Partial IgA-deficiency with increased Th2-type cytokines in TGF-beta 1 knockout mice.

Though it has been shown that TGF-beta 1 directs B cells to switch to IgA in vitro, no studies have assessed TGF-beta 1 effects on mucosal vs systemic immunity in vivo. When the B cell functions of TGF-beta 1 gene-disrupted (TGF-beta 1-/-) mice were analyzed, significantly decreased IgA levels and increased IgG and IgM levels in serum and external secretions were observed. Further, analysis of Ab forming cells (AFC) isolated from both mucosal and systemic lymphoid tissue showed elevated IgM, IgG, and IgE, with decreased IgA AFC. A lack of IgA-committed B cells was seen in TGF-beta 1-/- mice, especially in the gastrointestinal (GI) tract. Splenic T cells triggered via the TCR expressed elevated Th2-type cytokines and, consistent with this observation, a 31-fold increase in serum IgE was seen in TGF-beta 1-/- mice. Thus, uncontrolled B cell responses, which include elevated IgE levels, a lack of antiinflammatory IgA, and an excess of complement-binding IgG and IgM Abs, will promote inflammation at mucosal surfaces in TGF-beta 1-/- mice and likely contribute to pulmonary and GI tract lesions, ultimately leading to the early death of these mice.     

Purification and characterisation of a novel alkalophilic α-D-mannosidase from Pseudomonas fluorescens

An extracellular alkaline α-D-mannosidase in the cell culture of a marine bacterium Pseudomonas fluorescens JK-02 was purified to homogeneity with a 30.7-fold by ammonium sulphate fractionation, anion-exchange chromatography and gel-filtration chromatography. The molecular weight of the purified enzyme was estimated to be 50.5 kDa based on the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH and temperature of the purified enzyme were 8.5 and 30°C. The K_m and V_max values of the purified enzyme towards p-nitrophenyl-α-D-mannopyranoside were determined to be 77 µM and 0.23 µM min^?1mg^?1 of protein, respectively. The α-D-mannosidase showed higher substrate specificity to α-1,3-mannobiose than other isomeric substrates such as α-1,2- and α-1,6-mannobiose. In addition, molecular characterisation of this enzyme reveals that it belongs to a class II α-mannosidase from the glycosyl hydrolase family 38. To the best of our knowledge, this is the first report on the alkalophilic α-1,3 D-mannosidase of Pseudomonas species, which has selective algal-lytic activity against Alexandrium tamarense, Akashiwo sanguine, Gymnodinium catenatum, Gymnodinium mikimotoi and Prorocentrum dentatum.     

Identification of RUNX3 as a component of the MST/Hpo signaling pathway

Recent genetic screens of fly mutants and molecular analysis have revealed that the Hippo (Hpo) pathway controls both cell proliferation and cell death. Deregulation of its human counterpart (the MST pathway) has been implicated in human cancers. However, how this pathway is linked with the known tumor suppressor network remains to be established. RUNX3 functions as a tumor suppressor of gastric cancer, lung cancer, bladder cancer, and colon cancer. Here, we show that RUNX3 is a principal and evolutionarily conserved component of the MST pathway. SAV1/WW45 facilitates the close association between MST2 and RUNX3. MST2, in turn, stimulates the SAV1-RUNX3 interaction. In addition, we show that siRNA-mediated RUNX3 knockdown abolishes MST/Hpo-mediated cell death. By establishing that RUNX3 is an endpoint effector of the MST pathway and that RUNX3 is capable of inducing cell death in cooperation with MST and SAV1, we define an evolutionarily conserved novel regulatory mechanism loop for tumor suppression in human cancers.     

Lysophosphatidic acid activates TGFBIp expression in human corneal fibroblasts through a TGF-β1-dependent pathway

Granular corneal dystrophy type 2 (GCD2) is an autosomal dominant disease caused by a R124H point mutation in the transforming growth factor-β-induced gene (TGFBI). However, the cellular role of TGFBI and the regulatory mechanisms underlying corneal dystrophy pathogenesis are still poorly understood. Lysophosphatidic acid (LPA) refers to a small bioactive phospholipid mediator produced in various cell types, and binds G protein-coupled receptors to enhance numerous biological responses, including cell growth, inflammation, and differentiation. LPA levels are elevated in injured cornea and LPA is involved in proliferation and wound healing of cornea epithelial cells. Accumulating evidence has indicated a crucial role for LPA-induced expression of TGFBI protein (TGFBIp) through secretion of transforming growth factor-beta1 (TGF-β1). In the current study, we demonstrate that LPA induces TGFBIp expression in corneal fibroblasts derived from normal or GCD2 patients. LPA-induced TGFBIp expression was completely inhibited upon pretreatment with the LPA(1/3) receptor antagonists, VPC32183 and Ki16425, as well as by silencing LPA(1) receptor expression with small hairpin RNA (shRNA) in corneal fibroblasts. LPA induced secretion of TGF-β1 in corneal fibroblasts, and pretreatment with the TGF-β type I receptor kinase inhibitor SB431542 or an anti-TGF-β1 neutralizing antibody also inhibited LPA-induced TGFBIp expression. Furthermore, we show that LPA requires Smad2/3 proteins for the induction of TGFBIp expression. LPA elicited phosphorylation of Smad2/3, and Smad3 specific inhibitor SIS3 or siRNA-mediated depletion of endogenous Smad2/3 abrogates LPA-induced TGFBIp expression. Finally, we demonstrate that LPA-mediated TGFBIp induction requires JNK activation, but not ERK signaling pathways. These results suggest that LPA stimulates TGFBIp expression through JNK-dependent activation of autocrine TGF-β1 signaling pathways and provide important information for understanding the role of phospholipids involved in cornea related diseases.     

Synergistic activation of NF-kappaB by nontypeable H. influenzae and S. pneumoniae is mediated by CK2, IKKbeta-IkappaBalpha, and p38 MAPK

In review of the past studies on NF-kappaB regulation, most of them have focused on investigating how NF-kappaB is activated by a single inducer at a time. Given the fact that, in mixed bacterial infections in vivo, multiple inflammation inducers, including both nontypeable Haemophilus influenzae (NTHi) and Streptococcus pneumoniae, are present simultaneously, a key issue that has yet to be addressed is whether NTHi and S. pneumoniae simultaneously activate NF-kappaB and the subsequent inflammatory response in a synergistic manner. Here, we show that NTHi and S. pneumoniae synergistically induce NF-kappaB-dependent inflammatory response via activation of multiple signaling pathways in vitro and in vivo. The classical IKKbeta-IkappaBalpha and p38 MAPK pathways are involved in synergistic activation of NF-kappaB via two distinct mechanisms, p65 nuclear translocation-dependent and -independent mechanisms. Moreover, casein kinase 2 (CK2) is involved in synergistic induction of NF-kappaB via a mechanism dependent on phosphorylation of p65 at both Ser536 and Ser276 sites. These studies bring new insights into the molecular mechanisms underlying the NF-kappaB-dependent inflammatory response in polymicrobial infections and may lead to development of novel therapeutic strategies for modulating inflammation in mixed infections for patients with otitis media and chronic obstructive pulmonary diseases.     

Hydrothermal synthesis and crystal structure of a polyoxomolybdate aggregate constructed from hexamolybdate, octamolybdate, and cobalt(III) complex ion

A new polyoxomolybdate compound {Co(phen)₃}₂[Mo₆O₁₉][Mo₈O₂₆] · 2H₂O (1) containing hexacoordinated {Co(phen)₃}³⁺ (phen = 1,10-phenanthroline) as counter ion has been hydrothermally synthesized and characterized by the elemental analyses, IR spectrum, TG analysis, and the single crystal X-ray diffraction. The crystal structure consists of four independent molecular moieties: {Co(phen)₃}³⁺, {β-Mo₈O₂₆}⁴⁻, {Mo₆O₁₉}²⁻, and water molecules of crystallization.     

Presence of a putative vesicular inositol 1,4,5-trisphosphate-sensitive nucleoplasmic Ca2+ store

The inositol 1,4,5-trisphosphate receptors (IP(3)Rs) are widely localized in both the heterochromatin and euchromatin regions. We found recently the presence of nucleoplasmic complexes that are composed of phospholipids, IP(3)R/Ca(2+) channels, and Ca(2+) storage protein chromogranin B (CGB). Close examination and 3D image reconstruction of these complexes revealed numerous vesicular structures with an average diameter of approximately 50 nm that are primarily interspersed between the heterochromatins. IP(3) rapidly released Ca(2+) from these structures, but other inositol phosphates, inositol 1,4-bisphosphate, inositol 1,3,4-trisphosphate, and inositol 1,3,4,5-tetrakisphosphate, failed to release Ca(2+). Addition of heparin or IP(3)R antibody blocked the IP(3)-induced Ca(2+) releases, indicating the release of Ca(2+) through the IP(3)R/Ca(2+) channels. Given the presence of the IP(3)R/Ca(2+) channels and Ca(2+) storage protein CGB in these vesicular structures, we postulate that these vesicles are the IP(3)-sensitive nucleoplasmic Ca(2+) stores. Abundance of the vesicular Ca(2+) stores between the heterochromatins appeared to imply critical roles these vesicular Ca(2+) stores play in controlling the Ca(2+) concentrations of the chromosomes.     

Corneal Dystrophy-associated R124H Mutation Disrupts TGFBI Interaction with Periostin and Causes Mislocalization to the Lysosome

The 5q31-linked corneal dystrophies are heterogeneous autosomal-dominant eye disorders pathologically characterized by the progressive accumulation of aggregated proteinaceous deposits in the cornea, which manifests clinically as severe vision impairment. The 5q31-linked corneal dystrophies are commonly caused by mutations in the TGFBI (transforming growth factor-β-induced) gene. However, despite the identification of the culprit gene, the cellular roles of TGFBI and the molecular mechanisms underlying the pathogenesis of corneal dystrophy remain poorly understood. Here we report the identification of periostin, a molecule that is highly related to TGFBI, as a specific TGFBI-binding partner. The association of TGFBI and periostin is mediated by the amino-terminal cysteine-rich EMI domains of TGFBI and periostin. Our results indicate that the endogenous TGFBI and periostin colocalize within the trans-Golgi network and associate prior to secretion. The corneal dystrophy-associated R124H mutation in TGFBI severely impairs interaction with periostin in vivo. In addition, the R124H mutation causes aberrant redistribution of the mutant TGFBI into lysosomes. We also find that the periostin-TGFBI interaction is disrupted in corneal fibroblasts cultured from granular corneal dystrophy type II patients and that periostin accumulates in TGFBI-positive corneal deposits in granular corneal dystrophy type II (also known as Avellino corneal dystrophy). Together, our findings suggest that TGFBI and periostin may play cooperative cellular roles and that periostin may be involved in the pathogenesis of 5q31-linked corneal dystrophies.Corneal dystrophies are characterized by the progressive loss of corneal transparency as a result of extracellular amyloid and non-amyloid deposits, which accumulate in different layers of corneal tissues. 5q31-linked corneal dystrophies are pathologically heterogeneous, autosomal-dominant disorders caused by mutations in the TGFBI (transforming growth factor-β-induced) gene, which encodes the TGFBI protein (also known as keratoepithelin or Big-H3) (1, 2). To date, more than 30 different mutations leading to corneal dystrophies have been attributed to mutations in TGFBI, the most frequent of which are mutations within exons 4 and 12, which result in amino acid substitutions in Arg¹²⁴ and Arg⁵⁵⁵, respectively (3, 4). The different mutations in TGFBI cause clinically distinct types of corneal dystrophies, which are classified according to the accumulation patterns of the deposits, including lattice corneal dystrophies type I and IIIA, deep stromal lattice corneal dystrophy, granular corneal dystrophies (GCDs)² type I and II (also known as Avellino corneal dystrophy), Reis-Bucklers corneal dystrophy (also known as corneal dystrophy of Bowman''s layer type I), or Thiel-Behnke corneal dystrophy (also known as corneal dystrophy of Bowman''s layer type II) (reviewed in Refs. 5 and 6). Histological examinations of corneal tissues demonstrate the presence of amyloid deposits in lattice corneal dystrophies and GCD II, hyaline accumulations in GCDs, and subepithelial fibrous material in Reis-Bucklers corneal dystrophy and Thiel-Behnke corneal dystrophy (7–14).TGFBI was originally identified as a gene induced by transforming growth factor-β stimulation in adenocarcinoma cells and is expressed in many tissues (15). The human TGFBI consists of 683 amino acids, with the mature protein predicted to have a molecular mass of ∼68 kDa. As shown in Fig. 1A, TGFBI contains an NH₂-terminal signal peptide that targets it for insertion into the lumen of the endoplasmic reticulum (ER) for eventual secretion, a cysteine-rich EMI domain, four tandem repeats of fasciclin-1 like (FAS1) domains, and a COOH-terminal RGD sequence (15–19). The FAS1 domains of TGFBI display homology to the cell adhesion protein fasciclin-I in Drosophila, an axon guidance protein that is involved in neuronal development (20). Based on the presence of multiple FAS1 domains, TGFBI has been assigned to a larger family of proteins, which includes periostin, stabilin-1, and stabilin-2 (16, 21). To date, many TGFBI homologues have been reported in various vertebrates, including mouse, chicken, pig, and zebrafish, but no homologues have been identified in invertebrates (16, 19, 21). TGFBI has been shown to interact with a number of extracellular matrix (ECM) proteins, including fibronectin, biglycan, decorin, and several types of collagen (19, 22–25). Furthermore, TGFBI also functions as a ligand for several integrins, including α3β1, αvβ5, αvβ3, and αmβ2 (26–29). The COOH-terminal RGD domain of TGFBI is the putative integrin-binding motif. However, several studies have suggested that the interactions between TGFBI and integrins are mediated via the YH (tyrosine-histidine) motifs and DI (aspartate-isoleucine) motifs present in the TGFBI FAS1 domains (30). Although the precise roles of TGFBI are not fully understood yet, emerging evidence suggests a role for TGFBI as a secreted factor involved in cell adhesion, proliferation, and migration.Open in a separate windowFIGURE 1.Periostin is expressed in human cornea. A, schematic representation of TGFBI and periostin. TGFBI and periostin contain NH₂-terminal signal peptides, followed by a cysteine-rich EMI domain and four tandem FAS1 domains. TGFBI also contains a COOH-terminal RGD motif that is not present in periostin, which instead contains a COOH-terminal hydrophilic region. The following domains of TGFBI and periostin are indicated: EMI, FAS1, fasciclin 1, and Arg-Gly-Asp (RGD). Antigenic regions of antibodies used in this study (ab14041 and C-20) are represented by black lines. B, cell and tissue lysates were separated by SDS-PAGE and Western blotted using the indicated antibodies. Anti-periostin antibody (C-20) recognizes an ∼85-kDa protein in several cell lines and corneal fibroblast cell lines. In contrast, anti-periostin antibody recognizes a ∼60-kDa form in corneal epithelial cell lines and corneal epithelium tissues (top, black arrow). The specificity of periostin antibody (C-20) was confirmed by preabsorption with 10 μg of C-20 antigen peptide (second panel). WB, Western blot.TGFBI and periostin show a high degree of similarity in amino acid sequence and in overall domain structure, diverging primarily at the COOH terminus (Fig. 1A) (16, 21). Similar to TGFBI, periostin contains a NH₂-terminal secretory signal peptide followed by a cysteine-rich EMI domain, four tandem repeats of FAS1 domains, and a hydrophilic region in its COOH terminus (Fig. 1A) (16, 17, 31, 32). Periostin has been found to be ubiquitously expressed in multiple tissues in mammals (31, 33, 34). In addition, the expression of periostin has been implicated in the development of variety of cancers, including neuroblastoma, head and neck cancer, and non-small cell lung cancer, possibly by regulating the metastatic growth (32, 35). Periostin is also associated with epithelial-mesenchymal transition during cardiac development (36) and is induced during the proliferation of cardiomyocytes, thereby promoting cardiac repair after heart failure (37, 38). In addition, interlukin-4 and -13 have been found to induce the secretion of periostin from lung fibroblasts, implicating periostin in subepithelial fibrosis in bronchial asthma (39).Despite the similarities between TGFBI and periostin, it is not known whether periostin is involved in the pathogenesis of 5q31-linked corneal dystrophies. In this study, we find that periostin specifically interacts with TGFBI via the NH₂-terminal cysteine-rich EMI domain and colocalizes with TGFBI in the trans-Golgi network of COS-7 and corneal fibroblast cells. In addition, corneal dystrophy-linked mutations in TGFBI disrupt its subcellular localization and impair its interaction with periostin. Furthermore, we find that periostin accumulates in extracellular corneal deposits in GCD II patients bearing homozygous R124H mutations in TGFBI. These findings provide new insights into the pathogenic mechanisms of TGFBI mutations in 5q31-linked corneal dystrophies and have important implications for understanding and treating corneal dystrophies.