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. 2019 Oct 4:2:359.
doi: 10.1038/s42003-019-0600-6. eCollection 2019.

Mechanical strain promotes skin fibrosis through LRG-1 induction mediated by ELK1 and ERK signalling

Ya Gao #  1 , Jia Zhou #  1 , Zhibo Xie #  2 , Jing Wang  3 , Chia-Kang Ho  1 , Yifan Zhang  1 , Qingfeng Li  1
Affiliations

Mechanical strain promotes skin fibrosis through LRG-1 induction mediated by ELK1 and ERK signalling

Ya Gao et al. Commun Biol. .

"VSports注册入口" Abstract

Biomechanical force and pathological angiogenesis are dominant features in fibro-proliferative disorders. Understanding the role and regulation of the mechanical microenvironment in which pathological angiogenesis occurs is an important challenge when investigating numerous angiogenesis-related diseases. In skin fibrosis, dermal fibroblasts and vascular endothelial cells are integral to hypertrophic scar formation. However, few studies have been conducted to closely investigate their relationship. Here we show, that leucine-rich-alpha-2-glycoprotein 1 (LRG-1) a regulator of pathological angiogenesis, links biomechanical force to angiogenesis in skin fibrosis. We discover that LRG-1 is overexpressed in hypertrophic scar tissues, and that depletion of Lrg-1 in mouse skin causes mild neovascularization and skin fibrosis formation in a hypertrophic scarring model. Inhibition of FAK or ERK attenuates LRG-1 expression through the ELK1 transcription factor, which binds to the LRG-1 promoter region after transcription initiation by mechanical force. Using LRG-1 to uncouple mechanical force from angiogenesis may prove clinically successful in treating fibro-proliferative disorders. VSports手机版.

Keywords: Angiogenesis; Cell signalling; Mechanisms of disease. V体育安卓版.

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Conflict of interest statement

Competing interestsThe authors declare no competing interests.

Figures

Fig. 1
Fig. 1
LRG-1 is overexpressed in human hypertrophic scarring. a Images of normal skin, atrophic scar, and hypertrophic scar. b Images of H&E-stained sections of normal skin, atrophic scar, and hypertrophic scar. (Scale bar = 200 μm). c, d Images and quantitative analysis of immunohistochemistry staining of CD31 and LRG-1. (Scale bar = 50 μm). e, f The levels of LRG-1 mRNA and protein in different skin tissues were measured using RT-qPCR and Western blotting. Data are presented as mean ± SD. n = 20 biologically independent samples. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 2
Fig. 2
LRG-1 promotes HUVECs proliferation, migration, and angiogenesis. a EdU (green) proliferation assay was performed 24 h after the addition of 300 ng/mL and 500 ng/mL LRG-1 and the control group. DAPI-stained nuclei are blue. (Scale bar = 50 μm). b Apoptosis was detected after treating HUVECs with LRG-1 for 3 days by flow cytometry. c Transwell assay images and quantitative analysis for HUVEC migration after incubating with different concentrations of LRG-1. (Scale bar = 50 μm). d Matrigel tube-formation assay images and quantitative analysis of cumulative tube length. (Scale bar = 100 μm). Results were represented as means with standard errors (n = 3 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 3
Fig. 3
LRG-1 knock-down inhibits scar formation in a mechanic loading-induced mouse model. a Immunohistochemistry staining for LRG-1 in mouse scar tissues and expression level quantification. (Scale bar = 50 μm). b mRNA level of mouse skin of LRG-1 in loading group, loading with AAV5-shCtrl injection group and loading with AAV5-shLRG-1 injection group. c, d Immunohistochemistry staining for LRG-1 and CD31 of mouse scar tissues in three groups mentioned above. (Scale bar = 50 μm). e Gross pathology of scar tissue in three groups and gross scar areas quantification. The dashed lines outline the scar. (Scale bar = 3 mm). f Images of H&E stained sections and cross section size quantification. The dashed lines outline the scar. (Scale bar = 500 μm). Data are presented as mean ± SD. n = 15 biologically independent animals. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 4
Fig. 4
Mechanical stretch—not inflammation or TGF-β1—induces LRG-1 overexpression in human dermal fibroblasts (HDFs). a Immunohistochemistry staining images of α-SMA and LRG-1. (Scale bar = 200 μm). b LRG-1 levels were examined by Western blotting after HDFs and HUVECs were incubated with TGF-β1 in different concentration for 12 h. c LRG-1 expression after HDFs and HUVECs were incubated with LPS at different concentrations for 12 h. d mRNA expression of ANKRD1 were examined by RT-qPCR after HDFs and HUVECs were subjected to mechanical stretching at different strengths for 12 h. e LRG-1 expression after HDFs and HUVECs were subjected to mechanical stretch (10%) over different periods of time. f LRG-1 expressions after HDFs and HUVECs were subjected to mechanical stretch (12 h) at different strengths. Results were represented as means with standard errors (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 5
Fig. 5
Mechanic loading induces LRG-1 expression via FAK-ERK signaling pathway. a Expression level of phosphorylated and total FAK after HDFs were subjected to mechanical stretching (10%) over different periods of time. b Western blotting analysis of LRG-1 and phosphorylation and total FAK in HDFs subjected to mechanical stretching (10%) and treated (or not treated) with FAK inhibitor (FAK-I) PF573228. c Expression level of phosphorylated and total JNK, ERK, and p38 after HDFs were subjected to mechanical stretching (10%) over different periods of time. d Mechanic loading-induced LRG-1 expression with small molecule inhibition of JNK (SP600125), ERK (PD98059), or p38 (SB203580). e Western blotting analysis of phosphorylation and total ERK in HDFs subjected to mechanical stretching (10%) and treated (or not treated) with FAK-I. f Immunofluorescence staining for ERK and LRG-1 in HDFs subjected to mechanical stretching (10%) and treated (or not treatment) with FAK-I or ERK-I. ERK are labeled in red and LRG-1 in green. (Scale bar = 50 μm). Results were represented as means with standard errors (n = 3 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 6
Fig. 6
Immunohistochemistry staining of p-FAK (a) and p-ERK(b) in human skin tissues (n = 20) and mouse skin tissues (n = 15). (Scale bar = 50 μm). Data are presented as mean ± SD. **P < 0.01, ***P < 0.001
Fig. 7
Fig. 7
FAK or ERK inhibitor injection blocks mechanical loading-induced LRG-1 expression and attenuate scar formation. a, b Immunohistochemistry staining for LRG-1 and CD31 in mouse scar tissues of the loading group, loading with FAK inhibitor (PF573228) injection group, and loading with ERK inhibitor (PD98059) injection group and expression level quantification. (Scale bar = 50 μm). c Images of scars and gross area quantification at all examined time points. The dashed lines outline the scar. (Scale bar = 3 mm). d Images of H&E stained sections and cross-section size quantification. The dashed lines outline the scar. (Scale bar = 500 μm). Data are presented as mean ± SD. n = 15 biologically independent animals. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 8
Fig. 8
ELK1 controls LRG-1 expression. a Venn diagram showing the intersection of JASPAR and PROMO’s online prediction of possible TFs that bind to the LRG1 promoter region. b Expression level of phosphorylated and total ELK1 after HDFs were applied after mechanical stretching (10%) at different periods of time or different strengths for 2 h, n = 3 independent experiments. c Western blotting analysis of phosphorylation and total ELK1 in HDFs applied with mechanical stretching (10%) and treatment (or no treatment) with FAK-I, n = 3. d Immunofluorescence staining for ERK and p-ELK1 in HDFs after mechanical stretching (10%) and treatment (or no treatment) with FAK-I. ERK are labeled in green and p-ELK1 in red, n = 3. (Scale bar = 50 μm). e Western blotting analysis of phosphorylation and total ERK and ELK1 in HDFs after mechanical stretching (10%) and treatment (or no treatment) with ERK-I, n = 3. f Protein expression levels of total ELK1 analyzed by Western blotting 2 days after siELK1 transfection, n = 3. g Effect of ELK1 inhibition on LRG-1 expression at day 3 after mechanical stretching (10%) (or not) and transfection at day 1 with siELK1 or siCTL, n = 3. h Immunofluorescence staining for p-ELK1 and LRG-1 in HDFs after mechanical stretching (10%) and transfection with siELK1 or siCTL. p-ELK1 are labeled in green and LRG-1 in red. (Scale bar = 50 μm). i Luciferase reporter assay demonstrates that LRG-1 is a target gene of ELK1, n = 3. j Immunohistochemistry staining of p-ELK1 in human skin tissues (n = 20, upper) and in mouse scar tissues of the control group, loading group, loading with FAK inhibitor (PF573228) injection group, and loading with ERK inhibitor (PD98059) injection group (n = 15, lower). (Scale bar = 50 μm). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 9
Fig. 9
ELK1 binds to the LRG-1 promoter region. a Functional analysis of upregulated genes of ChIP-seq results with DNA immunoprecipitated by ELK1 antibody. The numbers of genes in each functional category are presented. b Tag distribution (using bigWIG metrics) across transcription start sites (TSS): X-axis indicates the distance to TSS and Y-axis indicates read count per million mapped reads. c UCSC genome browser visualization of ChIP-seq data generated at control group and loading group in HDFs. ChIP-seq analysis reveals the main difference distribution peak in chr19:4541300-4542400. Five binding motifs of ELK1 in the LRG-1 promoter region predicted by PROMO, marked with a black line. d Human LRG-1 locus including the position of the ELK1 consensus binding site (PROMO prediction), within a promoter region (FANTOM5 prediction) and positions detected by RT-qPCR for the ChIP assays, marked with red boxes. Numbers indicate positions relative to the TSS (+1). Ex1 means Exon 1. e ChIP assay confirmation of the binding of ELK1 in control and loading groups of HDFs (n = 3 independent experiments). DNA immunoprecipitated by ELK1 antibody or immunoglobulin G (IgG CTL) was amplified by RT-qPCR using primers flanking the putative ELK1 binding site in predicted LRG-1 promoter position. f Proposed model of mechanical force promotes hypertrophic scar formation through FAK/ELK1 mediated LRG-1 expression. Data are presented as mean ± SD. ***P < 0.001
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