Regulatory effects of the JAK3/STAT1 pathway on the release of secreted phospholipase A(2)-IIA in microvascular endothelial cells of the injured brain

BACKGROUND: Secreted phospholipase A(2)-IIA (sPLA(2)-IIA) is an inducible enzyme released under several inflammatory conditions. It has been shown that sPLA(2)-IIA is released from rat brain astrocytes after inflammatory stimulus, and lipopolysaccharide (LPS) and nitric oxide (NO) have been implicated in regulation of this release. Here, brain microvascular endothelial cells (BMVECs) were treated with LPS to uncover whether sPLA(2)-IIA was released, whether nitric oxide regulated this release, and any related signal mechanisms. METHODS: Supernatants were collected from primary cultures of BMVECs. The release of sPLA(2)-IIA, and the expression of inducible nitric oxide synthase (iNOS), phospho-JAK3, phospho-STAT1, total JAK3 and STAT1, β-actin, and bovine serum albumin (BSA) were analyzed by Western blot or ELISA. NO production was calculated by the Griess reaction. sPLA(2) enzyme activity was measured with a fluorometric assay. Specific inhibitors of NO (L-NAME and aminoguanidine, AG), JAK3 (WHI-P154,WHI), STAT1 (fludarabine, Flu), and STAT1 siRNA were used to determine the involvement of these molecules in the LPS-induced release of sPLA(2)-IIA from BMVECs. Nuclear STAT1 activation was tested with the EMSA method. The monolayer permeability of BMVECs was measured with a diffusion assay using biotinylated BSA. RESULTS: Treatment of BMVECs with LPS increased the release of sPLA(2)-IIA and nitrite into the cell culture medium up to 24 h. Pretreatment with an NO donor, sodium nitroprusside, decreased LPS-induced sPLA(2)-IIA release and sPLA(2) enzyme activity, and enhanced the expression of iNOS and nitrite generation after LPS treatment. Pretreatment with L-NAME, AG, WHI-P154, or Flu notably reduced the expression of iNOS and nitrite, but increased sPLA(2)-IIA protein levels and sPLA(2) enzyme activity. In addition, pretreatment of the cells with STAT1 siRNA inhibited the phosphorylation of STAT1, iNOS expression, and nitrite production, and enhanced the release of sPLA(2)-IIA. Pretreatment with the specific inhibitors of NOS, JAK2, and STAT3 decreased the permeability of BMVECs. In contrast, inhibition of sPLA(2)-IIA release increased cell permeability. These results suggest that sPLA(2)-IIA expression is regulated by the NO-JAK3-STAT1 pathway. Importantly, sPLA(2)-IIA augmentation could protect the LPS-induced permeability of BMVECs. CONCLUSION: Our results demonstrate the important action of sPLA(2)-IIA in the permeability of microvascular endothelial cells during brain inflammatory events. The sPLA(2) and NO pathways can be potential targets for the management of brain MVEC injuries and related inflammation.