Proteomic investigations revealed extensive perturbations in proteins associated with cytoskeletal processes
Thus, we performed iTRAQ-LC-MS/MS based proteomics investigation. In HD3, out of a total of 116 identified proteins, 47 were down-regulated and 5 were up-regulated. In HD7, out of a total of 119 identified proteins, 25 were down-regulated and 16 were up-regulated. In HD14, out of 116 identified proteins, 32 were down-regulated and 11 were up-regulated (Fig. 2a). Molecular and cellular functions that were dominant across all groups were Cellular movement, Cellular assembly and organization and Cell-to-cell signaling and interaction/Cellular function and maintenance (HD7) (Fig. 2b). We also observed that for a majority of proteins in the lung, their overall state of either up- or down- regulation continued to be similar across HD3, HD7 and HD14 as compared to Normoxia in the hierarchical clustergram (Fig. 2c &Table S1, Supplementary information ). Across groups, a larger number of down-regulated proteins indicates a trend of energy efficiency by down-regulating cell signaling, protein turnover and metabolic activities of the lung tissues. Most notable were increased hemoglobin subunits and carbonic anhydrase levels at HD7, indicating a major signaling impetus towards hematopoiesis and respiratory acidosis (Table S1, Supplementary information ). Upon IPA analysis of the lung proteome, we observed the top canonical pathways to be Remodeling of epithelial adherens junctions, signaling cytoskeleton signaling, Clathrin-mediated endocytosis signaling and Epithelial adherens junction signaling across HD3 and HD7. Among these, only Remodeling of epithelial adherens and Actin cytoskeleton signaling show directionality in terms of significant z-scores. In HD14, Signaling by Rho-family GTPases replaces Epithelial adherens junction signaling among the top four canonical pathways while also showing directionality (down-regulation) (Fig. 2d and Fig. S3, Supplementary information ).
The overall directionality of Remodeling of epithelial adherens junctions was neither up nor down-regulated. Thus, no further analysis of the pathway was carried out. Upon detailed investigation of the Actin cytoskeleton signaling pathway with expression levels overlaid from the dataset (Fig. 3a), we observed multiple perturbed cytoskeletal elements involved in processes related to actin polymerization, cytoskeleton re-organization, formation of adherens complex and focal complex assembly. We also assessed the key proteins involved in this canonical pathway across groups. Almost all the cytoskeletal elements were down-regulated in HD3, up-regulated in HD7 with either down-regulation or normalization observed in HD14.
Using IPA, an integrated network of interlinked proteins involved in different significant canonical pathways (Fig. 2d) as well as their upstream regulators was created. This network was then overlaid with protein expression data from the lung proteome (Fig. 3b). We observed STAT3 to be the main upstream protein. Based on the interactions observed among structural proteins (CLIC5, vimentin), antioxidant proteins (thioredoxin), housekeeping proteins (actin, tubulin, GAPDH), metabolism associated proteins (RXR, MDH) we immunoblotted certain proteins known to be associated with these processes in lung tissues (Fig. 3c & d). GAPDH levels were constant across all groups suggesting minimal effects on housekeeping functions. Thioredoxin levels were the highest in HD3, normalized in HD7 and again increased in HD14 indicating active antioxidant defense during extended low pO2 exposure. RXR, involved in lipid metabolism via LXR/RXR signaling, was also observed to up-regulated during low pO2 exposure especially in HD7 suggesting preferential utilization of lipids during extended exposure to low pO2. A concomitant and successive decline in malate dehydrogenase (MDH) levels during extended low pO2exposure marked a shift in energy metabolism away from TCA cycle. The increased impetus towards more efficient utilization of energy is also signified by declined expression of CLIC5 (Chloride Intracellular Channel 5) suggesting inhibition of energy intensive signaling and transport processes during extended low pO2 exposure. HIF-1a, the master regulator of hypoxia, was observed to have increased expression throughout the extended low pO2 exposure (particularly HD14). STAT3, identified as a major upstream regulator in our study, slightly decreased in HD3. In HD7 and HD14, STAT3 levels were higher than Normoxia with the highest levels being observed in HD7.