CLIC4 regulates apical exocytosis and renal tube luminogenesis through retromer- and actin-mediated endocytic trafficking.

Chloride intracellular channel 4 (CLIC4) is a mammalian homologue of EXC-4 whose mutation is associated with cystic excretory canals in nematodes. Here we show that CLIC4-null mouse embryos exhibit impaired renal tubulogenesis. In both developing and developed kidneys, CLIC4 is specifically enriched in the proximal tubule epithelial cells, in which CLIC4 is important for luminal delivery, microvillus morphogenesis, and endolysosomal biogenesis. Adult CLIC4-null proximal tubules display aberrant dilation. In MDCK 3D cultures, CLIC4 is expressed on early endosome, recycling endosome and apical transport carriers before reaching its steady-state apical membrane localization in mature lumen. CLIC4 suppression causes impaired apical vesicle coalescence and central lumen formation, a phenotype that can be rescued by Rab8 and Cdc42. Furthermore, we show that retromer- and branched actin-mediated trafficking on early endosome regulates apical delivery during early luminogenesis. CLIC4 selectively modulates retromer-mediated apical transport by negatively regulating the formation of branched actin on early endosomes.

The protein Zfand5 binds and stabilizes mRNAs with AU-rich elements in their 3'-untranslated regions.

AU-rich elements (AREs) in the 3'-UTR of unstable transcripts play a vital role in the regulation of many inflammatory mediators. To identify novel ARE-dependent gene regulators, we screened a human leukocyte cDNA library for candidates that enhanced the activity of a luciferase reporter bearing the ARE sequence from TNF (ARE(TNF)). Among 171 hits, we focused on Zfand5 (zinc finger, AN1-type domain 5), a 23-kDa protein containing two zinc finger domains. Zfand5 expression was induced in macrophages in response to IFNγ and Toll-like receptor ligands. Knockdown of Zfand5 in macrophages decreased expression of ARE class II transcripts TNF and COX2, whereas overexpression stabilized TNF mRNA by suppressing deadenylation. Zfand5 specifically bound to ARE(TNF) mRNA and competed with tristetraprolin, a protein known to bind and destabilize class II ARE-containing RNAs. Truncation studies indicated that both zinc fingers of Zfand5 contributed to its mRNA-stabilizing function. These findings add Zfand5 to the growing list of RNA-binding proteins and suggest that Zfand5 can enhance ARE-containing mRNA stability by competing with tristetraprolin for mRNA binding.

Role of CLIC4 in the host innate responses to bacterial lipopolysaccharide.

Chloride intracellular channel (CLIC) 4 has diverse functions in membrane trafficking, apoptosis, angiogenesis and cell differentiation. CLIC4 is abundantly expressed in macrophages, but its role in innate immune functions is unclear. Here, we show that primary murine macrophages express increased amounts of CLIC4 after exposure to bacterial lipopolysaccharide (LPS). Endogenous CLIC4 level was significantly elevated in the brain, heart, lung, kidney, liver and spleen after LPS injection of mice. Stable macrophage lines overexpressing CLIC4 produced more TNF, IL-6, IL-12 and CCL5 than mock transfectants when exposed to LPS. To explore the role of CLIC4 in vivo, we generated CLIC4-null mice. These mice were protected from LPS-induced death, and had reduced serum levels of inflammatory cytokines. Upon infection with Listeria monocytogenes, CLIC4-deficient mice were impaired in their ability to clear infection, and their macrophages responded to Listeria by producing less inflammatory cytokines and chemokines than the WT controls. When challenged with LPS in vitro, deletion of clic4 gene had little effect on MAPK and NF-κB activation, but led to a reduced accumulation of phosphorylated interferon response factor 3 (IRF3) within macrophages. Conversely, overexpression of CLIC4 enhanced LPS-mediated IRF3. Thus, these findings suggest that CLIC4 is an LPS-induced product that can serve as a positive regulator of LPS signaling.

Secretory leukocyte protease inhibitor plays an important role in the regulation of allergic asthma in mice.

Secretory leukocyte protease inhibitor (SLPI) is an anti-inflammatory protein that is observed at high levels in asthma patients. Resiquimod, a TLR7/8 ligand, is protective against acute and chronic asthma, and it increases SLPI expression of macrophages in vitro. However, the protective role played by SLPI and the interactions between the SLPI and resiquimod pathways in the immune response occurring in allergic asthma have not been fully elucidated. To evaluate the role of SLPI in the development of asthma phenotypes and the effect of resiquimod treatment on SLPI, we assessed airway resistance and inflammatory parameters in the lungs of OVA-induced asthmatic SLPI transgenic and knockout mice and in mice treated with resiquimod. Compared with wild-type mice, allergic SLPI transgenic mice showed a decrease in lung resistance (p < 0.001), airway eosinophilia (p < 0.001), goblet cell hyperplasia (p < 0.001), and plasma IgE levels (p < 0.001). Allergic SLPI knockout mice displayed phenotype changes significantly more severe compared with wild-type mice. These phenotypes included lung resistance (p < 0.001), airway eosinophilia (p < 0.001), goblet cell hyperplasia (p < 0.001), cytokine levels in the lungs (p < 0.05), and plasma IgE levels (p < 0.001). Treatment of asthmatic transgenic mice with resiquimod increased the expression of SLPI and decreased inflammation in the lungs; resiquimod treatment was still effective in asthmatic SLPI knockout mice. Taken together, our study showed that the expression of SLPI protects against allergic asthma phenotypes, and treatment by resiquimod is independent of SLPI expression, displayed through the use of transgenic and knockout SLPI mice.

Beneficial effects of secretory leukocyte protease inhibitor after spinal cord injury.

Secretory leukocyte protease inhibitor is a serine protease inhibitor produced by various cell types, including neutrophils and activated macrophages, and has anti-inflammatory properties. It has been shown to promote wound healing in the skin and other non-neural tissues, however, its role in central nervous system injury was not known. We now report a beneficial role for secretory leukocyte protease inhibitor after spinal cord injury. After spinal cord contusion injury in mice, secretory leukocyte protease inhibitor is expressed primarily by astrocytes and neutrophils but not macrophages. We show, using transgenic mice over-expressing secretory leukocyte protease inhibitor, that this molecule has an early protective effect after spinal cord contusion injury. Furthermore, wild-type mice treated for the first week after spinal cord contusion injury with recombinant secretory leukocyte protease inhibitor exhibit sustained improvement in locomotor control and reduced secondary tissue damage. Recombinant secretory leukocyte protease inhibitor injected intraperitoneally localizes to the nucleus of circulating leukocytes, is detected in the injured spinal cord, reduces activation of nuclear factor-kappaB and expression of tumour necrosis factor-alpha. Administration of recombinant secretory leukocyte protease inhibitor might therefore be useful for the treatment of acute spinal cord injury.

Selection of CC chemokine receptor 5-binding peptide from a phage display peptide library.

Human CC chemokine receptor (CCR) 5 is a G protein-coupled receptor involved in a broad range of human diseases that mediates HIV-1 viral entry into cells. Certain small molecule receptor antagonists to CCR5 have been useful in therapy for these diseases. In this study, CCR5-expressing CHO cells (CHO/CCR5 cells) were used to select CCR5-binding peptides from a phage-displayed 12-mers peptide library. All of the 30 clones selected from the library showed specific binding to CHO/CCR5 cells by enzyme linked immunosorbent assay (ELISA). Seventeen out of the 30 clones shared the amino acid motif AFDWTFVPSLIL. The motif-containing phages and synthetic peptide AFDWTFVPSLIL blocked the binding of mAb 2D7 to CHO/CCR5 cells and competitively inhibited the ability of chemokine regulated on activation normal T cell expressed and secreted (RANTES) binding to CHO/CCR5 cells. Furthermore, the peptide AFDWTFVPSLIL also inhibited RANTES induced increase in the intracellular Ca2+ level in CHO/CCR5 cells. These results suggest that the peptide AFDWTFVPSLIL was specific for CCR5 and that it might become a CCR5 antagonist.

Dynamic distribution and expression in vivo of human endostatin gene delivered by adenoviral vector.

Endostatin, a 20-kDa carboxyl-terminal fragment of collagen XVIII, is a potent inhibitor of endothelial cell proliferation and tumor angiogenesis. We have constructed replication-deficient recombinant adenovirus (Ad-rhE), which encoded secreted human endostatin, and our previous studies showed that Ad-rhE had a potent suppression of tumor growth in vivo. In the present study, we investigated the dynamic distribution and expression of human endostatin gene in vivo using fluorogenic real-time quantitative PCR and enzyme-linked immunosorbent assay(ELISA), respectively, with an injection of 2.0 x10(9)pfu of Ad-rhE. After injection, the Ad-rhE DNAs decreased sharply, but lasted a relative long-term at low concentration (10,000--20,000 copies/mg tissues). Whereas the expressed endostatin rose up rapidly, and reached to the top on day 5 after injection of Ad-rhE, and then decreased sharply, but endostatin in tumors sustained to over 9 days at a certain level. Both Ad-rhE DNAs and endostatin mainly enriched in tumors in vivo, and then in livers. These results suggest that endostatin gene delivered by adenoviral vector can generate a high expression in vivo, and both the metabolism pathways of Ad-rhE DNAs and endostatin in vivo are through the systems of livers.

The C-terminal domain of canstatin suppresses in vivo tumor growth associated with proliferation of endothelial cells.

Angiogenesis is crucial for the growth and metastasis of solid tumors with sizes larger than a few cubic millimeter Canstatin, the non-collagenous 1 (NC1) domain of alpha2 chain of type IV collagen, was previously shown to inhibit proliferation of endothelial cells in vitro and suppress in vivo tumor growth. Our previous studies showed that canstatin-N, the N-terminal 1-89 amino acid fragment of canstatin, inhibited the neovascularization in vivo, potently induced apoptosis of endothelial cells in vitro, and suppressed in vivo tumor growth in BALB/c mice. In the present study, we demonstrated that canstatin-C, the C-terminal 157-227 amino acid fragment of canstatin, also specifically inhibited in vitro the proliferation of human umbilical vein endothelial cells and induced apoptosis, but the apoptosis-inducing activity, while close to that of the full-length canstatin, was much lower than that of canstatin-N. Canstatin-C also suppressed in vivo tumor growth in BALB/c mice at a dosage of 10mg/kg/day. These results suggest that canstatin-C is an anti-angiogenic domain of canstatin mainly associated with the specific inhibition of proliferation of endothelial cells, whereas canstatin-N with the potential apoptosis-inducing activity on endothelial cells.

Canstatin-N fragment inhibits in vitro endothelial cell proliferation and suppresses in vivo tumor growth.

Type IV collagen is one of the components of vascular basement involved in regulation of angiogenesis. Canstatin, the non-collagenous 1 (NC1) domain of alpha2 chain of type IV collagen, was identified as an inhibitor of angiogenesis and tumor growth by Kamphaus et al. Our previous studies showed that canstatin-N, the N-terminal 1-89 amino acid fragment of canstatin, inhibited the neovascularization in a dose-dependent manner as tested by CAM assay. In the present study, we demonstrate that canstatin-N produced in Escherichia coli specifically inhibited in vitro the proliferation of human umbilical vein endothelial cells (ECV304) and significantly induced apoptosis. The apoptosis-inducing activity of canstatin-N was much stronger than that of canstatin, indicating that the apoptosis-inducing activity of canstatin is likely located within its N-terminal 1-89 amino acid fragment. Canstatin-N also suppressed in vivo growth of B(16) murine melanoma in BALB/c mice at a dosage of 10mg/kg/day. These results suggest that canstatin-N is a useful candidate molecule for inhibition of tumor growth.

[Expression and purification of the N-domain of human canstatin and its bioactivity].

Total RNA was extracted from placenta umbilical tissue. The canstatin cDNA was amplified from total RNA by net-RT-PCR technique and cloned into pSP72, and the resulted plasmid pSP72C was used as template to amplify its N-domain. The amplified 1-89 aa N-domain was then cloned into pET-3c. The resulted plasmid pET-CN was transformed into E. coli BL21(DE3). The N-domain was efficiently expressed after IPTG induction as a 10 kD band on SDS-PAGE. The expressed product accounted for approximately 35.3% of the total bacterial proteins, as estimated by densitometry and existed mainly as inclusion body. The inclusion bodies were washed, lysed and the reactivated proteins were purified by the Sephadex G-100 gel filtration to a purity of 92.6%. CAM assay showed that N-domain effectively inhibited the angiogenesis of chicken embryo microcapillary vessel.

[Cloning and expression of human canstatin and its purification and bioactivity].

Total RNA was extracted from placenta umbilical tissue and the canstatin cDNA was amplified from total RNA by net-RT-PCR technique. The amplified cDNA was cloned into pSP72 and sequenced. The canstatin cDNA was cut down from pSP72C with BamH I / Nde I and ligated into the vector pET-3c. The resultant plasmid pETC was then transformed into E. coli BL21 (DE3). The canstatin gene was efficiently expressed after IPTG induction as a 25 kD band on SDS-PAGE. The expressed product constituted approximately 27.9% of the total bacterial proteins estimated by densitometry and existed mainly as inclusion body. The inclusion bodies were washed, lysed, refolded and purified on the Sephadex G-75 gel filtration column to a purity of 91.4%. CAM assay showed that 10 microg purified canstatin is enough to inhibit the angiogenesis of chichen embryo microcapillary vessel.