Hypertonic Saline Primes Activation of the p53-p21 Signaling Axis in Human Small Airway Epithelial Cells That Prevents Inflammation Induced by Pro-inflammatory Cytokines

Abstract

Uncontrolled inflammatory responses underlie the etiology of acute lung injury and acute distress respiratory syndrome, the most common late complications in trauma, the leading cause of death under the age of 59. Treatment with HTS decreases lung injury in clinical trials, rat models of trauma and hemorrhagic shock and inflammation in lung cell lines, although the mechanisms underlying these responses are still incompletely understood. Transcriptomics (RNaseq), proteomics, and U-¹³C-glucose tracing metabolomics experiments were performed to investigate the mechanisms of cellular responses to HTS treatment in primary small airway epithelial cells in the presence or absence of inflammatory injury mediated by a cocktail of cytokines (10 ng/mL of IFNγ, IL-1β, and TNFα). Modestly hyperosmolar HTS has an anti-inflammatory effect, triggers the p53-p21 signaling axis, and deregulates mitochondrial metabolism while inducing minimal apoptosis in response to a second hit by cytokines. Decreased transcription of pro-inflammatory cytokines suggested a role for the tumor suppressor protein p53 in mediating the beneficial effects of the HTS treatment. The anti-inflammatory mechanisms induced by HTS involves p53 gene regulation, promotes cell cycle arrest, and prevents ROS formation and mitochondria depolarization. Pharmaceutical targeting of the p53-p21 axis may mimic or reinforce the beneficial effects mediated by HTS when sustained hypertonicity cannot be maintained.

Keywords: RNaseq; metabolomics; mitochondria; osmotic stress; proteomics.

Figure 1

RNaseq data suggestion of a role for HTS in mediating activation of the p53 signaling axis. Down-regulated (A) and up-regulated (B) pathways in the RNaseq screening of SAECs cells, either control or treated with hypertonic saline (HTS). Consistent with the tumor protein p53 signaling pathway ranking top in (B), significant up-regulation of TP53 and its downstream targets, p21 and TP53AIP1, was confirmed in the RNaseq data set (C). RT-PCR indicated transcriptional up-regulation of TP53 in response to cytomix (CMX) treatment, alone or in combination with HTS, while HTS alone was sufficient to elicit transcriptional expression of p21 (D). In (E), increased protein expression of TP53 and p21 was observed in response to HTS treatment.

Figure 2

HTS treatment priming of increases and nuclear translocation of p53 and preservation of the stability of the p53 pool through promotion of degradative phosphorylation at T155. Fluorescence microscopy images indicate that the total levels of cytosolic p53 and nuclear p53 increase after treatment with HTS and HTS+CMX (A–D,E). However, CMX treatment (either alone or in combination with HTS) decreased the percentage of cytosolic p53 phosphorylated at T155 (F–I), a post-translational modification that primes p53 degradation (J). Labeling: p53 (A–D) or pT155-p53 (F–I, red), cell nuclei (DAPI, blue), and membranes (WGA, green).

Figure 3

Dependency of cytosolic and nuclear localization of p21 on T145 phosphorylation and correlation with HTS inhibitory effect on proliferation. Expression of p21 in SAECs was enhanced by the HTS treatment (A–D). HTS treatment, alone or in combination with CMX, promoted phosphorylation of p21 on T145, which prevented nuclear translocation and promoted cytosolic localization (E–H). Increased p21 expression and phosphorylation after treatment with HTS inhibited cell proliferation over 24 h (I). Inhibition of proliferation mediated by HTS effect on p21 may result in either cell-cycle arrest or apoptosis, depending on cytosolic vs nuclear localization of p21, which is in turn dependent on phosphorylation of T145 of p21. Labeling: p21 (A–D) or pT145-p21 (E–H, red), cell nuclei (DAPI, blue), membranes (WGA, green).

Figure 4

Double treatment of HTS and CMX promotion of increased oxidative stress but only minor apoptosis. HTS treatment primes mitochondrial membrane potential uncoupling that prevents CMX-induced mitochondrial hyperpolarization. Minor apoptosis (~2% increase over untreated controls) as a function of caspase 3 and 7 activity upon HTS or CMX treatment or both in SAECs was observed at 24 h of combined treatment (A). No further increases were observed at 48 h (data not shown) Total reactive oxygen species (DCFDA, red) decreased in response to HTS treatment and increased in the combined treatment in comparison to controls (B–E,K). However, both CMX and HTS treatments decreased mitochondrial superoxide generation (F–I), suggestive of deregulated mitochondrial metabolism and mitochondrial dysfunction. Consistently, uncoupling of mitochondrial membrane potential was observed in response to all treatments, with CMX promoting membrane hyperpoplarization and HTS promoting mitochondrial membrane depolarization, especially in the combined treatment (J,K). A schematic representation of the p53-p21 cascade activation, mitochondrial membrane depolarization and minor induction of apoptosis is proposed in (L).

Figure 5

HTS-treated SAECs and decreased levels of electron transport chain complexes. Proteomics analyses were performed on SAECs by nanoUHPLC–MS/MS (A). Results confirmed Western blot data on TIGAR (B) and revealed that HTS treatment promoted the up-regulation of a wide series of membrane transporters and electron transport chain complex II components and down-regulation of enzymes involved in glutathione biosynthesis (C). In panels D and E, top pathways were down- or up-regulated upon treatment with HTS, as gleaned through GO term enrichment of >2-fold down- or up-regulated proteins with DAVID. These pathways included components of electron transport chain complexes and diseases, in which these components are altered (down, panel D), or proteasome, amino acid, and nucleoside (purine and pyrimidine) metabolism and succinate dehydrogenase complex components (up, panel E). The majority of the down-regulated proteins were part of mitochondrial electron transport chain complexes I, III, IV, and V, where the majority of the components decreased at least 2-fold (asterisks in panel F), as detailed in the panel.

Figure 6

HTS promotion of significant metabolic reprogramming UHPLC-MS metabolomics analyses were performed on SAECs cells and supernatants (n = 5, panel A). Partial least-squares-discriminant analysis shows significant alterations of the metabolic phenotype induced by the HTS and CMX treatments (B), as detailed by the heat maps for supernatants (C) and cells (D) showing impaired nucleotide metabolism and increased amino acid uptake and decreased amino acid catabolism in HTS-treated cells. In the heat map, Z-score normalized relative metabolite levels across samples are graphed in a blue-to-red (low-to-high) color coding. Western blot analysis showed that protein expression of TIGAR, a downstream target of the p53-p21 transcriptional axis, was induced by HTS alone (E). Specifically (F), this translated into increased steady-state levels of fructose bisphosphate, decreased glucose 6-phosphate, and increased ribose phosphate in HTS and HTS+CMX cells, suggestive of increased fluxes through the pentose phosphate pathway. The lowest levels of Krebs cycle intermediates were detected in HTS-treated cells, suggestive of depressed mitochondrial metabolism. Merging metabolomics data with results from proteomics analyses (G) indicated that HTS’s effect on glycolysis may be mediated by the up-regulation of glucose and lactate transporters (GLUT1 and MCT4), hexokinase, and biphosphoglycerate mutase. The effect on amino acid uptake in response to HTS treatment may be mediated by the increased protein expression of amino acid transporters (solute carriers SLC1, SLC6, SLC38, and large amino acid transporter 1 (LAT1, panel G). However, down-regulation of Krebs cycle (acetyl-coA lyase, citrate synthase, oxoglutarate dehydrogenase, and fumarate hydratase) and glutathione biosynthetic enzymes (γ glutamyl cysteine lyase and glutathione synthase) may contribute to explaining the metabolic observations on depressed Krebs cycle and decreased glutathione levels in response to HTS treatment (G).

Figure 7

Tracing experiments with U-¹³C-glucose and confirmation that HTS decreases glucose uptake and glucose catabolism through the TCA cycle, which is in turn promoted by CMX. Hypertonic saline treatment in combination with CMX promotes activation of the pentose phosphate pathway, as indicated by the accumulation of isotopologue M+5 of ribose phosphate. Experiments were performed by incubating SAECs with HTS, alone or in combination with CMX, in the presence of uniformly labeled glucose for up to 4 h (240 min). Lines indicate median of time course tracing experiments, color coded according to the legend in panel A (top right corner).