Bruna Arcce Lacerda

Vimentin phosphorylation as a target of cell signaling mechanisms induced by 1α,25-dihydroxyvitamin D3 in immature rat testes

The effects of 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] are mainly mediated by nuclear receptors modulating gene expression. However, there are increasing evidences of nongenomic mechanisms of this hormone associated with kinase- and calcium-activated signaling pathways. In this context, the aim of the present work was to investigate the signaling pathways involved in the mechanism of action of 1,25(OH)(2)D(3) on vimentin phosphorylation in 15-day-old rat testes. Results showed that 1,25(OH)(2)D(3) at concentrations ranging from 1 nM to 1 microM increased vimentin phosphorylation independent of protein synthesis. We also demonstrated that the mechanisms underlying the hormone action involve protein kinase C activation in a phospholipase C-independent manner. Moreover, we showed that the participation of protein kinase A, extracellular regulated protein kinase (ERK), and intra- and extracellular Ca(2+) mediating the effects of 1,25(OH)(2)D(3) on the cytoskeleton. In addition, we investigated the effect of different times of exposure to the hormone on total and phosphoERK1/2 or c-Jun N-terminal kinases 1/2 (JNK1/2) in immature rat testis. Results showed that the total levels of ERK1/2 and JNK1/2 were unaltered from 1 to 15 min exposure to 1,25(OH)(2)D(3). However, the phosphoERK1/2 levels significantly increased at 1 and 5 min 1,25(OH)(2)D(3) treatment. Furthermore, phosphoJNK1 levels were decreased at 10 and 15 min 1,25(OH)(2)D(3) exposure, while phosphoJNK 2 levels were diminished at 5, 10 and 15 min treatment with the hormone. These findings demonstrate that 1,25(OH)(2)D(3) may modulate vimentin phosphorylation through nongenomic Ca(2+)-dependent mechanisms in testis cells.

Hyperhomocysteinemia selectively alters expression and stoichiometry of intermediate filament and induces glutamate- and calcium-mediated mechanisms in rat brain during development

The aim of the present work was to investigate the actions of a chemically induced chronic hyperhomocysteinemia model on intermediate filaments (IFs) of cortical and hippocampal neural cells and explore signaling mechanisms underlying such effects. Results showed that in hyperhomocysteinemic rats the expression of neural IF subunits was affected. In cerebral cortex, glial fibrillary acidic protein (GFAP) expression was donwregulated while in hippocampus high and middle molecular weight neurofilament subunits (NF‐H and NF‐M, respectively) were up‐regulated. Otherwise, the immunocontent of IF proteins was unaltered in cerebral cortex while in hippocampus the immunocontent of cytoskeletal‐associated low molecular weight neurofilament (NF‐L) and NF‐H subunits suggested a stoichiometric ratio consistent with a decreased amount of core filaments enriched in lateral projections. These effects were not accompanied by an alteration in IF phosphorylation. In vitro results showed that 500 μM Hcy‐induced protein phosphatases 1‐, 2A‐ and 2B‐mediated hypophosphorylation of NF subunits and GFAP in hippocampal slices of 17‐day‐old rats without affecting the cerebral cortex, showing a window of vulnerability of cytoskeleton in developing hippocampus. Ionotropic and metabotropic glutamate receptors were involved in this action, as well as Ca2+ release from intracellular stores through ryanodine receptors. We propose that the mechanisms observed in the hippocampus of 17‐day‐old rats could support the neural damage observed in these animals.