Inhibitory Effect Of Retinoic Acid Research Articles

Retinoic acid inhibits the angiogenesis of human embryonic stem cell-derived endothelial cells by activating FBP1-mediated gluconeogenesis

BackgroundEndothelial cells are located in the inner lumen of blood and lymphatic vessels and exhibit the capacity to form new vessel branches from existing vessels through a process called angiogenesis. This process is energy intensive and tightly regulated. Glycolysis is the main energy source for angiogenesis. Retinoic acid (RA) is an active metabolite of vitamin A and exerts biological effects through its receptor retinoic acid receptor (RAR). In the clinic, RA is used to treat acne vulgaris and acute promyelocytic leukemia. Emerging evidence suggests that RA is involved in the formation of the vasculature; however, its effect on endothelial cell angiogenesis and metabolism is unclear.MethodsOur study was designed to clarify the abovementioned effect with human embryonic stem cell-derived endothelial cells (hESC-ECs) employed as a cell model.ResultsWe found that RA inhibits angiogenesis, as manifested by decreased proliferation, migration and sprouting activity. RNA sequencing revealed general suppression of glycometabolism in hESC-ECs in response to RA, consistent with the decreased glycolytic activity and glucose uptake. After screening glycometabolism-related genes, we found that fructose-1,6-bisphosphatase 1 (FBP1), a key rate-limiting enzyme in gluconeogenesis, was significantly upregulated after RA treatment. After silencing or pharmacological inhibition of FBP1 in hESC-ECs, the capacity for angiogenesis was enhanced, and the inhibitory effect of RA was reversed. ChIP-PCR demonstrated that FBP1 is a target gene of RAR. When hESC-ECs were treated with the RAR inhibitor BMS493, FBP1 expression was decreased and the effect of RA on angiogenesis was partially blocked.ConclusionsThe inhibitory role of RA in glycometabolism and angiogenesis is RAR/FBP1 dependent, and FBP1 may be a novel therapeutic target for pathological angiogenesis.

All-Trans Retinoic Acid Attenuates Blue Light-Induced Apoptosis of Retinal Photoreceptors by Upregulating MKP-1 Expression.

The study investigated the antiapoptotic effects of all-trans retinoic acid (RA) on retinal degeneration caused by exposure to blue light. Sprague-Dawley rats received intraperitoneal injections of RA and, if necessary, the mitogen-activated protein kinase phosphotase-1(MKP-1) inhibitor, (E)-2-benzylidene-3-(cyclohexylamino)-2, 3-dihydro-1H-inden-1-one (BCI), or the retinoic acid receptor (RAR) antagonist, AGN 193109. Retinal damage was induced by 24 h of continuous exposure to blue light. Haematoxylin and eosin staining and electroretinography were performed to measure retinal thickness and retinal function before and at 3 days and 7 days after light exposure. The retinal protein expression levels of phosphorylated c-Jun N-terminal kinase (JNK), phosphorylated nuclear factor-κB, MKP-1, Bim, Bax, and cleaved caspase-3 were also measured. Terminal-deoxynucleotidyl-transferase-mediated deoxyuridine triphosphate-biotin nick end labelling (TUNEL) staining and immunofluorescent staining of cleaved caspase-3 were also performed to evaluate photoreceptor apoptosis. The administration of RA significantly mitigated retinal dysfunction and the decrease in the outer nuclear layer (ONL) thickness at 3 days and 7 days after light exposure. RA also reduced the percentage of TUNEL-positive nuclei in the ONL and cleaved caspase-3 immunofluorescence intensity at 3 days after light exposure. Light exposure increased the retinal expression of proapoptotic proteins (Bim, Bax, and cleaved caspase-3), which was attenuated by RA. Moreover, RA enhanced the expression of MKP-1 and inhibited the phosphorylation of JNK, which were attenuated by the inhibition of RAR. The inhibitory effects of RA on blue light-induced photoreceptor apoptosis were abrogated by the MKP-1inhibitor. Our results indicate that RA alleviates photoreceptor loss following blue light exposure, at least partly, by the MKP-1/JNK pathway, which may serve as a therapeutic target for relieving retinal degeneration.

The Control of Stromal Vascular Cell Differetiation by Retinoic Acid and Calcium in Hanwoo Beef Cattle Adipose Tissue

Stromal vascular cells (SVC) from the perirenal adipose tissue of castrated Hanwoo beef cattle steers were isolated and cultured in medium containing different concentrations of calcium and retinoic acid. The cells were stimulated to differentiate in defined medium until lipid accumulation was apparent. The cell viability of adipose tissue SVC were not affected by the different concentrations of calcium and retinoic acid in the media. No observable cytotoxic effect was apparent among the cells treated with various levels of calcium and retinoic acid. Oil-red O staining of differentiated adipose tissue SVC shows that as the level of calcium and retinoic acid decreased, the degree of adipocyte differentiation increased. This shows that manipulating the extracellular level of calcium and retinoic acid in perirenal adipose tissue SVC can result in inhibition or stimulation of adipogenesis. RT-PCR and western blot analysis of adipogenic related genes and proteins clearly showed that the reduction in the level of calcium and retinoic acid in the media, stimulated adipogenesis. The expression level of PPARγ, C/EBPα, SREBP-1c and aP2 were significantly upregulated by the diminution of the calcium and retinoic acid. The expression of major calcium buffering protein calreticulin was also down regulated thus, diminishing its inhibitory effect on PPARγ. Retinoic acid related receptors RARα and CRABP-II were also down regulated upon the reduction of calcium and retinoic acid which may account for the decreased inhibitory effect of retinoic acid on adipocyte differentiation. These results suggest the possible mechanism of action for the increased adipocyte differentiation in Hanwoo beef cattle adipose stromal vascular cells upon decreasing the extracellular calcium and retinoic acid concentration. These findings may be applied in the future to further develop micronutrient manipulations to increase intramuscular fat accumulation in Hanwoo beef cattle.

Retinoic acid protects cardiomyocytes from high glucose‐induced apoptosis through inhibition of NF‐κB signaling Pathway

We have previously shown that retinoic acid (RA) has protective effects on high glucose (HG)-induced cardiomyocyte apoptosis. To further elucidate the molecular mechanisms of RA effects, we determined the interaction between nuclear factor (NF)-κB and RA signaling. HG induced a sustained phosphorylation of IKK/IκBα and transcriptional activation of NF-κB in cardiomyocytes. Activated NF-κB signaling has an important role in HG-induced cardiomyocyte apoptosis and gene expression of interleukin-6 (IL-6), tumor necrosis factor (TNF)-α, and monocyte chemoattractant protein-1 (MCP-1). All-trans RA (ATRA) and LGD1069, through activation of RAR/RXR-mediated signaling, inhibited the HG-mediated effects in cardiomyocytes. The inhibitory effect of RA on NF-κB activation was mediated through inhibition of IKK/IκBα phosphorylation. ATRA and LGD1069 treatment promoted protein phosphatase 2A (PP2A) activity, which was significantly suppressed by HG stimulation. The RA effects on IKK and IκBα were blocked by okadaic acid or silencing the expression of PP2Ac-subunit, indicating that the inhibitory effect of RA on NF-κB is regulated through activation of PP2A and subsequent dephosphorylation of IKK/IκBα. Moreover, ATRA and LGD1069 reversed the decreased PP2A activity and inhibited the activation of IKK/IκBα and gene expression of MCP-1, IL-6, and TNF-α in the hearts of Zucker diabetic fatty rats. In summary, our findings suggest that the suppressed activation of PP2A contributed to sustained activation of NF-κB in HG-stimulated cardiomyocytes; and that the protective effect of RA on hyperglycemia-induced cardiomyocyte apoptosis and inflammatory responses is partially regulated through activation of PP2A and suppression of NF-κB-mediated signaling and downstream targets.

Retinoic acid influences neuronal migration from the ganglionic eminence to the cerebral cortex

The ganglionic eminence contributes cells to several forebrain structures including the cerebral cortex, for which it provides GABAergic interneurons. Migration of neuronal precursors from the retinoic-acid rich embryonic ganglionic eminence to the cerebral cortex is known to be regulated by several factors, but retinoic acid has not been previously implicated. We found retinoic acid to potently inhibit cell migration in slice preparations of embryonic mouse forebrains, which was reversed by an antagonist of the dopamine-D(2) receptor, whose gene is transcriptionally regulated by retinoic acid. Histone-deacetylase inhibitors, which amplify nuclear receptor-mediated transcription, potentiated the inhibitory effect of retinoic acid. Surprisingly, when retinoic acid signalling was completely blocked with a pan-retinoic acid receptor antagonist, this also decreased cell migration into the cortex, implying that a minimal level of endogenous retinoic acid is necessary for tangential migration. Given these opposing effects of retinoic acid in vitro, the in vivo contribution of retinoic acid to migration was tested by counting GABAergic interneurons in cortices of adult mice with experimental reductions in retinoic acid signalling: a range of perturbations resulted in significant reductions in the numerical density of some GABAergic interneuron subpopulations. These observations suggest functions of retinoic acid in interneuron diversity and organization of cortical excitatory-inhibitory balance.

Action of retinoic acid receptor on EGFR gene transactivation and breast cancer cell proliferation: Interplay with the estrogen receptor

In the present report, we investigated the action of retinoic acid (RA) on the transactivation of the epidermal growth factor receptor (EGFR) gene promoter. In a previous study, we showed that the estrogen receptor (ER) α activated by 17β-estradiol (E 2) increased EGFR expression by enhancing the binding of the transcription factor Sp1 to the EGFR minimal promoter in HeLa cells. Here, we demonstrate that ligand-activated RA receptor (RAR) α inhibited EGFR transactivation by competing with Sp1 for binding to the same promoter fragment in the same cell model. When RARα and ERα were coexpressed, the inhibitory effect of RA on transactivation of the EGFR promoter counteracted the enhancement induced by E 2-activated ERα and became more pronounced in the presence of ligand-free ERα. In the MCF7 breast cancer cell line, which endogenously expresses RARα and ERα, RA exerted anti-proliferative effects in the presence of ligand-free ERα. Moreover, interplay between the pathways mediated by the two receptors was observed, as RA counteracted E 2-induced cell proliferation. Our results suggest that the interference with the activity of Sp1 on the EGFR promoter could be related to the observed RA-mediated growth suppression of breast cancer cells.

Transcription Factor Smad3 Is Required for the Inhibition of Adipogenesis by Retinoic Acid

The process of adipocyte differentiation is driven by a highly coordinated cascade of transcriptional events that results in the development of the mature adipocyte and in lipid accumulation. One of the early events of differentiation is the up-regulation of CCAAT/enhancer-binding protein beta (C/EBPbeta) expression. C/EBPbeta then acts to up-regulate the expression of adipogenic factors such as C/EBPalpha, which control the late stage of adipogenesis. Retinoic acid (RA) is a potent inhibitor of adipogenesis, and its action appears to block C/EBPbeta transcriptional potential early during differentiation. Using preadipocytes and mesenchymal stem cell models, we show that RA specifically blocks the occupancy of C/EBPbeta of the Cebpa promoter, thereby abrogating the differentiation process. RA does not act directly on C/EBPbeta but rather stimulates the expression of the transforming growth factor beta-effector protein Smad3, which can interact with C/EBPbeta via its Mad homology 1 domain and can interfere with C/EBPbeta DNA binding. The RA-induced increase in Smad3 expression results in increased cytoplasmic and nuclear Smad3, an important event as ectopic expression of Smad3 in preadipocytes in the absence of RA treatment only modestly inhibits adipogenesis and C/EBPbeta DNA binding, suggesting that Smad3 alone is not sufficient to completely recapitulate the effects of retinoic acid treatment during differentiation. However, in the absence of Smad3, RA is not able to inhibit adipocyte differentiation or to elicit a decrease in C/EBPbeta DNA occupancy suggesting that Smad3 is necessary to convey the inhibitory effects of retinoic acid during adipogenesis.

Abstract A30: NF1 is a tumor suppressor in neuroblastoma that determines retinoic acid response and disease outcome

Abstract Neuroblastoma is a malignancy of early childhood that derives from the developing neural crest and accounts for 15% of all paediatric oncology deaths. The vitamin A derivative retinoic acid (RA) controls neural development and promotes differentiation, cell cycle arrest and apoptosis of neuronal and neuroblastoma cells. These diverse effects of RA are exerted primarily through the ability to differentially regulate gene expression mediated by the retinoic acid receptors (RARs). RA is used in the clinic to treat high-risk neuroblastoma patients, however with variable success. This variability in clinical response to RA is enigmatic as no mutations in components of the RA signaling cascade have been found. By using large-scale RNAi-based genetic screens, we aimed to identify predictive biomarkers for RA sensitivity in neuroblastoma. We found an unexpected crosstalk between the tumor suppressor neurofibromin (NF1) and RA signaling. NF1 is a negative regulator of RAS signaling that promotes conversion of the active GTP bound form into the inactive GDP bound form of RAS. Activation of RAS-MEK signaling by loss of NF1 repressed ZNF423, a critical transcriptional co-activator of the retinoic acid receptors, and blocked RA induced differentiation and target gene activation. Restoration of ZNF423 levels resensitized NF1 knockdown cells to the inhibitory effect of RA. Moreover, ZNF423 and NF1 expression predicted sensitivity to RA in a large panel of neuroblastoma cell lines. Neuroblastomas with low levels of both NF1 and ZNF423 have extremely poor outcome. We found NF1 mutations in neuroblastoma cell lines and also in primary tumors. Inhibition of MEK signaling downstream of NF1 restored responsiveness to RA, suggesting a therapeutic strategy to overcome RA resistance in NF1 deficient neuroblastomas. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A30

Inhibitory effects of retinoic acid on invasiveness of human thyroid carcinoma cell lines in vitro

The prognosis of patients with metastasized thyroid carcinoma is not optimistic, necessitating the search for new treatment options. Beneficial effects of retinoic acid (RA) have been suggested in thyroid cancer differentiation and the present study was performed to investigate the anti-metastatic potential of RA in respect of important determinants of metastatic behavior in thyroid carcinoma, focusing on the role of invasion-associated proteins. Differentiated thyroid carcinoma cell lines FTC- 133 and XTC.UC1, and anaplastic thyroid cancer cell lines C643 and HTH74 were studied. All cell lines were cultured with alltrans- RA (ATRA) or the solvent ethanol. Invasion and adhesion potency in vitro was studied by transwell experiment and short-term adhesion assay. The involvement of invasion-associated proteins, urokinase type plasminogen activator (uPA), uPA receptor (uPAR), matrix metalloproteinase-2 (MMP-2) and E-cadherin, were investigated by semi-quantitative RT-PCR and Western blot. In vitro invasion assay revealed that ATRA treatment could reduce the invasive potency in all the thyroid cancer cell lines, with the most significant effect in anaplastic cancer cells. Short-term adhesion assay suggested that ATRA increases cancer cell adhesion to extracellular matrix (ECM) in C643, HTH74 and XTC.UC1, probably through a transcriptional and translational regulation of some attachment molecules. RT-PCR andWestern blot both revealed diminished expression of uPAR in all four carcinoma cell lines. In C643 and HTH74 cell lines, the expression of uPA was reduced and the expression of E-cadherin was increased, whereas the MMP-2 expression was not significantly down-regulated in ATRA-treated group. In ATRA-treated FTC-133 and XTC.UC1 cell lines, MMP-2 expression was decreased, but no significant changes in uPA and E-cadherin expression were observed. The present study demonstrates the influence of ATRA on both important determinants of metastatic behavior ("de-adhesion" and proteolysis) in thyroid carcinoma cell lines, especially in anaplastic cancer cells. These findings may add to the explanations for beneficial effects of RA in the treatment of metastatic thyroid carcinomas.

Opposing effects of retinoid signaling on astrogliogenesis in embryonic day 13 and 17 cortical progenitor cells

All-trans retinoic acid (RA) is a differentiation factor in many tissues. However, its role in astrogliogenesis has not been extensively studied. Here, we investigated the effect of RA on the regulation of astrogliogenesis at different cortical developmental stages. We prepared rat cortical progenitor cells from embryonic day (E) 13 and E17, which correspond to the beginning of neurogenic and astrogliogenic periods, respectively. Surprisingly, RA promoted astrogliogenesis at E17 but inhibited astrogliogenesis induced by ciliary neurotrophic factor (CNTF) at E13. The inhibitory effect of RA on astrogliogenesis at E13 was not due to premature commitment of progenitors to a neuronal or oligodendroglial lineage. Rather, RA retained more progenitors in a proliferative state. Furthermore, RA inhibition of astrogliogenesis at E13 was independent of STAT3 signaling and required the function of the alpha and beta isoforms of the RA receptors (RAR). Moreover, the differential response of E13 and E17 progenitors to RA was due to differences in the intrinsic properties of these cells that are preserved in vitro. The inhibitory effect of RA on cytokine-induced astrogliogenesis at E13 may contribute to silencing of any potential precocious astrogliogenesis during the neurogenic period.

On the role of transforming growth factor-β in the growth inhibitory effects of retinoic acid in human pancreatic cancer cells

BackgroundRetinoids are potent growth inhibitory and differentiating agents in a variety of cancer cell types. We have shown that retinoids induce growth arrest in all pancreatic cancer cell lines studied, regardless of their p53 and differentiation status. However, the mechanism of growth inhibition is not known. Since TGF-β2 is markedly induced by retinoids in other cancers and mediates MUC4 expression in pancreatic cancer cells, we investigated the role of TGF-β in retinoic acid-mediated growth inhibition in pancreatic cancer cells.ResultsRetinoic acid markedly inhibited proliferation of two cell lines (Capan-2 and Hs766T) in a concentration and time-dependent manner. Retinoic acid increased TGF-β2 mRNA content and secretion of the active and latent forms of TGF-β2 (measured by ELISA and bioassay). The concentrations of active and TGF-β2 secreted in response to 0.1 – 10 μM retinoic acid were between 1–5 pM. TGF-β2 concentrations within this range also inhibited proliferation. A TGF-β neutralizing antibody blocked the growth inhibitory effects of retinoic acid in Capan-2 cells and partially inhibitory the effects in Hs766T cells.ConclusionThese findings indicate that TGF-β can cause growth inhibition of pancreatic cancer cells, in a p53-independent manner. Furthermore, it demonstrates the fundamental role of TGF-β in growth inhibition in response to retinoic acid treatment is preserved in vitro.

Inhibitory effects of retinoic acid and IIF on growth, migration and invasiveness in the U87MG human glioblastoma cell line

Glioblastomas, the most malignant and prevalent brain tumors which remain incurable, are characterized by both extensive proliferation and invasive growth. We previously reported a remarkable antitumoral effect of the retinoid 6-OH-11-O-hydroxyphenantrene (IIF) on neuroblastoma, leukemia and colon carcinoma cells. In this study we examined the effect of IIF on proliferation, apoptosis and cell invasion in the human glioblastoma cell line U87MG, in comparison with all-trans-retinoic acid (RA). Our results showed that both retinoids induced cell growth inhibition and apoptosis in a dose- and time-dependent manner. We also demonstrated that the invasive ability of glioblastoma cells decreased after treatment with IIF or RA. Since cell invasion involves a complex system of tightly regulated proteases, matrix metalloproteinases (MMPs) and their specific inhibitors, tissue inhibitors of MMPs (TIMPs), we analysed the effect of IIF on MMP and TIMP expression in comparison with RA. Treatment with both retinoids resulted in a marked decrease of MMP2 and MMP9 expression and of lytic activity of MMP2. In addition, exposure to IIF led to enhanced expression of TIMP2. Collectively, our results demonstrated the effectiveness of both IIF and RA in inhibiting proliferation, cell migration, and the invasive potential of glioblastoma U87MG cells. Notably, the anticancer activity of IIF, on the whole, was more pronounced than that of RA. Therefore, these findings, besides providing further evidence that IIF may be a powerful tool in the development of cancer treatments, suggest that IIF may have therapeutic potential against the invasiveness of brain tumors.

5-Aza-2′-deoxycytidine induces retinoic acid receptor- β2 demethylation and growth inhibition in esophageal squamous carcinoma cells

Esophageal squamous cell carcinoma (ESCC) is one of the most common tumors in human. Previous studies showed that multiple genetic and epigenetic alterations involved in carcinogenesis of esophagus, whereas the molecular mechanisms are poorly understood. So far, more and more ESCC-related genes have been found and retinoic acid receptor β 2 ( RARβ 2) is such a gene which was recognized as a putative tumor suppressor gene since reduced RARβ 2 mRNA expression has been observed in several solid tumors, including ESCC. A growing evidence indicated that RARβ 2 was required for the growth inhibitory effect of retinoic acid (RA). However, the molecular mechanism of its inactivation remained obscure in ESCC. The RARβ2 methylation status was assessed by methylation-specific PCR (MSP) in 12 ESCC cell lines and compared with their mRNA and protein expression level. Bisulfite sequencing of RARβ 2 promoter region was performed to confirm the MSP results. After 5-aza-2′-deoxycytidine (5-aza-dc) treatment the expression of RARβ 2 was reversed in two RARβ 2-downregulated cell lines. Therefore, hypermethylation of the promoter regions of RARβ2 gene is a major mechanism of transcriptional inactivation and might be involved in tumor development of esophagus in some ESCC cell lines suggesting that multiple mechanisms contribute to the loss of RARβ 2 expression in ESCC cell lines. Furthermore, the methylation status of RARβ 2 promoter region and its expression was analyzed in 51 ESCC tissue samples with their adjacent normal epithelia and two normal esophageal epithelia. The results showed that there was a statistically significant correlation between methylation status of RARβ 2 and tumor grade; Moreover, a relationship between methylation status and decreased RARβ 2 expression was found only in G 2 stage tumors. After 5-aza-dc treatment, RARβ 2 restoration was accompanied by growth inhibition and this might be one of the mechanisms but not the only mechanism for the tumor cell growth inhibition by 5-aza-dc. This study may have clinical applications for ESCC therapy and prevention.

Mitogen-activated Protein Kinases and Mitogen-activated Protein Kinase Phosphatases Mediate the Inhibitory Effects of All-trans Retinoic Acid on the Hypertrophic Growth of Cardiomyocytes

All-trans retinoic acid (RA) has been implicated in mediation of cardiac growth inhibition in neonatal cardiomyocytes. However, the associated signaling mechanisms remain unclear. Utilizing neonatal cardiomyocytes, we demonstrated that RA suppressed the hypertrophic features induced by cyclic stretch or angiotensin II (Ang II). Cyclic stretch- or Ang II-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAP kinase) was dose- and time-dependently inhibited by RA. Significant inhibition was observed by 5 microm RA, from 8 to 24 h of pretreatment. This inhibitory effect was not mediated at the level of mitogen-activated protein kinase kinases (MKKs), because RA had no effect on stretch- or Ang II-induced phosphorylation of MEK1/2, MKK4, and MKK3/6. However, the phosphatase inhibitor vanadate reversed the inhibitory effect of RA on MAP kinases and protein synthesis. RA up-regulated the expression level of MAP kinase phosphatase-1 (MKP-1) and MKP-2, and the time course was correlated with the inhibitory effect of RA on activation of MAP kinases. Overexpression of wild-type MKP-1 inhibited the phosphorylation of JNK and p38 in cardiomyocytes. These data indicated that MKPs were involved in the inhibitory effect of RA on MAP kinases. Using specific RAR and RXR antagonists, we demonstrated that both RARs and RXRs were involved in regulating stretch- or Ang II-induced activation of MAP kinases. Our findings provide the first evidence that the anti-hypertrophic effect of RA is mediated by up-regulation of MKPs and inhibition of MAP kinase signaling pathways.

124 Peptidomimetic inhibitors of Stat3: structure—activity relationships and cellular activity

Retinoic acid (RA) strongly inhibits proliferation of the estrogen (E2)-dependent human breast cancer cell lines MCF7, T47D, and ZR75-1, but not the E2-independent and E2 receptor (ER)-negative lines MDA-MB231, MDA-MB468, BT20 and Hs578T. The specific sensitivity of the E2-dependent cell lines seems not to be caused by an inhibitory effect of RA on ER functioning since RA inhibited the proliferative response not only to E2 but also to insulin. Furthermore, endogenous RA receptors (RARs) hardly impaired transcriptional activation of an E2 responsive element-tk-CAT reporter construct.RARα mRNA was highly expressed in the RA-responsive lines, but not in the unresponsive lines, except BT20. With the exception of Hs578T, also RARβ mRNA expression was low in the unresponsive lines. While in the dependent lines and Hs578T RA activated RA responsive element-dependent transcriptional activity, this response was very low in MDA-MB231, MDA-MB468, and BT20, suggesting that the RA resistance of these latter three ER-negative lines is due to underexpression of functional RARs. Our results suggest that the loss of functional RARs may be a frequent event, leading to RA unresponsiveness of ER-negative breast cancer cells. This implies that both the steroid and retinoid receptor status of breast tumors may be used to predict a successful treatment with retinoids.

Retinoic acid inhibits chondrogenesis of mesenchymal cells by sustaining expression of N-cadherin and its associated proteins.

Retinoic acid (RA) is a well-known regulator of chondrocyte phenotype. RA inhibits chondrogenic differentiation of mesenchymal cells and also causes loss of differentiated chondrocyte phenotype. The present study investigated the mechanisms underlying RA regulation of chondrogenesis. RA treatment in chondrifying mesenchymal cells did not affect precartilage condensation, but blocked progression from precartilage condensation to cartilage nodule formation. This inhibitory effect of RA was independent of protein kinase C and extracellular signal-regulated protein kinase, which are positive and negative regulators of cartilage nodule formation, respectively. The progression from precartilage condensation to cartilage nodule requires downregulation of N-cadherin expression. However, RA treatment caused sustained expression of N-cadherin and its associated proteins including alpha- and beta-catenin suggesting that modulation of expression of these molecules is associated with RA-induced inhibition of chondrogenesis. This hypothesis was supported by the observation that disruption of the actin cytoskeleton by cytochalasin D (CD) blocks RA-induced sustained expression of cell adhesion molecules and overcomes RA-induced inhibition of chondrogenesis. Taken together, our results suggest RA inhibits chondrogenesis by stabilizing cell-to-cell interactions at the post-precartilage condensation stage.

Mechanism of retinoic acid induced attenuation of PTH action in UMR 106-01 cells.

Studies from our laboratory in osteoblast-like cells have shown that the increase in EGF receptor expression in response to PTH was cyclic AMP mediated and was blocked by treatment with retinoic acid (RA). The present studies investigate the mechanism for this effect of RA on PTH actions. UMR 106-01 cells were exposed to RA and were tested for cAMP response to PTH as well as for (125)I PTH binding. cAMP production in response to PTH was markedly decreased by RA (25.1 +/- 1.6% of control) whereas there was only a slight decrease in PTH binding in response to RA. For the study of adenylate cyclase activity, membranes were isolated from intact cells that had been exposed to RA. Treatment with RA decreased PTH-stimulated adenylate cyclase activity; however, forskolin-stimulated enzyme activity was unchanged. Treatment of intact cells with pertussis toxin, to inactivate Gi, did not alter the inhibitory effect of RA on PTH-stimulated adenylate cyclase activity. Addition of GppNHp, a non-hydrolyzable analogue of GTP, completely restored the response to PTH in the membranes. Therefore, we examined the activity of IMP dehydrogenase, the rate-limiting enzyme for GTP biosynthesis, and GMP reductase which counteracts the effect of the synthetic enzyme. Treatment with RA for 48 hours increased GMP reductase activity by 240.9 +/- 24.2% and decreased IMP dehydrogenase activity to 67.5 +/- 8.8% of control values. These data indicate that RA impairs the response to PTH in intact cells. This blunted response was preserved in membrane preparations but was corrected by GTP. The RA-induced alterations of enzymes involved in the GTP biosynthetic pathway in a direction that favors a decrease in GTP biosynthesis provide an explanation for the inhibitory effect of RA on PTH actions.

Loss of growth inhibitory effects of retinoic acid in human breast cancer cells following long-term exposure to retinoic acid.

Although retinoids are known to be inhibitory to breast cancer cell growth, a key remaining question is whether they would remain effective if administered long-term. We describe here the long-term effects of all-trans retinoic acid on two oestrogen-dependent human breast cancer cell lines MCF7 and ZR-75-1. Although both cell lines were growth inhibited by retinoic acid in the short-term in either the absence or the presence of oestradiol, prolonged culture with 1 μM all-trans retinoic acid resulted in the cells acquiring resistance to the growth inhibitory effects of retinoic acid. Time courses showed that oestrogen deprivation of the cell lines resulted in upregulation of the basal non-oestrogen stimulated growth rate such that cells learned to grow at the same rate without as with oestradiol, but the cells remained growth inhibited by retinoic acid throughout. Addition of 1 μM all-trans retinoic acid to steroid deprivation conditions resulted in reproducible loss of growth response to both retinoic acid and oestradiol, although the time courses were separable in that loss of growth response to retinoic acid preceded that of oestradiol. Loss of growth response to retinoic acid did not involve loss of receptors, ER as measured by steroid binding assay or RARα as measured by Northern blotting. Function of the receptors was retained in terms of the ability of both oestradiol and retinoic acid to upregulate pS2 gene expression, but there was reduced ability to upregulate transiently transfected ERE- and RRE-linked reporter genes. Despite the accepted role of IGFBP3 in retinoic acid-mediated growth inhibition, progression to retinoic acid resistance occurred irrespective of level of IGFBP3, which remained high in the resistant MCF7 cells. Measurement of AP1 activity showed that the two cell lines had markedly different basal AP1 activities, but that progression to resistance was accompanied in both cases by a lost ability of retinoic acid to reduce AP1 activity. These results warn of potential resistance which could arise on long-term treatment with retinoic acid in a clinical situation and echo the problems of progression to endocrine resistance. It seems that whatever the constraints imposed on growth, these cells have a remarkable ability to escape from growth inhibition. However, the ability of retinoic acid to delay progression to oestrogen resistance is encouraging for endocrine therapy, and the concentration-dependence of retinoic acid resistance suggests that progression is not absolute but could be manipulated by dose. © 2000 Cancer Research Campaign

Inhibitory effect of retinoic acid on the development of immature porcine granulosa cells to mature cells.

The present study investigated the effect of retinoic acid (RA) on the differentiation of granulosa cells prepared from porcine ovaries. The granulosa cells were precultured for 15 h, then cultured for 48 h with FSH and further treated for 24 h with LH in order to induce their transformation into luteal cells. After the cells had been exposed to 1 microM retinoids (RA, retinal and retinol) for 87 h, analysis of the LH receptor mRNA expression, an indicator of granulosa cell differentiation, was carried out by using semiquantitative RT-PCR. The results showed that there was a decrease in LH receptor mRNA levels, and that RA had a more potent effect on these levels than the other two retinoids. When cells were exposed to RA in the immature stage (before the addition of FSH) or the early stage of development (0-24 h after the addition of FSH), expression of LH receptor mRNA was greatly diminished. When the immature cells were cultured for 15 h with RA, then washed and cultured for 48 h with FSH and for 24 h with LH, the expression of LH receptor mRNA was not reversed. In the differentiated cells (24 h after the addition of FSH), however, RA no longer had any inhibitory effect. When the immature cells were exposed to RA, FSH-induced expression of c-fos mRNA was markedly decreased. In contrast, expression of c-jun and activating transcription factor-4 mRNAs remained constant. However, the expression of c-fos mRNA was not decreased by forskolin. The results indicate that RA is a potent inhibitor in the immature stage of porcine granulosa cell differentiation, probably through decreased expression of FSH receptor, but that RA does not inhibit differentiation in the mature stage of the cells.

Stage-dependent responses of the developing lung to retinoic acid signaling.

Morphological analysis of vitamin A-deficient rat fetuses and of retinoic acid receptor (RAR and RXR) mutant mice have demonstrated that retinoic acid (RA) is essential for lung development. To gainfurther insight into RA signaling pathways during primary lung budformation and lung branching, we have investigated the effects of RA and of a pan-RAR antagonist in cultures of whole embryos and lung explants. Treatment of E8.0 embryos with the pan-RAR antagonist inhibits the formation of the primitive respiratory system. On the other hand, treatment of E11.75 and E12.5 lung explants with RA inhibits branching morphogenesis, whereas treatment with the pan-RAR antagonist at the same developmental stages stimulates formation of distal buds. The inhibitory effect of RA on branching is strongly decreased in RARbeta null lungs, while enhancement of budding by the pan-RAR antagonist is not affected by an RARgamma null mutation. Additionally, cellular retinol binding protein one (CRBPI) null lungs are more sensitive than wild type lungs to the pan-RAR antagonist-induced stimulation of branching. These data indicate that retinoid signaling is indispensable for the formation of primary lung buds and the oesophagotracheal septum from the primitive foregut. They also suggest that at the pseudoglandular stage, RA signaling through RARbeta, but not RARgamma, inhibits distal bud formation thereby promoting the formation of conducting airways. Moreover, the level of CRBPI in the pseudoglandular lung appears to participate in the control of branching morphogenesis.