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THE ROLE OF TRPM7 IN CELL STRESS

by
Hsiang-Chin Chen
M.S., NATIONAL YANG-MING UNIVERSITY
TAIPEI, TAIWAN - 2004

Thesis Advisor Loren W. Runnels, Ph.D.
Graduate Program Cellular & Molecular Pharmacology

RWJMS Research Tower
4th floor Conference Room
Piscataway

Monday, December 19, 2011
10:30 a.m.


Abstract

TRPM7 is a remarkable ion channel in possession of its own functional kinase domain. A member of the transient receptor potential melastatin-like (TRPM) ion channel subfamily, the ubiquitously expressed TRPM7 is divalent permeable, allowing Mg2+ and Ca2+ as well as various trace metal ions to compose its small inward current. Various physiological as well as pathological functions have been ascribed to the channel-kinase, including Mg2+ and rare metal ion homeostasis, cell proliferation, melanophore maturation, sensing of sheer stress, synaptic vesicle fusion, thymopoiesis, gastrulation, polarized cell movements and ischemic brain injury.

Previous studies have demonstrated that depletion of the TRPM7 protein in rat hippocampus reduced ischemia-induced neuronal cell death. However, the precise mechanism by which the channel contributes to the demise of cells has remained unclear. We’ve been investigating the process by which TRPM7 contributes to cell death. An unexpected outgrowth of these studies has been the identification of 5-lipoxygenase inhibitors NDGA, AA861 and MK886 as TRPM7 channel blockers. Application of the blockers diminished the amount of cell death that occurred in response to a range of apoptotic stimuli, thereby demonstrating the feasibility of increasing cell survival through pharmacological blockade of the TRPM7 channel.

Cellular loss-of-function and gain-of-function experiments were pursued to further uncover the mechanisms by which the TRPM7 channel affects cell survival. Our results demonstrate a pivotal role for the channel and its control of cellular Mg2+ in the production of reactive oxygen species (ROS) during cell stress. Knockdown of TRPM7 in Swiss 3T3 fibroblasts as well as HEK-293 cells increased cellular viability in response to a range of apoptotic stimuli. Depletion of TRPM7 decreased apoptotic signaling as revealed by reduced cleavage of PARP and caspase-3. Moreover, knockdown of TRPM7 diminished the activation of stress-activated protein kinases (SAPKs) p38 and JNK in response to cell stress and increased the activation of the pro-survival molecule ERK. TRPM7-knockdown fibroblasts have lower levels of ROS than control cells in the absence of cell stress as well as in response to apoptotic stimuli. The decrease in ROS caused by depletion of the channel was reversed by re-expression of TRPM7 in knockdown cells. Surprisingly, re-expression of the Mg2+ transporter SLC41A2 in TRPM7 knockdown cells was equally effective in restoring levels of ROS as well as in re-sensitizing the cells to apoptotic stimuli. These results indicate that TRPM7 plays a central role in a cell stress, uncovering for the first time, Mg2+ as a key determinant of ROS production.


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