Background/Purpose: Paget´s disease of bone (PDB) is a chronic bone metabolic disorder. Currently, PDB is a focal disorder characterized by an increase in bone turnover in a disorganized way. Its cause is unknown, but it is clear that it is a multifactorial disease in which genetic and environmental factors are involved. The most important known genetic factor predisposing to PDB is mutation in Sequestosome1 (SQSTM1) gene. This gene encodes p62, a multifunctional protein involved in autophagy that binds ubiquitin and regulates activation of the nuclear factor kappa-B (NF-kB) signaling pathway, regulating autophagy. We detected for the first time the c.961C >T SQSTM1 gene mutation localized in exon 6 of SQSTM1 gene in three PDB patients. It causes the p.R321C mutation, localized in the LIR domain of p62 protein. The aim of this study is to characterize the effect of the p.R321C mutation in human osteoclasts.

Methods: First of all, the SQSTM1 cDNA was cloned into the EcoRI site of the pCEFL-Flag vector to generate the pCEFL-Flag-cSQSTM1-321wt construct. The p.R321C mutation was introduced into this construct by mutagenesis using primers designed to introduce C >T base change at position +961 to generate pCEFL-Flag-cSQSTM1-321C construct. After that, PBMCs were isolated from venous blood samples for generating human osteoclasts in vitro, using Ficoll.  Mononuclear cells were seeded in 6-well culture plates with glass cover slips and cultured with α-MEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37ºC in a humidified 5% CO2 atmosphere. Cells were differentiated with fresh α-MEM containing M-CSF and RANKL every 3 days for 3 weeks. After that, the cells were transiently transfected with total plasmid DNA (pCEFL-Flag-cSQSTM1-321wt and pCEFL-Flag-cSQSTM1-321C). 72 hours after transfection cells were fixed and we performed a Confocal Co-Immunofluorescence Assay, using as primary antibodies anti-Flag M2 and anti-p62. Also, cells were stained with DAPI (4’, 6-diamidino-2-phenylindole), to select the ones with at least 3 nuclei. Fluorescence images were captured with a confocal microscope.

Results: Our results showed that, while endogenous p62 remains scattered though the cytoplasm, p.R321C p62 is concentrated in the periphery of human osteoclasts. Transfection with WT p62 showed the same localization that endogenous WT p62. This change of localization caused by the mutation detected in PDB patients could result in an alteration in autophagy that can have an important role in the development of PDB, since autophagy is a catabolic process that occurs in the cytoplasm. In fact, previous studies have associated other mutations in autophagy genes with the physiopathology of PDB. To confirm the effect of this mutation in autophagy functional studies are ongoing.

Conclusion: The mutation p.R321C in the LIR domain of p62 protein leads to a change of localization of the protein in human osteoclasts, reinforcing the hypothesis that autophagy is involved in PDB.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-p-r321c-mutation-in-p62-associated-with-pagets-disease-of-bone-leads-to-a-change-of-localization-of-the-protein-in-human-osteoclasts/