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.     

Mapping of an inducible element in the T cell receptor V beta 2 promoter.

The murine V beta 2 promoter was analyzed for an element regulating phorbol ester inducibility of the TCR beta chain gene. In transient expression analysis of 5' nested deleted fragments of the V beta 2 promoter, the TPA-inducible element mapped between -85 and -42. The -85 to -62 oligo conferred 12-0-tetradecanoylphorbol-13-acetate (TPA) inducibility to the heterologous TPA-uninducible thymidine kinase promoter. The -85 to -62 region contained an AP-1 site (-85 to -72) and inverted repeat motif (-72 to -62). The AP-1 site required the 3' flanking inverted repeat region for conferring optimal inducibility. In vitro transcribed and translated jun/fos heterodimers bind to the V beta 2 AP-1 motif with a 16-fold lower affinity as compared to the collagenase AP-1 motif. This explains the inability of the V beta 2 AP-1 motif to confer optimal TPA inducibility by itself. The affinity of jun/fos heterodimers for the V beta 2 AP-1 motif was not increased by the presence in cis of the inverted repeat motif. The 3' flanking inverted repeat binds the ets transactivator but not jun/fos heterodimers. The demonstrated cooperativity between the AP-1 and the 3' flanking sequence to confer TPA inducibility can thus be explained by the individual contributions of jun/fos and ets transactivators.     

Characterization of an inverted repeat with a zero spacer (IR0)-type retinoic acid response element from the mouse nuclear orphan receptor TR2-11 gene.

An inverted repeat with zero nucleotides in the spacer (IR0, 5'-GGGTCA CGAACT-3') element was localized in the proximal promoter region of the mouse TR2-11 gene, and characterized as a functional retinoic acid response element (RARE). In gel mobility shift assays, heterodimers of retinoic acid receptor alpha (RARalpha) and retinoid X receptor beta (RXRbeta) bound specifically to this element. Neither receptor alone was able to bind to this element efficiently. The dissociation constant (Kd) with respect to RAR-RXR binding was estimated to be 8 nM. The biological activity of this IR0 element was assessed in a heterologous reporter system. The IR0-containing reporter was induced by RA in COS-1 cells in the presence of exogenously provided RARalpha and RXRbeta. In addition, the IR0 oligomers could be bound by nuclear extracts isolated from COS-1 cells harboring the expression vectors for RARalpha and RXRbeta, but not by extracts isolated from control COS-1 cells. RA responsiveness of this IR0 was further confirmed in P19 cells that expressed endogenous RARs and RXRs. Collectively, these data demonstrated, for the first time, the presence of a natural RARE of the IR0 type, and suggested a potential cross-talk between nuclear orphan receptor TR2-11 and RAR-RXR families.     

Retinoic acid response element in HOXA-7 regulatory region affects the rate,not the formation of anterior boundary expression

Since it is known that a 307 bp fragment of the position specific regulatory element of human HOXA-7 contains two (DR3 and DR5) retinoic acid response elements (RAREs) at its 3' end, we constructed several deletion constructs containing different numbers of RAREs and examined their effects in vitro and in vivo. The 5' deletion constructs, BM112 and OM213, retaining both RAREs, were highly responsible (about 8 fold induction) for RA in F9 teratocarcinoma cells, versus NM307 (4-5 fold). The construct NS218, with both RAREs deleted but retaining the 5' sequences lost RA responsibility completely, whereas NR271, with one RARE(DR5) deleted retained a 50% inducibility (2.5 fold). In vivo transgenic analysis revealed that the constructs NM307 and NR271, but not OM213 nor BM112, directed the position specific expression pattern. Sequence analysis revealed that HOXA-7 enhancer sequences, including the RARE repeat sequences, were well conserved in human, mouse and chick. Part of the RAREs overlaps with the CDX1 binding site, and sequences of the DR3 RAREs were identical in this species. Two GAGA binding sites were also found to be strictly conserved. Because OM213, which had one GAGA site disrupted but retaining both RAREs, did not direct anterior boundary formation in transgenic mice, these results suggest the importance of the 5' 94 bp region, including the GAGA binding site, in anterior boundary formation and the involvement of the RARE in the rate of expression not in anterior boundary formation.     

Characterization of a retinoic acid responsive element isolated by whole genome PCR.

We have used whole PCR in an attempt to isolate novel retinoic acid (RA) responsive genes. We cloned several small genomic fragments from total human DNA containing putative retinoic acid responsive elements (RAREs) selected by direct binding to the retinoic acid receptor alpha (RAR alpha). We report here that an oligonucleotide containing a sequence from one of the cloned human DNA fragments, and referred to as alpha 1, functions as an authentic RARE. It is shown that both RAR alpha and RAR beta produced in Cos cells as well as in vitro translated RAR alpha bind directly and sequence-specifically to the alpha 1RARE. By mutational analysis it is demonstrated that the alpha 1RARE consists of an imperfect direct repeat of the estrogen- and thyroid hormone-related AGGTCA half-site motif separated by a 5 bp spacer. The orientation and spacing of the half-site repeats are shown to play a critical role in RAR recognition. When cloned upstream of a TK-Luc reporter, the alpha 1RARE is shown to confer responsiveness to RA in an orientation-independent fashion in F9 and CV-1 cells. The magnitude of the RA response mediated by the alpha 1RARE differed in these cell lines.     

Functional characterization of a natural retinoic acid responsive element.

Retinoic acid receptor (RAR) and thyroid hormone receptor (T3R) are thought to bind as dimers to a T3 responsive element (T3REpal) comprised of inverted repeats of the half-site motif GGTCA. However, a RA responsive element (beta RARE) was previously identified in the promoter of the RAR beta 2 gene which contains two direct repeats of the motif GTTCA spaced by a six nucleotide gap. We now demonstrate the ability of RAR alpha, beta and gamma to bind to and transactivate through this element and that the two direct repeats comprise the beta RARE. Surprisingly, the GTTCA motifs rearranged to form a palindrome do not confer RA responsiveness to a heterologous promoter. Furthermore, no significant level of transactivation is detected by ligand-activated RAR through the Moloney murine leukaemia virus T3RE, which comprises two direct repeats of the sequence GGTCA/C spaced by a five nucleotide gap. Similarly, T3R does not induce gene expression through the beta RARE. This study establishes the preference of T3R to transactivate through direct repeats spaced by a five nucleotide gap as opposed to a six nucleotide gap. In contrast, RAR appears to be more flexible with respect to spacing requirements between repeats, although higher levels of transactivation are obtained through direct repeats spaced by a six nucleotide gap. Interestingly, although some elements mediate either RA or T3 induction, changing a single nucleotide in the MoMLV T3RE with a five nucleotide spacing creates a promiscuous RA/T3 responsive element.     

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.     

Murine hoxd4 expression in the CNS requires multiple elements including a retinoic acid response element

We have identified a retinoic acid response element (RARE) within a neural enhancer located 3' to the Hoxd4 gene. This RARE is required for the initiation and maintenance of Hoxd4 transgene expression in neurectoderm, and for full anteriorized expression upon retinoic acid (RA) treatment. Mutations within the sequence TTTTCTG, located 2 bp downstream of the RARE, posteriorized transgene activity. However, the onset of transgene expression and its response to RA were indistinguishable from wild type. While the TTTTCTG motif resembles a CDX binding site, human CDX1 protein did not interact with this element in vitro. Three additional regions were also shown to control transgene expression in neurectoderm, establishing that multiple elements constitute the Hoxd4 neural enhancer.