Kyoung-Ah Kim

Mechanism of silica-induced ROS generation in Rat2 fibroblast cells.

Reactive oxygen species (ROS) play an important role in cell signaling pathway. Previously, we found that silica induced immediate ROS generation and sequential cellular responses such as kinase activation in Rat2 cells as well as an increase of intracellular calcium concentration in A549 cells. However, the detailed mechanism underlying the immediate ROS generation induced by silica in fibroblast cells remains to be elucidated. Therefore, in the present study, we investigated the mechanism of ROS generation by silica within Rat2 fibroblast cells by examining the effects of a diverse group of inhibitors for the enzymes related with signal transduction events. Inhibitors for protein tyrosine kinase (PTK), phospholipase C (PLC), protein kinase C (PKC) and calmodulin (CaM) kinase II effectively suppressed ROS generation in silica-stimulated Rat2 cells, whereas those for protein kinase A and phospholipase A(2) did not. Diphenyleneiodonium chloride (DPI), an inhibitor for NADPH oxidase was also found to be effective in inhibiting silica-induced ROS generation. These results suggest that PTK, PLC, PKC, CaM kinase II, and NADPH oxidase are all involved in signal transduction pathways for ROS generation in silica-stimulated Rat2 cells.

Potential biomarker of coal workers’ pneumoconiosis

It is well known that various cytokines and growth factors secreted from macrophages/monocytes play the key role in the pathogenesis of pneumoconiosis. These can act as biosensors for the prediction of pneumoconiosis. To evaluate which cytokines can be used as sensitive biomarkers in pneumoconiosis, we measured tumor necrosis factor-alpha (TNF-alpha), interleukin-8 (IL-8) and platelet-derived growth factor-AA (PDGF-AA) in supernatant of monocytes with or without coal dust (5 mg/ml) and serum in 42 coal miners with pneumoconiosis and ten healthy control. The coal-stimulated release of TNF-alpha and IL-8 from blood monocytes was significant increased in pneumoconiosis patients compared with controls. The level of TNF-alpha and IL-8 in blood serum was higher in subjects with pneumoconiosis than in controls.

Activation of telomerase by silica in rat lung

By measuring the activity of telomerase in a silica-instilled rat lung, the study found a significant increase in telomerase activity compared to that of the control. Pneumoconiosis displays the characteristics of fibroblast-proliferation and accumulation of collagen, which finally causes the pathologic changes of irreversible and progressive fibrosis of the lung. On the basis of the hypothesis that cellular proliferation may trigger telomerase-activity, the experiment was carried out with telomerase-activation in silicosis. Silica-instilled rat lungs showed increased activity of telomerase, which was measured by TRAP (telomeric repeat amplification protocol) assay, at the time of the 1st, 5th and 8th week after intratracheal instillation of silica in vivo. However, no activity was shown in silica-co-cultured fibroblast in vitro. By summarizing these results, the activity of telomerase is thought to be a very sensitive marker for the evaluation of pathogenicity, showing cellular immortalization in an experimental silicosis model.

Effect of Bradykinin on Cultured Bovine Corneal Endothelial Cells

Purpose: To clarify the effect of bradykinin on cytosolic free calcium mobilization and cell proliferation in cultured bovine corneal endothelial cells (BCEC). Methods: The cytosolic free calcium concentration (Ca²⁺]_i) was measured with the InCa^TM Imaging System after the treatment of bradykinin (10^–11 to 10^–7 M) alone or with the pretreatments of EGTA, bradykinin receptor (Bk₁ and Bk₂) antagonists and an inhibition of phospholipase C (U-73122). Also, the effect of bradykinin on cell proliferation in BCEC was evaluated using cell counts. Results: In BCEC, [Ca²⁺]_i in the resting state was 87 ± 9 nM. Bradykinin induced an increment of [Ca²⁺]_i in a concentration-dependent manner and its 50% effective concentration was approximately 5 × 10^–11 M. A [Ca²⁺]_i increment at 10^–8 M bradykinin was inhibited with the pretreatment of EGTA, an extracellular calcium chelator. U-73122 (5 × 10^–6 M) attenuated the bradykinin-induced [Ca²⁺]_i increment. The pretreatment of HOE-140 (Bk₂ antagonist) almost attenuated the bradykinin (10^–8 M)-induced [Ca²⁺]_i increase, but des-Arg⁹-[Leu⁸]-bradykinin (Bk₁ antagonist) did not suppress it. To investigate the physiological effect of bradykinin, the effect of bradykinin on cell proliferation was studied. 10^–8 M of bradykinin produced a significant increase in cell numbers. This mitogenic effect of bradykinin was inhibited by the Bk₂ antagonist. Conclusions: Bradykinin-induced stimulation of the signal transduction pathway in BCEC is coupled with the Bk₂ type receptor. Furthermore, bradykinin produces the mitogenic effect in BCEC.

Silica induces nuclear factor-κB activation through TAK1 and NIK in Rat2 cell line

Silica has been known to be a factor in acute cell injury and chronic pulmonary fibrosis. In Rat2 fibroblasts, silica induced the activation of nuclear factor-kappa B (NF-kappaB), which plays a crucial role in regulating the expression of many genes involved in the subsequent inflammatory response. In addition, we observed that transforming growth factor-beta activated kinase 1 (TAK1) and NF-kappaB-inducing kinase (NIK) were involved in silica-mediated NF-kappaB activation in Rat2 cells. The dominant negative mutant forms of TAK1 and NIK inhibited the silica-induced NF-kappaB activation in Rat2 cells. Furthermore, we demonstrated that endogenous TAK1 is phosphorylated in silica-stimulated Rat2 cells. These results indicate that TAK1 functions as a critical mediator in the silica-induced signaling pathway.

Effect of Desferrioxamine on Silica-Induced Pulmonary Reaction

Abstract Surface iron on a mineral particle may be a major mediator of mineral-dust-induced toxicity, because iron on the surface of the particle acts as a Fenton catalyst to produce hydroxyl radical from hydrogen peroxide. Desferrioxamine (DF), an iron chelator, might inhibit the process of silica-induced pulmonary reaction. To test this assumption, we investigated the protective effect of DF on lipid peroxidation of cell membrane, production of inflammatory cytokine, and fibroblast proliferation by crystalline silica for an in vitro model. The Fenton activity of silica was decreased by preincubation with DF. Marked decreases in malondialde-hyde (MDA) levels were seen in the DF-treated silica group compared with the untreated group. DF inhibited not only silica-induced release of tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) from A549, but also fibroblast proliferation. The therapeutic effect of DF on experimental silicosis in rat was also studied using total cell count with differential percentage in bronchoaiveoiar lavage fluid, the amount of hydroxyproline in lung, and examination of a histologic section. DF significantly reduced inflammation and fibrosis compared with the untreated control. From these results, we concluded that DF might play a role in the inhibition of silica-induced pulmonary reaction.