Fibroblast growth factor signaling in myofibroblasts differs from lipofibroblasts during alveolar septation in mice - PubMed (original) (raw)
Fibroblast growth factor signaling in myofibroblasts differs from lipofibroblasts during alveolar septation in mice
Stephen E McGowan et al. Am J Physiol Lung Cell Mol Physiol. 2015.
Abstract
Pulmonary alveolar fibroblasts produce extracellular matrix in a temporally and spatially regulated pattern to yield a durable yet pliable gas-exchange surface. Proliferation ensures a sufficient complement of cells, but they must differentiate into functionally distinct subtypes: contractile myofibroblasts (MF), which generate elastin and regulate air-flow at the alveolar ducts, and, in mice and rats, lipofibroblasts (LF), which store neutral lipids. PDGF-A is required but acts in conjunction with other differentiation factors arising from adjacent epithelia or within fibroblasts. We hypothesized that FGF receptor (FGFR) expression and function vary for MF and LF and contributes to their divergent differentiation. Whereas approximately half of the FGFR3 was extracellular in MF, FGFR2 and FGFR4 were primarily intracellular. Intracellular FGFR3 localized to the multivesicular body, and its abundance may be modified by Sprouty and interaction with heat shock protein-90. FGF18 mRNA is more abundant in MF, whereas FGF10 mRNA predominated in LF, which also express FGFR1 IIIb, a receptor for FGF10. FGF18 diminished fibroblast proliferation and was chemotactic for cultured fibroblasts. Although PDGF receptor-α (PDGFR-α) primarily signals through phosphoinositide 3-kinase and Akt, p42/p44 MAP kinase (Erk1/2), a major signaling pathway for FGFRs, influenced the abundance of cell-surface PDGFR-α. Observing different FGFR and ligand profiles in MF and LF is consistent with their divergent differentiation although both subpopulations express PDGFR-α. These studies also emphasize the importance of particular cellular locations of FGFR3 and PDGFR-α, which may modify their effects during alveolar development or repair.
Keywords: PDGF receptor-α; adipocyte; cell differentiation; endosome; fibroblast.
Figures
Fig. 1.
PDGF receptor (PDGFR)-α green fluorescent protein (GFP)high and PDGFR-α-GFPlow fibroblasts (F) exhibit different kinetics of proliferation. A: proliferating (Ki67+, solid portion of bars) alveolar cells were enumerated and expressed relative to all cells within a particular population (GFPhigh, GFPlow, or GFPneg) on 3 different postnatal (P) days. The numbers within the bars represent the mean percentages of Ki67+ cells within the respective parent population (including the Ki67- cells, open portion of the bars). Error bars are 1 SE, n = 3 mice at each age, *P < 0.01 P4 vs. P8. B: mean proportions of GFPhigh, GFPlow, and GFPneg cells relative to all alveolar cells at each age. The mean percentages of each population based on PDGFR-α-GFP expression relative to all alveolar cells at a particular age are shown. **P < 0.01 P2 vs. P8. C: thymidine analog bromodeoxyuridine (BrdU) was administered to PDGFR-α-GFP mice during P2 through P4. The same mice received a second thymidine analog, 5-ethynyl-2′-deoxyuridine (EdU) on P8, when the lung F were isolated. The F were immediately fixed, permeabilized, and stained for BrdU, EdU, and CD45. Flow cytometry was used to separate the 3 subpopulations based on the intensity of GFP fluorescence and the absence of CD45. Means ± SE, n = 3, 2-way ANOVA, Student- Newman-Keuls post hoc test. *P < 0.01 compared with GFPneg, †P < 0.05 comparing GFPlow and GFPhigh.
Fig. 2.
Abundance and location of FGF receptors (FGFRs) in F subpopulations determined by flow cytometry. F isolated on P8 were fixed immediately after isolation and were either permeabilized (total) or not (surface) before staining for FGFR2 (CD332, n = 5) (A). B: representative dot plots for FGFR3 (CD333) showing isotype controls (b and c) and CD333 (e and f) in permeabilized (b and e) or unpermeabilized (c and f) groups. Combined data for FGFR3 (n = 4) (C) or FGFR4 (CD334, n = 4) (D) are shown. CD45+ cells were gated out, and data were expressed relative to the total number of cells in the respective subpopulations, based on the intensity of the PDGFR-α-GFP tag. Comparisons were made among the intracellular pools (open portion of bar) or for the extracellular receptors across (closed portions) the 3 subpopulations. Means ± SE, 2-way ANOVA, Student-Newman-Keuls post hoc test. *P < 0.05 compared with GFPneg, †P < 0.05 comparing GFPlow and GFPhigh. FSC, forward scatter; SSC, side scatter.
Fig. 3.
Quantitative real-time PCR showing abundance of FGFR mRNA in F. F were isolated and separated into 3 subpopulations by flow cytometric sorting based on the intensity of GFP and absence of CD45. Means ± SE. A: FGFR3, n = 6; FGFR4 n = 4. FGFR2IIIb (B, n = 5) and FGFR2IIIc (A, n = 5) splice forms were distinguished and are shown separately, whereas the splice forms of FGFR3 were not distinguished. 2-way ANOVA, Student-Newman-Keuls post hoc test. *P < 0.05 compared with GFPneg, †P < 0.05 comparing GFPlow and GFPhigh.
Fig. 4.
FGFR3 localizes to endosomes and complexes with heat shock protein 90 (HSP90). F were isolated from PDGFR-α-GFP+ mice, selected by adherence to plastic, replated on glass cover slides, and adhered overnight, which preserved the proportions of GFPhigh and GFPlow cells. A: using confocal microscopy, FGFR3 (red) was colocalized with various intracellular compartments by characteristic immunoreactive marker proteins for various vesicles: CHMP2B (blue), multivesicular bodies; GM130, Golgi apparatus; EEA1, early endosomes; Rab11, recycling endosomes. Cover slides were prepared from 3 separate F isolations and used in 3 separate staining sessions along with anti-FGFR3 for each marker. Bars are means ± SE. Mander's coefficients: M1 = ∑Ai coloc/∑Ai (A = FGFR3; i, pixel intensity; coloc, pixels with colocalization); M2 = ∑Bi coloc/∑Bi (B = organelle marker). B and C: proximity ligation assay for FGFR3 and HSP90 using 3 separate cell isolations counting 88 GFPhigh and 35 GFPlow F. The reaction product (red particles shown in B) overlying GFPlow and GFPhigh F were enumerated and compared within each field (6 fields for each isolation) using Student's _t_-test for paired variables (means ± SE **P < 0.01).
Fig. 5.
Sprouty (Spry)-4 mRNA is higher, and more Spry4 colocalizes to the multivesicular body in GFPhigh F. A: F were isolated and separated into 3 subpopulations by flow cytometric sorting based on the intensity of GFP and absence of CD45. Spry-4 (n = 3) but not Spry-2 (n = 3) mRNA was higher in GFPhigh F. Means ± SE; 2-way ANOVA, Student-Newman-Keuls post hoc test. *P < 0.05 compared with GFPneg, †P < 0.05 comparing GFPlow and GFPhigh. B: cover slides were prepared from 3 separate cell isolations as described in Fig. 4, and colocalization of Spry4 with CHMP2B was quantified for all F (total) or separately for the GFPlow and GFPhigh subpopulations. 88 GFPhigh and 45 GFPlow F were analyzed; *P < 0.05, 2-way ANOVA comparing GFPlow and GFPhigh.
Fig. 6.
FGF18 mRNA is more abundant in GFPhigh, and FGF18 complexes with FGFR3. A: F from 7 different litters were isolated and sorted into 3 subpopulations by flow cytometry, and FGF18 mRNA was quantified (without culture) using qRT-PCR 2-way ANOVA, Student-Newman-Keuls post hoc test. *P < 0.01 compared with GFPneg, †P < 0.05 comparing GFPlow and GFPhigh. B: proximity ligation assay of complexes between endogenous FGF18 and FGFR3 or FGFR4 in F after overnight adherence. The assay was conducted using 4 separate cell isolations counting 250 and 212 GFPhigh F for FGFR3 and FDFR4, respectively, and 54 and 53 GFPlow F for FGFR3 and FGFR4, respectively. Means ± SE, based on the intraexperiment means for each of the 4 cell isolations. (*P < 0.05), 2-way ANOVA, Student-Newman-Keuls post hoc test comparing FGFR3 and FGFR4.
Fig. 7.
PDGFR-α drives phosphoinositide 3-kinase-phospho S473-Akt but not phospho-Erk (phosphoT202/204 Erk1/2) signaling in myofibroblasts. F were isolated from PDGFR-α-GFP or PDGFR-α gene-deleted TGCre+/−;PDGFR-αF/F mice at P8, fixed and stained for α-smooth muscle actin (α-SMA), pAkt, pErk, and CD45 (only CD45- cells are shown), and analyzed by flow cytometry. A: larger proportion of PDGFR-α-GFPpos than GFPneg F contained pS473-Akt. Conditional deletion of PDGFR-α reduced pS473-Akt (B), but not phosphoT202/204 Erk1/2 (C) in the α-SMA+ (myofibroblast) subpopulation (means ± SE, n = 4, *P < 0.05, _t_-test for paired variables).
Fig. 8.
Cell-surface PDGFR-α (CD140a) correlates with intracellular phospho (p) Erk. Lung F were isolated from PDGFR-α-GFP mice at P8, an aliquot of live cells was stained for CD140a before fixation, and the remainder was fixed. After permeabilization, some cells were stained for CD140a and all with anti-pErk and were then subjected to fluorescence-activated cell sorting (FACS). Data are expressed relative to cells within a particular subpopulation. A: larger proportion of GFPhigh than GFPlow F exhibited PDGFR-α on their surface although similar proportions contained PDGFR-α intracellularly. B: larger proportion of GFPlow F contained pErk, **P < 0.01, 2-way ANOVA, n = 4. C: larger proportion of GFPhigh than GFPlow F displayed CD140a on the surface. Means ± SE, n = 4, 2-way ANOVA, Student-Newman-Keuls post-hoc test. *P < 0.05 compared with GFPneg, †P < 0.05 comparing GFPlow and GFPhigh. In both GFPlow and GFPhigh, cell surface CD140a was more prevalent in pErk+ F (**P < 0.05).
Fig. 9.
FGF8b and FGF18 are chemotactic for F. A: F were cultured to 60% confluence and exposed to FGF18 in the concentrations shown, and proliferating cells, which incorporated EdU, were enumerated using FACS. *P < 0.05, n = 3, 2-way ANOVA comparing FGF18 to control without FGF18. B: F were exposed to 200 ng FGF18/ml for 10 or 30 min or unexposed, 0 min (*P < 0.05, 0 vs. 10 min). Means ± SE, n = 4, 2-way ANOVA, Student-Newman-Keuls post hoc test. C_–_F: F were isolated from PDGFR-α-GFP+ mice and placed in microfluidic devices, which established a chemotactic gradient of either FGF8b or FGF18. Time-lapse imaging was conducted, and the x and y coordinates were plotted at 30-min intervals over 9 h. 3 experiments using cells from different primary isolations were performed for each chemotactic agent, and 15 cells were analyzed for the control and chemotaxin-stimulated migration conditions in each experiment. Speed (S) and persistence (P) were calculated for each cell using the equation D2 = S_2P2(T/P − 1 + e−_T/P). D2 is the square of the distance moved in the preceding 1 h (squared displacement in μm2); P is the persistence time in hours, T is time in hours, and S is speed or velocity in μm/h. The overall means ± SE for chemotaxin-stimulated migration from representative experiments for FGF8b or FGF18 are shown. *P < 0.01 for FGF8b compared with control.
Fig. 10.
GFPlow F express both FGF10 and the FGFR1 IIIb splice variant, a receptor for FGF10. RNA was isolated from F that were separated by flow cytometric sorting based on their levels of PDGFR-α gene expression (GFP fluorescence intensity). Quantitative RT-PCR was performed for FGF10 using a TaqMan probe (A, n = 7), Student-Newman-Keuls post hoc test compared with GFPneg; †P < 0.05 comparing GFPlow and GFPhigh. B and C: custom-designed primer and 5′-nuclease probe sets were used to uniquely detect either the FGFR1 IIIb or FGFR1 IIIc splice variants (n = 5). FGF10 and FGFR1 mRNA abundance was normalized to β2-microglobulin for each sample. (*P < 0.05, 2-way ANOVA).
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