Calcineurin Binding Protein Research Articles

Cabin1 involves in renal tubular epithelial cells mitochondrial dysfunction through SIRT1/p53 pathway

Background: Angiotensin II (AngII) induced Calcineurin binding protein 1 (Cabin1) protein expression significantly increased during Renal tubular epithelial cells (RTEC) injury. However, the detailed function of Cabin1 protein in RTEC was not characterized well. In this study, we aimed to explore the downstream target of Cabin1 in vitro model.Methods: Rat kidney epithelial cells were cultured and stimulated with AngII. Electron microscopy was performed to observe mitochondrial morphology change. Immunofluorescence staining was detected to observe the distribution of cytoskeleton and Cabin1. Mitochondrial morphology change and protein expression were detected by electrical microscopy and western blot.Results: AngII induced the disruption of cytoskeleton at 24 and 48 h. Western blot analysis showed AngII significantly induced the overexpression of Cabin1. AngII induced a great deal of small, long and irregular mitochondria in RTEC, aspect ratio which reflects the length-to-width ratio of mitochondria remarkably increased at 12 and 24 h. Knocking down Cabin1 aggravated mitochondrial morphological abnormality in AngII treated RTEC. In comparison with control, Cabin1, p53 and cyto C level were significantly increased in AngII treated cells, while SIRT1 level was obviously decreased. Knocked down Cabin1 plus AngII stimulated, SIRT1 was further decreased, while p53 and cyto C were significantly increased.Conclusions: Cabin1 involves in RTEC mitochondrial dysfunction through SIRT1/p53 pathway. Cabin1 may be used as a new marker for the mechanisms of RTEC injury.

Altering of host larval (Spodoptera exigua) calcineurin activity in response to ascovirus infection.

Calcineurin (CaN) is involved in numerous cellular processes and Ca2+ -dependent signal transduction pathways. According to our previous transcriptome studies, thousands of host larval (Spodoptera exigua) transcripts were downregulated after the infection of Heliothis virescent ascovirus 3h (HvAV-3h), while the Spodoptera exigua calcineurin genes (SeCaNs) were significantly upregulated. To understand the regulation of SeCaNs in S. exigua larvae during the infection of HvAV-3h, the functions of CaN subunit A (SeCaN-SubA) and CaN binding protein (SeCaN-BP) were analysed. The in vitro assays indicated that the bacterial expressed SeCaN-SubA is an acid phosphatase, but no phosphatase activity was detected with the purified SeCaN-BP. The transcription level of SeCaN-SubA was upregulated after HvAV-3h infection and the CaN activity was significantly increased after HvAV-3h infection in S. exigua larvae. Interestingly, the SeCaN-BP transcripts were only detectable in the HvAV-3h infected larvae. Further immunoblotting results consistently agree with those obtained by qPCR, indicating that the infection of HvAV-3h causes the upregulated expression of SeCaN-SubA and the appearance of SeCaN-BP. An interaction between the cleaved SeCaN-SubA and SeCaN-BP was detected by co-immunoprecipitation assays, and the expression of SeCaN-BP in Spodoptera frugiperda-9 (Sf9) cells can help to increase the CaN activity of SeCaN-SubA. Further investigations with CaN inhibitors suggested that HvAV-3h. Further investigations with CaN inhibitors suggested that the inhibition on host larval CaN activity can also inhibit the viral replication of HvAV-3h. The increase in CaN activity caused by HvAV-3h infection might be due to the upregulation of SeCaN-SubA and the induced expression of SeCaN-BP, and increased CaN activity is essential for ascoviral replication. © 2019 Society of Chemical Industry.

Dissecting the Roles of the Calcineurin Pathway in Unisexual Reproduction, Stress Responses, and Virulence in Cryptococcus deneoformans.

The Ca2+/calmodulin-dependent protein phosphatase calcineurin orchestrates sexual reproduction, stress responses, and virulence via branched downstream pathways in the opportunistic human fungal pathogen Cryptococcus neoformans The calcineurin-binding protein Cbp1, the calcineurin temperature suppressor Cts1, the calcineurin-responsive zinc finger transcription factor Crz1, and the calcineurin targets Pbp1, Tif3, and Puf4, all function downstream of calcineurin to orchestrate distinct cellular processes. To elucidate how the calcineurin pathway regulatory network governs unisexual reproduction, stress responses, and virulence, we have analyzed the self-filamentous C. deneoformans strain, XL280α, and generated double mutants of these calcineurin downstream genes. We demonstrated that calcineurin governs unisexual reproduction at different sexual developmental stages, in which the initiation of the yeast-hyphal morphological transition is independent of Crz1, whereas the sporulation process is dependent on Crz1. Calcineurin-dependent unisexual reproduction is independent of the pheromone response pathway. Crz1 synergistically interacts with different calcineurin downstream targets in responding to ER, high-calcium, and cell wall stresses. We observed a widespread synergy suggesting that these proteins function in complex branched pathways downstream of calcineurin with some functional redundancy, which may allow efficient signaling network rewiring within the pathway for prompt adaptation to changing environments. Finally, we showed that deletion of PBP1 or TIF3 in the cna1∆ mutant background conferred a modest level of growth tolerance at 37°, but that the cna1∆ pbp1∆ and cna1∆ tif3∆ double mutants were both avirulent, suggesting that calcineurin may control virulence via mechanisms beyond thermotolerance.

Knocking down Cabin1 induces glomerular podocyte injury.

Podocyte damage exerts a key role in proteinuria. We have demonstrated that calcineurin-binding protein 1 (Cabin1) upregulated during podocyte injury, yet its function in podocyte is still unclear. We established 5/6 nephrectomized rats and angiotensin II (AngII)-injured podocyte, as well as knocked down Cabin1 with siRNA in cultured podocytes. Rats were killed at 4 or 8weeks after 5/6 nephrectomy. The localization of podocyte cytoskeleton was detected after immunofluorescence staining. Podocyte mitochondrial morphology was observed under electron microscopy. Podocyte mitochondrial transmembrane potential (MMP) was measured with MitoCapture kit. Cabin1 and cytochrome c protein expression were detected by western blot. Massive proteinuria, as well as obvious segmental glomerular sclerosis, was found in rats at 8weeks after nephrectomy, accompanied with the disruption of synaptopodin. Moreover, mitochondria changed from large and ellipsoid shape to the small, long, and irregular shape in rats at 4weeks after operation. At 8weeks, mitochondria were swollen and cristae were remarkably dissolved. Compared to sham-operated rats, Cabin1 protein expression was obviously upregulated in rats at 8weeks. AngII induced the decrease in MMP, as well as the overexpression of Cabin1 and cytochrome c protein in podocytes. Knocking down Cabin1 induced the disruption of F-actin and overexpression of cytochrome c (1.81±0.21 in siRNA group vs. 0.86±0.11 in negative control group). Knocking down Cabin1 induces the disruption of cytoskeleton and mitochondrial dysfunction in podocyte. Cabin1 could be a crucial factor in podocyte damage.

Downregulation of miRNA-15a and miRNA-16 promote tumor proliferation in multiple myeloma by increasing CABIN1 expression.

Multiple myeloma (MM) is a malignant disorder characterized by the neoplastic growth of plasma cells in the bone marrow. MicroRNAs (miRNAs/miRs) modulate key regulatory cell pathways via their influence on target genes, and may serve a crucial function in tumorigenesis. Previous studies have indicated that the downregulation of miR-15a and miR-16 contributes to MM pathogenesis. However, the functional mechanisms of miR-15a and miR-16 in MM remain unclear. In the present study, potential target sites for miR-15a and miR-16 were identified on the calcineurin-binding protein 1 (CABIN1) mRNA sequence from analyses of previously published crosslinking, ligation and sequencing of hybrids data. Again-of-function study was also performed, which determined that miR-15a/16 directly targeted CABIN1 mRNA and negatively regulated the expression of CABIN1 at the mRNA and protein level in MM cells. A cell proliferation assay demonstrated that the upregulation of miR-15a and miR-16 inhibited the proliferation of MM cells via targeting CABIN1. miR-15a and miR-16 were significantly decreased in MM specimens, compared with in normal specimens, whereas CABIN1 mRNA levels were significantly higher in MM samples compared with in normal samples. CABIN1 mRNA levels were negatively correlated with miR-15a and miR-16 expression levels in MM tissues, as determined using Pearson's correlation coefficient analysis. The results of the present study indicate that the downregulation of miR-15a and miR-16 promotes tumor proliferation in MM by increasing CABIN1 expression. The present study may aid elucidation of the functions of miR-15a and miR-16 and their function in MM carcinogenesis.

The novel cardiac z-disc protein CEFIP regulates cardiomyocyte hypertrophy by modulating calcineurin signaling

The z-disc is a structural component at the lateral borders of the sarcomere and is important for mechanical stability and contractility of both cardiac and skeletal muscles. Of note, the sarcomeric z-disc also represents a nodal point in cardiomyocyte function and signaling. Mutations of numerous z-disc proteins are associated with cardiomyopathies and muscle diseases. To identify additional z-disc proteins that might contribute to cardiac disease, we employed an in silico screen for cardiac-enriched cDNAs. This screen yielded a previously uncharacterized protein named cardiac-enriched FHL2-interacting protein (CEFIP), which exhibited a heart- and skeletal muscle-specific expression profile. Importantly, CEFIP was located at the z-disc and was up-regulated in several models of cardiomyopathy. We also found that CEFIP overexpression induced the fetal gene program and cardiomyocyte hypertrophy. Yeast two-hybrid screens revealed that CEFIP interacts with the calcineurin-binding protein four and a half LIM domains 2 (FHL2). Because FHL2 binds calcineurin, a phosphatase controlling hypertrophic signaling, we examined the effects of CEFIP on the calcineurin/nuclear factor of activated T-cell (NFAT) pathway. These experiments revealed that CEFIP overexpression further enhances calcineurin-dependent hypertrophic signal transduction, and its knockdown repressed hypertrophy and calcineurin/NFAT activity. In summary, we report on a previously uncharacterized protein CEFIP that modulates calcineurin/NFAT signaling in cardiomyocytes, a finding with possible implications for the pathogenesis of cardiomyopathy.

O-linked N-acetylglucosamine transferase (OGT) interacts with the histone chaperone HIRA complex and regulates nucleosome assembly and cellular senescence

The histone chaperone HIRA complex, consisting of histone cell cycle regulator (HIRA), Ubinuclein1 (UBN1), and calcineurin binding protein 1 (CABIN1), deposits histone variant H3.3 to genic regions and regulates gene expression in various cellular processes, including cellular senescence. How HIRA-mediated nucleosome assembly of H3.3-H4 is regulated remains not well understood. Here, we show that O-linked N-acetylglucosamine (GlcNAc) transferase (OGT), an enzyme that catalyzes O-GlcNAcylation of serine or threonine residues, interacts with UBN1, modifies HIRA, and promotes nucleosome assembly of H3.3. Depletion of OGT or expression of the HIRA S231A O-GlcNAcylation-deficient mutant compromises formation of the HIRA-H3.3 complex and H3.3 nucleosome assembly. Importantly, OGT depletion or expression of the HIRA S231A mutant delays premature cellular senescence in primary human fibroblasts, whereas overexpression of OGT accelerates senescence. Taken together, these results support a model in which OGT modifies HIRA to regulate HIRA-H3.3 complex formation and H3.3 nucleosome assembly and reveal the mechanism by which OGT functions in cellular senescence.

Tacrolimus restores podocyte injury and stabilizes the expression of Cabin1 in 5/6 nephrectomized rats

Podocyte injury is a vital factor, which induces massive proteinuria. Studies have shown that tacrolimus (TAC) protected podocyte via stabilizing cytoskeleton. Our latest study indicates that calcineurin binding protein 1 (Cabin1) undergoes nuclear translocation during podocytes injury. Whether TAC targets on Cabin1 during podocyte injury is still not clear. This study establishes non-immunological proteinuric model. To observe the effect of the treatment of TAC on Cabin1 expression in 5/6 nephrectomized rats. Sprague–Dawley rats were injected with TAC (0.2 mg/kg/day) for 4–8 weeks after 5/6 nephrectomy. Then, rats were sacrificed in the eighth week after operation, renal tissues were processed for morphological studies under light and electrical microscope. Cabin1 expression and distribution were detected by western blot and indirect immunofluorescence staining. In 5/6 nephrectomized rats, urinary protein excretion reached 90.2 ± 30.1 mg/24 h, glomerular sclerosis index and tubulointerstitial fibrosis score were significantly increased, and widespread of podocyte foot processes fusion was found. Moreover, Cabin1 protein expression was markedly increased, and its distribution became much more obviously in podocytes nuclei. In TAC treated rats, urinary protein excretion significantly decreased (44.9 ± 22.5 mg/24 h), glomerular sclerosis and tubulointerstitial fibrosis were alleviated, and podocyte foot processes fusion was inhibited. Furthermore, TAC alleviated the increased protein expression and abnormal distribution of Cabin1. In conclusion, TAC restores podocyte injury and stabilizes the expression of Cabin1. Cabin1 may become a new target to demonstrate the mechanism of TAC in podocyte injury.

Effect of the knockdown of Cabin1 on p53 in glomerular podocyte

Calcineurin binding protein 1 (Cabin1) is a natural inhibitor of calcineurin (CN). Moreover, Cabin1 retards tumor cell apoptosis by regulating p53. This study was designed to observe the expression of Cabin1 during podocyte injury, as well as its relationship with p53. Sprague-Dawley rats were used for the establishment of 5/6 nephrectomized rat model. Sham-operated rats underwent ventral laparotomy without nephrectomy. Then, rats were sacrificed at 8 and 12 weeks after nephrectomy. WT-1, a podocyte nuclear protein, was used for indicating the localization of Cabin1 in glomeruli. As tacrolimus protects podocyte via inhibiting AngiotensinII (AngII) induced CN activation. Cultured podocytes were injured by AngII or restored by tacrolimus. The protein expression and localization was detected by western blot or immunofluorescence staining. Cabin1 was knocked down by siRNA in cultured podocytes. In 5/6 nephrectomized rats, the colocalization of Cabin1 and WT-1 became more obviously in podocyte nuclei. Cabin1 protein was markedly increased in rats at 8 and 12 weeks after nephrectomy, as well as in AngII injured podocytes at 48 h (0.99 ± 0.12 in AngII group versus 0.80 ± 0.16 in control group). Cabin1 and p53 colocalized in cultured podocyte nuclei, p53 expression was significantly decreased (0.21 ± 0.05 in siRNA group versus 0.31 ± 0.05 in negative control group) after Cabin1 was being knocked down. In conclusion, Cabin1 expression significantly increases during podocyte injury. Knockdown of Cabin1 induces p53 expression decrease in cultured podocyte. Cabin1 may provide a new target to investigate podocyte injury.

Cabin1 localizes in glomerular podocyte and undergoes nuclear translocation during podocyte injury

Context: Podocyte injury is related to increasing proteinuria and contributes to the progression of kidney disease. Calcineurin binding protein 1 (Cabin1) is a repressor of myocyte enhancer factor 2 (MEF2) and calcineurin-mediated transcription in the immune system. Moreover, Cabin1 interacts with p53 and negatively regulates p53 in tumor cells. However, its function in kidney is unknown. Objective: To explore the exact localization of Cabin1 in glomeruli, as well as the relationship between Cabin1 and podocyte injury. Methods: Sprague–Dawley rats were sacrificed to observe the localization and protein expression of Cabin1 in the kidney. Cabin1 localization and protein expression were detected by immunofluorescence staining and western blot, respectively. Mouse podocytes were cultivated at 33 °C to propagate, then cells were transferred to an incubator at 37 °C to allow differentiation. Differentiated podocytes were stimulated by angiotensin II (AngII) or AngII plus tacrolimus. Cells were harvested to detect the localization and protein expression of Cabin1. Cytoplasmic and nuclear protein were separated by protein extraction kit. Results: Cabin1 mainly localized in the nuclei of glomerular innate cells, it colocalized with WT-1 in podocytes nuclei. Western bolt showed Cabin1 protein remarkably expressed in renal cortex. AngII-induced Cabin1 nuclear protein significantly increased, accompanied by cytoskeleton disruption in cultured mouse podocytes. Conclusion: Cabin1 localizes in glomerular podocytes. AngII induces nuclear translocation of Cabin1 in cultured podocytes.

Identification and mutational analyses of phosphorylation sites of the calcineurin-binding protein CbpA and the identification of domains required for calcineurin binding in Aspergillus fumigatus

Calcineurin is a key protein phosphatase required for hyphal growth and virulence in Aspergillus fumigatus, making it an attractive antifungal target. However, currently available calcineurin inhibitors, FK506 and cyclosporine A, are immunosuppressive, limiting usage in the treatment of patients with invasive aspergillosis. Therefore, the identification of endogenous inhibitors of calcineurin belonging to the calcipressin family is an important parallel strategy. We previously identified the gene cbpA as the A. fumigatus calcipressin member and showed its involvement in hyphal growth and calcium homeostasis. However, the mechanism of its activation/inhibition through phosphorylation and its interaction with calcineurin remains unknown. Here we show that A. fumigatus CbpA is phosphorylated at three distinct domains, including the conserved SP repeat motif (phosphorylated domain-I; PD-I), a filamentous fungal-specific domain (PD-II), and the C-terminal CIC motif (Calcipressin Inhibitor of Calcineurin; PD-III). While mutation of three phosphorylated residues (Ser208, Ser217, Ser223) in the PD-II did not affect CbpA function in vivo, mutation of the two phosphorylated serines (Ser156, Ser160) in the SP repeat motif caused reduced hyphal growth and sensitivity to oxidative stress. Mutational analysis in the key domains in calcineurin A (CnaA) and proteomic interaction studies confirmed the requirement of PxIxIT motif-binding residues (352-NIR-354) and the calcineurin B (CnaB)-binding helix residue (V371) for the binding of CbpA to CnaA. Additionally, while the calmodulin-binding residues (442-RVF-444) did not affect CbpA binding to CnaA, three mutations (T359P, H361L, and L365S) clustered between the CnaA catalytic and the CnaB-binding helix were also required for CbpA binding. This is the first study to analyze the phosphorylation status of calcipressin in filamentous fungi and identify the domains required for binding to calcineurin.

The histone chaperone complex HIR maintains nucleosome occupancy and counterbalances impaired histone deposition in CAF-1 complex mutants.

Chromatin organization is essential for coordinated gene expression, genome stability, and inheritance of epigenetic information. The main components involved in chromatin assembly are specific complexes such as Chromatin Assembly Factor 1 (CAF-1) and Histone Regulator (HIR), which deposit histones in a DNA synthesis-dependent or -independent manner, respectively. Here, we characterize the role of the plant orthologs Histone Regulator A (HIRA), Ubinuclein (UBN) and Calcineurin Binding protein 1 (CABIN1), which constitute the HIR complex. Arabidopsis loss-of-function mutants for the various subunits of the complex are viable, but hira mutants show reduced fertility. We show that loss of HIRA reduces extractable histone H3 protein levels and decreases nucleosome occupancy at both actively transcribed genes and heterochromatic regions. Concomitantly, HIRA contributes to maintenance of silencing of pericentromeric repeats and certain transposons. A genetic analysis based on crosses between mutants deficient in subunits of the CAF-1 and HIR complexes showed that simultaneous loss of both the CAF-1 and HIR histone H3 chaperone complexes severely affects plant survival, growth and reproductive development. Our results suggest that HIRA partially rescues impaired histone deposition in fas mutants to preserve nucleosome occupancy, implying plasticity in histone variant interaction and deposition.

EBV‐Encoded LMP‐1 Sensitizes Nasopharyngeal Carcinoma Cells to Genotoxic Drugs by Down‐Regulating Cabin1 Expression

The oncogenic latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is involved in the pathogenesis of human nasopharyngeal carcinoma (NPC) and lymphoma. We and other authors have shown earlier that LMP1 induces apoptosis and inhibits xenograft tumor growth in mice, but the mechanism underlying these processes has not been investigated so far. In the present study, we show that knockdown of LMP1 renders the EBV-positive NPC cell line CG-1 resistant to various genotoxic drugs (cisplatin, etoposide, and adriamycin). LMP1 inhibits the expression of Cabin1, a Ca(2+) regulated protein shown earlier to inhibit calcineurin. Knockdown of calcineurin binding protein (Cabin1) with small hairpin RNA sensitizes CG-1 cells to genotoxic drugs. In contrast, LMP1 overexpression reduces Cabin1 level and renders both CG-1 cells and EBV-negative NPC cell lines sensitive to cisplatin. The c-Jun-N-terminal kinase (JNK) and ERK pathways are required for LMP1-induced suppression of Cabin1 at the transcriptional level. Chromatin immunoprecipitation assays further confirm that the JNK-activated transcription factor AP-1 mediates the LMP1-induced down-regulation of Cabin1 gene expression. LMP1 knockdown also increases the resistance of xenograph tumors to cisplatin in mice, therefore confirming the relevance of our findings in vivo. This study reveals the molecular mechanism underlying the pro-apoptotic activity of LMP1 during cisplatin-based NPC chemotherapy.

Chronic Unpredictable Stress (CUS)-Induced Anxiety and Related Mood Disorders in a Zebrafish Model: Altered Brain Proteome Profile Implicates Mitochondrial Dysfunction

Anxiety and depression are major chronic mood disorders, and the etiopathology for each appears to be repeated exposure to diverse unpredictable stress factors. Most of the studies on anxiety and related mood disorders are performed in rodents, and a good model is chronic unpredictable stress (CUS). In this study, we have attempted to understand the molecular basis of the neuroglial and behavioral changes underlying CUS-induced mood disorders in the simplest vertebrate model, the zebrafish, Danio rerio. Zebrafish were subjected to a CUS paradigm in which two different stressors were used daily for 15 days, and thorough behavioral analyses were performed to assess anxiety and related mood disorder phenotypes using the novel tank test, shoal cohesion and scototaxis. Fifteen days of exposure to chronic stressors appears to induce an anxiety and related mood disorder phenotype. Decreased neurogenesis, another hallmark of anxiety and related disorders in rodents, was also observed in this zebrafish model. The common molecular markers of rodent anxiety and related disorders, corticotropin-releasing factor (CRF), calcineurin (ppp3r1a) and phospho cyclic AMP response element binding protein (pCREB), were also replicated in the fish model. Finally, using 2DE FTMS/ITMSMS proteomics analyses, 18 proteins were found to be deregulated in zebrafish anxiety and related disorders. The most affected process was mitochondrial function, 4 of the 18 differentially regulated proteins were mitochondrial proteins: PHB2, SLC25A5, VDAC3 and IDH2, as reported in rodent and clinical samples. Thus, the zebrafish CUS model and proteomics can facilitate not only uncovering new molecular targets of anxiety and related mood disorders but also the routine screening of compounds for drug development.

Characterization of the Pearl Oyster (Pinctada martensii) Mantle Transcriptome Unravels Biomineralization Genes

Pearl oyster, Pinctada martensii, is a marine bivalve species widely distributed in tropic and subtropic marine coasts. Mantle is the special tissue of P. martensii that secretes biomineralization proteins inducing shell deposition as well as iridescent nacre both in the inner shell and artificial nucleus. The pearl oyster is very efficient for artificial pearl production and is therefore an ideal organism for studies into the processes of biomineralization. However, deficiency of transcriptome information limits the insight into biomineralization mechanisms and pearl formation. In this study, we sequenced and characterized the P. martensii mantle transcriptome using 454 pyrosequencing. A total of 25,723 unique transcripts were assembled from 220,824 quality reads, followed by annotation and Gene Ontology classification analysis. A total of 146 unique transcript segments homologous to 49 reference biomineralization genes were identified, including calcineurin-binding protein, amorphous calcium carbonate binding protein 1, calmodulin, calponin-like protein, carbonic anhydrase 1, glycine-rich shell matrix protein, lysine-rich matrix protein, mantle gene or protein, nacrein, pearlin, PIF, regucalcin, and shematrin. The sequence data enabled the identification of 10,285 potential single nucleotide polymorphism loci and 7,836 putative indels, providing a resource for molecular biomarker, population genetics, and functional genomic studies. A large number of candidate genes for biomineralization were identified, considerably enriching resources for the study of shell formation. These sequence data will notably advance biomineralization and transcriptome study in pearl oyster and other Pinctada species.

Regulation of inflammatory responses and fibroblast‐like synoviocyte apoptosis by calcineurin‐binding protein 1 in mice with collagen‐induced arthritis

Calcineurin-binding protein 1 (CABIN-1) regulates calcineurin phosphatase activity as well as the activation, apoptosis, and inflammatory responses of fibroblast-like synoviocytes (FLS), which actively participate in the chronic inflammatory responses in rheumatoid arthritis (RA). However, the mechanism of action of CABIN-1 in FLS apoptosis is not clear. This study was undertaken to define the regulatory role of CABIN-1 in FLS from mice with collagen-induced arthritis (CIA). Transgenic mice overexpressing human CABIN-1 in joint tissue under the control of a type II collagen promoter were generated. Expression of human CABIN-1 (hCABIN-1) in joints and FLS was determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. The expression of cytokines, matrix metalloproteinases (MMPs), and apoptosis-related genes in FLS was determined by enzyme-linked immunosorbent assay, gelatin zymography, and RT-PCR, respectively. Joints were stained with hematoxylin and eosin and with tartrate-resistant acid phosphatase for histologic analysis. Human CABIN-1-transgenic mice with CIA had less severe arthritis than wild-type mice with CIA, as assessed according to hind paw thickness and histologic features. The milder arthritis was accompanied by significantly enhanced apoptosis in transgenic mice, evidenced by a significantly greater number of TUNEL-positive cells in synovial tissue. Expression of inflammatory cytokines and MMPs in the transgenic mice with CIA was reduced, and they exhibited decreased Akt activation and increased expression of p53, caspase 3, caspase 9, and Bax. Our findings demonstrate that hCABIN-1 plays a critical role in promoting apoptosis of FLS and in attenuating inflammation and cartilage and bone destruction in RA. These results help elucidate the pathogenic mechanisms of RA and suggest that CABIN-1 is a potential target for treatment of this disease.

Ran and Calcineurin Can Participate Collaboratively in the Regulation of Spermatogenesis in Scallop

Calcineurin is a calcium/calmodulin-dependent protein phosphatase that plays important roles in the transduction of calcium signals in a variety of tissues. In addition, calcineurin has been implicated in the process of spermatogenesis. A novel calcineurin-binding protein, CaNBP75, has been identified in scallop testis. The C-terminal region of CaNBP75 is homologous to the C-terminal region of RanBP3, a Ran-binding domain-containing protein. A small G protein Ran has been involved in spermiogenesis by virtue of the fact that its localization in spermatids changes during spermiogenesis. The current study was performed to investigate the functions of Ran and CaNBP75 in the regulation of calcineurin in testis to further understand the basic functions of calcineurin during spermatogenesis. First, cloning and sequencing of a scallop Ran cDNA isolated from testis revealed that scallop Ran is well-conserved at the amino acid level. Secondly, direct binding of Ran to CaNBP75 was demonstrated in an in vitro pull-down assay. Thirdly, analysis of the tissue distribution of Ran, CaNBP75, and calcineurin showed that these proteins are abundantly expressed in testis. Fourthly, comparison of the expression profiles of Ran and CaNBP75 with that of calcineurin in scallop testis during the maturation cycle revealed that Ran and CaNBP75 mRNA levels increase during meiosis and spermiogenesis, similar to calcineurin. Finally, co-immunoprecipitation analysis suggests that Ran, CaNBP75, and calcineurin interact in scallop testis during maturation. These results suggest that Ran, CaNBP75, and calcineurin may act in a coordinated manner to regulate spermatogenesis.

Tissue-Specific Expression of Human Calcineurin-Binding Protein 1 in Mouse Synovial Tissue Can Suppress Inflammatory Arthritis

Calcineurin (CN) is a calcium- and calmodulin-dependent serine/threonine phosphatase. In immune cells, CN controls the activity of a wide range of transcription factors, including nuclear factor of activated T, nuclear factor-kappa B, c-fos, and Elk-1. CN plays an important role in synoviocyte activation and arthritis progression in vivo and this function is tightly linked to dysregulated intracellular Ca(2+) store and Ca(2+) response triggered by proinflammatory cytokines. In the present study, transgenic mice expressing human calcineurin-binding protein 1 (hCabin1) were generated, driven by type II collagen promoter, and the efficiency of these mice was investigated by experimental arthritis. These transgenic mice successfully expressed hCabin1 in joint tissue as well as other organs such as liver, heart, and brain. The overexpression of hCabin1 reduced the disease severity during collagen-induced arthritis. In fibroblast-like synoviocytes (FLSs) from hCabin1 transgenic mice, the productions of these cytokines, including interleukin (IL)-2, IL-4, and IFN-γ, were decreased and matrix metalloproteinases were also depressed in transgenic mice FLS. In addition, these effects were only found in the joint tissue, which is a major inflammation site. These findings will provide a better knowledge of the pathogenic mechanisms of rheumatoid arthritis and a potential animal model of the chronic inflammatory conditions, including atherosclerosis and transplantation.

Vacuolar Transport in Tobacco Leaf Epidermis Cells Involves a Single Route for Soluble Cargo and Multiple Routes for Membrane Cargo

We tested if different classes of vacuolar cargo reach the vacuole via distinct mechanisms by interference at multiple steps along the transport route. We show that nucleotide-free mutants of low molecular weight GTPases, including Rab11, the Rab5 members Rha1 and Ara6, and the tonoplast-resident Rab7, caused induced secretion of both lytic and storage vacuolar cargo. In situ analysis in leaf epidermis cells indicates a sequential action of Rab11, Rab5, and Rab7 GTPases. Compared with Rab5 members, mutant Rab11 mediates an early transport defect interfering with the arrival of cargo at prevacuoles, while mutant Rab7 inhibits the final delivery to the vacuole and increases cargo levels in prevacuoles. In contrast with soluble cargo, membrane cargo may follow different routes. Tonoplast targeting of an α-TIP chimera was impaired by nucleotide-free Rha1, Ara6, and Rab7 similar to soluble cargo. By contrast, the tail-anchored tonoplast SNARE Vam3 shares only the Rab7-mediated vacuolar deposition step. The most marked difference was observed for the calcineurin binding protein CBL6, which was insensitive to all Rab mutants tested. Unlike soluble cargo, α-TIP and Vam3, CBL6 transport to the vacuole was COPII independent. The results indicate that soluble vacuolar proteins follow a single route to vacuoles, while membrane spanning proteins may use at least three different transport mechanisms.

WITHDRAWN: USP14 stabilizes calcineurin and regulates NFAT/AP-1 dependent IL-4 transcription as a novel calcineurin-binding protein

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