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The Nuclear envelope (NE) is frequently challenged by mechanical stimuli involving cells passing through a tight space and such stress is known as “NE stress.” Various factors that cooperate to repair the NE have been identified, including endosomal sorting complex required for transport-III (ESCRT-III). Recently, vacuolar protein sorting 4 homolog B (VPS4B) has been reported to modulate the recycling of ESCRT-III during NE repair, but the regulatory mechanism remains unclear. Our previous study revealed that U251MG cells, derived from the glioblastoma (GBM), exhibited nuclear deformation followed by DNA damage upon mechanical NE stress while these phenotypes were not observed in U87MG, another GBM-derived cell line. Here, we found that VPS4B expression was lower in U251MG than in U87MG. Our functional analysis demonstrated that insufficient VPS4B triggers an inadequate response to NE stress and that VPS4B regulates the dynamics of ESCRT-III, uncovering the mechanism underlying the NE stress response in GBM.

Abstract The nuclear envelope (NE) is often challenged by various stresses (known as “NE stress”), leading to its dysfunction. Accumulating evidence has proven the pathological relevance of NE stress in numerous diseases ranging from cancer to neurodegenerative diseases. Although several proteins involved in the reassembly of the NE after mitosis have been identified as the NE repair factors, the regulatory mechanisms modulating the efficiency of NE repair remain unclear. Here, we showed that response to NE stress varied among different types of cancer cell lines. U251MG derived from glioblastoma exhibited severe nuclear deformation and massive DNA damage at the deformed nuclear region upon mechanical NE stress. In contrast, another cell line derived from glioblastoma, U87MG, only presented mild nuclear deformation without DNA damage. Time-lapse imaging demonstrated that repairing of ruptured NE often failed in U251MG, but not in U87MG. These differences were unlikely to have been due to weakened NE in U251MG because the expression levels of lamin A/C, determinants of the physical property of the NE, were comparable and loss of compartmentalization across the NE was observed just after laser ablation of the NE in both cell lines. U251MG proliferated more rapidly than U87MG concomitant with reduced expression of p21, a major inhibitor of cyclin-dependent kinases, suggesting a correlation between NE stress response and cell cycle progression. Indeed, visualization of cell cycle stages using fluorescent ubiquitination-based cell cycle indicator reporters revealed greater resistance of U251MG to NE stress at G1 phase than at S and G2 phases. Furthermore, attenuation of cell cycle progression by inducing p21 in U251MG counteracted the nuclear deformation and DNA damage upon NE stress. These findings imply that dysregulation of cell cycle progression in cancer cells causes loss of the NE integrity and its consequences such as DNA damage and cell death upon mechanical NE stress.

Objective: Although individual steps have been characterized, there is little understanding of the overall process whereby glucose co-ordinates the biosynthesis of insulin with its export out of the endoplasmic reticulum (ER) and incorporation into insulin secretory granules (ISGs). Here we investigate a role for the transcription factor CREB3L2 in this context. Methods: MIN6 cells and mouse islets were analysed by immunoblotting after treatment with glucose, fatty acids, thapsigargin and various inhibitors. Knockdown of CREB3L2 was achieved using si or sh constructs by transfection, or viral delivery. In vivo metabolic phenotyping was conducted after deletion of CREB3L2 in β-cells of adult mice using Ins1-CreER+. Islets were isolated for RNAseq and assays of glucose-stimulated insulin secretion (GSIS). Trafficking was monitored in islet monolayers using a GFP-tagged proinsulin construct that allows for synchronised release from the ER. Results: With a Km ≈3.5 mM, glucose rapidly (T1/2 0.9 h) increased full length (FL) CREB3L2 followed by a slower rise (T1/2 2.5 h) in its transcriptionally-active cleavage product, P60 CREB3L2. Glucose stimulation repressed the ER stress marker, CHOP, and this was partially reverted by knockdown of CREB3L2. Activation of CREB3L2 by glucose was not due to ER stress, however, but a combination of O-GlcNAcylation, which impaired proteasomal degradation of FL-CREB3L2, and mTORC1 stimulation, which enhanced its conversion to P60. cAMP generation also activated CREB3L2, but independently of glucose. Deletion of CREB3L2 inhibited GSIS ex vivo and, following a high-fat diet (HFD), impaired glucose tolerance and insulin secretion in vivo. RNAseq revealed that CREB3L2 regulated genes controlling trafficking to-and-from the Golgi, as well as a broader cohort associated with β-cell compensation during a HFD. Although post-Golgi trafficking appeared intact, knockdown of CREB3L2 impaired the generation of both nascent ISGs and proinsulin condensates in the Golgi, implying a defect in ER export of proinsulin and/or its processing in the Golgi. Conclusion: The stimulation of CREB3L2 by glucose defines a novel, rapid and direct mechanism for co-ordinating the synthesis, packaging and storage of insulin, thereby minimizing ER overload and optimizing β-cell function under conditions of high secretory demand. Upregulation of CREB3L2 also potentially contributes to the benefits of GLP1 agonism and might in itself constitute a novel means of treating β-cell failure.

Upregulation of transcription factor OASIS in podocytes contributes to podocyte and/or tubular injury through decreased PRKCi expression and is a critical event for the disturbance of kidney homeostasis.

Several microRNAs (miRNAs), including miR-23 and miR-27a have been reportedly involved in regulating myelination in the central nervous system. Although miR-23 and miR-27a form clusters in vivo and the clustered miRNAs are known to perform complementary functions, the role of these miRNA clusters in myelination has not been studied. To investigate the role of miR-23-27-24 clusters in myelination, we generated miR-23-27-24 cluster knockout mice and evaluated myelination in the brain and spinal cord. Our results showed that 10-week-old knockout mice had reduced motor function in the hanging wire test compared to the wild-type mice. At 4 weeks, 10 weeks, and 12 months of age, knockout mice showed reduced myelination compared to wild-type mice. The expression levels of myelin basic protein and myelin proteolipid protein were also significantly lower in the knockout mice compared to the wild-type mice. Although differentiation of oligodendrocyte progenitor cells to oligodendrocytes was not inhibited in the knockout mice, the percentage of oligodendrocytes expressing myelin basic protein was significantly lower in 4-week-old knockout mice than that in wild-type mice. Proteome analysis and western blotting showed increased expression of leucine-zipper-like transcription regulator 1 (LZTR1) and decreased expression of R-RAS and phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) in the knockout mice. In summary, loss of miR-23-27-24 clusters reduces myelination and compromises motor functions in mice. Further, LZTR1, which regulates R-RAS upstream of the ERK1/2 pathway, a signal that promotes myelination, has been identified as a novel target of the miR-23-27-24 cluster in this study.

Osteoarthritis (OA), the most prevalent aging-related joint disease, is characterized by insufficient extracellular matrix synthesis and articular cartilage degradation and is caused by various risk factors including aging and traumatic injury. Most microRNAs (miRNAs) have been associated with pathogenesis of osteoarthritis (OA) using in vitro models. However, the role of many miRNAs in skeletal development and OA pathogenesis is uncharacterized in vivo using genetically modified mice. Here, we focused on miR-23–27–24 clusters. There are two paralogous miR-23–27–24 clusters: miR-23a-27a-24–2 (miR-23a cluster) and miR-23b-27b-24–1 (miR-23b cluster). Each miR-23a/b, miR-24, and miR-27a/b is thought to function coordinately and complementary to each other, and the role of each miR-23a/b, miR-24, and miR-27a/b in OA pathogenesis is still controversial. MiR-23a/b clusters are highly expressed in chondrocytes and the present study examined their role in OA. We analyzed miRNA expression in chondrocytes and investigated cartilage-specific miR-23a/b clusters knockout (Col2a1-Cre; miR-23a/bflox/flox: Cart-miR-23clus KO) mice and global miR-23a/b clusters knockout (CAG-Cre; miR-23a/bflox/flox: Glob-miR-23clus KO) mice. Knees of Cart- and Glob-miR-23a/b clusters KO mice were evaluated by histological grading systems for knee joint tissues using aging model (12 and/or 18 month-old) and surgically-induced OA model. miR-23a/b clusters were among the most highly expressed miRNAs in chondrocytes. Skeletal development of Cart- and Glob-miR-23clus KO mice was grossly normal although Glob-miR-23clus KO had reduced body weight, adipose tissue and bone density. In the aging model and surgically-induced OA model, Cart- and Glob-miR-23clus KO mice exhibited mild OA-like changes such as proteoglycan loss and cartilage fibrillation. However, the histological scores were not significantly different in terms of the severity of OA in Cart- and Glob-miR-23clus KO mice compared with control mice. Together, miR-23a/b clusters, composed of miR-23a/b, miR-24, miR-27a/b do not significantly contribute to OA pathogenesis.