Background/Purpose:

Rheumatoid arthritis (RA) is characterised by massive bone erosions leading to irreversible joint destructions. In this context, the multi-adapter protein p62/SQSTM1 is of interest because apart from its involvement in autophagy, p62/SQSTM1 also interacts with TNFalpha signalling pathways and has the ability to bind directly to TRAF6 resulting in the stimulation of osteoclastogenesis. Here, we sought to investigate, whether based on these signalling characteristics, p62/SQSTM1 is functionally involved in human RA and in animal models of the disease.

Methods:

Tissue samples of RA (n=30) and OA (n=9) patients were analysed to identify correlations between p62 expression and RA diagnosis, disease duration and medication. Furthermore, mice were generated that carry a shortened, but functional mutant of p62 with defective signal transduction domains (p62^aaD69-251) for interbreeding studies with arthritic hTNFtg mice. The resulting genotypes (wt, hTNFtg, p62^aaD69-251 and hTNFtg/ p62^aaD69-251/wt) were scored for clinical parameters (paw swelling, grip strength, weight) for 14 weeks. To quantify the extent of inflammation, cartilage degradation and number of osteoclasts, joints of 14 wks old mice were embedded into paraffin and stained with toluidine blue and TRAP. In addition, bone marrow derived monocytes (BMDMs) were isolated from all genotypes and osteoclastogenesis was studied using an established osteoclast formation assay. To investigate the underlying signalling pathways, cells were treated with TNFalpha at different time points, and MAPK activation was studied by Western blot analyses.

Results:

A significant correlation of p62 expression with RA and prolonged disease duration and specific treatment was found (p<0.05). In our animal studies, histology revealed only minor changes in the number and size of osteoclasts between p62^aaD69-251 and wt animals suggesting that under physiological conditions, regulatory mechanisms compensate for the lack of the signal transduction domains of p62. Compared to wt cells, however, BMDMs of p62^aaD69-251 mice showed a significantly increased osteoclastogenic potential (+28,9% vs hTNFtg BMDMs), in particular when stimulated with TNFalpha in vitro (+30,6% vs hTNFtg BMDMs). Crossing of p62^aaD69-251 mice with hTNFtg animals resulted in a dramatic increase in the severity of joint damage in the hTNFtg/p62^aaD69-251/wt mice as determined clinically and by histomorphometry, showing a significant increase of inflammation area (+113,9% vs hTNFtg) and the number and size of osteoclasts in vivo (+278,5% vs hTNFtg). Interestingly, MAPK activation studies only showed minor differences between wt and p62^aaD69-251 BMDMs.

Conclusion:

In summary, our human expression data as well as the functional mouse data suggest that p62 is an important regulator of TNFalpha mediated joint damage. They indicate that the loss of the TRAF6 and aPKCs binding domains of p62 has important consequences for osteoclastogenesis under inflammatory conditions.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/p62sqstm1-modulates-bone-erosions-in-a-murine-model-of-rheumatoid-arthritis/