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Renal fibrosis is controlled by profibrotic and antifibrotic forces. Exploring anti-fibrosis factors and mechanisms is an attractive strategy to prevent organ failure. Here we identified the JNK-associated leucine zipper protein (JLP) as a potential endogenous antifibrotic factor. JLP, predominantly expressed in renal tubular epithelial cells (TECs) in normal human or mouse kidneys, was downregulated in fibrotic kidneys. Jlp deficiency resulted in more severe renal fibrosis in unilateral ureteral obstruction (UUO) mice, while renal fibrosis resistance was observed in TECs-specific transgenic Jlp mice. JLP executes its protective role in renal fibrosis via negatively regulating TGF-β1 expression and autophagy, and the profibrotic effects of ECM production, epithelial-to-mesenchymal transition (EMT), apoptosis and cell cycle arrest in TECs. We further found that TGF-β1 and FGF-2 could negatively regulate the expression of JLP. Our study suggests that JLP plays a central role in renal fibrosis via its negative crosstalk with the profibrotic factor, TGF-β1., Qi Yan et al. find that JNK-associated leucine zipper protein (Jlp) counteracts the profibrotic effects of TGF-β1 and autophagy on renal tubular epithelial cells and that TGF-β1 and FGF-2 can negatively regulate the expression of Jlp. These findings provide insights into the role of Jlp in kidney fibrosis.

Peptidylarginine deiminase 4 (PAD4), encoded by PADI4, plays critical roles in the immune system; however, its contribution to the pathogenesis of lupus nephritis remains controversial. The pathological roles of PAD4 were investigated in lupus model mice. An imiquimod (IMQ)-induced lupus model was analyzed in wild-type (WT) and Padi4-knockout (KO) mice. Proteinuria, serum anti-double stranded DNA (anti-dsDNA) antibody, and renal infiltrated cells were evaluated. Neutrophil migration and adhesion were assessed using adoptive transfer and adhesion assay. PAD4-regulated pathways were identified by RNA-sequencing of Padi4 KO neutrophils. Padi4 KO mice exhibited significant improvements in proteinuria progression compared with WT mice, whereas, serum anti-dsDNA antibody and immune complex deposition in the glomeruli showed no difference between both mice strains. Padi4 KO mice showed decreased neutrophil infiltration in the kidneys. Adoptively transferred Padi4 KO neutrophils showed decreased migration to the kidneys of IMQ-treated WT mice, and adhesion to ICAM-1 was impaired in Padi4 KO neutrophils. Padi4 KO neutrophils exhibited reduced upregulation of p38 mitogen-activated protein kinase (MAPK) pathways. Toll-like receptor 7 (TLR7)-primed Padi4 KO neutrophils demonstrated reduced phosphorylation of p38 MAPK and lower expression of JNK-associated leucine zipper protein (JLP), a p38 MAPK scaffold protein. Neutrophils from heterozygous Jlp KO mice showed impaired adhesion to ICAM-1 and decreased migration to the kidneys of IMQ-treated WT mice. These results indicated a pivotal role of PAD4-p38 MAPK pathway in renal neutrophil infiltration in TLR7 agonist-induced lupus nephritis, and the importance of neutrophil-mediated kidney inflammation. Inhibition of the PAD4-p38 MAPK pathway may help in formulating a novel therapeutic strategy against lupus nephritis.

Osteopontin (OPN) is involved in cell proliferation, migration, inflammation, and tumor progression in various tissues. OPN induces stemness by interacting with CD44, but the functional relevance of OPN-mediated interferon (IFN) signaling and hepatitis C virus (HCV) replication in stem cell populations remains unclear. In this study, we investigated the effect of OPN on HCV replication and IFN signaling in cancer stem cells (CSCs) positive for epithelial cell adhesion molecule (EpCAM) and CD44. We show that the EpCAM+/CD44+ CSCs show marked HCV replication when compared to EpCAM−/CD44− cells. In addition, OPN significantly enhances this HCV replication in EpCAM+/CD44+ CSCs and markedly suppresses IFN-stimulated gene expression. The GSK-3β inhibitor BIO increases the EpCAM+/CD44+ CSC population and OPN expression and impairs IFN signaling via STAT1 degradation. Taken together, our data suggest that OPN enhances HCV replication in the EpCAM+/CD44+ CSCs, while it also negatively regulates the IFN signaling pathway via inhibition of STAT1 phosphorylation and degradation. Therefore, OPN may represent a novel therapeutic target for treating HCV-related hepatocellular carcinoma.

Human papillomavirus (HPV) is the most common sexually transmitted agent worldwide and is etiologically linked to several cancers, including cervical and genital cancers. NKG2D, an activating receptor expressed by NK cells, plays an important role in cancer immune-surveillance. We analyzed the impact of a NKG2D gene variant, rs1049174, on the incidence of HPV-related cancers in Vietnamese patients and utilized various molecular approaches to elucidate the mechanisms of NKG2D receptor regulation by rs1049174. In a group of 123 patients with HPV+ anogenital cancers, the low cytotoxicity allele LNK was significantly associated with increased cancer susceptibility (p = 0.016). Similar results were also observed in a group of 153 women with cervical cancer (p = 0.05). In functional studies, NK cells from individuals with LNK genotype showed a lower NKG2D expression and displayed less efficient NKG2D-mediated functions than NK cells with HNK genotype. Notably, the rs1049174 variant occurs within a targeting site for miR-1245, a negative regulator of NKG2D expression. Compared with the higher cytotoxicity allele HNK, the LNK allele was more efficiently targeted by miR-1245 and thus determined lower NKG2D expression in NK cells with the LNK genotype. The NKG2D variants may influence cancer immunosurveillance and thus determine susceptibility to various malignancies, including HPV-induced cancers.

金沢大学医薬保健研究域医学系, In a previous study, we reported that Alzheimer's disease-associated presenilin-2 interacts with a LIM-domain protein, namely, DRAL/FHL2/SLIM3. In this study, we investigated whether DRAL modifies the metabolism of the amyloid precursor protein (APP). We used small interfering RNA (siRNA) to knockdown DRAL in COS7 and HEK293 cells that stably overexpress APP695. We found that the knockdown was accompanied by a decrease in the amount of secreted α-secretase-cleaved APP and the membrane-bound C-terminal fragment C83 and an increase in the amount of secreted β-amyloid peptide (Aβ) from the cells. We also found that in addition to a disintegrin and metalloprotease (ADAM)-17, DRAL binds to ADAM-10. Thus, DRAL may be involved in the processing of APP through the α-secretase pathway. © 2008 Elsevier Ireland Ltd. All rights reserved.

金沢大学がん研究所がん分子細胞制御, The mitogen-activated protein kinase (MAPK) signaling pathway, which is conserved from yeast to humans, is activated in response to a variety of extra- and intracellular stimuli, and plays key roles in multiple cellular processes, including proliferation, differentiation, and apoptosis. The MAPK pathway transmits its signal through the sequential phosphorylation of MAPK kinase kinase to MAPK kinase to MAPK. Specific and efficient activation of the MAPK cascades is crucial for proper cellular responses to stimuli. As shown in yeast, the mammalian MAPK signaling system may also employ scaffold proteins, in part, to organize the MAPK signaling components into functional MAPK modules, thereby enabling the efficient activation of specific MAPK pathways. This review article describes recent advances in the study of potential mammalian scaffold proteins that may help us understand the complex regulation, including the spatial and temporal control, of the mammalian MAPK signaling pathways.

金沢大学がん研究所がん分子細胞制御, FK506 is a powerful immunosuppressive drug currently in use that inhibits the activation of several transcription factors (nuclear factor (NF)-AT and NF-κB) critical for T cell activation. We show here that, contrary to the situation in T cells, FK506 activates transcription factor NF-κB in non-lymphoid cells such as fibroblasts and renal mesangial cells. We further show that FK506 induces NF-κB-regulated IL-6 production in vitro and in vivo, in particular in kidney. IL-6 has been shown previously to produce renal abnormalities in vivo, such as mesangioproliferative glomerulonephritis. Similar renal abnormalities were also observed in FK506-treated animals. These results thus suggest a causal relationship between FK506-induced NF-κB activation/IL-6 production and some of FK506-induced renal abnormalities.

金沢大学がん研究所がん分子細胞制御, The wild type Copia Gag precursor protein of Drosophila melanogaster expressed in Escherichia coli was shown to be processed autocatalytically to generate two daughter proteins with molecular masses of 33 and 23 kDa on SDS/PAGE. The active-site motif of aspartic proteinases, Asp-Ser-Gly, was present in the 23 kDa protein corresponding to the C-terminal half of the precursor protein. The coding region of this daughter protein (152 residues) in the copia gag gene was expressed in E. coli to produce the recombinant enzyme protein as inclusion bodies, which was then purified and refolded to create the active enzyme. Using the peptide substrate His-Gly-Ile-Ala-Phe-Met-Val-Lys-Glu-Val-Asn (cleavage site: Phe–Met) designed on the basis of the sequence of the cleavage-site region of the precursor protein, the enzymatic properties of the proteinase were investigated. The optimum pH and temperature of the proteinase toward the synthetic peptide were 4.0 and 70 °C respectively. The proteolytic activity was increased with increasing NaCl concentration in the reaction mixture, the optimum concentration being 2 M. Pepstatin A strongly inhibited the enzyme, with a Ki value of 15 nM at pH 4.0. On the other hand, the active-site residue mutant, in which the putative catalytic aspartic acid residue was mutated to an alanine residue, had no activity. These results show that the Copia proteinase belongs to the family of aspartic proteinases including HIV proteinase. The B-chain of oxidized bovine insulin was hydrolysed at the Leu15-–Tyr16 bond fairly selectively. Thus the recombinant Copia proteinase partially resembles HIV proteinase, but is significantly different from it in certain aspects.

Compared with the knowledge of maternal care, much less is known about the factors required for paternal parental care. Here we report that new sires of laboratory mice, though not spontaneously parental, can be induced to show maternal-like parental care (pup retrieval) using signals from dams separated from their pups. During this interaction, the maternal mates emit 38-kHz ultrasonic vocalizations to their male partners, which are equivalent to vocalizations that occur following pheromone stimulation. Without these signals or in the absence of maternal mates, the sires do not retrieve their pups within 5 min. These results show that, in mice, the maternal parent communicates to the paternal parent to encourage pup care. This new paradigm may be useful in the analysis of the parental brain during paternal care induced by interactive communication., Parental responsibilities in mice are usually carried out by the mother of the pups. In this study, the authors show that when mothers are separated from their mouse pups, they emit ultrasonic vocalizations to their male partners, who respond by administering paternal care to the pups.

Northern blot and nucleotide sequence analyses of copia RNA from a transfectant made by introducing a genomic copia into copia-free cells showed that the 2 kb RNA, one of the major transcripts of copia, is generated through splicing. Using the polymerase chain reaction (PCR), we have also found that the position of the splice sites used in Drosophila larvae and cultured cells originally containing copia is the same as that used in the transfectant. To investigate the function of the 2 kb RNA, we constructed mutant copias which harboured a single point mutation at the splice site or approximately 3 kb deletion of the internal region corresponding to the spliced out sequence. Analyses of transfectants made by introducing these mutant copias into copia-free cells demonstrated that the spliced 2 kb RNA contains sufficient information to make copia virus-like particles (VLPs). Furthermore, when copia RNA corresponding to the spliced RNA was translated in vitro, the major VLP protein was found to be released autocatalytically from its own precursor. A single amino acid substitution at the putative protease active site in the precursor prevented the processing, and resulted in accumulation of the mutant precursor in vitro. From these results, we conclude that copia VLPs are produced through autocatalytic processing of the precursor polyprotein encoded by the spliced copia RNA.

Drosophila copia protease is likely to be encoded in the gag gene. We have expressed copia gag polyprotein precursor in E. coli. The gag precursor was correctly processed to generate a unique laminate structure in E. coli. The processing was almost completely blocked by a mutation at the putative active site of copia protease, and resulted in accumulation of the precursor. Furthermore, the laminate structure was not found in E. coli expressing the mutant precursor. These results indicate that the protease is involved in cleaving the gag precursor itself. Also, the assembly of copia gag protein should correlate to the autoprocessing of copia gag polyprotein precursor.