SUMMARY The intracellular localization of the major capsid protein (ICP5) of herpes simplex virus was studied during virogenesis. Except for a brief period at the onset of synthesis, this protein was found almost exclusively inside the nucleus. Its localization was not at random since 80% was tightly bound to the nuclear matrix as early as 4 h after infection. Discrete modifications of the fluorescence pattern occurred in an orderly fashion during the progression of the infection. Immunoelectron microscopic studies using Protein A-gold labelling demonstrated that this protein is synthesized on cytoskeleton-bound polyribosomes and accumulates in the central part of the nucleus where formation of viral capsids occurs; no gold particles were found in association with the peripheral chromatin or with the nucleolus.
The epithelial sodium channel (ENaC) plays an important role in the alveolar epithelium by mediating Na + reabsorption, an essential process for resolution of pulmonary edema in acute respiratory distress syndrome (ARDS). Several proinflammatory conditions such as TNF‐α leads to a reduction of ENaC expression and activity that contributes to development of ARDS. To study how pro‐inflammatory conditions could modulate αENaC mRNA stability, we developped a Tet‐Off model that allows the specific inhibition of αENaC mRNA expression in presence of doxycycline. Using this model, we found that the half‐life (T 1/2 ) for the transcript was about 1 hr. TNF‐α, a proinflammatory cytokine found in broncho‐alveolar lavage of ARDS patients, decreased T 1/2 to 15 min, suggesting that destabilisation of αENaC transcript plays an important role in downregulation of ENaC expression in pro‐inflammatory conditions. The 3’ untranslated region (3’UTR) of a mRNA plays an important role in modulating mRNA stability. αENaC mRNA 3’UTR is very long (~30% of the transcript). Using a bioinformatic approach, we could define three domains (D1, D2, D3) able to form secondary structures that were highly conserved in different species. Alveolar epithelial cells were co‐transfected with 3’UTR deletion mutants inserted in pTRE‐tight vector along with the Tet‐Off vector. Deletion of the distal part of 3’UTR (D3) leaded to stabilization of the transcript with a T 1/2 of 2.6 hrs. A longer deletion to remove also D2 showed a T 1/2 of 4.6 hrs, while deletion of D1 decreased the half‐life to 0.5 hr. These results showed that the conserved domains have stabilizing (D1) and destabilizing (D2, D3) properties on αENaC mRNA expression. The hairpin structure of these domains could recruit RNA‐binding proteins responsible for modulation of αENaC mRNA stability. Altogether, our results suggest that modulation of αENaC mRNA stability via its 3’UTR could play an important role in regulation of ENaC expression during pro‐inflammatory conditions. Grant Funding Source : Supported by FRSQ, CFC and CIHR.
Pseudomonas aeruginosa is a gram-negative bacilli frequently encountered in human pathology. This pathogen is involved in a large number of nosocomial infections and chronic diseases. Herein we investigated the effects of polyunsaturated fatty acids (PUFA) in chronic Pseudomonas aeruginosa lung infection. C57BL/6 mice were fed for 5 wk with specifically designed diets with high contents in either omega-3 (omega-3) or omega-6 PUFA and compared to a control diet. P. aeruginosa included in agarose beads was then instilled intratracheally, and the animals were studied for 7 days. On the 4th day, the mice fed with the omega-3 diet had a higher lean body mass gain and a lower omega-6:omega-3 ratio of fatty acids extracted from the lung tissue compared with the other groups (P < 0.05). The omega-3 group had the lowest mortality. Distal alveolar fluid clearance (DAFC) as well as the inflammatory response and the cellular recruitment were higher in the omega-3 group on the 4th day. The effect on DAFC was independent of alpha-epithelial Na(+) channels (alpha-ENaC), beta-ENaC, and alpha(1)-Na-K-ATPase mRNA expressions, which were not altered by the different diets. In conclusion, a diet enriched in omega-3 PUFA can change lung membrane composition and improve survival in chronic pneumonia. This effect on survival is probably multifactorial involving the increased DAFC capacity as well as the optimization of the initial inflammatory response. This work suggests that a better control of the omega-6/omega-3 PUFA balance may represent an interesting target in the prevention and/or control of P. aeruginosa infection in patients.
The lamins are the major components of the nuclear matrix and are known as lamins A, B, and C with Mr 72,000, 68,000, and 62,000 when analysed by SDS PAGE. These three polypeptides are very similar, as determined by polypeptide mapping and immunological reactivity. Lamins A and C are so homologous that a precursor-product relationship has been proposed. Using an antiserum against nuclear matrix proteins that specifically immunoprecipitates the three lamins, we examined their synthesis in the rabbit reticulocytes lysate. Four bands of Mr 62,000, 68,000, 70,000, and 74,000 were specifically immunoprecipitated when polysomes or polyadenylated RNA were translated in vitro. By two-dimensional gel electrophoresis, the 68,000- and the 62,000-mol-wt proteins were identified as lamins B and C, respectively, and the 74,000-mol-wt polypeptide had properties of a precursor of lamin A. The mRNAs of lamin C and of the putative precursor of lamin A were completely separated by gel electrophoresis under denaturing conditions, and their respective sizes were determined. These results suggest that lamin A is not a precursor of lamin C.
Le transport ionique de sodium (Na+) par les cellules épithéliales des poumons permet de réguler la quantité de liquide des voies aériennes et permet la résorption du liquide du poumon à la naissance, ainsi que de l’oedème pulmonaire chez l’adulte. Ce transport est modulé en grande partie par le canal ionique ENaC (epithelial Na channel). Si l’augmentation de l’activité de ENaC dans les voies aériennes se traduit par une accumulation de mucus et une inflammation comme dans la fibrose kystique, l’inhibition de ENaC dans les alvéoles lors d’une infection pulmonaire pourrait contribuer à aggraver la sévérité d’un syndrome de détresse respiratoire. Les stratégies pharmacologiques qui modulent l’activité ou l’expression du canal ENaC pourraient donc être bénéfiques dans différentes pathologies pulmonaires.
The intratracheal instillation of Pseudomonas aeruginosa entrapped in agar beads in the mouse lung leads to chronic lung infection in susceptible mouse strains. As the infection generates a strong inflammatory response with some lung edema, we tested if it could modulate the expression of genes involved in lung liquid clearance, such as the α, β and γ subunits of the epithelial sodium channel (ENaC) and the catalytic subunit of Na+-K+-ATPase. Pseudomonas aeruginosa entrapped in agar beads were instilled in the lung of resistant (BalB/c) and susceptible (DBA/2, C57BL/6 and A/J) mouse strains. The mRNA expression of ENaC and Na+-K+-ATPase subunits was tested in the lung by Northern blot following a 3 hours to 14 days infection. The infection of the different mouse strains evoked regulation of α and β ENaC mRNA. Following Pseudomonas instillation, the expression of αENaC mRNA decreased to a median of 43% on days 3 and 7 after infection and was still decreased to a median of 45% 14 days after infection (p < 0.05). The relative expression of βENaC mRNA was transiently increased to a median of 241%, 24 h post-infection before decreasing to a median of 43% and 54% of control on days 3 and 7 post-infection (p < 0.05). No significant modulation of γENaC mRNA was detected although the general pattern of expression of the subunit was similar to α and β subunits. No modulation of α1Na+-K+-ATPase mRNA, the catalytic subunit of the sodium pump, was recorded. The distinctive expression profiles of the three subunits were not different, between the susceptible and resistant mouse strains. These results show that Pseudomonas infection, by modulating ENaC subunit expression, could influence edema formation and clearance in infected lungs.
Active sodium transport by the epithelial Na+ channel (ENaC) is important for fluid movement across the alveolar epithelium in the lungs and for the resolution of pulmonary oedema in acute respiratory distress syndrome (ARDS). It was shown that cellular stress induced by lipopolysaccharides (LPS) or cycloheximide (Chx) downregulate αENaC mRNA via different molecular mechanisms. The purpose of this project was to investigate the importance of the αENaC 3′UTR in the mRNA modulation of ENaC. Alveolar epithelial cells were co‐transfected with 3′UTR mutants (V5/ENaC±3′UTR and Luc±3′UTR) inserted in pTRE‐tight vector along with the Tet‐Off vector. The cells were treated with Chx (1.0μM), LPS (15μg/mL) or Actinomycin D (5μg/mL) along with Dox (1μg/mL) to inhibit transcription. We found that 3′UTR deletion caused a ~60% decrease of luciferase activity and expression compared to the complete recombinant. The T 1/2 of V5‐αENaC mRNA was 60 min and the deletion of 3′UTR reduced T 1/2 to 37min. Chx reduced αENaC mRNA T 1/2 to 33min whereas LPS had no effect. Actinomycin D, a transcription inhibitor, increased T 1/2 >; 120min. These results suggest that the αENaC mRNA half‐life is shorter then previously estimated and that 3′UTR seems to be important for the stability of αENaC mRNA. Supported by FRSQ, CFC and CIHR.
Although the amiloride-sensitive epithelial sodium channel (ENaC) plays an important role in the modulation of alveolar liquid clearance, the precise mechanism of its regulation in alveolar epithelial cells is still under investigation. Protein kinase C (PKC) has been shown to alter ENaC expression and activity in renal epithelial cells, but much less is known about its role in alveolar epithelial cells. The objective of this study was to determine whether PKC activation modulates ENaC expression and transepithelial Na + transport in cultured rat alveolar epithelial cells. Alveolar type II cells were isolated and cultured for 3 to 4 d before they were stimulated with phorbol 12-myristate 13-acetate (PMA 100 nmol/L) for 4 to 24 h. PMA treatment significantly decreased α, β, and γENaC expression in a time-dependent manner, whereas an inactive form of phorbol ester had no apparent effect. This inhibitory action was seen with only 5-min exposure to PMA, which suggested that PKC activation was very important for the reduction of αENaC expression. The PKC inhibitors bisindolylmaleimide at 2 µmol/L and Gö6976 at 2 µmol/L diminished the PMA-induced suppression of αENaC expression, while rottlerin at 1 µmol/L had no effect. PMA elicited a decrease in total and amiloride-sensitive current across alveolar epithelial cell monolayers. This decline in amiloride-sensitive current was not blocked by PKC inhibitors except for a partial inhibition with bisindolylmaleimide. PMA induced a decrease in rubidium uptake, indicating potential Na + -K + -ATPase inhibition. However, since ouabain-sensitive current in apically permeabilized epithelial cells was similar in PMA-treated and control cells, the inhibition was most probably related to reduced Na + entry at the apical surface of the cells. We conclude that PKC activation modulates ENaC expression and probably ENaC activity in alveolar epithelial cells. Ca 2+ -dependent PKC is potentially involved in this response.Key words: alveolar epithelial cells, Na + transport, Na + channel, ENaC, protein kinase C, Na + -K + -ATPase, amiloride, gene expression.
