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Increased FGF1-FGFRc expression in idiopathic pulmonary fibrosis.

MacKenzie B, Korfei M, Henneke I, Sibinska Z, Tian X, Hezel S, Dilai S, Wasnick R, Schneider B, Wilhelm J, El Agha E, Klepetko W, Seeger W, Schermuly R, Günther A, Bellusci S - Respir. Res. (2015)

Bottom Line: Therefore, the level and location of FGF/FGFR expression as well as the exogenous effect of the most highly expressed FGFR2b ligand, FGF1, was analyzed on human lung fibroblasts.While smooth muscle actin was unchanged, heparin + FGF1 decreased collagen production in IPF fibroblasts.The FGFR inhibitor (PD173074) attenuated these effects.

View Article: PubMed Central - PubMed

Affiliation: German Center for Lung Research, Excellence Cluster Cardio-Pulmonary System, Universities of Giessen and Marburg Lung Center, Giessen, Hessen, Germany.

ABSTRACT

Background: Recent clinical studies show that tyrosine kinase inhibitors slow the rate of lung function decline and decrease the number of acute exacerbations in patients with Idiopathic Pulmonary Fibrosis (IPF). However, in the murine bleomycin model of fibrosis, not all tyrosine kinase signaling is detrimental. Exogenous ligands Fibroblast Growth Factor (FGF) 7 and 10 improve murine lung repair and increase survival after injury via tyrosine kinase FGF receptor 2b-signaling. Therefore, the level and location of FGF/FGFR expression as well as the exogenous effect of the most highly expressed FGFR2b ligand, FGF1, was analyzed on human lung fibroblasts.

Methods: FGF ligand and receptor expression was evaluated in donor and IPF whole lung homogenates using western blotting and qPCR. Immunohistochemistry for FGF1 and FGFR1/2/3/4 were performed on human lung tissue. Lastly, the effects of FGF1, a potent, multi-FGFR ligand, were studied on primary cultures of IPF and non-IPF donor fibroblasts. Western blots for pro-fibrotic markers, proliferation, FACS for apoptosis, transwell assays and MetaMorph analyses on cell cultures were performed.

Results: Whole lung homogenate analyses revealed decreased FGFR b-isoform expression, and an increase in FGFR c-isoform expression. Of the FGFR2b-ligands, FGF1 was the most significantly increased in IPF patients; downstream targets of FGF-signaling, p-ERK1/2 and p-AKT were also increased. Immunohistochemistry revealed FGF1 co-localization within basal cell sheets, myofibroblast foci, and Surfactant protein-C positive alveolar epithelial type-II cells as well as co-localization with FGFR1, FGFR2, FGFR3, FGFR4 and myofibroblasts expressing the migratory marker Fascin. Both alone and in the presence of heparin, FGF1 led to increased MAPK-signaling in primary lung fibroblasts. While smooth muscle actin was unchanged, heparin + FGF1 decreased collagen production in IPF fibroblasts. In addition, FGF1 + heparin increased apoptosis and cell migration. The FGFR inhibitor (PD173074) attenuated these effects.

Conclusions: Strong expression of FGF1/FGFRs in pathogenic regions of IPF suggest that aberrant FGF1-FGFR signaling is increased in IPF patients and may contribute to the pathogenesis of lung fibrosis by supporting fibroblast migration and increased MAPK-signaling.

No MeSH data available.


Related in: MedlinePlus

Western blot and qPCR analyses of IPF and donor whole lung homogenate lysates and RNA revealed increased FGF1-FGFR expression. Western blots were performed on end-stage IPF (IPF) and non-IPF, donor lung homogenate lysates for FGF, MAPK and PI3/K signaling markers (a). Densitometry plots of arbitrary units indicated a significant increase in FGF1 (b), no increase in FGF7 (c), FGF10 (d). Receptors FGFR1 (e), FGFR2 (f), FGFR3 (g) but not FGFR4 (h) were increased in IPF samples as well as p-ERK1 (i), p-ERK2 (i’) and p-AKT (j). ACTA2 and COL1A1 transcripts were increased in IPF samples (k) as well as FGFR2b ligand transcripts: FGF1, FGF7, (l) and FGF10 (m). B-isoforms of FGFRs were decreased (n) while FGFR2 c-isoform was significantly increased (o). Expression of FGFR3 was variable and FGFR4 (p) was not changed between IPF and donor
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Fig1: Western blot and qPCR analyses of IPF and donor whole lung homogenate lysates and RNA revealed increased FGF1-FGFR expression. Western blots were performed on end-stage IPF (IPF) and non-IPF, donor lung homogenate lysates for FGF, MAPK and PI3/K signaling markers (a). Densitometry plots of arbitrary units indicated a significant increase in FGF1 (b), no increase in FGF7 (c), FGF10 (d). Receptors FGFR1 (e), FGFR2 (f), FGFR3 (g) but not FGFR4 (h) were increased in IPF samples as well as p-ERK1 (i), p-ERK2 (i’) and p-AKT (j). ACTA2 and COL1A1 transcripts were increased in IPF samples (k) as well as FGFR2b ligand transcripts: FGF1, FGF7, (l) and FGF10 (m). B-isoforms of FGFRs were decreased (n) while FGFR2 c-isoform was significantly increased (o). Expression of FGFR3 was variable and FGFR4 (p) was not changed between IPF and donor

Mentions: Western blots were performed for FGFR2b ligands FGF1, FGF7, and FGF10. A significant increase in FGF1 was observed in IPF patients (Fig. 1a,b). FGF7 and FGF10 protein levels were not significantly different (Fig. 1a,c,d). In addition, FGFR1 (Fig. 1a,e), FGFR2 (Fig. 1a,f) and FGFR3 (Fig. 1a,g) were upregulated in IPF lungs, while FGFR4 was unchanged. Next, downstream pathways activated by growth factor signaling, including FGFR-signaling, including: activated protein kinases (MAPK) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), [24] were analyzed. Both p-ERK1 and p-ERK2 were increased in IPF samples (Fig. 1a, I-I’) as well as, p-AKT (Fig. 1a,j). In addition, qPCR was performed on FGF ligands and receptor transcripts. IPF samples showed characteristic increases in Smooth muscle actin (ACTA2) and Collagen 1a1 (COL1A1) transcripts (Fig. 1k). A trend towards an increase in FGF1 transcript in IPF lungs was observed (Fig. 1l). FGF7 and FGF10 transcripts were also increased (Fig. 1m). While the overall trends in the direction of the changes of expression were similar, mRNA transcription profiles did not always correlate exactly with the protein expression profiles. Post-transcriptional regulation of RNA by microRNAs and/or heterogeneous homogenates may account for the discrepancies. As antibodies used against FGFR receptors were not isoform specific, qPCR was performed to determine which isoforms of FGFRs were increased. Epithelial b-isoform expression of FGFR1 and 2 were decreased in IPF homogenates while FGFR3b transcript expression was variable (Fig. 1n). The mesenchymally expressed c-isoform of FGFR2 and to some extent FGFR3, were increased while the expression of FGFR1c and FGFR4 were unchanged (Fig. 1o,p). These data suggest that despite the increase in FGF7 and FGF10, which have been shown to attenuate lung injury in mice, the low level of FGFR2b receptor suggests that epithelial FGFR2b-signaling may be reduced in IPF patients. In contrast, the abundant expression of FGFR c-isoform, and availability of FGF1 ligand suggested that FGF1-FGFRc signaling may be increased in IPF patients.Fig. 1


Increased FGF1-FGFRc expression in idiopathic pulmonary fibrosis.

MacKenzie B, Korfei M, Henneke I, Sibinska Z, Tian X, Hezel S, Dilai S, Wasnick R, Schneider B, Wilhelm J, El Agha E, Klepetko W, Seeger W, Schermuly R, Günther A, Bellusci S - Respir. Res. (2015)

Western blot and qPCR analyses of IPF and donor whole lung homogenate lysates and RNA revealed increased FGF1-FGFR expression. Western blots were performed on end-stage IPF (IPF) and non-IPF, donor lung homogenate lysates for FGF, MAPK and PI3/K signaling markers (a). Densitometry plots of arbitrary units indicated a significant increase in FGF1 (b), no increase in FGF7 (c), FGF10 (d). Receptors FGFR1 (e), FGFR2 (f), FGFR3 (g) but not FGFR4 (h) were increased in IPF samples as well as p-ERK1 (i), p-ERK2 (i’) and p-AKT (j). ACTA2 and COL1A1 transcripts were increased in IPF samples (k) as well as FGFR2b ligand transcripts: FGF1, FGF7, (l) and FGF10 (m). B-isoforms of FGFRs were decreased (n) while FGFR2 c-isoform was significantly increased (o). Expression of FGFR3 was variable and FGFR4 (p) was not changed between IPF and donor
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4495640&req=5

Fig1: Western blot and qPCR analyses of IPF and donor whole lung homogenate lysates and RNA revealed increased FGF1-FGFR expression. Western blots were performed on end-stage IPF (IPF) and non-IPF, donor lung homogenate lysates for FGF, MAPK and PI3/K signaling markers (a). Densitometry plots of arbitrary units indicated a significant increase in FGF1 (b), no increase in FGF7 (c), FGF10 (d). Receptors FGFR1 (e), FGFR2 (f), FGFR3 (g) but not FGFR4 (h) were increased in IPF samples as well as p-ERK1 (i), p-ERK2 (i’) and p-AKT (j). ACTA2 and COL1A1 transcripts were increased in IPF samples (k) as well as FGFR2b ligand transcripts: FGF1, FGF7, (l) and FGF10 (m). B-isoforms of FGFRs were decreased (n) while FGFR2 c-isoform was significantly increased (o). Expression of FGFR3 was variable and FGFR4 (p) was not changed between IPF and donor
Mentions: Western blots were performed for FGFR2b ligands FGF1, FGF7, and FGF10. A significant increase in FGF1 was observed in IPF patients (Fig. 1a,b). FGF7 and FGF10 protein levels were not significantly different (Fig. 1a,c,d). In addition, FGFR1 (Fig. 1a,e), FGFR2 (Fig. 1a,f) and FGFR3 (Fig. 1a,g) were upregulated in IPF lungs, while FGFR4 was unchanged. Next, downstream pathways activated by growth factor signaling, including FGFR-signaling, including: activated protein kinases (MAPK) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), [24] were analyzed. Both p-ERK1 and p-ERK2 were increased in IPF samples (Fig. 1a, I-I’) as well as, p-AKT (Fig. 1a,j). In addition, qPCR was performed on FGF ligands and receptor transcripts. IPF samples showed characteristic increases in Smooth muscle actin (ACTA2) and Collagen 1a1 (COL1A1) transcripts (Fig. 1k). A trend towards an increase in FGF1 transcript in IPF lungs was observed (Fig. 1l). FGF7 and FGF10 transcripts were also increased (Fig. 1m). While the overall trends in the direction of the changes of expression were similar, mRNA transcription profiles did not always correlate exactly with the protein expression profiles. Post-transcriptional regulation of RNA by microRNAs and/or heterogeneous homogenates may account for the discrepancies. As antibodies used against FGFR receptors were not isoform specific, qPCR was performed to determine which isoforms of FGFRs were increased. Epithelial b-isoform expression of FGFR1 and 2 were decreased in IPF homogenates while FGFR3b transcript expression was variable (Fig. 1n). The mesenchymally expressed c-isoform of FGFR2 and to some extent FGFR3, were increased while the expression of FGFR1c and FGFR4 were unchanged (Fig. 1o,p). These data suggest that despite the increase in FGF7 and FGF10, which have been shown to attenuate lung injury in mice, the low level of FGFR2b receptor suggests that epithelial FGFR2b-signaling may be reduced in IPF patients. In contrast, the abundant expression of FGFR c-isoform, and availability of FGF1 ligand suggested that FGF1-FGFRc signaling may be increased in IPF patients.Fig. 1

Bottom Line: Therefore, the level and location of FGF/FGFR expression as well as the exogenous effect of the most highly expressed FGFR2b ligand, FGF1, was analyzed on human lung fibroblasts.While smooth muscle actin was unchanged, heparin + FGF1 decreased collagen production in IPF fibroblasts.The FGFR inhibitor (PD173074) attenuated these effects.

View Article: PubMed Central - PubMed

Affiliation: German Center for Lung Research, Excellence Cluster Cardio-Pulmonary System, Universities of Giessen and Marburg Lung Center, Giessen, Hessen, Germany.

ABSTRACT

Background: Recent clinical studies show that tyrosine kinase inhibitors slow the rate of lung function decline and decrease the number of acute exacerbations in patients with Idiopathic Pulmonary Fibrosis (IPF). However, in the murine bleomycin model of fibrosis, not all tyrosine kinase signaling is detrimental. Exogenous ligands Fibroblast Growth Factor (FGF) 7 and 10 improve murine lung repair and increase survival after injury via tyrosine kinase FGF receptor 2b-signaling. Therefore, the level and location of FGF/FGFR expression as well as the exogenous effect of the most highly expressed FGFR2b ligand, FGF1, was analyzed on human lung fibroblasts.

Methods: FGF ligand and receptor expression was evaluated in donor and IPF whole lung homogenates using western blotting and qPCR. Immunohistochemistry for FGF1 and FGFR1/2/3/4 were performed on human lung tissue. Lastly, the effects of FGF1, a potent, multi-FGFR ligand, were studied on primary cultures of IPF and non-IPF donor fibroblasts. Western blots for pro-fibrotic markers, proliferation, FACS for apoptosis, transwell assays and MetaMorph analyses on cell cultures were performed.

Results: Whole lung homogenate analyses revealed decreased FGFR b-isoform expression, and an increase in FGFR c-isoform expression. Of the FGFR2b-ligands, FGF1 was the most significantly increased in IPF patients; downstream targets of FGF-signaling, p-ERK1/2 and p-AKT were also increased. Immunohistochemistry revealed FGF1 co-localization within basal cell sheets, myofibroblast foci, and Surfactant protein-C positive alveolar epithelial type-II cells as well as co-localization with FGFR1, FGFR2, FGFR3, FGFR4 and myofibroblasts expressing the migratory marker Fascin. Both alone and in the presence of heparin, FGF1 led to increased MAPK-signaling in primary lung fibroblasts. While smooth muscle actin was unchanged, heparin + FGF1 decreased collagen production in IPF fibroblasts. In addition, FGF1 + heparin increased apoptosis and cell migration. The FGFR inhibitor (PD173074) attenuated these effects.

Conclusions: Strong expression of FGF1/FGFRs in pathogenic regions of IPF suggest that aberrant FGF1-FGFR signaling is increased in IPF patients and may contribute to the pathogenesis of lung fibrosis by supporting fibroblast migration and increased MAPK-signaling.

No MeSH data available.


Related in: MedlinePlus