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Lipopolysaccharide-Induced Apoptosis in Transformed Bovine Brain Endothelial Cells and Human Dermal Microvessel Endothelial Cells: The Role of JNK¹

Hisae Karahashi^*,, Kathrin S. Michelsen^*, and Moshe Arditi^2,*

^* Division of Pediatrics Infectious Diseases and Immunology, Immunobiology Research Institute, Women’s Cancer Research Institute, and Inflammatory Bowel Disease Center, Cedars-Sinai Medical Center, David Geffen School of Medicine, University of California Los Angeles, CA 90048

Stimulation of transformed bovine brain endothelial cells (TBBEC) with LPS leads to apoptosis while human microvessel endothelial cells (HMEC) need the presence of cycloheximide (CHX) with LPS to induce apoptosis. To investigate the molecular mechanism of LPS-induced apoptosis in HMEC or TBBEC, we analyzed the involvement of MAPK and PI3K in TBBEC and HMEC. LPS-induced apoptosis in TBBEC was hallmarked by the activation of caspase 3, caspase 6, and caspase 8 after the stimulation of LPS, followed by poly(ADP-ribose) polymerase cleavage and lactate dehydrogenase release. We also observed DNA cleavage determined by TUNEL staining in TBBEC treated with LPS. Herbimycin A, a tyrosine kinase inhibitor, and SP600125, a JNK inhibitor, suppressed the activation of caspases and lactate dehydrogenase release. Moreover, a PI3K inhibitor (LY294002) suppressed activation of caspases and combined treatment with both SP600125 and LY294002 completely inhibited the activation of caspases. These results suggest that the JNK signaling pathway through the tyrosine kinase and PI3K pathways is involved in the induction of apoptosis in LPS-treated TBBEC. On the other hand, we observed sustained JNK activation in HMEC treated with LPS and CHX, and neither ERK1/2 nor AKT were activated. The addition of SP600125 suppressed phosphorylation of JNK and the activation of caspase 3 in HMEC treated with LPS and CHX. These results suggest that JNK plays an important role in the induction of apoptosis in endothelial cells.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grants AI 067995, AI 058128, and HL66436 (to M.A.).

² Address correspondence and reprint requests to Dr. Moshe Arditi, Division of Pediatrics Infectious Diseases and Immunology, Immunobiology Research Institute, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Room 4220, Los Angeles, CA 90048. E-mail address: moshe.arditi{at}cshs.org

³ Abbreviations used in this paper: EC, endothelial cell; HMEC, human microvessel EC; TBBEC, transformed bovine brain EC; Ac-DEVD-AMC, acetyl-DEVD-aminomethyl coumarin; Ac-DEVD-CHO, acetyl-DEVD-aldehyde; Ac-VEID-AMC, acetyl-VEID-aminomethyl coumarin; Ac-YVAD-AMC, acetyl-YVAD-aminomethyl coumarin; Ac-YVAD-CMK, acetyl-YVAD-chloromethyl ketone; AMC, 7-amino-4-methyl coumarin; CHX, cycloheximide; LDH, lactate dehydrogenase; PARP, poly(ADP-ribose) polymerase; Z-Asp-CH₂-DCB, carbobenzoxy-L-aspart-1-yl-[(2,6-dichlorobenzoyl)oxy]methane; Ac-IETD-AMC, acetyl-IETD- aminomethyl coumarin; Ac-WEHD-AMC, acetyl-WEHD- aminomethyl coumarin; ICE, IL-1β-converting enzyme.