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Hox5 genes direct elastin network formation during alveologenesis by regulating myofibroblast adhesion.

ABSTRACT: Hox5 genes (Hoxa5, Hoxb5, Hoxc5) are exclusively expressed in the lung mesenchyme during embryogenesis, and the most severe phenotypes result from constitutive loss of function of all three genes. Because Hox5 triple null mutants exhibit perinatal lethality, the contribution of this paralogous group to postembryonic lung development is unknown. Intriguingly, expression of all three Hox5 genes peaks during the first 2 weeks after birth, reaching levels far exceeding those measured at embryonic stages, and surviving Hoxa5 single and Hox5 AabbCc compound mutants exhibit defects in the localization of alveolar myofibroblasts. To define the contribution of the entire Hox5 paralogous group to this process, we generated an Hoxa5 conditional allele to use with our existing null alleles for Hoxb5 and Hoxc5 Postnatally, mesenchymal deletion of Hoxa5 in an Hoxb5/Hoxc5 double-mutant background results in severe alveolar simplification. The elastin network required for alveolar formation is dramatically disrupted in Hox5 triple mutants, while the basal lamina, interstitial matrix, and fibronectin are normal. Alveolar myofibroblasts remain Pdgfr?+/SMA+ double positive and present in normal numbers, indicating that the irregular elastin network is not due to fibroblast differentiation defects. Rather, we observe that SMA+ myofibroblasts of Hox5 triple mutants are morphologically abnormal both in vivo and in vitro with highly reduced adherence to fibronectin. This loss of adhesion is a result of loss of the integrin heterodimer Itga5b1 in mutant fibroblasts. Collectively, these data show an important role for Hox5 genes in lung fibroblast adhesion necessary for proper elastin network formation during alveologenesis.

SUBMITTER: Hrycaj SM

PROVIDER: S-EPMC6233103 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Hox5 genes direct elastin network formation during alveologenesis by regulating myofibroblast adhesion.

Hrycaj Steven M SM Marty-Santos Leilani L Cebrian Cristina C Rasky Andrew J AJ Ptaschinski Catherine C Lukacs Nicholas W NW Wellik Deneen M DM

Proceedings of the National Academy of Sciences of the United States of America 20181022 45

Hox5 genes (Hoxa5, Hoxb5, Hoxc5) are exclusively expressed in the lung mesenchyme during embryogenesis, and the most severe phenotypes result from constitutive loss of function of all three genes. Because Hox5 triple null mutants exhibit perinatal lethality, the contribution of this paralogous group to postembryonic lung development is unknown. Intriguingly, expression of all three Hox5 genes peaks during the first 2 weeks after birth, reaching levels fa ...[more]

PMID: 30348760

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Project description:BACKGROUND:Alveologenesis is the final stage of lung development to form air-exchanging units between alveoli and blood vessels. Genetic susceptibility or hyperoxic stress to perturb this complicated process can cause abnormal enlargement of alveoli and lead to bronchopulmonary dysplasia (BPD)-associated emphysema. Platelet-derived growth factor receptor ? (PDGFR?) signaling is crucial for alveolar myofibroblast (MYF) proliferation and its deficiency is associated with risk of BPD, but posttranscriptional mechanisms regulating PDGFR? synthesis during lung development remain largely unexplored. Cytoplasmic polyadenylation element-binding protein 2 (CPEB2) is a sequence-specific RNA-binding protein and translational regulator. Because CPEB2-knockout (KO) mice showed emphysematous phenotypes, we investigated how CPEB2-controlled translation affects pulmonary development and function. METHODS:Respiratory and pulmonary functions were measured by whole-body and invasive plethysmography. Histological staining and immunohistochemistry were used to analyze morphology, proliferation, apoptosis and cell densities from postnatal to adult lungs. Western blotting, RNA-immunoprecipitation, reporter assay, primary MYF culture and ectopic expression rescue were performed to demonstrate the role of CPEB2 in PDGFR? mRNA translation and MYF proliferation. RESULTS:Adult CPEB2-KO mice showed emphysema-like dysfunction. The alveolar structure in CPEB2-deficient lungs appeared normal at birth but became simplified through the alveolar stage of lung development. In CPEB2-null mice, we found reduced proliferation of MYF progenitors during alveolarization, abnormal deposition of elastin and failure of alveolar septum formation, thereby leading to enlarged pulmonary alveoli. We identified that CPEB2 promoted PDGFR? mRNA translation in MYF progenitors and this positive regulation could be disrupted by H2O2, a hyperoxia-mimetic treatment. Moreover, decreased proliferating ability in KO MYFs due to insufficient PDGFR? expression was rescued by ectopic expression of CPEB2, suggesting an important role of CPEB2 in upregulating PDGFR? signaling for pulmonary alveologenesis. CONCLUSIONS:CPEB2-controlled translation, in part through promoting PDGFR? expression, is indispensable for lung development and function. Since defective pulmonary PDGFR signaling is a key feature of human BPD, CPEB2 may be a risk factor for BPD.

Project description:Decorin, a small leucine-rich proteoglycan, is a natural inhibitor of transforming growth factor beta (TGFbeta). Myofibroblast and haze formation in the cornea have been attributed to TGFbeta hyperactivity released from corneal epithelium following injury to eye. This study tested the hypothesis that decorin-gene transfer inhibits TGFbeta-driven myofibroblast and haze formation in the cornea. Human corneal fibroblast (HSF) cultures generated from donor human corneas were used. Decorin cDNA was cloned into mammalian expression vector. Restriction enzyme analysis and DNA sequencing confirmed the nucleotide sequence of generated vector construct. The decorin gene cloned into mammalian expression vector was introduced into HSF with lipofectamine transfection kit. Expression of decorin in selected clones was characterized with RT-PCR, immunocytochemistry and western blotting. Phage contrast microscopy and trypan blue exclusion assay evaluated the effects of decorin-gene transfer on HSF phenotype and viability, respectively. Real-time PCR, western blot and immunocytochemistry were used to analyze inhibitory effects of decorin-gene transfer on TGFbeta-induced myofibroblast formation by measuring differential expression of alpha smooth muscle actin (SMA), a myofibroblast marker, mRNA and protein expression. Analysis of variance (ANOVA) and the Bonferroni-Dunn adjustment for repeated measures were used for statistical analysis. Our data indicate that decorin-gene transfer into HSF do not alter cellular phenotype or viability. Decorin over-expressing HSF clones grown in the presence of TGFbeta1 under serum-free conditions showed a statistically significant 80-83% decrease in SMA expression (p value < 0.01) compared to naked-vector transfected clones or un-transfected HSF controls. Decorin-transfected, naked-vector transfected and un-transfected HSF grown in the absence of TGFbeta1 showed no or extremely low expression of SMA. Furthermore, decorin over-expression did not affect HSF phenotype and decreased TGFbeta-induced RNA levels of profibrogenic genes such as fibronectin, collagen type I, III, and IV that play important role in stromal matrix modulation and corneal wound healing. The results of study suggest that decorin-gene transfer effectively prevents TGFbeta-driven transformation of keratocyte and corneal fibroblast to myofibroblasts. We postulate that decorin-gene therapy can be used to treat corneal haze in vivo.

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Hox5 genes direct elastin network formation during alveologenesis by regulating myofibroblast adhesion.

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<i>Hox5</i> genes direct elastin network formation during alveologenesis by regulating myofibroblast adhesion.

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