Autophagy and 3-Phosphoinositide-Dependent Kinase 1 (PDK1)-Related Kinome in Pagetic Osteoclasts

Abstract

In Paget's disease of bone (PDB), a major contributory factor are osteoclasts (OCs) that are larger, more numerous, resistant to apoptosis, and hyperactive. The aim of this human in vitro study was to identify kinase cascades involved in the OC phenotype and to determine their impact on downstream processes. Basal phosphorylation levels of Akt and ERK were found to be elevated in PDB OCs. Given our previous findings that 3-phosphoinositide-dependent protein kinase 1 (PDK1) associates with the crucial adaptor p62 in OCs, we hypothesized that PDK1 may play an important role in OC-related kinome regulation. The increased phosphorylation of Akt and its substrate GSK3b observed in PDB OCs was reduced significantly upon PDK1 inhibition, as well as that of 4EBP1 and Raptor. This suggests a PDK1/Akt-dependent activation of mammalian target of rapamycin complex 1 (mTORC1) in PDB OCs. The resistance to apoptosis and the bone resorption were also overcome upon PDK1 inhibition. Studying autophagy by LC3B expression, we found a less inducible autophagy compared with control cells, which was reversed by PDK1 inhibition. In addition, PBD OCs exhibited higher LC3B-II/LC3B-I ratios and numbers of p62 and LC3B puncta per OC area, which did not further increase in the presence of lysosomal protease inhibitors, suggesting an accumulation of non-degradative autophagosomes. Together these results indicate a strong potential regulatory role for PDK1 in OC stimulatory pathways (Akt, ERK) and autophagy induction (via mTORC1), which may contribute to the OC phenotype in PDB. We also identified defects in late autophagosome maturation in these cells, the mechanism of which remains to be determined.

Rab GTPases in Osteoclastic Bone Resorption and Autophagy

“…As inclusion bodies in pagetic osteoclasts resemble the sequestosome-1 or SQSTM1/p62 aggregates observed in diseases involving defective autophagy, the pathogenesis of PDB possibly involves the impairment of autophagy [ 86 ]. In previous studies, defects in autophagy flux were observed in PBD osteoclasts or Cos-1 cells harboring a PDB-associated p62 mutation, suggesting accumulation of non-degradative autophagosomes [ 87 , 88 ]. The activation of TBK1 (TANK binding kinase) and TBK1-induced IL-6 production may also contribute to the generation of PDB osteoclasts [ 89 ].…”

Rab GTPases in Osteoclastic Endomembrane Systems

“…Moreover, inhibition of autophagy by mTORC1 promoted OC survival and RANKL-induced formation and activation of OCs [ 51 , 52 ]. Dysfunctional autophagy has also been identified in the OC phenotype of Paget's bone disease, in which defects in autophagy induction and clearance of autophagosomes have been observed [ 53 ], and activated autophagy in OCs was associated with rheumatoid arthritis [ 54 ].…”

Exosomal lncRNAs NONMMUT000375.2 and NONMMUT071578.2 derived from titanium particle treated RAW264.7 cells regulate osteogenic differentiation of MC3T3‐E1 cells

“…NONMMUT000375.2 and NONMMUT071578.2 may bind to miR‐28a‐5p, and Pdk1 is a direct target of miR‐28a‐5p. It was demonstrated a regulatory role in osteoclast stimulatory pathways (McManus et al, 2016). In this case, the up‐regulated expressed NONMMUT000375.2 should increase the expression of Pdk1 and thereby enhanced osteoclast differentiation.…”