Heparan sulfate-FGF10 interactions during lung morphogenesis

Signaling by fibroblast growth factor 10 (FGF10) through FGFR2b is essential for lung development. Heparan sulfates (HS) are major modulators of growth factor binding and signaling present on cell surfaces and extracellular matrices of all tissues. Although recent studies provide evidence that HS are required for FGF-directed tracheal morphogenesis in Drosophila, little is known about the HS role in FGF10-mediated bud formation in the vertebrate lung. Here, we mapped HS expression in the early lung and we investigated how HS interactions with FGF10-FGFR2b influence lung morphogenesis. Our data show that a specific set of HS low in O-sulfates is dynamically expressed in the lung mesenchyme at the sites of prospective budding near Fgf10-expressing areas. In turn, highly sulfated HS are present in basement membranes of branching epithelial tubules. We show that disrupting endogenous gradients of HS or altering HS sulfation in embryonic lung culture systems prevents FGF10 from inducing local responses and markedly alters lung pattern formation and gene expression. Experiments with selectively sulfated heparins indicate that O-sulfated groups in HS are critical for FGF10 signaling activation in the epithelium during lung bud formation, and that the effect of FGF10 in pattern is in part determined by regional distribution of O-sulfated HS. Moreover, we describe expression of a HS 6-O-sulfotransferase preferentially at the tips of branching tubules. Our data suggest that the ability of FGF10 to induce local budding is critically influenced by developmentally regulated regional patterns of HS sulfation.     

Heparan sulfate in lung morphogenesis: The elephant in the room

Heparan sulfate (HS) is a structurally complex polysaccharide located on the cell surface and in the extracellular matrix, where it participates in numerous biological processes through interactions with a vast number of regulatory proteins such as growth factors and morphogens. HS is crucial for lung development; disruption of HS synthesis in flies and mice results in a major aberration of airway branching, and in mice, it results in neonatal death as a consequence of malformed lungs and respiratory distress. Epithelial–mesenchymal interactions governing lung morphogenesis are directed by various diffusible proteins, many of which bind to, and are regulated by HS, including fibroblast growth factors, sonic hedgehog, and bone morphogenetic proteins. The majority of research into the molecular mechanisms underlying defective lung morphogenesis and pulmonary pathologies, such as bronchopulmonary dysplasia and pulmonary hypoplasia associated with congenital diaphragmatic hernia (CDH), has focused on abnormal protein expression. The potential contribution of HS to abnormalities of lung development has yet to be explored to any significant extent, which is somewhat surprising given the abnormal lung phenotype exhibited by mutant mice synthesizing abnormal HS. This review summarizes our current understanding of the role of HS and HS‐binding proteins in lung morphogenesis and will present in vitro and in vivo evidence for the fundamental importance of HS in airway development. Finally, we will discuss the future possibility of HS‐based therapeutics for ameliorating insufficient lung growth associated with lung diseases such as CDH. Birth Defects Research (Part C) 90:32–44, 2010. © 2010 Wiley‐Liss, Inc.     

Pulmonary hypoplasia in mice lacking tumor necrosis factor-alpha converting enzyme indicates an indispensable role for cell surface protein shedding during embryonic lung branching morphogenesis

Many membrane-bound protein precursors, including cytokines and growth factors, are proteolytically shed to yield soluble intercellular regulatory ligands. The responsible protease, tumor necrosis factor-alpha converting enzyme (TACE/ADAM-17), is a transmembrane metalloprotease-disintegrin that cleaves multiple cell surface proteins, although it was initially identified for the enzymatic release of tumor necrosis factor-alpha (TNF-alpha). Mammalian lung growth and development are tightly controlled by cytokines and peptide growth factors. However, the biological function of the cell shedding mechanism during lung organogenesis is not understood. We therefore evaluated the role of TACE as a "sheddase" during lung morphogenesis by analyzing the developmental phenotypes of lungs in mice with an inactive TACE gene in both in vivo and ex vivo organ explant culture. Neonatal TACE-deficient mice had visible respiratory distress and their lungs failed to form normal saccular structures. These newborn mutant lungs had fewer peripheral epithelial sacs with deficient septation and thick-walled mesenchyme, resulting in reduced surface for gas exchange. At the canalicular stage of E16.5, the lungs of TACE mutant mice were impaired in branching morphogenesis, inhibited in epithelial cell proliferation and differentiation, and delayed in vasculogenesis. Embryonic TACE knockout mouse lungs (E12) branched poorly compared to wild-type lungs, when placed into serumless organ culture. Gene expression of both surfactant protein-C and aquaporin-5 were inhibited in cultured TACE-mutant embryonic lungs, indicating defects in both branching and peripheral epithelial cytodifferentiation in the absence of TACE protein. Furthermore, both the hypoplastic phenotype and the delayed cytodifferentiation in TACE-deficient lungs were rescued by exogenous addition of soluble stimulatory factors including either TNF-alpha or epidermal growth factor in embryonic lung culture. Thus, the impaired lung branching and maturation without TACE suggest a broad role for TACE in the processing of multiple membrane-anchored proteins, one or more of which is essential for normal lung morphogenesis. Taken together, our data indicate that the TACE-mediated proteolytic mechanism which enzymatically releases membrane-tethered proteins plays an indispensable role in lung morphogenesis, and its inactivation leads to abnormal lung development.     

Lung hypoplasia and neonatal death in Fgf9-null mice identify this gene as an essential regulator of lung mesenchyme

Mammalian lung develops as an evagination of ventral gut endoderm into the underlying mesenchyme. Iterative epithelial branching, regulated by the surrounding mesenchyme, generates an elaborate network of airways from the initial lung bud. Fibroblast growth factors (FGFs) often mediate epithelial-mesenchymal interactions and mesenchymal Fgf10 is essential for epithelial branching in the developing lung. However, no FGF has been shown to regulate lung mesenchyme. In embryonic lung, Fgf9 is detected in airway epithelium and visceral pleura at E10.5, but is restricted to the pleura by E12.5. We report that mice homozygous for a targeted disruption of Fgf9 exhibit lung hypoplasia and early postnatal death. Fgf9(-/-) lungs exhibit reduced mesenchyme and decreased branching of airways, but show significant distal airspace formation and pneumocyte differentiation. Our results suggest that Fgf9 affects lung size by stimulating mesenchymal proliferation. The reduction in the amount of mesenchyme in Fgf9(-/-) lungs limits expression of mesenchymal Fgf10. We suggest a model whereby FGF9 signaling from the epithelium and reciprocal FGF10 signaling from the mesenchyme coordinately regulate epithelial airway branching and organ size during lung embryogenesis.     

Iroquois genes influence proximo-distal morphogenesis during rat lung development

Lung development is a highly regulated process directed by mesenchymal-epithelial interactions, which coordinate the temporal and spatial expression of multiple regulatory factors required for proper lung formation. The Iroquois homeobox (Irx) genes have been implicated in the patterning and specification of several Drosophila and vertebrate organs, including the heart. Herein, we investigated whether the Irx genes play a role in lung morphogenesis. We found that Irx1-3 and Irx5 expression was confined to the branching lung epithelium, whereas Irx4 was not expressed in the developing lung. Antisense knockdown of all pulmonary Irx genes together dramatically decreased distal branching morphogenesis and increased distention of the proximal tubules in vitro, which was accompanied by a reduction in surfactant protein C-positive epithelial cells and an increase in beta-tubulin IV and Clara cell secretory protein positive epithelial structures. Transmission electron microscopy confirmed the proximal phenotype of the epithelial structures. Furthermore, antisense Irx knockdown resulted in loss of lung mesenchyme and abnormal smooth muscle cell formation. Expression of fibroblast growth factors (FGF) 1, 7, and 10, FGF receptor 2, bone morphogenetic protein 4, and Sonic hedgehog (Shh) were not altered in lung explants treated with antisense Irx oligonucleotides. All four Irx genes were expressed in Shh- and Gli(2)-deficient murine lungs. Collectively, these results suggest that Irx genes are involved in the regulation of proximo-distal morphogenesis of the developing lung but are likely not linked to the FGF, BMP, or Shh signaling pathways.     

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.     

Specific heparan sulfate structures modulate FGF10-mediated submandibular gland epithelial morphogenesis and differentiation

FGF10, a heparan sulfate (HS)-binding growth factor, is required for branching morphogenesis of mouse submandibular glands (SMGs). HS increases the affinity of FGF10 for FGFR2b, which forms an FGF10.FGFR2b.HS ternary signaling complex, and results in diverse biological outcomes, including proliferation and epithelial morphogenesis. Defining the HS structures involved in specific FGF10-mediated events is critical to understand how HS modulates growth factor signaling in specific developmental contexts. We used HS-deficient BaF3/FGFR2b cells, which require exogenous HS to proliferate, to investigate the HS requirements for FGF10-mediated proliferation and primary SMG epithelia to investigate the structural requirements of HS for FGF10-mediated epithelial morphogenesis. In BaF3/FGFR2b cells, heparin with at least 10 saccharides and 6-O-, 2-O-, and N-sulfates were required for maximal proliferation. During FGF10-mediated SMG epithelial morphogenesis, HS increased proliferation and end bud expansion. Defined heparin decasaccharide libraries showed that 2-O-sulfation with either an N-or 6-O-sulfate induced end bud expansion, whereas decasaccharides with 6-O-sulfation alone induced duct elongation. End bud expansion resulted from increased FGFR1b signaling, with increased FGFR1b, Fgf1, and Spry1 as well as increased Aqp5 expression, a marker of end bud differentiation. Duct elongation was associated with expression of Cp2L1, a marker of developing ducts. Collectively, these findings show that the size and sulfate patterns of HS modulate specific FGF10-mediated events, such as proliferation, duct elongation, end bud expansion, and differentiation, and provide mechanistic insight as to how the developmental localization of specific HS structures in tissues influences FGF10-mediated morphogenesis and differentiation.     

Levels of mesenchymal FGFR2 signaling modulate smooth muscle progenitor cell commitment in the lung

Fibroblast growth factor (FGF) signaling has been shown to regulate lung epithelial development but its influence on mesenchymal differentiation has been poorly investigated. To study the role of mesenchymal FGF signaling in the differentiation of the mesenchyme and its impact on epithelial morphogenesis, we took advantage of Fgfr2c(+/Delta) mice, which due to a splicing switch express Fgfr2b in mesenchymal tissues and manifest Apert syndrome-like phenotypes. Using a set of in vivo and in vitro studies, we show that an autocrine FGF10-FGFR2b signaling loop is established in the mutant lung mesenchyme, which has several consequences. It prevents the entry of the smooth muscle progenitors into the smooth muscle cell (SMC) lineage and results in reduced fibronectin and elastin deposition. Levels of Fgf10 expression are raised within the mutant mesenchyme itself. Epithelial branching as well as epithelial levels of FGF and canonical Wnt signaling is dramatically reduced. These defects result in arrested development of terminal airways and an "emphysema like" phenotype in postnatal lungs. Our work unravels part of the complex interactions that govern normal lung development and may be pertinent to understanding the basis of respiratory defects in Apert syndrome.     

小鼠胎肺分支形态发生的分子机制

哺乳动物在早期胚胎发育过程中,肺发育经历了气管分支的形态发生、树样结构上皮管道的形成,并伴随着血管的发育而发生的气体通路和肺泡的分化等过程.肺发生涉及到许多复杂的分子机制.肺形态学的变化受到一系列持家基因、激素、核转录因子、生长因子及其他因素的综合调控.目前已经发现决定肺分支形态发生的许多重要因子.本文根据目前最新研究进展,阐述了小鼠胚胎肺在分支形态发生过程中,上皮与间充质之间诱导的信号通路之间的相互作用及其对呼吸树形态建成的调控机制.     

Time-lapse observation of branching morphogenesis of the lung bud epithelium in mesenchyme-free culture and its relationship with the localization of actin filaments

It has been shown that branching morphogenesis of the lung bud is mediated by epithelial-mesenchymal interaction via such molecules as FGF10, BMP4 and Shh. However, a recent study showed that the isolated lung epithelium still undergoes branching morphogenesis in vitro even in the absence of mesenchyme (Nogawa and Ito, 1995). In the present study, we observed in vitro the dynamic movement of the isolated lung epithelium of the fetal mouse using time-lapse recording, and investigatedthe roles of actinfilaments in branching of the lung bud. First, time-lapse observation of the initial phase of lung branching morphogenesis revealed that at the sites of cleft formation, the epithelial surface was retracted inward from its original position. From this observation we assumed that there should be some structures which exert a physical force on the epithelium, and the localization and arrangement of actin fibers in the cultured lung epithelium were examined at various stages of branching morphogenesis. At the prebudding (6 h) and onset-budding (24 h) stages, no specific localization of actin filaments was observed in the lung bud epithelium, but at the postbudding stage (48 h) they were localized densely in the cells at the tip of the branched lung epithelium. The cell density was not different between the tip and cleft regions of the lung bud epithelium. When cultured with FGF-soaked beads, an actin-rich region was induced at the tip of the lung bud which was growing toward an FGF-soaked bead. These results indicate that actin fibers do not play a significant part in cleft formation but can be secondarily induced by FGF in the surrounding matrix and play some roles at later shaping of the branch in lung morphogenesis.     

Peristalsis of airway smooth muscle is developmentally regulated and uncoupled from hypoplastic lung growth

Prenatal airway smooth muscle (ASM) peristalsis appears coupled to lung growth. Moreover, ASM progenitors produce fibroblast growth factor-10 (FGF-10) for lung morphogenesis. Congenital diaphragmatic hernia (CDH) is associated with lung hypoplasia, FGF-10 deficiency, and postnatal ASM dysfunction. We hypothesized ASM dysfunction emerges in tandem with, and may contribute toward, the primordial lung hypoplasia that precedes experimental CDH. Spatial origin and frequency of ASM peristaltic waves were measured in normal and hypoplastic rat lungs cultured from day 13.5 of gestation (lung hypoplasia was generated by nitrofen dosing of pregnant dams). Longitudinal lung growth was assayed by bud counts and tracing photomicrographs of cultures. Coupling of lung growth and peristalsis was tested by stimulation studies using serum, FGF-10, or nicotine and inhibition studies with nifedipine or U0126 (MEK1/2 inhibitor). In normal lung, ASM peristalsis is developmentally regulated: proximal ASM becomes quiescent (while retaining capacity for cholinergic-stimulated peristalsis). However, in hypoplastic lung, spontaneous proximal ASM activity persists. FGF-10 corrects this aberrant ASM activity in tandem with improved growth. Stimulation and inhibition studies showed that, unlike normal lung, changes in growth or peristalsis are not consistently accompanied by parallel modulation of the other. ASM peristalsis undergoes FGF-10-regulated spatiotemporal development coupled to lung growth: this process is disrupted early in lung hypoplasia. ASM dysfunction emerges in tandem with and may therefore contribute toward lung hypoplasia in CDH.     

Heparan sulfates expressed in the distal lung are required for Fgf10 binding to the epithelium and for airway branching

Fibroblast growth factor (Fgf) 10 is a critical regulator of bud formation during lung morphogenesis. fgf10 is expressed in distal lung mesenchyme at sites of prospective budding from the earliest developmental stages and signals through its epithelial receptor Fgfr2b. Experiments in intact lung organ cultures demonstrate that Fgf10 is a chemotactic factor for distal, but not for proximal, epithelium. This differential response suggests the involvement of an additional mechanism regulating Fgf10-Fgfr2b interactions, because Fgfr2b is uniformly expressed throughout the respiratory tract. Here we use an immunohistochemistry-based binding assay to show that O-sulfated heparan sulfates (HS) are critical for Fgf10 binding to the distal epithelium. We show that altering endogenous gradients of HS sulfation with sodium chlorate or over-O-sulfated synthetic heparin in lung organ cultures dramatically decreases Fgf10 binding. Moreover, we show that under these conditions epithelial binding is not improved by providing exogenous FGF10. Our data suggest that, not only ligand availability, but also the presence of specific patterns of HS modification in the distal lung epithelium are critical determinants of Fgf10 binding to the epithelium and signaling.     

Cancer stem cells in solid tumors: elusive or illusive?

Background

The fibroblast growth factor receptor (FGFR) interprets concentration gradients of FGF ligands and structural changes in the heparan sulfate (HS) co-receptor to generate different cellular responses. However, whether the FGFR generates different signals is not known.

Results

We have previously shown in rat mammary fibroblasts that in cells deficient in sulfation, and so in HS co-receptor, FGF-2 can only stimulate a transient phosphorylation of p42/44^MAPK and so cannot stimulate DNA synthesis. Here we demonstrate that this is because in the absence of HS, FGF-2 fails to stimulate the phosphorylation of the adaptor FGFR substrate 2 (FRS2). In cells possessing the HS co-receptor, FGF-2 elicits a bell-shaped dose response: optimal concentrations stimulate DNA synthesis, but supramaximal concentrations (≥ 100 ng/mL) have little effect. At optimal concentrations (300 pg/mL) FGF-2 stimulates a sustained dual phosphorylation of p42/44^MAPK and tyrosine phosphorylation of FRS2. In contrast, 100 ng/mL FGF-2 only stimulates a transient early peak of p42/44^MAPK phosphorylation and fails to stimulate appreciably the phosphorylation of FRS2 on tyrosine.

Conclusions

These results suggest that the nature of the FGFR signal produced is determined by a combination of the HS co-receptor and the concentration of FGF ligand. Both the phosphorylation of the adaptor FRS2, the kinetics (sustained or transient) of phosphorylation of p42/44(MAPK) are varied, and so differing cellular responses are produced.     

In vivo therapeutic effect of CDH3/P-cadherin-targeting radioimmunotherapy

Purpose

We examined the possible efficacy of the yttrium-90 (⁹⁰Y)-labeled anti-CDH3/P-cadherin mouse monoclonal antibody (MAb-6) in radioimmunotherapy (RIT) for lung and colorectal cancers that express CDH3/P-cadherin.

Experimental design

MAb-6 was established using genetic immunization. The biodistribution of MAb-6 in nude mice with lung and colorectal cancers was examined by administering indium-111(¹¹¹In)-labeled MAb-6 to mice. The mice were prepared by inoculation of CDH3/P-cadherin-positive (EBC1, H1373, and SW948) and CDH3/P-cadherin-negative (A549 and RKO) tumor cells. Therapeutic effects and toxicity were investigated by administration of ⁹⁰Y-labeled MAb-6 (⁹⁰Y-MAb-6) to EBC, H1373, and SW948-inoculated mice.

Results

Our in vivo results confirmed the specific binding of MAb-6 to tumor cells after intravenous injections of ¹¹¹In-labeled MAb-6 to mice with tumors expressing CDH3/P-cadherin. A single intravenous injection of ⁹⁰Y-MAb-6 (100?μCi) significantly suppressed tumor growth in mice with tumors expressing CDH3/P-cadherin. Furthermore, two injections of ⁹⁰Y-MAb-6 led to complete tumor regression in H1373-inoculated mice without any detectable toxicity.

Conclusions

Our findings demonstrate that CDH3/P-cadherin-targeting RIT with ⁹⁰Y-MAb-6 is a promising strategy for the treatment for cancers expressing CDH3/P-cadherin.     

Phosphoinositide 3-Kinase Alpha-Dependent Regulation of Branching Morphogenesis in Murine Embryonic Lung: Evidence for a Role in Determining Morphogenic Properties of FGF7

Branching morphogenesis is a critical step in the development of many epithelial organs. The phosphoinositide-3-kinase (PI3K) pathway has been identified as a central component of this process but the precise role has not been fully established. Herein we sought to determine the role of PI3K in murine lung branching using a series of pharmacological inhibitors directed at this pathway. The pan-class I PI3K inhibitor ZSTK474 greatly enhanced the branching potential of whole murine lung explants as measured by an increase in the number of terminal branches compared with controls over 48 hours. This enhancement of branching was also observed following inhibition of the downstream signalling components of PI3K, Akt and mTOR. Isoform selective inhibitors of PI3K identified that the alpha isoform of PI3K is a key driver in branching morphogenesis. To determine if the effect of PI3K inhibition on branching was specific to the lung epithelium or secondary to an effect on the mesenchyme we assessed the impact of PI3K inhibition in cultures of mesenchyme-free lung epithelium. Isolated lung epithelium cultured with FGF7 formed large cyst-like structures, whereas co-culture with FGF7 and ZSTK474 induced the formation of defined branches with an intact lumen. Together these data suggest a novel role for PI3K in the branching program of the murine embryonic lung contradictory to that reported in other branching organs. Our observations also point towards PI3K acting as a morphogenic switch for FGF7 signalling.