Loss of the Endothelial Glucocorticoid Receptor Prevents the Therapeutic Protection Afforded by Dexamethasone after LPS

<div><p>Glucocorticoids are normally regarded as anti-inflammatory therapy for a wide variety of conditions and have been used with some success in treating sepsis and sepsis-like syndromes. We previously demonstrated that mice lacking the glucocorticoid receptor in the endothelium (GR ^{EC KO} mice) are extremely sensitive to low-dose LPS and demonstrate prolonged activation and up regulation of NF-κB. In this study we pre-treated these GR ^{EC KO} mice with dexamethasone and assessed their response to an identical dose of LPS. Surprisingly, the GR ^{EC KO} mice fared even worse than when given LPS alone demonstrating increased mortality, increased levels of the inflammatory cytokines TNF-α and IL-6 and increased nitric oxide release after the dexamethasone pre-treatment. As expected, control animals pre-treated with dexamethasone showed improvement in all parameters assayed. Mechanistically we demonstrate that GR ^{EC KO} mice show increased iNOS production and NF-κB activation despite treatment with dexamethasone.</p></div

Heightened inflammation in GR ^{EC KO} mice following DEX pre-treatment.

<p>(A) No differences in corticosterone level were observed between GR ^{EC KO} mice and controls for any of the conditions tested. (B) Total nitric oxide levels in GR ^{EC KO} mice are increased following DEX+LPS. (C) TNF-α and (D) IL-6 levels are significantly increased in GR ^{EC KO} mice following DEX+LPS treatment while they are nearly unchanged from baseline in controls. All blood samples were collected 8 hours after LPS treatment (and 10 hours after DEX pre-treatment, if applicable). *p<0.05 compared to similarly treated controls.</p

Impaired survival in GR ^{EC KO} mice after DEX.

<p>(A) GR ^{EC KO} mice show increased mortality after LPS treatment. (B) Mortality in GR ^{EC KO} mice is further increased in GR ^{EC KO} mice following DEX pre-treatment while controls are fully rescused following DEX+LPS. (C) Continuous blood pressure monitoring demonstrates hemodynamic instability in GR ^{EC KO} mice following pre-treatement with DEX while blood pressure is completely stabilized in control mice. *p<0.05</p

Increased iNOS expression and NF-κB activation in GR ^{EC KO} mice.

<p>Western blot of aortic homogenates from controls and GR ^{EC KO} mice treated with LPS alone or DEX+LPS and harvested at the indicated timepoints. Densitometry values are indicated below each lane. Activation of NF-κB was assayed in the same homogenates. (A) Control mice show decreased expression of iNOS when given DEX+LPS as compared to LPS alone and while (B) GR ^{EC KO} mice show increased iNOS levels following DEX+LPS as compared to LPS alone. (C) Activation of NF-κB is suppressed following DEX pre-treatement in control animals while in (D) GR ^{EC KO} mice increased activation of NF-κB is shown at every time point. *p<0.05 compared to similarly treated control. U = untreated, D = dexamethasone.</p

Expression profile of GRα and GRβ mRNA in endothelial cells.

<p>Real time PCR analysis was performed on mouse lung endothelial cells treated as described. Values were normalized to untreated control siRNA GRα levels and represent mean ± SEM for 3 independent samples. Cells were isolated from C57/BL6 mice. *p <0.05 compared to untreated control siRNA GRα levels.</p

EMMPRIN, GOLPH3 and eNOS are co-localized at the Golgi apparatus in endothelial cells.

<p>(<b>A</b>) Immunolabeling of EMMPRIN, eNOS, GOLPH3 and GM130 (a Golgi marker) in bovine aortic endothelial cells (BAECs). The upper panel shows EMMPRIN (red, left) and eNOS (green, center) and their merged image with DNA in blue (right). Arrows indicate where EMMPRIN and eNOS are located. The lower panel shows GOLPH3 (red, left), GM130 (green, center), and their merged image with nucleus in blue (right). Arrows indicate where GOLPH3 and GM130 are located. Scale bar; 10 µm. Images were taken using a Nikon E800 Microscope with a Plan-Fluorchromat 40×/0.75 objective (Nikon, Melville, NY). Shown are representative images from at least 3 independent experiments. (<b>B</b>) Immunolabeling of COS cells that were transfected with wild-type eNOS (blue, upper left panel), YFP-EMMPRIN (green, upper right panel) and HA-tagged GOLPH3 (red, lower left panel). The lower right panel shows their merged image. Scale bar; 10 µm. Arrows indicate eNOS-, EMMPRIN- and GOLPH3-rich areas. Scale bar; 10 µm. Shown are representative images from at least 3 independent transfection experiments. (<b>C</b>) Immunoprecipitation (IP) using anti-EMMPRIN (EMP) and total goat IgG from BAEC lysates. IP samples were blotted with eNOS and GOLPH3 antibodies. Input refers to eNOS levels in lysates before IP, indicating an equal amount of proteins in lysates used for IP. The blots are representative images from three independent experiments.</p

S-nitrosylation of EMMPRIN is increased in the aorta isolated from cirrhotic rats.

<p>Aorta samples were lysed in a lysis buffer. Three aorta samples were combined per group to obtain a sufficient amount of proteins for the biotin-switch assay. Lysates before the biotin-switch assay were blotted for EMMPRIN and a loading control, heat shock protein 90 (Hsp90) (input, left panel). Equal amounts of proteins (500 µg) in the lysates were used for the biotin-switch assay. Biotinylated proteins were captured by streptavidin agarose beads and blotted with an EMMPRIN antibody (right panel). The aorta from cirrhotic rats showed a higher level of S-nitrosylated EMMPRIN than that of normal rats. Interestingly, only those glycosylated EMMPRIN were S-nitrosylated in the aorta.</p

Proteins at the Golgi apparatus can be targets for S-nitrosylation.

<p>(<b>A</b>) Golgi membrane enrichment and its purity were assessed by Western blot with a Golgi marker (GM130), an endoplasmic reticulum (ER) marker (Calnexin) and a nuclear marker (Lamin A/C). Fraction III of the preparation showed a successful enrichment and purity of Golgi membranes and was used in this study. (<b>B</b>) S-nitrosylated Golgi membrane proteins were increased dose-dependently in response to the addition of an NO donor, S-nitroso-N-acetylpenicillamine (SNAP). Golgi membranes were treated with indicated concentrations of SNAP <i>in vitro</i> at room temperature (RM) for 30 min. Protein S-nitrosylation was assessed by the biotin-switch assay. Asterisks indicate endogenous biotin-containing proteins, thus considered as non-specific bands. Dithiothreitol (DTT), which cleaves nitroso-cysteine bonds, serves as a negative control. Shown are representative blots from 3 independent experiments. (<b>C</b>) S-nitrosylated Golgi membrane proteins were increased time-dependently in response to 100 µM SNAP <i>in vitro</i> for indicated incubation times (0, 30 and 60 min). Arrows indicate those proteins increased in response to SNAP. Asterisks indicate endogenous biotin-containing proteins, thus considered as non-specific bands. Shown are representative images from 3 independent experiments. (<b>D</b>) The sample quality was verified before performing a proteomic analysis. Successful biotinylation (i.e., S-nitrosylation) by the biotin-switch assay was determined by Western blot. Golgi membranes were incubated with or without 10 µM S-nitrosocysteine (Cys-NO) for 15 min at 37°C. Then, the biotin-switch assay was performed in the presence or absence of 2 mM ascorbic acid (AA) (right panel). Proteins were separated by SDS-PAGE and an equal protein loading to each lane was confirmed by Ponceau staining (left panel). Subsequent Western blotting using an anti-biotin antibody detected biotinylated proteins (right panel). Arrows indicate unique bands of biotinylated proteins that appeared in the presence of AA. Those biotinylated proteins increased by Cys-NO treatment (the 4^th lane in the right panel) were identified by mass spectrometry.</p

Putative S-nitrosylated Golgi membrane proteins in rats.

<p>Putative S-nitrosylated Golgi membrane proteins in rats.</p

NO donor dose dependently decreases cell number induced by PDGF-BB and blocks survivin expression and <i>in vitro</i>.

<p>(A), Rat SMC were treated or not with PDGF (10 ng/ml), in the absence or presence of the NO donor, DETA/NO (10, 30, and 100 µM) and cell number quantified after 48 h. (B), NO reduces PDGF induced survivin levels. VSM were treated with PDGF (10 ng/ml) with or withoutt DETA/NONO (10, 30 and 100 µM) and the levels of survivin assessed by Western blotting. DETA/NO dose dependently decreased survivin levels relative to Hsp90 (a protein loading control). Densitometric values of the ratio of survivin to Hsp90 are shown below the blots. * P<0.05, ** P<0.01 with one way ANOVA with Bonferroni posttest.</p