Abstract
Ca(2+) and β-catenin, a 92-kDa negatively charged transcription factor, transduce Wnt signaling via the non-canonical, Wnt/Ca(2+) and canonical, Wnt/β-catenin pathways independently. The nuclear envelope is a barrier to large protein entry, and this process is regulated by intracellular calcium [Ca(2+)]i and trans-nuclear potential. How β-catenin traverses the nuclear envelope is not well known. We hypothesized that Wnt/Ca(2+) and Wnt/β-catenin pathways act in a coordinated manner and that [Ca(2+)]i release facilitates β-catenin entry into the nucleus in mammalian cells. In a live assay using calcium dyes in PC3 prostate cancer cells, six Wnt peptides (3A, 4, 5A, 7A, 9B, and 10B) mobilized [Ca(2+)]i but Wnt11 did not. Based upon dwell time (range = 15-30 s) of the calcium waveform, these Wnts could be classified into three classes: short, 3A and 5A; long, 7A and 10B; and very long, 4 and 9B. Wnt-activated [Ca(2+)]i release was followed by an increase in intranuclear calcium and the depolarization of both the cell and nuclear membranes, determined by using FM4-64. In cells treated with Wnts 5A, 9B, and 10B, paradigm substrates for each Wnt class, increased [Ca(2+)]i was followed by β-catenin translocation into the nucleus in PC3, MCF7, and 253J, prostate, breast, and bladder cancer cell lines; both the increase in Wnt 5A, 9B, and 10B induced [Ca(2+)]i release and β-catenin translocation are suppressed by thapsigargin in PC3 cell line. We propose a convergent model of Wnt signaling network where Ca(2+) and β-catenin pathways may act in a coordinated, interdependent, rather than independent, manner.
Highlights
Cellular transducers of Wnt signaling, -catenin and Ca2ϩ, are considered to act via independent canonical and non-canonical pathways
The nuclear envelope is a barrier to large protein entry, and this process is regulated by intracellular calcium [Ca2؉]i and trans-nuclear potential
In cells treated with Wnts 5A, 9B, and 10B, paradigm substrates for each Wnt class, increased [Ca2؉]i was followed by -catenin translocation into the nucleus in PC3, MCF7, and 253J, prostate, breast, and bladder cancer cell lines; both the increase in Wnt 5A, 9B, and 10B induced [Ca2؉]i release and -catenin translocation are suppressed by thapsigargin in PC3 cell line
Summary
Cellular transducers of Wnt signaling, -catenin and Ca2ϩ, are considered to act via independent canonical and non-canonical pathways. Activation of the non-canonical, -catenin-independent, Wnt/Ca2ϩpathway results in the mobilization of free intracellular calcium [Ca2ϩ]i [11] that regulates many cellular processes [12, 13], including cytoskeleton and cell motility of prostate cancer cells [14]. By using a live fluorochrome assay and subsequent assessment of -catenin translocation to nucleoplasm in Wnt ligand-activated mammalian cancer cell lines (PC3, prostate, MCF7, breast, and 253J bladder cell lines), we demonstrate that a number of Wnt ligands increase intracellular and intranuclear Ca2ϩ, with distinct kinetics, depolarization of the NE and translocation of -catenin to the nucleus; both the increase in Wnt induced [Ca2ϩ]i release and -catenin translocation are suppressed by thapsigargin, an inhibitor of microsomal Ca2ϩ-ATPase [37]. A model in which Wnt/Ca2ϩ and Wnt/-catenin pathways are coupled rather than independent, as thought previously, is proposed
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