Involvement of kinase PKC-zeta in the p62/p62P392L-driven activation of NF-κB in human osteoclasts

Abstract

Mutations of the gene encoding sequestosome1 (SQSTM1/p62), clustering in or near the UBA domain, have been described in Paget's disease of bone (PDB); among these the P392L substitution is the most prevalent. Protein p62 mediates several cell functions, including the control of NF-κB signaling, and autophagy. This scaffolding protein interacts with atypical PKCζ in the RANKL-induced signaling complex. We have previously shown that osteoclasts (OCs) overexpressing the p62(P392L) variant were in a constitutively activated state, presenting activated kinase p-PKCζ/λ and activated NF-κB prior to RANKL stimulation. In the present study, we investigated the relationships between PKCζ and NF-κB activation in human OCs transfected with p62 variants. We showed that PKCζ and p-PKCζ/λ co-localize with p62, and that PKCζ is involved in the RANKL-induced NF-κB activation and in the RANKL-independent activation of NF-κB observed in p62(P392L)-transfected cells. We also observed a basal and RANKL-induced increase in IκBα levels in the presence of the p62(P392L) mutation that contrasted with the NF-κB activation. In this study we propose that PKCζ plays a role in the activation of NF-κB by acting as a p65 (RelA) kinase at Ser(536), independently of IκBα; this alternative pathway could be used preferentially in the presence of the p62(P392L) mutation, which may hinder the ubiquitin-proteasome pathway. Overall, our results highlight the importance of p62-associated PKCζ in the overactive state of pagetic OCs and in the activation of NF-κB, particularly in the presence of the p62(P392L) mutation.

Alternative splicing in osteoclasts and Paget’s disease of bone

“…To evaluate the expression of the encoded proteins, we used OCs derived from cord blood monocytes (CBMs), and investigated the protein-level expression by immunofluorescence and western blot using specific antibodies. Our objective was to confirm the expression of the proteins encoded by the selected genes in human OCs, for which the CBM-derived OCs represent a reliable model [8],[14],[22]. …”

“…Mutations in the SQSTM1 gene have been identified in a high proportion of PDB patients [6], the p62P392L substitution being the most frequent [7]. In PDB, p62P392L contributes at least in part to the induction of an activated stage in OCs by stimulating signaling pathways that can lead to NF-κB activation [5],[8]. In vivo , knock-in mice expressing p62 P394L (the murine equivalent of human p62 P392L ) develop focal osteolytic lesions, some of which resemble PDB lesions [9], although in another study, co-expression of the measles virus nucleocapsid gene in the OC lineage was required [4].…”

Unacylated ghrelin restores insulin and autophagic signaling in skeletal muscle of diabetic mice

“…5). PKCζ, besides binding with munc18c leading to the Glut 4 vesicle fusion to the plasma membrane [25], has also been demonstrated to interact with p62 in different cell lines and tissues [9,17,26,48]. Therefore, the binding between PKCζ and p62 might affect the interaction between PKCζ and munc18c.…”

Role of p62/SQSTM1 in liver physiology and pathogenesis

“…It has been suggested that p62 forms a complex with TRAF-6 and aPKC, which is critical for RANKL-induced NF-κB activation 6, 42, 43 . Mutations in the UBA domain results in loss of UBA function and also activate TRAF6–NF-kB signaling, which results in increased osteoclastogenesis 8, 39, 44 . p62 KO mice also show a similar phenotype reminiscent of Paget’s disease with impaired NF-κB activation 6 , further supporting that p62 is an important mediator regulating bone homeostasis.…”