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. 2017 Feb;14(2):214-222.
doi: 10.1038/cmi.2015.67. Epub 2015 Jul 20.

Inhibition of smoothened decreases proliferation of synoviocytes in rheumatoid arthritis

Shang-Ling Zhu  1   2 , Jian-Lin Huang  1   2 , Wei-Xiang Peng  1 , Dan-Chun Wu  1 , Min-Qi Luo  1 , Qiu-Xia Li  1 , Zhao-Xia Li  1 , Xiao-Xue Feng  1 , Fang Liu  1 , Ming-Xia Wang  1 , Wei-Qian Chen  3 , Nancy Olsen  3 , Song Guo Zheng  2   3
Affiliations

Inhibition of smoothened decreases proliferation of synoviocytes in rheumatoid arthritis

Shang-Ling Zhu et al. Cell Mol Immunol. 2017 Feb.

Abstract

Fibroblast-like synoviocytes (FLSs) contribute to synovial hyperplasia in rheumatoid arthritis (RA). Smoothened (Smo) is a key component of sonic hedgehog (Shh) signaling and contributes to tumor cell proliferation. The objective of this study was to investigate the role of Smo in RA synoviocyte proliferation. FLSs were isolated from RA synovium. Shh signaling was studied using a Smo antagonist (GDC-0449) and small interfering RNA (siRNA) targeting the Smo gene in FLSs. Cell proliferation was quantified by using kit-8 assay and cell cycle distribution and apoptosis were evaluated by flow cytometry. Cell cycle-related genes and proteins were detected by real-time PCR and western blot. FLSs treated with GDC-0449 or Smo-siRNA showed significantly decreased proliferation compared to controls (P < 0.05). Incubation with GDC-0449 or transfection with Smo-siRNA resulted in a significant increase of G1 phase cells compared to controls (P < 0.05). Cell cycle arrest was validated by the significant increase in cyclin D1 and E1 mRNA expression, decrease in cyclin-dependent kinase p21 mRNA expression in Smo-siRNA transfected cells (P < 0.05). Protein expression of cyclin D1 was also downregulated after Smo gene knockdown (P < 0.05). The results suggest that Shh signaling plays an important role in RA-FLSs proliferation in a Smo-dependent manner and may contribute to synovial hyperplasia. Targeting Shh signaling may help control joint damage in patients with RA.

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Figures

Figure 1
Figure 1
Smo agonist and antagonist regulate FLSs proliferation and cell cycle distribution. (a–e) FLSs were treated with GDC-0449 or purmorphamine at different concentrations (5 μM and 25 μM). After 48 hours of incubation, real-time PCR was used to determine Gli1 expression (a), and cell counting kit-8 (CCK-8) assay was performed to examine the proliferation rate of FLSs (b), and flow cytometry was used to analyze cells in different cycle phases (c, d, e). Data are represented as means ± S.D. of three independent experiments. *P <0.05 versus control group. **P <0.01 versus control group.
Figure 2
Figure 2
Knockdown of Smo by siRNA decreases FLSs proliferation and induces G1 cell cycle arrest. FLSs were transfected with a negative control siRNA (NC-siRNA) and siRNA directed against Smo for 48 hours. The expression of Smo was measured by western blot analyses and Smo expression was evaluated and normalized to GAPDH (ratio of Smo/GAPDH) (a). Cell proliferation (b) and cell cycle progression (c, d, e) were determined by CCK-8 assay and flow cytometry, respectively. Data are presented as means ± S.D. of three independent experiments. *P < 0.05 versus NC-siRNA group.
Figure 3
Figure 3
Smo agonist and antagonist do not affect FLSs apoptosis. FLSs were cultured and serum-starved for 18 hours before incubation with GDC-0449 (5 μM) or purmorphamine (5 μM) for 48 hours. Cell apoptosis was determined by Annexin V and PI staining. Populations in the lower-right quadrant of the flow cytometric graph represented early apoptotic cells (a). The percentage of apoptotic cells (b) and live cells (c) was quantified. Data are represented as means ± S.D. of three independent experiments. NS: not significant.
Figure 4
Figure 4
Knockdown of Smo by siRNA does not affect FLSs apoptosis. FLSs were cultured and transfected with small interfering RNAs (NC, Smo) for 48 hours. Cell apoptosis was determined by Annexin V and PI staining. Populations in the lower-right quadrant of the flow cytometric graph represented early apoptotic cells (a). The percentage of apoptotic cells (b) and live cells (c) was quantified. Data are represented as means ± S.D. of three independent experiments. *P < 0.05 versus control group. NS: not significant.
Figure 5
Figure 5
Knockdown of Smo by siRNA affects the mRNA and protein expression of cell cycle-related genes. The level of mRNA expression of cyclin D1 (a), cyclin E1 (b), and p21/CIP1 (c) were evaluated using real time-PCR. Relative quantification of gene expression was performed by the 2−ΔΔCt method. The level of protein expression of cyclin D1 (d) was estimated using western blot analysis. Expression of cyclin D1 protein was quantified using GAPDH as an internal control (e). Data are represented as means ± S.D. of three independent experiments. *P < 0.05 versus NC-siRNA group. p21/CIP1: p21/cyclin-dependent kinase-interacting protein 1.
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