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.