TGFB1 Governs Osteoblast Differentiation in Ankylosing Spondylitis Via Modulating BMP2

Abstract

Background/Purpose: Ankylosing spondylitis (AS) is characterized by inflammation, high osteoblastic activity, leading to ankyloses. Transforming growth factor beta 1 (TGFB1) has been known to be a proinflammatory cytokine in inflammatory disorder and is expressed in AS. This study was undertaken to investigate the role of TGFB1 on osteoblast differentiation, to determine where TGFB1 can stimulate the osteoblast-induced bone formation that is a hallmark of AS.

Methods: All experimental and clinical samples were male. Serum from 26 patients with AS, 23 patients with rheumatoid arthritis (RA), and 28 healthy controls (HC) were collected. Bone tissues were obtained at surgery from facet joints of 10 patients with AS and 26 patients with noninflammatory spinal disease from traffic trauma or spinal compression disease, who served as controls. Expression of TGFB1 in patient serum and bone tissues with AS was confirmed by ELISA and immunohistochemistry, respectively. Primary osteoprogenitor cells were cultured to assess osteoblastic activity, using intracellular alkaline phosphatase (ALP) activity and staining, calcium deposit, and hydroxyapatite staining. Additionally, we observed the effect of TGFB1 on the expression of BMP2 which was evaluated by qPCR, promoter assay, immunostaining, immunoblotting, and Chromatin Immunoprecipitation (ChIP) assay.

Results: Serum and bone tissues of AS patients exhibited higher level of TGFB1 than those of RA and controls. Moreover, treatment of TGFB1 with primary osteoprogenitor cells showed an increase in phos-smad2 and phos-smad3 proteins, intercellular ALP activity, and BMP2 transcript under non-osteoblastic stimuli. TGFB1 also activated phos-smad2 protein directly binds to proximal region at Smad protein biding element (SBE) in BMP2 promoter. Interestingly, a slight stimulation of TGFB1 in immature stage promises more rapid differentiation toward mature stage under osteogenic stimuli.

Conclusion: TGFB1 was highly expressed in AS patients, which were considered to promote osteoblastic activity and osteoblast differentiation by a direct activation of BMP2 signaling pathway. These findings suggest that activation of TGFB1 contribute to the pathogenesis of new bone formation and TGFB1 appears to be a novel therapeutic target in preventing progression of AS.