Estradiol and Testosterone Regulate Arginine-Vasopressin Expression in SH-SY5Y Human Female Neuroblastoma Cells Through Estrogen Receptors-α and -β

The expression of arginine-vasopressin (AVP) is regulated by estradiol and testosterone in different neuronal populations by mechanisms that are not yet fully understood. Estrogen receptors (ERs)have been shown to participate in the regulation of AVP neurons by estradiol. In addition, there is evidence of the participation of ER in the regulation of AVP expression exerted by testosterone via its metabolite 5- alpha-dihyihydrotestosterone (5-alpha DHT) and its further conversion in the androgen metabolite and ER ligand 3-diol. In this study we have explored the role of ERs in the regulation exerted by estradiol and testosterone on AVP expression, using the human neuroblastoma cell line SH-SY5Y. Estradiol treatment increased AVP mRNA levels in SH-SY5Y cells in comparison to cells treated with vehicle. The stimulatory effect of estradiol on AVP expression was imitated by the ER agonist PPT and blocked by the ER antagonist, ICI 182,780 and the ER antagonist MPP. In contrast, the ER beta agonist DPN reduced AVP expression, while the ER antagonist PHTPP enhanced the action of estradiol on AVP expression. Testosterone increased AVP expression in SH-SY5Y cells by a mechanism that was dependent on aromatase, but not on 5-reducatse activity. testosterone effect was not affected by blocking androgen receptor, was not imitated by the testosterone metabolite 5-DHT, and was blocked by the ER alpha antagonist MPP. In contrast, 5-alpha-DHT had a similar effect than the ER beta agonists DPN and 3-beta-diol, reducing AVP expression. These findings suggest that estradiol and testosterone regulate AVP expression in SH-SY5Y cells through ERs, exerting a stimulatory action via ER alpha and an inhibitory action via ER betaFil: Grassi, Daniela. Instituto Cajal; España. Università di Torino; ItaliaFil: Bellini, Maria Jose. Instituto Cajal; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Acaz Fonseca, Estefania. Instituto Cajal; EspañaFil: Panzica, GianCarlo. Università di Torino; Italia. Neuroscience Institute Cavalieri-Ottolenghi ; ItaliaFil: Garcia Segura, Luis Miguel. Instituto Cajal; Españ

Changes in cannabinoid receptors, aquaporin 4 and vimentin expression after traumatic brain injury in adolescent male mice. Association with edema and neurological deficit.

Traumatic brain injury (TBI) incidence rises during adolescence because during this critical neurodevelopmental period some risky behaviors increase. The purpose of this study was to assess the contribution of cannabinoid receptors (CB1 and CB2), blood brain barrier proteins (AQP4) and astrogliosis markers (vimentin) to neurological deficit and brain edema formation in a TBI weight drop model in adolescent male mice. These molecules were selected since they are known to change shortly after lesion. Here we extended their study in three different timepoints after TBI, including short (24h), early mid-term (72h) and late mid-term (two weeks). Our results showed that TBI induced an increase in brain edema up to 72 h after lesion that was directly associated with neurological deficit. Neurological deficit appeared 24 h after TBI and was completely recovered two weeks after trauma. CB1 receptor expression decreased after TBI and was negatively correlated with edema formation and behavioral impairments. CB2 receptor increased after injury and was associated with high neurological deficit whereas no correlation with edema was found. AQP4 increased after TBI and was positively correlated with edema and neurological impairments as occurred with vimentin expression in the same manner. The results suggest that CB1 and CB2 differ in the mechanisms to resolve TBI and also that some of their neuroprotective effects related to the control of reactive astrogliosis may be due to the regulation of AQP4 expression on the end-feet of astrocytes

Effects of TBI on body weight, brain edema and neurological deficit.

<p>A) Percentage of b.w. change at 24 h, 72 h and two weeks after TBI. B) Brain edema. C) Neurological score. D) Brain edema in animals classified according to neurological score. Data are mean±SEM. * p< 0.05 versus naïve group of same time; # p<0.05 versus two weeks of same treatment.</p

Effects of TBI on CB1 mRNA and protein levels.

<p>A) CB1 mRNA levels. B) Analysis of correlation between brain edema and CB1 mRNA levels. C) CB1 mRNA levels in animals classified according to neurological score. D) CB1 protein levels. E) Analysis of correlation between brain edema and CB1 protein levels. F) CB1 protein levels in animals classified according to neurological score. Data are mean±SEM. * p< 0.05 versus naïve group of same time.</p

Primary antibodies and dilutions used for Western Blot analyses.

<p>Primary antibodies and dilutions used for Western Blot analyses.</p

Effects of TBI on vimentin mRNA and protein levels.

<p>A) Vimentin mRNA levels. B) Analysis of correlation between brain edema and vimentin mRNA levels. C) Vimentin mRNA levels in animals classified according to neurological score. D) Vimentin protein levels. E) Analysis of correlation between brain edema and vimentin protein levels. F) Vimentin protein levels in animals classified according to neurological score. Data are mean±SEM. * p< 0.05 versus naïve group of same time; # p<0.05 versus two weeks of same treatment.</p

Primer sequences for quantitative real-time polymerase chain reaction.

<p>Primer sequences for quantitative real-time polymerase chain reaction.</p

Effects of TBI on CB2 mRNA and protein levels.

<p>A) CB2 mRNA levels. B) Analysis of correlation between brain edema and CB2 mRNA levels. C) CB2 mRNA levels in animals classified according to neurological score. D) CB2 protein levels. E) Analysis of correlation between brain edema and CB2 protein levels. F) CB2 protein levels in animals classified according to neurological score. Data are mean±SEM. * p< 0.05 versus naïve group of same time; # p<0.05 versus two weeks of same treatment.</p

Effects of TBI on AQP4 mRNA and protein levels.

<p>A) AQP4 mRNA levels. B) Analysis of correlation between brain edema and AQP4 mRNA levels. C) AQP4 mRNA levels in animals classified according to neurological score. D) AQP4 protein levels. E) Analysis of correlation between brain edema and AQP4 protein levels. F) AQP4 protein levels in animals classified according to neurological score. Data are mean±SEM. * p< 0.05 versus naïve group of same time.</p

Double-stranded oligonucleotides used for the construction of wild-type EGFP-NES1 and EGFP-NES2 constructs.

<p>Double-stranded oligonucleotides used for the construction of wild-type EGFP-NES1 and EGFP-NES2 constructs.</p