Article Figures & Tables
Figures
- Fig. 1.
Interaction between FREM1 and PDGFC. (A) Structural representations of the full-length FREM1, FLAG-tagged construct and truncated constructs: NV alone, NV-CSPG, CSPG and CalXβ-C-lectin domains are shown with the tags indicated. The MYC tag shown refers to a 3×MYC tag + Igκ secretion signal. (ab), location of immunising sequence for anti-FREM1 antibodies upstream of mutation. (B) E13.5 WT and bat embryo head skin sections stained for FREM1 (green), PDGFC (red) and nuclear dye DAPI (blue). (C) NIH3T3 fibroblasts expressing FREM1-FLAG and PDGFC-V5 and immunostained as indicated. (D) Coimmunoprecipitation of FREM1-FLAG and PDGFC-V5 in transfected HEK293 cells. (E,F) Co-immunoprecipitation of PDGFC-V5 with MYC-tagged FREM1 subdomains. (G) Co-immunoprecipitation of endogenous FREM1 and PDGFC from embryo extracts at E12.5. A rabbit pre-immune serum was included as a control. * IgG heavy chain; IP, immunoprecipitation antibody; WB, western blotting antibody.
- Fig. 2.
FREM1 regulates activation of AKT and MAPK upon PDGFC stimulation. (A) Representative western blotting of phosphorylation of AKT and MAPK ERK1/2 in MEFs from WT and bat mouse embryos stimulated with PDGFCC for the indicated time periods. (B) Relative quantification of AKT phosphorylation levels. WT cells 10 minutes after stimulation were assigned a value of 1 and all other samples are standardised against this value. Graph represents average of up to nine WT and 16 bat samples, performed across four independent experiments from at least three different cell lines for each genotype. Black bars: WT; white bars, bat mutant. (C) FREM1 mutation in bat mutants reduces phosphorylation of PDGFRα in response to the addition of exogenous PDGFCC. IP, immunoprecipitation antibody; WB, western blotting antibody. (D) E13.5 WT embryo head skin sections stained for PDGFC (green), PDGFRα (red) and nuclear dye DAPI (blue). Error bars represent standard error of the mean (s.e.m.); *P<0.05, **P<0.01, ***P<0.005.
- Fig. 3.
FREM1 regulates Timp1 transcription downstream of PDGFCC, through a mechanism that is dependent on PDGFRα, PI3K and MAPK activation. (A) qRT-PCR analysis of Timp1 mRNA levels in WT (black bars) and bat mutant (white bars) MEFs following PDGFCC stimulation for the indicated time points. Data was obtained from four independent experiments from at least three different cell lines for each genotype, and presented as fold increase relative to unstimulated WT cells. (B) Alterations in phosphorylation of AKT and ERK1/2 precede changes in Timp1 expression. (C) TIMP1 protein secretion is reduced in stimulated FREM1 bat mutant MEF cultures. (D) WT cells were pre-treated with either the PI3K inhibitor LY294002, AKT1/2 inhibitor, MAPK inhibitor UO126 or DMSO vehicle control prior to PDGFCC stimulation and analysed by immunoblotting with anti-phospho-AKT, anti-phospho-ERK1/2, total AKT or total ERK1/2. (E) qRT-PCR for Timp1 mRNA was performed on the same cells. Levels of Timp1 mRNA were presented as a fold increase relative to unstimulated cells. Experiments were performed three times using three different cell lines. (F) E13.5 WT and bat embryo head skin sections stained for COL1 (green), keratin 14 (K14; red) and nuclear dye DAPI (blue). Error bars represent s.e.m.; *P<0.05, **P<0.01.
- Fig. 4.
Summary model of FREM1 bat mutant basement membrane fragility. (Left) In WT mice, FREM1 forms a stabilising complex (1) with FRAS/FREM proteins to cross-link the lamina densa with the underlying dermis. Additionally, FREM1 binds keratinocyte-secreted PDGFC, which is presented to PDGFRα in the adjacent dermal fibroblasts, potentiating PDGFRα signalling and promoting ECM modelling events, including TIMP1 upregulation and COL1 deposition (2). (Right) In bat mice, FREM1 mutation removes the structural cross-link of the FRAS/FREM complex (1), but also reduces PDGFRα signalling, leading to lowered TIMP1 and diminished COL1 deposition (2), thereby further weakening part of the foundation of the basement membrane.
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