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Nickel, the most frequent contact allergy cause, is widely used for various metallic materials and medical devices. Autophagy is an intracellular protein degradation system and contributes to metal recycling. However, it is unclear the functions of nickel in autophagy. We here demonstrated that NiCl2 induced microtubule-associated protein 1 light chain 3 (LC3)-II and LC3 puncta, markers of autophagosomes. Bafilomycin A1 (BafA1) treatment did not enhance LC3 puncta under NiCl2 stimulation, suggesting that NiCl2 did not induce autophagic flux. In addition, NiCl2 promotes the accumulation of SQSTM1/p62 and increased SQSTM1/p62 colocalization with lysosomal-associated membrane protein 1 (LAMP1). These data indicated that NiCl2 attenuates autophagic flux. Interestingly, NiCl2 induced the expression of the high-molecular-weight (MW) form of SQSTM1/p62. Inhibition of NiCl2-induced reactive oxygen species (ROS) reduced the high-MW SQSTM1/p62. We also showed that NiCl2-induced ROS activate transglutaminase (TG) activity. We found that transglutaminase 2 (TG2) inhibition reduced high-MW SQSTM1/p62 and SQSTM1/p62 puncta under NiCl2 stimulation, indicating that TG2 regulates SQSTM1/p62 protein homeostasis under NiCl2 stimulation. Our study demonstrated that nickel ion regulates autophagy flux and TG2 restricted nickel-dependent proteostasis.

Genome-wide association studies have identified autophagy-related susceptibility genes for inflammatory bowel disease (IBD); however, whether autophagy regulators can be utilized as therapeutic targets remains unclear. To identify novel microtubule-associated protein 1 light chain 3 (LC3)-interacting proteins in intestinal epithelial cells (IECs), we isolated primary IECs from green fluorescent protein (GFP)-LC3 mice. We performed immunoprecipitation with a GFP antibody and then analyzed co-immunoprecipitates by mass spectrometry. HADHA was identified as an LC3-interacting protein from primary IECs. The HADHA gene encodes the alpha subunit of the mitochondrial trifunctional protein. Given that HADHA catalyzes the last three steps of mitochondrial beta-oxidation of long-chain fatty acids, we investigated whether long-chain fatty acids induce autophagy in IECs. We found that palmitic acid induced autophagy in DLD-1, HT29, and HCT116 cells. HADHA was expressed in not only the mitochondria but also the cytosol. LC3 puncta co-localized with HADHA, which were enhanced by palmitic acid stimulation. However, LC3 puncta did not co-localize with Tom20, suggesting that HADHA was induced to associate with LC3 puncta at sites other than the mitochondria. Thus, HADHA may have extra-mitochondrial functions. Furthermore, we found that palmitic acid induced cell death in IECs, which was accelerated by bafilomycin A and chloroquine. These findings suggested that palmitic acid-induced autophagy supports the survival of IECs. Taken together, these results suggested that HADHA is involved in long-chain fatty acid-induced autophagy in IECs, thus providing new insights into the pathology of IBD and revealing novel therapeutic targets of IBD.

Intestinal goblet cells are characterized by their unique morphology and specialized function to secrete mucins. Although it is known that they are a heterogeneous population of cells, there have been few studies that relate the expression of a particular gene with functionally distinct subpopulations of intestinal goblet cells. Here we show that CCN3, a gene encoding a member of the CCN family proteins, is induced by inhibition of Notch signaling in colonic epithelial cells and expressed in goblet cells in mice. We demonstrate that CCN3 expression is confined to a subpopulation of goblet cells in the lower crypt of the proximal and middle colon. In addition, CCN3+ cells in the colon correlate well with the cells that are positive for alcian blue (AB) staining but negative for high-iron diamine (HID) staining in histology. We also show that CCN3+ cells, which are absent in the normal distal colon, transiently and ectopically emerge in regenerating crypts during the repair phase of DSS-induced colitis model. Our study thus suggests that CCN3 labels a unique subpopulation of sulfomucin-nonproducing colonic goblet cells that function in both normal and diseased colonic epithelia.

Ubiquitin chains are formed with 8 structurally and functionally distinct polymers. However, the functions of each polyubiquitin remain poorly understood. We developed a polyubiquitin-mediated fluorescence complementation (PolyUb-FC) assay using Kusabira Green (KG) as a split fluorescent protein. The PolyUb-FC assay has the advantage that monoubiquitination is nonfluorescent and chain-specific polyubiquitination can be directly visualized in living cells without using antibodies. We applied the PolyUb-FC assay to examine K33-linked polyubiquitin. We demonstrated that SQSTM1/p62 puncta colocalized with K33-linked polyubiquitin and this interaction was modulated by the ZRANB1/TRABID-K29 and -K33 linkage-specific deubiquitinase (DUB). We further showed that the colocalization of K33-linked polyubiquitin and MAP1LC3/LC3 (microtubule associated protein 1 light chain 3) puncta was impaired by SQSTM1/p62 deficiency. Taken together, these findings provide novel insights into how atypical polyubiquitin is recruited by SQSTM1/p62. Finally, we developed an inducible-PolyUb-FC system for visualizing chain-specific polyubiquitin. The PolyUb-FC will be a useful tool for analyzing the dynamics of atypical polyubiquitin chain generation.

Ubiquitination is a crucial post-translational modification; however, the functions of ubiquitin-coding genes remain unclear. UBA52 encodes a fusion protein comprising ubiquitin at the N-terminus and ribosomal protein L40 (RPL40) at the C-terminus. Here we showed that Uba52-deficient mice die during embryogenesis. UBA52-deficient cells exhibited normal levels of total ubiquitin. However, UBA52-deficient cells displayed decreased protein synthesis and cell-cycle arrest. The overexpression of UBA52 ameliorated the cell-cycle arrest caused by UBA52 deficiency. Surprisingly, RPL40 expression itself is insufficient to regulate cyclin D expression. The cleavage of RPL40 from UBA52 was required for maintaining protein synthesis. Furthermore, we found that RPL40 formed a ribosomal complex with ubiquitin cleaved from UBA52. UBA52 supplies RPL40 and ubiquitin simultaneously to the ribosome. Our study demonstrated that the ubiquitin-coding gene UBA52 is not just an ubiquitin supplier to the ubiquitin pool but is also a regulator of the ribosomal protein complex. These findings provide novel insights into the regulation of ubiquitin-dependent translation and embryonic development.

Retinol (ROL), the alcohol form of vitamin A, is known to control cell fate decision of various types of stem cells in the form of its active metabolite, retinoic acid (RA). However, little is known about whether ROL has regulatory effects on colonic stem cells. We examined in this study the effect of ROL on the growth of murine normal colonic cells cultured as organoids. As genes involved in RA synthesis from ROL were differentially expressed along the length of the colon, we tested the effect of ROL on proximal and distal colon organoids separately. We found that organoid forming efficiency and the expression level of Lgr5, a marker gene for colonic stem cells were significantly enhanced by ROL in the proximal colon organoids, but not in the distal ones. Interestingly, neither retinaldehyde (RAL), an intermediate product of the ROL-RA pathway, nor RA exhibited growth promoting effects on the proximal colon organoids, suggesting that ROL-dependent growth enhancement in organoids involves an RA-independent mechanism. This was confirmed by the observation that an inhibitor for RA-mediated gene transcription did not abrogate the effect of ROL on organoids. This novel role of ROL in stem cell maintenance in the proximal colon provides insights into the mechanism of region-specific regulation for colonic stem cell maintenance.