Expression Of CRABP-II Research Articles

Targeting CRABP-II overcomes pancreatic cancer drug resistance by reversing lipid raft cholesterol accumulation and AKT survival signaling

BackgroundResistance to standard therapy is a major reason for the poor prognosis of pancreatic ductal adenocarcinoma (PDAC). Developing novel therapy to overcome PDAC drug-resistance is urgently needed. CRABP-II was highly expressed in all PDAC but not expressed in normal pancreatic tissues and chronic pancreatitis. CRABP-II was shown to promote PDAC migration and metastasis while its potential role in promoting PDAC drug-resistance was not known.MethodsA paired cohort of human primary and relapsing PDAC tissues was assessed for CRABP-II expression by immunohistochemistry. CRISPR/cas9 gene editing was used to establish CRABP-II knockout cell lines and MTT assays were performed to assess gemcitabine sensitivity in vitro. Cleaved caspase-3/PARP blots and Annexin V staining were conducted to detect cell apoptosis. Gene expression microarray, Q-PCR, western blots, Co-IP and RNA-IP were used to study the molecular function of CRABP-II. Sucrose gradient ultracentrifugation was applied to isolate lipid rafts and LC–MS-MS was used to assess cholesterol content. Both subcutaneous CDX models and orthotopic PDX models were established to examine the efficacy of SNIPER-11 and the synergistic effect between SNIPER-11 and gemcitabine in vivo.ResultsA higher expression of CRABP-II was found in relapsing PDAC tissue and was associated with poor prognosis. Gemcitabine-resistant cell lines exhibited increased level of CRABP-II, while CRABP-II knockout resensitized PDAC cells to gemcitabine. Mechanistically, aberrant expression of CRABP-II increased the stability of SREBP-1c mRNA through cooperation with HuR and upregulated the downstream genes of SREBP-1c to favor cholesterol uptake and accumulation in lipid rafts. Increased lipid raft cholesterol accumulation facilitated ATK survival signaling and PDAC drug resistance. The small compound SNIPER-11 treatment effectively induced CRABP-II protein degradation, induced apoptosis, and suppressed tumor growth. Combination of SNIPER-11 and gemcitabine significantly reduced the lipid raft cholesterol content in CDX/PDX and profoundly inhibited tumor progression.ConclusionsThese findings identified CRABP-II as a novel regulator of cholesterol metabolism and suggested that CRABP-II is a selective target for overcoming PDAC drug resistance.

Differential CRABP-II and FABP5 expression patterns and implications for medulloblastoma retinoic acid sensitivity.

Medulloblastoma (MB) cells exhibit different responses to retinoid acid (RA) for reasons that are poorly understood. RA signaling can be transduced by two approaches that are mediated by cellular retinoic acid-binding protein 2 (CRABP-II) as a tumor-suppressive pathway, and by fatty acid-binding protein 5 (FABP5) as a tumor-promoting pathway. The biological effects of RA on cancer cells are largely determined by the patterns of CRABP-II and FABP5 expression. This study aims to profile the statuses of CRABP-II and FABP5 expression in MB and to evaluate their correlation with RA sensitivities using RA-sensitive (Med-3) and RA-insensitive (UW228-2, UW228-3) MB cells. Our results show that CRABP-II is distinctly expressed and the level of FABP5 is extremely low in Med-3 cells, while the patterns of CRABP-II and FABP5 expression are reversed in UW228-2 and UW228-3 cells. RA up-regulates CRABP-II expression in Med-3 cells, whereas it up-regulates FABP5 expression in the other two cell lines. The FABP5-specific inhibitor BMS309403 increases the RA sensitivity of UW228-2 cells (p < 0.01). Tissue microarray-based immunohistochemical staining showed CRABP-II/FABP5 expression patterns in MB that were variable (CRABP-II−/FABP5−, CRABP-II−/FABP5+, CRABP-II+/FABP5− and CRABP-II+/FABP5+) and imbalanced (CRABP-II↑/FABP5↓ and CRABP-II↓/FABP5↑). MB cases exhibited patterns ofCRABP-II−/FABP5− (12.24%, 6/49), CRABP-II−/FABP5+ (30.61%, 15/49) or CRABP-II↓/FABP5↑ (12.24%, 6/49), implicating unresponsiveness or insensitivity to RA. In conclusion, the ratios of CRABP-II/FABP5 levels are closely related to the RA sensitivities of MB cells. The differential CRABP-II and FABP5 expression patterns are prospective parameters, and of potential value in personalized RA therapy for MB.

RIS-1/psoriasin expression in epithelial skin cells indicates their selective role in innate immunity and in inflammatory skin diseases including acne.

ABSTRACTObjective: RIS-1/psoriasin/S100A7 is an epithelial antimicrobial peptide, whose expression is upregulated in inflammatory skin diseases and is induced by retinoids. Its molecular expression was investigated in skin cell cultures and in skin specimens to better understand its role in inflammatory procedures of the pilosebaceous unit. Methods: rtPCR and northern blotting of RIS-1/psoriasin and the retinoid-metabolizing genes CYP26AI and CRABP-II were performed in cells cultures (keratinocytes, sebocytes, fibroblasts, endothelial cells, melanocytes, lymphocytes and prostate cells; native and treated with retinoids) and in situ hybridization in normal and inflamed skin (acne, psoriasis). Results: a) RIS-1/psoriasin is expressed in keratinocytes and fibroblasts in vitro and in keratinocytes of the stratum granulosum in vivo. Retinoids in vitro and inflammatory conditions in vivo increase the levels of RIS-1/psoriasin in keratinocytes (both), sebocytes (inflammation only) and fibroblasts (retinoids). Sebocytes and fibroblasts are the metabolically most active skin cells, since they can upregulate the expression of CRABP-II and CYP26AI, genes responsible for retinoid metabolism. Inflammation modifies the compartmentation of RIS-1/psoriasin in sebaceous glands and the follicular root sheaths. Conclusion: The present data indicate that anti-inflammatory treatment targeting the epithelial compartments of the skin, including such with antibacterial peptides, may be promising for inflammatory skin diseases.

Loss of CRABP-II Characterizes Human Skin Poorly Differentiated Squamous Cell Carcinomas and Favors DMBA/TPA-Induced Carcinogenesis

Retinol and its derivatives play an important role in epidermal growth and differentiation and represent chemopreventive agents in nonmelanoma skin cancer. Retinoic acid binding protein II (CRABP-II) is a cytoplasmic receptor that critically regulates all-trans-retinoic acid (ATRA) trafficking. We documented the marked reduced expression of CRABP-II and its promoter methylation in human poorly differentiated squamous cell carcinomas. To investigate the role of CRABP-II in skin carcinogenesis we used skin lesion induction by dimethylbenz[a]anthracene/12-O-tetradecanoyl-phorbol-13-acetate in CRABP-II-knockout C57BL/6 mice. We observed earlier and more diffuse epidermal dysplasia, greater incidence and severity of tumors, reduced expression of cytokeratin 1/cytokeratin 10 and involucrin, increased proliferation, and impaired ATRA inhibition of tumor promotion compared with wild-type animals. CRABP-II-transfected HaCaT, FaDu, and A431 cells showed expression of differentiation markers, retinoic acid receptor-β/-γ signaling, ATRA sensitivity, and suppression of EGFR/v-akt murine thymoma viral oncogene homolog 1 (AKT) pathways in a fatty acid binding protein 5/peroxisome proliferator-activated receptor-β/-δ-independent manner. The opposite was true in keratinocytes isolated from CRABP-II-knockout mice. Finally, CRABP-II accumulation induced ubiquitination-associated reduction of EGFR. Our results showed reduced CRABP-II expression in human poorly differentiated squamous cell carcinomas, and its gene deletion favored experimental skin carcinogenesis and impaired ATRA antitumor efficacy, likely modulating EGFR/AKT pathways and retinoic acid receptor-β/-γ signaling. Therapeutic interventions aimed at restoring CRABP-II-mediated signaling may amplify therapeutic retinoid efficacy in nonmelanoma skin cancer.

CRABP-II- and FABP5-independent responsiveness of human glioblastoma cells to all-trans retinoic acid.

Glioblastomas respond differently to all-trans retinoic acid (RA) for unknown reasons. Because CRABP-II and FABP5 mediate RA intracellular signaling respectively and lead to distinct biological consequences, their expression patterns in different grades of astrocytomas and the glioblastoma cells lines LN18, LN428 and U251 were examined to identify potential correlations with RA sensitivities. The response of glioblastoma cells to RA, decitabine or the FABP5 competitive inhibitor, BMS309403, was analyzed. CRABP-II and FABP5 were expressed to varying degrees by the 84-astrocytoma cases examined. Treatment of LN428, U251 and LN18 cells with RA failed to suppress their growth; however, U251 proliferation was inhibited by decitabine. The combination of decitabine and RA suppressed the growth of all three cell lines and induced significant apoptosis of LN428 and U251 cells. Both CRABP-II and FABP5 were transcribed in the three cell lines but FABP5 proteins were undetectable in U251 cells. The ratio of CRABP-II to FABP5 was not altered after RA, decitabine or RA and decitabine treatment and the resistance of cells to RA was not reversed by BMS309403 treatment. In conclusion, CRABP-II and FABP5 expression patterns are neither related to the tumor grades nor correlated with RA sensitivity. Additional molecular factors may be present that determines the sensitivity of glioblastoma cells to RA. Dicitabine may improve the sensitivity of glioblastoma cells to RA, however, its underlying mechanism and its in vivo feasibility need to be investigated.

Anti-Epileptic Drug Targets Ewing Sarcoma.

Ewing Sarcoma (ES) is a rare form of bone cancer that most commonly affects children and adolescents. Chromosomal translocations are fundamental to the development of Ewing Sarcoma, linked to the changes in gene expression affecting transcription factors. Histone acetyl transferases (HATs) and histone deacetylases (HDACs) regulate transcription by modifying acetylation of both histones and transcription factors. Despite the use of multimodal therapeutic approaches current therapies are associated with significant short and long-term side effects. Hence, new therapeutic approaches are needed. In this study, we show that ERG/EWS-ERG, inhibits transcriptional activation properties of RXRα. These results suggest that ERG/EWS-ERG/EWS-Fli-1 may target transcriptional co-activators and transcriptional repressors and thereby regulate RXRα transcriptional activity. To understand the molecular mechanism of action, how the fusion protein targets nuclear receptor function, and to provide a clue for the cancer health disparity seen in Ewing Sarcoma, we hypothesized that the aberrant fusion protein, EWS-ERG/EWS-Fli-1 regulates HDACs-mediated repressor complex and inhibits the binding of transcriptional activator complex causing transcriptional repression of RXRα activity. Since it is known that HDACs regulate nuclear receptors, we proposed that HDAC inhibitor, valproic acid (VPA), an anti-epileptic drug, may reverse the inhibitory properties of EWS-ERG/EWS-Fli-1 oncoprotein on RXRα transcriptional activity and might therefore be used as therapeutic agent in ES. We demonstrate that VPA reverses the inhibitory effect of EWSERG/EWS-Fli-1 on RXRα transcriptional activity and also inhibits the cell growth. Furthermore, VPA induces apoptosis and restored the expression of RXRα target genes RARβ, CRABPII and p21 activity and repressed the expression of aberrant fusion proteins, EWS-ERG and EWS-Fli-1 in Ewing Sarcoma cells. Thus, therapeutic regulation of transcriptional repressor properties of EWS-ERG/EWS-Fli-1 with an anti-epileptic drug with a promising new potential might have a profound impact on prevention, management and treatment of Ewing Sarcoma. Therapeutic use of VPA in minority patients may help reduce the health disparity.

CRABP-II is a highly sensitive and specific diagnostic molecular marker for pancreatic ductal adenocarcinoma in distinguishing from benign pancreatic conditions

CRABP-II, a retinoic acid binding protein, shuffles retinoic acid from cytoplasm into nucleus and forms a complex with nuclear retinoic acid receptor to facilitate transcriptional activities of retinoic acid. In this study, we studied the expression patterns of CRABP-II in pancreatic ductal adenocarcinoma (PDAC) compared with those in normal pancreas, chronic pancreatitis, and precancerous lesions. We showed no detectable expressions of CRABP-II in normal pancreatic parenchyma, normal ductal epithelium, and chronic pancreatitis. In contrast, the expression of CRABP-II was readily detected in all PDACs including metastatic PDACs. CRABP-II staining was also observed and progressively increased from pancreatic intraepithelial neoplasia 1 to 3. In addition, when fine needle aspiration specimens were evaluated from patients with PDAC, CRABP-II was positive in 55.6% cases if cytology diagnosis was "atypia," and in 87.5% cases, if "malignancy." Our study suggests that CRABP-II is highly and specifically expressed in PDAC and is more commonly expressed in high-grade precursor cancerous lesions than in low-grade lesions. Therefore, overexpression of CRABP-II is a late event of pancreatic carcinogenesis, and it could be used as a diagnostic marker to distinguish PDAC from other benign pancreatic conditions in both resection and cytology specimens.

All-trans retinoic acid inhibits craniopharyngioma cell growth: study on an explant cell model

The ratio between FABP5 and CRABPII determines cellular response to physiological level of retinoic acid; tumor cells undergo proliferation with high level of FABP5 and apoptosis with high level of CRABPII. We intended to study FABP5 and CRABPII expression in craniopharyngiomas, to establish craniopharyngioma cell model using explants method, and to study the effect of pharmacological dose of retinoic acid on craniopharyngioma cells. Expression of FABP5 and CRABPII in craniopharyngioma tissue from 20 patients was studied using immunohistochemistry. Primary craniopharyngioma cell cultures were established using tissue explants method. Craniopharyngioma cells were treated using various concentrations of all-trans retinoic acid, and cell growth curve, apoptosis, expression of FABP5, CRABPII and NF-κB were assayed in different groups. FABP5/CRABPII ratio was significantly higher in adamatinomatous group than that in papillary group. Cell cultures were established in 19 cases (95 %). Pharmacological level retinoic acid inhibited cell growth and induced cellular apoptosis in dose dependent manner, and apoptosis rate cells treated with 30 μM retinoic acid for 24 h was 43 %. Also, retinoic acid increased CRABPII, and decreased FABP5 and NF-κB expression in craniopharyngioma cells. High FABP5/CRABPII ratio is observed in adamatinomatous craniopharyngioma. Retinoic acid at pharmacological level induced craniopharyngioma cell apoptosis via increasing FABP5/CRABPII ratio and inhibiting NF-κB signaling pathway. Our study demonstrated that all-trans retinoic acid might be a candidate for craniopharyngioma adjuvant chemotherapy in future.

CRABP-II methylation: A critical determinant of retinoic acid resistance of medulloblastoma cells

Medulloblastoma cells exhibit varied responses to therapy by all-trans retinoic acid (RA). The underlying mechanism for such diverse effects however remains largely unclear. In this study, we attempted to elucidate the molecular basis of RA resistance through the study of RA signaling components in both RA-sensitive (Med-3) and RA-resistant (UW228-2 and UW228-3) medulloblastoma cells. The results revealed that RARα/β/γ and RXRα/β/γ were found in the three cell lines. Expression of CRABP-I and CRABP-II was seen in Med-3 cells, up-regulated when treated with RA, but was absent in UW228-2 and UW228-3 cells regardless of RA treatment. Bisulfite sequencing revealed 8 methylated CG sites at the promoter region of CRABP-II in UW228-2 and UW228-3 but not in Med-3 cells. Demethylation by 5-aza-2′-deoxycytidine recovered CRABP-II expression. Upon restoration of CRABP-II expression, both UW228-2 and UW228-3 cells responded to RA treatment by forming neuronal-like differentiation, synaptophysin expression, β-III tubulin upregulation, and apoptosis. Furthermore, CRABP-II specific siRNA reduced RA sensitivity in Med-3 cells. Tissue microarray-based immunohistochemical staining showed variable CRABP-II expression patterns among 104 medulloblastoma cases, ranging from negative (42.3%), partly positive (14.4%) to positive (43.3%). CRABP-II expression was positively correlated with synaptophysin (rs = 0.317; p = 0.001) but not with CRABP-I expression (p > 0.05). In conclusion, aberrant methylation in CRABP-II reduces the expression of CRABP-II that in turn confers RA resistance in medulloblastoma cells. Determination of CRABP-II expression or methylation status may enable a personalized RA therapy in patients with medulloblastomas and other types of cancers.

CRABP‐II‐ and FABP5‐independent all‐trans retinoic acid resistance in COLO 16 human cutaneous squamous cancer cells

The effect of all-trans retinoic acid (ATRA) on cutaneous squamous cell carcinomas (c-SCC) has been poorly described. Because the imbalance of CRABP-II-mediated anticancer signalling and FABP5-mediated growth-promoting signalling was supposed to be related with ATRA sensitivities of cancer cells, COLO16 human c-SCC cell line was selected to check underlying mechanism leading to ATRA resistance by multiple experimental approaches. The results revealed that COLO 16 cells were resistant to 15 μm ATRA treatment. FABP5 as well as the elements related with CRABP-II signalling (CYP26A1, CYP26B1, CRABP-I, RARα/β/γ and RXRα/β/γ) and with FABP5 signalling (PPARβ/δ) were expressed, but CRABP-II was undetectable in COLO 16 cells. 5-Aza treatment enhanced CRABP-II expression but further bisulfite sequencing PCR-DNA sequencing revealed no methylation in CRABP-II promoter region. Transfection of CRABP-II-expressing plasmids or FABP5 siRNA or both successfully manipulated the level(s) of target gene expression but failed to overcome ATRA resistance in the transfectants. In conclusion, CRABP-II and FABP5 expression were imbalanced in ATRA-resistant COLO 16 cells. 5-Aza-enhanced CRABP-II expression and unmethylation in CRABP-II promoter region suggest the methylation of certain CRABP-II regulatory gene(s) in COLO 16 cells. As neither restoration of CRABP-II expression nor the increased CRABP-II versus FABP5 ratio can overcome ATRA resistance of COLO 16 cells, additional ATRA-resistant mechanism(s) may present in human c-SCCs and COLO 16 cells would be of value in addressing this issue.

Research on retinoic acid signals in retina of guinea pig eyes with different monochromatic illumination

Retinoic acid (RA) is the only extrinsic biochemical candidate that could act as a growth controller, the present study was designed to evaluate the retinoic acid signals in retina of guinea pig eyes with different monochromatic illumination. Ninety 2-week-old guinea pigs were divided into 3 groups randomly and were raised under green (530 nm) (n = 30), blue (430 nm) (n = 30) or white lighting (5000K) (n = 30) conditions. The axial length was measured with Cine scan A/B ultrasonography before, 2 weeks, 4 weeks, 8 weeks during experiment. After the biometrical measurement finished, 9 guinea pigs were sacrificed randomly in each group and retina was dissected. The RA level in retina was detected by High Performance Liquid Chromatography (HPLC). The expressions of retinoic acid-binding proteins I (CRABP-I) and retinoic acid receptor-β (RAR-β) protein and mRNA were assayed by Western-blot and Real-time PCR, respectively. The experiment and use of animals followed the Standard of Association for Research in Vision and Ophthalmology. 1. The axial length among the three groups showed a significant difference at 4 weeks during experiment (F = 3.946; P < 0.05), the green lighting group showed the fastest growing, the axial length of the three groups are (8.36 ± 0.11) mm (green), (7.88 ± 0.49) mm (blue), 7.98 ± 0.32 mm (white) respectively at 8 weeks during experiment; 2. The RA levels demonstrated a same trend with the axial length, at 8 weeks during experiment the RA levels are 4.846 ± 0.56 µg/g (green), 3.583 ± 0.26 µg/g (blue), 4.419 ± 0.36 µg/g (white) respectively; 3. Changes of the expression of CRABP-I and RAR-β occurred at different times, a significant difference of RAR-β among three groups appeared earlier, the green lighting group showed a highest level. The different levels of retinoic acid signals were showed in retina of guinea pig eyes under different monochromatic illumination. The eye growth was significantly faster in green group than in blue group and white group, meanwhile, the RA level, the expressions of CRABP-I and RAR-β in the retina were higher in green group than the other two groups. These findings suggest that RA may act as a messenger in eye growth promoted by different monochromatic illumination.

Cloning, Expression, and Regulation of Bovine Cellular Retinoic Acid-binding Protein-II (CRABP-II) during Adipogenesis

The mammalian cellular retinoic acid-binding proteins, CRABP-I and CRABP-II, bind retinoic acid which acts as an inducer of differentiation in several biological systems. To investigate a possible role for CRABP-II in bovine adipogenesis, we have cloned bovine CRABP-II cDNA and the coding region for CRABP-I. The predicted amino acid sequences of CRABP-II were highly conserved among several animal species (human, mouse, and rat at 97%, 93%, and 93%, respectively). The expression pattern of bovine CRABP-II was examined in greater details by applying RT-PCR to various bovine tissues. CRABP-II mRNA was expressed in most adipose-containing tissues. Moreover, the expression of CRABP-I and -II mRNA dramatically increased during the differentiation of adipocytes from bovine intramuscular fibroblast-like cells. The effects of retinoic acid on adipocyte differentiation of bovine intramuscular fibroblast-like cells were concentration-dependent. Retinoic acid activated the formation of lipid droplets at a level of 1 nM, whereas inhibition was observed at a level of 1 μM. CRABP-I gene was up-regulated and CRABP-II gene down-regulated by retinoic acid during adipocyte differentiation. These results suggest that CRABPs may play an important role in the regulation of intracellular retinoic acid concentrations during adipogenesis.

Molecular mechanism of teratogenic effects induced by the fungicide triadimefon: Study of the expression of TGF-β mRNA and TGF-β and CRABPI proteins during rat in vitro development

Azole derivatives are teratogenic in rats and mice in vitro and in vivo. The postulated mechanism for the dysmorphogenetic effects is the inhibition of retinoic acid (RA)-degrading enzyme CYP26. Azole-related abnormalities are confined to structures controlled by RA, especially the neural crest cells, hindbrain, cranial nerves, and craniofacial structures, through a complex signal cascade. The aim of this work is to study the expression of signal molecules activated by RA (TGF-βs) or involved in the modulation of cellular RA concentrations (CRABPI). E9.5 (9.5 day post coitum old embryos) rat embryos, exposed in vitro to triadimefon (FON) for 24 h, were examined or cultured in normal serum for extra 4, 16, and 24 h. RT-PCR was performed to quantify TGF-β1, TGF-β2, TGF-β3, TGF-βRI, TGF-βRII, and TGF-βRIII mRNA in the hindbrain after 24 h of culture. TGF-β1, TGF-β2, and TGF-βRI were found significantly decreased by FON exposure, and consequently their protein expression was analyzed by Western blot and immunohistochemistry. In both controls and FON-exposed embryos, TGF-β1 and TGF-βRI were detected at 24 and 24 + 4 h; TGF-β2 was present only at 24 h. Only TGF-β1 was expressed at the level of hindbrain and branchial tissues. After quantization, TGF-β1 was reduced in the FON group. The expression of CRABPI was observed at all developmental stages. However, in FON-exposed embryos, it was increased at 24 and 24 + 4 h. The hindbrain distribution of CRABPI-positive cells was abnormal in FON-exposed embryos. The results show that the two RA-related molecules (TGF-β1 and CRABPI) are altered by FON exposure in vitro.

Overexpression of CRABPI in suprabasal keratinocytes enhances the proliferation of epidermal basal keratinocytes in mouse skin topically treated with all-trans retinoic acid

We investigated whether ectopic expression of CRABPI, a cellular retinoic acid binding protein, influenced the actions of all-trans retinoic acid (ATRA) in transgenic (TG) mice. We targeted CRABPI to the basal vs. suprabasal layers of mouse epidermis by using the keratin 14 (K14) and keratin 10 (K10) promoters, respectively. Greater CRABPI protein levels were detected in the epidermis of adult transgenic(+) mice than in transgenic(−) mice for both transgenes. In adult mouse skin CRABPI overexpression in the basal or suprabasal keratinocytes did not cause morphological abnormalities, but did result in decreased CRABPII mRNA levels. Ectopically overexpressed CRABPI in suprabasal keratinocytes, but not in basal keratinocytes, enhanced the thickening of the epidermis induced by topical ATRA treatments (10 μM, 400 μl for 4 days) by 1.59 ± 0.2-fold ( p < 0.05). ATRA treatment (10 μM) resulted in a 59.9 ± 9.8% increase ( p < 0.05) in the BrdU labeling index in K10/FLAG-CRABPI TG(+) mice vs. TG(−) mice. Retinoid topical treatments reduced p27 and CYP26A1 mRNA levels in TG(+) and TG(−) mouse skin in K14 and K10/FLAG-CRABPI transgenic mice. As epidermal basal keratinocyte proliferation is stimulated by paracrine growth factors secreted by ATRA activated suprabasal keratinocytes, our results indicate that CRABPI overexpression in suprabasal keratinocytes enhances the physiological functions of ATRA.

AP2 Transcription Factors Regulate Expression of CRABPII in Hormone Responsive Breast Carcinoma

The AP2 transcription factor family is a set of developmentally regulated, retinoic acid (RA) inducible genes, which regulate expression of estrogen receptor-alpha (ERalpha) in breast carcinoma. We hypothesized that AP2 factors regulate a set of genes characteristic of the hormone responsive breast cancer phenotype. To better understand the role of AP2 factors in hormone responsive breast cancer, we sought to identify AP2-target genes in breast epithelial cells. Overexpression of AP2 factors was achieved in human mammary epithelial cells (HMECs) using adenoviral vectors. AP2 target genes were identified by comparative hybridization to cDNA microarrays containing 30,000 human genes. Expression patterns were confirmed by Northern and Western blot and by elimination of AP2 using siRNA. Potential regulatory elements in promoters of target genes were identified by DNase I hypersensitive site mapping. Comparative cDNA microarray hybridization identified a set of genes induced by overexpression of AP2alpha and AP2gamma in HMECs. The up-regulation of cellular retinoic acid-binding protein 2 (CRABPII), EST-1, and ECM1 was induced by overexpression of AP2alpha, AP2gamma, or a chimeric AP2 factor in which the activation domain of AP2alpha was replaced by the activation domain of herpesvirus VP16. Interestingly, hormone unresponsive MDA-MB-231 cells were resistant to CRABPII induction by any of the AP2 factors. Elimination of AP2gamma in MCF7 cells resulted in a significant reduction in CRABPII expression. AP2alpha induced DNase I hypersensitive sites in the promoter of the CRABPII gene at -5000 bp, which corresponds to the site of action of RAR/RXR factors. AP2 factors regulate CRABPII expression in HMECs and breast cancer cells and accounts for the associated expression of ERalpha and CRABPII in hormone responsive breast cancer. Because CRABPII mediates growth suppressive effects of RA in breast cancer, the data suggest that AP2 factors have the ability to mediate RA responsiveness through the regulation of CRABP II expression.

Mutation of Cellular Retinoic Acid-Binding Protein-II (Crabp-II) Does Not Account for Resistance to All-Trans Retinoic Acid (ATRA) in Relapse Acute Promyelocytic Leukemia (APL).

An increase in CRABP-II has frequently been invoked as a cause of resistance to ATRA in APL due to cytoplasmic sequestration and catabolism of ATRA. However, recent evidence indicates that CRABP-II has a positive rather than a negative role in ATRA activity by facilitating delivery of ATRA to retinoic acid receptor-alpha (RARα) associated with ATRA target genes in the cell nucleus or/and by serving as a co-activator of RARα-regulated transcription. This implies that if CRABP-II has a role in the development of ATRA resistance in APL, this would more likely occur through a deficiency rather than from an increase in CRABP-II. We previously reported that CRABP-II is constitutively expressed at similar levels in pretreatment and relapse APL cells (Zhou, et al, Cancer Res 58, 5770, 1998), suggesting that putative CRABP-II deficiency is not due to the loss of CRABP-II expression. To investigate the alternative possibility that CRABP-II deficiency might arise through inactivating mutations, we sequenced the entire coding region of CRABP-II from 18 cases of APL who had relapsed from prior ATRA-containing treatment regimens. In 8 cases RNA was transcribed by reverse transcriptase to cDNA and amplified by polymerase chain reaction (PCR), using primers anchored in the 5′ and 3′ untranslated region of mRNA; in 10 cases, genomic DNA was PCR amplified for sequence analysis, using primers anchored in the introns between the 4 exons of the gene. No CRABP-II mutations were identified. The samples tested included 11 first-relapse cases, 2 of whom were refractory to reinduction therapy with intravenous liposomal-ATRA, and 7 multiple relapse cases, all of whom were clinically refractory to ATRA and had, additionally, relapsed from arsenic trioxide therapy. Also, no mutations were found in 3 APL patients who had relapsed from chemotherapy-induced remissions or in 3 APL cell lines (NB4, UF-1 and AP-1060). Two heterozygous base substitutions were incidentally identified in CRABP-II intron 2 in a chemotherapy-only treated patient. These results indicate that CRABP-II mutations rarely, if ever, contribute to ATRA-resistance or disease relapse in APL.

249BOVINE GRANULOSA CELLS MRNA EXPRESSION OF PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-± AND THE PROTO-ONCOGENE C-FOS

PPARα and c-Fos are involved in regulation of gene expression and are known to be dependent on retinoic acid (RA), which in turn influences oocyte growth and developmental competence (Duque et al., 2002 Hum. Reprod. 17, 2706–2714; Hidalgo et al., 2003. Reproduction 125, 409–416), probably acting in part through granulosa cells. Peroxisome proliferator-activated receptor-α (PPARα) heterodimerizes with the retinoid receptor X (RXR), while c-Jun/c-Fos heterodimerizes with liganded retinoic acid receptors (RARs), then preventing formation of transcription factor activator protein 1 (AP-1) complexes capable of DNA binding. Cellular retinoic acid binding protein (CRABP) limits RA excess and regulates the transcriptional potential of RA;; CRABPII has been detected in rat granulosa cells from mature follicles and luteal cells. The aim of this study was to investigate PPARα, c-Fos and CRABPII mRNA expression in bovine granulosa cells. In parallel, other genes whose expression can be influenced by RA were analyzed: luteinizing hormone receptor (LHr), follicle stimulating hormone receptor (FSHr), aromatase and growth hormone (GH). Ovaries were collected at a local abattoir and kept in saline at 30–35°C. Granulosa cells were obtained by aspirating 2- to 7-mm antral follicle contents, pelleted at 700g for 4min and resuspended in RNA-later (Ambion®). Total RNA was isolated with a NucleoSpin® RNAII kit (Macherey-Nagel), and mRNA was reverse transcribed into single-stranded cDNA using a 1st Strand cDNA Synthesis Kit for RT-PCR (AMV) (Roche). A PCR standard method was made using 1μL of the cDNA as a template. All PCR primer couples were designed on the basis of the bovine sequence, but c-Fos and CRABPII primers were designed based on the human-murine sequences. Primers within the couple were located in different exons to distinguish DNA from RNA amplification. CRABPII was further investigated in bovine whole ovary, corpus luteum (CL) and liver, in a search for positive controls. Bovine β-actin, 18S and 28S were examined in each sample as positive controls for RNA isolation and cDNA synthesis efficiency. TenμL of product were loaded into an agarose 2% gel in TBE buffer containing ethidium bromide, and were separated by horizontal electrophoresis. Gels were visualized with ultraviolet light and photographed using a digital camera. Gene expression in granulosa was demonstrated for PPARα, c-Fos, LHr, FSHr, aromatase, GH and controls (β-actin, 18S and 28S) but CRABPII gene did not express in granulosa cells, whole ovary, CL or liver under our experimental conditions. While lacking CRABPII expression remains intriguing, the expressed genes support a role of retinoid pathway within granulosa cells under both in vivo and in vitro conditions, because granulosa cells used in the present experiments were derived from follicles providing oocytes for IVM-IVF. Grant support: Spanish Ministry of Science and Technology (AGL-2002-01175).

Elevation of CRABP-I in the cerebrospinal fluid of patients with Moyamoya disease.

The etiology of moyamoya disease (MMD) remains obscure. This study was undertaken to identify specific proteins associated with the pathogenesis of MMD. We studied cerebrospinal fluid (CSF) from 20 patients with angiographically confirmed MMD (4 boys and 16 girls; age range, 3 to 13 years; mean, 7.5 years) and 4 control patients with cerebral palsy who underwent selective dorsal rhizotomy (2 boys and 2 girls; age range, 5 to 10 years; mean, 7.3 years). CSF proteins were analyzed by 2-dimensional polyacrylamide gel electrophoresis, and protein identification was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The presence of specific CSF protein in patients with MMD was confirmed by Western blotting. In addition, cerebral CSF was also tested in 7 patients who had other brain diseases but no MMD (2 boys and 5 girls; age range, 1 to 12 years; mean, 6.9 years). We identified 1 polypeptide spot (Mr of 13 to 15 kDa and isoelectric point of 5 to 5.5) that was differentially expressed in the CSF samples of MMD patients (mean optical density intensity, 0.36+/-0.24; range, 0.05 to 0.92) and control spinal CSF samples (mean, 0.03+/-0.04; range, 0 to 0.08; P=0.002). This polypeptide was identified as cellular retinoic acid-binding protein (CRABP)-I. High levels of expression of CRABP-I in the CSF from 17 MMD children were confirmed by Western blotting. The analysis of the CSF of MMD patients reveals high CRABP-I expression. The present study suggests that the elevation of CRABP-I in CSF may be a candidate for pathogenesis of MMD.

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.

Direct channeling of retinoic acid between cellular retinoic acid-binding protein II and retinoic acid receptor sensitizes mammary carcinoma cells to retinoic acid-induced growth arrest.

Cellular retinoic acid-binding protein II (CRABP-II) is an intracellular lipid-binding protein that associates with retinoic acid with a subnanomolar affinity. We previously showed that CRABP-II enhances the transcriptional activity of the nuclear receptor with which it shares a common ligand, namely, the retinoic acid receptor (RAR), and we suggested that it may act by delivering retinoic acid to this receptor. Here, the mechanisms underlying the effects of CRABP-II on the transcriptional activity of RAR and the functional consequences of these effects were studied. We show that CRABP-II, a predominantly cytosolic protein, massively undergoes nuclear localization upon binding of retinoic acid; that it interacts with RAR in a ligand-dependent fashion; and that, in the presence of retinoic acid, the CRABP-II-RAR complex is a short-lived intermediate. The data establish that potentiation of the transcriptional activity of RAR stems directly from the ability of CRABP-II to channel retinoic acid to the receptor. We demonstrate further that overexpression of CRABP-II in MCF-7 mammary carcinoma cells dramatically enhances their sensitivity to retinoic acid-induced growth inhibition. Conversely, diminished expression of CRABP-II renders these cells retinoic acid resistant. Taken together, the data unequivocally establish the function of CRABP-II in modulating the RAR-mediated biological activities of retinoic acid.