Genetic Targets of Hydrogen Sulfide in Ventilator-Induced Lung Injury – A Microarray Study

<div><p>Recently, we have shown that inhalation of hydrogen sulfide (H₂S) protects against ventilator-induced lung injury (VILI). In the present study, we aimed to determine the underlying molecular mechanisms of H₂S-dependent lung protection by analyzing gene expression profiles in mice. C57BL/6 mice were subjected to spontaneous breathing or mechanical ventilation in the absence or presence of H₂S (80 parts per million). Gene expression profiles were determined by microarray, sqRT-PCR and Western Blot analyses. The association of Atf3 in protection against VILI was confirmed with a Vivo-Morpholino knockout model. Mechanical ventilation caused a significant lung inflammation and damage that was prevented in the presence of H₂S. Mechanical ventilation favoured the expression of genes involved in inflammation, leukocyte activation and chemotaxis. In contrast, ventilation with H₂S activated genes involved in extracellular matrix remodelling, angiogenesis, inhibition of apoptosis, and inflammation. Amongst others, H₂S administration induced Atf3, an anti-inflammatory and anti-apoptotic regulator. Morpholino mediated reduction of Atf3 resulted in elevated lung injury despite the presence of H₂S. In conclusion, lung protection by H₂S during mechanical ventilation is associated with down-regulation of genes related to oxidative stress and inflammation and up-regulation of anti-apoptotic and anti-inflammatory genes. Here we show that Atf3 is clearly involved in H₂S mediated protection.</p></div

Venn diagram (A) showing the intersections between the lists of differentially regulated genes in response to 6 h mechanical ventilation (effect of ventilation, S1), mechanical ventilation with supplemented H₂S (effect of H₂S ventilation, S2) and, genes found differentially regulated in both groups (S3).

<p>The lists of effect-specific and intersection genes are shown in supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102401#pone.0102401.s001" target="_blank">tables S1</a> to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102401#pone.0102401.s003" target="_blank">S3</a>. Only genes with statistical relevance after unpaired Bayer T-test p≤0.005, false discovery rate (FDR) ≤10% and effect size ≥1.7 were included in the diagram (created using Genedata Analyst 2.2.6b software). Gene enrichment analysis (GEA) (B+C) of significant genes induced during mechanical ventilation (S1) and mechanical ventilation with H₂S (S2). Effect size was transformed to log2 ratio prior GEA performed with the MetaCore software. Only significant events, p<0.02, FDR<15% and including minimum of 5 genes GO processes (B) and GO processes networks (C) are shown. Shades of red (up-regulated) and blue (down-regulated) coded the degree of significance of the corresponding annotation.</p

Effect of reduced Atf3 protein synthesis on VILI.

<p>(A) Representative pictures of H&E stained lung tissue from control animals and animals mechanically ventilated with synthetic air in the presence or absence of H₂S. Control-Morpholino or Atf3-Morpholino treated mice were ventilated with supplemented H₂S. (B) Ventilator-induced lung injury (VILI) score was measured from the histology samples and (C) the relative amount of neutrophils was determined by cytospin analysis of BALF. Data represent means ±SEM for n = 4/group. Analysis of variance (Student–Newman–Keuls post hoc test), *P<0.05 vs. control; ^#P<0.05 vs. H₂S control-Morpfolino; ^§P<0.05 vs. air-ventilated group.</p

IIS balances anti-proliferating (tumor-suppressive) and pro-proliferating (oncogenic) activities across tissue boundaries.

<p>(A) Model of the cell-autonomous and non-cell-autonomous functions of DAF-16. IIS inhibits nuclear entry and activity of DAF-16/FOXO, BMP promotes nuclear entry and activates R-SMAD proteins SMA-2 and SMA-3. Both DAF-16 and SMA-2/3 have individual roles and regulate transcription of distinct target genes [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006801#pgen.1006801.ref019" target="_blank">19</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006801#pgen.1006801.ref042" target="_blank">42</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006801#pgen.1006801.ref050" target="_blank">50</a>]. For example, BMP activity in the hypodermis controls body size, oocyte and germline quality maintenance independently of IIS. We currently have no evidence for germline activity of BMP signaling. In the hypodermis DAF-16 interacts with SMA-2/3 in the nucleus to activate mTORC1 at transcriptional level to promote germline proliferation (green arrow). DAF-16 also activates <i>hpo-11</i> transcription independent of BMP signaling to promote cell proliferation. In addition, BMP signaling in the somatic gonad also positively regulate germline proliferation, while DAF-16 in the germline has been proposed to inhibit cell proliferation (inhibitory activity in red) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006801#pgen.1006801.ref005" target="_blank">5</a>]. (B) Hypodermal and germline DAF-16 activities need to be tightly balanced to prevent hyperproliferation. Signaling activities of at least two tissues contribute to IIS homeostasis and may explain, why DAF-16 hyperactivity (in the hypodermis) may cause germline tumors in <i>daf-2(+)</i> background, whereas <i>daf-2(-)</i> (or starvation) decreases DAF-16 oncogenic potential, although in a simplistic view, it is thought to activate DAF-16.</p

Effect of ventilation and hydrogen sulfide (H₂S) treatment on lung injury.

<p>(A) Representative pictures of H&E stained lung tissue from animals mechanically ventilated (vent)(12 ml/kg, 6 h) in the absence or presence of supplemental H₂S (80 ppm) and their corresponding non-ventilated controls. (B) Ventilator-induced lung injury (VILI) score was measured from the histology samples and (C) the relative amount of neutrophils was determined by cytospin analysis of BALF. Data represent means ±SEM for n = 5/group. Analysis of variance (Student–Newman–Keuls post hoc test), *P<0.05 vs. control; ^#P<0.05 vs. H₂S control; ^§P<0.05 vs. H₂S-ventilated group.</p

Verification of the microarray expression profiles of Socs3, Atf3 and Gadd45a by semi-quantitative RT-PCR.

<p>(A) Microarray expression levels of the control group (spontaneously air breathing animals) were used to calculate the effect of mechanical ventilation, spontaneous inhalation of H₂S, and ventilation in the presence of H₂S. (B) RT-PCR expression values for each gene were normalized to GAPDH. Data represent median of fold change ±SEM for n = 5/group. Analysis of variance (Student–Newman–Keuls post hoc test), *P<0.05 vs. control; ^#P<0.05 vs. H₂S control; ^§P<0.05 vs. air-ventilated group.</p

DAF-16 and BMP signaling do not cooperate in regulation of lifespan or body size.

<p>(A) Lifespan extension resulting from DAF-16 activation upon reduced IIS does not require SMA-6. Since <i>sma-6</i> mutant animals have a strong egg-laying defect, lifespan was performed in a temperature sensitive <i>fer-15</i> mutant background, in which animals become sterile at 25°C due to defective sperm formation [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006801#pgen.1006801.ref033" target="_blank">33</a>]. Mean lifespan and statistical analysis are summarized in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006801#pgen.1006801.s013" target="_blank">S3 Table</a>. (B) DAF-16 and BMP signaling regulate body size independently. Average body length are presented as mean ± SD. N2: 1.10 ± 0.09 mm (n = 17); <i>daf-2(e1370)</i>: 1.22 ± 0.06 mm (n = 14); <i>daf-16(mu86);daf-2(e1370)</i>: 1.08 ± 0.06 mm (n = 19); <i>sma-6(wk7)</i>: 0.70 ± 0.06 mm (n = 15); <i>sma-6(wk7);daf-2(e1370)</i>: 0.94 ± 0.07 mm (n = 10); <i>daf-16(mu86);sma-6(wk7);daf-2(e1370)</i>: 0.59 ± 0.04 mm (n = 21). (C) <i>sma-6</i> mutation does not affect nuclear localization of DAF-16(4A)::GFP in <i>shc-1;Ex[daf-16(4A)</i>::<i>GFP]</i> animals. Arrows: DAF-16::GFP in the hypodermal (h), the muscle (m) and intestinal nuclei (i). (D) <i>sma-6</i> mutation strongly suppresses the gonadal basement membrane defect in <i>shc-1;Ex[daf-16(4A)</i>::<i>GFP]</i> animals.</p

DAF-16 and BMP signaling positively regulate germline proliferation.

<p>(A) DAF-16 promotes germline proliferation in a non-cell-autonomous way. (B) BMP signaling promotes germline proliferation in a non-cell-autonomous way. (C) Hypodermis-specific RNAi against <i>rsks-1</i>, <i>rheb-1</i>, <i>daf-15</i> and <i>hpo-11</i> reduces mitotic germ cell numbers. (D) Hypodermis-specific RNAi against <i>rsks-1</i>, <i>rheb-1</i>, <i>daf-15</i> and <i>hpo-11</i> in <i>daf-16</i> mutant animals. (E) Hypodermis-specific RNAi against <i>rsks-1</i>, <i>rheb-1</i>, <i>daf-15</i> and <i>hpo-11</i> in <i>sma-6</i> mutant animals. The average numbers of germ cell nuclei in the proliferation zone of day one adult animals ± SEM and P-values are summarized in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006801#pgen.1006801.s019" target="_blank">S9 Table</a>.</p

<i>C</i>. <i>elegans</i> DAF-16/FOXO interacts with TGF-ß/BMP signaling to induce germline tumor formation via mTORC1 activation

<div><p>Activation of the FOXO transcription factor DAF-16 by reduced insulin/IGF signaling (IIS) is considered to be beneficial in <i>C</i>. <i>elegans</i> due to its ability to extend lifespan and to enhance stress resistance. In the germline, cell-autonomous DAF-16 activity prevents stem cell proliferation, thus acting tumor-suppressive. In contrast, hypodermal DAF-16 causes a tumorous germline phenotype characterized by hyperproliferation of the germline stem cells and rupture of the adjacent basement membrane. Here we show that cross-talk between DAF-16 and the transforming growth factor ß (TGFß)/bone morphogenic protein (BMP) signaling pathway causes germline hyperplasia and results in disruption of the basement membrane. In addition to activating MADM/NRBP/<i>hpo-11</i> gene alone, DAF-16 also directly interacts with both R-SMAD proteins SMA-2 and SMA-3 in the nucleus to regulate the expression of mTORC1 pathway. Knocking-down of BMP genes or each of the four target genes in the hypodermis was sufficient to inhibit germline proliferation, indicating a cell-non-autonomously controlled regulation of stem cell proliferation by somatic tissues. We propose the existence of two antagonistic DAF-16/FOXO functions, a cell-proliferative somatic and an anti-proliferative germline activity. Whereas germline hyperplasia under reduced IIS is inhibited by DAF-16 cell-autonomously, activation of somatic DAF-16 in the presence of active IIS promotes germline proliferation and eventually induces tumor-like germline growth. In summary, our results suggest a novel pathway crosstalk of DAF-16 and TGF-ß/BMP that can modulate mTORC1 at the transcriptional level to cause stem-cell hyperproliferation. Such cell-type specific differences may help explaining why human FOXO activity is considered to be tumor-suppressive in most contexts, but may become oncogenic, e.g. in chronic and acute myeloid leukemia.</p></div

mTORC1 and BMP signaling pathways contribute to the DAF-16 dependent tumorous germline phenotype.

<p>(A) Representative pictures of MitoTracker stained gonadal basement membrane. The white arrows point to disrupted gonadal basement membrane. (B) RNAi knock-down of <i>sma-6</i>, <i>hpo-11</i> and the components of mTORC1 signaling suppresses the defects in the gonadal integrity in <i>shc-1;Is[daf-16</i>::<i>GFP]</i> animals. (C) Anti-PGL-1 germline antibody staining of <i>shc-1;Is[daf-16</i>::<i>GFP]</i> and <i>shc-1;sma-6;Is[daf-16</i>::<i>GFP]</i> L3 larvae. The white arrow points to hyperplasia in the anterior gonad arm of a representative <i>shc-1;Is[daf-16</i>::<i>GFP]</i> animal (top). In <i>sma-6</i> mutant background, hyperproliferation is suppressed (bottom). (D) <i>sma-6</i> mutation significantly decreases the elevated number of the germ cells in the anterior gonad arms of <i>shc-1;Is[daf-16</i>::<i>GFP]</i> L3 animals. Numbers of germ cells per gonad arm ± SEM: wild-type: 47 ± 2 (n = 13); <i>shc-1;Is[daf-16</i>::<i>GFP]</i>: 67 ± 4 (n = 23); <i>shc-1;sma-6;Is[daf-16</i>::<i>GFP]</i>: 49 ± 2 (n = 21) (P<0.0001 compared to <i>shc-1;Is[daf-16</i>::<i>GFP]</i> animals). (E) RNAi knock-down of the components of mTORC1 signaling and <i>hpo-11</i> significantly decreases the elevated number of the germ cells in <i>shc-1;Is[daf-16</i>::<i>GFP]</i> L3 animals. Numbers of germ cells per gonad arm ± SEM: L4440 control: 90 ± 6 (n = 32); <i>let-363</i> RNAi: 64 ± 4 (n = 28); <i>rheb-1</i> RNAi: 57 ± 6 (n = 20); <i>rsks-1</i> RNAi: 54 ± 5 (n = 22); <i>hpo-11</i> RNAi: 61 ± 4 (n = 32) (P<0.0001 compared to <i>shc-1;Is[daf-16</i>::<i>GFP]</i> + RNAi L4440 control animals). (F) Inactivation of genes in the BMP signaling pathway completely abolishes gonad disruption in <i>shc-1;Is[daf-16</i>:: <i>GFP]</i> L3 animals. (G) Inactivation of genes in the BMP signaling pathway strongly suppresses gonad disruption in <i>shc-1;Is[daf-16</i>:: <i>GFP]</i> day one adult animals. Scale bar: 10 μm. In this and the following figures, percentages of basement membrane defects are presented as mean ± SD; the mean values and statistical analysis are summarized in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006801#pgen.1006801.s012" target="_blank">S2 Table</a>; the asterisks represent: * P<0.01, ** P<0.001, and *** P<0.0001.</p