SQSTM1 Knockout Cell Lysate

Paget's disease of bone (PDB) is characterized by focal or multifocal increase in bone turnover. One of the most well-established candidate genes for susceptibility to PDB is Sequestosome 1 (SQSTM1). Mutations inSQSTM1have been documented among Western-European, British and American patients with PDB. However, there is no information onSQSTM1mutation status in PDB patients from the Central- and Eastern-European regions. In this study, we conducted a mutation screening forSQSTM1gene variants in 82 PDB patients and 100 control participants in Hungary. Mutations ofSQSTM1were detected in 18 PDB patients (21.95%); associations between genotype and clinical characteristics were also analyzed. Altogether, six different exonic alterations, including two types of UTR variants in theSQSTM1gene, were observed in our PDB patients. Similarly, to previous genetic studies on Paget's disease, our most commonly detected variant was the c.1175C > T (p.Pro392Leu) in nine cases (four in monostotic and five in polyostotic form). We have surveyed the germlineSQSTM1variant distribution among Hungarian patients with PDB. We also highlighted that the pattern of the analyzed disease-associated pathophysiological parameters could partially discriminate PDB patients with normal or mutantSQSTM1genotype. However, our findings also underline and strengthen that not solelySQSTM1stands in the background of the complex PDB etiology.

Podocyte loss is a detrimental feature and major cause of proteinuria in diabetic nephropathy (DN). Our previous study revealed that hepatocyte growth factor (HGF) prevented high glucose-induced podocyte injury via en-hancing autophagy. In the current study, we aimed to assess the role of HGF on podocyte homeostasis in DN and clarify its mechanisms further. Diabetic mice treated with HGF had markedly reduced ratio of kidney weight to body weight, urinary albumin excretion, podocyte loss and matrix expansion compared with that in the non treated counterpart. Simultaneously, HGF-treated diabetic mice exhibited increased autophagy activity as indicated by the decreased accumulation of sequestosome 1 (SQSTM1/ p62) and increased microtubule-associated proteins 1 light chains 3 (LC3) II/LC3I ratio. These beneficial effects of HGF were blocked by HGF/c-Met inhibitor Crizotinib or phosphatidylinositide 3-kinases (PI3K) inhibitor LY294002. Moreover, HGF treatment obviously prevented inactivation of the protein kinase B (Akt)-glycogen synthase kinase 3 beta (GSK3 beta)-transcription factor EB (TFEB) axis in high glucose-stimulated podocytes, which was associated with improved lysosome function and autophagy. Accordingly, adenovirus vector encoding constitutively active GSK3 beta (AdGSK3 beta-S9A) offset whereas small interfering RNA against GSK3 beta (GSK3 beta siRNA) recapitulated salutary effects of HGF on lysosome number and autophagy in podocytes. These results suggested that HGF protected against diabetic nephropathy through restoring podocyte autophagy, which at least partially involved PI3K/Akt-GSK3 beta TFEB axis-mediated lysosomal function improvement.