Control of HIF-1{alpha} and vascular signaling in fetal lung involves cross talk between mTORC1 and the FGF-10/FGFR2b/Spry2 airway branching periodicity clock

Abstract

Lung development requires coordinated signaling between airway and vascular growth, but the link between these processes remains unclear. Mammalian target of rapamycin complex-1 (mTORC1) can amplify hypoxia-inducible factor-1α (HIF-1α) vasculogenic activity through an NH(2)-terminal mTOR binding (TOS) motif. We hypothesized that this mechanism coordinates vasculogenesis with the fibroblast growth factor (FGF)-10/FGF-receptor2b/Spry2 regulator of airway branching. First, we tested if the HIF-1α TOS motif participated in epithelial-mesenchymal vascular signaling. mTORC1 activation by insulin significantly amplified HIF-1α activity at fetal Po(2) (23 mmHg) in human bronchial epithelium (16HBE14o-) and induced vascular traits (Flk1, sprouting) in cocultured human embryonic lung mesenchyme (HEL-12469). This enhanced activation of HIF-1α by mTORC1 was abolished on expression of a HIF-1α (F99A) TOS-mutant and also suppressed vascular differentiation of HEL-12469 cocultures. Next, we determined if vasculogenesis in fetal lung involved regulation of mTORC1 by the FGF-10/FGFR2b/Spry2 pathway. Fetal airway epithelium displayed distinct mTORC1 activity in situ, and its hyperactivation by TSC1(-/-) knockout induced widespread VEGF expression and disaggregation of Tie2-positive vascular bundles. FGF-10-coated beads grafted into fetal lung explants from Tie2-LacZ transgenic mice induced localized vascular differentiation in the peripheral mesenchyme. In rat fetal distal lung epithelial (FDLE) cells cultured at fetal Po(2), FGF-10 induced mTORC1 and amplified HIF-1α activity and VEGF secretion without induction of ERK1/2. This was accompanied by the formation of a complex between Spry2, the cCBL ubiquitin ligase, and the mTOR repressor, TSC2, which abolished GTPase activity directed against Rheb, the G protein inducer of mTORC1. Thus, mTORC1 links HIF-1α-driven vasculogenesis with the FGF-10/FGFR2b/Spry2 airway branching periodicity regulator.

Fig. 1.

mTOR amplifies hypoxia-inducible factor-1α (HIF-1α) activity and vasculogenic signaling in HBE-HELMF cocultures. A: the bicameral culture method for examining epithelial-mesenchymal trophic signaling. Human bronchial epithelial cells (HBE) were cultured on permeable supports suspended above a monolayer of HEL-12469 human embryonic lung fibroblast cells (HELMF) in serum-free MEM (SFM). Single culture was performed by culturing HBE and HELMF in SFM separately. B: HIF-1α-driven luciferase reporter gene expression in HELMF at alveolar (100 mmHg) or fetal (23 mmHg) Po₂. Values are expressed as fold change over untreated cells at alveolar Po₂, n = 4, *P < 0.05. C: HIF-1α activity in HBE; n = 4, *P < 0.05. D: VEGF secretion in HBE is elevated at fetal Po₂ and is inhibited by rapamycin and LY-294002. VEGF production was undetectable from HELMF cells (data not shown); *P < 0.05, n = 4. E: Flk-1 expression at fetal Po₂ in HELMF cocultured with HBE (top) or alone (HELMF single culture, bottom). Representative of 3 independent experiments.

Fig. 2.

The HIF-1α F99A TOS mutation suppresses trophic signaling between HBE and HELMF. A, top blots: conditional phenotype of HBE cells used to generate conditioned medium for HELMF culture. Rapamycin (100 nM) was used to suppress mTOR signaling. Bottom blots: expression of Flt1 and Flk1 [nascent (150 kDa) and glycosylated (200 kDa) forms shown] in HELMF following 18-h culture in HBE-conditioned medium. Blots are representative of 4 experiments. B: top histogram shows the actin-corrected change in Flk1 protein abundance in HELMF cultured in HBE-conditioned SFM. *P < 0.05, n = 5. Bottom histogram shows the activity of HIF-1α in the HBE used to generate the conditioned medium. Activity is expressed as fold change over empty vector (pRK7) control. *P < 0.05 relative to control group; n = 5. C: sprouting from HELMF spheroids in Matrigel following 48-h culture in medium obtained from the HBE in A. Scale bar: 182 μm. Box plots at bottom show the data distribution of sprout length. (From bottom: 5th percentile, smallest nonoutlier, lower quartile, median, top quartile, 95th percentile. Bars indicate the data range.) *P < 0.05 relative to wild-type transfected control group; **P < 0.001 relative to wild-type rheb-transfected group; n = 4. One-way ANOVA on ranks with Dunn's post hoc test.

Fig. 3.

mTORC1 activity in the pseudoglandular rat lung in vivo. Gestation day 19 fetal rat lungs were fixed and stained with a phospho-antibody against the mTORC1 substrate, p70S6K1-T389. A: low-power (×40) image of fetal lung in situ. p70S6K-T389 phosphorylation is apparent in the nascent airway epithelium (brown stain). B: control section treated with nonimmune IgG at same magnification. C: mid-power (×200) image showing airway (a), mesenchyme (m), and endothelial tube (et) structures. Note apparent polarization of mTORC1 activity around the tube (white arrows). D: high-power (×400) image showing mTOR activity extending laterally from the epithelium indicating activity within a possible branching node. Localized activity in cells of the mesenchyme is evident (black arrows). E: high mTOR activity in the mesothelial lung lining (mth), a site of FGF9 expression (56). White arrow indicates regional activity in an epithelial lateral branch node. F and G: ×200 and ×400 images of sections treated with nonimmune IgG.

Fig. 4.

Endogenous mTOR activity is critical for morphogenesis of fetal rat lung explants. A: influence of culture Po₂ and rapamycin on airway surface complexity [ASC; perimeter/√airway area (46)] in lung explants from gestation day (G) 14 fetal rats. Circles, Control; triangles, 0.1 μM rapamycin. *P < 0.05 23 mmHg control vs. 100 mmHg control, n = 15; †P < 0.05 rapamycin vs. control at 23 mmHg, n = 15. B: box plots showing data distribution of epithelial thickness in G14 explants maintained for 48 h in the presence or absence of rapamycin (0.1 μM) at the indicated Po₂. Measurements from G16 lung in situ are included for comparison (from bottom: 5th percentile, smallest nonoutlier, lower quartile, median, top quartile, 95th percentile. Bars indicate the data range). *P < 0.05, n = 4. One-way ANOVA on ranks with Dunn's post hoc test. C: endogenous activity of mTOR [αS6K-(P)-T389] in explants cultured in SFM for 48 h at fetal or alveolar Po₂. Insulin (20 nM) was used as a positive control, and rapamycin (0.1 μM) was used to block mTOR activity. Representative of 4 replicates. D: growth rate of fetal rat lung fibroblasts determined over 48 h at fetal or alveolar Po₂ in the presence of rapamycin or following overexpression of a kinase dead (D2357E) mutant of mTOR. *P < 0.05 relative to 23 mmHg treatment, n = 6.

Fig. 5.

Vascular differentiation is induced by FGF-10 in E12 fetal murine lung explants. Beads coated in FGF-10 or PBS (control) were grafted onto E12 murine fetal lung explants from Tie2-LacZ transgenic mice. Explants were maintained at fetal Po₂ for 48 h, fixed, and then stained for LacZ reporter expression. Grayscale images on left show bead location and morphogenesis over 48 h; color images on right show subsequent expression of Tie2-LacZ reporter gene with bead excised. Bead position is indicated as: F (FGF-10), P (PBS). Combined treatment indicates PBS and FGF-10-coated beads grafted onto the same explant. Note lack of Tie2-LacZ expression in the peripheral mesenchyme (PM) shown in bottom image. Findings shown in these images were replicated across explants isolated from 3 independent litters.

Fig. 6.

Knockout of the upstream repressor of mTOR, TSC1, alters vasculogenic signaling in the murine lung. Wild-type (A–C) and TSC1^−/− (D–F) embryonic day (E) 12.5 murine fetuses were sectioned and stained for mTOR activity [S6K1 (T389) phosphorylation], VEGF, and Tie2 expression. Arrows in A indicate raised mTOR activity at sites of airway epithelial outgrowth in wild-type animals. Arrows in C indicate Tie2 receptor expression corresponding to nascent vascular bundles. Images are representative of 4 fetuses from independent litters. Bar = 200 μm.

Fig. 7.

FGF-10 positively modulates HIF-1α activity via mTORC1 in polarized, primary FDLE cells. A: top graph shows HIF-1α activity measured from FDLE transfected with pHRE₃-TK-GL2 and pRL (8:1) and cultured at fetal (23 mmHg; closed symbols) or alveolar (100 mmHg; open symbols) Po₂ for 6 h in the presence of increasing concentrations of FGF-10. Rapamycin (0.1 μM; triangles) was added to a parallel set of cells to determine mTORC1-sensitive HIF-1α activity. Values are normalized to 0 μg·ml⁻¹ FGF-10 treatment at fetal Po₂. Bottom graph shows the level of VEGF secreted into the medium beneath the permeable support from the same cells. *P < 0.05 relative to control at fetal Po₂; †P < 0.05 relative to paired rapamycin treatment, n = 5. B: representative blots showing mTORC1 [αS6K-(p)-T389 phosphorylation] and ERK1/2 activation (T202/Y204 and T185/Y187 phosphorylation) and Spry2 protein abundance measured in samples from A. *Contrast is enhanced separately from total ERK blot to show decline in ERK1/2 phosphorylation with FGF-10. C: densitometry of pooled experimental blots represented in B. Open/closed symbols are as described in A; triangles represent ERK2 values. *P < 0.05 relative to control, n = 4. D: the MEK1 inhibitor U0126 (10 μM) does not prevent mTORC1 activation (S6K1-T389 phosphorylation) by 0.1 μg/ml⁻¹ FGF-10 in FDLE at fetal Po₂. Rapamycin (0.1 μM) was added to show inhibition of mTORC1. Histogram (right) shows pooled densitometry data for mTORC1 activity and ERK1/2 phosphorylation. *P < 0.05 relative to control; †not significantly different from FGF-10 treatment. ND, not detectable; n = 5.

Fig. 7.

FGF-10 positively modulates HIF-1α activity via mTORC1 in polarized, primary FDLE cells. A: top graph shows HIF-1α activity measured from FDLE transfected with pHRE₃-TK-GL2 and pRL (8:1) and cultured at fetal (23 mmHg; closed symbols) or alveolar (100 mmHg; open symbols) Po₂ for 6 h in the presence of increasing concentrations of FGF-10. Rapamycin (0.1 μM; triangles) was added to a parallel set of cells to determine mTORC1-sensitive HIF-1α activity. Values are normalized to 0 μg·ml⁻¹ FGF-10 treatment at fetal Po₂. Bottom graph shows the level of VEGF secreted into the medium beneath the permeable support from the same cells. *P < 0.05 relative to control at fetal Po₂; †P < 0.05 relative to paired rapamycin treatment, n = 5. B: representative blots showing mTORC1 [αS6K-(p)-T389 phosphorylation] and ERK1/2 activation (T202/Y204 and T185/Y187 phosphorylation) and Spry2 protein abundance measured in samples from A. *Contrast is enhanced separately from total ERK blot to show decline in ERK1/2 phosphorylation with FGF-10. C: densitometry of pooled experimental blots represented in B. Open/closed symbols are as described in A; triangles represent ERK2 values. *P < 0.05 relative to control, n = 4. D: the MEK1 inhibitor U0126 (10 μM) does not prevent mTORC1 activation (S6K1-T389 phosphorylation) by 0.1 μg/ml⁻¹ FGF-10 in FDLE at fetal Po₂. Rapamycin (0.1 μM) was added to show inhibition of mTORC1. Histogram (right) shows pooled densitometry data for mTORC1 activity and ERK1/2 phosphorylation. *P < 0.05 relative to control; †not significantly different from FGF-10 treatment. ND, not detectable; n = 5.

Fig. 8.

FGF-10 destabilizes Spry2 in FDLE. A: abundance of Spry2 declines with increasing FGF-10 concentration at fetal (closed circles) and alveolar (open circles) Po₂. Densitometry performed on full-length Spry2 in ratio to actin. *P < 0.05 relative to control, n = 4. B: Spry2 becomes unstable in the presence of FGF-10. FDLE were treated with 100 μM cycloheximide (CHX) and 0.1 μg·ml⁻¹ FGF-10 as indicated at fetal Po₂. Representative blot is accompanied by graph showing full-length Spry2 abundance measured in ratio to actin. Circles, control; triangles, 0.1 μg·ml⁻¹ FGF-10. *P < 0.05, n = 4 relative to time 0. Error bars may be within symbols. C: Spry2 cleavage is proteasomal. FDLE were treated with 10 μM MG132 and 0.1 μg·ml⁻¹ FGF-10 at fetal Po₂ for 3 h as indicated. Proteins were separated by 10% SDS-PAGE to resolve both full-length and cleaved Spry2 product. Blot is representative of n = 3.

Fig. 9.

Spry2 associates with cCBL and TSC2. A: immunoprecipitation (IP) of cCBL from FDLE at fetal Po₂ treated with FGF-10 (0.1 μg·ml⁻¹) or MG132 (10 μM) as indicated. Immunoblots (IB) were performed to detect TSC2, cCBL, and Spry2. Histograms on right show ratio of TSC2 or Spry2 to cCBL. *P < 0.05 relative to control, n = 5. B: effect of FGF-10 on TSC1/2 GAP activity towards the Rheb guanine nucleotide binding protein in FDLE at fetal or ambient Po₂. TLC phosphorimage shows resolution of ³²P-GTP and -GDP immunoprecipitating with FLAG-Rheb in cells treated with FGF-10. Immunoblot beneath shows even recovery of FLAG-Rheb by IP. Histogram shows ratio of GTP:GDP as a percentage of total radioactivity eluting with Rheb. The superimposed line graph shows fold change in total [³²P]-labeled guanine nucleotide phosphates eluting with Rheb, *P < 0.05 relative to control at fetal Po₂, n = 4.

Fig. 10.

Overexpression of Spry2 in FDLE abolishes FGF-10-evoked Rheb-GTP binding and is relieved by mutation of the Spry2 (Y55) cCBL docking site. A: TSC1/2 GAP activity (ratio of GTP:GDP bound Rheb), HIF-1α activity, and VEGF secretion in FDLE transfected with empty vector, wild-type, or Y55F Spry2. For GAP assays, the transfection mix included FLAG-Rheb (ratio of 1:4 Rheb to test vector), whereas for HIF-1α activity assays and VEGF secretion, the transfection mix included pHRE₃-TK-GL2 (1:4). All experiments were performed at fetal Po₂. *P < 0.05 relative to EV control; n = 4 (Rheb GAP assay), n = 6 (HIF-1α luciferase and VEGF secretion). B: representative blot showing TSC2, Spry2, and actin expression levels. Note that the Spry2 blot shows bands for the endogenous full-length and cleaved forms of Spry2 in EV lanes, which are superimposed by overexpressed Spry2 in other lanes. Histogram shows TSC2 abundance corrected for actin. *P < 0.05 relative to respective EV and Spry2 (Y55F) treatment, n = 6 independent transfections.

Fig. 11.

Model of integrated airway and vasculogenesis in the developing lung. Model is represented next to a late embryonic stage (E12) rat lung showing formation of airway branches extending into the mesenchyme tissue surrounded by blood-filled vascular structures proximal to the airway tip. Foregut (Fg) is shown displaced to left of lung. Zone A: FGF-10 expressed in the mesenchyme ahead of nascent airway buds induces Spry2 cleavage by an unknown ubiquitin ligase (possibly SIAH2 or cCBL) in the progenitor epithelial cells of the airway tip. This cleavage event, although necessary for mTORC1 activation, is accompanied by the formation of a complex between Spry2, cCBL, and TSC2 (Figs. 7 and 8), which dislocates the TSC1/2 complex through the proteolytic cleavage of TSC2 (Fig. 10). This disrupts TSC1/2 GTPase activating protein (GAP) activity allowing GTP interaction with Rheb and activation of mTORC1. Zones B and C: trophic signaling, involving VEGF release by airway epithelium into the mesenchyme and subsequent vasculogenesis, proceeds proximal to the airway tip by binding of mTORC1 to the HIF-1α TOS motif and subsequent interaction with the p300 transcriptional initiation complex. This interaction locally amplifies the transcriptional activity of HIF-1α causing increased release of VEGF and the appearance of vascular structures behind the growing tip of the airway. Our model acknowledges that there are 2 oxygen-dependent points of control that are critical for proper lung morphogenesis. These are: 1) the proliferation of mesenchyme tissue that requires low-oxygen tension and mTOR activity for sustained growth, and 2) hypoxic priming of HIF-1α transcriptional activity, which can be positively modulated by locally expressed growth factors. The ERK1/2 activation by FGF-10 reported by Tefft et al. (53) and observed by us in HBE (Supplementary Figs. S4 and S5) is represented by a dotted line. Our study shows that the magnitude of this response is also oxygen sensitive as it is suppressed at fetal compared with alveolar Po₂, leading us to speculate a secondary role for oxygen in regulating ERK1/2 signaling in the fetal lung. This phenomenon could be linked to the conservation of progenitor cell populations during lung morphogenesis (50).