Nora Butta

Role of transcription factor Sp1 and CpG methylation on the regulation of the human podocalyxin gene promoter

BackgroundPodocalyxin (podxl) is a heavily glycosylated transmembrane protein mainly found on the apical membrane of rat podocytes and also in endothelial, hematopoietic, and tumor cells. Despite of its interest no much is known about the transcriptional regulation of podxl in different cells. Thus, we aimed at studying the functional features of the 5-regulatory region of the human Podxl gene.ResultsThe promoter region of the human Podxl gene has been cloned and its structure and function were analyzed. The primary DNA sequence is rich in G+C and is devoid of TATA or CAAT boxes. The sequence contains recognition sites for several putative transcription factors; however, the basic promoter activity seems to rely entirely on Sp1 transcription factor since supershift analysis was positive only for this factor. The region encompassed by 66 to -111 nts conferred the minimal transcriptional activity that increases as the number of Sp1 sites augmented with the length of the promoter fragment. In Sp1-lacking insect cells the Podxl promoter constructs showed activity only if cotransfected with an Sp1 expression plasmid. Finally, mutation of the Sp1 sites reduced the promoter activity.We analyzed whether methylation of the CpG dinucleotides present in the first ~600 nts of the promoter region of Podxl could explain the variable rates of expression in different types of cells. Inactivation of methyltransferases by 5-aza-2deoxicitidine showed a dose-dependent increase in the podxl content. Moreover, in vitro methylation of the promoter constructs -111,-181 and -210 led to an almost complete reduction of the promoter activity. A correlation was found between the degree of methylation of the CpG promoter dinucleotides and the rate of podxl expression in different cell lines.ConclusionOur results indicate that transcriptional regulation of Podxl is supported primarily by Sp1 site(s) and that DNA-methylation of the CpG promoter islands contributes to control the tissue specific expression of podxl.

Podocalyxin enhances the adherence of cells to platelets

Abstract.Podocalyxin (PODXL) is a mucin protein of the CD34 family expressed in kidney glomerular podocytes, vascular endothelium, progenitor bone marrow and tumor cells. It is assumed that PODXL plays an anti-adherent role in kidney podocytes. CHO cells stably expressing human PODXL (CHO-PODXL) or human tumor cells (Tera-1) inherently expressing PODXL showed increased adherence to platelets. The adherence of cells was inhibited (70%) by blockers of platelet P-selectin, prevented by the soluble ectodomain of human PODXL (PODXL-Δ) or by the arginine-glycine-aspartate (RGDS) peptide and partially impeded by inhibition of integrin αVβ3/αVβ5, suggesting a coordinated action of P-selectin and integrins. Colocalization of platelet P-selectin and PODXL expressed on CHO cells was demonstrated by confocal immunofluorescence. No adherence to platelets was observed when PODXL was expressed in glycomutant CHO cells deficient in sialic acid.

A 1063G-->A mutation in exon 12 of glycoprotein (GP)IIb associated with a thrombasthenic phenotype: mutation analysis of [324E]GPIIb.

We report the molecular, genetic and functional analysis of a case of thrombasthenic phenotype. The proband showed absence of platelet glycoprotein (GP)IIb and very low content of GPIIIa, and both his parents showed a marked reduction in the levels of platelet GPIIb‐IIIa. Single‐stranded conformational polymorphism‐polymerase chain reaction (SSCP‐PCR) analysis and direct sequencing of PCR‐amplified GPIIb exon‐12 revealed the presence of a G→A transition at position 1063 with the expected substitution of glutamate 324 with lysine (K). This mutation did not alter the level of GPIIb mRNA. Co‐expression of normal or mutant [324K] GPIIb with normal human GPIIIa in Chinese hamster ovary (CHO) cells failed to show surface exposure of [324K]GPIIb‐IIIa complexes. Pulse‐chase and immunoprecipitation analysis demonstrated that [324K]GPIIb cDNA was translated into proGPIIb, but neither mutant GPIIb heavy chain (GPIIbH) nor [324K]GPIIb‐GPIIIa complexes were detected, suggesting that this mutation is the underlying molecular basis for the thrombasthenic phenotype. Mutation analysis demonstrated that 324E of GPIIb could be replaced by other negatively charged or polar amino acids (AAs) without impairing the surface expression of GPIIb‐IIIa. However, substitution of 324E of GPIIb for a positively charged AA other than K prevented the expression of GPIIb‐IIIa complexes. These observations suggest that a domain encompassing 324E of GPIIb is essential for heterodimerization with GPIIIa and its substitution for a positively charged residue precludes normal subunit association.

A novel homozygous splice junction mutation in GPIIb associated with alternative splicing, nonsense-mediated decay of GPIIb-mRNA, and type II Glanzmann's thrombasthenia

Summary.u2002 This work reports the study of a patient suffering a bleeding disorder clinically diagnosed as Glanzmanns thrombasthenia (GT). Immunoblotting and flow cytometric analysis showed a low (≤u200310% of control) platelet content of GPIIb–IIIa, confirming it was indeed a type II GT. The molecular genetic analysis of the proband revealed the presence of a homozygous G188A transition in GPIIb. This mutation alters the consensus sequence of the splice donor site of intron 1 changing arginine 63 for lysine (R63K). No other mutation than [G188A]GPIIb was found in the proband and her parents after complete analysis of GPIIb and GPIIIa coding sequences, and the promoter, 3′‐UTR, and intronic flanking regions of GPIIb. The GT phenotype of the proband is the result of a limited availability of GPIIb‐mRNA. The etiopathogenic role of the [G188A]GPIIb mutation is supported by the following observations: (i) both parents, who are heterozygous for the [G188A]GPIIb mutation, show a marked decrease in the platelet content of GPIIb‐mRNA; (ii) exontrap analysis demonstrated that the G188A mutation leads to a marked reduction in the steady‐state level of GPIIb‐mRNA. The reduced availability of platelet GPIIb‐mRNA associated with the G188A mutation seems to be caused by either inefficient RNA splicing or a preferred utilization of alternative intronic donor sites that generate an in‐frame STOP codon with the result of activation of nonsense‐mediated mRNA decay, or both.

Characterization of the α1-adrenoceptor-mediated responses in perfused rat liver

Abstract The present work aimed to further characterise the hepatic α1-adrenergic actions by studying the influence of nutritional status and/or extracellular medium composition in the α1-adrenoceptor-induced responses. The experiments were performed in a non-recirculating liver-perfusion system featuring continuous monitoring of vascular resistance, as well as the effluent perfusate changes in pO2, pCa2+, pK+ and pH. The α1-adrenoceptor activation produced biphasic responses to most parameters studied. The acute phase lasted for about 3 min and it was followed by a phase of sustained stimulation that lasted as long as the receptor activation was maintained. Our data indicate that there is not a single pattern of α1-adrenergic responses but variable patterns depending on the nutritional status and the experimental conditions. Gluconeogenic substrates alone produced reciprocal changes in the outflow perfusate pH and Ca2+ activity. The magnitude of these changes indicates that the diversity of α1-adrenoceptor responses are the result of the superposed effects of different rates of substrates and/or metabolites transport. The sustained α1-adrenoceptor stimulation produced extracellular acidification and increases in respiration, vascular resistance and Ca2+ release. These responses required physiological extracellular [Ca2+]. At low extracellular [Ca2+], the α1-adrenoceptor activation failed to acidify the extracellular medium, suggesting that receptor-induced H+ efflux demands normal rates of Ca2+ influx. The correlation between α1-adrenergic-induced increase in O2 uptake and Ca2+ release indicates that the increased energy production can be accounted for by the energy cost of Ca2+ release. The α1-agonist concentration-response studies have shown significant differences in the [α1-agonist]0.5 for each type of response, suggesting the existence of multiple α1-adrenoceptor-coupled signal-transduction pathways.

Modulation of the hepatic α1-adrenoceptor responsiveness by colchicine: dissociation of free cytosolic Ca2+-dependent and independent responses

1 . The cytoskeletal depolymerizing agent, colchicine, prevents the hepatic α1‐adrenoceptor‐mediated stimulation of respiration, H+ and Ca2+ release to the effluent perfusate, intracellular alkalosis, and glycogenolysis. Unlike the other parameters, colchicine does not perturb the α1‐agonist‐induced stimulation of gluconeogenesis or phosphorylase ‘a’ activation, and enhances the increase in portal pressure response. The lack of effect of colchicine on the hepatic α2‐adrenoceptor‐mediated effects indicates that its actions are α1‐specific. 2 . Colchicine enhances the acute α1‐adrenoceptor‐mediated intracellular Ca2+ mobilization and prevents the activation of protein kinase C. This differential effect on the two branches of the α1‐adrenoceptor signalling pathway is a distinctive feature of the colchicine action. 3 . The lack of effect of colchicine in altering the α1‐adrenoceptor ligand binding affinity suggests that it might interact with some receptor‐coupled regulatory element(s). 4 . The acuteness of the colchicine effect and the ability of its isomer β‐lumicolchicine to prevent all the α1‐adrenoceptor‐mediated responses but the increase in vascular resistance, indicate that its action cannot be merely ascribed to its effects in depolymerizing tubulin. 5 . Colchicine perturbs the hepatic responses to vasoactive peptides. It enhances the vasopressin‐induced rise of cytosolic free Ca2+ in isolated hepatocytes and prevents the sustained decrease of Ca2+ in the effluent perfusate. It also inhibits the stimulation of glycogenolysis, without altering the stimulation of gluconeogenesis. 6 . It is concluded that there are at least two major α1‐adrenoceptor signalling pathways. One is colchicine‐sensitive, independent of variations in free cytosolic Ca2+, and protein kinase C‐independent; the other one is colchicine‐insensitive, dependent on variations in free cytosolic Ca2+, and protein kinase C‐independent.

Role of the α-subunit326GRV sequence in the surface expression of fibrinogen and vitronectin receptors

The platelet GPIIb-GPIIIa heterodimer (integrin αIIbβ3) binds fibrinogen with high affinity in response to activation by agonists, leading to platelet aggregation and formation of a hemostatic plug. The 326GRV motif in GPIIb is highly conserved in the α-subunit of other integrins, suggesting that it might play an important functional role. Moreover, Arg327→His substitution in GPIIb has been associated with defective platelet surface expression of GPIIb-IIIa and thrombasthenic phenotype. This work aimed at elucidating whether the absence of Arg327 or its substitution by His was responsible for the impaired surface expression of GPIIb-IIIa complexes. Transfection of cDNA encoding [Ala327]GPIIb, [Gln327]GPIIb, or [Phe327]GPIIb into Chinese hamster ovary cells inherently expressing GPIIIa permitted surface exposure of GPIIb-IIIa complexes, whereas [Glu327]GPIIb did not. These observations indicate that it is not the loss of [Arg327]GPIIb but the presence of His327 or a negatively charged residue like Glu at position 327 of GPIIb that prevents the surface exposure of GPIIb-IIIa heterodimers. In contrast, changing Gln344, the homologue to Arg327 in the α-subunit of the vitronectin receptor, to His did not prevent the surface expression of αv-GPIIIa complexes. Thus the conformational constraint imposed by His327 seems to be rather specific for the heterodimerization and/or processing of GPIIb-IIIa complexes.

Effect of phenylarsine oxide on hepatic α1-adrenoreceptor responsiveness. dissociation between ionotropic and metabolic responses

The present studies analyze the effect of the tervalent arsenical compound phenylarsine oxide (PAO) on hepatic response to alpha 1-adrenoreceptor stimulation. PAO, while not significantly altering the rate of glycogen breakdown, was found to inhibit many characteristic alpha 1-adrenoreceptor mediated responses including H+ and Ca2+ release, increased energy production, and vascular smooth muscle contraction. PAO inhibited basal gluconeogenesis but failed to inhibit the alpha 1-agonist induced stimulation of glucose production. These data suggest that alpha 1-adrenoreceptor mediated stimulation of metabolism and rates of ion flux across the plasma membrane are separate processes and that exchange in ion homeostasis is not essential to elicit the receptor-mediated metabolic responses. The selective effect of PAO offers an interesting tool for studying the alpha 1-adrenoreceptor signaling mechanisms.