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.     

Neurotoxicity of microglial cathepsin D revealed by secretome analysis

Microglia-driven inflammatory responses have both neuroprotective and neurotoxic effects in the CNS. The excessive and chronic activation of microglia, however, may shift the balance towards neurotoxic effects. In this regard, proteins secreted from activated microglia likely play a key role in the neurotoxic effects. To characterize secreted proteins of activated microglia, conditioned media obtained from BV-2 mouse microglia cells were analyzed by two-dimensional gel electrophoresis or liquid chromatography coupled with tandem mass spectrometry. Among many proteins identified in the secretome of activated microglia, an aspartic endoprotease cathepsin D has been found to mediate microglial neurotoxicity based on the following results: (i) the expression of cathepsin D protein was markedly increased in lipopolysaccharide/interferon-γ-stimulated microglia compared with resting microglia as determined by western blot analysis of conditioned media; (ii) knockdown of cathepsin D expression in microglia using short hairpin RNA diminished the neurotoxicity in the coculture of microglia and neuroblastoma cells and (iii) recombinant procathepsin D protein exerted cytotoxic effects toward cultured neurons. In conclusion, cathepsin D appears to play a central role in the microglial neurotoxicity, and could be a potential biomarker or drug target for the diagnosis and treatment of neurodegenerative diseases that are associated with excessive microglial activation and subsequent neurotoxic inflammation.     

Characterization and immunostimulating activity of a water-soluble polysaccharide isolated from <Emphasis Type="Italic">Haematococcus lacustris</Emphasis>

A water-soluble polysaccharide was isolated and purified from the culture filtrate of the photosynthetic green microalgae Haematococcus lacustris by 75% ethanol precipitation and Sepharose CL-6B column chromatography. The molecular mass of the purified polysaccharide (named HCP) was estimated to be approximately 135 kDa by size-exclusion HPLC and its monosaccharide composition was galactose, glucose and mannose at a relative molar ratio of 2.0, 1.0, and 4.1, respectively, suggesting that HCP is a galactomannan. Fourier-transform infrared and elemental analysis revealed that the purified HCP contains sulfate esters by 1.08% (in mass) and no detectable level of protein. The HCP significantly stimulated murine macrophage RAW264.7 cells to secrete the pro-inflammatory cytokine, TNF-α, in a dose-dependent manner and also enhanced the expression of COX-2 and iNOS genes at a concentration of lower than 10 μg/mL HCP. These results indicated that the sulfated HCP of H. lacustris has potent early innate immune stimulating activities.     

Preparation of self-assembled silk sericin nanoparticles

Silk sericin (SS) possessing moisture-retaining property was reacted with activated poly(ethylene glycol) (PEG) to obtain self-assembled SS nanoparticles. The aliphatic and aromatic hydroxyl groups of serine and tyrosine residues as the reaction sites in SS were clarified by amino acid analysis and 1H NMR spectroscopy, respectively. From IR and circular dichroism (CD) measurements, introduction of PEG into SS induced the conformational change from random coil to beta-sheet. DSC thermogram of sericin-PEG conjugate suggests that mutual miscibility between PEG and SS chains was poor. Nanoparticles of sericin-PEG conjugate with sizes measured by dynamic light scattering ranging about 200-400 nm in diameter, were prepared by the diafiltration method. Shape of sericin-PEG conjugate nanoparticles observed by scanning and transmission electron microscopes was spherical. The results suggest that sericin-PEG conjugates are self-associated to form spherical nanoparticles through hydrophobic interaction.     

Wet spinning of silk polymer. I. Effect of coagulation conditions on the morphological feature of filament

Regenerated silk fibroin (SF) filaments were prepared by the wet spinning technique. The effect of coagulation conditions, such as coagulant type and coagulation temperature, was investigated on the morphological feature of SF filaments and a theoretical approach was also performed to understand the coagulation phenomena. SEM observation revealed that as the R group size of alcoholic coagulant (ROH) increased, the cross-sectional shape deviated more from a circular form. This is attributed to the fact that as the R group size increased, the mass transfer rate difference increased, but the coagulation rate decreased due to a reduced diffusion rate. Most non-alcoholic coagulants exhibited this circular cross-sectional shape, except dioxane, which showed a clover shape. As the coagulation bath temperature increased, the cross-section deviated less from a circular form, with the reduction of fiber diameter. When methanol/water mixture was used as a coagulant, an ellipse or a dog-bone shape was obtained with higher water content in methanol, which was attributed to the decrease of coagulation rate. Although methanol exhibited a positive value of mass transfer rate difference, a circular shape of cross-section was obtained due to the density difference of the coagulated and uncoagulated parts in the coagulating SF filament.     

Preparation of semi-interpenetrating polymer networks composed of silk fibroin and poloxamer macromer

A system was designed to utilize silk fibroin (SF) as a matrix for wound dressing. For this system, we prepared a sponge type of porous semi-interpenetrating networks (SIPNs) hydrogel composed of SF and poloxamer 407 macromer to enhance the mechanical and functional properties of SF. The thermal and mechanical properties of the hydrogels as well as their swelling behaviors were studied by means of differential scanning calorimetry, compressive modulus measurement, and gravimetric method, respectively. The morphology and crystalline structure of these SIPN hydrogels were also investigated by scanning electron microscopy (SEM) and wide-angle diffractometry, respectively. Conformational change of SF from random coil to beta-sheet structure was accelerated by formation of SIPNs with poloxamer. The melting temperature of poloxamer in the SIPNs decreased due to the prevention of crystallization by the incorporation of SF. The mechanical strength of SIPNs hydrogel was much higher than those of SF itself or SF/poloxamer blend and increased with the poloxamer content. The equilibrium water content of SF was remarkably increased by formation of SIPNs with poloxamer due to the hydrophilicity of poloxamer. The crystallinity and morphology of SIPNs hydrogel were affected by SIPNs hydrogel composition.     

Impaired autophagy and delayed autophagic clearance of transforming growth factor β-induced protein (TGFBI) in granular corneal dystrophy type 2

Granular corneal dystrophy type 2 (GCD2) is an autosomal dominant disease characterized by a progressive age-dependent extracellular accumulation of transforming growth factor β-induced protein (TGFBI). Corneal fibroblasts from GCD2 patients also have progressive degenerative features, but the mechanism underlying this degeneration remains unknown. Here we observed that TGFBI was degraded by autophagy, but not by the ubiquitin/proteasome-dependent pathway. We also found that GCD2 homozygous corneal fibroblasts displayed a greater number of fragmented mitochondria. Most notably, mutant TGFBI (mut-TGFBI) extensively colocalized with microtubule-associated protein 1 light chain 3β (MAP1LC3B, hereafter referred to as LC3)-enriched cytosolic vesicles and CTSD in primary cultured GCD2 corneal fibroblasts. Levels of LC3-II, a marker of autophagy activation, were significantly increased in GCD2 corneal fibroblasts. Nevertheless, levels of SQSTM1/p62 and of polyubiquitinated protein were also significantly increased in GCD2 corneal fibroblasts compared with wild-type (WT) cells. However, LC3-II levels did not differ significantly between WT and GCD2 cells, as assessed by the presence of bafilomycin A₁, the fusion blocker of autophagosomes and lysosomes. Likewise, bafilomycin A₁ caused a similar change in levels of SQSTM1. Thus, the increase in autophagosomes containing mut-TGFBI may be due to inefficient fusion between autophagosomes and lysosomes. Rapamycin, an autophagy activator, decreased mut-TGFBI, whereas inhibition of autophagy increased active caspase-3, poly (ADP-ribose) polymerase 1 (PARP1) and reduced the viability of GCD2 corneal fibroblasts compared with WT controls. These data suggest that defective autophagy may play a critical role in the pathogenesis of GCD2.     

Isolation of a glucosamine-specific kinase, a unique enzyme of Vibrio cholerae

We showed previously that chitin catabolism by the marine bacterium Vibrio furnissii involves at least three signal transduction systems and many genes, several of which were molecularly cloned, and the corresponding proteins were characterized. The predicted amino acid sequences of these proteins showed a high degree of identity to the corresponding proteins from Vibrio cholerae, whose complete genomic sequence has recently been determined. We have therefore initiated studies with V. cholerae. We report here a novel ATP-dependent glucosamine kinase of V. cholerae encoded by a gene designated gspK. The protein, GspK (31.6 kDa), was purified to apparent homogeneity from recombinant Escherichia coli. The product of the reaction was shown to be GlcN-6-P by matrix-assisted laser desorption/ionization-time of flight (MALDI mass spectrometry) and NMR. The K(m) values for GlcN, ATP, and MgCl(2) were 0.45, 2.4, and 2.2 mm, respectively, and the V(max) values were in the range 180-200 nmol/microg/min (approximately 6 nmol/pmol/min). Kinase activity was not observed with any other sugar, including: galactosamine, mannosamine, Glc, GlcNAc, GalNAc, mannose, 2-deoxyglucose, and oligosaccharides of chitosan. The enzyme is also ATP-specific. The kinase can be used to specifically determine micro quantities of GlcN in acid hydrolysates of glycoconjugates. The physiological function of this enzyme remains to be determined.     

Clonal expansion of double-positive intraepithelial lymphocytes by MHC class I-related chain A expressed in mouse small intestinal epithelium

Expression of a distant homologue MHC class I molecule, MHC class I-related chain A (MICA), has been found to be stress inducible and limited to the intestinal epithelium. This nonclassical MHC molecule is associated with various carcinomas in humans. To understand the biological consequences of MICA expression in the gut, we generated transgenic (Tg) mice (T3(b)-MICA Tg) under the control of the T3(b) promoter. The T3(b)-MICA Tg mice expressed MICA selectively in the intestine and had an increased number of TCRalphabeta CD4CD8alphaalpha, double-positive (DP) intraepithelial lymphocytes (IELs) in the small bowel. These MICA-expanded DP IELs exhibited a bias to Vbeta8.2 and overlapped motifs of the complementarity-determining region 3 region among various Tg mice. Hence, the overexpression of MICA resulted in a clonal expansion of DP IELs. Studies in model of inflammatory bowel disease showed that transgenic MICA was able to attenuate the acute colitis induced by dextran sodium sulfate administration. Therefore, this unique in vivo model will enable investigation of possible influences of stress-inducible MICA on the gut immune surveillance.