Andrea Vecchiola

PIASγ Represses the Transcriptional Activation Induced by the Nuclear Receptor Nurr1

Nurr1 is a transcription factor essential for the development of ventral dopaminergic neurons. In search for regulatory mechanisms of Nurr1 function, we identified the SUMO (small ubiquitin-like modifier)-E3 ubiquitin-protein isopeptide ligase, PIASγ, as an interaction partner of Nurr1. Overexpressed PIASγ and Nurr1 co-localize in the nuclei of transfected cells, and their interaction is demonstrated through co-immunoprecipitation and glutathione S-transferase pulldown assays. Co-expression of PIASγ with Nurr1 results in a potent repression of Nurr1-dependent transcriptional activation of an artificial NGFI-B response element (NBRE) reporter as well as of a reporter driven by the native tyrosine hydroxylase promoter. We identified two consensus sumoylation sites in Nurr1. The substitution of lysine 91 by arginine in one SUMO site enhanced the transcriptional activity of Nurr1, whereas the substitution of lysine 577 by arginine in the second SUMO site decreased transcriptional activity of Nurr1. Interestingly, PIASγ-induced repression of Nurr1 activity does not require the two sumoylation sites, because each mutant is repressed as efficiently as the wild type Nurr1. In addition, the mutations do not alter Nurr1 nuclear localization. Finally, we provide evidence that Nurr1 and PIASγ co-exist in several nuclei of the rodent central nervous system by demonstrating the co-expression of Nurr1 protein and PIASγ mRNA in the same cells. In conclusion, our studies identified PIASγ as a transcriptional co-regulator of Nurr1 and suggest that this interaction may have a physiological role in regulating the expression of Nurr1 target genes.

Insulin restores glucose inhibition of adenosine transport by increasing the expression and activity of the equilibrative nucleoside transporter 2 in human umbilical vein endothelium.

L‐Arginine transport and nitric oxide (NO) synthesis (L‐arginine/NO pathway) are stimulated by insulin, adenosine or elevated extracellular D‐glucose in human umbilical vein endothelial cells (HUVEC). Adenosine uptake via the human equilibrative nucleoside transporters 1 (hENT1) and 2 (hENT2) has been proposed as a mechanism regulating adenosine plasma concentration, and therefore its vascular effects in human umbilical veins. Thus, altered expression and/or activity of hENT1 or hENT2 could lead to abnormal physiological plasma adenosine level. We have characterized insulin effect on adenosine transport in HUVEC cultured in normal (5 mM) or high (25 mM) D‐glucose. Insulin (1 nM) increased overall adenosine transport associated with higher hENT2‐, but lower hENT1‐mediated transport in normal D‐glucose. Insulin increased hENT2 protein abundance in normal or high D‐glucose, but reduced hENT1 protein abundance in normal D‐glucose. Insulin did not alter the reduced hENT1 protein abundance, but blocked the reduced hENT1 and hENT2 mRNA expression induced by high D‐glucose. Insulin effect on hENT1 mRNA expression in normal D‐glucose was blocked by NG‐nitro‐L‐arginine methyl ester (L‐NAME, NO synthase inhibitor) and mimicked by S‐nitroso‐N‐acetyl‐L,D‐penicillamine (SNAP, NO donor). L‐NAME did not block insulin effect on hENT2 expression. In conclusion, insulin stimulation of overall adenosine transport results from increased hENT2 expression and activity via a NO‐independent mechanism. These findings could be important in hyperglycemia‐associated pathological pregnancies, such as gestational diabetes, where plasma adenosine removal by the endothelium is reduced, a condition that could alter the blood flow from the placenta to the fetus affecting fetus growth and development. J. Cell. Physiol. 209: 826–835, 2006.

High D-glucose reduces SLC29A1 promoter activity and adenosine transport involving specific protein 1 in human umbilical vein endothelium.

High D‐glucose reduces human equilibrative nucleoside transporter 1 (hENT1)‐mediated adenosine uptake involving endothelial nitric oxide synthase (eNOS), mitogen‐activated protein (MAP) kinase kinases 1 and 2/MAP kinases p42/44 (MEK/ERKs), and protein kinase C (PKC) activation in human umbilical vein endothelium (HUVEC). Since NO represses SLC29A1 gene (hENT1) promoter activity we studied whether D‐glucose‐reduced hENT1‐adenosine transport results from lower SLC29A1 expression in HUVEC primary cultures. HUVEC incubation (24 h) with high D‐glucose (25 mM) reduced hENT1‐adenosine transport and pGL3‐hENT1−1114 construct SLC29A1 reporter activity compared with normal D‐glucose (5 mM). High D‐glucose also reduced pGL3‐hENT1−1114 reporter activity compared with cells transfected with pGL3‐hENT1−795 construct. NG‐nitro‐L‐arginine methyl ester (L‐NAME, NOS inhibitor), PD‐98059 (MEK1/2 inhibitor), and/or calphostin C (PKC inhibitor) blocked D‐glucose effects. Insulin (1 nM) and phorbol 12‐myristate 13‐acetate (PMA, 100 nM, PKC activator), but not 4α‐phorbol 12,13‐didecanoate (4αPDD, 100 nM, PMA less active analogue) reduced hENT1‐adenosine transport. L‐NAME and PD‐98059 blocked insulin effects. L‐NAME, PD‐98059, and calphostin C increased hENT1 expression without altering protein or mRNA stability. High D‐glucose increased Sp1 transcription factor protein abundance and binding to SLC29A1 promoter, phenomena blocked by L‐NAME, PD‐98059, and calphostin C. Sp1 overexpression reduced SLC29A1 promoter activity in normal D‐glucose, an effect reversed by L‐NAME and further reduced by S‐nitroso‐N‐acetyl‐L,D‐penicillamine (SNAP, NO donor) in high D‐glucose. Thus, reduced hENT1‐mediated adenosine transport in high D‐glucose may result from increased Sp1 binding to SLC29A1 promoter down‐regulating hENT1 expression. This phenomenon depends on eNOS, MEK/ERKs, and PKC activity, suggesting potential roles for these molecules in hyperglycemia‐associated endothelial dysfunction. J. Cell. Physiol. 215: 645–656, 2008.

Role of the Renin-Angiotensin-Aldosterone System beyond Blood Pressure Regulation: Molecular and Cellular Mechanisms Involved in End-Organ Damage during Arterial Hypertension

Arterial hypertension is a common condition worldwide and an important predictor of several complicated diseases. Arterial hypertension can be triggered by many factors, including physiological, genetic, and lifestyle causes. Specifically, molecules of the renin-angiotensin-aldosterone system not only play important roles in the control of blood pressure, but they are also associated with the genesis of arterial hypertension, thus constituting a need for pharmacological interventions. Chronic high pressure generates mechanical damage along the vascular system, heart, and kidneys, which are the principal organs affected in this condition. In addition to mechanical stress, hypertension-induced oxidative stress, chronic inflammation, and the activation of reparative mechanisms lead to end-organ damage, mainly due to fibrosis. Clinical trials have demonstrated that renin-angiotensin-aldosterone system intervention in hypertensive patients lowers morbidity/mortality and inflammatory marker levels as compared to placebo patients, evidencing that this system controls more than blood pressure. This review emphasizes the detrimental effects that a renin-angiotensin-aldosterone system (RAAS) imbalance has on health considerations above and beyond high blood pressure, such as fibrotic end-organ damage.

Differential regulation of μ-opioid receptor mRNA in the nucleus accumbens shell and core accompanying amphetamine behavioral sensitization

Repeated amphetamine (AMPH) administration results in behavioral sensitization. To investigate the participation of the opioid system in this phenomenon, we examined the effects of acute and repeated AMPH administration on mu-opioid receptor (MOR) mRNA levels in the nucleus accumbens (NAc) and striatum (STR) of rats, by quantitative non-radioactive in situ hybridization. Five injections of d-AMPH (1.5 mg kg-1, i.p., once every other day), resulted in a sensitization response profile and a significant down-regulation of MOR mRNA levels in the NAc shell, whereas no change was observed in MOR mRNA levels in the NAc core compared to the saline controls. Conversely, MOR mRNA levels were up-regulated in the rostral STR of AMPH-sensitized rats compared to saline controls. No changes in MOR mRNA levels were observed after acute AMPH treatment in any of the brain regions studied. These results suggest that the opioid system participates in the neurobiological underpinnings of behavioral sensitization and that opioid receptor (OR) expression in the STR and NAc shell and core is differentially modulated by repeated AMPH exposure.

D-glucose stimulation of L-arginine transport and nitric oxide synthesis results from activation of mitogen-activated protein kinases p42/44 and Smad2 requiring functional type II TGF-β receptors in human umbilical vein endothelium

Elevated extracellular D‐glucose increases transforming growth factor β1 (TGF‐β1) release from human umbilical vein endothelium (HUVEC). TGF‐β1, via TGF‐β receptors I (TβRI) and TβRII, activates Smad2 and mitogen‐activated protein kinases p44 and p42 (p42/44mapk). We studied whether D‐glucose‐stimulation of L‐arginine transport and nitric oxide synthesis involves TGF‐β1 in primary cultures of HUVEC. TGF‐β1 release was higher (∼1.6‐fold) in 25 mM (high) compared with 5 mM (normal) D‐glucose. TGF‐β1 increases L‐arginine transport (half maximal effect ∼1.6 ng/ml) in normal D‐glucose, but did not alter high D‐glucose‐increased L‐arginine transport. TGF‐β1 and high D‐glucose increased hCAT‐1 mRNA expression (∼8‐fold) and maximal transport velocity (Vmax), L‐[3H]citrulline formation from L‐[3H]arginine (index of NO synthesis) and endothelial NO synthase (eNOS) protein abundance, but did not alter eNOS phosphorylation. TGF‐β1 and high D‐glucose increased p42/44mapk and Smad2 phosphorylation, an effect blocked by PD‐98059 (MEK1/2 inhibitor). However, TGF‐β1 and high D‐glucose were ineffective in cells expressing a truncated, negative dominant TβRII. High D‐glucose increases L‐arginine transport and eNOS expression following TβRII activation by TGF‐β1 involving p42/44mapk and Smad2 in HUVEC. Thus, TGF‐β1 could play a crucial role under conditions of hyperglycemia, such as gestational diabetes mellitus, which is associated with fetal endothelial dysfunction. J. Cell. Physiol. 212:626–632, 2007.

EL GENOTIPO D/D DEL GEN DE LA ENZIMA CONVERSIVA DE LA ANGIOTENSINA COMO FACTOR DE RIESGO DE REESTENOSIS POST-STENT CORONARIO

Introduccion Un numero significativo de los pacientes en quienes se implanta un stent coronario experimentan reestenosis, la cual es secundaria a proliferacion tisular. El sistema renina-angiotensina esta muy involucrado en la hiperplasia neointimal. Se conoce que la enzima conversiva de la angiotensina esta regulada en parte por el polimorfismo I/D del gen de esta enzima y sus concentraciones plasmaticas son mayores en los pacientes homozigotos D/D. En este trabajo se estudio la posibilidad de que el polimorfismo I/D pudiera estar relacionado con la reestenosis intra-stent. Metodos Estudiamos el polimorfismo I/D del gen de la enzima conversiva de la angiotensina en 48 pacientes consecutivos en quienes se implanto un stent en forma electiva, en arterias nativas y que tuvieran un reestudio angiografico a los 6 meses. Encontramos reestenosis (definida como estenosis > 50% respecto del lumen del vaso de referencia) en 23 de los 48 pacientes. Los grupos con y sin reestenosis no diferian en variables como diabetes, colesterol plasmatico o resultados inmediatos de la angioplastia. Resultados Encontramos que el 22,9% de los pacientes presentaba genotipo D/D; el 14,5% de los pacientes fueron I/I y el 62,5% de los pacientes eran heterozigotos I/D. Se pudo establecer que el 81,8% de los pacientes D/D desarrollo reestenosis intra-stent, mientras que solo el 40,0% de los pacientes heterozigotos I/D presentaron reestenosis. Los homozigotos I/I presentaron reestenosis muy raramente (14,2%). La diferencia de incidencia de reestenosis entre los grupos fue estadisticamente significativa (p Conclusiones De acuerdo con estos resultados, el polimorfismo D/D del gen de la enzima conversiva de la angiotensina puede ser un factor de riesgo de reestenosis intra-stent, mientras que los individuos con genotipo I/I practicamente no desarrollaron reestenosis.

Aldosterone Production and Signaling Dysregulation in Obesity

In the past decades, we have extended the view of aldosterone effects beyond epithelial tissues. New evidence regarding the aldosterone/mineralocorticoid receptor (MR) pathway in active metabolic tissues, including adipose tissue, has confirmed its pathogenic role in systemic inflammation, endothelial dysfunction, insulin resistance, and dyslipidemia. Obesity, a current epidemic worldwide, increases aldosterone production by several adipocyte factors such as leptin but is also associated with local aldosterone production. In addition, obesity can modulate MR activation leading to signaling dysregulation and a pro-inflammatory profile of adipocytes. Current knowledge have deciphered that this phenotypical differences of obesity may be explained, at least in part, by novel non-genomic activation of MR, new inducers of aldosterone synthesis, and probably by several epigenetic modifications. In addition, with the understanding of the complex interplay of obesity, hormones, and receptors, targeted pharmacological therapy is expected and is currently under active research.

PIASγ enhanced SUMO-2 modification of Nurr1 activation-function-1 domain limits Nurr1 transcriptional synergy.

Nurr1 (NR4A2) is a transcription factor that belongs to the orphan NR4A group of the nuclear receptor superfamily. Nurr1 plays key roles in the origin and maintenance of midbrain dopamine neurons, and peripheral inflammatory processes. PIASγ, a SUMO-E3 ligase, represses Nurr1 transcriptional activity. We report that Nurr1 is SUMOylated by SUMO-2 in the lysine 91 located in the transcriptional activation function 1 domain of Nurr1. Nurr1 SUMOylation by SUMO-2 is markedly facilitated by overexpressing wild type PIASγ, but not by a mutant form of PIASγ lacking its first LXXLL motif (PIASγmut1). This PIASγmut1 is also unable to interact with Nurr1 and to repress Nurr1 transcriptional activity. Interestingly, the mutant PIASγC342A that lacks SUMO ligase activity is still able to significantly repress Nurr1-dependent transcriptional activity, but not to enhance Nurr1 SUMOylation. A SUMOylation-deficient Nurr1 mutant displays higher transcriptional activity than the wild type Nurr1 only in promoters harboring more than one Nurr1 response element. Furthermore, lysine 91, the major target of Nurr1 SUMOylation is contained in a canonical synergy control motif, indicating that SUMO-2 posttranslational modification of Nurr1 regulates its transcriptional synergy in complex promoters. In conclusion, PIASγ can exert two types of negative regulations over Nurr1. On one hand, PIASγ limits Nurr1 transactivation in complex promoters by SUMOylating its lysine 91. On the other hand, PIASγ fully represses Nurr1 transactivation through a direct interaction, independently of its E3-ligase activity.

Modulation of Immunity and Inflammation by the Mineralocorticoid Receptor and Aldosterone

The mineralocorticoid receptor (MR) is a ligand dependent transcription factor. MR has been traditionally associated with the control of water and electrolyte homeostasis in order to keep blood pressure through aldosterone activation. However, there is growing evidence indicating that MR expression is not restricted to vascular and renal tissues, as it can be also expressed by cells of the immune system, where it responds to stimulation or antagonism, controlling immune cell function. On the other hand, aldosterone also has been associated with proinflammatory immune effects, such as the release of proinflammatory cytokines, generating oxidative stress and inducing fibrosis. The inflammatory participation of MR and aldosterone in the cardiovascular disease suggests an association with alterations in the immune system. Hypertensive patients show higher levels of proinflammatory mediators that can be modulated by MR antagonism. Although these proinflammatory properties have been observed in other autoimmune and chronic inflammatory diseases, the cellular and molecular mechanisms that mediate these effects remain unknown. Here we review and discuss the scientific work aimed at determining the immunological role of MR and aldosterone in humans, as well as animal models.