血清对全反式视黄酸抑制肺癌细胞生长的影响

 研究不同浓度的血清对全反式视黄酸 (ATRA)抑制肺癌细胞生长的影响 .当细胞培养在 10 %血清中 ,ATRA不能抑制肺癌细胞生长 ,但是当细胞培养在 1%血清中 ,ATRA能够有效地抑制肺癌细胞生长 .视黄酸受体RARβ介导视黄酸的抗癌作用 .Northern印迹分析表明 ,在高浓度血清中AT RA不能诱导RARβ表达 ,但在低浓度血清中ATRA可以诱导RARβ表达 ,并且瞬时转染和CAT测定证实是通过激活RABβ启动子转录活性而诱导RARβ表达的 .孤生受体Nur77受到血清生长因子刺激后会大量表达 ,具有抗视黄酸活性的作用 .肺癌细胞培养在低浓度血清中 ,Nur77mRNA低水平表达和Nur77蛋白不表达 .然而在高浓度血清中 ,Nur77mRNA和蛋白高水平表达 .另外 ,在无血清条件下 ,EGF也可以诱导Nur77表达 .结果提示 ,血清中的生长因子可能拮抗ATRA抑制肺癌细胞生长的作用 ,其作用途径可能是通过刺激细胞中Nur77表达 ,或者通过下调RARβ启动子的转录活性而抑制RARβ的表达     

E2F1 impairs all-trans retinoic acid-induced osteogenic differentiation of osteosarcoma via promoting ubiquitination-mediated degradation of RARα

All-trans retinoic acid (ATRA) is a widely used differentiation drug that can effectively induce osteogenic differentiation of osteosarcoma cells, but the underlying mechanism remains elusive, which limits the clinical application for ATRA in osteosarcoma patients. In this study, we identified E2F1 as a novel regulator involved in ATRA-induced osteogenic differentiation of osteosarcoma cells. We observed that osteosarcoma cells are coupled with individual differences in the expression levels of E2F1 in patients, and E2F1 impairs ATRA-induced differentiation of osteosarcoma cells. Moreover, remarkable anti-proliferative and differentiation-inducing effects of ATRA treatment are only observed in E2F1 low to negative expressed primary osteosarcoma cultures. These results strongly suggested that E2F1 may serve as a potent indicator for the effectiveness of ATRA treatment in osteosarcoma. Interestingly, E2F1 is found to downregulate retinoic acid receptor α (RARα), a key factor determines the effectiveness of ATRA. E2F1 specifically binds to RARα and promotes its ubiquitination-mediated degradation; as a consequence, RARα-mediated differentiation is inhibited in osteosarcoma. Therefore, our studies present E2F1 as a potent biomarker, as well as a therapeutic target for ATRA-based differentiation therapeutics, and raise the hope of using differentiation-based approaches for osteosarcoma patients.     

胃癌细胞中视黄酸受体抑制AP-1活性的不同方式

 研究胃癌细胞中视黄酸受体RARα和RARβ抑制活化蛋白 1(activatorprotein 1,AP 1)活性的不同方式及其与全反式视黄酸 (ATRA)作用的相关性 .瞬时转染RARβ表达载体到MKN 4 5细胞后 ,佛波脂 (TPA)诱导的AP 1活性受到明显抑制 ,且与RARβ浓度正相关 ,与ATRA存在与否无关 ;相反 ,RARα转染细胞后 ,对TPA诱导的AP 1活性的抑制不仅与RARα的浓度相关 ,而且依赖于AT RA .凝胶阻抑测定表明 ,TPA可以显著加强AP 1结合活性 ,当ATRA处理不表达RARβ和低表达RARα的MKN 4 5细胞后 ,AP 1结合活性不受影响 ;然而 ,表达RARα和RARβ的BGC 82 3细胞经AT RA处理后 ,TPA诱导的AP 1结合活性则受到抑制 .另外 ,分析与抗AP 1活性相关的RARβ功能区表明 ,DNA结合区的缺失导致RARβ抑制AP 1活性作用的丧失 ,而配体结合区对于RARβ抑制AP 1活性则是非必需的 .以上结果证实 ,有胃癌细胞中 ,RARβ可能是AP 1活性的抑制因子 ,RARα则可能是ATRA作用的靶向 .尽管它们的作用方式有所不同 ,但最终都可以通过抑制AP 1活性来抑制胃癌细胞生长     

Retinoic acid receptor signalling directly regulates osteoblast and adipocyte differentiation from mesenchymal progenitor cells

Low and high serum retinol levels are associated with increased fracture risk and poor bone health. We recently showed retinoic acid receptors (RARs) are negative regulators of osteoclastogenesis. Here we show RARs are also negative regulators of osteoblast and adipocyte differentiation. The pan-RAR agonist, all-trans retinoic acid (ATRA), directly inhibited differentiation and mineralisation of early osteoprogenitors and impaired the differentiation of more mature osteoblast populations. In contrast, the pan-RAR antagonist, IRX4310, accelerated differentiation of early osteoprogenitors. These effects predominantly occurred via RARγ and were further enhanced by an RARα agonist or antagonist, respectively. RAR agonists similarly impaired adipogenesis in osteogenic cultures. RAR agonist treatment resulted in significant upregulation of the Wnt antagonist, Sfrp4. This accompanied reduced nuclear and cytosolic β-catenin protein and reduced expression of the Wnt target gene Axin2, suggesting impaired Wnt/β-catenin signalling. To determine the effect of RAR inhibition in post-natal mice, IRX4310 was administered to male mice for 10 days and bones were assessed by µCT. No change to trabecular bone volume was observed, however, radial bone growth was impaired. These studies show RARs directly influence osteoblast and adipocyte formation from mesenchymal cells, and inhibition of RAR signalling in vivo impairs radial bone growth in post-natal mice.     

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 .     

Upregulation of transient receptor potential melastatin 6 channel expression by rosiglitazone and all-trans-retinoic acid in erlotinib-treated renal tubular epithelial cells

Anti-epidermal growth factor receptor (EGFR) drugs including erlotinib cause a side effect of hypomagnesemia. In lung adenocarcinoma A549 cells, anticancer agents such as cisplatin and doxorubicin dose-dependently increased toxicity, but the effects were significantly suppressed by culturing the cells in low Mg²⁺-containing media. To obtain the maximum effect in cancer chemotherapy, it should be necessary to prevent the reduction of body Mg ²⁺ content. Anti-EGFR drugs inhibit EGF-induced elevation of transient receptor potential melastatin 6 (TRPM6) Mg ²⁺ channel in renal tubular epithelial NRK-52E cells. Here, we found that rosiglitazone, an antidiabetic drug, and all- trans-retinoic acid (ATRA), a vitamin A derivative, increase the messenger RNA (mRNA) level of TRPM6 in the presence of erlotinib. The rosiglitazone- and ATRA-induced elevation of mRNA level, Mg ²⁺ influx, and promoter activity of TRPM6 were inhibited by GW-9662, a potent antagonist of peroxisome proliferator-activated receptor (PPAR)γ, and LE135, a retinoic acid receptor (RAR) antagonist, respectively. Rosiglitazone increased the phosphorylation and nuclear localization levels of PPARγ, which were inhibited by GW-9662. In contrast, RAR was mainly distributed in the nuclei under control conditions, which was unchanged by ATRA and LE135. The promoter activity of TRPM6 was inhibited by a mutation in the peroxisome proliferator hormone response element (PPRE). A chromatin immunoprecipitation assay revealed that PPARγ and RAR bind to the PPRE, which was blocked by GW-9662 and LE135, respectively. These results suggest that rosiglitazone and ATRA reverse the reduction in Mg ²⁺ reabsorption caused by anti-EGFR drugs.     

Expression and function of a retinoic acid receptor in budding ascidians

 Retinoic acid is thought to induce transdifferentiation of multipotent epithelial stem cells in the developing buds of the ascidian Polyandrocarpa misakiensis. We isolated a cDNA clone from this species, named PmRAR, encoding a retinoic acid receptor (RAR) homologue. PmRAR clusters with other RARs on phylogenetic trees constructed by three different methods. Within the cluster, PmRAR is on a separate branch from all the subtypes of RARs, suggesting that RAR subtypes arose in the ancestral vertebrates after divergence of vertebrates and urochordates. The embryos of another ascidian species Ciona intestinalis were co-electroporated with a mixture of a PmRAR expression vector and a lacZ reporter plasmid containing vertebrate-type retinoic acid response elements. The expression of lacZ depended on the presence of both retinoic acid and PmRAR, suggesting that PmRAR is a functional receptor. PmRAR mRNA is expressed in the epidermis and mesenchyme cells of the Polyandrocarpa developing bud. The mRNA is not detectable in the mesenchyme cells in the adult body wall, but its expression can be induced by retinoic acid in vitro. These results suggest that the PmRAR is a mediator of retinoic acid signalling in transdifferentiation during asexual reproduction of protochordates. Received: 6 April 1998 / Accepted: 27 July 1998     

Evidence for species differences in the regulation of MMPs by all-trans retinoic acid in cytokine-stimulated chondrocytes

In inflammatory conditions, chondrocytes produce large amounts of matrix metalloproteases (MMP) and nitric oxide (NO) thought to contribute to joint degradation. We tested the ability of all-trans retinoic acid (ATRA, a retinoic acid receptor (RAR) agonist) to modulate these inflammatory genes in chondrocytes from humans or rats, chosen as representative of animal models of arthritis. All RAR subtypes and RXR-alpha or -beta were expressed at the mRNA level in both species, although IL-1beta (10 ng/ml) inhibited RAR subtypes more markedly in rat than in human cells. ATRA (300 or 1000 nM) inhibited IL-1-induced expression of iNOS and nitrites level in both species, although the NO pathway was induced maximally in rat cells. ATRA displayed controversial effects on MMPs between rat and human chondrocytes, especially for MMP-9 expression. The effects of ATRA were irrelevant to the nuclear translocation of AP-1. The present data underlines that retinoids have a species-dependent impact on IL-1-induced responses in chondrocytes, suggesting that extrapolation of their pharmacological properties from animal cells has a poor relevance to clinical situation.     

Pharmacological evidence for the role of RAR in axon guidance and embryonic development of a protostome species

Retinoic acid (RA), the active metabolite of vitamin A, functions through nuclear receptors, one of which is the retinoic acid receptor (RAR). Though the RAR is essential for various aspects of vertebrate development, little is known about the role of RAR in nonchordate invertebrates. Here, we examined the potential role of an invertebrate RAR in mediating chemotropic effects of retinoic acid. The RAR of the protostome Lymnaea stagnalis is present in the growth cones of regenerating cultured motorneurons, and a synthetic RAR agonist (EC23), was able to mimic the effects of retinoic acid in inducing growth cone turning. We also examined the ability of the natural retinoids, all‐trans RA and 9‐cis RA, as well as the synthetic RAR agonists, to disrupt embryonic development in Lymnaea. Developmental defects included delays in embryo hatching, arrested eye, and shell development, as well as more severe abnormalities such as halted development. Developmental defects induced by some (but not all) synthetic RAR agonists were found to mimic those induced by addition of high concentrations of the natural retinoid isomers. These pharmacological data support a possible physiological role for the RAR in axon guidance and embryonic development of an invertebrate protostome species.     

Retinoid-dependent growth inhibition, differentiation and apoptosis in acute promyelocytic leukemia cells. Expression and activation of caspases

In the NB4 model of acute promyelocytic leukemia (APL), ATRA, 9-cis retinoic acid (9-cis RA), the pan-RAR and RARalpha-selective agonists, TTNPB and AM580, induce growth inhibition, granulocytic differentiation and apoptosis. By contrast, two RXR agonists, a RARbeta agonist and an anti-AP1 retinoid have very limited activity, ATRA- and AM580-dependent effects are completely inhibited by RAR antagonistic blockade, while 9-cis RA-induced cell-growth-inhibition and apoptosis are equally inhibited by RAR and RXR antagonists. ATRA, 9-cis RA and AM580 cause upregulation of the mRNAs coding for pro-caspase-1, -7, -8, and -9, which, however, results in increased synthesis of only pro-caspase-1 and -7 proteins. These phenomena are associated with activation of pro-caspase-6, -7 and -8, cytochrome c release from the mitochondria, inversion of Bcl-2/Bax ratio and degradation of PML-RARalpha. Caspase activation is fundamental for retinoid-induced apoptosis, which is suppressed by the caspase-inhibitor z-VAD.     

Extracellular transglutaminase 2 induces myotube hypertrophy through G protein-coupled receptor 56

Skeletal muscle secretes biologically active proteins that contribute to muscle hypertrophy in response to either exercise or dietary intake. The identification of skeletal muscle-secreted proteins that induces hypertrophy can provide critical information regarding skeletal muscle health. Dietary provitamin A, β-carotene, induces hypertrophy of the soleus muscle in mice. Here, we hypothesized that skeletal muscle produces hypertrophy-inducible secretory proteins via dietary β-carotene. Knockdown of retinoic acid receptor (RAR) γ inhibited the β-carotene-induced increase soleus muscle mass in mice. Using RNA sequencing, bioinformatic analyses, and literature searching, we predicted transglutaminase 2 (TG2) to be an all-trans retinoic acid (ATRA)-induced secretory protein in cultured C2C12 myotubes. Tg2 mRNA expression increased in ATRA- or β-carotene-stimulated myotubes and in the soleus muscle of β-carotene-treated mice. Knockdown of RARγ inhibited β-carotene-increased mRNA expression of Tg2 in the soleus muscle. ATRA increased endogenous TG2 levels in conditioned medium from myotubes. Extracellular TG2 promoted the phosphorylation of Akt, mechanistic target of rapamycin (mTOR), and ribosomal p70 S6 kinase (p70S6K), and inhibitors of mTOR, phosphatidylinositol 3-kinase, and Src (rapamycin, LY294002, and Src I1, respectively) inhibited TG2-increased phosphorylation of mTOR and p70S6K. Furthermore, extracellular TG2 promoted protein synthesis and hypertrophy in myotubes. TG2 mutant lacking transglutaminase activity exerted the same effects as wild-type TG2. Knockdown of G protein-coupled receptor 56 (GPR56) inhibited the effects of TG2 on mTOR signaling, protein synthesis, and hypertrophy. These results indicated that TG2 expression was upregulated through ATRA-mediated RARγ and that extracellular TG2 induced myotube hypertrophy by activating mTOR signaling-mediated protein synthesis through GPR56, independent of transglutaminase activity.