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Inhibition of ICMT Induces Endothelial Cell Apoptosis through GRP94

Vascular Research Laboratory, Providence Veterans Affairs Medical Center; and Department of Medicine, Brown Medical School, Providence, Rhode Island

Correspondence and requests for reprints should be addressed to Sharon Rounds, M.D., Providence VA Medical Center, Pulmonary/Critical Care Medicine Section, 830 Chalkstone Avenue, Providence, RI 02908. E-mail: Sharon_Rounds{at}brown.edu

Abstract

Isoprenylcysteine-O-carboxyl methyltransferase (ICMT) catalyzes methylation of proteins containing a C-terminal CAAX motif. We have previously shown that chemical inhibition of ICMT caused endothelial cell apoptosis, an effect correlated with decreased Ras and RhoA carboxyl methylation and GTPase activities. In the current study, proteomic analysis of pulmonary artery endothelial cells (PAEC) exposed to the ICMT inhibitor, N-acetyl-geranylgeranyl-cysteine (AGGC), demonstrated a shift in the isoelectric points (pI) of the glucose-regulated protein (GRP) 94. Two-dimensional PAGE and immunoblot analysis further documented that ICMT inhibition caused multiple changes in the pI of GRP94. GRP94 is an endoplasmic reticulum molecular chaperone, a component of the unfolded protein response (UPR), and is involved in apoptosis. Immunofluorescence analyses revealed redistribution and aggregation of GRP94 after 3 h exposure to AGGC. A similar finding was noted with calnexin. In addition, GRP94 protein levels were significantly diminished upon 18 h AGGC exposure or ICMT suppression. The effects of ICMT inhibition on changes in GRP94 subcellular localization and protein content were blunted by overexpression of constitutively active RhoA or a caspase inhibitor. Furthermore, GRP94 depletion augmented endothelial cell apoptosis induced by ICMT inhibition. These results indicate that ICMT inhibition leads to GRP94 relocalization, aggregation, and degradation; effects were dependent upon the activities of RhoA and caspases. We speculate that changes in the pI, subcellular localization, and protein level of GRP94 cause endothelial cell apoptosis, possibly through UPR dysfunction. These studies suggest a novel link between RhoA GTPases and the UPR.

Key Words: ICMT • GRP94 • RhoA GTPase • unfolded protein response • apoptosis

CLINICAL RELEVANCE Endothelial cell apoptosis plays important roles in homeostasis and diseases. This study has potential importance in the treatment of lung diseases in which abnormal endothelial apoptosis occurs, such as emphysema, primary pulmonary hypertension, and cancer.

 

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