Expression Of Cellular Retinol-binding Protein Research Articles

Expression of cellular retinol-binding protein-1 during myofibroblastic differentiation in hepatic stellate cells and portal fibroblasts: Kozue Uchio, GREF, INSERM E9917, Universite Bordeaux 2, Bordeaux France; Beatriz Tuchweber, Department of Nutrition, University of Montreal, Montreal Canada; Noboru Manabe, Department of Animal Sciences, University of Kyoto, Kyoto Japan; Giulio Gabbiani, Department of Pathology, Centre Medical Universitaire, Geneva Switzerland; Jean Rosenbaum, Alexis Desmouliere,

Expression of cellular retinol-binding protein-1 during myofibroblastic differentiation in hepatic stellate cells and portal fibroblasts: Kozue Uchio, GREF, INSERM E9917, Universite Bordeaux 2, Bordeaux France; Beatriz Tuchweber, Department of Nutrition, University of Montreal, Montreal Canada; Noboru Manabe, Department of Animal Sciences, University of Kyoto, Kyoto Japan; Giulio Gabbiani, Department of Pathology, Centre Medical Universitaire, Geneva Switzerland; Jean Rosenbaum, Alexis Desmouliere,

Cellular retinol‐binding protein‐1 expression in endometrial stromal cells: physiopathological and diagnostic implications

Cellular retinol-binding protein-1 (CRBP-1) contributes to the maintenance of the differentiated state of the endometrium through retinol bioavailability regulation. The aim was to analyse CRBP-1 expression in endometrial stromal cells at eutopic and ectopic sites in different physiopathological conditions. Antibodies to CRBP-1, CD10 and alpha-smooth muscle actin were applied to proliferative (n = 10), secretory (n = 9) and atrophic (n = 7) endometrium, decidua (n = 4), adenomyosis (n = 5), endometriosis (n = 10), endometrial polyps (n = 9), simple endometrial hyperplasia (n = 6), well-differentiated endometrioid carcinoma (n = 6) and submucosal leiomyomas (n = 5). In some cases, Western blotting and reverse transcription-polymerase chain reaction were also applied. CRBP-1 was expressed by eutopic and ectopic endometrial stromal cells more markedly during the late secretory phase and in decidua of pregnancy. CRBP-1 expression was low in the stroma of atrophic endometrium and absent in myometrium, leiomyomas and cervical stroma. CD10 immunoreactivity was weak in atrophic endometrium and in decidua. CRBP-1 expression characterizes endometrial stromal cells at eutopic and ectopic sites and appears to be more specific than CD10. The level of CRBP-1 varies in intensity according to hormonal variations, reaching its maximum in predecidua and decidua. Thus, immunodetection of CRBP-1 may help to elucidate the physiopathological changes which occur in endometrial stroma and can also be applied as an adjuvant stromal marker.

Aberrant cellular retinol binding protein 1 (CRBP1) gene expression and promoter methylation in prostate cancer

Aims: Retinoids are involved in cell growth, differentiation, and carcinogenesis. Their effects depend on cytosolic transport and binding to nuclear receptors. CRBP1 encodes a protein involved in this process. Because...

Cellular retinol-binding protein-1 expression in normal and fibrotic/cirrhotic human liver: different patterns of expression in hepatic stellate cells and (myo)fibroblast subpopulations

Background/Aims Cellular retinol-binding protein-1 (CRBP-1) which is involved in vitamin A metabolism is highly expressed in liver cells, particularly in hepatic stellate cells (HSCs). In this work, the CRBP-1 expression was studied by immunohistochemistry in the different liver cell populations, including HSCs and portal fibroblasts, of normal liver and of fibrotic and cirrhotic liver. Methods Normal liver, fibrotic liver in different stages and cirrhotic liver sections were studied. Immunohistochemistry was performed using antibodies against CRBP-1, α-smooth muscle actin (SMA), CD 68 and CD 34. Results In normal liver, quiescent HSCs expressed CRBP-1, while portal fibroblasts did not. In fibrotic or cirrhotic liver, activated HSCs co-expressed CRBP-1 and α-SMA; a variable proportion of portal and septal (myo)fibroblasts, more important in cirrhosis, neo-expressed both CRBP-1 and α-SMA. Biliary epithelial cells both in normal and pathological situations expressed CRBP-1. Neither Kupffer cells, nor endothelial cells showed CRBP-1 expression. Conclusions Our study demonstrates that CRBP-1 is a good marker to identify HSC in normal human liver. Furthermore, in fibrotic or cirrhotic liver, the different patterns of expression for CRBP-1 and α-SMA allow the distinction of different subsets of fibroblastic cells involved in fibrogenesis and septa formation.

Cellular retinol-binding protein-1 in hepatocellular carcinoma correlates with β-catenin, Ki-67 index, and patient survival

The cellular retinol-binding protein-1 (CRBP-1) plays a key role in the esterification and intercellular transfer of retinol. By in situ hybridization, immunohistochemistry, and confocal laser scanning microscopy (CLSM), we show that, in normal liver, CRBP-1 is strongly expressed in the cytoplasm of hepatic stellate cells (HSCs) and myofibroblasts (MFs) with only low CRBP-1 levels in hepatocytes. By contrast, in 196 hepatocellular carcinoma (HCC) specimens CRBP-1 expression in MFs was down-regulated in 83%. Patients with high CRBP-1 expression in MFs had a significantly higher 2-year survival as compared with patients with low CRBP-1 expression (52% vs. 29%, respectively; P = .034). An aberrant nuclear CRBP-1 accumulation resulting from cytoplasmic invagination was found in 29% of HCCs. Nuclear CRBP-1 staining correlated positively with a favorable tumor stage (Okuda stage I; P = .01) and negatively with the Ki-67 + proliferation fraction (PF). A Ki-67 + PF of ≥10% was associated with a lower 2-year survival probability as compared with patients with a Ki-67 + PF of <10% (12% vs. 40%, respectively; P = .015). Prognosis did not correlate with the nuclear β-catenin expression. There was, however, a close correlation between nuclear CRBP-1 inclusions and nuclear β-catenin staining in HCCs ( P = .008), suggesting a cross talk between CRBP-1 and the Wnt/wingless signal transduction pathway. In conclusion, our findings demonstrate that CRBP-1 detection may be useful for the discrimination between nonneoplastic and neoplastic liver cells and suggest that modulation of CRBP-1 expression in HCCs contributes to tumor growth and progression via retinoid-mediated signaling and disruption of cellular vitamin A homeostasis. (H epatology 2003;38:470-480.)

Expression of retinol-binding protein and cellular retinol-binding protein in the bovine ovary.

Retinol (vitamin A) is essential for reproduction, and retinoids have been suggested to play a role in ovarian steroidogenesis, oocyte maturation, and early embryonic development. Retinol is transported systemically and intercellularly by retinol-binding protein (RBP). Within the cell, cellular retinol-binding protein (CRBP) functions in retinol accumulation and metabolism. Since the actions of retinoids are mediated, in part, by retinoid-binding proteins, the objective of this study was to investigate cell-specific expression of RBP and CRBP in the bovine ovary. Immunocytochemical analysis (ICC) localized RBP to the thecal and granulosa cell layers of antral and preantral follicles with the most intense staining in the cells of large, healthy follicles. The tunica adventitia of arterial blood vessels also exhibited RBP staining. Immunostaining of CRBP was most intense in the granulosa cells of preantral follicles and present, but diminished, in thecal and granulosa cells of antral follicles. Within the corpus luteum, both proteins were observed in large luteal cells, but only RBP was observed in small luteal cells. Northern blot analysis demonstrated that thecal and granulosa cells from antral follicles and luteal tissue expressed RBP and CRBP mRNA. Synthesis and secretion of RBP by thecal cells, granulosa cells, and luteal cells were demonstrated by immune-complex precipitation of radiolabeled RBP from the medium of cultured cells or explants, followed by SDS-PAGE and fluorography. Follicular fluid was collected from small (<5 mm) and large (8-14 mm) follicles, pooled according to follicular size, and analyzed for retinol, RBP, estradiol-17beta, and progesterone. Concentrations of retinol, RBP, and estradiol were greater in the fluid of large follicles. Results demonstrate retinoid-binding protein expression by bovine ovaries and provide physical evidence that supports the concept that retinoids play a role in ovarian function.

Cellular Retinol-Binding Protein-1 Expression and Modulation during In Vivo and In Vitro Myofibroblastic Differentiation of Rat Hepatic Stellate Cells and Portal Fibroblasts

Cellular retinol-binding protein-1 (CRBP-1) is involved in vitamin A metabolism because it mediates both retinol esterification to retinyl esters and retinol oxidation to retinal and retinoic acid. CRBP-1 is highly expressed in the liver, particularly in hepatic stellate cells (HSC). In this study, we investigated the liver expression of CRBP-1 during experimental fibrogenesis. We also studied the regulation of CRBP-1 expression in cultured HSC and portal fibroblasts, two fibroblastic cell types involved in liver fibrogenesis. Fibrosis was induced in rats by carbon tetrachloride (CCl4) or bile duct ligation. Immunohistochemical staining was performed for CRBP-1 and α-smooth muscle (SM) actin, an activation marker of fibrogenic cells. CRBP-1 and α-SM actin expression was studied by Western blotting and/or Northern blot in primary cultures of HSC isolated by conventional methods and in portal fibroblasts that were obtained by outgrowth from the biliary tree after enzymatic digestion. In normal liver, contrary to HSC, portal fibroblasts did not express CRBP-1. After CCl4 injury, CRBP-1 expression was maintained in myofibroblastic α-SM actin-positive HSC. After bile duct ligation, portal fibroblasts (which proliferated around ductular structures) acquired expression of both CRBP-1 and α-SM actin. During HSC activation in culture, CRBP-1 expression gradually increased until Day 5 when α-SM actin expression was obvious. Cultured portal fibroblasts developed both CRBP-1 and α-SM actin expression. In both cell populations, transforming growth factor-β1 treatment increased CRBP-1 expression. Thus, in normal liver, CRBP-1 expression was different among fibroblastic cells, a finding that adds to the concept of heterogeneity of liver fibrogenic cells. Furthermore, during myofibroblastic differentiation, HSC that lost their stores of retinol maintained a high level of CRBP-1 expression, whereas portal fibroblasts acquired CRBP1 expression. Together, these data suggest a correlation between CRBP-1 expression and myofibroblastic differentiation.

Topical retinoic acid alters the expression of cellular retinoic acid-binding protein-I and cellular retinoic acid-binding protein-II in non-lesional but not lesional psoriatic skin.

Therapeutic retinoids have profound effects on psoriatic skin pathology but their interactions with various retinoid-binding proteins in lesional vs non-lesional skin have not been investigated. Using quantitative real-time PCR the mRNA expression of cellular retinol-binding protein I (CRBPI) and retinoic acid-binding protein I/II (CRABPI/CRABPII) was studied in psoriatic and healthy control (=normal) skin after 4 days of occlusive RA/vehicle treatment (n=6). Untreated psoriatic lesions showed a markedly elevated CRABPII/CRABPI ratio, while the CRBPI level was reduced in lesional and non-lesional skin as compared to normal skin. In RA-treated normal and non-lesional skin, the mRNA expression of CRBPI was unaltered while that of CRABPI and CRABPII was reduced by approximately 80% and increased approximately 5-fold, respectively, as compared to vehicle-treated skin. In contrast, lesional skin exposed to RA showed an almost 90% increase in CRBPI transcripts but unaltered expression of CRABPI and CRABPII, yet, the mRNA expression of several inflammatory mediators, e.g. inducible nitric oxide synthase, interferon-gamma and interleukin-1beta, was clearly reduced. Immunohistochemistry localized CRABPII to suprabasal keratinocytes in normal skin and revealed markedly elevated levels in lesional skin. RA treatment induced CRABPII protein expression in normal and non-lesional skin, to similar levels as in untreated lesions. The results indicate that the effects of RA differ in normal/non-lesional psoriatic skin and lesional skin. Whether the high expression of CRABPII in psoriatic skin lesions is due to increased amounts of endogenous retinoids in lesional skin or reflects an abnormal regulation of the CRABPII gene in psoriasis remains to be studied.

High levels of cellular retinol binding protein-1 expression in leiomyosarcoma: possible implications for diagnostic evaluation

Retinoid bioavailability is regulated by the activity of specific cytoplasmic receptors. High levels of cellular retinol binding protein-1 (CRBP-1) have been documented during experimental arterial and wound-healing processes, but data concerning neoplastic smooth muscle tissues are scarce and/or controversial. This study reports that the expression of CRBP-1 is markedly higher in uterine and gastrointestinal leiomyosarcomas than in leiomyomas and normal myometrium; CRBP-1 was practically absent in normal gastrointestinal smooth muscle tissue. CRBP-1 positivity was particularly elevated in the epithelioid variant of leiomyosarcoma; it was associated with increased proliferative and apoptotic rates and inversely related to smooth muscle differentiation evaluated by alpha- and gamma-smooth muscle actin and desmin expression. Western blotting and reverse-transcription polymerase chain reaction confirmed the observations concerning CRBP-1 and actin isoform expression and revealed higher NF-kappa-Bp65 and RAR alpha and lower Bax protein levels in leiomyosarcoma than in the other conditions. These findings document that a high CRBP-1 expression is associated with smooth muscle malignancy and suggest that CRBP-1 expression represents a new useful marker for the classification of unusual variants of smooth muscle tumors.

Long-Term Marginal Intakes of Zinc and Retinol Affect Retinol Homeostasis without Compromising Circulating Levels during Lactation in Rats

Marginal zinc or vitamin A intake is more common than previously thought in industrialized and developing countries, with pregnant and lactating women believed to be particularly at risk. However, the lack of sensitive indicators of zinc and vitamin A status precludes accurate assessment of marginal nutriture. Concurrent deficiencies in zinc and vitamin A intake often coexist, and the interaction between zinc deficiency and vitamin A metabolism may confound results from epidemiologic or intervention studies. To investigate effects of a maternal diet chronically restricted in zinc or vitamin A intake on indices of vitamin A metabolism, we fed rats a control diet (C) or a diet marginal in zinc (ZD), marginal in vitamin A (AD), marginal in both (DD) or pair-fed to DD (PF), preconception through lactation. Plasma retinol (ROH) was greater and retinol binding protein (RBP) was lower in rats fed ZD, AD and DD compared with those fed C. Hepatic cellular retinol binding protein (CRBP) expression was greater than controls in rats fed ZD and AD and lower in those fed DD, whereas RBP expression was greater in the DD- and PF-fed groups compared with rats C. Mammary gland CRBP and RBP expression were not affected by the diets. Milk ROH was lower in rats fed AD, and milk RBP was lower in those fed ZD and DD compared with rats fed C. In summary, chronic, marginal intake of zinc or vitamin A resulted in alterations in tissue retinol metabolism and milk retinol levels without decreasing plasma zinc, retinol or ROH:RBP during lactation. These observations are of concern because these parameters, which are commonly used to assess zinc and vitamin A status, may lead to misassessment of marginal zinc or vitamin A nutriture in some human populations.

CRBP suppresses breast cancer cell survival and anchorage-independent growth.

We showed earlier that cellular retinol-binding protein (CRBP) expression is downregulated in a subset of human breast cancers. We have now investigated the outcome of ectopic CRBP expression in MTSV1-7 cells, a SV40 T antigen-transformed human breast epithelial cell line devoid of endogenous CRBP expression. We found that: (i) CRBP did not inhibit adherent cell growth but suppressed foci formation in post-confluent cultures and colony formation in soft agar; (ii) this effect was due to CRBP inhibition of cell survival, as demonstrated by viability and TUNEL assays of cells in soft-agar or plated on polyHEMA-coated dishes; (iii) CRBP inhibited protein kinase B/Akt activation in cells in suspension but not in adherent cells and the CRBP suppression of anchorage-independent growth was mimicked by cell treatment with the phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002; (iv) CRBP enhanced retinyl ester formation and storage but did not regulate retinoic acid synthesis or retinoic acid receptor activity. Ectopic CRBP-mediated inhibition of anchorage-independent cell survival and colony formation in the absence of significantly altered responses to either retinol or retinoic acid was also documented in T47D human breast cancer cells. In conclusion, the data suggest two novel and linked CRBP functions in mammary epithelial cells: inhibition of the PI3K/Akt survival pathway and suppression of anchorage-independent growth.

Prenatal Ethanol Consumption Increases Retinol and Cellular Retinol-Binding Protein Expression in the Rat Fetal Snout

Fetal exposure to ethanol disrupts normal craniofacial development, resulting in characteristic features of fetal alcohol syndrome (FAS). One mechanism that could result in some anomalies of this syndrome is through ethanol disrupting the regulatory role played by vitamin A in fetal development, thereby inducing morphological alterations which manifest as FAS. This work begins to explore a possible interaction of ethanol with vitamin A in craniofacial development. Retinoid levels and the expression of cellular retinol-binding protein (CRBP) and retinoic acid receptor (RAR) mRNA were determined in snouts of 20-day fetuses exposed to ethanol throughout gestation, compared to controls. Snout retinol and retinyl palmitate levels were elevated in fetuses of ethanol-treated rats, but retinoic acid levels were unaffected. The expression of CRBP mRNA, as determined by Northern analysis, was greater in snouts of fetuses exposed to ethanol, but there was no change in RAR α, β, γ or retinoid X receptor β mRNA. These results demonstrate that prenatal ethanol consumption can alter certain markers of vitamin A metabolism and function in the fetal snout.

Regulation of α‐smooth muscle actin and CRBP‐1 expression by retinoic acid and TGF‐β in cultured fibroblasts

We have reported that Cellular Retinol Binding Protein-1 (CRBP-1) is expressed de novo during skin wound healing by a proportion of fibroblastic cells which then differentiate into myofibroblasts and express alpha-smooth muscle actin. In fibroblasts cultured from different tissues we have shown that alpha-smooth muscle actin expression, mainly controlled by Transforming Growth Factor-beta (TGF-beta), is also regulated by retinoic acid and that CRBP-1, known to be a retinoic acid-responsive gene, is modulated by TGF-beta. The aim of the present study has been to investigate the relationships between retinoic acid and TGF-beta in regulating the expression of CRBP-1 and alpha-smooth muscle actin in cultured rat subcutaneous tissue fibroblasts. We have observed that the TGF-beta-induced, but not the retinoic acid-induced, alpha-smooth muscle actin expression is associated with a modulation of endogenous TGF-beta and TGF-beta receptors, suggesting that the action of retinoic acid on alpha-smooth muscle actin expression is not mediated by TGF-beta. The expression of CRBP-1 is regulated at the transcriptional level by TGF-beta and retinoic acid but not synergistically, suggesting a possible common pathway. However, retinoic acid, but not TGF-beta, increases the transcription of a transiently transfected chimeric construct containing the retinoic acid response element of the CRBP-1 promoter, indicating that TGF-beta does not influence CRBP-1 through the retinoic acid pathway. Our results indicate that distinct pathways regulate the genes involved in the appearance and evolution of the myofibroblastic cells. The characterization of these pathways will be helpful for the design of drugs influencing wound healing.

Regulation of Hepatic Vitamin A Storage in a Rat Model of Controlled Vitamin A Status during Aging

It is currently unknown whether the capacity of the liver to esterify and store vitamin A (VA) changes as a function of long-term VA intake or age. The objective of this study was to investigate whether age and/or VA status are factors for the hepatic expression of cellular retinol-binding protein (CRBP), the esterification of retinol by lecithin:retinol acyltransferase (LRAT) and the accumulation of VA and lipids in liver. Two factors, VA intake and age, were studied in a 3x3 design. Diets denoted as VA-marginal, control and supplemented contained 0.35, 4 and 25 mg retinol equivalents/kg diet, respectively; male Lewis rats were fed these diets from weaning until the ages of 2-3 mo (young), 8-10 mo (middle-aged) and 18-20 mo (old) (n = 6/group. Liver CRBP mRNA differed (two-way ANOVA) with dietary VA (P<0.0001) and age (P<0.05). Hepatic LRAT activity increased with dietary VA (P<0.0001). Age was not a factor (P = 0.47) although there was an interaction of age and dietary VA (P<0.0001). Hepatic LRAT activity was correlated (r = 0.633, P<0.0001) with plasma retinol at physiologic concentrations. In VA-supplemented rats of all ages, the plasma molar ratio of total retinol:retinol-binding protein (RBP) exceeded 1, and liver VA and total lipid concentrations were elevated. However, tests of liver function had previously been shown to be within normal values. Thus, the capacity of the liver for retinol esterification by LRAT was not diminished by age or the accumulation of VA and other lipids. We conclude the following: 1) hepatic LRAT activity is regulated across a broad, physiologic range of dietary VA; 2) LRAT activity is regulated throughout life; and 3) the capacity for hepatic VA storage is high throughout life.

Evidence for independent control at the mRNA and protein levels of cellular retinol binding protein 1 in rat Sertoli cells.

Cellular retinol binding protein 1 (CRBP1) is the cytosolic carrier for retinol. It is expressed in many tissues, but the concentrations vary considerably. In Sertoli cells from immature rat testis, CRBP1 is highly expressed. The results of the present study show that regulation of CRBP1 expression at the protein level appears to be independent of regulation at the mRNA level. In Sertoli cells from prepubertal 19-day-old rats, CRBP1 mRNA is strongly induced for up to 72 h by the presence of serum factors. In contrast, treatment of the cells with cAMP analogues led to a rapid reduction in mRNA to quantities less than 5% of control values. However, the changes in CRBP1 mRNA did not lead to similar changes in the concentration of CRBP1 protein during 72 h of observation. Similarly, treatment of cells from 32- and 44-day-old rats with serum led to increased CRBP1 mRNA, whereas cAMP treatment resulted in a decrease in CRBP1 mRNA. Again, no changes were observed in the concentration of CRBP1 protein. Furthermore, co-incubation of Sertoli cells from 19-day-old rats with purified pachytene spermatocytes or round spermatids resulted in an increase in mRNA for CRBP1. However, comparable changes in CRBP1 protein concentrations were not observed. Neither cAMP nor serum changed the fraction of CRBP1 mRNA that was associated with polysomes. As a possible explanation for some of the results, pulse-chase experiments showed that the rate of CRBP1 degradation in cultured Sertoli cells is decreased by cAMP. It is proposed that these changes at the level of protein turnover contribute to the maintenance of stable concentrations of CRBP1 even when the corresponding mRNA concentrations vary markedly.

Evidence for independent control at the mRNA and protein levels of cellular retinol binding protein 1 in rat Sertoli cells

Cellular retinol binding protein 1 (CRBP1) is the cytosolic carrier for retinol. It is expressed in many tissues, but the concentrations vary considerably. In Sertoli cells from immature rat testis, CRBP1 is highly expressed. The results of the present study show that regulation of CRBP1 expression at the protein level appears to be independent of regulation at the mRNA level. In Sertoli cells from prepubertal 19-day-old rats, CRBP1 mRNA is strongly induced for up to 72 h by the presence of serum factors. In contrast, treatment of the cells with cAMP analogues led to a rapid reduction in mRNA to quantities less than 5% of control values. However, the changes in CRBP1 mRNA did not lead to similar changes in the concentration of CRBP1 protein during 72 h of observation. Similarly, treatment of cells from 32- and 44-day-old rats with serum led to increased CRBP1 mRNA, whereas cAMP treatment resulted in a decrease in CRBP1 mRNA. Again, no changes were observed in the concentration of CRBP1 protein. Furthermore, co-incubation of Sertoli cells from 19-day-old rats with purified pachytene spermatocytes or round spermatids resulted in an increase in mRNA for CRBP1. However, comparable changes in CRBP1 protein concentrations were not observed. Neither cAMP nor serum changed the fraction of CRBP1 mRNA that was associated with polysomes. As a possible explanation for some of the results, pulse-chase experiments showed that the rate of CRBP1 degradation in cultured Sertoli cells is decreased by cAMP. It is proposed that these changes at the level of protein turnover contribute to the maintenance of stable concentrations of CRBP1 even when the corresponding mRNA concentrations vary markedly.

Cellular retinol-binding protein expression and breast cancer.

The biologic activity of vitamin A depends, in part, on its metabolism to active nuclear receptor ligands, chiefly retinoic acid. The cellular retinol-binding protein (CRBP) binds vitamin A with high affinity and is postulated to regulate its uptake and metabolism. In this report, we analyze the expression of CRBP in normal and malignant breast tissues. We evaluated CRBP expression by in situ hybridization in six reduction mammoplasty specimens and 49 human breast carcinoma specimens by use of digoxigenin-labeled RNA probes and in nine cultured mammoplasty specimens by northern or western blot analysis. Statistical significance was evaluated with the chi(2) test or Fisher's exact test if the sample sizes were small. All P values are from two-sided tests. CRBP was expressed in all 15 mammoplasty specimens (normal breast tissue) and in 33 of 35 available specimens of normal tissue adjacent to carcinoma. In contrast, 12 (24%) of 49 carcinoma lesions were uniformly negative for CRBP (P =.023 for comparison with adjacent normal breast tissue). The loss of CRBP expression was as frequent in ductal carcinoma in situ (six [27%] of 22) as in invasive lesions (six [22%] of 27), suggesting that it is a relatively early event in carcinogenesis and not associated with patient age, tumor grade, and expression of steroid receptors or c-Myc. Preliminary experiments did not find an association between CRBP and retinoic acid receptor beta loss, but most (four of five) CRBP-negative tumors were also retinoic acid receptor beta negative. CRBP is underexpressed in 24% (95% confidence interval = 12.5%-36.5%) of human breast carcinomas, implying a link between cellular vitamin A homeostasis and breast cancer. We hypothesize that the loss of CRBP restricts the effects of endogenous vitamin A on breast epithelial cells.

Retinoic acid synthesis and expression of cellular retinol-binding protein and cellular retinoic acid-binding protein type II are concurrent with decidualization of rat uterine stromal cells.

Decidualization of stromal cells at the site of embryo implantation in the rat uterus is accompanied by expression of cellular retinol-binding protein and cellular retinoic acid-binding protein [CRABP(II)], whose presence has been shown to correlate with gain of ability to synthesize retinoic acid in other cells. Here we examined whether decidual cells also acquired the ability to synthesize retinoic acid, which would have important implications for understanding the implantation process. Decidual cells were isolated from the uterus on day 8 of pregnancy and cultured. When provided with retinol, they indeed synthesized and released retinoic acid to the medium. To follow acquisition of this ability more closely, artificial induction of decidualization was exploited. Ovariectomized rats were placed on a hormonal regimen that allows decidualization to occur in vivo, with oil stimulation, or in vitro, if cells are isolated on day 5 of the regimen and then cultured. Decidualization in vivo reproduced the expression of cellular retinol-binding protein and CRABP(II) seen during pregnancy. Stromal cells isolated on regimen day 2 synthesized little retinoic acid and expressed little alkaline phosphatase, a marker of decidualization. Stromal cells isolated on regimen day 5 had elevated levels of alkaline phosphatase, increasing during the 3 days of culture examined. The ability of the stromal cells to synthesize retinoic acid showed the same pattern: a substantially elevated production from that previously observed, on day 2, with production increasing significantly over the next 2 culture days. Thus, expression of CRABP(II) was correlated with gain of ability to synthesize retinoic acid. Retinoid signaling may be an important part of the process of embryo implantation.

Retinoic Acid Synthesis and Expression of Cellular Retinol-Binding Protein and Cellular Retinoic Acid-Binding Protein Type II Are Concurrent with Decidualization of Rat Uterine Stromal Cells

Retinoic Acid Synthesis and Expression of Cellular Retinol-Binding Protein and Cellular Retinoic Acid-Binding Protein Type II Are Concurrent with Decidualization of Rat Uterine Stromal Cells

Spatial and temporal patterns of expression of cellular retinol-binding protein and cellular retinoic acid-binding proteins in rat uterus during early pregnancy.

Retinoic acid, perhaps the most potent hormonal form of the naturally occurring retinoids (retinol and derivatives), is required in vivo for the maintenance of normal pregnancy and embryo development. However, little is known about the specific sites of action and metabolism in the uterus during pregnancy. In this study we describe the pattern of temporal and cell-specific expression of cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding proteins type I and type II (CRABP and CRABP[II], respectively) in the rat uterus during the periimplantation period (Day 1 to Day 7 of pregnancy; Day 1 = presence of vaginal plug). Immunohistochemical studies showed that there were dramatic and rapid changes in expression pattern of the retinoid-binding proteins after mating as early as Day 1, as well as a differential expression of these proteins when the mesometrial side and antimesometrial side of the uterus were examined during the periimplantation period. CRABP(II), whose presence has been previously shown to correlate with retinoic acid synthesis in the uterine epithelium, was specifically localized to the luminal epithelium at Day 1, being stronger on the mesometrial side, and then fell to lower levels. CRBP was also expressed in the luminal epithelium on the mesometrial side at Day 1 as well as in some stromal cells, declining at these sites over the next several days. CRABP was localized to some of the stromal cells at Day 1, overlapping CRBP expression. Embryonic implantation was accompanied by the appearance of CRBP and CRABP(II) in the decidual cells. CRBP and CRABP were also present in both smooth muscle layers of the uterus. The changes in the temporal and cell-specific distribution of retinoid-binding proteins imply a multifunctional role of vitamin A in uterine cell proliferation, differentiation, and embryonic implantation. The presence of CRABP(II) suggests that local generation of retinoic acid is important in these processes.