J Rheum Dis.  2023 Oct;30(4):243-250. 10.4078/jrd.2023.0024.

Elevated BMPR2 expression amplifies osteoblast differentiation in ankylosing spondylitis

• Jo S
• Lee SH
• Jeon C
• Jo HR
• Ko E
• Whangbo M
• Kim TJ
• Park YS
• Kim TH

• KMID: 2546240
• DOI: http://doi.org/10.4078/jrd.2023.0024

Abstract

Objective
Bone morphogenetic protein receptor type 2 (BMPR2) has been associated with radiographic changes in ankylosing spondylitis (AS), but further characterization of the cellular signaling pathway in osteoprogenitor (OP) is not clearly understood. The aim of this study was to investigate the expression of BMPR2 and bone morphogenetic protein 2 (BMP2)-mediated responsibility in AS.
Methods
We collected 10 healthy control (HC) and 14 AS-OPs derived from facet joints. Subsequently, we then conducted RNA sequencing with two samples per group and selected BMP-related genes. Facet joint tissues and derived primary OPs were evaluated by validation of selected RNA sequencing data, immunohistochemistry, and comparison of osteogenic differentiation potential.
Results
Based on RNA-sequencing analysis, we found that BMPR2 expression is higher in AS-OPs compared to in HC-OPs. We also validated the increased BMPR2 expression in facet joint tissues with AS and its derived OPs in messenger RNA and protein levels. Additionally, primary AS-OPs showed much greater response to osteogenic differentiation induced by BMP2 and a higher capacity for smad1/5/8-induced RUNX2 expression compared to HCs.
Conclusion
The expression of BMPR2 was found to be significantly increased in facet joint tissues of patients with AS. These findings suggest that BMPR2 may play a role in the BMP2-mediated progression of AS.

Keyword

Ankylosing spondylitis; Bone morphogenetic protein 2; Bone morphogenetic protein receptor type 2; Osteoprogenitors

Figure

• 

  Fig. 1 BMPR2 is highly expressed in patients with AS. (A) Two healthy control (HC)-OPs and two AS-OPs conducted RNA sequencing. Heatmap with the BMP-related genes were selected and shown. (B) BMPR1A, BMPR1B, BMPR2, ALK1, and ALK2 expressions were validated by RT-qPCR (HC-OPs: n=8; AS-OPs: n=8). BMPR2 and ALK1 expressions were validated by (C) immunoblotting (HC-OPs: n=2, AS-OPs: n=6) and (D) immunohistochemistry (HC: n=6; AS: n=6). BMPR2-positive cells in bone-lining cells were counted. Representative images are shown from two individual samples per group. (D) The scale bar showed is 200 μm. AS: ankylosing spondylitis, OP: osteoprogenitor. Values are the mean±standard error of the mean. Statistical significance was determined with *p<0.05, **p<0.01, by Mann–Whitney U test.

• 

  Fig. 2 AS-OPs had a greater capacity for induction of RUNX2 by BMP2. (A) AS-OPs were treated with exogenous BMP2 as indicated dose for a day and subjected to immunoblotting (upper) and RT-qPCR (lower). (B) AS-OPs were treated with 50 ng/mL BMP2 as indicated time and subjected to immunoblotting. (C) Both HC-OPs and AS-OPs were stimulated with 50 ng/mL BMP2 as indicated time and subjected to immunoblotting. HC: healthy control, AS: ankylosing spondylitis, OP: osteoprogenitor. Values are the mean±standard deviation. Statistical significance was determined with ***p<0.01, by Mann–Whitney U test.

• 

  Fig. 3 AS-OPs revealed a greater capacity for RUNX2 induction by BMP2. Both HC-OPs and AS-OPs were stimulated with 50 ng/mL BMP2 as indicated days and subjected to (A) ALP staining at 3 and 7 days, (B) ALP activity, (C) ARS, VON, and HA staining at 21 days, (D) quantification data of ARS, VON, and HA staining, (E) RT-qPCR for RUNX2 and OCN expressions. AS: ankylosing spondylitis, OP: osteoprogenitor, HC: healthy control, ALP: alkaline phosphatase, OM: osteogenic medium. Values are the mean±standard error of the mean. Statistical significance was determined with *p<0.05, **p<0.01, ***p<0.001, by Mann–Whitney U test.

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