Sphingolipid regulation of apoptosis through the mitogen -activated protein kinase pathways in C3H10T ½ cells

Sharon Sianna Castillo, Purdue University

Abstract

The purpose of the present study was to determine how sphingolipids regulate apoptosis in C3H10T ½ (10T ½) murine fibroblasts. The sphingolipids ceramide and sphingosine-1-phosphate (SSP) are important lipid signaling molecules involved in cell growth, differentiation, apoptosis, and cell survival. Sphingolipids have potential anticancer properties and are found in a variety of foods. Thus understanding the molecular mechanisms of these lipids may aid in the prevention of cancer. Ceramide induces apoptosis through activation of the stress activated protein kinase (SAPK) pathway in many cell types, yet does not in 10T ½ cells. SAPK is a member of the mitogen activated protein kinase (MAPK) family, which also includes the extracellular signal-regulated protein kinase (ERK). Both ERK and SAPK activity are down-regulated by the MAPK phosphatase-1 (MKP-1). The hypothesis of this project is that 10T ½ cells are resistant to ceramide induced apoptosis due to the conversion of ceramide to SSP, which then inhibits SAPK activity via MKP-1. Labeling studies demonstrated ceramide was converted to SSP and this conversion is essential for cell survival. Either preventing the conversion of ceramide to SSP or the use of a non-metabolizable analog of ceramide, ceramine (CN), resulted in an apoptotic response. Apoptosis was inhibited in both conditions by the addition of SSP. To determine the mechanism by which SSP prevented apoptosis, ERK and SAPK activities were analyzed. CN alone and in combination (CB) with SSP significantly increased SAPK activation. Only CB treatment significantly increased ERK activity and increased MKP-1 levels. ERK activation was required for SSP to prevent CN induced apoptosis for when an inhibitor blocked ERK activation, the cells underwent apoptosis. Similarly, MKP-1 was essential for the inhibition of apoptosis for in the presence of a dominant negative MKP-1 mutant with CB treatment, the cells underwent apoptosis. Further studies with the MKP-1 mutant revealed MKP-1 preferentially down-regulates the SAPK pathway over the ERK pathway in 10T ½ cells. Therefore, 10T ½ cells are resistant to ceramide-induced apoptosis due to the cells' metabolism to SSP. Once formed, SSP prevents apoptosis through the activation of ERK and increases in MKP-1 levels, which down-regulates the SAPK pathway.

Degree

Ph.D.

Advisors

Teegarden, Purdue University.

Subject Area

Nutrition|Cellular biology

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