Background/Purpose: Posttranslational modification of proteins by SUMO has been shown for a number of target molecules including transcription factors and is involved in a variety of cellular processes, including protein localization, transcriptional regulation, protein stability, cell survival and death. Previously, we have shown that the increased expression of SUMO-1 contributes to the inflammatory response in RA. Here, we investigated the role of SUMO-1 in osteoclastogenesis and studied the skeletal phenotype of SUMO-1^-/- mice.

Methods:  The skeletal phenotype of 8-week old  SUMO-1^-/- and wild type mice was investigated by µCT-analysis of trabecular bone in the lumbar spine and femora. L5 vertebral bodies from these mice mice were embedded into methylmetacrylate and analyzed using van Kossa and tartrate-resistant acid phosphatase (TRAP) staining. For in vitro experiments, bone marrow macrophages (BMMs) were isolated from SUMO-1^-/- mice and wild-type controls. The cells were differntiated into osteoclasts in the presence of macrophage colony-stimulating factor and receptor activator of nuclear factor k-B ligand (NF-kB). Osteoclast differentiation was characterized by staining for TRAP. Using PCR, the expression levels of DC-STAMP, Cathepsin K and Integrin ß3 were analyzed. Proliferation and cell viability of BMMs was determined using CyQuant proliferation assay and MMT test. Osteoclast resorption capacity was analyzed using a calcium phosphate bone resorption assay. 

Results:  8-weeks old SUMO-1^-/- mice had a 20% higher trabecular bone volume fraction compared with wt mice. Moreover, trabecular thickness was higher and trabecular separation was lower in SUMO-1^-/- mice. In addition, histological analyses revealed a significantly reduced number of osteoclasts in SUMO-1^-/- mice in vivo. The loss of SUMO-1 was associated with impaired osteoclast differentiation and with impaired bone resorption capacity in vitro. In PCR analysis, we found a decreased expression of DC-STAMP, Cathepsin K and Integrin ß3 in osteoclasts differentiated from SUMO-1^-/- compared to wt mice. Proliferation and cell viability of BMMs were not affected by loss of SUMO-1. Using western blot analysis we found no differences in activation of MAPK p38 and p44/42 as well as in activation of NF-kB signaling in BMMs and osteoclasts in SUMO-1^-/- and wt mice. 

Conclusion: In our study, we found that SUMO-1^-/- mice have high bone mass owing to a decrease in number and function of osteoclasts. These finding are most likely due to decreased expression of osteoclast markers contributing to osteoclast fusion and to osteoclast resorption capacity. These data suggest that SUMO-1 is involved predominantly in the regulation of bone mass by osteoclast formation and activity, and therefore may be an interesting target for treating diseases associated with bone loss.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-small-ubiquitin-related-modifier-1-sumo-1-regulates-osteoclast-differentiation-in-vitro-and-in-vivo/