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A novel, retinoic acid-induced gene, GRP1-associated scaffold protein (GRASP), was isolated from P19 embryonal carcinoma cells using a subtractive screening strategy. GRASP was found to be highly expressed in brain and exhibited lower levels of expression in lung, heart, embryo, kidney, and ovary. The predicted amino acid sequence of GRASP is characterized by several putative protein-protein interaction motifs, suggesting that GRASP may be a component of a larger protein complex in the cell. Although GRASP does not harbor a predicted membrane spanning domain(s), the protein was observed to be associated with the plasma membrane of transiently transfected mammalian cells. Yeast two-hybrid screening revealed that GRASP interacted strongly with the General Receptor for Phosphoinositides 1 (GRP1), a brefeldin A-insensitive guanine nucleotide exchange factor for the ADP-ribosylation factor family of proteins. GRASP. GRP1 interactions were also demonstrated in vitro and in mammalian cells in which GRASP was shown to enhance GRP1 association with the plasma membrane. Furthermore, GRASP colocalized with endogenous ADP-ribosylation factors at the plasma membrane in transfected cells, suggesting that GRASP may modulate signaling by this family of small GTPases.

The cardiac Na+-Ca2+ exchanger (NCX1) is the principal Ca2+ efflux mechanism in cardiocytes. The exchanger is up-regulated in both cardiac hypertrophy and failure. In this report, we identify the cis-acting elements that control cardiac expression and alpha-adrenergic up-regulation of the exchanger gene. Deletion analysis revealed that a minimal cardiac promoter fragment from -184 to +172 is sufficient for cardiac expression and alpha-adrenergic stimulation. Mutational analysis revealed that both the CArG element at -80 and the GATA element at -50 were required for cardiac expression. Gel mobility shift assay supershift analysis demonstrated that the serum response factor binds to the CArG element and GATA-4 binds to the GATA element. Point mutations in the -172 E-box demonstrated that it was required for alpha-adrenergic induction. In addition, deletion analysis revealed one or more enhancer elements in the first intron (+103 to +134) that are essential for phenylephrine up-regulation but bear no homology to any known transcription element. Therefore, this work demonstrates that SRF and GATA-4 are critical for NCX1 expression in neonatal cardiomyocytes and that the -172 E-box in addition to a novel enhancer element(s) are required for phenylephrine up-regulation of NCX1 and may mediate its hypertrophic up-regulation.

Retinoic acid receptors (RARs) and retinoid X receptors (RXRs) mediate the effects of retinoids on gene expression by binding to response elements in retinoid-sensitive genes. RAR- but not RXR-selective retinoids were found in many previous studies to suppress the growth of various cells, implicating RXR-RAR in these effects. Using a co-expression vector for identifying cells that expressed retinoid receptors transiently and 5'-bromo-2'-deoxyuridine incorporation for labeling DNA-synthesizing cells, we found that RXR-selective retinoids inhibited DNA synthesis in squamous carcinoma 1483 cells transfected with RXRalpha but not with RARs. Ligand-induced transcription of the reporter luciferase gene via the activation of RXR-RXR but not RXR-RAR correlated with growth suppression. Studies with RXRalpha deletion mutants indicated that the DNA binding and the ligand binding domains are essential for mediating growth inhibition. A point mutation in the ligand binding domain (L430F) that decreased RXRalpha homodimerization compromised its growth inhibitory function. Further, RXRalpha mutant (F313A), which functions as a constitutively active receptor, inhibited DNA synthesis in the absence of ligand. These results demonstrate that RXR homodimer activation leads to growth inhibition and suggest that transfection of RXRalpha and treatment with RXR-selective retinoids or the transfection of constitutively activated RXRalpha mutant alone may have a therapeutic potential.

Naturally occurring retinoids, like all-trans retinoic acid and 9-cis retinoic acid, are known to affect proliferation and differentiation of sensitive neuroblastoma cell lines. Cellular responsiveness to retinoic acid depends on its interaction with two distinct classes of receptors, the retinoic acid receptors (RARs) and the retinoic X receptors (RXRs). Both receptor classes have three different subtypes (RARalpha, RARbeta, and RARgamma and RXRalpha, RARbeta, and RARgamma) that act as ligand-dependent transcription factors. To examine the involvement of the different receptor classes and subtypes in the biological responses of neuroblastoma cells to retinoids, we analyzed the effects of a panel of receptor-selective retinoids on cell growth, differentiation, and gene expression on in vitro cultured KCNR cells. Any association of per se inactive RXR-selective with RAR-selective ligands efficiently regulates growth inhibition, differentiation (neurite extension), and expression of RARbeta, TrkB, and N-myc. SR11383 alone, a very potent retinoid, entirely reproduces the pattern of biological responses induced by naturally occurring retinoids. In contrast to other tumor cell lines, the growth of neuroblastoma cell lines is not altered using AP1-antagonistic retinoids. These studies raise the possibility that three distinct RXR/RAR heterodimers mediate the effects of retinoids on neuroblastoma cells through an AP-1 antagonism-independent mechanism.

The Na+-Ca2+ exchanger (NCX1) plays a major role in calcium efflux and therefore in the control and regulation of intracellular calcium in the heart. The exchanger has been shown to be regulated at several levels including transcription. NCX1 mRNA levels are up-regulated in both cardiac hypertrophy and failure. In this work, the 5'-end of the ncx1 gene has been cloned to study the mechanisms that mediate hypertrophic stimulation and cardiac expression. The feline ncx1 gene has three exons that encode 5'-untranslated sequences that are under the control of three tissue-specific promoters. The cardiac promoter drives expression in cardiocytes, but not in mouse L cells. Although it contains at least one enhancer (-2000 to -1250 base pairs (bp)) and one or more negative elements (-1250 to -250 bp), a minimum promoter (-250 to +200 bp) is sufficient for cardiac expression and alpha-adrenergic stimulation.

Apolipoprotein D (apoD) is a human plasma protein, belonging to the lipocalin superfamily, that is produced by a specific subtype of highly differentiated breast carcinomas and that is strongly up-regulated by retinoic acid (RA) in breast cancer cells. In this work, we have examined the molecular mechanisms mediating the induction of apoD gene expression by retinoids in T-47D human breast cancer cells. Northern blot analysis revealed that Ro40-6055, a synthetic retinoid that selectively binds and activates the retinoic acid receptor RARalpha, induced the accumulation of apoD mRNA in breast cancer cells in a time- and dose-dependent manner. The time course analysis demonstrated that apoD mRNA was induced 14-fold over control cells after 48 h of incubation with 10(-8) M Ro40-6055. As little as 10(-11) M of this retinoid induced apoD mRNA 5-fold over the control, whereas incubation with 10(-7) M Ro40-6055 induced maximally 15-fold over control cells. RARalpha-selective antagonists counteracted the inductive effects of all-trans-RA, 9-cis-RA, and Ro40-6055 on the expression of apoD, when present at the same concentration as the retinoid agonists. By contrast, RARbeta-, RARgamma-, and RXR-selective retinoids did not affect apoD gene expression. The retinoid agonist Ro40-6055 had an antiproliferative effect on T-47D cells, with maximal growth inhibition of approximately 60% obtained after 7 days of incubation with 10(-7) M. This antiproliferative effect could be counteracted by a 100-fold excess of the antagonist Ro41-5253. Treatment of the cells with retinoids that do not bind the nuclear retinoic acid receptors did not affect apoD expression, despite the fact that they did have a strong antiproliferative effect on T-47D cells. On the basis of these results, a role for RARalpha on apoD gene expression induction by retinoids in breast cancer cells is proposed.

Retinoids regulate a broad range of biological processes through two subfamilies of nuclear retinoid receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Recently, we reported a novel type of retinoic acid antagonist (SR11335) and showed that this compound can inhibit retinoic acid (RA)-induced activation of a human immunodeficiency virus type 1 (HIV-1) promoter construct that contains a special RA response element (RARE). We have now further characterized the antagonism mediated by SR11335 and of newly synthesized structurally related compounds. Two compounds, SR11330 and SR11334, which are poor transactivators, also showed antagonist activities, inhibiting all-trans-RA (tRA) and 9-cis-RA. The retinoids inhibited transcriptional activation of RAR/RXR heterodimers effectively, while inhibition of RXR homodimers was less efficient. Inhibition was observed on several RAREs, including the TREpal, betaRARE, apoAI-RARE, and CRBPI-RARE. In addition, the antagonists inhibited tRA-induced differentiation of HL-60 cells. The antagonist did not interfere with DNA binding of the receptors. In limited proteolytic digestion assays, SR11335 induced resistance of the receptors to proteolysis, but the pattern of the degradation was not altered from that induced by tRA, suggesting that these antagonists induce their biological effects by competing with agonists for binding to RARs, thereby preventing the induction of conformational changes of the receptors necessary for transcriptional activation.

Retinoids, including retinol and retinoic acid derivatives, inhibit the growth of normal human bronchial epithelial (HBE) cells. The signaling pathways through which retinoids mediate this effect have not been defined. Normal HBE cell growth is stimulated by treatment with a variety of growth factors that increase mitogen-activated protein (MAP) activity. In this study, we examined MAP kinase-dependent pathways as potential targets of retinoid signaling and the role of MAP kinases in retinoid-induced c-fos gene regulation. All-trans-retinoic acid (t-RA) inhibited Jun N-terminal kinase (JNK) and, to a lesser extent, extracellular signal-regulated kinase activity in normal HBE cells. t-RA reduced c-fos mRNA and protein levels by decreasing c-fos gene transcription. The c-fos promoter was activated by co-transfection with a constitutively active JNK kinase (SEK)-1 and suppressed by a dominant negative JNK kinase kinase (MEKK)-1. Furthermore, c-fos expression was inhibited by agonists of retinoic acid receptors (RARs) or retinoid X receptors (RXRs), and suppression of c-fos promoter activity by t-RA was abrogated by treatment with antagonists of RAR-alpha or of all the RXRs. These findings provide the first evidence that t-RA inhibits JNK activity and demonstrate a potential role of JNK-dependent pathways in the suppression of c-fos expression by t-RA. Furthermore, c-fos expression was inhibited through activation of RAR- and RXR-dependent signaling pathways. In light of the growth activation induced by JNK/SEK-dependent pathways in a variety of cells, these data support further investigation into the role of JNK-dependent signaling in the growth-suppressive effects of retinoids.

In this study, we show that all-trans-retinoic acid (RA) is a potent inducer of tissue transglutaminase (TGase II) and apoptosis in the rat tracheobronchial epithelial cell line SPOC-1. We demonstrate that these cells express the retinoid receptors RAR alpha, RAR gamma, and RXR beta. To identify which of these receptors are involved in regulating these processes, we analyzed the effects of several receptor-selective agonists, an antagonist, and a dominant-negative RAR alpha. We show that the RAR-selective retinoid SRI-6751-84 strongly increased TGase II expression at both the protein and mRNA levels, whereas the RXR-selective retinoid SR11217 had little effect. The RAR alpha-selective retinoid Ro40-6055 was also able to induce TGase II, whereas the RAR gamma-selective retinoid CD437 was inactive. The induction of TGase II by the RAR-selective retinoid was completely inhibited by the RAR alpha-antagonist Ro41-5253. Overexpression of a truncated RAR alpha gene with dominant-negative activity also inhibited the induction of TGase II expression. The increase in TGase II is associated with an induction of apoptosis as revealed by DNA fragmentation and the generation of apoptotic cells. We demonstrate that apoptosis is affected by retinoids in a manner similar to TGase II. Our results suggest that the induction of TGase II expression and apoptosis in SPOC-1 cells are mediated through an RAR alpha-dependent signaling pathway.