RARα mRNA和RARβ mRNA在过量RA致神经管畸形发生中的表达

目的探讨过量RA对金黄地鼠胚胎神经管RARα和RARβ mRNA表达的影响。方法采用原位杂交及图像分析,半定量分析正常及RA致畸金黄地鼠胚胎神经管中RARα和RARβ mRNA表达水平的变化。结果过量RA可使RARα和RARβ mRNA在金黄地鼠神经管中的表达水平呈现短时下降,随后又大幅上升的变化。结论过量RA引起的RARα和RARβ mRNA表达水平的变化与RA致金黄地鼠神经管畸形相关。     

RARα and RARγ reciprocally control K5+ progenitor cell expansion in developing salivary glands

Understanding the mechanisms of controlled expansion and differentiation of basal progenitor cell populations during organogenesis is essential for developing targeted regenerative therapies. Since the cytokeratin 5-positive (K5⁺) basal epithelial cell population in the salivary gland is regulated by retinoic acid signaling, we interrogated how isoform-specific retinoic acid receptor (RAR) signaling impacts the K5⁺ cell population during salivary gland organogenesis to identify RAR isoform-specific mechanisms that could be exploited in future regenerative therapies. In this study, we utilized RAR isoform-specific inhibitors and agonists with murine submandibular salivary gland organ explants. We determined that RARα and RARγ have opposing effects on K5⁺ cell cycle progression and cell distribution. RARα negatively regulates K5⁺ cells in both whole organ explants and in isolated epithelial rudiments. In contrast, RARγ is necessary but not sufficient to positively maintain K5⁺ cells, as agonism of RARγ alone failed to significantly expand the population. Although retinoids are known to stimulate differentiation, K5 levels were not inversely correlated with differentiated ductal cytokeratins. Instead, RARα agonism and RARγ inhibition, corresponding with reduced K5, resulted in premature lumenization, as marked by prominin-1. With lineage tracing, we demonstrated that K5⁺ cells have the capacity to become prominin-1⁺ cells. We conclude that RARα and RARγ reciprocally control K5⁺ progenitor cells endogenously in the developing submandibular salivary epithelium, in a cell cycle-dependent manner, controlling lumenization independently of keratinizing differentiation. Based on these data, isoform-specific targeting RARα may be more effective than pan-RAR inhibitors for regenerative therapies that seek to expand the K5⁺ progenitor cell pool. Summary statement: RARα and RARγ reciprocally control K5⁺ progenitor cell proliferation and distribution in the developing submandibular salivary epithelium in a cell cycle-dependent manner while regulating lumenization independently of keratinizing differentiation.     

Targeting the Acute Promyelocytic Leukemia-Associated Fusion Proteins PML/RARα and PLZF/RARα with Interfering Peptides

In acute promyelocytic leukemia (APL), hematopoietic differentiation is blocked and immature blasts accumulate in the bone marrow and blood. APL is associated with chromosomal aberrations, including t(15;17) and t(11;17). For these two translocations, the retinoic acid receptor alpha (RARα) is fused to the promyelocytic leukemia (PML) gene or the promyelocytic zinc finger (PLZF) gene, respectively. Both fusion proteins lead to the formation of a high-molecular-weight complex. High-molecular-weight complexes are caused by the “coiled-coil” domain of PML or the BTB/POZ domain of PLZF. PML/RARα without the “coiled-coil” fails to block differentiation and mediates an all-trans retinoic acid-response. Similarly, mutations in the BTB/POZ domain disrupt the high-molecular-weight complex, abolishing the leukemic potential of PLZF/RARα. Specific interfering polypeptides were used to target the oligomerization domain of PML/RARα or PLZF/RARα. PML/RARα and PLZF/RARα were analyzed for the ability to form high-molecular-weight complexes, the protein stability and the potential to induce a leukemic phenotype in the presence of the interfering peptides. Expression of these interfering peptides resulted in a reduced replating efficiency and overcame the differentiation block induced by PML/RARα and PLZF/RARα in murine hematopoietic stem cells. This expression also destabilized the PLZF/RARα-induced high-molecular-weight complex formation and caused the degradation of the fusion protein. Targeting fusion proteins through interfering peptides is a promising approach to further elucidate the biology of leukemia.     

Recent advances in the design of RAR α and RAR β agonists as orally bioavailable drugs. A review

Retinoic acid receptors (RARs) α, β, and γ are members of the nuclear receptor superfamily. Compounds which bind to and activate the RARs are termed retinoids which regulate a wide variety of biological processes such as vertebrate embryonic morphogenesis and organogenesis, cell growth arrest, differentiation, and apoptosis, as well as their disorders. Although many synthetic selective RARα, RARβ, and RARγ agonists have been designed and prepared, these have generally been lipophilic acids without good drug-like properties and with low oral bioavailability. Recently this has been changing and drug design approaches to highly potent and selective RARα and RARβ agonists with low lipophilicity that are orally bioavailable and less toxic have been developed, that have a range of potential therapeutic uses. This review covers these new advances.     

RARβ在胃癌细胞生长调节中的作用

为探讨 RARβ受体介导全反式视黄酸 ( ATRA)抑制胃癌细胞生长的作用机理 ,用 Northern印迹测定 RARβ m RNA表达水平 ,脂质体介导的转染方法将含有 RARβ基因的表达载体转染MKN- 45细胞并稳定表达 ,MTT和软琼脂集落形成等实验测定细胞生长速率和生长状态 ,氯霉素乙酰转移酶活性 ( CAT)测定视黄酸应答元件βRARE的转录活性以及 AP- 1 ( activator protein- 1 )活性 .RARβ在 ATRA敏感细胞株 MGC80 - 3、BGC- 82 3和 SGC- 790 1中表达 ,而在 ATRA抗性细胞株 MKN- 45中不表达 .当 RARβ基因转染 MKN- 45细胞时 ,细胞变为 ATRA敏感 ,由此导致ATRA抑制 MKN- 45细胞生长和软琼脂集落形成 .ATRA可以加强诱导 MGC80 - 3、BGC- 82 3和SGC- 790 1细胞βRARE的转录活性 ,但对 MKN- 45细胞影响不大 ,不能抑制细胞 AP- 1活性 .当RARβ基因转染 MKN- 45细胞后 ,ATRA则能够诱导细胞 βRARE的转录活性 ,并抑制细胞的 AP-1活性 .RARβ表达与 ATRA抑制胃癌细胞生长密切相关 .ATRA诱导 βRARE转录活性和抑制AP- 1活性可能是其调控胃癌细胞生长的机制之一 .     

Detection of variable levels of RAR�� and RAR�� proteins in pluripotent and differentiating mouse embryonal carcinoma and mouse embryonic stem cells

Pluripotent mouse embryonal carcinoma (mEC) and mouse embryonic stem (mES) cells differentiate into several cell lineages upon retinoic acid (RA) addition. Differentiation is facilitated, in part, by RA activation of nuclear RA receptors (RARs) that bind to DNA response elements located in the promoters of target genes. The purpose of the studies reported here was to immunolocalize RARα and RARγ protein in mEC and mES cells and in their RA-induced differentiated progeny. Fixed cells were reacted with three different RARα antibodies and one RARγ antibody. Pluripotent and differentiated mEC and mES cells showed positive nuclear immunoreactivity with all antibodies tested. Two RARα antibodies also showed positive reactivity in the cytoplasm. Surprisingly, our results revealed variability in immunofluorescence intensity and in RARα and RARγ distribution from one cell to the other, suggesting that RARα and RARγ protein levels were not synchronous throughout the cell population. The results indicate that RARα and RARγ are present in pluripotent and differentiating mEC and mES cells and suggest that the expression of these proteins is dynamic.     

Sulindac sulfide reverses aberrant self-renewal of progenitor cells induced by the AML-associated fusion proteins PML/RARα and PLZF/RARα

Chromosomal translocations can lead to the formation of chimeric genes encoding fusion proteins such as PML/RARα, PLZF/RARα, and AML-1/ETO, which are able to induce and maintain acute myeloid leukemia (AML). One key mechanism in leukemogenesis is increased self renewal of leukemic stem cells via aberrant activation of the Wnt signaling pathway. Either X-RAR, PML/RARα and PLZF/RARα or AML-1/ETO activate Wnt signaling by upregulating γ-catenin and β-catenin. In a prospective study, a lower risk of leukemia was observed with aspirin use, which is consistent with numerous studies reporting an inverse association of aspirin with other cancers. Furthermore, a reduction in leukemia risk was associated with use of non-steroidal anti-inflammatory drug (NSAID), where the effects on AML risk was FAB subtype-specific. To better investigate whether NSAID treatment is effective, we used Sulindac Sulfide in X-RARα-positive progenitor cell models. Sulindac Sulfide (SSi) is a derivative of Sulindac, a NSAID known to inactivate Wnt signaling. We found that SSi downregulated both β-catenin and γ-catenin in X-RARα-expressing cells and reversed the leukemic phenotype by reducing stem cell capacity and increasing differentiation potential in X-RARα-positive HSCs. The data presented herein show that SSi inhibits the leukemic cell growth as well as hematopoietic progenitors cells (HPCs) expressing PML/RARα, and it indicates that Sulindac is a valid molecular therapeutic approach that should be further validated using in vivo leukemia models and in clinical settings.     

HDAC2 phosphorylation-dependent Klf5 deacetylation and RARα acetylation induced by RAR agonist switch the transcription regulatory programs of p21 in VSMCs

Abnormal proliferation of vascular smooth muscle cells (VSMCs) occurs in hypertension, atherosclerosis and restenosis after angioplasty, leading to pathophysiological vascular remodeling. As an important growth arrest gene, p21 plays critical roles in vascular remodeling. Regulation of p21 expression by retinoic acid receptor (RAR) and its ligand has important implications for control of pathological vascular remodeling. Nevertheless, the mechanism of RAR-mediated p21 expression in VSMCs remains poorly understood. Here, we show that, under basal conditions, RARα forms a complex with histone deacetylase 2 (HDAC2) and Krüppel-like factor 5 (Klf5) at the p21 promoter to inhibit its expression. Upon RARα agonist stimulation, HDAC2 is phosphorylated by CK2α. Phosphorylation of HDAC2, on the one hand, promotes its dissociation from RARα, thus allowing the liganded-RARα to interact with co-activators; on the other hand, it increases its interaction with Klf5, thus leading to deacetylation of Klf5. Deacetylation of Klf5 facilitates its dissociation from the p21 promoter, relieving its repressive effect on the p21 promoter. Interference with HDAC2 phosphorylation by either CK2α knockdown or the use of phosphorylation-deficient mutant of HDAC2 prevents the dissociation of Klf5 from the p21 promoter and impairs RAR agonist-induced p21 activation. Our results reveal a novel mechanism involving a phosphorylation-deacetylation cascade that functions to remove the basal repression complex from the p21 promoter upon RAR agonist treatment, allowing for optimum agonist-induced p21 expression.     

胃癌细胞中AP-1活性与RARα介导的相关性

为确定全反式视黄酸 ( ATRA)抑制胃癌细胞 AP- 1活性过程中 RARα介导的作用机理 ,利用 Northern印迹和 Western印迹测定 RARα基因和 c Jun、c Fos蛋白表达水平 ;氯霉素乙酰转移酶活性 ( CAT)分析 AP- 1活性 ;以及 MTT法检测细胞生长速率 .结果表明 ,ATRA能够诱导 RARα表达 ,抑制 c Jun和 c Fos蛋白表达和 AP- 1活性 ,由此导致胃癌细胞生长抑制 .结果证实 ,ATRA抑制胃癌细胞 AP- 1活性是抑制细胞生长的重要途径之一 ,并与 RARα介导密切相关 .     

Retinoic acid receptor isoform RARγ 1: an antagonist of the transactivation of the RARβ RARE in epithelial cell lines and normal human keratinocytes

The diversity of isoforms of retinoic acid (RA) receptors (RARs) and of DNA sequences of retinoic acid-responsive elements (RAREs) suggests the existence of selectivities in the RAR/RARE recognition or in the subsequent gene modulation. Such selectivities might be particularly important for RAREs involved in positive feedback, eg. the RAR RARE. In the present work we found that in several epithelial cell lines, reporter constructs containing the RAR RARE linked to the HSV-tk promoter were transactivated in the presence of RA by endogenous RARs and co-transfected RAR₁ and RAR₂ isoforms, but not by RAR₁. On the contrary, this latter isoform behaved towards the RAR RARE as an inhibitor of the transactivation produced by endogenous RARs and by cotransfected RAR₁ and RAR₂. RAR₁ also behaved as an antagonist of the transactivation produced by cotransfected RXR. The natural RAR gene promoter or RAR RARE tk constructs were not activated by the endogenous receptors of normal human keratinocytes (NHK), which are known to contain predominantly RAR₁. It was, however, possible to activate to a certain extent RAR RARE-reporter constructs in NHK by co-transfecting RAR₁, RAR₂ or RXR. The antagonist behavior of RAR₁ towards the RAR RARE may explain why in certain cell types such as keratinocytes, RAR is neither expressed nor induced by RA.Abbreviations DMEM Dulbecco's modified Eagle medium - DMSO dimethyl sulfoxide - FCS fetal calf serum - MEM minimal Eagle medium - NHK normal human keratinocyte - RA retinoic acid - RAR retinoic acid receptor - RARE retinoic acid responsive element - TRE thyroid responsive element - VDRE vitamin D response element - RXR retinoid X receptor     

Amplified Inhibition of Stellate Cell Activation Pathways by PPAR-γ, RAR and RXR Agonists

Peroxisome proliferator activator receptors (PPAR) ligands such as 15-Δ12,13-prostaglandin L(2) [PJ] and all trans retinoic acid (ATRA) have been shown to inhibit the development of liver fibrosis. The role of ligands of retinoic X receptor (RXR) and its ligand, 9-cis, is less clear. The purpose of this study was to investigate the effects of combined treatment of the three ligends, PJ, ATRA and 9-cis, on key events during liver fibrosis in rat primary hepatic stellate cells (HSCs). We found that the anti-proliferative effect of the combined treatment of PJ, ATRA and 9-cis on HSCs was additive. Further experiments revealed that this inhibition was due to cell cycle arrest at the G₀/G₁ phase as demonstrated by FACS analysis. In addition, the combined treatment reduced cyclin D1 expression and increased p21 and p27 protein levels. Furthermore, we found that the three ligands down regulated the phosphorylation of mTOR and p70^S6K. The activation of HSCs was also inhibited by the three ligands as shown by inhibition of vitamin A lipid droplets depletion from HSCs. Studies using real time PCR and western blot analysis showed marked inhibition of collagen Iα1 and αSMA by the combination of the three ligands. These findings suggest that the combined use of PJ, ATRA and 9-cis causes inhibition of cell proliferation by cell cycle arrest and down-regulation of fibrotic markers to a greater extent compared to each of the ligands alone.     

维甲酸核受体RARα真核表达载体的构建、突变纠正及表达

目的构建维甲酸核受体RARα真核表达载体,并检测其在人肺腺癌细胞A549中表达。方法从小鼠巨噬细胞RAW264.7中提取总RNA,以RT-PCR法扩增RARαcDNA,克隆至真核表达载体pDsRed1-C1中,测序结果显示RARα第1040位A→G,导致其编码蛋白的氨基酸发生改变。通过二次PCR将其纠正,重组载体RedC1-RARα转化大肠埃希菌Top10,筛选阳性克隆做酶切及测序鉴定。脂质体瞬时转染A549细胞,在荧光显微镜下观察RARα的表达。RT-PCR法检测RARα的mRNA水平表达。结果通过RT-PCR及二次PCR得到RARαcDNA,构建其真核表达载体,脂质体瞬时转染A549细胞得到了成功表达,RARα基因产物定位于细胞核内。结论成功构建维甲酸核受体RARα真核表达载体,且证实RARα编码蛋白定位于细胞核内,本研究结果为进一步探讨结核分枝杆菌固有免疫机制奠定了基础。     

腺病毒介导siRNA抑制全反式维甲酸诱导的骨髓间充质干细胞RARβ表达

构建携带针对大鼠维甲酸受体β(Retinoic acid receptorβ,RARβ)基因的siRNA重组腺病毒,并感染全反式维甲酸(All-trans retinoic acid,ATRA)处理的骨髓间充质干细胞(Mesenchymal stem cells,MSCs),检测其对RARβ的表达及MSCs成神经分化的影响。设计针对大鼠RARβ的4对siRNA的DNA序列,体外退火形成双链,定向克隆至含有U6/H1双启动子的腺病毒穿梭质粒pSES-HUS,随后与腺病毒骨架质粒pAd-Easy1在BJ5183细菌中同源重组,并在HEK293细胞中包装获得重组腺病毒Ad-siRARβ。腺病毒感染大鼠MSCs后经ATRA处理24 h,Real-time、Western blotting及免疫荧光检测RARβ的表达情况。改良神经诱导培养基(Modified neuronal induction medium,MNM)诱导MSCs神经分化,Real-time PCR及免疫荧光检测神经相关蛋白表达。PCR、酶切及测序鉴定均证实siRNA正确克隆至腺病毒质粒中,腺病毒感染大鼠MSCs后可观察到60%以上的细胞有红色荧光蛋白(Red fluorescent protein,RFP)表达。经ATRA处理24 h,Real-time、Westernblotting及免疫荧光检测发现RARβ表达定位于细胞核,ATRA作用后MSCs中RARβ表达增高16.5±2.34倍(P<0.05),有3组siRNA能有效抑制ATRA诱导的RARβ表达增强,抑制率分别为(66.26±9.12)%、(48.70±5.78)%、(64.09±0.53)%(P<0.05),且以pool组效果最强,抑制率为(78.09±4.24)%(P<0.01)。ATRA联合MNM诱导MSCs成神经样细胞,表达相关神经特异蛋白Nestin、NSE、MAP-2、Tau,免疫荧光结果显示神经标志蛋白Nestin、NSE、Tju1表达阳性细胞率为(50-88)%,而腺病毒介导的siRARβ能有效抑制MSCs的神经标志物表达水平及阳性细胞率(P<0.05)。成功构建了携带针对大鼠RARβ基因的siRNA重组腺病毒,能有效感染MSCs并显著抑制ATRA诱导的RARβ表达增强和MSCs的神经分化。     

COUP—TE和RARβ受体的表达影响癌细胞对视黄酸的敏感性

视黄酸(RA)通过其受体RARs和RXRs发挥作用。Northern blot显示,RARβ的表达介导RA对癌细胞的生长抑制作用。然而,其它的结果表明,RARβ的表达还不能完全驱动癌细胞对RA的应答,由此提示,癌细胞的RA敏感性除了受RARβ影响外,还受其它因子影响。COUP-TF则是其中的一个因子。凝胶阻抑测定和CAT测定表明,COUP-TF通过降低βRARE的基础转录活性来加强癌细胞的RA敏感性。结果证实,RARβ和COUP-TF受体的表达能够调节癌细胞对RA的敏感性。     

Detection of RASSF1A and RAR? Hypermethylation in Serum DNA from Breast Cancer Patients

Breast cancer is fast emerging as the leading cancer amongst females, especially in young females in metropolitan cities in India. The epigenetic alterations involved in the onset and progression of breast cancer may serve as biomarkers for early detection and prognosis of the disease. Furthermore, using body fluids such as serum offers a non-invasive method to procure multiple samples for such analyses. In this study, we examined methylation status of two normally unmethylated but biologically significant cancer genes, RAS association domain family protein 1A (RASSF1A) and Retionic acid receptor ? (RAR?) by Methylation Specific PCR (MSP) in invasive ductal carcinomas of the breast and paired serum DNA. RASSF1A was found to be methylated in 17 of 20 (85%) breast tumors; while sera from 15 of 20 (75%) of the patients showed concordant methylated RASSF1A, with a sensitivity of 88%. RAR? was methylated in 2/20 (10%) breast tumors. A gene unmethylated in the tumor DNA was always found to be unmethylated in the matched serum DNA for both RASSF1A and RAR? genes; hence specificity was 100%. Immunohistochemical analysis of RAR? protein in 15 breast carcinoma patients harboring unmethylated RAR? in tumors and serum DNA showed the expression of RAR? protein in tumors and paired normal breast tissues, confirming the MSP findings, suggesting that RAR? promoter is functional in these cases. This study underscores the potential utility of DNA methylation based screening of serum, a readily accessible body fluid, as a surrogate marker for early detection of breast cancer.