Arnaud Mailleux

Evidence that SPROUTY2 functions as an inhibitor of mouse embryonic lung growth and morphogenesis

Experimental evidence is rapidly emerging that the coupling of positive regulatory signals with the induction of negative feedback modulators is a mechanism of fine regulation in development. Studies in Drosophila and chick have shown that members of the SPROUTY family are inducible negative regulators of growth factors that act through tyrosine kinase receptors. We and others have shown that Fibroblast Growth Factor 10 (FGF10) is a key positive regulator of lung branching morphogenesis. Herein, we provide direct evidence that mSprouty2 is dynamically expressed in the peripheral endoderm in embryonic lung and is downregulated in the clefts between new branches at E12.5. We found that mSprouty2 was expressed in a domain restricted in time and space, adjacent to that of Fgf10 in the peripheral mesenchyme. By E14.5, Fgf10 expression was restricted to a narrow domain of mesenchyme along the extreme edges of the individual lung lobes, whereas mSprouty2 was most highly expressed in the subjacent epithelial terminal buds. FGF10 beads upregulated the expression of mSprouty2 in adjacent epithelium in embryonic lung explant culture. Lung cultures treated with exogenous FGF10 showed greater branching and higher levels of mSpry2 mRNA. Conversely, Fgf10 antisense oligonucleotides reduced branching and decreased mSpry2 mRNA levels. However, treatment with exogenous FGF10 or antisense Fgf10 did not change Shh and FgfR2 mRNA levels in the lungs. We investigated Sprouty2 function during lung development by two different but complementary approaches. The targeted overexpression of mSprouty2 in the peripheral lung epithelium in vivo, using the Surfactant Protein C promoter, resulted in a low level of branching, lung lobe edges abnormal in appearance and the inhibition of epithelial proliferation. Transient high-level overexpression of mSpry2 throughout the pulmonary epithelium by intra-tracheal adenovirus microinjection also resulted in a low level of branching. These results indicate for the first time that mSPROUTY2 functions as a negative regulator of embryonic lung morphogenesis and growth.

Molecular Mechanisms of Early Lung Specification and Branching Morphogenesis

The “hard wiring” encoded within the genome that determines the emergence of the laryngotracheal groove and subsequently early lung branching morphogenesis is mediated by finely regulated, interactive growth factor signaling mechanisms that determine the automaticity of branching, interbranch length, stereotypy of branching, left-right asymmetry, and finally gas diffusion surface area. The extracellular matrix is an important regulator as well as a target for growth factor signaling in lung branching morphogenesis and alveolarization. Coordination not only of epithelial but also endothelial branching morphogenesis determines bronchial branching and the eventual alveolar-capillary interface. Improved prospects for lung protection, repair, regeneration, and engineering will depend on more detailed understanding of these processes. Herein, we concisely review the functionally integrated morphogenetic signaling network comprising the critical bone morphogenetic protein, fibroblast growth factor, Sonic hedgehog, transforming growth factor-β, vascular endothelial growth factor, and Wnt signaling pathways that specify and drive early embryonic lung morphogenesis.

Fgf10 expression identifies parabronchial smooth muscle cell progenitors and is required for their entry into the smooth muscle cell lineage.

Lineage formation in the lung mesenchyme is poorly understood. Using a transgenic mouse line expressing LacZ under the control of Fgf10 regulatory sequences, we show that the pool of Fgf10-positive cells in the distal lung mesenchyme contains progenitors of the parabronchial smooth muscle cells. Fgf10 gene expression is slightly repressed in this transgenic line. This allowed us to create a hypomorphic Fgf10 phenotype by expressing the LacZ transgene in a heterozygous Fgf10 background. Hypomorphic Fgf10 mutant lungs display a decrease inβ -galactosidase-positive cells around the bronchial epithelium associated with an accumulation of β-galactosidase-expressing cells in the distal mesenchyme. This correlates with a marked reduction of α smooth muscle actin expression, thereby demonstrating that FGF10 is mostly required for the entry of mesenchymal cells into the parabronchial smooth muscle cell lineage. The failure of exogenous FGF10 to phosphorylate its known downstream targets ERK and AKT in lung mesenchymal cultures strongly suggests that FGF10 acts indirectly on the progenitor population via an epithelial intermediate. We provide support for a role of epithelial BMP4 in mediating the formation of parabronchial smooth muscle cells.

Gli3-mediated somitic Fgf10 expression gradients are required for the induction and patterning of mammary epithelium along the embryonic axes.

Little is known about the regulation of cell fate decisions that lead to the formation of five pairs of mammary placodes in the surface ectoderm of the mouse embryo. We have previously shown that fibroblast growth factor 10 (FGF10) is required for the formation of mammary placodes 1, 2, 3 and 5. Here, we have found that Fgf10 is expressed only in the somites underlying placodes 2 and 3, in gradients across and within these somites. To test whether somitic FGF10 is required for the formation of these two placodes, we analyzed a number of mutants with different perturbations of somitic Fgf10 gradients for the presence of WNT signals and ectodermal multilayering, markers for mammary line and placode formation. The mammary line is displaced dorsally, and formation of placode 3 is impaired in Pax3ILZ/ILZ mutants, which do not form ventral somitic buds. Mammary line formation is impaired and placode 3 is absent in Gli3Xt-J/Xt-J and hypomorphic Fgf10 mutants, in which the somitic Fgf10 gradient is shortened dorsally and less overall Fgf10 is expressed, respectively. Recombinant FGF10 rescued mammogenesis in Fgf10-/- and Gli3Xt-J/Xt-J flanks. We correlate increasing levels of somitic FGF10 with progressive maturation of the surface ectoderm, and show that full expression of somitic Fgf10, co-regulated by GLI3, is required for the anteroposterior pattern in which the flank ectoderm acquires a mammary epithelial identity. We propose that the intra-somitic Fgf10 gradient, together with ventral elongation of the somites, determines the correct dorsoventral position of mammary epithelium along the flank.

Identification of Periplakin as a New Target for Autoreactivity in Idiopathic Pulmonary Fibrosis

RATIONALEnInjury to alveolar epithelial cells is central to the pathophysiology of idiopathic pulmonary fibrosis (IPF). An abnormal autoimmune response directed against antigens of the alveolar epithelium may contribute to the disease.nnnOBJECTIVESnTo detect circulating autoantibodies (autoAbs) directed against epithelial structures.nnnMETHODSnWe performed immunoblot by separating human placental amnion extract or alveolar epithelial cell (A549 cell line) proteins on polyacrylamide gels, blotting on nitrocellulose membranes, and incubating with serum from patients with IPF (n = 40) or healthy subjects (n = 40). Proteomic analysis and mass spectrometry characterized the target protein. Inhibition experiments performed with the correspondent recombinant protein confirmed our results.nnnMEASUREMENTS AND MAIN RESULTSnWe identified IgG autoAbs recognizing a 200-kD protein in the serum of patients with IPF. Proteomic analysis identified this protein as human periplakin (PPL), a component of desmosomes. Anti-PPL Abs were found by immunoblot in both serum and bronchoalveolar lavage in patients with IPF: 16/40 (40%) of them were positive versus none of the control subjects. Immunohistochemistry revealed that PPL was strongly expressed in bronchial and alveolar epithelium, but that PPL exhibited changes in intracellular localization among normal and fibrotic alveolar epithelium. In an alveolar epithelial wound repair assay, an anti-PPL IgG decreased cell migration. Recombinant PPL induced bronchoalveolar lavage T lymphocyte proliferation. Patients with IPF with anti-PPL Abs had a more severe respiratory disease, despite no difference in survival.nnnCONCLUSIONSnWe found a new circulating autoAb directed against PPL in patients with IPF, associated with a more severe disease.

Targeting the Hedgehog–Glioma-Associated Oncogene Homolog Pathway Inhibits Bleomycin-Induced Lung Fibrosis in Mice

Idiopathic pulmonary fibrosis has been associated with the reactivation of developmental pathways, notably the Hedgehog-Glioma-associated oncogene homolog (GLI) pathway. In this study, we determined whether the Hedgehog pathway was activated in bleomycin-induced lung injury in mice, and whether targeting the Hedgehog-Gli pathway could decrease bleomycin-induced lung fibrosis. After intratracheal injection of bleomycin on Day 0, C57Bl6 mice received GDC-0449 (an inhibitor of Smoothened, the transducer of the pathway), or 2,2-[[Dihydro-2-(4-pyridinyl)-1,3(2H,4H)-pyrimidinediyl]bis(methylene)]bis[N,N dimethylbenzenamine (GANT61; an inhibitor of GLI transcription factors in the nucleus), from Day 7 to Day 13. At Day 14, whole-lung homogenates were obtained for morphological analysis, assessment of cell apoptosis and proliferation, collagen quantification, and evaluation of profibrotic (transforming growth factor-β, connective tissue growth factor, plasminogen activator inhibitor 1, vascular endothelial growth factor-A) and proinflammatory mediators (IL-1β) expression. We showed that the Hedgehog pathway was activated in bleomycin-induced lung fibrosis on Day 14 after injury, with an increased lung expression of the ligand, Sonic Hedgehog, and with increased messenger RNA expression and nuclear localization of GLI1 and GLI2. Inhibition of Smoothened with GDC-0449 did not influence the development of bleomycin-induced lung fibrosis. By contrast, the inhibition of GLI activity with GANT61 decreased lung fibrosis and lung collagen accumulation, and promoted an antifibrotic and anti-inflammatory environment. Our results identify the hedgehog-Gli pathway as a profibrotic pathway in experimental fibrosis. Inhibition of the Hedgehog-Gli pathway at the level of GLI transcriptional activity could be a therapeutic option in fibrotic lung diseases.

Hepatocyte Growth Factor and Lung Fibrosis

Idiopathic pulmonary fibrosis is currently believed to be driven by alveolar epithelial cells, with abnormally activated alveolar epithelial cells accumulating in an attempt to repair injured alveolar epithelium (1). Thus, targeting the alveolar epithelium to prevent or inhibit the development of pulmonary fibrosis might be an interesting therapeutic option in this disease. Hepatocyte growth factor (HGF) is a growth factor for epithelial and endothelial cells, which is secreted by different cell types, especially fibroblasts and neutrophils. HGF has mitogenic, motogenic, and morphogenic properties and exerts an antiapoptotic action on epithelial and endothelial cells. HGF has also proangiogenic effect. In vitro, HGF inhibits epithelial-to-mesenchymal cell transition and promotes myofibroblast apoptosis. In vivo, HGF has antifibrotic properties demonstrated in experimental models of lung, kidney, heart, skin, and liver fibrosis. Hence, the modulation of HGF may be an attractive target for the treatment of lung fibrosis.

FGF9 and FGF18 in idiopathic pulmonary fibrosis promote survival and migration and inhibit myofibroblast differentiation of human lung fibroblasts in vitro

Idiopathic pulmonary fibrosis (IPF) is characterized by an accumulation of extracellular matrix proteins and fibroblasts in the distal airways. Key developmental lung signaling pathways are reactivated in IPF. For instance, fibroblast growth factor 9 (FGF9) and FGF18, involved in epithelial-mesenchymal interactions, are critical for lung development. We evaluated the expression of FGF9, FGF18, and FGF receptors (FGFRs) in lung tissue from controls and IPF patients and assessed their effect on proliferation, survival, migration, and differentiation of control and IPF human lung fibroblasts (HLFs). FGF9, FGF18, and all FGFRs were present in the remodeled alveolar epithelium close to the fibroblast foci in IPF lungs. FGFR3 was generally detected in fibroblast foci by immunohistochemistry. In vitro, HLFs mainly expressed mesenchyme-associated FGFR isoforms (FGFR1c and FGFR3c) and FGFR4. FGF9 did not affect fibroblast proliferation, whereas FGF18 inhibited cell growth in control fibroblasts. FGF9 and FGF18 decreased Fas-ligand-induced apoptosis in control but not in IPF fibroblasts. FGF9 prevented transforming growth factor β1-induced myofibroblast differentiation. FGF9 and FGF18 increased the migratory capacities of HLF, and FGF9 actively modulated matrix metalloproteinase activity. In addition, FGFR3 inhibition by small interfering RNA impacted p-ERK activation by FGF9 and FGF18 and their effects on differentiation and migration. These results identify FGF9 as an antiapoptotic and promigratory growth factor on HLF, maintaining fibroblasts in an undifferentiated state. The biological effects of FGF9 and FGF18 were partially driven by FGFR3. FGF18 was a less potent molecule. Both growth factors likely contribute to the fibrotic process in vivo.

Alveolar fluid in acute respiratory distress syndrome promotes fibroblast migration: role of platelet-derived growth factor pathway*.

Objectives:Fibroblast migration is an initiating step in fibroproliferation; its involvement during acute lung injury and acute respiratory distress syndrome remains poorly understood. The aims of this study were: 1) to determine whether bronchoalveolar lavage fluids from patients with acute lung injury/acute respiratory distress syndrome modulate lung fibroblast migration; 2) to assess lung fibroblast migration’s clinical relevance; and 3) to evaluate the role of the platelet-derived growth factor pathway in this effect. Design:Prospective cohort study. Setting:Three intensive care units of a large tertiary referral center. Patients:Ninety-three ventilated patients requiring bronchoalveolar lavage fluids were enrolled (48 with acute respiratory distress syndrome, 33 with acute lung injury, and 12 ventilated patients without acute lung injury/acute respiratory distress syndrome). Interventions:After bronchoalveolar lavage fluids collection during standard care, the patients were followed up for 28 days and clinical outcomes were recorded. Migration assays were performed by using a Transwell model; bronchoalveolar lavage fluids platelet-derived growth factor and soluble platelet-derived growth factor receptor-&agr; were characterized by Western blot and measured by ELISA. Measurements and Main Results:Most of the bronchoalveolar lavage fluids inhibited basal fibroblast migration. Bronchoalveolar lavage fluids chemotactic index increased with severity of lung injury (28% in patients without acute lung injury/acute respiratory distress syndrome and with acute lung injury vs. 91% in acute respiratory distress syndrome patients; p = .016). In acute lung injury/acute respiratory distress syndrome patients, inhibition of basal fibroblast migration by bronchoalveolar lavage fluids below 52% was independently associated with a lower 28-day mortality (odds ratio [95% confidence interval] 0.313 [0.10–0.98], p = .046). Platelet-derived growth factor–related peptides and soluble platelet-derived growth factor-R&agr; were detected in all bronchoalveolar lavage fluids from acute lung injury/acute respiratory distress syndrome patients. The effect of bronchoalveolar lavage fluids stimulating migration was inhibited by a specific platelet-derived growth factor receptor inhibitor (AG1296). Bronchoalveolar lavage fluids inhibiting migration reversed the effect of rh-platelet-derived growth factor-BB and reduced by 40% the binding of 125I-platelet-derived growth factor-BB to fibroblast cell surface in favor of a role for platelet-derived growth factor-sR&agr;. Conclusions:Together, our results suggest that during acute lung injury, fibroblast migration is modulated by bronchoalveolar lavage fluids through a platelet-derived growth factor/platelet-derived growth factor-sR&agr; balance. Migration is associated with clinical severity and patient 28-day mortality.

The pro-apoptotic BAX protein influences cell growth and differentiation from the nucleus in healthy interphasic cells

ABSTRACT It has become more and more evident that the BCL-2 family proteins mediate a wide range of non-apoptotic functions. The pro-apoptotic BAX protein has been reported in interphasic nuclei. Whether the nuclear form of BAX could be involved in non-apoptotic function is still unknown. Our study showed for the first time that BAX was associated with chromatin in vitro. Next, we used gain and loss of function approaches to decipher the potential role of nuclear BAX in non-apoptotic cells. In vitro, nuclear BAX promoted cell proliferation in lung epithelial cells and primary human lung fibroblasts by modulating CDKN1A expression. Interestingly, BAX occupancy of CDKN1A promoter was specifically enriched close to the transcription-starting site. Nuclear BAX also modulated the basal myofibroblastic differentiation and migration of primary human lung fibroblasts. Finally, BAX nuclear localization was associated in vivo with the remodelling of lung parenchyma during development, tumorigenesis as well as fibrosis compared to control adult human lungs. Hence, our study established for the first time, a strong link between the nuclear localization of the pro-apoptotic BAX protein and key basic cellular functions in the non-apoptotic setting.