Preferential FGF18/FGFR activity in pseudoglandular versus canalicular stage human lung fibroblasts

Abstract

Fibroblast growth factor (FGF) signaling is necessary for proper lung branching morphogenesis, alveolarization, and vascular development. Dysregulation of FGF activity has been implicated in various lung diseases. Recently, we showed that FGF18 promotes human lung branching morphogenesis by regulating mesenchymal progenitor cells. However, the underlying mechanisms remain unclear. Thus, we aimed to determine the role of FGF18 and its receptors (FGFR) in regulating mesenchymal cell proliferation, migration, and differentiation from pseudoglandular to canalicular stage. We performed siRNA assays to identify the specific FGFR(s) associated with FGF18-induced biological processes. We found that FGF18 increased proliferation and migration in human fetal lung fibroblasts (HFLF) from both stages. FGFR2/FGFR4 played a significant role in pseudoglandular stage. HFLF proliferation, while FGFR3/FGFR4 were involved in canalicular stage. FGF18 enhanced HFLF migration through FGFR2 and FGFR4 in pseudoglandular and canalicular stage, respectively. Finally, we provide evidence that FGF18 treatment leads to reduced expression of myofibroblast markers (ACTA2 and COL1A1) and increased expression of lipofibroblast markers (ADRP and PPARγ) in both stages HFLF. However, the specific FGF18/FGFR complex involved in this process varies depending on the stage. Our findings suggest that in context of human lung development, FGF18 tends to associate with distinct FGFRs to initiate specific biological processes on mesenchymal cells.

Keywords: FGF18; FGFR; lung development; mesenchyme; progenitor cells.

FIGURE 1

Activation of FGFR2/FGFR4 in pseudoglandular stage and FGFR3/FGFR4 in canalicular stage mediate FGF18-induced proliferation during human lung development. IF staining of KI67 (green) of pseudoglandular (A) or canalicular stage (C) human fetal lung fibroblasts (HFLF) treated or not with rhFGF18 alone, or 10 nM siRNA FGFR2 (siFGFR2), siRNA FGFR3 (siFGFR3) and siRNA FGFR4 (siFGFR4) supplemented or not with rhFGF18. Scramble siRNA (CTL SCR) was used as control. Quantification of total KI67-positive cells in pseudoglandular (B) and canalicular stage (D) HFLF. Results are shown as dot plot with mean ±SEM, n = 4 for each group. RT-qPCR for ETV4 (E) and ETV5 (F) in HFLF from pseudoglandular stage. RT-qPCR for ETV4 (G) and ETV5 (H) in HFLF from canalicular stage. Results are shown as dot plot with mean ±SEM, *p < 0.05, n = 3 for each group.

FIGURE 2

FGF18 enhances mesenchymal cell migration within lung development. Representative images of a scratch assay taken 24 h after 10 nM siRNA treatment for FGFR2 (siFGFR2), FGFR3 (siFGFR3) or FGFR4 (siFGFR4) supplemented or not with rhFGF18 (A, C, E). The percentage of wound closure was quantified at 24 h (B, D, F). Results are shown as dot plots with mean ±SEM, *p < 0.05, **p < 0.01, n = 4 for each group.

FIGURE 3

Specific FGF18/FGFR activity regulates mesenchymal cell differentiation. RT-qPCR for ACTA2 in pseudoglandular (A) and canalicular stage (B) HFLF treated or not with 10 nM siRNA for FGFR2 (siFGFR2), FGFR3 (siFGFR3) or FGFR4 (siFGFR4) complemented or not with rhFGF18 (in red). Representative western blots for ACTA2 and GAPDH in HFLF treated or not with siRNA and rhFGF18 (C). IF staining of ACTA2 of pseudoglandular (D) or canalicular stage (F) HFLF treated or not with siRNA and rhFGF18. Relative fluorescent quantification was assessed (respectively (E,G)). RT-qPCR for ADRP in pseudoglandular (H) and canalicular stage (I) HFLF. Representative western blots for ADRP and GAPDH in HFLF (J). Results are shown as dot plots with mean ±SEM, *p < 0.05, **p < 0.01, n = 4 for each group.

FIGURE 4

Schematic and table summarizing the role of FGF18 through its different receptors during the pseudoglandular and canalicular stage of human lung development.