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. 2018 Dec 5:9:2847.
doi: 10.3389/fimmu.2018.02847. eCollection 2018.

Sonic Hedgehog Signaling Pathway Mediates Proliferation and Migration of Fibroblast-Like Synoviocytes in Rheumatoid Arthritis via MAPK/ERK Signaling Pathway

Fang Liu  1   2 , Xiao Xue Feng  1 , Shang Ling Zhu  1 , Hong Yu Huang  3 , Ying Di Chen  1 , Yun Feng Pan  2 , Rayford R June  4 , Song Guo Zheng  4 , Jian Lin Huang  1
Affiliations

Sonic Hedgehog Signaling Pathway Mediates Proliferation and Migration of Fibroblast-Like Synoviocytes in Rheumatoid Arthritis via MAPK/ERK Signaling Pathway

Fang Liu et al. Front Immunol. .

Abstract

Fibroblast-like synoviocytes (FLSs) are the major effector cells that lead to rheumatoid arthritis (RA) synovitis and joint destruction. Our previous studies showed that Sonic Hedgehog (SHH) signaling pathway is involved in aberrant activation of RA-FLSs and inhibition of SHH pathway decreases proliferation and migration of RA-FLSs. The objective of this study was to investigate if the SHH pathway mediates proliferation and migration of RA-FLSs via the mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPK/ERK) signaling pathway. SHH signaling was studied by using SHH agonist (Purmorphamine) and antagonist (Cyclopamine) targeting the Smoothened (SMO) in FLSs. U0126-EtOH was used to inhibit the MAPK/ERK signaling pathway. The phosphorylation of ERK 1/2 (p-ERKl/2) was examined by western blot. Cell viability was detected using cell proliferation and cytotoxicity kit-8 (CCK8), and cell cycle distribution and proliferating cells were evaluated by the flow cytometry. Cell migration was examined by Transwell assay. Results showed that, compared with the control group, Purmorphamine increased the levels of p-ERK1/2 in concentration-and time-dependent manners (P < 0.01). Co-treated with Purmorphamine and U0126-EtOH or Cyclopamine both decreased the levels of p-ERK1/2 (P < 0.05). RA-FLSs treated with Purmorphamine resulted in alteration of cell cycle distribution, increasing of proliferating cells, cell viability, and migration cells compared to controls (P < 0.01). However, the above phenomenon can be abolished by U0126-EtOH (P < 0.05). The findings suggest that SHH signaling pathway mediates proliferation and migration of RA-FLSs via MAPK/ERK pathway and may contribute to progression of RA. Targeting SHH signaling may have a therapeutic potential in patients with RA.

Keywords: MAPK/ERK; Sonic Hedgehog; arthritis; cell migration; cell proliferation; mitogen-activated protein kinases; rheumatoid; signal transduction.

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Figures

Figure 1
Figure 1
The cell morphological characters and surface markers expression. Under the light microscope, RA-FLSs crawled out from synovial tissues (A), and RA-FLSs at passages 3 cells were spindle-shaped and braided growth (D). In (B,C,E,F), The left was isotype control map; the right was surface mark map. Cells were identified by CD68 (B) and CD14 (C) negative staining, coupled with positive staining for CD90 (E), CD55 (F) staining.
Figure 2
Figure 2
Purmorphamine and Cyclopamine regulate the activation of SHH signaling in RA-FLSs. FLSs were treated with Purmorphamine (1 μM) or Cyclopamine (10 μM). After 48 h of incubation, real-time PCR was used to determine GLI1 (A) and SMO mRNA expression (B). Relative quantification of gene expression was performed by the 2−ΔΔCt method. The expression of SMO protein was examined by western blot analysis and normalized to expression of GAPDH (C). The results represent the mean ± SD based on ≥3 replicates.*P < 0.05 vs. control group. **P < 0.01 vs. control group.
Figure 3
Figure 3
Purmorphamine and Cyclopamine regulate MAPK/ERK phosphorylation in RA-FLSs. FLSs were stimulated with 1 μM Purmorphamine for the indicated time (A) and various concentrations of Purmorphamine for 15 min (B). The Purmorphamine-induced increases in MAPK/ERK phosphorylation were abolished in the presence of U0126-EtOH (10 μM) for 15 min (C). Addition of Cyclopamine (10 μM) for 15 min abolishes the MAPK/ERK phosphorylation (D). Expression of ERK1/2 was detected by western blot analysis and the levels of p-ERK1/2 were normalized to expression of total ERK1/2. The results represent the mean ± SD median values or interquartile ranges (IQR) based on ≥3 replicates. *P < 0.05 vs. control group. **P < 0.01 vs. control group.
Figure 4
Figure 4
U0126-EtOH inhibit cell viability and regulate cell cycle distribution of RA-FLSs induced by SHH signaling. Cells were treated with vehicle (DMSO in DMEM supplemented with 10% FBS), Purmorphamine (1 μM), Cyclopamine (10 μM), or co-treated with U0126-EtOH and Purmorphamine cultured for another 48 h, respectively. Cell counting kit-8 (CCK-8) assay was performed to examine the cell viability (A), and flow cytometry was used to analyze cells distribution (B–F). The results represent the mean ± SD based on ≥3 replicates. **P < 0.01 vs. control group.
Figure 5
Figure 5
U0126-EtOH decrease the proliferating cells induced by SHH signaling. (A) Cells of the blank control group. (B) Cells were treated with vehicle (DMSO in DMEM supplemented with 10% FBS). (C) Cells were treated with Purmorphamine (1 μM). (D) Cells were co-treated with U0126-EtOH (10 μM) and Purmorphamine(1 μM). (E) Cells were treated with Cyclopamine (10 μM). The proliferating cells were detected by the flow cytometry and the ratio of EdU positive cells were calculated (F). The results represent the mean ± SD based on ≥3 replicates. *P < 0.05 vs. control group. **P < 0.01 vs. control group.
Figure 6
Figure 6
U0126-EtOH decrease the numbers of migration RA-FLSs induced by SHH signaling. Representative images of RA-FLSs migration after being treated with vehicle (A), Purmorphamine (1 μM) (B), co-treated with U0126-EtOH (10 μM) and Purmorphamine (1 μM) (C), or Cyclopamine (10 μM) (D) were displayed (100 magnifications). Cell migration was evaluated using Transwell assays and the numbers of migrated cells were calculated (× 104 cells/ml) (E). The results represent the mean ± SD based on ≥3 replicates. **P < 0.01 vs. control group.
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