Transcriptomics of acute injured lungs reveals IGF1R action on DNA damage response, metabolic reprogramming, mitochondrial homeostasis and epigenetics

doi:10.21203/rs.3.rs-5224854/v1

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Transcriptomics of acute injured lungs reveals IGF1R action on DNA damage response, metabolic reprogramming, mitochondrial homeostasis and epigenetics

https://doi.org/10.21203/rs.3.rs-5224854/v1

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Background.

Acute lung injury (ALI), ARDS and COVID-19 usually involve a “cytokine storm”. Insulin-like growth factor receptor 1 (IGF1R) maintains lung homeostasis and is implicated in these pulmonary inflammatory diseases. In mice, widespread IGF1R deficiency was reported to counteract respiratory inflammation and alveolar damage after bleomycin (BLM)-induced ALI.

Methods.

To explore the molecular mechanisms mediated by IGF1R signaling after BLM challenge, we performed RNA-sequencing in lungs of IGF1R-deficient mice after BLM or saline instillation, followed by differential expression and functional enrichment analyses. To further explore the findings, we performed protein immunodetection and DNA methylation measurements on lung sections and extracts, and analyses of primary MEFs lacking IGF1R.

Results.

Transcriptomic analysis identified differentially expressed genes between BLM-challenged and untreated control lungs, detecting biological processes and signaling pathways involved in ALI pathobiology. IGF1R depletion in BLM-challenged mice reversed large part of the transcriptional changes triggered by BLM, counteracting the transcriptomic profile of the inflammatory "cytokine storm". Data mining also identified changes in the expression of gene clusters with key roles in DNA damage, metabolic reprogramming, mitochondrial homeostasis, and epigenetics. These functional groups were deeply explored and further validated. IGF1R depleted MEFs exhibited decreased mitochondrial respiration and were protected against BLM-mediated morphological alterations, nuclear impairment and mitochondrial accumulation. Lung genomic DNA methylation levels in IGF1R-deficient BLM-challenged mice were found increased.

Conclusions.

These findings provide new insights into the molecular mechanisms underlying the attenuating effect of IGF1R deficiency on ALI, reinforce the important role of IGF1R in promoting ALI and postulate it as an epigenetic regulator in ARDS.

Acute lung injury

IGF1R

bleomycin mouse model

cytokine storm

epigenetics

Inflammation is a critical factor in many respiratory diseases including acute respiratory distress syndrome (ARDS) and COVID-19 [1, 2]. ARDS is primarily caused by acute and diffuse inflammatory damage of the alveolar-capillary barrier, which leads to pulmonary edema and reduced lung compliance, thus compromising gas exchange and causing refractory hypoxemia [3]. ARDS has been strongly associated with severe COVID-19 pneumonia cases, especially those that require mechanical ventilation [4]. In this regard, it has been proposed that a prominent factor related to COVID-19 severity and mortality is the development of a "cytokine storm" following SARS-CoV-2 infection, where overproduction of proinflammatory cytokines exacerbates ARDS and can lead to extensive tissue damage, multiple organ failure, and death [5].

Acute lung injury (ALI) has been experimentally studied as a model of ARDS using the bleomycin (BLM) mouse model [6, 7]. BLM treatment induces reactive oxygen species (ROS) generation, oxidative stress and subsequent DNA damage in the lung [8]. In mice, BLM causes alveolar damage and inflammation, leading to ALI within one week [9, 10]. In the early inflammatory phase, activation of both alveolar macrophages and neutrophils promotes the release of proinflammatory mediators and ROS, leading to apoptosis or necrosis of alveolar type 1 cells as well as increased permeability of the alveolar-capillary barrier, pulmonary edema, and reduction of surfactant production [11, 12]. Thus, exploring the molecular basis of the cytokine storm in the BLM mouse model could be of relevance to explore novel therapies for the clinical “cytokine storm”.

The insulin-like growth factor 1 receptor (IGF1R) is a ubiquitously expressed trans-membrane tyrosine kinase that is activated by its ligands, IGF1 and IGF2. IGF1R signaling regulate multiple cellular functions such as growth, proliferation, differentiation, apoptosis, adhesion and migration. IGF signaling is tightly regulated by several high-affinity IGF binding proteins (IGFBPs), IGFBP specific proteases (i.e. pappalysins) and their inhibitors (i.e. stanniocalcins) [13, 14]. IGF signaling has been widely reported to play a role in multiple lung diseases with an inflammatory component [15]. More specifically, there is substantial evidence for the relevance of IGFs in ARDS [16, 17]. More recently, proteins of the IGF system were postulated as components of the ‘cytokine release syndrome’ and intermediaries in long-COVID19 patients [18–20]. In addition, IGF1R was identified as an outcome biomarker in critical COVID-19 patients to predict mortality [21].

By using the IGF1R-deficient conditional mutant mice UBC-CreERT2; Igf1r^fl/fl (CreERT2) [22], we previously investigated the involvement of IGF1R in the ALI process mediated by BLM. IGF1R widespread deficiency conferred resistance to BLM-mediated ALI, improving survival within a week. Three days after injury, IGF1R-deficient mice presented attenuated lung inflammation and protection against alveolar damage [23]. In order to gain new insights into the molecular mechanisms involved in IGF modulation of acute murine lung inflammation as a model of the pulmonary “cytokine storm” in patients, we performed high-throughput RNA-seq in lung tissue of CreERT2 and Igf1r^fl/fl control mice three days after BLM or saline (SAL) intra-tracheal instillation. Transcriptomic analysis of acute injured lungs revealed IGF1R actions on DNA damage response, metabolism, mitochondrial homeostasis and epigenetics, which were further tested at the protein or functional level. In summary, our results provide new insights into the cellular and molecular mechanisms underlying acute lung inflammation and the associated “cytokine storm” mediated by IGF1R function.

Generation of IGF1R-deficient mice, establishment of the BLM-induced acute lung injury murine model and tissue collection

UBC-CreERT2; Igf1r ^fl/fl mice were crossed with Igf1r^fl/fl mice to directly generate descendants in equal proportions in the same litter. Tamoxifen (TMX) (Sigma) was daily administered for five consecutive days to four-week-old mice of both genotypes to induce a postnatal Igf1r gene conditional deletion in UBC-CreERT2; Igf1r^fl/fl mice in the so-called CreERT2 mutants [22]. Six-week-old Igf1r^fl/fl (control) and CreERT2 (Igf1r-deficient) female mice were used for SAL or BLM treatment, as previously reported [23], to generate the four experimental groups: Igf1r^fl/fl, CreERT2, Igf1r^fl/fl-BLM and CreERT2-BLM. Left lungs were used for histopathological evaluation or immunohistochemistry, and right lung lobes for RNA, DNA and protein extraction (Fig. 1A). For additional details, see Additional file 1.

RNA isolation, cDNA library preparation and high-throughput sequencing

Total RNA was isolated from right lung lobes and assessed for quantity and quality. Sequencing libraries were prepared by following the TruSeq Stranded mRNA Library Preparation Kit (Illumina Inc.). Then libraries were assessed for concentration and size, and run in a HiSeq 1500 PE100 lane to obtain high-throughput sequencing data. For additional details, see Additional file 1.

Bioinformatic analysis

After quality assessment, trimming and filtering, RNA-seq reads were aligned to the Mus musculus GRCm38.p6 reference genome. Resulting alignment files were then quality-assessed, sorted and indexed. Reads on gene features were counted and three differential expression comparisons were established between experimental groups (Fig. 1B). Next, read count normalization, quantitative differential expression analysis, principal component analysis (PCA) and hierarchical clustering were performed for each comparison. To control the False Discovery Rate (FDR), p-values were amended by Benjamini-Hochberg (BH) multiple testing correction and those features with FDR adjusted p-values below a 0.05 threshold and Fold Change ≥ 1.5 / ≤ 0.67 (│log2 FC│ ≥ 0.58) were considered as differentially expressed genes (DEGs). Gene Ontology (GO), KEGG and Reactome gene set enrichment analysis (GSEA) was then performed. Raw reads and read counts were submitted to Gene Expression Omnibus (accession number GSE274184). For additional details, see Additional file 1.

Histology, immunohistochemistry and immunofluorescence

Hematoxylin and eosin (H&E) staining was performed for histopathological evaluation. CC10 (1:100; clone E-11; cat. no. sc-365992; Santa Cruz Biotech. Inc., Dallas, TX), Pro-SPC (1:100; clone M-20; cat. no. sc-7706; Santa Cruz Biotech. Inc) and H3K9ac (1:200; clone C5B11; cat.no. 9649; Cell Signalling Technology, Danvers, MA) antibodies were used for fluorescence immunostaining on lung sections. For DAB immunohistochemical analyses (Novolink Polymer Detection System; Novocastra Laboratories Ltd), p21 (1:100; clone M19; cat. no. sc-471; Santa Cruz Biotech. Inc.), and LC3 A/B (1:150; cat. no. 4108; Cell Signaling Technology) antibodies were used. For additional details, see Additional file 1.

Generation of Igf1r KO mice and isolation and culture of mouse embryonic fibroblasts

Heterozygous Igf1r^fl/− mice, originated from TMX-treated UBC-CreERT2; Igf1r^fl/fl matings, were inter-crossed to generate embryos for isolation of primary mouse embryonic fibroblasts (MEFs). At day 16 of gestation, mouse embryos were dissected and homozygous control Igf1r^fl/fl and Igf1r KO (Igf1r^−/−) embryo littermates were used to establish primary cultures of MEFs. For additional information, see Additional file 1.

MEFs treatments and immunofluorescence

Igf1r ^fl/fl and Igf1r^−/− MEFs were treated with either 20 µM BLM in DMEM or DMEM for 2h, washed and maintained in culture. For short-term experiments of BLM-induced DNA damage and mitochondrial homeostasis disruption, cells were fixed after 48 h in culture and stained with TOM20 (1:1000; cat.no. 11802-1-AP; Proteintech) or γH2AX (1:100; cat.no. 05-636; Sigma) primary antibodies, Alexa Fluor secondary antibodies and fluorochrome-conjugated Phaloidin Dylight 488 (1:500; Thermo Fisher Scientific Inc.). For longer-term experiments of induced senescence, cells were maintained in culture for 7 days and then used for protein extraction and ß-Gal staining (Cell Signaling Technology kit #9860). For additional details, see Additional file 1.

Mitochondrial morphology and network connectivity analysis.

Igf1r ^fl/fl and Igf1r^−/− MEFs stained for TOM20, actin/phalloidin and DAPI were imaged on a confocal microscope (Stellaris 8, Leica Microsystems). The ImageJ Mitochondria Analyzer plugin developed by Chaundhry et al. was then used for 3D mitochondrial object segmentation and morphological and network connectivity analysis [24]. Maximal projections of z-stack images were used for illustration purposes. For additional details, see Additional file 1.

Measurement of mitochondrial respiration by Seahorse analyzer

Igf1r ^fl/fl and Igf1r^− l− MEFs were seeded on XF24 plates coated with poly-D-lysine and grown overnight at 37 ºC under 5% CO₂. 24 h after seeding, culture medium was washed and replaced by pre-warmed Seahorse XF-DMEM medium (pH 7.4) containing 10 mM glucose, 2 mM glutamine, 2% FBS and with or without 1 mM sodium pyruvate. After incubation at 37°C for 45 min in a non-CO₂ incubator, oxygen consumption rate (OCR) was repeatedly measured under basal conditions and after the sequential injection of oligomycin, FCCP and antimycin/rotenone (all from Sigma) using a XF24 Seahorse analyzer (Agilent). Hoechst was then injected to stain cell nuclei, and photos of all wells taken for data normalization. Finally, normalized OCR values, basal respiration, proton leak, ATP production-coupled respiration, maximal respiration, % coupling efficiency, % spare respiratory capacity and non-mitochondrial OCR were calculated using the Seahorse Analytics software (Agilent). For additional details, see Additional file 1.

γH2AX foci and nuclear morphology analysis

Igf1r ^fl/fl and Igf1r^−/− MEFs stained for γH2AX and DAPI were imaged on a confocal microscope (Stellaris 8, Leica Microsystems). The ImageJ software was then used for image processing, γH2AX foci counting and nuclear morphology analysis. Finally, nuclear atypia classification into “normal”, “mis-shapen”, “micronuclei” and “multinucleated” categories was performed as previously described [25]. For additional details, see Additional file 1.

Measurement of Reactive Oxygen Species (ROS)

ROS measurement on cultured MEFs was performed using the ROS-sensitive dye 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA) (Invitrogen). Briefly, Igf1r^−/− and Igf1r^fl/fl MEFs were cultured in a 96-well plate for 24 h, washed and incubated in HBSS-H2DCFDA (10 µM) with increasing concentrations of H₂O₂. Finally, fluorescence intensity was monitored at 485/535 nm (excitation/emission) in a microplate reader. The experiment was repeated three times with five replicates per condition. For additional details, see Additional file 1.

Western blotting

Superior mouse lung lobes were solubilized in RIPA lysis buffer supplemented with protease and phosphatase inhibitor cocktails. Protein samples were then separated in 4–20% Bis-Tris gels (GenScript Biotech) and transferred to nitrocellulose membranes (Bio-Rad). Following blocking, membranes were incubated overnight with p21 (1:500; clone M19; cat. no. sc-471; Santa Cruz Biotech. Inc.), LC3 A/B (1:1000; cat. no. 4108; Cell Signaling Technology), TOM20 (1:2000; cat. no. 11802-1-AP; Proteintech) primary antibodies. After washing, membranes were incubated with horseradish peroxidase-conjugated antibodies and incubated in ECL substrate. Finally, chemiluminescence signals were detected in a ChemiDoc Imager (Bio-Rad), quantified and normalized to total protein signals. For additional details, see Additional file 1.

Lung genomic DNA extraction and global DNA methylation assay

Lung genomic DNA was extracted from the accessory lung lobes. Global DNA methylation levels were obtained with an ELISA commercial kit (5-mC DNA ELISA Kit, Zymo Research; D5325) as previously described [26]. According to manufacturer’s protocol, absorbance was measured at 450 nm in a plate reader and quantification of global DNA methylation was obtained by calculating the amount of methylated cytosines in the samples (5-mC) relative to a standard curve, including negative and positive controls. For additional details, see Additional file 1.

Statistical analysis

Following a Shapiro-Wilk normality test, statistical significance was determined using the Student’s t-test or Mann-Whitney U test for pairwise comparisons, or two-way ANOVA/complex model for two-variable analyses. Statistical analyses were carried out using GraphPad Prism 8 (GraphPad Software Inc.). For all analyses, a p-value < 0.05 was considered statistically significant.

Whole transcriptome differential expression and functional enrichment analyses in lungs of BLM-challenged and IGF1R-deficient mice

To determine the impact of IGF1R deficiency in combination with BLM injury on global lung gene expression, RNA-Seq was performed on lungs of the four experimental groups of mice indicated in Fig. 1A-B, and differential expression was analyzed in three comparisons (Fig. 1B). A threshold of fold discovery rate adjusted p-value (FDR adj. p-value) < 0.05 and Fold Change (FC) ≥ 1.5 / ≤ 0.67 (│log₂ FC│ ≥ 0.58) was set to consider differentially expressed genes (DEGs). DEGs are represented in volcano plots in Fig. 1C-E, and differential expression data and description for all DEGs is shown in Tables S1-S3. In the CreERT2 vs. Igf1r^fl/fl comparison (“mutation”), down-regulated genes (378) tended to show higher differential expression than up-regulated genes (203) (Fig. 1C and Table S1). In Igf1r^fl/fl-BLM vs. Igf1r^fl/fl lungs (“injury”), 989 up- and 811 down-regulated DEGs were detected, with greater changes predominating in up-regulated genes (Fig. 1D and Table S2). Finally, in CreERT2-BLM vs. Igf1r^fl/fl-BLM lungs (“attenuation”), 1005 up- and 1550 down-regulated DEGs were identified, the latter showing much greater expression changes overall. Of note, the number of DEGs elicited by IGF1R deficiency under BLM challenge is 1.4 times greater than those caused by BLM (Fig. 1E and Table S3). A Venn diagram indicating the overlap of DEGs between the three comparisons is shown in Fig. 1F. Interestingly, 952 genes were counter-regulated between the “attenuation” and “injury” comparisons, accounting for more than half of the 1800 genes differentially expressed as a consequence of BLM challenge. Among them, 74 genes were also differentially expressed in CreERT2 mice in basal conditions (“mutation”). Moreover, it should be noted the very consistent overlap observed in genes shared by the “mutation” and “attenuation” comparisons while not differentially expressed in the “injury” comparison. Thus, 179 down-regulated and 50 up-regulated genes match, whereas only 3 genes appeared counter-regulated. Principal component analysis (PCA) graphs and hierarchical clustering heatmaps of RNA-Seq groups are shown and described in Fig. S1. Altogether, these data highlight not only the dependence on IGF1R signaling to mediate BLM-induced acute lung damage but also to maintain basal lung homeostasis.

On the whole, gene set enrichment analysis (GSEA) was performed using the GO, KEGG and REACTOME databases. Regarding the effect of IGF1R deficiency in basal conditions, functional enrichment of CreERT2 vs. Igf1r^fl/fl (“mutation”) differential gene expression identified down-regulated pathways and processes mainly involved in cell cycle regulation, extracellular matrix (ECM) organization, and DNA damage response. Interestingly, the “Regulation of IGF transport and uptake by IGF Binding Proteins” REACTOME term was also observed. All pathways and processes spotted regarding IGF1R deficiency in naïve lungs showed a down-regulation trend (Table S4). As expected, the functional clusters with the most significant transcriptional changes in the “injury” comparison included up-regulated pathways/processes involved in the pathobiology of BLM-induced ALI, including mainly inflammation but also ECM, vascular homeostasis, DNA damage response and cellular senescence/cell cycle arrest. Remarkably, the “Regulation of IGF transport and uptake by IGF Binding Proteins” REACTOME term was also detected in this comparison, as well as several signaling pathways acting downstream of IGF signaling (Table S5). Conversely, the majority of these biological processes and pathways appeared as significant and in the opposite direction (down-regulated) in the “attenuation” comparison. In addition, GSEA detected down-regulated processes/pathways involved in cell cycle regulation, not identified in the “injury” comparison but in notable agreement with the “mutation” comparison. Furthermore, several metabolic processes were significantly dysregulated, mostly down-regulated (ROS, nitric oxide, alpha-amino acid, arginine and proline, alcohol, pyrimidine) but with three up-regulated, namely “Regulation of lipid metabolic process”, “Acetyl-CoA metabolic process” and “Cellular ketone metabolic process” (Table S6).

IGF1R deficiency counteracts the inflammatory “cytokine storm” transcriptomic profile elicited by BLM and protects against acute bronchiolar injury

The overlap of DEGs between the “injury” and “attenuation” comparisons is shown in the Venn Diagram on Fig. 2A, with a striking number of 952 genes counter-regulated, which are listed in Table S7. Out of the top 50 counter-regulated genes with the smallest FDR adj. p-value in CreERT2-BLM vs. Igf1r^fl/fl-BLM mice, 47 of them (94%) were down-regulated, as shown in the heatmap of Fig. 2B, and thus up-regulated in Igf1r^fl/fl-BLM vs. Igf1r^fl/fl mice. Most of them are inflammatory response-related genes, corresponding with a clear “cytokine storm” profile. Among them, the acute inflammatory marker Serum amyloid A3 gene (Saa3) and genes of the CCL (C-C motif) and CXCL (C-X-C motif) chemokine families (Ccl9, Cxcl10 and Cxcl13) are noteworthy. Furthermore, there is a strong presence of genes involved in the interferon anti-viral response. These include some genes codifying for 2’ – 5’ oligoadenylate synthases (Oas1a, Oas1g, Oas2, Oas3 and Oasl2) and other interferon-induced proteins (Ifit1, Ifit2, Ifitm3, Ifi44, Ifi204, Ifi27l2a, Isg15 and Mx1), as well as genes with central roles in the regulation of the interferon response (Irf7, Zbp1 and Usp18). There was also notable presence of genes involved in ECM remodeling (Timp1, Serpina3m, Serpina3n, Mmp12, Lgals3bp and Fn1).

Similar to Fig. 2A, the overlap of DEGs between the "mutation" and "attenuation" comparisons are shown in the Venn Diagram in Fig. 2C, with only 12 shared genes counter-regulated, but with 259 shared down-regulated and 65 up-regulated genes, totaling 324 genes which share direction of change between the two comparisons, genes that are strictly dependent on IGF1R deficiency. These genes are listed in Table S8 and the top 50 with the smallest FDR adj. p-value in CreERT2-BLM vs. Igf1r^fl/fl-BLM mice are shown in the heatmap of Fig. 2D. Here it is particularly striking that several of the genes counter-regulated in the “injury” and “attenuation” comparisons (Cxcl10, Oas1a, Oas1g, Oas3, Oasl2, Ifit1, Ifit3, Ifi44, Ifi204, Isg15, Mx1, Irf7, Zbp1,Timp1, Serpina3n, Mmp12, Lgals3bp) were also down-regulated in CreERT2 vs. Igf1r^fl/fl lungs, without the effect of any insult other than SAL instillation. Transcriptional changes of genes with proinflammatory function including Tnf, Il1b, Il6, Cxcl1 and Adgre1, as well as anti-inflammatory Cd209 were previously validated by qPCR, and in case of TNF also by ELISA [23].

According to previous data [23], lung inflammation was highly reduced in CreERT2-BLM compared to Igf1r^fl/fl-BLM lungs (Fig. S2A-D) and mRNA expression of the surfactant protein C precursor gene (Sftpc) was significantly reduced in Igf1r^fl/fl-BLM vs. Igf1r^fl/fl lungs, while increased in CreERT2-BLM vs. Igf1r^fl/fl-BLM lungs. Furthermore, a similar expression profile was found for the marker of bronchiolar club cells Scgb1a1 (Tables S2 and S3). These results were further investigated by fluorescence immunohistochemistry (see details in Fig. S2). Interestingly, while pro-SPC staining was rare in the bronchioles of Igf1r^fl/fl and CreERT2 lungs (Fig. S2I-J), it was greatly increased in Igf1r^fl/fl-BLM bronchioles (Fig. S2K), with pro-SPC⁺ staining in a subpopulation of bronchiolar cells which were not CC10⁺ cells (Fig. S2O). Cells of this type were recently identified as AT2 proliferating precursors in BLM-challenged lungs [27]. Of note, expression of pro-SPC in bronchioles of CreERT2-BLM mice was restored to basal levels (Fig. S2L). Altogether, these results demonstrate that IGF1R deficiency very efficiently protects epithelial cells against BLM-mediated injury in the alveolar and bronchiolar lung compartments.

Differential mRNA expression of IGF/Insulin signaling pathway genes in lungs of IGF1R-deficient mice and following BLM challenge

Screening for specific DEGs of the IGF/Insulin family revealed significant changes of different components on the three comparisons examined (Fig. 3). In the absence of BLM challenge (“mutation” comparison), CreERT2 mice showed notable down-regulation of Igf1 and Stc2. On the contrary, Igfbp6 and Irs2 were up-regulated. (Fig. 3A). Remarkably, several IGF/insulin signaling components were dysregulated upon BLM challenge (“injury” comparison). Thus, four of the six IGFBP genes were differentially expressed, with Igfbp3, Igfbp5 and Igfbp6 down-regulated and Igfbp4 up-regulated. Interestingly, the Pappalysin 1 gene (Pappa), a metalloproteinase with IGFBP proteolytic activity, exhibited a high increase in expression, whereas the Stanniocalcin 1 gene (Sct1), a PAPPA 1 and 2 inhibitor, was down-regulated (Fig. 3B). In contrast, IGF1R deficiency in CreERT2-BLM mice counter-regulated most of these IGF system genes, exhibiting up-regulation of Igfbp3, Igfbp5, Igfbp6, Insr and Irs1 and down-regulation of Igf1, Igfbp4, Ig2fbp2, Pappa2, Igf2r and Ide. (Fig. 3C).

It should be noted that differential expression analysis did not show a significant decrease in Igf1r normalized counts in either CreERT2 vs. Igf1r^fl/fl or CreERT2-BLM vs. Igf1r^fl/fl-BLM lungs. However, assessment of read coverage along the Igf1r transcribed region and exon-specific FPKM quantification showed that both CreERT2 samples presented similar levels of reads throughout the entire Igf1r mRNA sequence compared to both Igf1r^fl/fl samples, but a very diminished amount of reads mapping to exon 3, corresponding to the floxed Igf1r gene region where TMX-induced Cre-mediated deletion occurs (Fig. S3A-B). Inspection of the exon junctions detected by the same analysis corroborated that in CreERT2 mice an effective alternative splicing occurs between exon 2 and exon 4 of the Igf1r consensus mRNA, generating “full length” mRNAs with an efficient deletion of exon 3 (Fig. S3A). However, translation of these mutant mRNAs in CreERT2 mice could only generate a putative non-functional truncated α chain polypeptide (see details in Fig. S4). Besides, a large reduction in IGF1R protein expression was previously confirmed in the lungs of CreERT2 and CreERT2-BLM mice [23, 28].

Transcriptomic analysis of lungs from BLM-challenged IGF1R-deficient mice corroborates previous findings and uncovers novel biological functions of IGF1R

In order to gain knowledge on the altered molecular mechanisms mediated by IGF1R deficiency in lungs from BLM challenged mice, intensive data mining based on functional annotations in databases (MGI, GO, KEGG, REACTOME, GeneCards and UniProt) was performed in the RNA-seq “attenuation” comparison. DEGs in CreERT2-BLM vs. Igf1r^fl/fl-BLM lungs were functionally grouped and represented as up- and down-regulated genes in the reported functional categories. The most relevant functional categories, ordered according to the number of genes involved from highest to lowest were: inflammation, metabolism, cell cycle, mitochondrial homeostasis, ECM organization, vascular homeostasis, DNA damage response, autophagy, IGF signaling and epigenetics. All of them, with the exception of epigenetics comprised genes mostly down-regulated (Fig. 4). Expression data and description of the genes featured in each functional group are shown in Tables S9-S18, respectively. In sum, these results reinforce the concept that IGF1R deficiency causes a generalized inhibition of transcription in the lung in the context of a BLM challenge, affecting multiple biological functions.

IGF1R depletion strongly affects expression of DNA damage-related genes and protects against BLM-mediated cell cycle arrest, nuclear impairment and ROS

Since BLM induces DNA damage in the lung and GSEA identified DNA damage response-related pathways and processes in the three RNA-Seq comparisons (Tables S4-S6), we decided to explore the effect of IGF1R deficiency on DNA damage-related functions in depth. Overall, data mining analysis in CreERT2-BLM vs. Igf1r^fl/fl-BLM lungs showed 138 DEGs with DNA damage-related functions (Fig. 4 and Table S15). Among them stand several genes with key functions in DNA damage responses, including DNA repair, genomic stability maintenance and cell cycle arrest (Fig. 5A). Remarkably, the cyclin-dependent kinase inhibitor 1 (commonly known as p21) gene (Cdkn1a), a well recognized marker of cell cycle arrest [29], showed greatly reduced expression (6.0 times) in CreERT2-BLM vs. Igf1r^fl/fl-BLM lungs. In view of these data, we decided to evaluate p21 protein expression by Western blot and IHC. Thus, Western blot analysis on lung extracts showed a significant increase of p21 protein expression after BLM injury both in Igf1r^fl/fl-BLM and CreERT2-BLM lungs compared to controls. Although there was not a statistically significant difference between genotypes, a tendency to decreased p21 protein was observed in CreERT2-BLM vs. Igf1r^fl/fl-BLM lungs (Fig. 5B-C). IHQ for p21 in bronchiolar epithelial cells showed a similar profile. Quantification of p21⁺ bronchiolar cells revealed an increase in both genotypes of BLM-treated mouse lungs compared to controls, and, in addition, a significant decrease in CreERT2-BLM vs. Igf1r^fl/fl-BLM lungs (Fig. 5D-E).

To evaluate the effect of BLM treatment on DNA damage and ROS detoxification in the context of IGF1R deficiency, primary MEFs were used. With this goal, Igf1r^fl/fl and Igf1r^−/− MEFs treated with 20 µM BLM for 2 h were immunostained against Phospho-H2AX (γ-H2AX) as a marker of DNA damage after 48 h (Fig. 5F). Quantification of γH2AX⁺ foci per nucleus did not show significant differences. However, nuclear morphology assessment showed lower nuclear perimeter as well as higher nuclear circularity in Igf1r^−/− MEFs (Fig. 5G). In addition, frequencies of nuclei with distinct nuclear abnormalities changed in Igf1r^−/− MEFS (Fig. 5H). Since p21 also contributes to the induction of cellular senescence in the long term, we determined p21 expression levels in untreated or BLM-treated MEFs by Western blot 7 days post-treatment. Although non-treated MEFs did not show differences between genotypes, BLM induced a significant increase in p21 content in Igf1r^fl/fl, an increase that was not observed in Igf1r^−/− MEFs (Fig. 5I-J). However, when we looked for a reduction in SA-β-gal staining in CreERT2 BLM-treated MEFs respect to Igf1r^fl/fl, we did not find differences (data not shown). Subsequently, ROS detoxification capacity was assessed by treating primary MEFs with increasing concentrations of H₂O₂ using a ROS-sensitive probe as an indicator. Of note, ROS levels were found significantly reduced in Igf1r^−/− MEFs when compared to Igf1r^fl/fl controls (Fig. S5). Taken together, these data suggest that IGF1R deficiency could protect MEFs from BLM-mediated DNA damage, nuclear impairment, cell cycle arrest and ROS.

IGF1R deficiency elicits transcriptomic changes in the lungs that suggest a metabolic shift from glycolysis- to fatty acid-derived energy supply after the BLM-mediated ALI, and Igf1r^−/− MEFs exhibit decreased mitochondrial respiration

A substantial number DEGs in CreERT2-BLM vs. Igf1r^fl/fl-BLM lungs were involved in central metabolic processes (Fig. 4 and Table S10). On GSEA data for this RNA-seq comparison, “Regulation of lipid metabolic process”, “Acetyl-CoA metabolic process” and “Cellular ketone metabolic process” stand out as GO terms with an up-regulation tendency (Table S6). More specifically, several genes encoding for enzymes involved in acetate conversion to Acetyl-CoA (Acss1, Acss3), fatty acid beta-oxidation (Acaa2) and ketogenesis (Hmgcs2) highlight as up-regulated genes (Fig. 6A). Conversely, many of the enzymes of the glycolysis pathway exhibited decreased expression (Hk2, Hk3, Gapdh, Pkm, Pgam1, Eno1), with the exception of the muscle-specific phosphofructokinase (Pfkm), which was up-regulated. Furthermore, the GLUT1 constitutive glucose transporter gene (Slc2a1) was down-regulated, whereas the insulin-inducible GLUT4 gene (Slc2a4) was up-regulated. Of note, Pdk1 and Pdk2, which encode pyruvate dehydrogenase kinases 1 and 2, enzymes that inactivate pyruvate dehydrogenase (PDH), were up-regulated. A schematic representation of these transcriptional changes is shown in Fig. 6B.

Next, we decided to evaluate the real-time mitochondrial respiration profile of primary Igf1r^fl/fl and Igf1r^− l− MEFs in either presence (standard Seahorse mito-stress test conditions) or absence of pyruvate. Thus, using the Seahorse mitochondrial stress test, oxygen consumption rate (OCR) was measured under basal conditions and after addition of electron transport chain modulators (Fig. 6C). A significant decrease in basal and ATP production-linked mitochondrial respiration was observed in Igf1r^−/− MEFs, both in presence or absence of pyruvate. Additionally, the presence of pyruvate significantly decreased these two parameters in Igf1r^−/− but not Igf1r^fl/fl MEFs (Fig. 6D-E). No statistically significant differences were found in non-mitochondrial respiration, proton leak, reserve respiratory capacity or maximal respiration across all conditions, although the latter showed a similar trend to basal and ATP-linked respiration (see details in Fig. S6).

IGF1R deficiency impacts expression of mitochondrial homeostasis-related genes and prevents mitochondrial accumulation after BLM challenge

Extensive data mining identified 228 DEGs with functions in the mitochondria in CreERT2-BLM vs. Igf1r^fl/fl-BLM lungs (Fig. 4 and Table S12), including a particularly relevant group DEGs with key roles in modulating mitochondrial function and dynamics (Fig. 7A). Among them, three up-regulated genes with central roles in mitochondrial quality control and mitophagy highlight: PTEN Induced Kinase 1 (Pink1), E3 ubiquitin-protein ligase PARKIN (Park2) and BCL2 interacting protein 3 (Bnip3). In addition, the uncoupling protein 4 gene (Slc25a27) and another solute carrier with a putative role in electron transport chain uncoupling (Slc25a47) were also up-regulated. Furthermore, three genes encoding cytochrome C oxidase structural subunits (Cox6a2, Cox7a1 and Cox8b) were up-regulated, as well as the mitochondrially encoded cytochrome oxidase II (mt-Co2) and ATP synthase 6 (mt-Atp6). Some remarkable down-regulated genes were the mitochondrial fission regulator 2 (Mtfr2), as well as lamin A/C (Lmna) and BCL2 Antagonist/Killer 1 (Bak1), which mediate changes in mitochondrial membrane stability, and two stress-responsive chaperones for mitochondrial proteins (Hspa5 and Hsp90aa1). Since transcriptomic data showed changes in genes of such importance in the regulation of mitochondrial homeostasis, we assessed the effect of IGF signaling and BLM treatment on mitochondrial mass and morphology in primary MEFs. Thus, Igf1r^fl/fl and Igf1r^−/− MEFs treated or non-treated with 20 µM BLM for 48 h were first evaluated for the abundance of the mitochondrial mass marker TOM20 by Western blot, with no differences between any groups (Fig. 7B-C). In contrast, fluorescent immunolabeling of mitochondria (Fig. 7D) and subsequent analysis of mitochondrial volume, morphology and network connectivity revealed insightful data. Thus, Igf1r^fl/fl MEFs exposed to BLM had higher total mitochondrial volume and count per cell than their controls, while Igf1r^−/− MEFs showed a reduction compared to Igf1r^fl/fl (Fig. 7E). However, it should be noted that the cells themselves differed in size, with BLM-treated Igf1r^fl/fl MEFs being generally more enlarged and with more aberrant morphology than Igf1r^−/− MEFs (Fig. 7D). No notable differences were found in other morphological parameters analyzed (Fig. 7E).

IG1R deficiency does not alter LC3 I/II levels in the lungs of BLM-challenged mice despite their autophagy-related transcriptomic footprint

Since multiple studies have shown that IGF1R regulates autophagy in various tissues and conditions [30, 31], we explored the expression profile of genes involved in the regulation and activity of autophagy in the lungs of IGF1R-deficient mice. A considerable number of autophagy-related genes were detected in CreERT2-BLM vs. Igf1r^fl/fl-BLM mouse lungs (Fig. 4 and Table S16). However, an unclear, mixed differential expression pattern occurs, as genes with central roles in both positive and negative regulation of autophagy were up- and down-regulated (Fig. S7A). To elucidate whether IGF1R deficiency has an effect on autophagy under BLM-induced ALI conditions, the protein expression of the autophagy marker LC3 was assessed by Western blot and IHC. LC3 II, the active isoform present in autophagosomes, was barely detectable by Western blot relative to isoform I (Fig. S7B). In addition, LC3 I levels did not vary between lungs from any groups (Fig. S7C). In agreement with this, IHC showed scarce presence of LC3 puncta, with mainly diffuse and unchanged staining in all four groups (Fig. S7D).

IGF1R deficiency impacts epigenetic regulation in BLM-challenged mice

In-depth screening of differential expression data revealed 22 genes with important roles in histone-related epigenetic regulation and DNA accessibility (Fig. 8A) as part of the abovementioned epigenetics functional gene cluster (Fig. 4 and Table S18). Thus, some prominent class II (Hdac5, Hdac10) and class IV (Hdac11) histone deacetylases (HDACs) were up-regulated. It is also worth noting the up-regulation of Akap8 and Akap8l, which encode two paralog proteins (A-kinase anchoring protein 8 and A-kinase anchoring protein 8-like) which recruit HDAC3 and possibly other HDACs to the vicinity of chromatin. Furthermore, some genes encoding histone methylases were up-regulated, including Ezh1, Smyd1 and Prdm6. In addition, two H2 histone genes were up-regulated (H2afv and Hist1h2be), both replication-dependent histones. Among the downregulated genes stand the P-element-induced wimpy testis genes Piwil2 and Piwil4, which play an important role in RNA interference and transposon silencing.

Based on these data of IGF1R involvement in epigenetic regulation and given the increased transcriptional levels of genes involved in histone deacetylation, we decided to study the levels of acetylated histone H3 (H3K9ac) in lung sections. Immunofluorescence staining for H3K9ac in Igf1r^fl/fl-BLM and CreERT2-BLM mice showed a heterogeneous, non-uniform patchy pattern throughout the lungs of both genotypes, with accumulation of cells presenting high H3K9ac fluorescence intensity on certain regions, particularly surrounding blood vessels (Fig. S8A). However, quantification of fluorescence intensity did not show significant differences between genotypes either when measured in the whole lung or only in alveolar areas (Fig. S8B-C). Finally, we decided to evaluate lung genomic DNA methylation levels in BLM-challenged mice. Of note, we found a significant increase in percentage (20%) of 5-methyl cytosine (5-mC) in genomic lung DNA of CreERT2-BLM mice relative to Igf1r^fl/fl-BLM mice (Fig. 8B). Taken together, these results suggest a role of IGF1R in regulating epigenetic events in the context of ALI.

Here we report the lung transcriptome profile of a BLM-induced lung injury mouse model in the context of IGF1R deficiency. We performed RNA-seq in lungs from IGF1R-deficient and control mice three days after BLM or saline intra-tracheal instillation to explore the molecular mechanisms mediated by IGF/IGF1R signaling in pulmonary homeostasis and acute inflammation associated with the BLM-mediated “cytokine storm”. As expected, in part based on our previous reports [23], IGF1R deficiency caused a general inhibition of gene expression in the lung, counteracting the massive inflammatory profile and protecting against ALI. Notably, transcriptomic analysis of lungs from BLM-challenged IGF1R-deficient mice brings to front IGF1R action on DNA damage response, cell cycle arrest, metabolic reprogramming, mitochondrial homeostasis and epigenetics.

Lung transcriptome data on untreated CreERT2 lungs show that down-regulated genes in the “mutation” comparison were higher in number and tended to show more marked differential expression than up-regulated genes. This tendency was also previously noted in transcriptomic studies in lungs of both CreERT2 mice (using a different analysis pipeline) and Igf1-deficient prenatal mice [23, 32]. Interestingly, an even higher transcriptional blockade was observed in the “attenuation” comparison in BLM-challenged lungs. These results clearly indicate that deficiency of IGF/IGF1R signaling results in a generalized transcriptional inhibition in the lung. It is very well known that IGFs have multiple roles in numerous cell types, and different healthy and diseased tissues regulating multiple cellular responses [13]. In the murine lung, the relevance of IGF1R signaling was demonstrated by the fact that it shows the highest activation levels of all organs tested after IGF1 exposure [33]. Therefore, it is not surprising that lack of IGF1R causes generalized transcriptional repression in CreERT2-BLM lungs, and specifically in all reported biological functions except epigenetics.

GSEA identified the “IGF signaling” gene cluster as common to the three comparisons. Igf1r^fl/fl-BLM lungs showed lower expression of Igfbp-6, -5 and − 3 and increased Igfbp4 than unchallenged controls. If we add to this the increased expression of the IGFBP protease Pappa and the reduction of its inhibitor Stc1 [14], it could be concluded that an overall activation of the IGF system takes place in response to BLM challenge. Interestingly, most of these genes were found counter-regulated in CreERT2-BLM lungs. Of note, Igfbp3, Insr, Igfbp5 and Igf1 DEGs in this comparison were previously validated by qPCR [23]. Changes in several IGFBPs and other IGF signaling mediators were previously reported in BLM-challenged rodents, although at later stages during the establishment of lung fibrosis [34–36]. Taken together, these results demonstrate that the expression of IGF/Ins system proteins in the mouse injured lung is highly counterbalanced, forming a tightly regulated complex network, which further reinforces the relevance of IGF/IGF1R signaling in this context. Alterations in IGF signaling genes are also relevant in patients with ARDS, resembling data from animal models [16, 17, 37]. More recently, Iosef et al. described plasma signatures of IGF proteins during the COVID-19 “cytokine release syndrome” [18]. Furthermore, blood content of IGF1, IGF1R and IGFBP2 were found associated with COVID-19 severity and/or risk of mortality [20, 21, 38]. These reports strongly suggest that IGF/IGF1R signaling is also implicated in pulmonary diseases involving a “cytokine storm”, and that IGF proteins should be further tested as candidate biomarkers in respiratory diseases with this context.

Our results demonstrate the strong anti-inflammatory effect that IGF1R deficiency has in the context of ALI, by largely counteracting the strong “cytokine storm” resulting from BLM-induced injury. Of note, Cxcl10, one of the most counter-regulated genes in BLM-challenged lungs, while also basally down-regulated by IGF1R deficiency, has been regarded as one of the key cytokines mediating the COVID-19 “cytokine storm” [39]. In addition to acute interstitial and intra-alveolar injury, BLM also causes bronchiolar damage [8, 40], which we also found counteracted in CreERT2-BLM mice. The role of IGF1/IGF1R signaling abnormalities in inflammatory lung diseases has been previously reviewed [15]. In fact, mice with compromised signaling of IGF1R were reported to be protected against inflammation in the lung tumor microenvironment, as well as upon allergic responses and ALI in different respiratory models [28, 41–46]. These data indicate that IGF1R is an important player in promoting ALI and blocking its action could blunt many of ALI fatal consequences in lung diseases with an inflammatory component.

Mechanistically, BLM causes cell damage by generating ROS and single- and double-stranded DNA breaks [8, 47, 48]. Our GSEA on the Igf1r^fl/fl-BLM vs. Igf1r^fl/fl (BLM “injury”) comparison identified a “DNA damage response” functional cluster with DEGs mainly upregulated. Remarkably, IGF1R deficiency reversed the expression of most of these genes in the CreERT2 vs. Igf1r^fl/fl-BLM (“attenuation”) comparison. Furthermore, DNA-damage response pathways were also found in the CreERT2 vs. Igf1r^fl/fl (“mutation”) comparison, including the REACTOME pathways “DNA repair” and “DNA damage bypass”, with all genes down-regulated. This suggests that IGF1R is involved in positively regulating genes that promote DNA repair and other responses to DNA damage after lung injury but also in basal conditions. In this sense, IGF1R has been reported to be located in the nucleus and involved in DNA repair and DNA damage tolerance in various cell lines [49–53]. Nevertheless, down-regulation of DNA damage response genes could also be explained by reduced upstream DNA damage. In this regard, it was recently reported that IGF1R deficiency in AT2 cells protects against radiation-induced lung injury in mice [54]. Altogether, these findings further support the notion that lack of IGF1R could protect lung cells from DNA damage. In addition, data obtained from primary IGF1R KO MEFs show a reduction in ROS levels following H₂O₂ treatment, indicating an enhanced ability to alleviate oxidative stress and supporting a positive effect of the lack of IGF1R on cellular redox status as another possible mechanism that attenuates lung damage. To clarify this thoroughly, further investigations are needed to better understand the role of IGF1R in relation to DNA damage and ROS buffering in the injured lung.

Transcriptome data would suggest that IGF1R deficiency elicits a metabolic shift from glycolysis- to fatty acid-derived energy source in the BLM-challenged lung. Thus, we found a remarkable pattern of DEGs implicated in differential substrate usage, including the up-regulation of Pdk1 and Pdk2 in IGF1R-deficient lungs. These two PDH kinases play a pivotal role in the regulation of glucose and fatty acid metabolism by inhibiting the formation of Acetyl-CoA from pyruvate and down-regulating aerobic respiration [55, 56]. These transcriptomic changes resemble those of a caloric restriction/ketogenic diet characterized by downregulated glucose metabolic pathway but upregulated ketogenic pathway to render acetyl-CoA [57–59]. Seahorse real-time OCR showed that Igf1r^−/− MEFs had decreased ATP-linked mitochondrial respiration compared to Igf1r^fl/fl MEFs. This is consistent with previous studies reporting a stimulatory effect of IGF signaling on energy production in different cell types including primary human lymphoblasts, rat neurons, primary mouse astrocytes and muscle cells [60–63]. Given that all this revolves around pyruvate supply and conversion to acetyl-CoA by the PDH complex, we performed the Seahorse mito-stress test both in standard conditions and without pyruvate in the media. Interestingly, pyruvate absence significantly decreased basal and especially ATP-linked respiration in Igf1r^−/− MEFs but not in control Igf1r^fl/fl MEFs. Of note, these results are in line with a recent study with human and rat cardiomyocytes where stimulation with IGF1 increased mitochondrial respiration along with a reduction in PDH phosphorylation [64]. Disruption of IGF1R function was previously associated with several beneficial metabolic alterations [57–59]. Moreover, we have recently reported that CreERT2 middle aged mice are able to counteract the negative effects of a high fat diet in glucose metabolism, adiposity and inflammation [65]. Transcriptomic changes altering the metabolic profile could also be due to the accumulation of proinflammatory immune cells in the lungs of Igf1r^fl/fl-BLM mice since they are known to preferentially use glycolysis and high lactate production pathways [66, 67]. Further studies on IGF1R- dependent differential substrate use and energy production should be carried out to further explore these mechanisms.

In our transcriptomic data, several key genes involved in the positive regulation of mitophagy and mitochondrial quality control were found to be up-regulated (Pink1, Parkin and Bnip3). Depending on the severity of mitochondrial dysfunction, PINK1 mediates the decision between promoting mitophagy or preventing apoptosis through PARKIN phosphorylation [68, 69] and BNIP3 has also been described to be highly involved in mitochondrial quality control and suppresses PINK1 proteolytic cleavage to promote mitophagy [70]. Thus, upregulation of Pink1, Parkin and Bnip3 would suggest a positive regulation of mitophagy in CreERT2-BLM lungs. In contrast, most of the literature refers IGF/IGF1R signaling as protector of mitochondria, improving mitophagy, mitochondrial function and biogenesis [71–73]. Our study on mitochondrial mass, morphology and network connectivity in BLM-treated MEFs did not show significant differences in mitochondrial morphology or fragmentation. However, these MEFs do show a higher mitochondrial volume paired with senescence-like cell enlargement in BLM-treated Igf1r^fl/fl MEFs that was partially reversed in Igf1r^−/− MEFs. Thus, Igf1r^−/− MEFs presented a milder senescence-like morphology in response to BLM challenge, which includes abnormal accumulation of mitochondria but without demonstrating a reduction in the expression of SA-β-gal staining in the long term.

In-depth exploration of DEGs involved in autophagy did not reveal a clear effect of IGF/IGF1R signaling on autophagy-related functions in the injured lung but rather contradictory or bidirectional roles, in the same way that is profusely reflected in the literature [74–77]. These contradictory results could indicate that IGF1R effects on autophagy are tissue-, cell type- or even cell status-dependent. The different cellular composition present in the injured lungs could also explain the complexity of DEGs related to autophagy in the “attenuation” comparison. In any case, we did not found differences in LC3 protein expression, isoform II/I ratio or presence of LC3 puncta between CreERT2-BLM and Igf1r^fl/fl-BLM lungs.

Importantly, we also identified predominantly up-regulated DEGs involved in epigenetic mechanisms in CreERT2-BLM vs. Igf1r^fl/fl-BLM lungs, including histones, histone methylases and HDACs. Of note, many of them are involved in mediating gene repression by chromatin condensation [78–84]. Moreover, the fact that CreERT2-BLM mice show increased 5-methyl cytosine (5-mC) in genomic lung DNA supports the massive gene transcription repression in BLM-challenged IGF1R-deficient lungs since DNA methylation increases chromatin condensation, favoring repression of gene expression [85]. In addition, since the mechanism of action of BLM is by generating DNA damage, the DNA of IGF1R-deficient lungs could be more protected against physical and chemical DNA damage by higher chromatin condensation [86]. Furthermore, as abovementioned, there is evidence that IGF1R-deficient mice and cells are more resistant to different types of damage [41, 43, 54, 59]. There are some evidences that IGF1/IGF1R signaling regulates expression of histone deacetylases and alters gene activation by histone modifications [87, 88]. Moreover, it has been shown that IGF1R phosphorylates H3 to induce gene expression and chromatin remodeling [89]. Furthermore, loss of IGF1R and insulin receptor (IR) in absence of their ligands was demonstrated to induce a major decrease in the expression of multiple imprinted genes and miRNAs concomitant with an increase in DNA methylation, which was not associated with changes in expression of DNA methylases, suggesting a non-canonical IGF1R/IR-mediated mechanism [90]. In this sense, IGF1R would be acting as an unblocker of the epigenetic load after the aggressive damage of BLM to the lungs. Altogether, these data suggest that IGF1R could be a key component that regulates epigenetics in ARDS.

Herein we analyzed differential gene expression due to IGF1R depletion in BLM-challenged mouse lungs. Our results identified significant changes in gene clusters with key roles in IGF signaling mediators, metabolism, cell cycle, mitochondrial homeostasis, DNA damage, and epigenetics in the lung, providing new insights into the molecular mechanisms underlying the “cytokine storm” during acute pulmonary inflammation and the involvement of IGF1R. These findings allow a more comprehensive view of IGF signaling-mediated regulation at the transcriptomic level and reinforce IGF1R as an important player in promoting acute lung inflammation.

5-mC

5-methylcytosine

ALI

acute lung injury

ARDS

acute respiratory distress syndrome

AT2

alveolar type 2

BLM

bleomycin

BNIP3

BCL2 interacting protein 3

CC10

club cells 10 kDa secretory protein

DEGs

differentially expressed genes

ECM

extracellular matrix

fold change

FDR

false discovery rate

Gene Ontology

GSEA

gene set enrichment analysis

H2DCFDA

2’,7’-dichlorodihydrofluorescein diacetate

H3K9ac

acetylated histone H3 at lysine 9

H&E

hematoxylin and eosin

HDACs

histone deacetylases

IHC

immunohistochemistry

IGFBPs

insulin-like growth factor binding proteins

IGF1

insulin-like growth factor 1

IGF1R

insulin-like growth factor receptor 1

insulin receptor

KEGG

Kyoto Encyclopedia of Genes and Genomes

LC3

microtubule-associated protein 1a/1b light chain 3

MEFs

mouse embryonic fibroblasts

MGI

Mouse Genome Informatics

OCR

oxygen consumption rate

PARKIN

E3 ubiquitin-protein ligase

PCA

principal component analysis

PDH

pyruvate dehydrogenase

PINK1

PTEN-induced kinase 1

pro-SPC

pulmonary-associated surfactant protein C

p21

cyclin-dependent kinase inhibitor 1

RNA-seq

RNA sequencing

ROS

reactive oxygen species

SAL

saline

SA-β-gal

senescence-associated beta-galactosidase

TMX

tamoxifen

TOM20

translocase of outer mitochondrial membrane 20

γ-H2AX

phospho-histone H2AX

Ethics approval and consent to participate

All experiments and animal procedures were carried out following the guidelines of the European Communities Council Directive on animal experiments EU Directive of 22 September 2010 laid down by the European Union Council Directive of 22 September 2010 (2010/63/EU). Animal procedures were revised and approved by the Institutional Animal Care & Use Committee (IACUC) from the Center of Biomedical Research of La Rioja (CIBIR, Spain) (refs. 03/12 and 13/12), in accordance to the present legislation and recommendations on animal welfare and ethical principles. All animals were bred and maintained under specific pathogen-free (SPF) conditions in laminar flow caging at the CIBIR animal facility.

Consent for publication

Not applicable.

Availability of data and materials

All data supporting the conclusions of this article are available upon request to the corresponding author.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by grants PGC2018-097397-B-I00 and PID2021-127860OB-I00) funded by MCIN/AEI/10.13039/501100011033 (Spain) and by “ERDF a way of making Europe” (EU) to JGP.

Authors’ contributions

SPH, IPL and JGP developed the animal model and obtained mouse samples. AU, IPL and JGP conceptualized the RNA-seq study and designed the experiments. AU, EAA, IPL and MC performed the experiments. AU, MdT, IPL and JGP analyzed the data. AU, IPL and JGP wrote the manuscript, with input from the rest of authors.

Acknowledgements

We are grateful to Drs J. Brüning (University of Cologne, Germany) and E. Brown (U Penn School of Medicine, PA) for providing Igf1r^fl/fland UBC-CreERT2 mouse lines, respectively. The authors are deeply grateful to Alvaro Perez-Sala (CIBIR, Logroño) for technical assistance with computer support. A.U. thanks the Sistema Riojano de Innovación (Gobierno de La Rioja, Spain) for a PhD grant.

Savin IA, Zenkova MA, Sen'kova AV. Pulmonary Fibrosis as a Result of Acute Lung Inflammation: Molecular Mechanisms, Relevant In Vivo Models, Prognostic and Therapeutic Approaches. Int J Mol Sci. 2022;23(23):14959.
Wong RSY. Inflammation in COVID-19: from pathogenesis to treatment. Int J Clin Exp Pathol. 2021;14(7):831–44.
Rezoagli E, Fumagalli R, Bellani G. Definition and epidemiology of acute respiratory distress syndrome. Ann Transl Med. 2017;5(14):282.
Yang X, Yu Y, Xu J, Shu H, Xia J, Liu H, et al. Clinical course and outcomes of critically ill patients with SARS-CoV-2 pneumonia in Wuhan, China: a single-centered, retrospective, observational study. Lancet Respir Med. 2020;8(5):475–81.
Chen R, Lan Z, Ye J, Pang L, Liu Y, Wu W, et al. Cytokine Storm: The Primary Determinant for the Pathophysiological Evolution of COVID-19 Deterioration. Front Immunol. 2021;12:589095.
Li L, Zhang H, Min D, Zhang R, Wu J, Qu H, et al. Sox9 activation is essential for the recovery of lung function after acute lung injury. Cell Physiol Biochem. 2015;37(3):1113–22.
Li LF, Liu YY, Kao KC, Wu CT, Chang CH, Hung CY, et al. Mechanical ventilation augments bleomycin-induced epithelial-mesenchymal transition through the Src pathway. Lab Invest. 2014;94(9):1017–29.
Della Latta V, Cecchettini A, Del Ry S, Morales MA. Bleomycin in the setting of lung fibrosis induction: From biological mechanisms to counteractions. Pharmacol Res. 2015;97:122–30.
Yoon YM, Hrusch CL, Fei N, Barron GM, Mills KAM, Hollinger MK, et al. Gut microbiota modulates bleomycin-induced acute lung injury response in mice. Respir Res. 2022;23(1):337.
Zhan P, Lu X, Li Z, Wang WJ, Peng K, Liang NN et al. Mitoquinone alleviates bleomycin-induced acute lung injury via inhibiting mitochondrial ROS-dependent pulmonary epithelial ferroptosis. Int Immunopharmacol. 2022;113(Pt A):109359.
Blazquez-Prieto J, Lopez-Alonso I, Huidobro C, Albaiceta GM. The Emerging Role of Neutrophils in Repair after Acute Lung Injury. Am J Respir Cell Mol Biol. 2018;59(3):289–94.
Laskin DL, Malaviya R, Laskin JD. Role of Macrophages in Acute Lung Injury and Chronic Fibrosis Induced by Pulmonary Toxicants. Toxicol Sci. 2019;168(2):287–301.
LeRoith D, Holly JMP, Forbes BE. Insulin-like growth factors: Ligands, binding proteins, and receptors. Mol Metab. 2021;52:101245.
Oxvig C, Conover CA, The Stanniocalcin-PAPP-A-IGFBP-IGF, Axis. J Clin Endocrinol Metab. 2023;108(7):1624–33.
Wang Z, Li W, Guo Q, Wang Y, Ma L, Zhang X. Insulin-Like Growth Factor-1 Signaling in Lung Development and Inflammatory Lung Diseases. Biomed Res Int. 2018;2018:6057589.
Ahasic AM, Zhai R, Su L, Zhao Y, Aronis KN, Thompson BT, et al. IGF1 and IGFBP3 in acute respiratory distress syndrome. Eur J Endocrinol. 2012;166(1):121–9.
Chen C, Shi L, Li Y, Wang X, Yang S. Disease-specific dynamic biomarkers selected by integrating inflammatory mediators with clinical informatics in ARDS patients with severe pneumonia. Cell Biol Toxicol. 2016;32(3):169–84.
Iosef C, Martin CM, Slessarev M, Gillio-Meina C, Cepinskas G, Han VKM, et al. COVID‐19 plasma proteome reveals novel temporal and cell‐specific signatures for disease severity and high‐precision disease management. J Cell Mol Med. 2022;27(1):141–57.
Iosef C, Knauer MJ, Nicholson M, Van Nynatten LR, Cepinskas G, Draghici S, et al. Plasma proteome of Long-COVID patients indicates HIF-mediated vasculo-proliferative disease with impact on brain and heart function. J Transl Med. 2023;21(1):377.
Mester P, Räth U, Schmid S, Amend P, Keller D, Krautbauer S, et al. Serum Insulin-like Growth Factor-Binding Protein-2 as a Prognostic Factor for COVID-19 Severity. Biomedicines. 2024;12(1):125.
Fraser DD, Cepinskas G, Patterson EK, Slessarev M, Martin C, Daley M, et al. Novel Outcome Biomarkers Identified With Targeted Proteomic Analyses of Plasma From Critically Ill Coronavirus Disease 2019 Patients. Crit Care Explor. 2020;2(9):e0189.
Lopez IP, Rodriguez-de la Rosa L, Pais RS, Piñeiro-Hermida S, Torrens R, Contreras J, et al. Differential organ phenotypes after postnatal Igf1r gene conditional deletion induced by tamoxifen in UBC-CreERT2; Igf1r fl/fl double transgenic mice. Transgenic Res. 2015;24(2):279–94.
Piñeiro-Hermida S, Lopez IP, Alfaro-Arnedo E, Torrens R, Iniguez M, Alvarez-Erviti L, et al. IGF1R deficiency attenuates acute inflammatory response in a bleomycin-induced lung injury mouse model. Sci Rep. 2017;7(1):4290.
Chaudhry A, Shi R, Luciani DS. A pipeline for multidimensional confocal analysis of mitochondrial morphology, function, and dynamics in pancreatic β-cells. Am J Physiol Endocrinol Metab. 2020;318(2):E87–101.
Hart M, Adams SD, Draviam VM. Multinucleation associated DNA damage blocks proliferation in p53-compromised cells. Commun Biol. 2021;4(1):451.
Ivorra C, Fraga MF, Bayón GF, Fernández AF, Garcia-Vicent C, Chaves FJ, et al. DNA methylation patterns in newborns exposed to tobacco in utero. J Transl Med. 2015;13(1):25.
Nabhan AN, Webster JD, Adams JJ, Blazer L, Everrett C, Eidenschenk C, et al. Targeted alveolar regeneration with Frizzled-specific agonists. Cell. 2023;186(14):2995–3012.
Piñeiro-Hermida S, Gregory JA, Lopez IP, Torrens R, Ruiz-Martinez C, Adner M, et al. Attenuated airway hyperresponsiveness and mucus secretion in HDM-exposed Igf1r-deficient mice. Allergy. 2017;72(9):1317–26.
Piñeiro-Hermida S, Bosso G, Sánchez-Vázquez R, Martínez P, Blasco MA. Telomerase deficiency and dysfunctional telomeres in the lung tumor microenvironment impair tumor progression in NSCLC mouse models and patient-derived xenografts. Cell Death Differ. 2023;30(6):1585–600.
Renna M, Bento CF, Fleming A, Menzies FM, Siddiqi FH, Ravikumar B, et al. IGF-1 receptor antagonism inhibits autophagy. Hum Mol Genet. 2013;22(22):4528–44.
Stalnecker CA, Coleman MF, Bryant KL. Susceptibility to autophagy inhibition is enhanced by dual IGF1R and MAPK/ERK inhibition in pancreatic cancer. Autophagy. 2022;18(7):1737–39.
Pais RS, Moreno-Barriuso N, Hernández-Porras I, López IP, De Las Rivas J, Pichel JG. Transcriptome Analysis in Prenatal IGF1-Deficient Mice Identifies Molecular Pathways and Target Genes Involved in Distal Lung Differentiation. PLoS ONE. 2013;8(12):e83028.
Moody G, Beltran PJ, Mitchell P, Cajulis E, Chung Y-A, Hwang D, et al. IGF1R blockade with ganitumab results in systemic effects on the GH–IGF axis in mice. J Endocrinol. 2014;221(1):145–55.
Kheirollahi V, Khadim A, Kiliaris G, Korfei M, Barroso MM, Alexopoulos I, et al. Transcriptional Profiling of Insulin-like Growth Factor Signaling Components in Embryonic Lung Development and Idiopathic Pulmonary Fibrosis. Cells. 2022;11(12):1973.
Kotarkonda LK, Kulshrestha R, Ravi K. Role of insulin like growth factor axis in the bleomycin induced lung injury in rats. Exp Mol Pathol. 2017;102(1):86–96.
Xiao H, Huang X, Wang S, Liu Z, Dong R, Song D, et al. Metformin ameliorates bleomycin-induced pulmonary fibrosis in mice by suppressing IGF-1. Am J Transl Res. 2020;12(3):940–49.
Schnapp LM, Donohoe S, Chen J, Sunde DA, Kelly PM, Ruzinski J, et al. Mining the acute respiratory distress syndrome proteome: identification of the insulin-like growth factor (IGF)/IGF-binding protein-3 pathway in acute lung injury. Am J Pathol. 2006;169(1):86–95.
Fan X, Yin C, Wang J, Yang M, Ma H, Jin G, et al. Pre-diagnostic circulating concentrations of insulin-like growth factor-1 and risk of COVID-19 mortality: results from UK Biobank. Eur J Epidemiol. 2021;36(3):311–18.
Zhang N, Zhao YD, Wang XM. CXCL10 an important chemokine associated with cytokine storm in COVID-19 infected patients. Eur Rev Med Pharmacol Sci. 2020;24(13):7497–505.
Foskett AM, Bazhanov N, Ti X, Tiblow A, Bartosh TJ, Prockop DJ. Phase-directed therapy: TSG-6 targeted to early inflammation improves bleomycin-injured lungs. Am J Physiol Lung Cell Mol Physiol. 2014;306(2):L120–31.
Ahamed K, Epaud R, Holzenberger M, Bonora M, Flejou J-F, Puard J, et al. Deficiency in type 1 insulin-like growth factor receptor in mice protects against oxygen-induced lung injury. Respir Res. 2005;6(1):31.
Choi J-E, Lee S-s, Sunde DA, Huizar I, Haugk KL, Thannickal VJ, et al. Insulin-like Growth Factor-I Receptor Blockade Improves Outcome in Mouse Model of Lung Injury. Am J Respir Crit Care Med. 2009;179(3):212–19.
Kim T-H, Chow Y-H, Gill SE, Schnapp LM. Effect of Insulin-Like Growth Factor Blockade on Hyperoxia-Induced Lung Injury. Am J Respir Cell Mol Biol. 2012;47(3):372–78.
Piñeiro-Hermida S, Alfaro-Arnedo E, Gregory JA, Torrens R, Ruiz-Martinez C, Adner M, et al. Characterization of the acute inflammatory profile and resolution of airway inflammation after Igf1r-gene targeting in a murine model of HDM-induced asthma. PLoS ONE. 2017;12(12):e0190159.
Alfaro-Arnedo E, Lopez IP, Piñeiro-Hermida S, Ucero AC, Gonzalez-Barcala FJ, Salgado FJ, et al. IGF1R as a Potential Pharmacological Target in Allergic Asthma. Biomedicines. 2021;9(8):912.
Alfaro-Arnedo E, Lopez IP, Piñeiro-Hermida S, Canalejo M, Gotera C, Sola JJ, et al. IGF1R acts as a cancer-promoting factor in the tumor microenvironment facilitating lung metastasis implantation and progression. Oncogene. 2022;41(28):3625–39.
Burger RM, Peisach J, Horwitz SB. Activated bleomycin. A transient complex of drug, iron, and oxygen that degrades DNA. J Biol Chem. 1981;256(22):11636–44.
Matute-Bello G, Frevert CW, Martin TR. Animal models of acute lung injury. Am J Physiol Lung Cell Mol Physiol. 2008;295(3):L379–99.
Chitnis MM, Lodhia KA, Aleksic T, Gao S, Protheroe AS, Macaulay VM. IGF-1R inhibition enhances radiosensitivity and delays double-strand break repair by both non-homologous end-joining and homologous recombination. Oncogene. 2013;33(45):5262–73.
Iwasa T, Okamoto I, Suzuki M, Hatashita E, Yamada Y, Fukuoka M, et al. Inhibition of insulin-like growth factor 1 receptor by CP-751,871 radiosensitizes non-small cell lung cancer cells. Clin Cancer Res. 2009;15(16):5117–25.
Liu X, Chen H, Xu X, Ye M, Cao H, Xu L, et al. Insulin-like growth factor-1 receptor knockdown enhances radiosensitivity via the HIF-1alpha pathway and attenuates ATM/H2AX/53BP1 DNA repair activation in human lung squamous carcinoma cells. Oncol Lett. 2018;16(1):1332–40.
Soni UK, Jenny L, Hegde RS. IGF-1R targeting in cancer – does sub-cellular localization matter? J Exp Clin Cancer Res. 2023;42(1):273.
Waraky A, Lin Y, Warsito D, Haglund F, Aleem E, Larsson O. Nuclear insulin-like growth factor 1 receptor phosphorylates proliferating cell nuclear antigen and rescues stalled replication forks after DNA damage. J Biol Chem. 2017;292(44):18227–39.
Chung EJ, Kwon S, Reedy JL, White AO, Song JS, Hwang I, et al. IGF-1 Receptor Signaling Regulates Type II Pneumocyte Senescence and Resulting Macrophage Polarization in Lung Fibrosis. Int J Radiat Oncol Biol Phys. 2021;110(2):526–38.
Atas E, Oberhuber M, Kenner L. The Implications of PDK1–4 on Tumor Energy Metabolism, Aggressiveness and Therapy Resistance. Front Oncol. 2020;10:583217.
Roche TE, Baker JC, Yan X, Hiromasa Y, Gong X, Peng T, et al. Distinct regulatory properties of pyruvate dehydrogenase kinase and phosphatase isoforms. Prog Nucleic Acid Res Mol Biol. 2001;70:33–75.
Benito M, Kahn CR, Fernández S, García G, García-Aguilar A, Guillén C, et al. Essential Role of IGFIR in the Onset of Male Brown Fat Thermogenic Function: Regulation of Glucose Homeostasis by Differential Organ-Specific Insulin Sensitivity. Endocrinology. 2016;157(4):1495–511.
Drabińska N, Wiczkowski W, Piskuła MK. Recent advances in the application of a ketogenic diet for obesity management. Trends Food Sci Technol. 2021;110:28–38.
Holzenberger M, Dupont J, Ducos B, Leneuve P, Géloën A, Even PC, et al. IGF-1 receptor regulates lifespan and resistance to oxidative stress in mice. Nature. 2002;421(6919):182–87.
Naia L, Ferreira IL, Cunha-Oliveira T, Duarte AI, Ribeiro M, Rosenstock TR, et al. Activation of IGF-1 and Insulin Signaling Pathways Ameliorate Mitochondrial Function and Energy Metabolism in Huntington’s Disease Human Lymphoblasts. Mol Neurobiol. 2014;51(1):331–48.
Aghanoori M-R, Smith DR, Shariati-Ievari S, Ajisebutu A, Nguyen A, Desmond F, et al. Insulin-like growth factor-1 activates AMPK to augment mitochondrial function and correct neuronal metabolism in sensory neurons in type 1 diabetes. Mol Metab. 2019;20:149–65.
Logan S, Pharaoh GA, Marlin MC, Masser DR, Matsuzaki S, Wronowski B, et al. Insulin-like growth factor receptor signaling regulates working memory, mitochondrial metabolism, and amyloid-β uptake in astrocytes. Mol Metab. 2018;9:141–55.
Bhardwaj G, Penniman CM, Klaus K, Weatherford ET, Pan H, Dreyfuss JM, et al. Transcriptomic Regulation of Muscle Mitochondria and Calcium Signaling by Insulin/IGF-1 Receptors Depends on FoxO Transcription Factors. Front Physiol. 2022;12:779121.
Sánchez-Aguilera P, López-Crisosto C, Norambuena-Soto I, Penannen C, Zhu J, Bomer N, et al. IGF-1 boosts mitochondrial function by a Ca2 + uptake-dependent mechanism in cultured human and rat cardiomyocytes. Front Physiol. 2023;14:1106662.
Pérez-Matute P, López IP, Íñiguez M, Recio-Fernández E, Torrens R, Piñeiro-Hermida S, et al. IGF1R is a mediator of sex-specific metabolism in mice: Effects of age and high-fat diet. Front Endocrinol. 2022;13:1033208.
Soto-Heredero G, Gómez de las Heras MM, Gabandé‐Rodríguez E, Oller J, Mittelbrunn M. Glycolysis – a key player in the inflammatory response. FEBS J. 2020;287(16):3350–69.
Xu Y, Chen Y, Zhang X, Ma J, Liu Y, Cui L, et al. Glycolysis in Innate Immune Cells Contributes to Autoimmunity. Front Immunol. 2022;13:920029.
Vives-Bauza C, Zhou C, Huang Y, Cui M, de Vries RLA, Kim J, et al. PINK1-dependent recruitment of Parkin to mitochondria in mitophagy. Proc Natl Acad Sci U S A. 2009;107(1):378–83.
Xiong H, Wang D, Chen L, Choo YS, Ma H, Tang C, Parkin, et al. PINK1, and DJ-1 form a ubiquitin E3 ligase complex promoting unfolded protein degradation. J Clin Invest. 2009;119(3):650–60.
Zhang J, Ney PA. Role of BNIP3 and NIX in cell death, autophagy, and mitophagy. Cell Death Differ. 2009;16(7):939–46.
Sádaba MC, Martín-Estal I, Puche JE, Castilla-Cortázar I. Insulin-like growth factor 1 (IGF-1) therapy: Mitochondrial dysfunction and diseases. Biochim Biophys Acta Mol Basis Dis. 2016;1862(7):1267–78.
Lyons A, Coleman M, Riis S, Favre C, O'Flanagan CH, Zhdanov AV, et al. Insulin-like growth factor 1 signaling is essential for mitochondrial biogenesis and mitophagy in cancer cells. J Biol Chem. 2017;292(41):16983–98.
Ribeiro M, Rosenstock TR, Oliveira AM, Oliveira CR, Rego AC. Insulin and IGF-1 improve mitochondrial function in a PI-3K/Akt-dependent manner and reduce mitochondrial generation of reactive oxygen species in Huntington’s disease knock-in striatal cells. Free Radic Biol Med. 2014;74:129–44.
Kasprzak A. Autophagy and the Insulin-like Growth Factor (IGF) System in Colonic Cells: Implications for Colorectal Neoplasia. Int J Mol Sci. 2023;24(4):3665.
Abdellatif M, Madeo F, Kroemer G, Sedej S. Spermidine overrides INSR (insulin receptor)-IGF1R (insulin-like growth factor 1 receptor)-mediated inhibition of autophagy in the aging heart. Autophagy. 2022;18(10):2500–02.
García-Mato Á, Cervantes B, Rodríguez-de la Rosa L, Varela-Nieto I. IGF-1 Controls Metabolic Homeostasis and Survival in HEI-OC1 Auditory Cells through AKT and mTOR Signaling. Antioxidants. 2023;12(2):233.
Li J, Zhang Y, Wang L, Li M, Yang J, Chen P, et al. FOXA1 prevents nutrients deprivation induced autophagic cell death through inducing loss of imprinting of IGF2 in lung adenocarcinoma. Cell Death Dis. 2022;13(8):711.
Grozinger CM, Hassig CA, Schreiber SL. Three proteins define a class of human histone deacetylases related to yeast Hda1p. Proc Natl Acad Sci U S A. 1999;96(9):4868–73.
Marzluff WF, Gongidi P, Woods KR, Jin J, Maltais LJ. The Human and Mouse Replication-Dependent Histone Genes. Genomics. 2002;80(5):487–98.
Davis CA, Haberland M, Arnold MA, Sutherland LB, McDonald OG, Richardson JA, et al. PRISM/PRDM6, a transcriptional repressor that promotes the proliferative gene program in smooth muscle cells. Mol Cell Biol. 2006;26(7):2626–36.
Li Y, Kao GD, Garcia BA, Shabanowitz J, Hunt DF, Qin J, et al. A novel histone deacetylase pathway regulates mitosis by modulating Aurora B kinase activity. Genes Dev. 2006;20(18):2566–79.
Shen X, Liu Y, Hsu YJ, Fujiwara Y, Kim J, Mao X, et al. EZH1 mediates methylation on histone H3 lysine 27 and complements EZH2 in maintaining stem cell identity and executing pluripotency. Mol Cell. 2008;32(4):491–502.
Ross RJ, Weiner MM, Lin H. PIWI proteins and PIWI-interacting RNAs in the soma. Nature. 2014;505(7483):353–59.
Rueda-Robles A, Audano M, Alvarez-Mercado AI, Rubio-Tomas T. Functions of SMYD proteins in biological processes: What do we know? An updated review. Arch Biochem Biophys. 2021;712:109040.
Siegfried Z, Eden S, Mendelsohn M, Feng X, Tsuberi BZ, Cedar H. DNA methylation represses transcription in vivo. Nat Genet. 1999;22(2):203–6.
Minami K, Iida S, Maeshima K. Chromatin organization and DNA damage. Enzymes. 2022;51:29–51.
De Bruyne E, Bos TJ, Schuit F, Van Valckenborgh E, Menu E, Thorrez L, et al. IGF-1 suppresses Bim expression in multiple myeloma via epigenetic and posttranslational mechanisms. Blood. 2010;115(12):2430–40.
Pizzo SV, Jiang W, Block ME, Boosani CS. Short communication: TNF-α and IGF-1 regulates epigenetic mechanisms of HDAC2 and HDAC10. PLoS ONE. 2022;17(2):e0263190.
Warsito D, Lin Y, Gnirck A-C, Sehat B, Larsson O. Nuclearly translocated insulin-like growth factor 1 receptor phosphorylates histone H3 at tyrosine 41 and inducesSNAI2expressionviaBrg1 chromatin remodeling protein. Oncotarget. 2016;7(27):42288–302.
Boucher J, Charalambous M, Zarse K, Mori MA, Kleinridders A, Ristow M, et al. Insulin and insulin-like growth factor 1 receptors are required for normal expression of imprinted genes. Proc Natl Acad Sci U S A. 2014;111(40):14512–17.

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Transcriptomics of acute injured lungs reveals IGF1R action on DNA damage response, metabolic reprogramming, mitochondrial homeostasis and epigenetics

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Abstract

Background.

Methods.

Results.

Conclusions.

Figures

Background

Materials and Methods

RNA isolation, cDNA library preparation and high-throughput sequencing

Bioinformatic analysis

Histology, immunohistochemistry and immunofluorescence

MEFs treatments and immunofluorescence

Measurement of mitochondrial respiration by Seahorse analyzer

γH2AX foci and nuclear morphology analysis

Measurement of Reactive Oxygen Species (ROS)

Western blotting

Lung genomic DNA extraction and global DNA methylation assay

Statistical analysis

Results

Whole transcriptome differential expression and functional enrichment analyses in lungs of BLM-challenged and IGF1R-deficient mice

IGF1R deficiency impacts expression of mitochondrial homeostasis-related genes and prevents mitochondrial accumulation after BLM challenge

IGF1R deficiency impacts epigenetic regulation in BLM-challenged mice

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1