Year of Publication

2009

Degree Name

Doctor of Philosophy (PhD)

Document Type

Dissertation

College

Medicine

Department

Physiology

First Advisor

Dr. Mariana N. Nikolova-Karakashian

Abstract

Acid Sphingomyelinase (ASMase) activity has been proposed to mediate LPS signaling in various cell types. This study shows that in macrophages, ASMase is a negative regulator of LPS-induced TNFα secretion. ASMasedeficient (asm-/-) mice and isolated peritoneal macrophages produce several fold more TNFα than their wild-type (asm+/+) counterparts when stimulated with LPS. The mechanism for these differences however is not transcriptional but post-translational.

The TNFα converting enzyme (TACE) catalyzes the maturation of the 26kD precursor (proTNFα) to the active 17kD form (sTNFα). In mouse peritoneal macrophages, the activity of TACE rather than the rate of TNFα mRNA synthesis was the rate-limiting factor regulating TNFα production. Substantial portion of the translated proTNFα was not processed to sTNFα; instead it was rapidly internalized and degraded in the lysosomes. TACE activity was 2 to 3 fold higher in asm-/- macrophages as compared to asm+/+ macrophages and was suppressed when cells were treated with exogenous ceramide and SMase. In asm-/- but not in asm+/+macrophages, indirect immunofluorescence experiments revealed distinct TNFα-positive structures in close vicinity of the plasma membrane. Asm-/- cells also had higher number of EEA1-positive early endosomes. Co-localization experiments that involved inhibitors of TACE and/or lysosomal proteolysis suggest that in asm-/-cells a significant portion of proTNFα is sequestered within the early endosomes, and instead of undergoing lysosomal proteolysis it is recycled to the plasma membrane and processed to sTNFα.

Share

COinS