Expression and promoter analysis of a highly restricted integrin alpha gene in vascular smooth muscle.

Full genome annotation requires gene expression analysis and elucidation of promoter activity. Here, we analyzed the expression and promoter of a highly restricted integrin gene, Itga8. RNase protection and quantitative RT-PCR showed Itga8 to be expressed most abundantly in vascular smooth muscle cells (SMC). Transcription start site mapping of Itga8 revealed the immediate 5' promoter region to be poorly conserved with orthologous sequences in the human genome. Further comparative sequence analysis showed a number of conserved non-coding sequence modules around the Itga8 gene. The immediate promoter region and an upstream conserved sequence module were each found to contain a CArG box, which is a binding site for serum response factor (SRF). Luciferase reporter assays revealed activity of several Itga8 promoter constructs with no apparent restricted activity to SMC types. Further, neither SRF nor its coactivator, Myocardin (MYOCD), was able to induce several distinct Itga8 promoter constructs. Transgenic mouse studies failed to reveal Itga8 promoter activity, indicating distal regulatory elements likely control this gene's in vivo expression profile. Interestingly, although the promoter was unresponsive to SRF/MYOCD, the endogenous Itga8 gene showed increases in expression upon ectopic MYOCD expression even though knockdown of SRF both in vitro and in vivo failed to demonstrate a corresponding change in Itga8. Collectively, these data demonstrate that Itga8 expression is CArG-SRF independent, but MYOCD dependent through an as yet unknown sequence module that is distal from the promoter region.

Retinoid-induced expression and activity of an immediate early tumor suppressor gene in vascular smooth muscle cells.

Retinoids are used clinically to treat a number of hyper-proliferative disorders and have been shown in experimental animals to attenuate vascular occlusive diseases, presumably through nuclear receptors bound to retinoic acid response elements (RARE) located in target genes. Here, we show that natural or synthetic retinoids rapidly induce mRNA and protein expression of a specific isoform of A-Kinase Anchoring Protein 12 (AKAP12ß) in cultured smooth muscle cells (SMC) as well as the intact vessel wall. Expression kinetics and actinomycin D studies indicate Akap12ß is a retinoid-induced, immediate-early gene. Akap12ß promoter analyses reveal a conserved RARE mildly induced with atRA in a region that exhibits hyper-acetylation. Immunofluorescence microscopy and protein kinase A (PKA) regulatory subunit overlay assays in SMC suggest a physical association between AKAP12ß and PKA following retinoid treatment. Consistent with its designation as a tumor suppressor, inducible expression of AKAP12ß attenuates SMC growth in vitro. Further, immunohistochemistry studies establish marked decreases in AKAP12 expression in experimentally-injured vessels of mice as well as atheromatous lesions in humans. Collectively, these results demonstrate a novel role for retinoids in the induction of an AKAP tumor suppressor that blocks vascular SMC growth thus providing new molecular insight into how retiniods may exert their anti-proliferative effects in the injured vessel wall.

Splice variants of the human ZC3H14 gene generate multiple isoforms of a zinc finger polyadenosine RNA binding protein.

The human ZC3H14 gene encodes an evolutionarily conserved Cys(3)His zinc finger protein that binds specifically to polyadenosine RNA and is thus postulated to modulate post-transcriptional gene expression. Expressed sequence tag (EST) data predicts multiple splice variants of both human and mouse ZC3H14. Analysis of ZC3H14 expression in both human cell lines and mouse tissues confirms the presence of multiple alternatively spliced transcripts. Although all of these transcripts encode protein isoforms that contain the conserved C-terminal zinc finger domain, suggesting that they could all bind to polyadenosine RNA, they differ in other functionally important domains. Most of the alternative transcripts encode closely related proteins (termed isoforms 1, 2, 3, and 3 short) that differ primarily in the inclusion of three small exons, 9, 10, and 11, resulting in predicted protein isoforms ranging from 82 to 64 kDa. Each of these closely related isoforms contains predicted classical nuclear localization signals (cNLS) within exons 7 and 11. Consistent with the presence of these putative nuclear targeting signals, these ZC3H14 isoforms are all localized to the nucleus. In contrast, an additional transcript encodes a smaller protein (34 kDa) with an alternative first exon (isoform 4). Consistent with the absence of the predicted cNLS motifs located in exons 7 and 11, ZC3H14 isoform 4 is localized to the cytoplasm. Both EST data and experimental data suggest that this variant is enriched in testes and brain. Using an antibody that detects endogenous ZC3H14 isoforms 1-3 reveals localization of these isoforms to nuclear speckles. These speckles co-localize with the splicing factor, SC35, suggesting a role for nuclear ZC3H14 in mRNA processing. Taken together, these results demonstrate that multiple transcripts encoding several ZC3H14 isoforms exist in vivo. Both nuclear and cytoplasmic ZC3H14 isoforms could have distinct effects on gene expression mediated by the common Cys(3)His zinc finger polyadenosine RNA binding domain.

The mating response cascade does not modulate changes in the steady-state level of target mRNAs through control of mRNA stability.

Many extracellular signals trigger changes in gene expression by altering the steady-state level of target transcripts. This modulation of transcript levels is typically ascribed to changes in transcription of target genes; however, there are numerous examples of changes in mRNA processing and stability that contribute to the overall change in transcript levels following signalling pathway activation. The alpha-factor-stimulated mating pathway in Saccharomyces cerevisiae is a receptor-operated MAP kinase cascade that results in increased levels of a large number of target mRNA transcripts when stimulated acutely. A previous study identified many of the transcripts modulated in response to alpha-factor and argued, based on genetic studies, that the response occurred solely at the level of gene transcription (Roberts et al., 2000). We directly examined whether enhanced mRNA stability contributes to the increase in the steady-state level of alpha-factor target transcripts by exploiting a temperature-sensitive RNA Polymerase II mutant, a Ste12 transcription factor import mutant, and tet-regulated synthetic mating factor minigene reporters. Examination of a panel of alpha-factor-responsive transcripts reveals no change in mRNA stability in response to alpha-factor stimulation, providing direct evidence that this signal transduction pathway in S. cerevisiae does not function through modulating transcript stability.

Platelet-derived growth factor-induced stabilization of cyclooxygenase 2 mRNA in rat smooth muscle cells requires the c-Src family of protein-tyrosine kinases.

Cyclooxygenases (COXs) are crucial rate-limiting enzymes required for the biosynthesis of prostaglandins. COX-2 is an inducible isoform of this enzyme, which is believed to play important roles in the development of atherosclerotic vascular disease. We found that COX-2 expression rapidly increases in response to various signaling events, including activation of the platelet-derived growth factor (PDGF) pathway. Activation of PDGF receptor (PDGFR) in rat aortic vascular smooth muscle cells leads to c-Src-dependent stabilization of COX-2 mRNA requiring an AU-rich region within the 3'-untranslated region of this transcript. This regulation correlates with tyrosine phosphorylation of the RNA-associated protein, CUG-binding protein 2 (CUGBP2), which appears to enhance its interaction with COX-2 mRNA. Site-directed mutagenesis of putative tyrosine phosphorylation sites in CUGBP2 identified tyrosine 39 as a c-Src target, and a CUGBP2 with a mutated tyrosine 39 displayed an attenuated ability to bind COX-2 mRNA. We further show that silencing of CUGBP2 with specific small interference RNAs significantly reduces PDGF-dependent induction of COX-2 at both mRNA and protein levels. Furthermore, forced expression of CUGBP2 or constitutively active c-Src leads to stabilization of co-expressed COX-2 mRNA. Finally, in vitro RNA decay assay demonstrates that CUGBP2 is functionally required for the stabilization of COX-2 mRNA. Therefore, our data suggest that tyrosine phosphorylation of CUGBP2 is an important underlying mechanism for the ability of PDGFR/c-Src signaling to control the stability of COX-2 mRNA.

Recognition of polyadenosine RNA by zinc finger proteins.

Messenger RNA transcripts are coated from cap to tail with a dynamic combination of RNA binding proteins that process, package, and ultimately regulate the fate of mature transcripts. One class of RNA binding proteins essential for multiple aspects of mRNA metabolism consists of the poly(A) binding proteins. Previous studies have concentrated on the canonical RNA recognition motif-containing poly(A) binding proteins as the sole family of poly(A)-specific RNA binding proteins. In this study, we present evidence for a previously uncharacterized poly(A) recognition motif consisting of tandem CCCH zinc fingers. We have probed the nucleic acid binding properties of a yeast protein, Nab2, that contains this zinc finger motif. Results of this study reveal that the seven tandem CCCH zinc fingers of Nab2 specifically bind to polyadenosine RNA with high affinity. Furthermore, we demonstrate that a human protein, ZC3H14, which contains CCCH zinc fingers homologous to those found in Nab2, also specifically binds polyadenosine RNA. Thus, we propose that these proteins are members of an evolutionarily conserved family of poly(A) RNA binding proteins that recognize poly(A) RNA through a fundamentally different mechanism than previously characterized RNA recognition motif-containing poly(A) binding proteins.

Multiple promoters direct expression of three AKAP12 isoforms with distinct subcellular and tissue distribution profiles.

A Kinase Anchoring Protein 12 (AKAP12; also known as src-suppressed C kinase substrate (SSeCKS) and Gravin) is a multivalent anchoring protein with tumor suppressor activity. Although expression of AKAP12 has been examined in a number of contexts, its expression control remains to be elucidated. Herein, we characterize the genomic organization of the AKAP12 locus, its regulatory regions, and the spatial distribution of the proteins encoded by the AKAP12 gene. Using comparative genomics and various wet-lab assays, we show that the AKAP12 locus is organized as three separate transcription units that are governed by non-redundant promoters coordinating distinct tissue expression profiles. The proteins encoded by the three AKAP12 isoforms (designated alpha, beta, and gamma) share >95% amino acid sequence identity but differ at their N termini. Analysis of the targeting of each isoform reveals distinct spatial distribution profiles. An N-terminal myristoylation motif present in AKAP12alpha is shown to be necessary and sufficient for targeted expression of this AKAP12 isoform to the endoplasmic reticulum, a novel subcellular compartment for AKAP12. Our results demonstrate heretofore unrecognized complexity within the AKAP12 locus and suggest a mechanism for genetic control of signaling specificity through distinct regulation of alternately targeted anchoring protein isoforms.

Myocardin: a component of a molecular switch for smooth muscle differentiation.

A hallmark of smooth muscle cells (SMCs) in culture and the injured vessel wall is their phenotypic modulation from a differentiated state to one of heightened growth, migration, and matrix synthesis. The transcriptional mechanisms underlying this altered genetic program have yet to be elucidated. Serum response factor (SRF) has emerged as a critical regulator of SMC-restricted gene expression via its interaction with proximal CArG elements; however, levels of SRF protein do not change during SMC phenotypic modulation, suggesting a role for other factors or events in this process. One such factor could be myocardin, a novel SRF coactivator recently cloned from cardiac tissue. Levels of myocardin are abundantly expressed in rat aortic media along with key SMC-restricted genes. In several SMC lines, myocardin mRNA levels decrease in parallel with the loss or attenuation of SMC marker expression. Transient transfection experiments with CMV-driven myocardin in both SMC and non-SMC reveal CArG-dependent transactivation of the SM-Calp promoter-enhancer. Several additional CArG-dependent SMC promoters show variable activation in a cell-and promoter-context dependent manner. To determine whether myocardin could activate an endogenous program of SMC differentiation, we stably transfected L6 myoblasts and assessed SMC marker expression and growth. Results reveal the expression of several SMC markers concomitant with a lower growth potential. Collectively, these studies suggest that myocardin is an important component of a molecular switch for the SMC differentiation program.

Expression of human smooth muscle calponin in transgenic mice revealed with a bacterial artificial chromosome.

Defining regulatory elements governing cell-restricted gene expression can be difficult because cis-elements may reside tens of kilobases away from start site(s) of transcription. Artificial chromosomes, which harbor hundreds of kilobases of genomic DNA, preserve a large sequence landscape containing most, if not all, regulatory elements controlling the expression of a particular gene. Here, we report on the use of a bacterial artificial chromosome (BAC) to begin understanding the in vivo regulation of smooth muscle calponin (SM-Calp). Long and accurate polymerase chain reaction, sequencing, and in silico analyses facilitated the complete sequence annotation of a BAC harboring human SM-Calp (hSM-Calp). RNase protection, in situ hybridization, Western blotting, and immunohistochemistry assays showed the BAC clone faithfully expressed hSM-Calp in both cultured cells and transgenic mice. Moreover, expression of hSM-Calp mirrored that of endogenous mouse SM-Calp suggesting that all cis-regulatory elements governing hSM-Calp expression in vivo were contained within the BAC. These BAC mice represent a new model system in which to systematically assess regulatory elements governing SM-Calp transcription in vivo.