Melanosomal localization is required for GIF-2115/2250 to inhibit melanogenesis in B16F10 melanoma cells.

OBJECTIVE: Tyrosinase inhibitors suppress melanogenesis in melanocytes. During a screening for tyrosinase inhibitors, however, we noticed some discrepancies in inhibitory efficacies between melanocytes and in vitro assays. The compound (S)-N-{3-[4-(dimethylamino)phenyl]propyl}-N-methyl-indan-1-amine (GIF-2115) exerts antioxidative stress activity upon accumulation in late endosomes and lysosomes. GIF-2115 was also identified as a potent antimelanogenic reagent in B16F10 mouse melanoma cells. GIF-2115 inhibited the activity of mushroom tyrosinase and the lysates of B16F10 cells. However, structure-activity relationship studies indicated that GIF-2238, which lacks the benzene ring in the aminoindan structure of GIF-2115, inhibited tyrosinase activity in vitro but did not inhibit melanogenesis in B16F10 cells. The aim of the present study is to show the importance of the intracellular distribution of tyrosinase inhibitors in exerting their antimelanogenic activity in melanocytes. METHODS: The intracellular distribution of compounds was monitored by linking with the fluorescent group of 7-nitro-2,1,3-benzoxadiazole (NBD). To mislocalize GIF-2115 to mitochondria, the mitochondria-preferring fluoroprobe ATTO565 was used. RESULTS: We reconfirmed the localization of GIF-2250 (GIF-2115-NBD) not only to matured but also to early-stage melanosomes. Although GIF-2286 (GIF-2238-NBD) maintained tyrosinase inhibitory activity, it did not show specific intracellular localization. Moreover, when GIF-2115 was linked with ATTO565, the resultant compound GIF-2265 did not inhibit melanogenesis in B16F10 cells, despite its strong tyrosinase inhibitory activity. CONCLUSION: These results suggest that melanosomal localization is essential for the antimelanogenic activity of GIF-2115, and GIF-2115 derivatives may be new guides for drugs to endosomes and lysosomes as well as melanosomes.

OBJECTIF: Les inhibiteurs de la tyrosinase suppriment la mélanogenèse dans les mélanocytes. Lors d'un criblage d'inhibiteurs de la tyrosinase, cependant, nous avons remarqué des différences dans les efficacités inhibitrices entre les mélanocytes et les essais in vitro. Le composé (S)-N-{3-[4-(diméthylamino)phényl]propyl}-N-méthyl-indan-1-amine (GIF-2115) exerce une activité antioxydante en cas de stress lors de l'accumulation dans les endosomes tardifs et les lysosomes. GIF-2115 a également été identifié comme un puissant réactif antimélanogène dans les cellules de mélanome murin B16F10. GIF-2115 a inhibé l'activité de la tyrosinase de champignon et les lysats des cellules B16F10. Cependant, des études de relation structure-activité ont indiqué que GIF-2238, à qui il manque l'anneau benzénique dans la structure aminoindan de GIF-2115, inhibait l'activité de la tyrosinase in vitro mais n'inhibait pas la mélanogenèse dans les cellules B16F10. L'objectif de la présente étude est de montrer l'importance de la distribution intracellulaire des inhibiteurs de la tyrosinase dans l'exercice de leur activité antimélanogène dans les mélanocytes. MÉTHODES: La distribution intracellulaire des composés a été surveillée en les liant au groupe fluorescent de la 7-nitro-2,1,3-benzoxadiazole (NBD). Pour délocaliser GIF-2115 vers les mitochondries, le fluorophore ATTO565 préférant les mitochondries a été utilisé. RÉSULTATS: Nous avons confirmé la localisation de GIF-2250 (GIF-2115-NBD) non seulement dans les mélanosomes matures mais aussi dans les mélanosomes à un stade précoce. Bien que GIF-2286 (GIF-2238-NBD) ait maintenu une activité inhibitrice de la tyrosinase, il n'a pas montré de localisation intracellulaire spécifique. De plus, lorsque GIF-2115 a été lié à ATTO565, le composé résultant GIF-2265 n'a pas inhibé la mélanogenèse dans les cellules B16F10, malgré son activité inhibitrice de la tyrosinase forte. CONCLUSION: Ces résultats suggèrent que la localisation dans les mélanosomes est essentielle pour l'activité antimélanogène de GIF-2115, et que les dérivés de GIF-2115 peuvent être de nouveaux guides pour les médicaments vers les endosomes et les lysosomes ainsi que les mélanosomes.

Abnormal glomerular basement membrane maturation impairs mesangial cell differentiation during murine postnatal nephrogenesis.

Although mesangial cell-glomerular basement membrane (GBM) connections play a key role in maintaining the glomerular capillary loop structure, information remains limited about how these connections are formed during glomerulogenesis. We have previously shown that weakened podocyte-GBM interactions owing to tensin 2 (Tns2) deficiency lead to abnormal GBM maturation during postnatal glomerulogenesis. Here, we investigated whether abnormal GBM maturation affected mesangial cell-GBM connections and mesangial cell differentiation. Histological analysis of the outer cortical glomeruli in Tns2-deficient mice revealed that GBM materials overproduced by stressed immature podocytes accumulated in the mesangium and interrupted the formation of mesangial cell-GBM connections, resulting in fewer capillary loops compared with that of normal glomeruli. In addition, expression of α-smooth muscle actin, an immature mesangial cell marker, persisted in mesangial cells of Tns2-deficient outer cortical glomeruli even after glomerulogenesis was completed, resulting in mesangial expansion. Furthermore, analysis of mouse primary mesangial cells revealed that mesangial cell differentiation depended on the type of extracellular matrix components to which the cells adhered, suggesting the participation of mesangial cell-GBM connections in mesangial cell differentiation. These findings suggest that abnormal GBM maturation affects mesangial cell differentiation by impairing mesangial cell-GBM connections.NEW & NOTEWORTHY Mesangial cell-glomerular basement membrane (GBM) connections play an important role in maintaining the structural integrity of the glomerular tuft. However, information remains scarce about how GBM maturation affects the formation of these connections during glomerular development. Here, we show that abnormal GBM maturation due to tensin 2 deficiency affects mesangial cell differentiation by impairing mesangial cell-GBM connections during postnatal glomerulogenesis.

Molecular characterization of wild-type and HSAN2B-linked FAM134B.

BACKGROUND: Family with sequence similarity 134, member B (FAM134B), also known as Reticulophagy regulator 1 (RETREG1), is an ER-phagy receptor involved in ER homeostasis. Congenital mutations in the FAM134B gene have been reported to be associated with hereditary sensory and autonomic neuropathy type 2B (HSAN2B); however, the molecular differences between wild-type and HSAN2B-linked FAM134B are not fully understood. METHODS AND RESULTS: We prepared several human FAM134B constructs, such as the HSAN2B-linked mutant, and compared their features with those of wild-type FAM134B by transfecting these constructs into FAM134B-deficient Neuro2a cells. Although intrinsic FAM134B protein expression in wild-type Neuro2a cells was affected by the supply of amino acids in the culture medium, the expression of each HSAN2B-linked mutant FAM134B protein was hardly affected by serum and amino acid deprivation. On the other hand, the intracellular localization of GFP-tagged HSAN2B-linked mutants, except for P7Gfs133X, overlapped well with ER-localized SP-RFPKDEL and did not differ from that of GFP-tagged wild-type FAM134B. However, analysis of protein‒protein interactions using the NanoBiT reporter assay revealed the difference between wild-type and C-terminal truncated mutant FAM134B. Furthermore, this NanoBiT assay demonstrated that both wild-type and G216R FAM134B interacted with LC3/GABARAPL1 to the same extent, but the FAM134B construct with mutations near the LC3-interacting region (LIR) did not. Similar to the NanoBiT assay, the C-terminal-truncated FAM134B showed lower ER-phagy activities, as assessed by the cotransfection of GFP-tagged reporters. CONCLUSIONS: We showed that wild-type and HSAN2B-linked FAM134B have different molecular characteristics by transfecting cells with various types of constructs. Thus, this study provides new insights into the molecular mechanisms underlying HSAN2B as well as the regulation of ER-phagy.

Intermittent inhibition of FYVE finger-containing phosphoinositide kinase induces melanosome degradation in B16F10 melanoma cells.

BACKGROUND: Melanosomes are lysosome-related organelles that contain melanogenic factors and synthesize melanin as they mature. FYVE finger-containing phosphoinositide kinase (PIKfyve) regulates late endosome and lysosome morphology, vesicle trafficking, and autophagy. In melanocytes, PIKfyve inhibition has been reported to induce hypopigmentation due to impairments in the metabolism of early-stage melanosomes. METHODS AND RESULTS: Here, we report a new type of melanosome metabolism: post-PIKfyve inhibition, which was found during the characterization of the endosome/lysosome fluoroprobe GIF-2250. In B16F10 mouse melanoma cells, GIF-2250 highlighted vesicles positive for lysosomal-associated membrane protein 1 (lysosome marker) and other endosome/lysosome markers (CD63 and Rab7/9). When cells were continuously treated with PIKfyve inhibitors, intracellular vacuoles formed, while GIF-2250 fluorescence signals diminished and were diffusely distributed in the vacuoles. After removal of the PIKfyve inhibitors, the GIF-2250 signal intensity was restored, and some GIF-2250-positive vesicles wrapped the melanosomes, which spun at high speed. In addition, intermittent PIKfyve inhibition caused melanin diffusion in the vacuoles and possible leakage into the cytoplasmic compartments, and melanosome degradation was detected by a transmission electron microscope. Melanosome degradation was accompanied by decreased levels of melanin synthesis enzymes and increased levels of the autophagosome maker LC3BII, which is also associated with early melanosomes. However, the protein levels of p62, which is degraded during autophagy, were increased, suggesting an impairment in autophagy flux during intermittent PIKfyve inhibition. Moreover, the autophagy inhibitor 3-methyladenine does not affect these protein levels, suggesting that the melanosome degradation by the intermittent inhibition of PIKfyve is not mediated by canonical autophagy. CONCLUSIONS: In conclusion, disturbance of PIKfyve activity induces melanosome degradation in a canonical autophagy-independent manner.

Elucidation of OSW-1-Induced Stress Responses in Neuro2a Cells.

OSW-1, a steroidal saponin isolated from the bulbs of Ornithogalum saundersiae, is a promising compound for an anticancer drug; however, its cytotoxic mechanisms have not been fully elucidated. Therefore, we analyzed the stress responses triggered by OSW-1 in the mouse neuroblastoma cell line Neuro2a by comparing it with brefeldin A (BFA), a Golgi apparatus-disrupting reagent. Among the Golgi stress sensors TFE3/TFEB and CREB3, OSW-1 induced dephosphorylation of TFE3/TFEB but not cleavage of CREB3, and induction of the ER stress-inducible genes GADD153 and GADD34 was slight. On the other hand, the induction of LC3-II, an autophagy marker, was more pronounced than the BFA stimulation. To elucidate OSW-1-induced gene expression, we performed a comprehensive gene analysis using a microarray method and observed changes in numerous genes involved in lipid metabolism, such as cholesterol, and in the regulation of the ER-Golgi apparatus. Abnormalities in ER-Golgi transport were also evident in the examination of secretory activity using NanoLuc-tag genes. Finally, we established Neuro2a cells lacking oxysterol-binding protein (OSBP), which were severely reduced by OSW-1, but found OSBP deficiency had little effect on OSW-1-induced cell death and the LC3-II/LC3-I ratio in Neuro2a cells. Future work to elucidate the relationship between OSW-1-induced atypical Golgi stress responses and autophagy induction may lead to the development of new anticancer agents.

Hypomorphic mutation of PEX3 with peroxisomal mosaicism reveals the oscillating nature of peroxisome biogenesis coupled with differential metabolic activities.

Impaired peroxisome assembly caused by mutations in PEX genes results in a human congenital metabolic disease called Zellweger spectrum disorder (ZSD), which impacts the development and physiological function of multiple organs. In this study, we revealed a long-standing problem of heterogeneous peroxisome distribution among cell population, so called "peroxisomal mosaicism", which appears in patients with mild form of ZSD. We mutated PEX3 gene in HEK293 cells and obtained a mutant clone with peroxisomal mosaicism. We found that peroxisomal mosaicism can be reproducibly arise from a single cell, even if the cell has many or no peroxisomes. Using time-lapse imaging and a long-term culture experiment, we revealed that peroxisome biogenesis oscillates over a span of days; this was also confirmed in the patient's fibroblasts. During the oscillation, the metabolic activity of peroxisomes was maintained in the cells with many peroxisomes while depleted in the cells without peroxisomes. Our results indicate that ZSD patients with peroxisomal mosaicism have a cell population whose number and metabolic activities of peroxisomes can be recovered. This finding opens the way to develop novel treatment strategy for ZSD patients with peroxisomal mosaicism, who currently have very limited treatment options.

Expression analysis and functional characterization of thioredoxin domain-containing protein 11.

BACKGROUNDS: The endoplasmic reticulum (ER) is a crucial organelle that regulates both the folding, modification and transport of many proteins and senses certain stimuli inside and outside of cells. ER-associated degradation (ERAD), including SEL1L is a crucial mechanism to maintain homeostasis. In this study, we performed comparative proteome analysis in wild-type (wt) and SEL1L-deficient cells. METHODS AND RESULTS: We found constitutively high expression of thioredoxin domain-containing protein 11 (TXNDC11) mRNA and protein in our SEL1L-deficient HEK293 cells by RT-PCR and Western blot analysis. The TXNDC11 gene possesses a well-conserved unfolded protein response element (UPRE) around its transcription start site, and ER stress increased TXNDC11 mRNA and luciferase reporter activity via this putative UPRE in HEK293 cells. The amounts of TXNDC11 protein in wild-type and SEL1L-deficient cells with or without thapsigargin (Tg) treatment were parallel to their mRNAs in these cells, which was almost proportional to spliced XBP1 (sXBP1) mRNA expression. The establishment and characterization of TXNDC11-deficient HEK293 cells revealed that the expression of three different ER resident stress sensors, ATF6α, CREB3 and CREB3L2, is regulated by TXNDC11. The rate of disappearance of the three proteins by CHX treatment in wt cells was remarkably different, and the full-length CREB3L2 protein was almost completely degraded within 15 min after CHX treatment. TXNDC11 deficiency increased the expression of each full-length form under resting conditions and delayed their disappearance by CHX treatment. Interestingly, the degree of increase in full-length CREB3/CREB3L2 by TXNDC11 deficiency was apparently higher than that in full-length ATF6α. The increase in these proteins by TXNDC11 deficiency was hardly correlated with the expression of each mRNA. Treatment with ER stress inducers influenced each full-length mature form, and the difference in each full-length form observed in wt and TXNDC11-deficient cells was smaller. CONCLUSION: This study demonstrated that TXNDC11 is an ER stress-inducible gene regulated by the IRE1-sXBP1 pathway. In addition, TXNDC11 is involved in the regulation of ATF6α, CREB3 and CREB3L2 protein expression, although the contribution to the stability of these proteins is quite variable. Therefore, its further characterization will provide new insights for understanding protein homeostasis in ER physiology and pathology.

The new live imagers MitoMM1/2 for mitochondrial visualization.

Mitochondria are eukaryotic organelles that consist of outer and inner bilayer membranes with a positive potential (H+) in the intermembrane space. This organelle plays an important role in ATP production and apoptosis. To observe the mitochondria in living cells, several fluorescent dyes (such as MitoTracker® [a standard mitochondrial imager] or rhodamine 123) have been developed. However, these reagents are unstable and exhibit a wide range of emission spectra, thereby hampering double staining results. Using recombinant DNA techniques, green or red fluorescent protein (GFP or RFP)-tagged proteins are now available for multi-color labeling of mitochondria. Here, we have discussed the development of the novel mitochondrial live imagers MitoMM1/2, derivatives of ATTO565; furthermore, MitoMM1/2 are sensitive to the membrane potential, resistant to detergents, and the fluorescence of MitoMM1/2 does not overlap with green fluorescence.

Transcriptional regulation of the ER stress-inducible gene Sec16B in Neuro2a cells.

Endoplasmic reticulum (ER) stress responses have been demonstrated to play important roles in maintaining various cellular functions and to underlie many tissue dysfunctions. In this study, we identified Sec16B as an ER stress-inducible gene by microarray analysis of brefeldin A (BFA)-inducible genes in a mouse neuroblastoma cell-line, Neuro2a. Sec16B mRNA was induced by treatment with the ER stress-inducing reagents thapsigargin (Tg) and brefeldin A in a time-dependent manner. In the genomic sequence of the mouse Sec16B gene, we found an unfolded protein response element (UPRE), which is well conserved between humans and mice. Using luciferase reporter analyses, we showed that the UPRE in the mouse Sec16B gene was functional and responded well to ER stress-inducing stimuli and spliced XBP1 (sXBP1)-overexpression. In addition, a unique ATF4-responsive sequence within the first intron of the mouse Sec16B gene was characterized. Our study may help to elucidate the regulation of trafficking through the ER-Golgi apparatus and the biogenesis of ER-derived intracellular organelles.

Elucidation of brefeldin A-induced ER and Golgi stress responses in Neuro2a cells.

Brefeldin A (BFA) disrupts the structure of the Golgi apparatus to trigger ER stress signaling pathways. On the other hand, treatment with BFA induces the activation of CREB3, the protein structure of which is similar to that of ATF6. In this study, we established Neuro2a cells in which three different transcription factors, namely, ATF4, ATF3 and CREB3, were deficient using the CRISPR/Cas9 approach, and we investigated the BFA-induced ER and Golgi stress response in these cells. BFA treatment rapidly induced ATF4, ATF3, Herp and GADD153 protein expression in Neuro2a cells. ATF4-deficient Neuro2a cells exhibited significantly decreased mRNA and protein expression of ATF3 and Herp but not GADD153; however, cells deficient in ATF3 exhibited minimal effects on GADD34, GADD153 and Herp expression. The cleavage of CREB3 in Neuro2a cells was triggered by BFA; however, the expression of several ER and Golgi stress-related factors was hardly influenced by the CREB3 deficiency in these Neuro2a cells. This study shows that CREB3 minimally associates with typical ER stress-inducible responses in Neuro2a cells. Therefore, identification and characterization of the downstream transcriptional targets of CREB3 is required to clarify not only Golgi stress response but also its relationship with ER stress signaling pathways.

Molecular characterization of mouse CREB3 regulatory factor in Neuro2a cells.

We performed expression and functional analysis of mouse CREB3 regulatory factor (CREBRF) in Neuro2a cells by constructing several expression vectors. Overexpressed full-length (FL) CREBRF protein was stabilized by MG132; however, the intrinsic CREBRF expression in Neuro2a cells was negligible under all conditions. On the other hand, N- or C-terminal deletion of CREBRF influenced its stability. Cotransfection of CREBRF together with GAL4-tagged FL CREB3 increased luciferase reporter activity, and only the N-terminal region of CREBRF was sufficient to potentiate luciferase activity. Furthermore, this positive effect of CREBRF was also observed in cells expressing GAL4-tagged cleaved CREB3, although CREBRF hardly influenced the protein stability of NanoLuc-tagged cleaved CREB3 or intracellular localization of EGFP-tagged one. In conclusion, this study suggests that CREBRF, a quite unstable proteasome substrate, positively regulates the CREB3 pathway, which is distinct from the canonical ER stress pathway in Neuro2a cells.

Comparative Analysis of CREB3 and CREB3L2 Protein Expression in HEK293 Cells.

We performed a comparative analysis of two ER-resident CREB3 family proteins, CREB3 and CREB3L2, in HEK293 cells using pharmacological and genome editing approaches and identified several differences between the two. Treatment with brefeldin A (BFA) and monensin induced the cleavage of full-length CREB3 and CREB3L2; however, the level of the full-length CREB3 protein, but not CREB3L2 protein, was not noticeably reduced by the monensin treatment. On the other hand, treatment with tunicamycin (Tm) shifted the molecular weight of the full-length CREB3L2 protein downward but abolished CREB3 protein expression. Thapsigargin (Tg) significantly increased the expression of only full-length CREB3L2 protein concomitant with a slight increase in the level of its cleaved form. Treatment with cycloheximide and MG132 revealed that both endogenous CREB3 and CREB3L2 are proteasome substrates. In addition, kifunensine, an α-mannosidase inhibitor, significantly increased the levels of both full-length forms. Consistent with these findings, cells lacking SEL1L, a crucial ER-associated protein degradation (ERAD) component, showed increased expression of both full-length CREB3 and CREB3L2; however, cycloheximide treatment downregulated full-length CREB3L2 protein expression more rapidly in SEL1L-deficient cells than the full-length CREB3 protein. Finally, we investigated the induction of the expression of several CREB3 and CREB3L2 target genes by Tg and BFA treatments and SEL1L deficiency. In conclusion, this study suggests that both endogenous full-length CREB3 and CREB3L2 are substrates for ER-associated protein degradation but are partially regulated by distinct mechanisms, each of which contributes to unique cellular responses that are distinct from canonical ER signals.

Characterization of IRE1α in Neuro2a cells by pharmacological and CRISPR/Cas9 approaches.

IRE1 is the most conserved endoplasmic reticulum (ER)-resident stress sensor. Its activation not only splices XBP1 but also participates in a variety of cell signaling. We elucidated the role of IRE1α in Neuro2a cells by establishing IRE1α-deficient cells and applying four IRE1 inhibitors. IRE1α deficiency prevented almost all spliced XBP1 (sXBP1) protein expression by treatment with thapsigargin (Tg) and tunicamycin (Tm); these phenomena paralleled the values measured by our two Nanoluciferase-based IRE1 assays. However, cell viability and protein expression of other ER stress-responsive factors in the IRE1α-deficient cells were comparable to those in the parental wild-type cells with or without Tm treatment. Next, we elucidated the IRE1 inhibitory actions and cytotoxicity of four compounds: STF083010, KIRA6, 4µ8C, and toyocamycin. KIRA6 attenuated IRE1 activity in a dose-dependent manner, but it showed severe cytotoxicity even in the IRE1α-deficient cells at a low concentration. The IRE1α-deficient cells were slightly resistant to KIRA6 at 0.1 µM in both the presence and absence of ER stress; however, resistance was not observed at 0.02 µM. Treatment with only KIRA6 at 0.1 µM for 12 h remarkably induced LC3 II, an autophagic marker, in both parental and IRE1α-deficient cells. Co-treatment with KIRA6 and Tm induced LC3 II, cleaved caspase-9, and cleaved caspase-3; however, IRE1α-deficiency did not abolish the expression of these two cleaved caspases. On the other hand, KIRA6 prohibited Tm-induced ATF4 induction in an IRE1-independent manner; however, co-treatment with KIRA6 and Tm also induced LC3 II and two cleaved caspases in the ATF4-deficient Neuro2a cells. Thus, we demonstrate that IRE1α deficiency has little impact on cell viability and expression of ER stress-responsive factors in Neuro2a cells, and the pharmacological actions of KIRA6 include IRE1-independent ways.

Functional validation of ATF4 and GADD34 in Neuro2a cells by CRISPR/Cas9-mediated genome editing.

Activating transcription factor 4 (ATF4), which is ubiquitously expressed, plays a crucial role in regulating various stress-responsive genes under pathophysiological conditions. Further, growth arrest and DNA damage-inducible gene 34 (GADD34), a downstream target of ATF4, has been reported to negatively regulate ATF4 expression. To understand the relationship between intrinsic ATF4 and GADD34 under resting and ER stress conditions, we used a novel gene editing approach, CRISPR/Cas9, to integrate antibiotic-resistant genes into the target genes, ATF4 and GADD34. First, we manipulated the ATF4 gene in the mouse neuroblastoma cell line, Neuro2a, and compared the ER stress responses between parental and ATF4-edited Neuro2a cells. Next, we established Neuro2a cells with edited GADD34 and ATF4/GADD34 genes and found that ATF4 acts as a proapoptotic factor, but GADD34 depletion did not attenuate the expression of cleaved caspase-3 induced by tunicamycin treatment. These findings provide new insights into the ATF4 signaling cascades. Additionally, the rapid establishment of cells lacking multiple genes using this CRISPR/Cas9 system will be a powerful tool for exploring various cellular issues under pathophysiological conditions.

Elucidating post-translational regulation of mouse CREB3 in Neuro2a cells.

CREB3 is an ER membrane-bound transcription factor; however, post-translational regulation of CREB3, including expression, processing, and activation, is not fully characterized. We therefore constructed several types of mouse CREB3 expression genes and elucidated their expression in Neuro2a cells by treatment with stimuli and co-transfection with genes associated with ER-Golgi homeostasis, such as mutant Sar1 [H79G], GRP78, and KDEL receptor 1 (KDELR1). Interestingly, treatment of Neuro2a cells expressing Flag-tagged full-length CREB3 with monensin and nigericin induced the expression of the approximately 50 kDa N-terminal fragment; however, its cleavage was not parallel to the levels of GADD153 and LC3-II. Co-transfection of full-length CREB3 together with Sar1 [H79G], GRP78, or KDELR1 showed that only Sar1 [H79G] induced expression of the cleaved form, and KDELR1 dramatically decreased the expression of the full-length form. Accordingly, Sar1 [H79G]- and KDELR1-overexpression influenced GAL4-CREB3-dependent luciferase activities. To understand the activation of CREB3 under more pathophysiological conditions, we focused on the effect of metal ions on CREB3 cleavage in Neuro2a cells. Among the six metal ions we tested, only copper ion stabilized full-length CREB3 expression. Copper ion also increased its N-terminal form and GAL4-CREB3-dependent luciferase activity, which was accompanied by the increase in the ubiquitinated proteins in Neuro2a cells. Taken together, CREB3 expression is regulated by multiple ER-Golgi resident factors in a post-translational manner, but its processing is not directly associated with ER stress and autophagic dysfunction. This finding is especially true for the unique action of the copper ion on CREB3 stabilization and processing in parallel to aberration of ubiquitin-proteasome system, which might provide new insights into understanding the mechanisms of intractable disorders.

Elucidating the rapid action of 2-(2-chlorophenyl)ethylbiguanide on HT-29 cells under a serum- and glucose-deprived condition.

We recently demonstrated the cytotoxic action of a novel phenformin derivative, 2-(2-chlorophenyl)ethylbiguanide (2-Cl-Phen), on HT-29 cells under a serum- and glucose-deprived condition. In that study, we showed that the ATF6 arm of the ER stress pathway and c-Myc expression were downregulated 12 h after the treatment with 2-Cl-Phen. Through characterization of intracellular events at the early phase of the 2-Cl-Phen treatment before noticeable morphological changes, we found rapid fluctuations in the c-Myc and ATF4 proteins but not in their mRNAs in 2-Cl-Phen-treated HT-29 cells under the serum- and glucose-deprived condition. The 2-Cl-Phen-mediated downregulation of ATF4 protein was not paralleled by the phosphorylation status of PERK and eIF2α. Reduction of c-Myc expression by 2-Cl-Phen was more profound than that of ATF4 expression, and phosphorylated c-Myc was downregulated within 2 h. Pharmacological studies on the expression of c-Myc and ATF4 proteins showed that this decrease was mediated through proteasomal degradation but not by autophagy. Interestingly, treatment with lithium chloride, which is a well-known inhibitor of GSK3ß, partially recovered the expression of ATF4 protein, but its effect on the level of total c-Myc protein was negligible. Treatment with 2-Cl-Phen increased the expression of phosphorylated AMPK, but Compound C, an AMPK inhibitor, did not influence the expression of c-Myc protein in HT-29 cells. Finally, we observed that 2-Cl-Phen partially attenuated the gene expression of integrin subunit α1 (ITGA1), a downstream target of c-Myc. Taken together, these results show that 2-Cl-Phen rapidly downregulated the expression of c-Myc in addition to ER stress responses in a post-translational manner. Further elucidation and improvement of this multi-target-directed compound will provide new insights for developing therapeutic strategies against cancer.

Application of NanoLuc to monitor the intrinsic promoter activity of GRP78 using the CRISPR/Cas9 system.

In this study, we applied a highly sensitive small luciferase, NanoLuc, to establish a knock-in cell line using the CRISPR/Cas9 system and characterized the endogenous promoter activity of the glucose-regulated protein 78 (GRP78) gene. The N-terminal region of the human GRP78 gene was fused to the NanoLuc gene and aligned with the puromycin-resistant gene through the 2A peptide sequence and used as a knock-in vector. The selected cells responded to both pharmacological and genetic ER stress and show NanoLuc-based CRISPR/Cas9 system is a very useful tool to isolate gene-edited cells and to characterize the endogenous promoter activity for genes of interest.

Acquired Exchange Protein Directly Activated by Cyclic Adenosine Monophosphate Activity Induced by p38 Mitogen-activated Protein Kinase in Primary Afferent Neurons Contributes to Sustaining Postincisional Nociception.

BACKGROUND: The molecular mechanisms responsible for sustained pain after tissue injury are largely unknown. The aim of this study was to clarify the role of exchange protein directly activated by cyclic adenosine monophosphate (EPAC) in sustained postincisional nociception, using tissue injury-induced nociceptor priming, and involvement of p38 mitogen-activated protein kinase (p38MAPK) in EPAC-mediated nociceptor priming. METHODS: Plantar incisions were made in the hind paws of Sprague-Dawley rats (n = 144). Nociceptor priming was confirmed by behavior testing followed by prostaglandin E2 injection 14 to 21 days after the incision. ESI-09, a selective EPAC inhibitor, was administered to assess its effects on nociceptor priming. Expression of two isoforms of EPAC (EPAC1/EPAC2) in dorsal root ganglions from naive rats and those 14 days after the incision was detected by immunohistochemistry and Western blotting. Separately, FR167653, a selective p38MAPK inhibitor, was administered to assess its effect on EPAC1/EPAC2 expression and the development of nociceptor priming. RESULTS: Prostaglandin E2 injection 14 to 21 days after the plantar incision induced persistent mechanical hyperalgesia for 7 days. EPAC1/EPAC2 expression in dorsal root ganglion neurons was trivial in naive rats (7.7 ± 4.8% for EPAC1; 6.3 ± 4.1% for EPAC2) but markedly increased 14 days after the incision (21.0 ± 9.4% and 20.1 ± 3.8%, respectively). ESI-09 treatment inhibited prostaglandin E2-induced persistent mechanical hypersensitivity but had no effect on incision-induced acute nociceptive hypersensitivity. Treatment with FR167653 before the incision inhibited the development of nociceptor priming and incision-induced EPAC1/EPAC2 expression (8.5 ± 5.4% and 7.6 ± 3.3%, respectively). CONCLUSIONS: Transient inflammatory stimulation causes long-lasting nociceptive hypersensitivity via nociceptor priming during the subacute period after incision. Acquired EPAC activity by p38MAPK in the dorsal root ganglion neurons is a key for this event.

Elucidation of a novel phenformin derivative on glucose-deprived stress responses in HT-29 cells.

Recently, we developed a variety of phenformin derivatives as selective antitumor agents. Based on previous findings, this study evaluated a promising compound, 2-(2-chlorophenyl)ethylbiguanide (2-Cl-Phen), on the basis of stress responses in the human colon cancer cell line HT-29 under a serum- and glucose-deprived condition. 2-Cl-Phen triggered morphological changes such as shrinkage and plasma membrane disintegration, as well as a decrease in mitochondrial activity and an increase in LDH leakage. To understand intracellular issues relating to 2-Cl-Phen, this study focused on the expression levels of ER stress-inducible genes and several oncogenic genes. Serum and glucose deprivation significantly induced a variety of ER stress-inducible genes, but a 12-h treatment of 2-Cl-Phen down-regulated expression of several ER stress-related genes, with the exception of GADD153. Interestingly, the expression levels of ATF6α, GRP78, MANF, and CRELD2 mRNA were almost completely decreased by 2-Cl-Phen. This study also observed that a 24-h treatment of 2-Cl-Phen attenuated the expression levels of GRP78, GADD153, and c-Myc protein. The decrease in c-Myc protein occurred before the fluctuation of GRP78 protein, while the expression of c-Myc mRNA showed little change with cotreatment of serum and glucose deprivation with 2-Cl-Phen. To further understand the 2-Cl-Phen-induced down-regulation of ATF6-related genes, this study investigated the stability of ATF6α and GRP78 proteins using NanoLuc-tagged constructs. The expression levels of NanoLuc-tagged ATF6α and GRP78 were significantly down-regulated by 2-Cl-Phen in the presence or absence of the translation inhibitor cycloheximide. Taken together, our novel phenformin derivative 2-Cl-Phen has the unique characteristic of diminishing tumor adaptive responses, especially the expression of ATF6-related genes, as well as that of c-Myc protein, in a transcriptional and posttranscriptional manner under a serum- and glucose-deprived condition. Further characterization of cytotoxic mechanisms related to phenformin derivatives may give new insights into developing additional promising anticancer agents.

SOD1 dimerization monitoring using a novel split NanoLuc, NanoBit.

In the present study, we applied a highly sensitive NanoLuc-based technology to understand the status of superoxide dismutase 1 (SOD1) within mammalian cells. Two fragments of NanoLuc (NanoBit), large N-terminal and small C-terminal regions, were fused with wild-type (wt) and mutant human SOD1 (hSOD1) genes and transfected into cells. Luciferase activity through NanoBit assembly was only detected in NanoBit-tagged wtSOD1-expressing cells. Furthermore, the developed NanoLuc system was used to investigate the role of protein-protein interactions in the pathogenesis of amyotrophic lateral sclerosis (ALS). In addition to SOD1, we also applied this NanoBit system for detecting the dimerization of wild-type, M337V-mutated human TAR-binding protein 43 kDa (hTDP43) and its cleaved C-terminal fragment (TDP25M337V ) as well as their interactions with SOD1. Luciferase activities of NanoBit-tagged mutant SOD1, TDP43, or TDP25 were negligible. Finally, we found that a zinc chelator partially reduced the luciferase activity of NanoBit-wtSOD1. Collectively, these results show that the present assay is sensitive and convenient to appreciate ALS and to develop useful agents for the modulation of SOD1 conformation.