BackgroundInsulin-like Growth Factor 1 (IGF1) is a multifunctional regulator of somatic growth and development throughout evolution. IGF1 signaling through IGF type 1 receptor (IGF1R) controls cell proliferation, survival and differentiation in multiple cell types. IGF1 deficiency in mice disrupts lung morphogenesis, causing altered prenatal pulmonary alveologenesis. Nevertheless, little is known about the cellular and molecular basis of IGF1 activity during lung development.Methods/Principal FindingsPrenatal Igf1−/− mutant mice with a C57Bl/6J genetic background displayed severe disproportional lung hypoplasia, leading to lethal neonatal respiratory distress. Immuno-histological analysis of their lungs showed a thickened mesenchyme, alterations in extracellular matrix deposition, thinner smooth muscles and dilated blood vessels, which indicated immature and delayed distal pulmonary organogenesis. Transcriptomic analysis of Igf1−/− E18.5 lungs using RNA microarrays identified deregulated genes related to vascularization, morphogenesis and cellular growth, and to MAP-kinase, Wnt and cell-adhesion pathways. Up-regulation of immunity-related genes was verified by an increase in inflammatory markers. Increased expression of Nfib and reduced expression of Klf2, Egr1 and Ctgf regulatory proteins as well as activation of ERK2 MAP-kinase were corroborated by Western blot. Among IGF-system genes only IGFBP2 revealed a reduction in mRNA expression in mutant lungs. Immuno-staining patterns for IGF1R and IGF2, similar in both genotypes, correlated to alterations found in specific cell compartments of Igf1−/− lungs. IGF1 addition to Igf1−/− embryonic lungs cultured ex vivo increased airway septa remodeling and distal epithelium maturation, processes accompanied by up-regulation of Nfib and Klf2 transcription factors and Cyr61 matricellular protein.Conclusions/SignificanceWe demonstrated the functional tissue specific implication of IGF1 on fetal lung development in mice. Results revealed novel target genes and gene networks mediators of IGF1 action on pulmonary cellular proliferation, differentiation, adhesion and immunity, and on vascular and distal epithelium maturation during prenatal lung development.

Insulin-like growth factor type 1 receptor (IGF1R) is a ubiquitously expressed tyrosine kinase that regulates cell proliferation, differentiation and survival. It controls body growth and organ homeostasis, but with specific functions depending on developmental time and cell type. Human deficiency in IGF1R is involved in growth failure, microcephaly, mental retardation and deafness, and its overactivation is implicated in oncogenesis. Igf1r-deficient mice die at birth due to growth retardation and respiratory failure. Although multiple Igf1r tissue-specific mutant lines have been analyzed postnatally, using Igf1r-floxed (Igf1r (fl/fl) ) mice mated with diverse cell-type recombinase Cre-expressing transgenics, no mouse models for the study of generalized Igf1r deficiency in adults have been reported. To this end we generated UBC-CreERT2; Igf1r (fl/fl) transgenic mice with an inducible deletion of Igf1r activated by tamoxifen. Tamoxifen administration to 4 week-old prepuberal male mice delayed their growth, producing a distinct impact on organ size 4 weeks later. Whereas testes were smaller, spleen and heart showed an increased organ to body weight ratio. Mosaic Igf1r genomic deletions caused a significant reduction in Igf1r mRNA in all organs analyzed, resulting in diverse phenotypes. While kidneys, spleen and cochlea had unaltered gross morphology, testes revealed halted spermatogenesis, and liver and alveolar lung parenchyma showed increased cell proliferation rates without affecting apoptosis. We demonstrate that UBC-CreERT2 transgenic mice efficiently delete Igf1r upon postnatal tamoxifen treatment in multiple mouse organs, and corroborate that IGF1R function is highly dependent on cell, tissue and organ type.

Regeneration of lung epithelium is vital for maintaining airway function and integrity. An imbalance between epithelial damage and repair is at the basis of numerous chronic lung diseases such as asthma, COPD, pulmonary fibrosis and lung cancer. IGF (Insulin-like Growth Factors) signaling has been associated with most of these respiratory pathologies, although their mechanisms of action in this tissue remain poorly understood. Expression profiles analyses of IGF system genes performed in mouse lung support their functional implication in pulmonary ontogeny. Immuno-localization revealed high expression levels of Igf1r (Insulin-like Growth Factor 1 Receptor) in lung epithelial cells, alveolar macrophages and smooth muscle. To further understand the role of Igf1r in pulmonary homeostasis, two distinct lung epithelial-specific Igf1r mutant mice were generated and studied. The lack of Igf1r disturbed airway epithelial differentiation in adult mice, and revealed enhanced proliferation and altered morphology in distal airway club cells. During recovery after naphthalene-induced club cell injury, the kinetics of terminal bronchiolar epithelium regeneration was hindered in Igf1r mutants, revealing increased proliferation and delayed differentiation of club and ciliated cells. Amid airway restoration, lungs of Igf1r deficient mice showed increased levels of Igf1, Insr, Igfbp3 and epithelial precursor markers, reduced amounts of Scgb1a1 protein, and alterations in IGF signaling mediators. These results support the role of Igf1r in controlling the kinetics of cell proliferation and differentiation during pulmonary airway epithelial regeneration after injury.

AEC1 del inglés alveolar epithelial type 1 cells AEC2 del inglés alveolar epithelial type 2 cells ABC del inglés Avidin-Biotin Peroxidase Comples ADC Adenocarcinoma ADN Ácido Desoxirribonucelico ADNc Ácido Desoxirribonucelico complementario al zona alveolar ARNm Ácido Ribonucleico mensajero Aqp5 del inglés Aquaporin 5 ARN Ácido Ribonucleico ATF1 del inglés activating transcription factor 1 BAC del inglés Bacterial artificial chromosome BADJ del inglés bronchial alveolar conduct junction BPD del inglés Bronchopulmonary displasia BrdU Bromodeoxiuridina BSA del inglés Bovine Serum Albumin bt bronquiolo terminal CCSP del inglés Clara cell secretory protein CC10 del inglés Clara cells 10 kDa secretory protein CE Carcinoma Epidermoide CGRP del inglés calcitonin gene-related peptide ClaraV del inglés variant clara cells CNTF del inglés Ciliary Neurotrophic Factor COPD del inglés Chronic obstructive pulmonary disease Cre Cre recombinase CTGF del inglés Connective Tissue Growth Factor CYR61 del inglés Cysteine-Rich 61 DAB 3,3'-Diaminobencidina DABCO 1,4-diazabiciclo[2.2.2]octano DAPI 4',6-diamidino-2-fenilindol DEPC Dietilpirocarbonato dNTP Didesoxinucleótido trifosfato DPT del inglés dermatopontin DTT Ditiotreitol E día de desarrollo embrionario ECL del inglés Enhanced Chemiluminescence EDTA del inglés ethylenediaminetetraacetic acid EGF del inglés Epidermal Growth Factor EGFP del inglés Enhanced Green Fluorescent Protein EGFR del inglés Epidermal Growth Factor Receptor EGR1 del inglés Early Growth Response 1 ERK del inglés Extracellular signal-Regulated Kinase FDR del inglés False Discovery Rate FGF del inglés Fibroblast Growth Factor FGFR del inglés Fibroblast Growth Factor Receptor FGF18 fibroblast growth factor 18 fl del inglés floxed con un gen o fragmento de ADN flanqueado por dos secuencias loxP Abreviaturas IV Gas5 del inglés growth arrest specific 5 GH del inglés Growth Hormone GRB del inglés Growth factor Receptor-Bound protein Abreviaturas V NSCLC del inglés Non-Small Cell Lung Cancer Nur77 del inglés nuclear hormone receptor NUR/77 Nkx2.1 del inglés homeobox protein Nkx-2.1 P dia postnatal (referido a la edad de los ratones) p38 del inglés p38 kinasa pAKT del inglés phosphorylated AKT pERK del inglés phosphorylated Extracellular Signal-Regulated Kinase PBST del inglés Phosphate Buffer Saline Tween pb pares de bases PCNA del inglés Proliferating Cell Nuclear Antigen PCR del inglés Polymerase Chain Reaction PDGF del inglés platelet-derived growth factor beta PECAM del inglés Platelet/Endothelial Cell Adhesion Molecule PFA Paraformaldehído PI3K del inglés Phosphoinositide-3 Kinase PGP9,5 del inglés neuron cytoplasmic protein 9.5 Plat del inglés plasminogen activator PH del inglés Plekstrin Homologue PL del inglés placental lactogen ProSPC del inglés Propeptide Surfactant Protein C PVDF del inglés Polyvinylidene Fluoride pSRC del inglés phosphorylated SRC pSTAT-3 del inglés phosphorylated STAT-3 pSTAT del inglés phosphorylated Signal Transducer and Activator of Transcription qRT-PCR del inglés quantitative RT-PCR...