In this study, we identified a population of MSC-enriched CD45− cells derived from the BMSCs of mice with TNF-induced chronic arthritis with significantly reduced osteogenic capacity. We found that expression levels of the ubiquitin E3 ligase Wwp1 were increased in TNF-Tg MSC-enriched CD45− cells, and that knocking down Wwp1 corrected the osteogenic differentiation defect in TNF-Tg cells. Furthermore, we showed that increased Wwp1 in TNF-Tg CD45− cells is accompanied by decreased protein levels of the AP1 protein JunB, a transcription factor critical for osteoblast formation. Wwp1 promotes the ubiquitination and proteasomal degradation of JunB protein. Wwp1 deficiency prevented TNF from impairing MSC differentiation into osteoblasts and inducing JunB ubiquitination. Taken together, these data indicate that chronic TNF exposure decreases the osteogenic potential of MSCs by increasing Wwp1-mediated JunB protein degradation, and this may be one of the molecular mechanisms underlying systemic bone loss seen in patients with chronic inflammation.
Accumulating evidence suggests that MSCs may decrease destructive inflammation, reduce tissue loss, and enhance the tissue repair process. In mice with collagen-induced arthritis, intravenous injection of WT MSCs reduces joint inflammation [21
]. With greater uptake of MSC-based therapeutics in various diseases including autoimmune diseases, it will be important to determine whether the MSCs derived from patients with chronic inflammatory disorders, most of them being autoimmune in etiology, function normally. Here, we demonstrate that MSCs obtained from mice subjected to chronic TNF exposure have significantly reduced osteogenic potential. This finding is consistent with a recent study using MSCs from a mouse model of systemic lupus erythematosus. BMSCs from systemic lupus erythematosus mice have significantly reduced osteoblast differentiation [22
]. However, our findings differ from published results using short-term TNF in vitro treatment of MSCs, in which TNF increases ALP activity [23
], induces a chemotactic effect, and stimulates production of anti-inflammatory and growth factors, such as hepatocyte growth factor [24
], insulin growth factor, and vascular endothelial growth factor [25
]. Thus, endogenous chronic TNF exposure may have a different effect on MSC function than does in vitro TNF treatment.
Wwp1 is a member of the C2-WW-HECT family of E3 ligases. We have reported previously that Smurf1, another member of this family, negatively regulates the function of mature osteoblasts in TNF-Tg mice [8
] and speculated that a chronic inflammatory environment increases the function or expression of a subset of E3 ligases, leading to the degradation of a group of positive regulators of osteogenesis. In Shn3−/−
mice, Wwp1 is proposed to be an E3 ligase which mediates Runx2 degradation by binding to Shn3, explaining the osteosclerosis phenotypes observed in Shn3−/−
]. However, this mechanism may not be operational in MSCs. MSCs express very low levels of Runx2, and we detected no difference in Runx2 protein levels between MSC-enriched CD4‒
cells derived from TNF-Tg mice when compared with WT littermates, suggesting that, at this stage, the expression of Runx2 protein is suppressed. Therefore, this protein modification mechanism likely does not apply to Runx2 at this stage. Alternatively, Runx2 is also known to be regulated by JunB [26
], which is expressed at relatively high levels in MSCs. Further, JunB can be regulated via Smurf1-mediated ubiquitination and proteasomal degradation [15
]. Unlike Runx2, we found that JunB protein levels are reduced in TNF-Tg CD45−
cells and this is accompanied by an elevation in Wwp1 (). However, Smurf1 levels are comparable in MSC-enriched CD45−
cells derived from TNF-Tg or from WT mice () although its expression is increased in mature osteoblasts of TNF-Tg mice [9
]. Thus it is possible that increased levels of Wwp1 inhibit the osteogenic potential of MSCs while elevated Smurf1 reduces the function of mature osteoblasts. Together, these inhibitory mechanisms result in severe systemic bone loss in TNF-Tg mice and patients with RA.
Ubiquitin protein ligases have largely been considered to be constitutively active and regulated only at the level of target binding. However, it has recently become evident that HECT-type E3 ligases can be regulated by other mechanisms, including phosphorylation of the ligase or substrate, utilization of adaptor proteins, or intramolecular and intermolecular interactions [27
]. A few E3 ligases are regulated at the transcriptional levels. TNF and glucocorticoid dexmethasone upregulate the expression of MuRF1/MAFbx expression (two muscle-specific ubiquitin ligases) [30
], possibly via the activation of NF-κ
]. Induction of these ligases is essential for TNF-induced muscle loss in mice [32
]. We reported that TNF increases the expression of Smurf1 in osteoblasts, but this takes 3 days, suggesting an indirect mechanism [9
]. Currently, we do not know the mechanism of TNF-induced Wwp1
expression, which needs to be studied further.
Several signaling pathways have been implicated in TNF-mediated inhibition of osteoblast function, including BMP/Smads, NF-κB, and Wnt/β-catenin. Although it has not been extensively studied as to whether these same pathways also affect MSC function in vivo under chronic TNF exposure condition, it is likely that Wwp1 is not the only mechanism for inhibiting the osteogenic potential of MSCs. Furthermore, ubiquitin E3 ligases usually have multiple target proteins and likely influence multiple pathways. In the future, it will be interesting to determine if post-translational modifications mediated by Wwp1 influences key signaling pathways in both MSCs and osteoblasts.