The role of integrin alpha8beta1 in fetal lung morphogenesis and injury.
ABSTRACT: Prenatal inflammation prevents normal lung morphogenesis and leads to bronchopulmonary dysplasia (BPD), a common complication of preterm birth. We previously demonstrated in a bacterial endotoxin mouse model of BPD that disrupting fibronectin localization in the fetal lung mesenchyme causes arrested saccular airway branching. In this study we show that expression of the fibronectin receptor, integrin alpha8beta1 is decreased in the lung mesenchyme in the same inflammation model suggesting it is required for normal lung development. We verified a role for integrin alpha8beta1 in lung development using integrin alpha8-null mice, which develop fusion of the medial and caudal lobes as well as abnormalities in airway division. We further show in vivo and in vitro that alpha8-null fetal lung mesenchymal cells fail to form stable adhesions and have increased migration. Thus we propose that integrin alpha8beta1 plays a critical role in lung morphogenesis by regulating mesenchymal cell adhesion and migration. Furthermore, our data suggest that disruption of the interactions between extracellular matrix and integrin alpha8beta1 may contribute to the pathogenesis of BPD.
Project description:Epithelio-mesenchymal interactions during kidney organogenesis are disrupted in integrin alpha8 beta1-deficient mice. However, the known ligands for integrin alpha8 beta1-fibronectin, vitronectin, and tenascin-C-are not appropriately localized to mediate all alpha8 beta1 functions in the kidney. Using a method of general utility for determining the distribution of unknown integrin ligands in situ and biochemical characterization of these ligands, we identified osteopontin (OPN) as a ligand for alpha8 beta1. We have coexpressed the extracellular domains of the mouse alpha8 and beta1 integrin subunits as a soluble heterodimer with one subunit fused to alkaline phosphatase (AP) and have used the alpha8 beta1-AP chimera as a histochemical reagent on sections of mouse embryos. Ligand localization with alpha8 beta1-AP in developing bone and kidney was observed to be overlapping with the distribution of OPN. In "far Western" blots of mouse embryonic protein extracts, bands were detected with sizes corresponding to fibronectin, vitronectin, and unknown proteins, one of which was identical to the size of OPN. In a solid-phase binding assay we demonstrated that purified OPN binds specifically to alpha8 beta1-AP. Cell adhesion assays using K562 cells expressing alpha8 beta1 were used to confirm this result. Together with a recent report that anti-OPN antibodies disrupt kidney morphogenesis, our results suggest that interactions between OPN and integrin alpha8 beta1 may help regulate kidney development and other morphogenetic processes.
Project description:Integrins are transmembrane alphabeta heterodimer receptors that function as structural and functional bridges between the cytoskeleton and ECM (extracellular matrix) molecules. The RGD (arginine-glycine-aspartate tripeptide motif)-dependent integrin alpha8beta1 has been shown to be involved in various cell functions in neuronal and mesenchymal-derived cell types. Its role in epithelial cells remains unknown.Integrin alpha8beta1 was found to be expressed in the crypt cell population of the human intestine but was absent from differentiating and mature epithelial cells of the villus. The function of alpha8beta1 in epithelial crypt cells was investigated at the cellular level using normal HIECs (human intestinal epithelial cells). Specific knockdown of alpha8 subunit expression using an shRNA (small-hairpin RNA) approach showed that alpha8beta1 plays important roles in RGD-dependent cell adhesion, migration and proliferation via a RhoA/ROCK (Rho-associated kinase)-dependent mechanism as demonstrated by active RhoA quantification and pharmacological inhibition of ROCK. Moreover, loss of alpha8beta1, through RhoA/ROCK, impairs FA (focal adhesion) complex integrity as demonstrated by faulty vinculin recruitment.Integrin alpha8beta1 is expressed in epithelial cells. In intestinal crypt cells, alpha8beta1 is closely involved in the regulation of adhesion, migration and cell proliferation via a predominant RhoA/ROCK-dependent mechanism. These results suggest an important role for this integrin in intestinal crypt cell homoeostasis.
Project description:Bronchopulmonary dysplasia (BPD) is a frequent complication of preterm birth. This chronic lung disease results from arrested saccular airway development and is most common in infants exposed to inflammatory stimuli. In experimental models, inflammation inhibits expression of fibroblast growth factor-10 (FGF-10) and impairs epithelial-mesenchymal interactions during lung development; however, the mechanisms connecting inflammatory signaling with reduced growth factor expression are not yet understood. In this study we found that soluble inflammatory mediators present in tracheal fluid from preterm infants can prevent saccular airway branching. In addition, LPS treatment led to local production of mediators that inhibited airway branching and FGF-10 expression in LPS-resistant C.C3-Tlr4(Lpsd)/J fetal mouse lung explants. Both direct NF-?B activation and inflammatory cytokines (IL-1? and TNF-?) that activate NF-?B reduced FGF-10 expression, whereas chemokines that signal via other inflammatory pathways had no effect. Mutational analysis of the FGF-10 promoter failed to identify genetic elements required for direct NF-?B-mediated FGF-10 inhibition. Instead, NF-?B activation appeared to interfere with the normal stimulation of FGF-10 expression by Sp1. Chromatin immunoprecipitation and nuclear coimmunoprecipitation studies demonstrated that the RelA subunit of NF-?B and Sp1 physically interact at the FGF-10 promoter. These findings indicate that inflammatory signaling through NF-?B disrupts the normal expression of FGF-10 in fetal lung mesenchyme by interfering with the transcriptional machinery critical for lung morphogenesis.
Project description:Mutation of the functionally redundant Hoxa 11/Hoxd 11 genes gives absent or rudimentary kidneys resulting from a dramatic reduction of the growth and branching of the ureteric bud. To understand better the molecular mechanisms of Hoxa 11/Hoxd 11 function in kidney development, it is necessary to identify the downstream target genes regulated by their encoded transcription factors. To this end, we conducted a screen for Hoxa 11-responsive genes in two kidney cell lines. HEK293 cells, which usually do not express Hoxa 11, were modified to allow inducible Hoxa 11 expression. The mK10 cells, derived specifically for this study from Hoxa 11/Hoxd 11 double-mutant mice, were also modified to give cell populations with and without Hoxa 11 expression. Differential display, Gene Discovery Arrays, and Affymetrix genechip probe arrays were used to screen for genes up- or down-regulated by Hoxa 11. Nine genes, PDGF A, Cathepsin L, annexin A1, Mm.112139, Est2 repressor factor, NrCAM, ZNF192, integrin-associated protein, and GCM1, showed reproducible 3-fold or smaller changes in gene expression in response to Hoxa 11. One gene, the Integrin alpha8, was up-regulated approximately 20-fold after Hoxa 11 expression. The Integrin alpha8 gene is expressed together with Hoxa 11 in metanephric mesenchyme cells, and mutation of Integrin alpha8 gives a bud-branching morphogenesis defect very similar to that observed in Hoxa 11/Hoxd 11 mutant mice. In situ hybridizations showed a dramatic regional reduction in Integrin alpha8 expression in the developing kidneys of Hoxa 11/Hoxd 11 mutant mice. This work suggests that the Integrin alpha8 gene may be a major effector of Hoxa 11/Hoxd 11 function in the developing kidney.
Project description:The epithelial-mesenchymal interactions required for kidney organogenesis are disrupted in mice lacking the integrin alpha8beta1. None of this integrin's known ligands, however, appears to account for this phenotype. To identify a more relevant ligand, a soluble integrin alpha8beta1 heterodimer fused to alkaline phosphatase (AP) has been used to probe blots and cDNA libraries. In newborn mouse kidney extracts, alpha8beta1-AP detects a novel ligand of 70-90 kD. This protein, named nephronectin, is an extracellular matrix protein with five EGF-like repeats, a mucin region containing a RGD sequence, and a COOH-terminal MAM domain. Integrin alpha8beta1 and several additional RGD-binding integrins bind nephronectin. Nephronectin mRNA is expressed in the ureteric bud epithelium, whereas alpha8beta1 is expressed in the metanephric mesenchyme. Nephronectin is localized in the extracellular matrix in the same distribution as the ligand detected by alpha8beta1-AP and forms a complex with alpha8beta1 in vivo. Thus, these results strongly suggest that nephronectin is a relevant ligand mediating alpha8beta1 function in the kidney. Nephronectin is expressed at numerous sites outside the kidney, so it may also have wider roles in development. The approaches used here should be generally useful for characterizing the interactions of novel extracellular matrix proteins identified through genomic sequencing projects.
Project description:Epithelial-mesenchymal interactions play a crucial role in branching morphogenesis, but very little is known about how endothelial cells contribute to this process. Here, we examined how anti-angiogenic miR-221 and pro-angiogenic miR-130a affect airway and vascular development in the fetal lungs. Lung-specific effects of miR-130a and miR-221 were studied in mouse E14 whole lungs cultured for 48 hours with anti-miRs or mimics to miR-130a and miR-221. Anti-miR 221 treated lungs had more distal branch generations with increased Hoxb5 and VEGFR2 around airways. Conversely, mimic 221 treated lungs had reduced airway branching, dilated airway tips and decreased Hoxb5 and VEGFR2 in mesenchyme. Anti-miR 130a treatment led to reduced airway branching with increased Hoxa5 and decreased VEGFR2 in the mesenchyme. Conversely, mimic 130a treated lungs had numerous finely arborized branches extending into central lung regions with diffusely localized Hoxa5 and increased VEGFR2 in the mesenchyme. Vascular morphology was analyzed by GSL-B4 (endothelial cell-specific lectin) immunofluorescence. Observed changes in airway morphology following miR-221 inhibition and miR-130a enhancement were mirrored by changes in vascular plexus formation around the terminal airways. Mouse fetal lung endothelial cells (MFLM-91U) were used to study microvascular cell behavior. Mimic 221 treatment resulted in reduced tube formation and cell migration, where as the reverse was observed with mimic 130a treatment. From these data, we conclude that miR-221 and miR-130a have opposing effects on airway and vascular morphogenesis of the developing lung.
Project description:Integrin-dependent interactions between cells and extracellular matrix regulate lung development; however, specific roles for ?1-containing integrins in individual cell types, including epithelial cells, remain incompletely understood. In this study, the functional importance of ?1 integrin in lung epithelium during mouse lung development was investigated by deleting the integrin from E10.5 onwards using surfactant protein C promoter-driven Cre. These mutant mice appeared normal at birth but failed to gain weight appropriately and died by 4?months of age with severe hypoxemia. Defects in airway branching morphogenesis in association with impaired epithelial cell adhesion and migration, as well as alveolarization defects and persistent macrophage-mediated inflammation were identified. Using an inducible system to delete ?1 integrin after completion of airway branching, we showed that alveolarization defects, characterized by disrupted secondary septation, abnormal alveolar epithelial cell differentiation, excessive collagen I and elastin deposition, and hypercellularity of the mesenchyme occurred independently of airway branching defects. By depleting macrophages using liposomal clodronate, we found that alveolarization defects were secondary to persistent alveolar inflammation. ?1 integrin-deficient alveolar epithelial cells produced excessive monocyte chemoattractant protein 1 and reactive oxygen species, suggesting a direct role for ?1 integrin in regulating alveolar homeostasis. Taken together, these studies define distinct functions of epithelial ?1 integrin during both early and late lung development that affect airway branching morphogenesis, epithelial cell differentiation, alveolar septation and regulation of alveolar homeostasis.
Project description:Integrin alpha8beta1 interacts with a variety of Arg-Gly-Asp (RGD)-containing ligands in the extracellular matrix. Here, we examined the binding activities of alpha8beta1 integrin toward a panel of RGD-containing ligands. Integrin alpha8beta1 bound specifically to nephronectin with an apparent dissociation constant of 0.28 +/- 0.01 nm, but showed only marginal affinities for fibronectin and other RGD-containing ligands. The high-affinity binding to alpha8beta1 integrin was fully reproduced with a recombinant nephronectin fragment derived from the RGD-containing central "linker" segment. A series of deletion mutants of the recombinant fragment identified the LFEIFEIER sequence on the C-terminal side of the RGD motif as an auxiliary site required for high-affinity binding to alpha8beta1 integrin. Alanine scanning mutagenesis within the LFEIFEIER sequence defined the EIE sequence as a critical motif ensuring the high-affinity integrin-ligand interaction. Although a synthetic LFEIFEIER peptide failed to inhibit the binding of alpha8beta1 integrin to nephronectin, a longer peptide containing both the RGD motif and the LFEIFEIER sequence was strongly inhibitory, and was approximately 2,000-fold more potent than a peptide containing only the RGD motif. Furthermore, trans-complementation assays using recombinant fragments containing either the RGD motif or LFEIFEIER sequence revealed a clear synergism in the binding to alpha8beta1 integrin. Taken together, these results indicate that the specific high-affinity binding of nephronectin to alpha8beta1 integrin is achieved by bipartite interaction of the integrin with the RGD motif and LFEIFEIER sequence, with the latter serving as a synergy site that greatly potentiates the RGD-driven integrin-ligand interaction but has only marginal activity to secure the interaction by itself.
Project description:Reciprocal signaling between an epithelium and its surrounding mesenchyme is common during morphogenesis. These epithelial-mesenchymal interactions are particularly evident in tissues that undergo branching morphogenesis, such as the airway epithelium of the lung. Here, we found that reciprocal interactions between the epithelium and mesenchyme drive remodeling of the extracellular matrix (ECM) during morphogenesis of the embryonic chicken lung. RNA-Seq analysis revealed changes in the expression of genes associated with integrin signaling and ECM remodeling. Consistently, we found that prior to branching, the basement membrane is a spatially uniform sheath that wraps the airway epithelium. After branch initiation, however, the basement membrane is significantly depleted from the tip of extending branches. Culturing embryonic lung explants revealed that this basement membrane thinning is mediated by matrix metalloproteinase-2 (MMP2), which is expressed in the mesenchyme. Inhibiting MMP activity suppresses branch extension but has no effect on branch initiation. As branches extend, we found that tenascin-C (TNC) accumulates in the mesenchyme several cell diameters away from the branch tip. Despite its pattern of accumulation, this mesenchymal ECM protein is expressed exclusively by airway epithelial cells, which activate focal adhesion kinase (FAK) to induce TNC expression. We found that branch extension coincides with the deformation of adjacent mesenchymal cells into elongated geometries, which correlates with an increase in the fluidity of the mesenchyme at branch tips. This local increase in mesenchymal movement transports TNC from the epithelial surface into the mesenchyme. These data reveal novel epithelial-mesenchymal interactions that direct ECM remodeling during airway branching morphogenesis. Overall design: Three biological repilcates from two different stages of chicken lung development (E5 and E6, a total of six samples) were used in this analysis. Fold changes in transcript levels between E5 and E6 lungs were generated referencing transcript counts in E5 lungs.
Project description:<h4>Background</h4>Intrauterine exposure to amniotic fluid (AF) cytokines is thought to predispose to bronchopulmonary dysplasia (BPD). We evaluated the effects of GBS exposure on RNA expression in fetal lung tissue to determine early molecular pathways associated with fetal lung injury that may progress to BPD.<h4>Methods</h4>Ten chronically catheterized pregnant monkeys (Macaca nemestrina) at 118-125 days gestation (term?=?172 days) received choriodecidual inoculation of either: 1) Group B Streptococcus (n?=?5) or 2) saline (n?=?5). Cesarean section and fetal necropsy was performed in the first week after GBS or saline inoculation regardless of labor. RNA was extracted from fetal lungs and profiled by microarray. Results were analyzed using single gene, Gene Set, and Ingenuity Pathway Analysis. Validation was by RT-PCR and immunohistochemistry.<h4>Results</h4>Despite uterine quiescence in most cases, fetal lung injury occurred in four GBS cases (intra-alveolar neutrophils, interstitial thickening) and one control (peri-mortem hemorrhage). Significant elevations of AF cytokines (TNF-?, IL-8, IL-1?, IL-6) were detected in GBS versus controls (p<0.05). Lung injury was not directly caused by GBS, because GBS was undetectable by culture and PCR in the AF and fetal lungs. A total of 335 genes were differentially expressed greater than 1.5 fold (p<0.05) with GBS exposure associated with a striking upregulation of genes in innate and adaptive immunity and downregulation of pathways for angiogenesis, morphogenesis, and cellular growth and development.<h4>Conclusions</h4>A transient choriodecidual infection may induce fetal lung injury with profound alterations in the genetic program of the fetal lung before signs of preterm labor. Our results provide a window for the first time into early molecular pathways disrupting fetal lung angiogenesis and morphogenesis before preterm labor occurs, which may set the stage for BPD. A strategy to prevent BPD should target the fetus in utero to attenuate alterations in the fetal lung genetic program.