Promoter Characterization and Role of cAMP/PKA/CREB in the Basal Transcription of the Mouse ORMDL3 Gene

Orosomucoid 1-like 3 (ORMDL3) gene was strongly linked with the development of asthma in genetic association studies, and its expression could be significantly induced by allergen in airway epithelial cells of mice. However, the expression mechanism of ORMDL3 was still unclear. Here we have identified and characterized the mouse ORMDL3 gene promoter. Deletion constructs of the 5′ flanking region were fused to a luciferase reporter gene. After transient transfection in mouse fibroblast cell line NIH3T3, a CRE (−27/−20) binding CREB was identified in the core promoter region. Deletion or mutation of the CRE consensus sequence resulted in a significant loss of the promoter activity. EMSA and ChIP assays demonstrated the binding of CREB to the core promoter. Knocking down endogenous CREB led to a reduction in ORMDL3 expression. Conversely, overexpression of CREB up-regulated ORMDL3 expression. Moreover, forskolin, a PKA activator, could facilitate the phosphorylation of CREB, which in turn heightens ORMDL3 expression. H-89, a PKA-specific inhibitor, could significantly inhibit ORMDL3 expression. This study delineates the characterization of mouse ORMDL3 gene promoter and shows signaling pathway cAMP/PKA/CREB plays an important role in regulating ORMDL3 expression, which will be helpful for future animal model studies regarding the regulation or function of ORMDL3 gene.     

Retinoic acid activation and thyroid hormone repression of the human alcohol dehydrogenase gene ADH3.

Mammalian alcohol dehydrogenase (ADH) catalyzes the oxidation of retinol to retinaldehyde, the rate-limiting step in the synthesis of retinoic acid. There exists a family of ADH isozymes encoded by unique genes, and it is unclear which isozymes are most important for regulation of retinoic acid synthesis during differentiation or development. A region in the human ADH3 promoter from -328 to -272 base pairs was shown previously to function as a retinoic acid response element (RARE), prompting an hypothesis for a positive feedback mechanism controlling retinoic acid synthesis (Duester, G., Shean, M. L., McBride, M. S., and Stewart, M. J. (1991) Mol. Cell. Biol. 11, 1638-1646). The ADH3 RARE contains three direct AGGTCA repeats which constitute the critical nucleotides of RAREs present in other genes. We dissected the ADH3 RARE and determined that receptor binding as well as transactivation are dependent upon only the two downstream AGGTCA motifs separated by 5 base pairs, a structure noticed previously for a RARE in the promoter for the retinoic acid receptor beta (RAR beta) gene. ADH3 and RAR beta RAREs functioned similarly in transfection assays, suggesting that the feedback mechanisms controlling ADH3 and RAR beta utilize a common RARE. We also found that the normal functioning of the ADH3 RARE was abrogated by thyroid hormone receptor in the presence of thyroid hormone. A negative thyroid hormone response element in the human ADH3 promoter was found to colocalize with the RARE. Since ADH production in rat liver is known to be repressed by thyroid hormone, these findings suggest that human ADH production may also be subject to thyroid hormone repression and that the mechanism involves an interference with retinoic acid induction.     

Retinoic acid response element in the human alcohol dehydrogenase gene ADH3: implications for regulation of retinoic acid synthesis.

Retinoic acid regulation of one member of the human class I alcohol dehydrogenase (ADH) gene family was demonstrated, suggesting that the retinol dehydrogenase function of ADH may play a regulatory role in the biosynthetic pathway for retinoic acid. Promoter activity of human ADH3, but not ADH1 or ADH2, was shown to be activated by retinoic acid in transient transfection assays of Hep3B human hepatoma cells. Deletion mapping experiments identified a region in the ADH3 promoter located between -328 and -272 bp which confers retinoic acid activation. This region was also demonstrated to confer retinoic acid responsiveness on the ADH1 and ADH2 genes in heterologous promoter fusions. Within a 34-bp stretch, the ADH3 retinoic acid response element (RARE) contains two TGACC motifs and one TGAAC motif, both of which exist in RAREs controlling other genes. A block mutation of the TGACC sequence located at -289 to -285 bp eliminated the retinoic acid response. As assayed by gel shift DNA binding studies, the RARE region (-328 to -272 bp) of ADH3 bound the human retinoic acid receptor beta (RAR beta) and was competed for by DNA containing a RARE present in the gene encoding RAR beta. Since ADH catalyzes the conversion of retinol to retinal, which can be further converted to retinoic acid by aldehyde dehydrogenase, these results suggest that retinoic acid activation of ADH3 constitutes a positive feedback loop regulating retinoic acid synthesis.     

Regulation of the murine alpha(2)(I) collagen promoter by retinoic acid and retinoid X receptors

Retinoic acid decreases collagen production by hepatic stellate cells. This study investigated the effects of retinoic acid receptor beta (RARbeta) and retinoid X receptor alpha (RXRalpha) on the regulation of the alpha(2)(I) collagen promoter. Retinoic acid and the RARbeta and RXRalpha expression vectors suppressed the promoter in transfected stellate cells with maximal suppression obtained when combined. Mutation of the retinoic acid response element (RARE) at -879 to -874 (site 1) enhanced promoter activity and diminished but did not eliminate the suppression by RARbeta and RXRalpha. Mutation of another RARE site (site 2), at -930 to -911, resulted in low activity that was inhibited by retinoic acid. Mutation of the AP-2-binding site enhanced promoter activity that was inhibited by retinoic acid. This study shows that the suppressive effect of retinoic acid on the promoter is maximal with a combination of RARbeta and RXRalpha and occurs at more than one RARE site. The effect of retinoic acid is not mediated by AP-2.     

Convergence of cAMP, TGF-beta and retinoic acid signaling pathways in cells of the embryonic palate.

We have previously described bi-directional cross-talk between the retinoic acid (RA) and transforming growth factor beta (TGF-beta) signal transduction pathways in primary cultures of murine embryonic palate mesenchymal (MEPM) cells. In this paper we identify interactions between the TGF-beta1, cyclic adenosine 3', 5'-monophosphate (cAMP) and RA signaling systems. TGF-beta1 and forskolin, an activator of the cAMP pathway, inhibited RA-induced expression of RAR-beta mRNA in MEPM cells, though only TGF-beta1 inhibited RA-induced RAR-beta protein expression. Forskolin, but not TGF-beta1, abrogated RA-induced expression of a reporter construct containing 900 base pair (bp) of the RAR-beta gene promoter, transfected into MEPM cells, suggesting that this portion of the promoter contains the forskolin-responsive, but not the TGF-beta-responsive, element. Thus, a putative TGF-beta Inhibitory Element (TIE) adjacent to the retinoic acid response element (RARE) in the RAR-beta promoter is either non-functional, or requires promoter/enhancer elements not present in the promoter construct used in these experiments. These studies further clarify the complex interactions among signal transduction pathways in the regulation of retinoic acid receptor gene expression.     

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     

All‐trans retinoic acid upregulates the expression of ciliary neurotrophic factor in retinal pigment epithelial cells

Retinopathy of prematurity, a leading cause of visual impairment in low birth‐weight infants, remains a crucial therapeutic challenge. Ciliary neurotrophic factor (CNTF) is a promyelinating trophic factor that promotes rod and cone photoreceptor survival and cone outer segment regeneration in the degenerating retina. Ciliary neurotrophic factor expression is regulated by many factors such as all‐trans retinoic acid (ATRA). In this study, we found that ATRA increased CNTF expression in mouse retinal pigment epithelial (RPE) cells in a dose‐ and time‐dependent manner, and PKA signaling pathway is necessary for ATRA‐induced CNTF upregulation. Furthermore, we showed that ATRA promoted CNTF expression through CREB binding to its promoter region. In addition, CNTF levels were decreased in serum of retinopathy of prematurity children and in retinal tissue of oxygen‐induced retinopathy mice. In mouse RPE cells cultured with high oxygen, CNTF expression and secretion were decreased, but could be recovered after treatment with ATRA. In conclusion, our data suggest that ATRA administration upregulates CNTF expression in RPE cells.     

Induction of human aquaporin-1 gene by retinoic acid in human erythroleukemia HEL cells

Retinoids have been implicated in the control of cell proliferation, differentiation, and developmental processes. We report here that aquaporin-1 (AQP1) is specifically induced by retinoic acid (RA) in human erythroleukemia HEL cells. Both all-trans-RA (ATRA) and 9-cis-RA (9CRA) strongly induced the AQP1 mRNA and protein in a dose-dependent manner. AQP1 protein was mainly expressed in plasma membrane in cells induced by RAs. To identify the RA response element (RARE) in the human AQP1 promoter, the 5(')-flanking region of AQP1 promoter was isolated and transient transfection experiment in HEL cells was performed. Deletion analysis of the AQP1 promoter revealed that one putative DR5-like RARE with five spaces was located in the region from -2218 to -2202; AGGGCAgggacAGGTGA. Electrophoretic mobility shift assay (EMSA) experiment demonstrated that two slowly migrated complexes (C1 and C2) capable of binding the RARE sequence were present in nuclear extracts prepared from cells and the complex C1 was strongly increased in nuclear extracts by RA stimulation. The complexes C1 and C2 were significantly abolished by an excess unlabeled probe. These results indicate that RAs strongly stimulate the human AQP1 gene expression through the RARE and define a novel role in the regulation of erythropoiesis.     

T-box binding protein type two (TBX2) is an immediate early gene target in retinoic-acid-treated B16 murine melanoma cells

Retinoic acid induces growth arrest and differentiation in B16 mouse melanoma cells. Using gene arrays, we identified several early response genes whose expression is altered by retinoic acid. One of the genes, tbx2, is a member of T-box nuclear binding proteins that are important morphogens in developing embryos. Increased TBX2 mRNA is seen within 2 h after addition of retinoic acid to B16 cells. The effect of retinoic acid on gene expression is direct since it does not require any new protein synthesis. We identified a degenerate retinoic acid response element (RARE) between -186 and -163 in the promoter region of the tbx2 gene. A synthetic oligonucleotide spanning this region was able to drive increased expression of a luciferase reporter gene in response to retinoic acid; however, this induction was lost when a point mutation was introduced into the RARE. This oligonucleotide also specifically bound RAR in nuclear extracts from B16 cells. TBX2 expression and its induction by retinoic acid was also observed in normal human and nonmalignant mouse melanocytes.     

Regulation of niemann-pick c1 gene expression by the 3'5'-cyclic adenosine monophosphate pathway in steroidogenic cells

The Niemann Pick-C1 (NPC-1) protein is essential for intracellular transport of cholesterol derived from low-density lipoprotein import in mammalian cells. The role of the protein kinase A (PKA) pathway in regulation of expression of the NPC-1 gene was investigated. NPC-1 promoter activity was induced by treatment with dibutryl cAMP (dbcAMP), alone or in combination with the cAMP response element (CRE) binding protein (CREB) overexpressed in adrenal Y-1 cells. When the catalytic subunit of PKA was overexpressed in Y-1 cells, there were similar increases in NPC-1 promoter activity in the presence of CREB. Responses were attenuated by blockade of the PKA pathway, and in the Kin-8 cell line deficient in PKA. Promoter deletion analysis revealed that this response was present in promoter fragments of 186 bp and larger but not present in the 121-bp fragment. Two promoter regions, one at -430 and one at -120 upstream of the translation initiation site, contained CRE consensus sequences. These bound recombinant CREB in EMSA, confirming their authenticity as CREB response elements. Promoters bearing mutations of both CRE displayed no response to dbcAMP. The orphan nuclear receptor, steroidogenic factor-1 (SF-1), was implicated in NPC-1 transactivation by the presence of SF-1 target sequence that formed a complex with recombinant SF-1 in EMSA. Furthermore, transfection of a plasmid that overexpressed SF-1 into ovarian granulosa cells increased promoter activity in response to dbcAMP, an effect abrogated by mutation of the SF-1 target sequence. Chromatin immunoprecipitation assays demonstrated that the CRE region of the endogenous and transfected NPC-1 promoter associated with both acetylated and phosphorylated histone H-3 and that this association was increased by dbcAMP treatment. Treatment with dbcAMP also increased the association of the CRE region of the promoter with CREB binding protein, which has histone acetyltransferase activity. Together, these results demonstrate a mechanism of regulation of NPC-1 expression by the cAMP-PKA pathway that includes PKA phosphorylation of CREB, recruitment of the coactivator CREB binding protein and the phosphorylation and acetylation of histone H-3 to transactivate the NPC-1 promoter.     

ATRA-regulated Asb-2 gene induced in differentiation of HL-60 leukemia cells

Suppressors of cytokine signaling (SOCS) proteins possess common structures, a SOCS box at the C-terminus and a SH2 domain at their center. These suppressors are inducible in response to cytokines and act as negative regulators of cytokine signaling. The ASB proteins also contain the SOCS box and the ankyrin repeat sequence at the N-terminus, but do not have the SH2 domain. Although Socs genes are directly induced by several cytokines, no Asb gene inducers have been identified. In this study, we screened the specific genes expressed in the course of differentiation of HL-60 cells, and demonstrated that ASB-2, one of the ASB proteins, was rapidly induced by all-trans retinoic acid (ATRA). Typical retinoid receptors (RARs) or retinoid X receptors (RXRs) binding element (RARE/RXRE) were presented in the promoter of the Asb-2 gene. We showed that RARalpha, one of the RARs, binds to the RARE/RXRE in the Asb-2 promoter. In addition, we demonstrated by luciferase reporter assay that this element was a functional RARE/RXRE. These findings indicate that ASB-2 is directly induced by ATRA and may act as a significant regulator, underlying such physiological processes as cell differentiation.     

Effect of retinoic acid on the enhancing effect of acetaldehyde on mouse type I collagen expression

Acetaldehyde alone and retinoic acid alone have been shown to increase and decrease, respectively, collagen production by stellate cells in culture. In this study the effects of retinoic acid on alpha(1)(I) and alpha(2)(I) collagen expression and its influence on the enhancing effects of acetaldehyde were determined. Retinoic acid decreased the activation of the alpha(2)(I) collagen promoter and decreased the message of alpha(2)(I) collagen in cultured stellate cells, but had no effect on either the activation of the alpha(1)(I) collagen promoter or on the alpha(1)(I) collagen message. This depressant effect of retinoic acid was also evident in the transfected alpha(2)(I) collagen promoter mutated at the retinoic acid response element (RARE). The activation of the alpha(2)(I) collagen promoter by acetaldehyde was not decreased significantly by retinoic acid, but was suppressed by the retinoic acid receptor (RAR) selective retinoid SRI-6751-84. Retinoic acid, however, decreased the acetaldehyde-induced enhancement of the alpha(1)(I) and alpha(2)(I) collagen messages. Acetaldehyde also resulted in a decrease in RAR beta message and RARbeta protein. This study shows that retinoic acid depresses alpha(2)(I) collagen gene expression but that this effect is less pronounced when the expression of this collagen is enhanced by acetaldehyde, which also decreases RARbeta message and protein. Furthermore, the action of retinoic acid in inhibiting alpha(2)(I) collagen gene expression occurs at sites other than the RARE site.