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Inflammation leads to systemic osteoporosis or local bone destruction, however, the underlying molecular mechanisms are still poorly understood. In this study, we report that PRL2 is a negative regulator of osteoclastogenesis and bone absorption. Mice with PRL2 deficiency exhibit a decrease in bone volume and an increase in osteoclast numbers. PRL2 negatively regulates RANKL-induced reactive oxygen species production through the activation of RAC1, thus PRL2 deficient osteoclast precursors have both increased osteoclast differentiation ability and bone resorptive capacity. During inflammation, oxidized PRL2 is a selected substrate of HSC70 and conditions of oxidative stress trigger rapid degradation of PRL2 by HSC70 mediated endosomal microautophagy and chaperone-mediated autophagy. Ablation of PRL2 in mouse models of inflammatory bone disease leads to an increase in the number of osteoclasts and exacerbation of bone damage. Moreover, reduced PRL2 protein levels in peripheral myeloid cells are highly correlated with bone destruction in a mouse arthritis model and in human rheumatoid arthritis, while the autophagy inhibitor hydroxychloroquine blocked inflammation-induced PRL2 degradation and bone destruction in vivo. Therefore, our findings identify PRL2 as a new regulator in osteoimmunity, providing a link between inflammation and osteoporosis. As such, PRL2 is a potential therapeutic target for inflammatory bone disease and inhibition of HSC70 mediated autophagic degradation of PRL2 may offer new therapeutic tools for the treatment of inflammatory bone disease.

Multiple sources contribute membrane and protein machineries to construct functional macroautophagic/autophagic structures. However, the underlying molecular mechanisms remain elusive. Here, we show that RAB2 connects the Golgi network to autophagy pathway by delivering membrane and by sequentially engaging distinct autophagy machineries. In unstressed cells, RAB2 resides primarily in the Golgi apparatus, as evidenced by its interaction and colocalization with GOLGA2/GM130. Importantly, autophagy stimuli dissociate RAB2 from GOLGA2 to interact with ULK1 complex, which facilitates the recruitment of ULK1 complex to form phagophores. Intriguingly, RAB2 appears to modulate ULK1 kinase activity to propagate signals for autophagosome formation. Subsequently, RAB2 switches to interact with autophagosomal RUBCNL/PACER and STX17 to further specify the recruitment of HOPS complex for autolysosome formation. Together, our study reveals a multivalent pathway in bulk autophagy regulation, and provides mechanistic insights into how the Golgi apparatus contributes to the formation of different autophagic structures. Abbreviations: ACTB: actin beta; ATG9: autophagy related 9A; ATG14: autophagy related 14; ATG16L1: autophagy related 16 like 1; BCAP31: B cell receptor associated protein 31; BECN1: beclin 1; Ctrl: control; CQ: chloroquine; CTSD: cathepsin D; DMSO: dimethyl sulfoxide; EBSS: Earle’s balanced salt solution; EEA1: early endosome antigen 1; GDI: guanine nucleotide dissociation inhibitor; GFP: green fluorescent protein; GOLGA2: golgin A2; HOPS: homotypic fusion and protein sorting complex; IP: immunoprecipitation; KD: knockdown; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LC3: microtubule-associated protein 1 light chain 3; OE: overexpression; PtdIns3K: class III phosphatidylinositol 3-kinase; SQSTM1/p62: sequestosome 1; RAB2: RAB2A, member RAS oncogene family; RAB7: RAB7A, member RAS oncogene family; RAB11: RAB11A, member RAS oncogene family; RUBCNL/PACER: rubicon like autophagy enhancer; STX17: syntaxin 17; TBC1D14: TBC1 domain family member 14; TFRC: transferrin receptor; TGOLN2: trans-golgi network protein 2; TUBB: tubulin beta class I; ULK1: unc-51 like autophagy activating kinase 1; VPS41: VPS41, HOPS complex subunit; WB: western blot; WT: wild type; YPT1: GTP-binding protein YPT1.

MLN4924 is a recently discovered small molecule inhibitor of NEDD8-Activating Enzyme (NAE). Because cullin RING ligase (CRL), the largest family of E3 ubiquitin ligase, requires cullin neddylation for its activity, MLN4924, therefore, acts as an indirect inhibitor of CRL by blocking cullin neddylation. Given that CRLs components are up-regulated, whereas neddylation modification is over-activated in a number of human cancers, MLN4924 was found to be effective in growth suppression of cancer cells. Whether MLN4924 is effective against gastric cancer cells, however, remains elusive. Here we showed that in gastric cancer cells, MLN4924 rapidly inhibited cullin 1 neddylation and remarkably suppressed growth and survival as well as migration in a dose-and time-dependent manner. Mechanistic studies in combination with siRNA knockdown-based rescue experiments revealed that MLN4924 induced the accumulation of a number of CRL substrates, including CDT1/ORC1, p21/p27 and PHLPP1 to trigger DNA damage response and induce growth arrest at the G2/M phase, to induce senescence, as well as autophagy, respectively. MLN4924 also significantly suppressed migration by transcriptionally activating E-cadherin and repressing MMP-9. Taken together, our study suggest that neddylation modification and CRL E3 ligase are attractive gastric cancer targets and MLN4924 might be further developed as a potent therapeutic agent for the treatment of gastric cancer.

The accumulation of ubiquitin-positive protein aggregates has been implicated in the pathogenesis of neurodegenerative diseases, heart disease and diabetes. Emerging evidence indicates that the autophagy lysosomal pathway plays a critical role in the clearance of ubiquitin aggregates, a process that is mediated by the ubiquitin binding protein p62. In addition to binding ubiquitin, p62 also interacts with LC3 and transports ubiquitin conjugates to autophagosomes for degradation. The exact regulatory mechanism of this process is still largely unknown. Here we report the identification of Keap1 as a binding partner for p62 and LC3. Keap1 inhibits Nrf2 by sequestering it in the cytosol and preventing its translocation to the nucleus and activation of genes involved in the oxidative stress response. In this study, we found that Keap1 interacts with p62 and LC3 in a stress-inducible manner, and that Keap1 colocalizes with LC3 and p62 in puromycin-induced ubiquitin aggregates. Moreover, p62 serves as a bridge between Keap1 and ubiquitin aggregates and autophagosomes. Finally, genetic ablation of Keap1 leads to the accumulation of ubiquitin aggregates, increased cytotoxicity of misfolded protein aggregates, and defective activation of autophagy. Therefore, this study assigns a novel positive role of Keap1 in upregulating p62-mediated autophagic clearance of ubiquitin aggregates.

Autophagy delivers many proteins and cellular components to the lysosome for degradation via selective or nonselective mechanisms. By controlling the stability of defined protein factors, autophagy might regulate cellular processes in a precise and finely-tuned manner. In this study, we demonstrated that autophagy positively regulates the biogenesis of the primary cilium, an antenna-like organelle that senses the environment and transduces signals. Defects in the function or structure of cilia cause a number of human diseases called "ciliopathies." We found that the autophagosome membrane anchored protein LC3 interacts with OFD1 (oral-facial-digital syndrome 1) and removes it from the centriolar satellite upon serum starvation to initiate primary cilium biogenesis. OFD1 regulation and primary cilium formation are defective in autophagy-deficient cells, and reducing OFD1 protein levels through RNA interference rescues primary cilium formation. More strikingly, knockdown of OFD1 induces primary cilium formation in unstressed cells as well as in a human breast cancer cell that was previously reported to have lost the ability to form primary cilia. These findings therefore suggest an unexpected link among autophagy, ciliogenesis, ciliopathy, and cancers.