Cylindromatosis suppression enhances rheumatoid arthritis fibroblast-like synoviocyte-induced osteoclastogenesis and bone resorption by promoting nuclear factor-κB and c-Jun N-terminal kinase activation

doi:10.21203/rs.3.rs-1497210/v1

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Cylindromatosis suppression enhances rheumatoid arthritis fibroblast-like synoviocyte-induced osteoclastogenesis and bone resorption by promoting nuclear factor-κB and c-Jun N-terminal kinase activation

https://doi.org/10.21203/rs.3.rs-1497210/v1

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Background: Rheumatoid arthritis (RA) is a systemic autoimmune disease. Cylindromatosis (CYLD) is a tumor suppressor that negatively regulates nuclear factor-κB (NF-κB) activation. We aimed to investigate both the effect and mechanism of CYLD-dependent RA-fibroblast-like synoviocyte (FLS)-induced osteoclastogenesis and bone resorption.

Results: (1) The CYLD mRNA of peripheral blood mononuclear cell (PBMC）was significantly downregulated in the RA group compared with that in the healthy control group. (2) Co-culture with RA-FLSs induced PBMC osteoclastogenesis and bone resorption. The mRNA and protein levels of CYLD, TRAP, c-fos, CTSK, CALCR, MMP-9, and NFATc1 were increased in osteoclast precursors and osteoclasts. (3) RANKL-induced CYLD expression during osteoclastogenesis was shown to be NF-κB-dependent. (4) Downregulation of CYLD expression in PBMCs could in turn significantly increase the number of osteoclasts, enhance the resorption lacunae area on bone slices, and exaggerate TRAP activity, when compared with that of the negative control. CYLD suppression could significantly increase the mRNA and protein levels of TRAP, c-fos, CTSK, CALCR, MMP-9, and NFATc1, as well as the phosphorylation of NF-κB and c-Jun N-terminal kinase (JNK) in PBMCs and osteoclasts.

Conclusions: Our results highlight CYLD as a negative regulator of RA-FLS-induced osteoclastogenesis and bone resorption.

rheumatoid arthritis

cylindromatosis

peripheral blood mononuclear cells

34 osteoclastogenesis

bone resorption

Table 1. Primers for Reverse-transcription-polymerase chain reaction (RT-PCR)

β-actin	sense：5'-GGACTTCGA GCAAGAGATGG -3'
	Antisense：5'-TGTGTTGGCGTACAGGTCTTTG-3'
CYLD	sense：5’-ACGCCACAATCTTCATCACACT-3’
	Antisense：5’-AGGTCGTGGTCAAGGTTTCACT-3’
TRAP	sense：5’- TGGTCCAGGAGCTTAACTGC-3’
	Antisense：5’- GTCAGGAGTGGGAGCCATATG-3’
c-fos	sense：5’-CCGGGGATAGCCTCTCTTACT-3’
	Antisense ：5’-CCAGGTCCGTGCAGAAGTC-3’
cathepsin K	sense: 5’- GTGGGTGTTCAAGTTTCTGC-3’
	Antisense：5’- GGTGAGTCTTCTTCCATAGC-3’
calcitonin receptor	sense：5’- CTCCAACAAGGTGCTTGGGA-3’
	Antisense：5’- GAAGCAGTAGATAGTCGCCA-3’
MMP-9	sense：5’- AGACCTGGGCAGATTCCAAAC-3’
	Antisense：5’- CGGCAAGTCTTCCGAGTAGT-3’
NFATc1	sense：5'-TGTGCCGGAATCCTGAAACTC-3'
	Antisense：5'-GAGCATTCGATGGGGTTGGAG-3'

Table 2. Baseline demographic and clinical features of RA patients

Characteristic	RA patients(n=25)
Demographic
Age, yrs, median (IQR)	53(37~68)
Female, n (%)	22(88)
Disease status
Disease duration, mo, median (IQR)	40(18 to 118)
ESR (mm/h), median (IQR)	79.76(18~141)
CRP (mg/dl), median (IQR)	4.74(1.23~6.87)
Rheumatoid factor-positive, n (%)	23(92)
ACPA-positive, n (%)	21(84)
DAS28, median (IQR)	5.09(3.04~6.84)
Previous medications, n (%)
Corticosteroids	9(36)
Methotrexate	11(44)
Leflunomide	2(8)
Sulfasalazine	2(8)
Hydroxychloroquine	4(16)
Etanercept	1(4)

^▲ESR, erythrocyte sedimentation rate; CRP, C-reactive protein; RF, rheumatoid factor; ACPA, anti-cyclic citrullinated peptide antibodies; DAS28, Disease Activity Score 28-joint assessment; n, number of patients; RA, rheumatoid arthritis; SD, standard deviation.

No competing interests reported.

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Cylindromatosis suppression enhances rheumatoid arthritis fibroblast-like synoviocyte-induced osteoclastogenesis and bone resorption by promoting nuclear factor-κB and c-Jun N-terminal kinase activation

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