Mickey M. Martin

Alternative splicing: a novel mechanism to fine-tune the expression and function of the human AT1 receptor

Activation of the angiotensin II type 1 (AT(1)) receptor is closely involved in the pathogenesis of cardiovascular diseases; therefore, aberrant regulation of the production of this receptor might play a role in these disorders. Currently, there is strong evidence to suggest that the predominant mechanism regulating the number of AT(1) receptors is the modulation of mRNA stability. Here, we discuss the importance of alternative splicing as an additional post-transcriptional mechanism regulating human AT(1) receptor number and function.

A functional comparison of the rat type-1 angiotensin II receptors (AT1AR and AT1BR)

To evaluate and functionally compare the rat AT1A and AT1B receptor subtypes, stable Chinese hamster ovary (CHO) cell lines expressing either recombinant receptor in approximately equal numbers were generated. Radioligand binding data suggests that the recombinant AT1A receptor is pharmacologically similar to the recombinant AT1B receptor. Functional studies indicate that both receptor subtypes can independently activate the phospholipase C/IP3 and the dihydropyridine-sensitive voltage-dependent Ca2+ channel signal transduction pathways with equal efficiency, but are unable to modulate cAMP accumulation under our experimental conditions. Furthermore, both receptors can be directly involved in the cellular growth properties of AII. Slot-blot experiments clearly demonstrate that these receptors are expressed in a tissue-specific manner. A sequence comparison of the 5 flanking regions of these two genes shows that they have very little sequence homology (approximately 36%), suggesting that although the AT1A and AT1B receptors appear to be pharmacologically and functionally similar, the control of their expression seems to be governed by distinct transcription factors.

Human phosducin-like protein (hPhLP) messenger RNA stability is regulated by cis-acting instability elements present in the 3'-untranslated region.

Phosducin (Pd) and phosducin-like protein (PhLP) have been shown to regulate G-protein signaling by binding G beta gamma subunits. To better define the function and regulation of PhLP, and to begin to investigate its potential role in human pathophysiological states, we have cloned the human PhLP (hPhLP) cDNA. The hPhLP shows 92% identity with the rat PhLP (rPhLP). However, unlike the rPhLP, no evidence of hPhLP isoforms were detected in the human tissues investigated. Additionally, unlike the rPhLP, alternative polyadenylation sites were detected in hPhLP cDNA clones which corresponded with two distinct mRNA transcripts, 1.2 kb and 3.1 kb, respectively. Interestingly, the predominantly expressed long transcript contains multiple AU-rich elements (AREs) in its 3-untranslated region (3-UTR) which have been shown to correlate with rapid mRNA turnover and translational control. This study shows that the hPhLP AREs are functional both in vitro and in vivo, with the long transcript exhibiting a much shorter mRNA half-life. We also demonstrate that subcloning of either the full-length 3-UTR or the ARE-rich region of the long transcript immediately following the stop codon of luciferase reporter gene confers instability to the luciferase mRNA and results in a ninefold reduction of luciferase activity in the cell types investigated. Taken together, these findings suggest that the AREs present in the long hPhLP mRNA may play a critical role in the regulation of hPhLP gene expression.

Regulation of Angiotensin II-induced G Protein Signaling by Phosducin-like Protein

Phosducin-like protein (PhLP) is a broadly expressed member of the phosducin (Pd) family of G protein βγ subunit (Gβγ)-binding proteins. Though PhLP has been shown to bind Gβγ in vitro, little is known about its physiological function. In the present study, the effect of PhLP on angiotensin II (Ang II) signaling was measured in Chinese hamster ovary cells expressing the type 1 Ang II receptor and various amounts of PhLP. Up to 3.6-fold overexpression of PhLP had no effect on Ang II-stimulated inositol trisphosphate (IP3) formation, whereas further increases caused an abrupt decrease in IP3 production with half-maximal inhibition occurring at 6-fold PhLP overexpression. This threshold level for inhibition corresponds to the cellular concentration of cytosolic chaperonin complex, a recently described binding partner that preferentially binds PhLP over Gβγ. Results of pertussis toxin sensitivity, GTPγS binding, and immunoprecipitation experiments suggest that PhLP inhibits phospholipase Cβ activation by dual mechanisms: (i) steric blockage of Gβγ activation of PLCβ and (ii) interference with Gβγ-dependent cycling of Gqα by the receptor. These results suggest that G protein signaling may be regulated through controlling the cellular concentration of free PhLP by inducing its expression or by regulating its binding to the chaperonin.

The transcription factors Sp1 and Sp3 are required for human angiotensin II type 1 receptor gene expression in H295-R cells.

The peptide hormone angiotensin II regulates a variety of physiological responses which are mediated by its interaction with high affinity G protein-coupled receptors localized on the surface of target cells. Our previous studies have demonstrated that a 145 bp sequence within the promoter region was required for basal level expression of the human angiotensin II type 1 receptor (hAT(1)R) gene. In the present study, deletional analysis of the hAT(1)R promoter localized the major regulatory sequence to two overlapping GC boxes harbored within the -105 to -85 bp region relative to the transcription start site in H295-R cells. Electrophoretic mobility shift assays (EMSAs) using a double-stranded (ds) oligonucleotide corresponding to this region and H295-R cell nuclear extract resulted in five specific DNA-protein complexes. EMSAs performed with competitive ds-oligonucleotides which harbored the consensus binding site for Sp1 prevented the formation of the DNA-protein complexes. Supershift EMSAs also demonstrated that Sp1 and Sp3 could bind to the GC boxes present within the -105 to -85 bp region of the hAT(1)R promoter. Transactivation experiments utilizing Drosophila SL2 cells, which lack endogenous Sp family transcription factors, demonstrated that Sp1 and Sp3 activated the hAT(1)R promoter and that maximal activation was only achieved when both GC boxes were present. Taken together, these findings suggest that Sp1 and Sp3 are necessary for the expression of the hAT(1)R gene in H295-R cells.

Translation of the human angiotensin II type 1 receptor mRNA is mediated by a highly efficient internal ribosome entry site.

Activation of the angiotensin II type 1 receptor (AT1R) is closely involved in the pathogenesis of cardiovascular disease. The human AT1R (hAT1R) mRNA splice variants have long 5-untranslated regions (5-UTRs) ranging from 272 to 414 bp that have the potential to form stable secondary structures. In this study, we show that the 5-UTR of hAT(1)R mRNAs contains an internal ribosome entry site (IRES) located within the first 40 bp of the proximal end of exon 1. Experiments utilizing the hAT1R 5-UTR as a molecular decoy demonstrate a reduction in IRES activity of approximately 50%. This inhibition is most efficient for the hAT1R IRES suggesting that a defined set of trans-factors are required to initiate translation through this cis-element. Translation initiation from the hAT1R IRES appears to be physiologically relevant since IRES activity was maintained during serum starvation, a cellular stress known to inhibit cap-dependent translation. These results suggest that cap-independent translation initiation by internal ribosome entry may represent an important mechanism for the regulation of hAT1R expression.

Identification and characterization of functional angiotensin II type 1 receptors on immortalized human fetal aortic vascular smooth muscle cells

Studies investigating the mechanisms that govern the expression of the human angiotensin II type 1 receptor (hAT(1)R) gene have progressed slowly due to the lack of human cell lines that express the AT(1)R. Recently, however, an immortalized human fetal aortic vascular smooth muscle cell line (FLTR) was generated using an amphotropic recombinant retroviral construct containing the E6/E7 open reading frames of the human papillomavirus type 16. Radioligand binding studies were undertaken to determine whether angiotensin II (Ang II) receptors were expressed on these cells. FLTR cell membranes were shown to express high-affinity Ang II receptors having a B(max) value of 324+/-43 fmol/mg protein and a K(d) of 0.36+/-0.1 nM. In both membranes and intact cells, Ang II, Ang III and the selective AT(1)R antagonist, Losartan, all had a high affinity for the receptor, suggesting that FLTR cells express the AT(1)R subtype. The expression of the hAT(1)R was validated by Northern and Western blot and RT-PCR experiments. In intact FLTR cells, Ang II (100 nM) evoked an increase in intracellular calcium ([Ca(2+)](i)) and induced hyperplasia. Additionally, our results demonstrated that FLTR cells were readily transfected, and hAT(1)R promoter luciferase constructs exhibited robust promoter activity (i.e. approximately 22-fold increase over pGL3-Basic only). Finally, our results demonstrated that the hAT(1)R gene is differentially regulated in FLTR cells vs. H295-R cells, a human adrenocarcinoma cell line that also abundantly expresses the AT(1)R. Taken together, our results suggest that FLTR cells express functional AT(1)Rs and will provide an excellent model system in which to investigate hAT(1)R gene regulation.

Human AT1 Receptor Gene Regulation

The peptide hormone angiotensin II (AII), the biologically active component of the renin-angiotensin system, regulates a variety of physiological responses including fluid homeostasis, aldosterone production, renal function and contraction of vascular smooth muscle (1). In addition, AII has actions in the central nervous system (2) and may play a role in development (3). AII binds to specific receptors that mediate intracellular Ca2+ mobilization through stimulation of phospholipase C and production of inositol trisphosphate (4), activation of Ca2+ channels (5), and inhibition of adenylate cyclase through a pertussis toxin-sensitive G-protein (6). Given the diversity of AII-mediated events and the differential signal transduction mechanisms, it has been proposed that multiple AII receptor subtypes exist (1,7).

Basal level transcriptional regulation of the human angiotensin II type 1 receptor gene.

The peptide hormone angiotensin II regulates a variety of physiological responses which are mediated by its interaction with high affinity G protein-coupled receptors localized on the surface of target cells. To gain insights into the transcriptional regulation of the human angiotensin II type 1 receptor (hAT(1)R) gene, we have isolated 1 kb of the 5-flanking sequence of this gene. Expression constructs containing various 5-deletions of the hAT(1)R promoter region, fused upstream to the luciferase reporter gene, were transiently transfected into H295-R, HEC-1B and A549 cells. It was demonstrated that a 145 bp sequence within the promoter region was required for basal level expression of the hAT(1)R gene in all of the three cell lines investigated. Computer analysis indicated the existence of numerous putative transcription factor binding sites in this region. Further detailed deletion data suggested essential transcription factor binding sites between -98 and -79 bp. Electrophoretic mobility shift assays revealed that four protein-DNA complexes were formed within the -98 to -79 bp region of the hAT(1)R gene when incubated with H295-R cell nuclear extract. Site-directed mutagenesis experiments showed that a putative Sp1 binding site was critical for the basal level expression of the hAT(1)R gene.

Integrando la historia clínica ambiental en el consejo prenatal y cuidado de 2 casos de gastrosquisis

INTRODUCTIONnGastroschisis is a malformation with an unknown aetiology, likely involving genetic and environmental risk factors (RF). The aim of this paper is to develop the paediatric environmental clinical history (PECH) of two patients with gastroschisis.nnnPATIENTS AND METHODSnReview of the medical literature using Pubmed and the Developmental and Reproductive Toxicology Database. Search teratogenic substances using the Hazardous Substances Data Bank. Keywords used were: Gastroschisis and Gastroschisis and Risk Factor.nnnRESULTSnAmong the RFs known and present in both cases were: short cohabitation, unintended pregnancies of relatively young mothers, recent change of paternity, excessive alcohol intake, important nutritional deficiencies, and active and passive smoking. Additionally, one of the cases was exposed to cocaine, cannabis smoke and ionizing radiation from an orthopantography during pregnancy.nnnCONCLUSIONSn1. The PECH should be obtained in all patients with gastroschisis. 2. A thorough PECH requires a proper review of the related RFs and basic training to characterise and quantify environmental exposures. 3. Following these steps, useful recommendations to improve patient care and family advice in future pregnancies are provided.