AIbZIP/CREB3L4 Promotes Cell Proliferation via the SKP2-p27 Axis in Luminal Androgen Receptor Subtype Triple-Negative Breast Cancer.

Breast cancer ranks first in incidence and fifth in cancer-related deaths among all types of cancer globally. Among breast cancer, triple-negative breast cancer (TNBC) has few known therapeutic targets and a poor prognosis. Therefore, new therapeutic targets and strategies against TNBC are required. We found that androgen-induced basic leucine zipper (AIbZIP), also known as cyclic AMP-responsive element-binding protein 3-like protein 4 (CREB3L4), which is encoded by Creb3l4, is highly upregulated in a particular subtype of TNBC, luminal androgen receptor (LAR) subtype. We analyzed the function of AIbZIP through depletion of AIbZIP by siRNA knockdown in LAR subtype TNBC cell lines, MFM223 and MDAMB453. In AIbZIP-depleted cells, the proliferation ratios of cells were greatly suppressed. Moreover, G1-S transition was inhibited in AIbZIP-depleted cells. We comprehensively analyzed the expression levels of proteins that regulate G1-S transition and found that p27 was specifically upregulated in AIbZIP-depleted cells. Furthermore, we identified that this p27 downregulation was caused by protein degradation modulated by the ubiquitin-proteasome system via F-box protein S-phase kinase-associated protein 2 (SKP2) upregulation. Our findings demonstrate that AIbZIP is a novel p27-SKP2 pathway-regulating factor and a potential molecule that contributes to LAR subtype TNBC progression. IMPLICATIONS: This research shows a new mechanism for the proliferation of LAR subtype TNBC regulated by AIbZIP, that may provide novel insight into the LAR subtype TNBC progression and the molecular mechanisms involved in cell proliferation.

CREB3L1/OASIS: cell cycle regulator and tumor suppressor.

Cell cycle checkpoints detect DNA errors, eventually arresting the cell cycle to promote DNA repair. Failure of such cell cycle arrest causes aberrant cell proliferation, promoting the pathogenesis of multiple diseases, including cancer. Endoplasmic reticulum (ER) stress transducers activate the unfolded protein response, which not only deals with unfolded proteins in ER lumen but also orchestrates diverse physiological phenomena such as cell differentiation and lipid metabolism. Among ER stress transducers, cyclic AMP-responsive element-binding protein 3-like protein 1 (CREB3L1) [also known as old astrocyte specifically induced substance (OASIS)] is an ER-resident transmembrane transcription factor. This molecule is cleaved by regulated intramembrane proteolysis, followed by activation as a transcription factor. OASIS is preferentially expressed in specific cells, including astrocytes and osteoblasts, to regulate their differentiation. In accordance with its name, OASIS was originally identified as being upregulated in long-term-cultured astrocytes undergoing cell cycle arrest because of replicative stress. In the context of cell cycle regulation, previously unknown physiological roles of OASIS have been discovered. OASIS is activated as a transcription factor in response to DNA damage to induce p21-mediated cell cycle arrest. Although p21 is directly induced by the master regulator of the cell cycle, p53, no crosstalk occurs between p21 induction by OASIS or p53. Here, we summarize previously unknown cell cycle regulation by ER-resident transcription factor OASIS, particularly focusing on commonalities and differences in cell cycle arrest between OASIS and p53. This review also mentions tumorigenesis caused by OASIS dysfunctions, and OASIS's potential as a tumor suppressor and therapeutic target.

Independent activation of CREB3L2 by glucose fills a regulatory gap in mouse ß-cells by co-ordinating insulin biosynthesis with secretory granule formation.

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 ß-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 ß-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 ß-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 ß-cell failure.

Impact of cell cycle on repair of ruptured nuclear envelope and sensitivity to nuclear envelope stress in glioblastoma.

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.

p53-independent tumor suppression by cell-cycle arrest via CREB/ATF transcription factor OASIS.

CREB/ATF transcription factor OASIS/CREB3L1 is upregulated in long-term-cultured astrocytes undergoing cell-cycle arrest due to loss of DNA integrity by repeated replication. However, the roles of OASIS in the cell cycle remain unexplored. We find that OASIS arrests the cell cycle at G2/M phase after DNA damage via direct induction of p21. Cell-cycle arrest by OASIS is dominant in astrocytes and osteoblasts, but not in fibroblasts, which are dependent on p53. In a brain injury model, Oasis-/- reactive astrocytes surrounding the lesion core show sustained growth and inhibition of cell-cycle arrest, resulting in prolonged gliosis. We find that some glioma patients exhibit low expression of OASIS due to high methylation of its promoter. Specific removal of this hypermethylation in glioblastomas transplanted into nude mice by epigenomic engineering suppresses the tumorigenesis. These findings suggest OASIS as a critical cell-cycle inhibitor with potential to act as a tumor suppressor.

Deficiency of MicroRNA-23-27-24 Clusters Exhibits the Impairment of Myelination in the Central Nervous System.

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.

Upregulation of OASIS/CREB3L1 in podocytes contributes to the disturbance of kidney homeostasis.

Podocyte injury is involved in the onset and progression of various kidney diseases. We previously demonstrated that the transcription factor, old astrocyte specifically induced substance (OASIS) in myofibroblasts, contributes to kidney fibrosis, as a novel role of OASIS in the kidneys. Importantly, we found that OASIS is also expressed in podocytes; however, the pathophysiological significance of OASIS in podocytes remains unknown. Upon lipopolysaccharide (LPS) treatment, there is an increase in OASIS in murine podocytes. Enhanced serum creatinine levels and tubular injury, but not albuminuria and podocyte injury, are attenuated upon podocyte-restricted OASIS knockout in LPS-treated mice, as well as diabetic mice. The protective effects of podocyte-specific OASIS deficiency on tubular injury are mediated by protein kinase C iota (PRKCI/PKCι), which is negatively regulated by OASIS in podocytes. Furthermore, podocyte-restricted OASIS transgenic mice show tubular injury and tubulointerstitial fibrosis, with severe albuminuria and podocyte degeneration. Finally, there is an increase in OASIS-positive podocytes in the glomeruli of patients with minimal change nephrotic syndrome and diabetic nephropathy. Taken together, OASIS in podocytes contributes to podocyte and/or tubular injury, in part through decreased PRKCI. The induction of OASIS in podocytes is a critical event for the disturbance of kidney homeostasis.

Impact of Nuclear Envelope Stress on Physiological and Pathological Processes in Central Nervous System.

The nuclear envelope (NE) separates genomic DNA from the cytoplasm and provides the molecular platforms for nucleocytoplasmic transport, higher-order chromatin organization, and physical links between the nucleus and cytoskeleton. Recent studies have shown that the NE is often damaged by various stresses termed "NE stress", leading to critical cellular dysfunction. Accumulating evidence has revealed the crucial roles of NE stress in the pathology of a broad spectrum of diseases. In the central nervous system (CNS), NE dysfunction impairs neural development and is associated with several neurological disorders, such as Alzheimer's disease and autosomal dominant leukodystrophy. In this review, the structure and functions of the NE are summarized, and the concepts of NE stress and NE stress responses are introduced. Additionally, the significant roles of the NE in the development of CNS and the mechanistic connections between NE stress and neurological disorders are described.

Advances in understanding the mechanisms of repairing damaged nuclear envelop.

The nuclear envelope (NE) separates genomic DNA from the cytoplasm in eukaryotes. The structure of the NE is dynamically altered not only in mitotic disassembly and reassembly but also during interphase. Recent studies have shown that the NE is frequently damaged by various cellular stresses that degenerate NE components and/or disrupt their functional interactions. These stresses are referred to as 'NE stress'. Accumulating evidence has demonstrated that NE stress potentially causes severe cellular dysfunctions, such as cell death and genome instability. In this review, the concept of NE stress, the processes repairing damage of the NE caused by NE stress, and the molecular mechanisms by which NE stress contributes to disease pathogenesis are introduced.

miR-23a/b clusters are not essential for the pathogenesis of osteoarthritis in mouse aging and post-traumatic models.

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.

Loss of Function of Mutant IDS Due to Endoplasmic Reticulum-Associated Degradation: New Therapeutic Opportunities for Mucopolysaccharidosis Type II.

Mucopolysaccharidosis type II (MPS II) results from the dysfunction of a lysosomal enzyme, iduronate-2-sulfatase (IDS). Dysfunction of IDS triggers the lysosomal accumulation of its substrates, glycosaminoglycans, leading to mental retardation and systemic symptoms including skeletal deformities and valvular heart disease. Most patients with severe types of MPS II die before the age of 20. The administration of recombinant IDS and transplantation of hematopoietic stem cells are performed as therapies for MPS II. However, these therapies either cannot improve functions of the central nervous system or cause severe side effects, respectively. To date, 729 pathogenetic variants in the IDS gene have been reported. Most of these potentially cause misfolding of the encoded IDS protein. The misfolded IDS mutants accumulate in the endoplasmic reticulum (ER), followed by degradation via ER-associated degradation (ERAD). Inhibition of the ERAD pathway or refolding of IDS mutants by a molecular chaperone enables recovery of the lysosomal localization and enzyme activity of IDS mutants. In this review, we explain the IDS structure and mechanism of activation, and current findings about the mechanism of degradation-dependent loss of function caused by pathogenetic IDS mutation. We also provide a potential therapeutic approach for MPS II based on this loss-of-function mechanism.

OASIS/CREB3L1 is a factor that responds to nuclear envelope stress.

The nuclear envelope (NE) safeguards the genome and is pivotal for regulating genome activity as the structural scaffold of higher-order chromatin organization. NE had been thought as the stable during the interphase of cell cycle. However, recent studies have revealed that the NE can be damaged by various stresses such as mechanical stress and cellular senescence. These types of stresses are called NE stress. It has been proposed that NE stress is closely related to cellular dysfunctions such as genome instability and cell death. Here, we found that an endoplasmic reticulum (ER)-resident transmembrane transcription factor, OASIS, accumulates at damaged NE. Notably, the major components of nuclear lamina, Lamin proteins were depleted at the NE where OASIS accumulates. We previously demonstrated that OASIS is cleaved at the membrane domain in response to ER stress. In contrast, OASIS accumulates as the full-length form to damaged NE in response to NE stress. The accumulation to damaged NE is specific for OASIS among OASIS family members. Intriguingly, OASIS colocalizes with the components of linker of nucleoskeleton and cytoskeleton complexes, SUN2 and Nesprin-2 at the damaged NE. OASIS partially colocalizes with BAF, LEM domain proteins, and a component of ESCRT III, which are involved in the repair of ruptured NE. Furthermore, OASIS suppresses DNA damage induced by NE stress and restores nuclear deformation under NE stress conditions. Our findings reveal a novel NE stress response pathway mediated by OASIS.

Transcription factor old astrocyte specifically induced substance is a novel regulator of kidney fibrosis.

Prevention of kidney fibrosis is an essential requisite for effective therapy in preventing chronic kidney disease (CKD). Here, we identify Old astrocyte specifically induced substance (OASIS)/cAMP responsive element-binding protein 3-like 1 (CREB3l1), a CREB/ATF family transcription factor, as a candidate profibrotic gene that drives the final common pathological step along the fibrotic pathway in CKD. Although microarray data from diseased patient kidneys and fibrotic mouse model kidneys both exhibit OASIS/Creb3l1 upregulation, the pathophysiological roles of OASIS in CKD remains unknown. Immunohistochemistry revealed that OASIS protein was overexpressed in human fibrotic kidney compared with normal kidney. Moreover, OASIS was upregulated in murine fibrotic kidneys, following unilateral ureteral obstruction (UUO), resulting in an increase in the number of OASIS-expressing pathological myofibroblasts. In vitro assays revealed exogenous TGF-ß1 increased OASIS expression coincident with fibroblast-to-myofibroblast transition and OASIS contributed to TGF-ß1-mediated myofibroblast migration and increased proliferation. Significantly, in vivo kidney fibrosis induced via UUO or ischemia/reperfusion injury was ameliorated by systemic genetic knockout of OASIS, accompanied by reduced myofibroblast proliferation. Microarrays revealed that the transmembrane glycoprotein Bone marrow stromal antigen 2 (Bst2) expression was reduced in OASIS knockout myofibroblasts. Interestingly, a systemic anti-Bst2 blocking antibody approach attenuated kidney fibrosis in normal mice but not in OASIS knockout mice after UUO, signifying Bst2 functions downstream of OASIS. Finally, myofibroblast-restricted OASIS conditional knockouts resulted in resistance to kidney fibrosis. Taken together, OASIS in myofibroblasts promotes kidney fibrosis, at least in part, via increased Bst2 expression. Thus, we have identified and demonstrated that OASIS signaling is a novel regulator of kidney fibrosis.

Toxic effects of endoplasmic reticulum stress transducer BBF2H7-derived small peptide fragments on neuronal cells.

Aggregation, fibril formation, and deposition of amyloid ß (Aß) protein are believed to be the central pathogeneses of Alzheimer's disease (AD). Numerous studies have shown that fibril formation is promoted by preformed seeds at the beginning of the aggregation process. Therefore, aggregated molecules that promote fibrillization of Aß protein as seeds could affect the pathology. We recently found that approximately 40 amino acid hydrophobic peptides, BBF2H7-derived small peptide (BSP) fragments, are generated via intramembranous cleavage under endoplasmic reticulum (ER) stress conditions. Interestingly, similar to Aß protein, the fragments exhibit a high aggregation propensity and form fibril structures. It has been noted that ER stress is involved in the pathogenesis of AD. In this study, we examined the effect of BSP fragments on aggregation and cytotoxicity of Aß1-40 protein, which is generated as a major species of Aß protein, but has a lower aggregative property than Aß1-42 protein. We demonstrated that BSP fragments promote aggregation of Aß1-40 protein. Aggregates of Aß1-40 protein mediated by BSP fragments also exhibited potent neurotoxicity. Our findings suggest the possibility that BSP fragments affect accumulation of Aß proteins and are involved in the pathogenesis of AD.

The Role of Tissue-Specific Ubiquitin Ligases, RNF183, RNF186, RNF182 and RNF152, in Disease and Biological Function.

Ubiquitylation plays multiple roles not only in proteasome-mediated protein degradation but also in various other cellular processes including DNA repair, signal transduction, and endocytosis. Ubiquitylation is mediated by ubiquitin ligases, which are predicted to be encoded by more than 600 genes in humans. RING finger (RNF) proteins form the majority of these ubiquitin ligases. It has also been predicted that there are 49 RNF proteins containing transmembrane regions in humans, several of which are specifically localized to membrane compartments in the secretory and endocytic pathways. Of these, RNF183, RNF186, RNF182, and RNF152 are closely related genes with high homology. These genes share a unique common feature of exhibiting tissue-specific expression patterns, such as in the kidney, nervous system, and colon. The products of these genes are also reported to be involved in various diseases such as cancers, inflammatory bowel disease, Alzheimer's disease, and chronic kidney disease, and in various biological functions such as apoptosis, endoplasmic reticulum stress, osmotic stress, nuclear factor-kappa B (NF-κB), mammalian target of rapamycin (mTOR), and Notch signaling. This review summarizes the current knowledge of these tissue-specific ubiquitin ligases, focusing on their physiological roles and significance in diseases.

Production of BBF2H7-derived small peptide fragments via endoplasmic reticulum stress-dependent regulated intramembrane proteolysis.

Intramembrane cleavage of transmembrane proteins is a fundamental cellular process to produce important signals that elicit biological responses. These proteolytic events are known as regulated intramembrane proteolysis (RIP). ATF6 and BBF2H7 are transmembrane basic leucine zipper transcription factors and are subjected to RIP by site-1 protease (S1P) and site-2 protease (S2P) sequentially in response to endoplasmic reticulum (ER) stress. However, the detailed mechanisms responsible for RIP of the transcription factors, including the precise cutting sites, are still unknown. In this study, we demonstrated that S1P cleaves BBF2H7 just before the RXXL S1P recognition motif. Conversely, S2P cut at least three different sites in the membrane (next to Leu380, Met381, and Leu385), indicating that S2P cleaves the substrates at variable sites or via a multistep process. Interestingly, we found BBF2H7-derived small peptide (BSP) fragments located between the S1P and S2P cleavage sites in cells exposed to ER stress. Major type of BSP fragments was composed of 45 amino acid including partial transmembrane and luminal regions and easily aggregates like amyloid ß (Aß) protein. These results advance the understanding of poorly characterized ER stress-dependent RIP. Furthermore, the aggregable peptides produced by ER stress could link to the pathophysiology of neurodegenerative disorders.

Hypertonicity-responsive ubiquitin ligase RNF183 promotes Na, K-ATPase lysosomal degradation through ubiquitination of its ß1 subunit.

We previously reported that RNF183, a member of the RING finger (RNF) protein family, is specifically expressed in the renal collecting duct and that RNF183 mRNA is induced by the activity of nuclear factor of activated T cells 5 (NFAT5), which regulates the transcription of essential proteins for adaptation to hypertonic conditions. The renal medulla is the only tissue that is continuously hypertonic; therefore, RNF183 possibly plays an important role in adaptation to continuous hypertonic conditions. However, the mechanism of how cells adapt to long-term hypertonicity via RNF183 remains unclear. In this study, the Na, K-ATPase α1 subunit was identified as a candidate substrate of RNF183 by the BirA proximity-biotinylation technique. The Na, K-ATPase α1 subunit acts as an ion transporter along with the Na, K-ATPase ß1 subunit at the plasma membrane. We confirmed that RNF183 interacted with both α1 and ß1 subunits; however, we found that RNF183 ubiquitinated only the ß1 subunit, not the α1 subunit. Furthermore, RNF183 translocated both α1 and ß1 subunits from the plasma membrane to lysosomes. In addition, the expression levels of α1 and ß1 subunits in HEK293 cells stably expressing RNF183 were significantly decreased compared with mock control cells, and were restored by siRNA-mediated knockdown of RNF183. Moreover, in RNF183-expressing cells, chloroquine treatment increased the protein levels of the α1 and ß1 subunits. Therefore, our results suggest that Na, K-ATPase α1 and ß1 subunits are degraded in lysosomes by RNF183-mediated ubiquitination of ß1 subunit.

Decreased Activity of the Ghrhr and Gh Promoters Causes Dominantly Inherited GH Deficiency in Humanized GH1 Mouse Models.

Isolated growth hormone deficiency type II (IGHD2) is mainly caused by heterozygous splice-site mutations in intron 3 of the GH1 gene. A dominant-negative effect of the mutant GH lacking exon 3 on wild-type GH secretion has been proposed; however, the molecular mechanisms involved are elusive. To uncover the molecular systems underlying GH deficiency in IGHD2, we established IGHD2 model mice, which carry both wild-type and mutant copies of the human GH1 gene, replacing each of the endogenous mouse Gh loci. Our IGHD2 model mice exhibited growth retardation along with intact cellular architecture and mildly activated endoplasmic reticulum stress in the pituitary gland, caused by decreased GH-releasing hormone receptor (Ghrhr) and Gh gene promoter activities. Decreased Ghrhr and Gh promoter activities were likely caused by reduced levels of nuclear CREB3L2, which was demonstrated to stimulate Ghrhr and Gh promoter activity. To our knowledge, this is the first in vivo study to reveal a novel molecular mechanism of GH deficiency in IGHD2, representing a new paradigm that differs from widely accepted models.

Renal medullary tonicity regulates RNF183 expression in the collecting ducts via NFAT5.

Nuclear factor of activated T-cells 5 (NFAT5) directly binds to the promoter of the RING finger protein 183 (RNF183) gene and induces its transcription under hypertonic conditions in mouse inner-medullary collecting duct (mIMCD-3) cells. However, there is no specific anti-RNF183 antibody for immunostaining; therefore, it is unclear whether NFAT5 regulates RNF183 expression in vivo and where RNF183 is localized in the kidney. This study investigated NFAT5-regulated in vivo RNF183 expression and localization using CRISPR/Cas9-mediated RNF183-green fluorescent protein (RNF183-GFP) knock-in mice. GFP with linker sequences was introduced upstream of an RNF183 open reading frame in exon 3 by homologous recombination through a donor plasmid. Immunofluorescence staining using GFP antibody revealed that GFP signals gradually increase from the outer medulla down to the inner medulla and colocalize with aquaporin-2. Furosemide treatment dramatically decreased RNF183 expression in the renal medulla, consistent with the decrease in NFAT5 protein and target gene mRNA expression. Furosemide treatment of mIMCD-3 cells did not affect mRNA expression and RNF183 promoter activities. These results indicated that RNF183 is predominantly expressed in the renal medullary collecting ducts, and that decreased renal medullary tonicity by furosemide treatment decreases RNF183 expression by NFAT5 downregulation.

Calnexin promotes the folding of mutant iduronate 2-sulfatase related to mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (MPS II) is one of the most common mucopolysaccharidoses, which is caused by mutation of the gene encoding iduronate 2-sulfatase (IDS). The loss of function of IDS leads to the accumulation of heparan sulfate and dermatan sulfate of glycosaminoglycans throughout the body, resulting in skeletal deformities, mental retardation, rigid joints, and thick skin. Recently, enzyme replacement therapy has become a common strategy for treating this condition. However, its effectiveness on the central nervous system (CNS) is limited because intravenously administered recombinant IDS (rIDS) cannot pass through the blood brain barrier. Therefore, several methods for delivering rIDS to the CNS, using anti-human transferrin receptor antibody and adeno-associated virus 9, have been explored. To investigate additional approaches for treatment, more cognition about the intracellular dynamics of mutant IDS is essential. We have already found that mutant IDS accumulated in the endoplasmic reticulum (ER) and was degraded by ER-associated degradation (ERAD). Although the dynamics of degradation of mutant IDS was revealed, the molecular mechanism related to the folding of mutant IDS in the ER remained unclear. In this research, we confirmed that mutant IDS retained in the ER would be folded by binding with calnexin (CNX). Thus, knockdown of CNX reduced the translocation of mutant IDS from ER to lysosome and its enzyme activity, indicating that the correct folding of this protein via interaction with CNX ensures its functional activity. These findings reveal the possibility that modifying the interaction of mutant IDS and CNX could contribute to alternative therapeutic strategies for MPS II.