Woon Kyu Lee

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.

The merlin tumor suppressor interacts with Ral guanine nucleotide dissociation stimulator and inhibits its activity

Neurofibromatosis type 2 (NF2) is the most commonly mutated gene in benign tumors of the human nervous system such as schwannomas and meningiomas. The NF2 gene encodes a protein called schwannomin or merlin, which is involved in regulating cell growth and proliferation through protein–protein interactions with various cellular proteins. In order to better understand the mechanism by which merlin exerts its function, yeast two-hybrid screening was performed and Ral guanine nucleotide dissociation stimulator (RalGDS), a downstream molecule of Ras, was identified as a merlin-binding protein. The direct interaction between merlin and RalGDS was confirmed both in vitro and in the NIH3T3 cells. The domain analyses revealed that the broad C-terminal region of merlin (aa 141–595) is necessary for the interaction with the C-terminal Ras-binding domain (RBD) of RalGDS. Functional studies showed that merlin inhibits the RalGDS-induced RalA activation, the colony formation and the cell migration in mammalian cells. These results suggest that merlin can function as a tumor suppressor by inhibiting the RalGDS-mediated oncogenic signals.

Merlin facilitates ubiquitination and degradation of transactivation-responsive RNA-binding protein

The Nf2 tumor suppressor codes for merlin, a protein whose function is largely unknown. We have previously demonstrated a novel interaction between merlin and TRBP, which inhibits the oncogenic activity of TRBP. In spite of the significance of their functional interaction, its molecular mechanism still remains to be elucidated. In this report, we investigated how merlin inhibits the oncogenic activity of TRBP in association with cell growth conditions. In the human embryonic kidney 293 cell line, the level of endogenous merlin increased, whereas that of endogenous TRBP significantly decreased along with the increase in cell confluence. We demonstrated that the carboxyl-terminal region of TRBP was responsible for this phenomenon using stable cell lines expressing deletion mutants of TRBP. The overexpression of merlin decreased the protein level of TRBP, and the ubiquitin-like subdomain of merlins FERM domain was important for this activity. We also demonstrated that TRBP is ubiquitinylated and the ubiquitinylated forms of TRBP are accumulated by ectopically expressed merlin or cell confluence in the presence of MG132, a proteasome inhibitor. Furthermore, we showed that the regulation of TRBP in response to cell confluence was abolished upon knockdown of merlin expression by specific small interfering RNA. Finally, we showed that ectopically expressed merlin restored cell–cell contact inhibition in cells stably expressing TRBP but not in TRBPΔc. These results suggest that merlin is involved in the regulation of TRBP protein level by facilitating its ubiquitination in response to such cues as cell–cell contacts.

Tumor necrosis factor-α gene promoter polymorphism in coal workers’ pneumoconiosis

Tumor necrosis factor-alpha (TNF-a) is believed to play a central role in the pathogenesis of pneumoconiosis. TNF2, a polymorphism in the TNF-a gene promoter, has been associated with an increase in TNF-a production and airway inflammation. To investigate the frequency of TNF2 in patients who have coal workers’ pneumoconiosis (CWP) and to determine whether it is associated with development of a large opacity in CWP, we investigated the expression of the TNF2 allele in 80 patients who had CWP and in 54 healthy controls using restriction fragment length polymorphism (RFLP). Compared to controls (10.2%), the frequency of the TNF2 allele was greater in the CWP patients (20.6%). Furthermore, the TNF2 allele was very common in patients who had a large opacity (28.2%) in comparison with 13.4% in those with simple CWP. From these data, we suggest that the TNF2 allele is associated with the development of a large opacity in CWP. (Mol Cell Biochem 234/235: 205–209, 2002)

Different sensitivity to nephrotoxic agents and osmotic stress in proximal tubular and collecting duct cell lines derived from transgenic mice.

We established six renal tubular cell lines from definite tubular areas of the kidney of transgenic mice harboring tsSV40 large T-antigen gene. Three are proximal tubular cell lines prepared from the S(1), S(2) and S(3) segments of the proximal tubule and the others are collecting duct cell lines obtained from cortical, outer medullary and inner medullary collecting ducts (CCD, OMCD and IMCD, respectively). To verify the growth properties of these cell lines under different temperature conditions (33 and 39 degrees C), two representative cells were chosen from the proximal tubule (S(1) cells) and from the collecting duct (IMCD cells). From these cells, a daily change in cell number was evaluated as a parameter of cell growth. As might be expected, cell numbers of these cells increased only at 33 degrees C. Similar patterns were also observed with the other cell lines. To observe the different sensitivity to nephrotoxic agents in proximal tubular cell lines, the cells were exposed to nephrotoxic agent, gentamicin, ochratoxin A or cisplatin. Gentamicin (1 mg/ml) dose-dependently decreased cellular ATP levels of the S(1) cells only. In contrast, the effect of ochratoxin A (10(-6) M) was most pronounced in the S(2) cells, and that of cisplatin (10 microg/ml) in the S(3) cells. To characterize collecting duct cell lines, a hyperosmotic challenge of 700 or 1100 mOsm/l was applied to the cells. At an isoosmotic condition of 300 mOsm/l, the number of cells from the collecting ducts, regardless of their origin, increased continuously during the culture period of 4 days. At an osmotic concentration of 700 mOsm/l, the number of CCD cells decreased, while OMCD cells showed a gradual but a significant increase in cell numbers throughout the culture period. IMCD cells, however, proliferated even at a concentration as high as 1100 mOsm/l, although an initial decrease in cell number was noted on the first day of culture. For confirmation of intracellular free calcium ([Ca(2+)](i)) mobilization, cells were treated with ATP and bradykinin. The [Ca(2+)](i) was increased significantly and immediately by ATP (10(-4) M) in S(1) cells and bradykinin (10(-7) M) in IMCD cells. From the results obtained, it is indicated that renal tubular cell lines from transgenic mice have different sensitivities to nephrotoxic or osmotic stress showing the conservation of the functional characters of the definite part it originated from.

Cellular distribution of isozymes of protein kinase C in septal olfactory epithelium of mice

To determine the presence of protein kinase C (PKC) isozymes in the septal olfactory epithelium of mice (mSOE), western blotting and immunohistochemistry were performed using antibodies against PKC isozymes. With the exception of PKC-betaI, all of the PKC isozymes were detected in the whole lysate of septal tissue layer and apparent molecular weights for each isoform were found. PKC-alpha, PKC-gamma and PKC-epsilon were detected in the olfactory glandular cells of the lamina propria, and PKC-betaI and PKC-betaII were located in the microvillar cells. Neither novel PKC nor atypical PKC was detected in olfactory glandular cells or microvillar cells, except for PKC-epsilon. PKC-lambda was localized in the mucous layer of the mSOE. Meanwhile, PKC-delta and PKC-xi were distributed in the receptor cells in the mSOE. These data demonstrate the isoform-specific expression of PKC in mSOE and suggest a role for the novel and atypical types of PKC in olfactory transduction.