Eui Yul Choi

Induction of matrix metalloproteinase-9 (MMP-9) in lipopolysaccharide-stimulated primary astrocytes is mediated by extracellular signal-regulated protein kinase 1/2 (Erk1/2).

In the present study, we investigated whether the activation of protein kinase C (PKC) and extracellular signal‐regulated kinase 1/2 (Erk1/2) are involved in the induction of MMP‐9 in lipopolysaccharide (LPS)–stimulated primary astrocytes. The expression of MMP‐9 but not MMP‐2 was increased by LPS. LPS treatment induced activation of Erk1/2 within 30 min, which was dose‐dependently inhibited by PD98059, a specific inhibitor of the Erk kinase (MEK). In this condition, PD98059 blocked the increase in MMP‐9 protein and mRNA level as well as gelatin‐digesting activity. Inhibition of PKC activity blocked the LPS‐induced activation of Erk1/2 as well as MMP‐9 expression. In addition, activation of PKC by phorbol myristoyl acetate (PMA) activated Erk1/2 with concomitant increase in MMP‐9 production. Moreover, treatment of PD98059 dose‐dependently decreased the PMA‐induced MMP‐9 expression. The results from the present study suggest that induction of MMP‐9 by LPS in rat primary astrocytes is mediated, at least in part, by the sequential activation of PKC and Erk1/2. The Erk1/2‐mediated MMP‐9 induction may provide insights into the regulation of MMP‐9 production in CNS, which may occur in vivo in pathological situations such as CNS inflammation. GLIA 41:15–24, 2003.

Calixarene derivative as a tool for highly sensitive detection and oriented immobilization of proteins in a microarray format through noncovalent molecular interaction

One important factor in fabricating protein microarray is to immobilize proteins without losing their activity on a solid phase. To keep them functional, it is necessary to immobilize proteins in a way that preserve their folded structural integrity. In a previous study, we developed novel Calixarene derivatives for the immobilization of proteins on the surface of a glass slide (1). In this study, we compared the sensitivity and the specificity of the linker molecules with those of five other protein attachment agents on glass slides using a prostate‐specific antigen and its antibodies as a model system. The Calixcrown‐coated protein chip showed a superior sensitivity and a much lower detection limit than those chips prepared by other methods. When we tested the capability of Calixcrown to immobilize antibody molecules, it appeared that Calixcrown makes arrangement of antibody be more regular with the vertical orientation than the covalent‐bond agent. We also observed that the Calixcrown chip could be used for the diagnostic application with clinical samples from prostate cancer and HIV patients. Finally, we applied the Calixcrown chip using an antibody microarray to identify up‐ or down‐regulated proteins in specific tissue and detected several up‐ or down‐regulated proteins from a rat liver by administering toxin. Thus, the Calixcrown chip can be used as a powerful tool with a wide range of applications, including protein‐protein interaction, protein‐DNA interaction, and an enzyme activity assay.

Role of p38 MAPK on the down-regulation of matrix metallo-proteinase-9 expression in rat astrocytes

In spite of their pathophysiological importance in neuro-inflammatory diseases, little is known about the signal transduction pathways that lead to the induction of matrix metalloproteinases (MMPs) in the central nervous system. We reported previously that lipopolysaccharide (LPS) induced MMP-9 expression through ERK1/2 pathway in rat primary astrocytes(Glia 41:15–24, 2003). Here, we investigated the role of other MAPK pathways, including p38 and JNK/SAPK, on the regulation of MMP-9 expression in LPS-stimulated rat primary astrocytes. LPS activated both p38 and JNK in astrocytes. Treatment with a specific p38 MAPK inhibitor SB203580, but not JNK inhibitor SP600125, increased the LPS-stimulated MMP-9 expression in a concentration-dependent manner. Anti-inflammatory cytokines, including IFN-γ and IL-4, activated p38 MAPK and decreased MMP-9 production in LPS-stimulated astrocytes. When p38 MAPK activation was blocked by SB203580, the inhibitory effects of these cytokines on MMP-9 induction were abolished. Finally, direct injection of SB203580 into the lateral ventricle of rat brain increased the LPS-induced MMP-9 activity in cerebral cortex. Altogether, these results suggest that p38 activation down-regulates the inflammatory stimulation-induced over-expression of MMP-9, both in primary astrocytes and in cerebral cortex. The elaborate interplay between ERK1/2 and p38 pathways provides a more sophisticated mechanism for regulating MMP-9 activity in neuroinflammatory diseases.

Immunological characterization of a mucin-associated protein from hamster tracheal epithelial cell culture.

Airway mucins are high molecular mass (>10(6) dalton) glycoproteins with various types of associated molecules including glycoproteins, lipoproteins, and lipids. The study of mucin-associated proteins is limited largely due to the lack of specific probes. In this study, we produced a monoclonal antibody, MAbHT10, against a 190-kDa mucin associated-protein by immunizing mice with hamster airway mucin purified in nondissociative condition. Using HT10, the 190-kDa mucin-associated protein was characterized immunologically. The 190-kDa mucin-associated protein is glycoprotein and HT10 recognized carbohydrate containing portion of the protein. The association of 190-kDa protein with mucin is strong enough that heat and detergent treatment is required to dissociate it from mucin as evidenced by gel filtration chromatography, Western blot, enzyme-linked immunoadsorbent assay (ELISA), and co-immunoprecipitation. The expression of the 190-kDa protein is increased with the development of hamster tracheal epithelial cells in culture, but showed differences with the pattern of the regulation of mucin expression. Adenosine triphosphate (ATP), a known strong mucin secretagogue, dose-dependently increased mucin release but caused only marginal increase in the release of the 190-kDa protein. The MAb should be useful in the structural and functional analysis of the 190-kDa mucin-associated proteins in physiological and pathological situations such as chronic airway diseases.

Cross-species immunoreactivity of airway mucin as revealed by monoclonal antibodies directed against mucins from human, hamster, and rat.

Airway mucin plays crucial role in host-defense and has been implicated in pathophysiology of various airway diseases including asthma and cystic fibrosis. The analysis of airway mucin has been hampered mostly by the lack of specific and efficient methods for the detection of mucin. Recent production of antibodies against airway mucin from several species and also the development of immunoassay procedures make it more efficient to study the airway mucin. However, the cross-species immunoreactivity of antibodies against airway mucin has not been clearly demonstrated and this prompted us to investigate the cross-species immunoreactivity of monoclonal antibodies against human (HM02), hamster (HTA), and rat airway mucin (RT03), which is three most widely used species in the study of mucin. All the monoclonal antibodies (MAbs) used in this study is IgM isotype and recognizes N-acetyl-galactosamine-linked carbohydrate core or backbone portion of airway mucin. In enzyme-linked immunoadsorbent assay (ELISA), Western blot, immunoprecipitation, and immunohistochemical staining experiments, it was demonstrated that human and hamster airway mucin showed strong cross-species immunoreactivity. However, rat airway mucin did not show any cross-species immunoreactivity against human and hamster airway mucin. Endotoxin-induced secretory cell metaplasia and hence the increase in mucin release from hamster airway mucin could be detected with antibodies against hamster and human airway mucin in vivo and in vitro. However, the same increase from rat airway could only be detected with antibody against rat airway mucin but not with antibodies against human and hamster airway mucin. In addition, the increase in mucin release from asthmatic patients could be detected with antibodies against human and hamster airway mucin but not with the antibody against rat airway mucin. The data from the present study implicates that the carbohydrate chain of human and hamster airway mucin, but not that of rat airway mucin, share common antigenic structure. In case of the interspecies use of the antibodies against airway mucin, it would be more desirable to clearly identify the cross-species immunoreactivity otherwise might lead to erroneous results.