Sphingosylphosphorylcholine (SPC) is an important bioactive molecule, involved in many cellular processes. We have recently identified two specific, high-affinity receptors for SPC (OGRI and GPR4). In Swiss 3T3 cells. SPC induces growth inhibition through OGR1 and growth stimulation through GPR4. In HEY ovarian cancer cells, SPC induces inhibition of both growth and Akt kinase activity. Our overall objective is to investigate the signaling mechanisms controlling the different cellular effects induced by SPC through its receptors. We hypothesize that p38 MAP kinase plays a critical role in mediating the growth inhibitory effect of SPC. p38 is differentially regulated through different SPC receptors. Inhibition of Akt may be involved in growth inhibition and apoptosis. These hypotheses will be achieved through two Specific Aims:
Specific Aim 1. To determine the role and regulatory mechanisms of p38 MAP kinase and Akt in the growth inhibitory/stimulatory effects of SPC through OGR1 and GPR4 in Swiss 3T3 cells. We will 1.1) identify the respective G protein(s) coupled to OGR1 and GPR4, those that mediate the growth inhibitory and stimulatory effects of SPC; 1.2) investigate whether p38 MAP kinase is necessary and/or sufficient for SPC-induced growth inhibition; 1.3) determine the role of ERK in SPC-induced growth regulation and p38 activation; 1.4) examine the role of PLC-PKC in SPC-induced growth regulation; and 1.5) determine whether Akt is differentially regulated by GPR4 and OGR1 and its role in SPC-induced growth regulation.
Specific Aim 2. To determine the role and signaling mechanisms of p38 MAP kinase and Akt in growth and cell survival regulated by SPC in HEY ovarian cancer cells. We postulate that SPC inhibits Akt activation by inhibiting phosphorylation at T308, leading to either inhibition of growth, prevention of cell survival, and/or induction of apoptosis. We will test this hypothesis by 2.1) determining whether ERK, p38, PLC, and PKC are required for the growth inhibitory effect of SPC; 2.2) investigating the signaling mechanisms of SPC-induced inhibition of the Akt kinase activity and determining whether Akt kinase inhibition is involved in SPC-induced growth inhibition; and 2.3) determining whether SPC-induced Akt activation induces chemo-sensitization. The focus of this study is to determine how p38 MAP kinase and Akt are differentially regulated in Swiss 3T3 and HEY cells by SPC through its two receptors and how these differential activations of p38 and Akt lead to distinct cellular effects. Our long-term goals are to understand the physiological and pathological roles of SPC and its receptors and to discover new target(s) or signaling pathways affecting tumor cell growth and/or metastasis. These studies may lead to novel therapies to treat highly malignant ovarian cancer, as well as other tumors.
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