BBA - Molecular Basis of Disease (v.1762, #8)

Evidence for a protective role for adiponectin in osteoarthritis by Tsu-Hsin Chen; Linda Chen; Ming-Shium Hsieh; Chih-Peng Chang; Der-Tsay Chou; Shu-Huei Tsai (711-718).
Obesity has been associated with an increased risk of osteoarthritis (OA). However, the mechanism by which obesity contributes to OA remains uncertain. Adiponectin, an adipocyte-derived hormone, has shown anti-diabetic and anti-atherogenic properties. In the present study, we aimed to investigate the potential role of adiponectin in OA disease. We demonstrated that adiponectin was present in OA synovial fluid (SF) and its expression level was almost 100-fold decrease compared with that in OA plasma. FPLC and ELISA studies revealed the distribution and abundance of the adiponectin complexes in plasma and SF from patients with OA. The percentage of high molecular weight (HMW) per total adiponectin in OA SF was lower than in OA plasma, while that of the hexamer form was similar and the trimer form was higher. The expression levels of adiponectin receptors AdipoR1 and AdipoR2 were examined in human OA tissues by RT-PCR. AdipoR1 was abundantly expressed in cartilage, bone and synovial tissues, whereas AdipoR2 was rarely detected. Finally, the effects of adiponectin on primary chondrocyte functions were studied by using antibody-based protein array and RT-PCR. The patterns of mRNA expression and protein production strongly indicate that adiponectin is involved in the modulation of cartilage destruction in chondrocytes by up-regulating TIMP-2 and down-regulating IL-1β-induced MMP-13. Together these findings clearly indicate that the adiponectin may act as a protective role in the progression of OA, and this also provide new thinking on the relationship between obesity and OA.
Keywords: Adiponectin; Osteoarthritis; MMP-13;

Many females develop bone diseases such as osteoporosis, and joint diseases such as osteoarthritis after menopause when estrogen levels decline. As estrogen receptors (ER) are present in such tissues, it is possible that the loss of estrogen at menopause influences the expression of enzymes such as members of the MMP family of proteinases to affect bone and connective tissue metabolism. The present study was undertaken to assess a possible relationship between ER-α and MMP-13 expression at the promoter level, and to determine how such a relationship could be modulated by ligands such as estrogen. Using a rabbit synovial cell line lacking endogenous ER, a transient transfection system with an ER-α construct, and a series of MMP-13 promoter-luciferase constructs of varying lengths and with specific mutations in transcription factor binding sites, it was found that ER-α can significantly enhance MMP-13 promoter activity via the AP-1 site, with modulatory influences by the Runx and PEA-3 sites on this ER-α dependent enhancement of the promoter activity. This enhancement by ER-α was significantly depressed in the presence of 17-ß-estradiol in a dose dependent manner. The influence of tamoxifen and raloxifen on the activity of the ER-α was consistent with their known agonist/antagonist activity. These findings indicate that loss of estrogen in vivo could potentially lead to enhanced expression of MMP-13, a proteinase that has been implicated in both osteoporosis and osteoarthritis, and thus contribute to the development and progression of these conditions.
Keywords: OA; MMP-13; ER-α; Estrogenic ligand; AP-1 Site; Regulation;

Reactive oxygen species (ROS) can participate in cellular signaling and have been shown to modulate activation of the transcriptional regulatory factor NF-κB. However, the effects of ROS can differ in various cell populations. To examine the role of superoxide in neutrophil activation, we exposed resting neutrophils and neutrophils stimulated with LPS to paraquat, an agent that specifically increases intracellular superoxide concentrations. Culture of resting neutrophils with paraquat resulted in increased production of the proinflammatory cytokines TNF-α and MIP-2, enhanced degradation of IκB-α, and increased nuclear accumulation of NF-κB. Such effects of paraquat were due to intracellular superoxide (O2 ) since they were blocked by the non-specific antioxidant N-acetyl cysteine and the cell permeable superoxide scavenger Tiron, but not by catalase, which facilitates the conversion of H2O2 to H2O and O2. Similar potentiating effects of paraquat were found in LPS-stimulated neutrophils. Exposure of neutrophils to paraquat also enhanced phosphorylation of Ser536 in the p65 subunit of NF-κB an event associated with increased transcriptional activity. Examination of kinases critical for LPS-stimulated gene expression showed that addition of paraquat to resting or LPS exposed neutrophils enhanced activation of p38 MAPK, but not that of Akt or ERK1/2. The potentiation of NF-κB translocation and proinflammatory cytokine production, but not of Ser536 p65 phosphorylation, by paraquat was dependent on activation of p38 MAPK. These results demonstrate that increased intracellular superoxide concentrations are proinflammatory in neutrophils, acting through a p38 MAPK dependent mechanism that results in enhanced nuclear accumulation of NF-κB and increased expression of NF-κB dependent proinflammatory cytokines.
Keywords: Reactive oxygen species; Neutrophils; Inflammation; NF-kappa B; Superoxide; Cytokines;

Protective role of Cop in Rip2/Caspase-1/Caspase-4-mediated HeLa cell death by Xin Wang; Malini Narayanan; Jean-Marie Bruey; Dorotea Rigamonti; Elena Cattaneo; John C. Reed; Robert M. Friedlander (742-754).
CARD only protein (Cop) was recently identified as a protein with significant homology with the CARD of caspase-1. We have conducted functional studies on Cop and report on its role as an inhibitor of cell death in a broad range of cell death paradigms. A notable exception in the ability of Cop to inhibit cell death pertains to its inability to inhibit ER stress-mediated cell death. Furthermore, in addition to the known interaction of Cop and caspase-1, we demonstrated a novel interaction of Cop with caspase-4. We propose that Cop's action to prevent TNF-α-induced cell death may operate independently of the mitochondrial death pathway. Furthermore, Cop overexpression inhibits Bid cleavage. In summary, Cop inhibition of cell death, at least to a certain extent, results from its interference with the activation of caspase-1 and caspase-4. Understanding the mechanistic details modulating caspase cell death pathways should provide important information for the development of therapies for diseases featuring aberrant caspase activation. Cop, as an inhibitor of an important apical caspase cell death axis, may provide a tool for modulating pathological cell death.
Keywords: Cop; Caspase-1; Caspase-4; Rip2; Apoptosis;

Adhesion contact kinetics of HepG2 cells during Hepatitis B virus replication: Involvement of SH3-binding motif in HBX by Tuan Lin Tan; Zhiqin Feng; Yi Wei Lu; Vincent Chan; Wei Ning Chen (755-766).
It has been shown that Hepatitis B virus (HBV) replication directly alters the expression of key cytoskeleton-associated proteins which play key roles in mechanochemical signal transduction. Nevertheless, little is known on the correlation between HBV replication and the subsequent adhesion mechanism of HBV-replicating cells. In this study, it is demonstrated that the lag time of adhesion contact evolution of HepG2 cells with HBV replication is significantly increased by two times compared to that of normal HepG2 cell on collagen coated substrate. During the initial 20 min of cell seeding, only diffuse forms of vinculin was detected in HBV replicating cells while vinculin-associated focal complexes were found in normal and control cells. Similar delay in cell adhesion in HBV-replicating cells was observed in cells transfected with HBX, the smallest HBV protein, suggesting its involvement in this cellular process. In addition, a proline rich region found in many SH3 binding proteins was identified in HBX. HBX was found to interact with the focal adhesion protein, vinexin-β, through the SH3 binding. Furthermore, HepG2 cells with HBV replication showed evidence of cell rounding up, possibly resulting from cytoskeletal reorganizations associated with interaction between HBX and vinexin-β. Taken together, our results suggest that HBX is involved in the cytoskeletal reorganization in response to HBV replication.
Keywords: Cell adhesion; Kinetics; Hepatitis B virus; Extracellular matrix; HBX; Vinexin β; SH3;

Two severe Class I human glucose-6-phosphate dehydrogenase (G6PD, EC1.1.1.49) mutations, G6PDWisconsin (nt1177 C → G, R393G) and G6PDNashville (nt1178 G → A, R393H), affect the same codon, altering a residue in the dimer interface close to the “structural” NADP+ site. These mutations are predicted to influence interaction with the bound “structural” NADP+, long supposed to be crucial for enzyme stability. Recombinant proteins corresponding to these mutants have been constructed, expressed and purified to homogeneity. Steady-state kinetic parameters of the mutant enzymes were comparable to those of normal human G6PD, indicating that the mutations do not alter catalytic efficiency drastically. However, investigations of thermostability, urea denaturation, protease digestion, and hydrophobic exposure demonstrated that G6PD R393H is less stable than normal G6PD or R393G, and stability was more NADP+-dependent. Apoenzymes were prepared by removal of “structural” NADP+. Again the G6PDNashville protein was markedly less stable, and its dissociation constant for “structural” NADP+ is ∼ 500 nM, about 10 times higher than values for R393G (53 nM) and normal G6PD (37 nM). These results, together with structural information, suggest that the instability of the R393H protein, enhanced by the weakened binding of “structural” NADP+, is the likely cause of the severe clinical manifestation observed for G6PDNashville. They do not, however, explain the basis of disease in the case of G6PDWisconsin.
Keywords: Glucose 6-phosphate dehydrogenase deficiency; Site-directed mutagenesis; Steady-state kinetic; Structural NADP+; Dissociation constant; Stability;

Matrix metalloproteinase (MMP)-8 has been associated with the progression of periodontitis, a common inflammatory disease of the supporting structures of the teeth, and with other degradative diseases. Tobacco smokers are at high risk of developing periodontitis that may progress more rapidly and respond poorly to treatment. Therefore, MMP-8 expression was determined by immunofluorescence staining in 60 random, computer-selected fields in the excised periodontal tissues of smokers and non-smokers, balanced for age, gender, and periodontal status. Immunofluorescence intensity, representing MMP-8 expression, in the periodontal tissues of smokers (30 fields from 6 subjects, mean 1154 ± 124 units) was significantly higher than that in the periodontal tissues of non-smokers (30 fields from 6 subjects, mean 817 ± 60 units; p  < 0.05). Serum MMP-8 concentrations were measured by ELISA and compared in a larger group of smokers (n  = 20) and age- and gender-balanced non-smokers (n  = 20). Systemic MMP-8 concentrations in smokers and non-smokers were not significantly different (p  > 0.05). A local tobacco-related increase in MMP-8 burden may contribute to periodontal disease progression in tobacco smokers. This finding may also have relevance to other tobacco-induced inflammatory diseases, such as vascular and pulmonary diseases.
Keywords: Collagen; MMP-8; Periodontitis; Smoking; Tobacco;

Erratum to “The role of PDE4 in pulmonary inflammation and goblet cell hyperplasia in allergic rats” [Biochem. Biophys. Acta 1762 (2006) 525–532] by Hui-Fang Tang; Ji-Qiang Chen; Qiang-Min Xie; Xu-Yang Zheng; Yi-Liang Zhu; Ian Adcock; Xiangdong Wang (781).