Chernet BT and Levin M (2013), Transmembrane voltage potential is an essential...

XB-ART-46781

Dis Model Mech 2013 May 01;63:595-607. doi: 10.1242/dmm.010835.

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Transmembrane voltage potential is an essential cellular parameter for the detection and control of tumor development in a Xenopus model.

Chernet BT , Levin M .

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Understanding mechanisms that orchestrate cell behavior into appropriately patterned tissues and organs within the organism is an essential element of preventing, detecting and treating cancer. Bioelectric signals (resting transmembrane voltage potential gradients in all cells) underlie an important and broadly conserved set of control mechanisms that regulate pattern formation. We tested the role of transmembrane potential in tumorigenesis mediated by canonical oncogenes in Xenopus laevis. Depolarized membrane potential (Vmem) was a characteristic of induced tumor-like structures (ITLSs) generated by overexpression of Gli1, Kras(G12D), Xrel3 or p53(Trp248). This bioelectric signature was also present in precursor ITLS sites. Vmem is a bioelectric marker that reveals ITLSs before they become histologically and morphologically apparent. Moreover, voltage was functionally important: overexpression of hyperpolarizing ion transporters caused a return to normal Vmem and significantly reduced ITLS formation in vivo. To characterize the molecular mechanism by which Vmem change regulates ITLS phenotypes, we performed a suppression screen. Vmem hyperpolarization was transduced into downstream events via Vmem-regulated activity of SLC5A8, a sodium-butyrate exchanger previously implicated in human cancer. These data indicate that butyrate, a histone deacetylase (HDAC) inhibitor, might be responsible for transcriptional events that mediate suppression of ITLSs by hyperpolarization. Vmem is a convenient cellular parameter by which tumors induced by human oncogenes can be detected in vivo and represents a new diagnostic modality. Moreover, control of resting membrane potential is functionally involved in the process by which oncogene-bearing cells depart from normal morphogenesis programs to form tumors. Modulation of Vmem levels is a novel and promising strategy for tumor normalization.

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Species referenced: Xenopus laevis
Genes referenced: cacna1b gli1 hdac3 kcnj10 kras mrc1 rel slc5a8 slc6a4l tbx2 tp53
GO keywords: ion transmembrane transport [+]

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	Fig. 1. Overexpression of canonical oncogenes or a mutant tumor suppressor results in the formation of ITLSs in Xenopus laevis embryos. (A) Unperturbed embryo showing normal development at stage 34. (B) Embryos showing the presence of ITLS (red arrowheads). To generate ITLSs, 16-cell stage embryos were injected with 0.5 ng of Xrel3, Gli1, KrasG12D or p53Trp248 mRNA into a single blastomere. Injected embryos were raised to tail-bud stages (∼280), scored for the presence of ITLSs and imaged using brightfield microscopy. Scale bars: 1 mm.
	Fig. 2. Induced foci exhibit hallmark characteristics of tumors. (A,B) To analyze proliferation in ITLS regions, embryos were fixed, embedded in agarose, sectioned (as shown in the schematic), and processed for immunohistochemistry with anti-H3B-P antibody: (A) Control section and (B) section through ITLS showing signals of H3B-P-positive cells. The regions traced in white dashes and denoted by ois a region dorsal to the gut; the region traced in yellow dashes is an ITLS region. (C) Quantification of H3B-P signals per pixel area showed significantly higher numbers of proliferative cells in ITLS regions than in dorsal-to-the-gut regions of the same sections or control embryo sections (n=9); P<0.01, *P<0.001 (one-way ANOVA, Bonferroni post hoc analysis). (D) Unperturbed embryo sectioned through the red axis (see schematic); (E) Xrel3-induced ITLS sectioned through the same axis. High-magnification analysis reveals that ITLS (red arrowheads) induced by our method can affect not only the epidermis but can also form in internal tissues of the animal (red circular traces). (F) Xrel3-induced ITLS in the tail region showing the ability to attract vasculature (blue arrowheads), as would most real tumors. Red arrowheads represent normal tail vasculature. Scale bars: 100 μm (A,B,D,E); 500 μm (F).
	Fig. 3. ITLSs exhibit depolarized Vmem. (A) Brightfield image of a Gli1-induced ITLS (outlined in yellow dashes) on the tail of a stage 40 embryo. (B) Depolarization of ITLSs regions was observed when the same embryo was imaged in DiBAC4(3), a fluorescent reporter of Vmem, using the FITC filter set of a fluorescent microscope. (C) Overlay of brightfield and DiBAC4(3) images showing a depolarized Vmem signature exclusive to the ITLS region. Scale bars: 1 mm.
	Fig. 4. Depolarization predicts the formation of Xrel3 ITLSs. (A) Experimental design for predictive testing: 0.5 ng of Xrel3 mRNA was injected into a single blastomere of 16-cell stage embryos; injected embryos were raised to stage 15 and imaged with the voltage-sensitive dye pair DiBAC4(3) and CC2-DMPE using fluorescent confocal microscopy; imaged embryos were sorted into two categories based on the presence (or lack of) depolarized foci; and embryos were raised to stage 35 and scored for ITLSs to assess success rate of depolarization as a predictor of ITLS. Data were used to calculate true positives (cases in which depolarized signal was present and an ITLS formed), false positives (cases in which depolarized signal was present but no ITLS formed), false negatives (ITLS formation that was not preceded by depolarized signal), and sensitivity and specificity values (calculated as described in Materials and Methods). (B) Images showing positive outcome of predictive testing. At stage 15 (left), when there are still no histological or morphological signs of neoplasm, Xrel3-injected embryos show a depolarized area (red arrowhead), which later forms an ITLS at stage 34 (right). Depolarization along the neural tube (yellow arrowhead) is a bioelectric signature of neurulation (Vandenberg et al., 2011). Scale bars: 150 μm (left); 1 mm (right). (C) Depolarized foci were present in a significantly higher number of embryos injected with the oncogene mRNA (χ2 test compared with uninjected embryos, *P<0.001), >51% of which (red bar) formed morphologically apparent ITLSs by stage 35 (χ2 test compared with uninjected embryos, #P<0.001). Although depolarization was present in a small percentage of embryos injected with control mRNAs, no ITLSs were observed.
	Fig. 5. Hyperpolarization suppresses the formation of Xrel3 ITLSs. (A) To visualize and track ITLSs, mRNA encoding a fusion construct of Xrel3 and tdTomato was injected into a single blastomere of 16-cell stage embryos. (Ai,Aii) ITLSs resulting from Xrel3-2A-tdTomato injections were highly fluorescent when visualized under a TRITC filter set. (Aiii) Overlay of brightfield (BF) and TRITC images shows colocalization of ITLS and red fluorescent signal, confirming that foci of oncogene expression are the cells that form ITLS. (B) To test whether hyperpolarization (driving depolarized Vmem back to the normal state) suppresses ITLS formation, a constitutively open, glycine gate chloride channel (GlyRF99A-GFP3) was co-injected with Xrel3-2A-tdTomato into a single blastomere of a 16-cell stage embryo. (Bi) Morphologically apparent ITLSs were often missing from these embryos. (Bii,Biii) Nevertheless, there was expression of Xrel3 protein evidenced by the presence of tdTomato signal. (Biv,Bv) Expression of hyperpolarizing channel protein (GlyRF99A-GFP3) was driven in the same region (Bvi) as Xrel3-2A-tdTomato expressing cells. (C) Fold change in ITLS for embryos co-injected with Xrel3 plus channel mRNA compared with Xrel3-only injected embryos. Xrel3-only injected embryos with ITLS have a ratio of one; ratio values below and above one represent fewer and more embryos with ITLS, respectively. Two very different hyperpolarizing channels (based on Cl− and K+) both resulted in suppression of ITLSs, demonstrating that this effect is due to change in Vmem and not tied to one channel protein or one ion. Fold decreases of 1.54 and 1.62 in the number of embryos were observed for Kir4.1- (K+ hyperpolarizing channel) and GlyRF99A-injected embryos, respectively (one sample t-test, *P<0.01 compared with a normalized value of 1 that represents ITLS incidence in Xrel3-only injected embryos). The number of embryos with ITLS was also increased (1.4-fold) by further depolarizing Xrel3-only injected embryos via ivermectin (one sample t-test, P=0.05 compared with Xrel3-only injected embryos). Scale bars: 1 mm
	Fig. 6. Suppression screen is employed to probe biophysical mechanisms that transduce Vmem changes into transcriptional responses. (A) Hyperpolarization can be transduced into transcriptional events and cell behavior changes by processes including calcium signaling via voltage-gated Ca2+ channels, Vmem-dependent transport of signaling molecules (e.g. serotonin), electrophoresis of morphogens through gap junctions and Vmem-guided movement of butyrate (tumor suppressor) via SLC5A8 (Levin, 2007; Levin, 2012a). 5HT, serotonin; GJC, gap junction; VGGC, voltage-gated calcium channels; SLC5A8, sodium/butyrate exchanger. To identify a biophysical transduction mechanism responsible for hyperpolarization-mediated tumor suppression, these processes were independently blocked in the ion channel ITLS suppression assay using pharmacological or molecular reagents: 10 μM fluoxetine, which blocks the voltage-gated serotonin transporter SERT; 1.7 mM lindane, which blocks GJC; 0.1 mM cadmium chloride; and 1 ng Vito-C mRNA injection, which inactivates native SLC5A8 transporters. (B) Fold change in the number of embryos with ITLSs: the number of embryos injected only with KrasG12D that exhibited ITLSs was normalized to 1. The embryos co-injected with Kir4.1 show a 1.3-fold decrease in the number of embryos with ITLS (one sample t-test, *P<0.01 compared with a normalized value of 1 that represents ITLS incidence in KrasG12D-only injected embryos). The observed significant decrease (ITLS suppression) was blocked only when Vito-C was injected together with KrasG12D and Kir4.1, implicating native SLC5A8 transporters in Vmem-mediated suppression of ITLS.
	Fig. 7. Model for Vmem-mediated control of cell dynamics during normal development and neoplastic transformation. (A) In unperturbed embryos, polarized Vmem is generated and maintained by several ion channels and pumps present in the plasma membrane; this condition allows moderate amounts of butyrate to influx through SLC5A8 and inhibits HDACs. This epigenetically regulates transcription machinery, thereby maintaining baseline level proliferation and differentiation compatible with normal somatic morphostasis. (B) Expression of oncogenes, or other physiological events [e.g. non-genetically induced depolarization (Morokuma et al., 2008; Blackiston et al., 2011)], results in the inability of SLC5A8 to import butyrate. Higher HDAC activity then leads to overproliferation and other neoplastic changes leading to appearance of tumor-like structures. (C) The effect can be blocked by forced hyperpolarization via molecular and/or pharmacological targeting of H+, K+ or Cl− ion translocators. Forced hyperpolarization of the overall transmembrane potential efficiently powers the uptake of Na+ through SLC5A8. This energetically favorable intake of Na+ drives the inward flux of butyrate through SLC5A8. High levels of butyrate continually block HDAC, which leads to hyperacetylation of important genes, resulting in cell cycle arrest and suppression of ITLS formation.
	Fig. S1. Vmem changes quantified via electrophysiology. Fertilized 1-cell stage embryos were divided into five treatment categories: uninjected controls in 0.1x MMR, uninjected controls in high Cl-, GlyRF99A injected embryos in high Cl-, Xrel3 injected embryos, and Xrel3 and GlyRF99A co-injected embryos in high Cl-. Standard electrophysiology was used to measure Vmem from embryos in each category: uninjected control group in 0.1x MMR averaged −22.6 mV (n=5), uninjected controls in high Cl- averaged −25.6 mV (n=5), and GlyRF99A injected embryos, when compared to the two control groups, showed a significantly hyperpolarized Vmem (−42.06 mV; n=5). Consistent with data obtained via voltage-sensitive dye imaging, Xrel3-injected embryos are significantly depolarized (average of −11.4 mV; n=5) compared to controls, while co injecting the GlyRF99A channel together with Xrel3 significantly counteracts the depolarization (average of −24.7 mV; n=12). (One-way ANOVA with Bonferroni post hoc analysis, ***P<0.001).
	Fig. S2. Hyperpolarization is detected by the voltage sensitive dye CC2-DMPE. (A) Schematic of forcing hyperpolarization by a constitutively open Cl- channel: embryos were co-injected with GlyRF99A mRNA and dextran-conjugated Alexa Fluor 555 (lineage dye) into 1 cell of a 2-cell embryo, allowing the uninjected side to serve as an internal control. Embryos were raised to stage 18 in a high (70 mM) Cl- medium. (B) At stage 18, embryos were soaked in 5 μM CC2 dye in 0.1x MMR, and imaged using a CC2 filter set (EX 405/20; BS 425; EM 460/50). The injected left half of the embryo was highly fluorescent, indicating relative hyperpolarization compared to the right half of the embryo. (C) Imaging of the same embryo using a TRITC filter set confirmed the presence of injected mRNA mix, evidenced by the red fluorescent signal of the lineage tracer. (D) Overlay of CC2 and TRITC images showing the co-localization of hyperpolarization signal and hyperpolarizing channel mRNA.

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