. Pulmonary tissue sections with HE and MT staining showed a remarkable lung structural remodeling. The content of pulmonary tissue collagen at the 30(rd) was higher than in the sham group (8.4% ± 3.6 vs 4.5% ± 2.6, P < 0.001). CRBP-1 expression was detected on the alveolar septa at the 3(rd) day after operation, and peaked at the 15(th) day (43.8 ± 7.4). Then the CRBP-1 expression decreased, and arrived at the level of the 3(rd) day. We demonstrate that CRBP-1 is expressed temporal on the alveolar septa after rat MI. It indicates a potential relationship between CRBP-1 and lung structure remodeling process after MI.

Correlation between expression of cellular retinol-binding protein 1 and its methylation status in larynx cancer

AimsThe authors have previously reported that cellular retinol-binding protein 1 (CRBP1) gene gain and its expression correlated significantly with survival in laryngeal carcinoma patients. The authors hypothesised that inactivation of...

Increased expression of cellular retinol-binding protein 1 in laryngeal squamous cell carcinoma

To investigate the genomic alterations in larynx carcinomas (LaCa) tissues and its prognostics values in predicting survival. To analyse the aberrations in the genome of LaCa patients, we used array comparative genomic hybridization in 19 human laryngeal tumour samples. DNA samples were also subjected to detect human papillomavirus (HPV) sequences by polymerase chain reaction (PCR). Copy number gain was confirmed by real-time PCR. The cellular retinol-binding protein 1 (CRBP-1) gene expression was also confirmed by immunohistochemistry assay on LaCa tissues. To identify prognostic feature, CRBP-1 gene gain was correlated to patient survival. The most common gains were detected for CRBP-1 and EGFR genes, while DNA lost in RAF-1 gene. Immunohistochemistry assay was revealed strong expression of CRBP1 protein in those cases with CRBP-1 gene gain. The CRBP-1 gene gain and its expression correlated significantly with survival (P = 0.003). Cox regression analysis indicated that CRBP-1 expression level was a factor of survival (P = 0.008). HPV sequences were detected in 42% of the samples, and did not show any relationship with specific gene alterations. Our data shows that CRBP-1 gene gain can be determined by immunohistochemistry on routinely processed tissue specimens, and could support as a potential novel marker for long-term survival in laryngeal squamous cell carcinoma.

Chmp 1A is a mediator of the anti-proliferative effects of All-trans Retinoic Acid in human pancreatic cancer cells

BackgroundWe recently have shown that Charged multivesicular protein/Chromatin modifying protein1A (Chmp1A) functions as a tumor suppressor in human pancreatic tumor cells. Pancreatic cancer has the worst prognosis of all cancers with a dismal 5-year survival rate. Preclinical studies using ATRA for treating human pancreatic cancer suggest this compound might be useful for treatment of pancreatic cancer patients. However, the molecular mechanism by which ATRA inhibits growth of pancreatic cancer cells is not clear. The objective of our study was to investigate whether Chmp1A is involved in ATRA-mediated growth inhibition of human pancreatic tumor cells.ResultsWe performed microarray studies using HEK 293T cells and discovered that Chmp1A positively regulated Cellular retinol-binding protein 1 (CRBP-1). CRBP-1 is a key regulator of All-trans retinoic acid (ATRA) through ATRA metabolism and nuclear localization. Since our microarray data indicates a potential involvement of Chmp1A in ATRA signaling, we tested this hypothesis by treating pancreatic tumor cells with ATRA in vitro. In the ATRA-responsive cell lines, ATRA significantly increased the protein expression of Chmp1A, CRBP-1, P53 and phospho-P53 at serine 15 and 37 position. We found that knockdown of Chmp1A via shRNA abolished the ATRA-mediated growth inhibition of PanC-1 cells. Also, Chmp1A silencing diminished the increase of Chmp1A, P53 and phospho-P53 protein expression induced by ATRA. In the ATRA non-responsive cells, ATRA did not have any effect on the protein level of Chmp1A and P53. Chmp1A over-expression, however, induced growth inhibition of ATRA non-responsive cells, which was accompanied by an increase of Chmp1A, P53 and phospho-P53. Interestingly, in ATRA responsive cells Chmp1A is localized to the nucleus, which became robust upon ATRA treatment. In the ATRA-non-responsive cells, Chmp1A was mainly translocated to the plasma membrane upon ATRA treatment.ConclusionCollectively our data provides evidence that Chmp1A mediates the growth inhibitory activity of ATRA in human pancreatic cancer cells via regulation of CRBP-1. Our results also suggest that nuclear localization of Chmp1A is important in mediating ATRA signaling.

Vitamin A metabolism is impaired in human ovarian cancer

Objectives We have previously reported that loss in expression of a protein considered critical for vitamin A homeostasis, cellular retinol-binding protein 1 (CRBP1), is an early event in ovarian carcinogenesis. The aim of the present study was to determine if loss of vitamin A metabolism also occurs early in ovarian oncogenesis. Methods We assessed CRBP1 expression by immunohistochemistry in ovaries prophylactically removed from women with a genetic risk for ovarian cancer. Furthermore, we investigated the ability of normal, immortalized but nontumorigenic, and tumorigenic human ovarian epithelial cells to synthesize retinoic acid and retinaldehyde when challenged with a physiological dose of retinol, and determined expression levels of the retinoid-related genes, RARα, RXRα, CRABP1, CRABP2, RALDH1 and RALDH2 in these cells. Results Immunohistochemistry revealed loss of CRBP1 expression in potentially preneoplastic lesions in prophylactic oophorectomies. HPLC analysis of vitamin A metabolism showed production of retinoic acid in four independent, normal human ovarian surface epithelial (HOSE) cell cultures upon exposure to retinol. However, only one of two SV40-immortalized HOSE cell lines made RA, while none of the ovarian carcinoma cell lines produced detectable RA due to complete loss of RALDH2. Conclusions The impaired conversion of retinol to RA in ovarian cancer cells and decreased CRBP1 protein expression in prophylactic oophorectomies support our hypothesis that concomitant losses of vitamin A metabolism and CRBP1 expression contribute to ovarian oncogenesis.

Cellular retinol-binding protein-1 is expressed by fibroblasts in rat myocardial infarction

Background: It has been reported that cellular retinol-binding protein-1 (CRBP-1) is transiently expressed in granulation tissue. To understand the mechanism of ventricular remodeling of heart after myocardial infarction (MI), we have examined the expression of CRBP-1 in rat MI model.

Chlormethiazole treatment prevents reduced hepatic vitamin A levels in ethanol-fed rats.

Chronic ethanol intake results in decreased hepatic vitamin A levels through both enhanced degradation of vitamin A via a cytochrome P450 enzyme (CYP)-dependent process and increased mobilization of vitamin A from the liver into the circulation. This study investigated whether treatment with chlormethiazole, a CYP inhibitor, restores vitamin A in the livers of ethanol-fed rats. Ethanol-exposed and non-ethanol-exposed rats were treated with or without chlormethiazole (10 and 100 mg/kg body weight) for 1 month. Liver and plasma levels of retinol and retinyl palmitate were analyzed by high-performance liquid chromatography. Expressions of hepatic lecithin:retinol acyltransferase (LRAT) and cellular retinol-binding protein were analyzed with reverse transcription-polymerase chain reaction. Hepatic retinol esterification by LRAT was examined by using incubations of the microsomal fractions of livers with exogenous sources of retinol. Ethanol-feeding in rats for a month resulted in lower hepatic levels of retinol and retinyl palmitate than those found in controls and the occurrence of several polar retinoid metabolites. In contrast, treatment with chlormethiazole at two different doses in ethanol-fed rats completely blocked the formation of hepatic retinoid polar metabolites and restored hepatic levels of retinol and hepatic retinyl palmitate in a dose-dependent manner. Furthermore, increased plasma concentrations of retinyl palmitate in rats fed with ethanol, which indicate increased mobilization of vitamin A, were partially inhibited by chlormethiazole treatment. However, neither ethanol nor chlormethiazole treatment altered the expression and activity of LRAT in the liver of rats. Hepatic expression of cellular retinol-binding protein increased significantly in ethanol-fed rats with or without chlormethiazole treatment compared with control rats. These data suggest that chlormethiazole can restore both hepatic retinol and retinyl ester concentrations to normal levels in ethanol-fed rats through blocking enhanced both degradation of vitamin A and mobilization of vitamin A from the liver into the circulation.

Cellular retinol binding protein-1 expression in endometrial hyperplasia and carcinoma: diagnostic and possible therapeutic implications

Cellular retinol binding protein-1 (CRBP-1) contributes to the maintenance of the differentiative state of endometrial glandular cells through the regulation of bioavailability of retinol and derivatives, but its role in endometrial oncogenetic process remains unclear. Antibodies to CRBP-1, estrogen and progesterone receptors (ER and PR) were applied to paraffin sections of proliferative (n=10) and secretory endometrium (n=9), and to endometrial polyps (n=6), simple (n=7), complex (n=3) and atypical endometrial hyperplasias (n=9) as well as to 47 endometrioid carcinomas of different histological grade (G) (G1, n=18; G2, n=19; G3, n=10). Four serous and two clear cell carcinomas were also examined. In glandular cells, CRBP-1 positivity was mainly cytoplasmic and rarely in the nuclei. CRBP-1 immunodetection was weakly positive in proliferative and low and focal in secretory endometrium and higher in atypical as compared to simple and complex hyperplasias. CRBP-1 expression in G1 endometrioid carcinomas was similar to that in atypical hyperplasias. In the latter, the highest CRBP-1 expression was observed in areas of squamous differentiation. Semiquantitative evaluation revealed a significant decrease of cytoplasmic CRBP-1 immunoreactivity with the increase of tumor grade. Among G3 endometrioid carcinomas, 60% were CRBP-1 negative, whereas the remaining cases showed a very low and focal positivity. Serous carcinomas were also CRBP-1 negative. When areas of different grading were present within the same tumor, less differentiated areas retained a lower CRBP-1 immunoreaction. The progressive decrease of CRBP-1 paralleled that of ER and PR immunodetection. RT-PCR in eight endometrioid carcinomas suggested a decrease of CRBP-1 with the increase of tumor grade also at transcriptional level. Our results indicate that CRBP-1 immunodetection may constitute an additional tool for histological grading of endometrial carcinoma. The CRBP-1 loss during the progression of endometrial cancer suggests a new potential target for pharmacological strategies aimed to counteract its progression by increased intracellular retinol bioavailability.

DNA methylation of genes linked to retinoid signaling in squamous cell carcinoma of the esophagus: DNA methylation of CRBP1 and TIG1 is associated with tumor stage

Hypermethylation of CpG islands is associated with the silencing of various tumor suppressor genes. Retinoic acid receptor-beta (RAR-beta), cellular retinol-binding protein 1 (CRBP1), and tazarotene-induced gene 1 (TIG1) have been linked to retinoic acid signaling. Little is known about the involvement of these three genes in esophageal squamous cell carcinoma (ESCC). In this study, we investigated the methylation status of these genes and analyzed the role of methylation of their DNA in ESCC. Methylation-specific polymerase chain reaction (PCR) was performed to study the methylation of CpG islands in 28 ESCC (stages I, II, and III) and 10 samples of corresponding non-neoplastic mucosa. The mRNA expression levels of the three genes were measured by quantitative reverse transcription-PCR. DNA hypermethylation of RAR-beta was found in seven (25.0%) of the 28 ESCC, of CRBP1 in five (17.9%), and of TIG1 in five (17.9%). DNA methylation of RAR-beta was identified in one of 10 samples of corresponding non-neoplastic mucosa (10.0%), whereas no DNA methylation of CRBP1 or TIG1 was detected. In total, at least one of the three genes was hypermethylated in 12 (42.9%) ESCC. Reduced expression of RAR-beta, CRBP1, and TIG1 was found in 14 (50.0%), 15 (53.6%), and 13 (46.4%) ESCC, respectively. DNA methylation of each gene was significantly associated with reduced expression of the respective mRNA. No correlation was found between the DNA methylation status of RAR-beta and clinicopathological factors such as depth of invasion, lymph node metastasis, or tumor stage. In contrast, DNA methylation of both CRBP1 and TIG1 was observed only in stage III ESCC. These results show that inactivation of the retinoic acid signaling-associated genes RAR-beta, CRBP1, and TIG1 by DNA methylation occurs frequently in ESCC.