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The Role of TRPM7 and Ion Homeostasis in Cell Growth and Proliferation
by
Omayra Gonzalez-Pagan
B.S. University of Puerto Rico - May 2007
Thesis Advisor: Loren Runnels, PhD
Graduate Program in Cellular & Molecular Pharmacology
Pharmacology Department 4th floor Conference Room
RWJMS Research Tower
Piscataway
Monday, December 3, 2012
2:30 p.m.
Abstract
TRPM7 is a ubiquitously expressed channel-kinase permeable to a range of divalent metal cations, with a permeability sequence of Zn2+~Ni2+>>Ba2+>Co2+>Mg2+¡İMn2+¡İSr2+¡İCd2+¡İCa2+. The channel¡¯s unusual permeability profile raises the possibility that it may be regulating the cellular homeostasis of one or more of these essential cations. Knockout of TRPM7 in the DT40 B cell line uncovered an essential role for the channel in cell proliferation and cellular Mg2+ homeostasis. Despite these advances, TRPM7¡¯s role in Mg2+ homeostasis as well as the mechanism(s) by which the channel-kinase influences cell proliferation still remains controversial, as conditional knockout of TRPM7 in other cell types fails to produce any changes in cell proliferation or in Mg2+ homeostasis. To better understand the role of the channel in ion homeostasis we¡¯ve generated recombinant adenoviruses that express TRPM7 channel-dead (TRPM7-E1047K) and TRPM7 channel/kinase-dead (TRPM7-E1047K/G1618D) mutants. Expression of these mutants in mouse embryonic fibroblasts (MEFS) potently inhibits cell proliferation and arrests cells in the G2-phase of the cell cycle. In addition, expression of the mutants lowers cellular free Mg2+ and Zn2+ uptake. Consistent with a role for TRPM7 in Mg2+ and Zn2+ homeostasis, overexpression of the Mg2+ transporter SLC41A2 or supplementation of cell growth medium with high Mg2+ or Zn2+ stimulates the growth of cells expressing the TRPM7 mutants. Expression of the TRPM7 mutants also interferes with mTORC1 signaling, causing a decrease in phosphorylation of ERK, AKT and mTORC1 substrates. To confirm these results we employed MEFS derived from heterozygous conditional channel-dead TRPM7-E1047K mice (TRPM7-E1047K-MEFS), which are capable of expressing the TRPM7-E1047K mutant in Cre recombinase dependent manner. Viral transduction of Cre recombinase into TRPM7-E1047K-MEFS inhibited cell proliferation compared to wildtype MEFS transduced with Cre. In addition, supplementation of the cell growth medium with high Mg2+ or Zn2+ suppressed the proliferation defect observed in TRPM7-E1047K-MEFS transduced with Cre. We conclude that TRPM7 is playing a critical role in both Mg2+ and Zn2+ cellular homeostasis and that the channel¡¯s regulation of these cations is vital to its ability to control cell growth and proliferation.
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