TRPM7-mediated spontaneous Ca2+ entry regulates the proliferation and differentiation of human leukemia cell line K562

Kiriko Takahashi, Chisato Umebayashi, Tomohiro Numata, Akira Honda, Jun Ichikawa, Yaopeng Hu, Ken Yamaura, Ryuji Inoue

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Continuous Ca2+ influx is essential to maintain intracellular Ca2+ homeostasis and its dysregulation leads to a variety of cellular dysfunctions. In this study, we explored the functional roles of spontaneous Ca2+ influx for the proliferation and differentiation of a human erythromyeloid leukemia cell line K562. mRNA/protein expressions were assessed by the real-time RT-PCR, western blotting, and immunocytochemical staining. Intracellular Ca2+ concentration ([Ca2+]i) and ionic currents were measured by fluorescent imaging and patch clamping techniques, respectively. Cell counting/viability and colorimetric assays were applied to assess proliferation rate and hemoglobin synthesis, respectively. Elimination of extracellular Ca2+ decreased basal [Ca2+]i in proliferating K562 cells. Cation channel blockers such as SK&F96365, 2-APB, Gd3+, and FTY720 dose dependently decreased basal [Ca2+]i. A spontaneously active inward current (Ispont) contributive to basal [Ca2+]i was identified by the nystatin-perforated whole-cell recording. Ispont permeated Ca2+ comparably to Na+, and was greatly eliminated by siRNA targeting TRPM7, a melastatin member of the transient receptor potential (TRP) superfamily. Consistent with these findings, TRPM7 immune reactivity was detected by western blotting, and immunofluorescence representing TRPM7 was found localized to the K562 cell membrane. Strikingly, all these procedures, that is, Ca2+ removal, TRPM7 blockers and siRNA-mediated TRPM7 knockdown significantly retarded the growth and suppressed hemin-induced γ-globin and hemoglobin syntheses in K562 cells, respectively, both of which appeared associated with the inhibition of ERK activation. These results collectively suggest that spontaneous Ca2+ influx through constitutively active TRPM7 channels may critically regulate both proliferative and erythroid differentiation potentials of K562 cells.

Original languageEnglish
Article numbere13796
JournalPhysiological Reports
Volume6
Issue number14
DOIs
Publication statusPublished - Jul 2018
Externally publishedYes

Fingerprint

K562 Cells
Leukemia
Cell Line
Small Interfering RNA
Hemoglobins
Western Blotting
Nystatin
Hemin
Globins
Patch-Clamp Techniques
Constriction
Fluorescent Antibody Technique
Cations
Real-Time Polymerase Chain Reaction
Cell Survival
Homeostasis
Cell Membrane
Staining and Labeling
Messenger RNA
Growth

All Science Journal Classification (ASJC) codes

  • Physiology
  • Physiology (medical)

Cite this

Takahashi, K., Umebayashi, C., Numata, T., Honda, A., Ichikawa, J., Hu, Y., ... Inoue, R. (2018). TRPM7-mediated spontaneous Ca2+ entry regulates the proliferation and differentiation of human leukemia cell line K562. Physiological Reports, 6(14), [e13796]. https://doi.org/10.14814/phy2.13796

TRPM7-mediated spontaneous Ca2+ entry regulates the proliferation and differentiation of human leukemia cell line K562. / Takahashi, Kiriko; Umebayashi, Chisato; Numata, Tomohiro; Honda, Akira; Ichikawa, Jun; Hu, Yaopeng; Yamaura, Ken; Inoue, Ryuji.

In: Physiological Reports, Vol. 6, No. 14, e13796, 07.2018.

Research output: Contribution to journalArticle

Takahashi, Kiriko ; Umebayashi, Chisato ; Numata, Tomohiro ; Honda, Akira ; Ichikawa, Jun ; Hu, Yaopeng ; Yamaura, Ken ; Inoue, Ryuji. / TRPM7-mediated spontaneous Ca2+ entry regulates the proliferation and differentiation of human leukemia cell line K562. In: Physiological Reports. 2018 ; Vol. 6, No. 14.
@article{8e426515462f437f8710b6f5a93110e3,
title = "TRPM7-mediated spontaneous Ca2+ entry regulates the proliferation and differentiation of human leukemia cell line K562",
abstract = "Continuous Ca2+ influx is essential to maintain intracellular Ca2+ homeostasis and its dysregulation leads to a variety of cellular dysfunctions. In this study, we explored the functional roles of spontaneous Ca2+ influx for the proliferation and differentiation of a human erythromyeloid leukemia cell line K562. mRNA/protein expressions were assessed by the real-time RT-PCR, western blotting, and immunocytochemical staining. Intracellular Ca2+ concentration ([Ca2+]i) and ionic currents were measured by fluorescent imaging and patch clamping techniques, respectively. Cell counting/viability and colorimetric assays were applied to assess proliferation rate and hemoglobin synthesis, respectively. Elimination of extracellular Ca2+ decreased basal [Ca2+]i in proliferating K562 cells. Cation channel blockers such as SK&F96365, 2-APB, Gd3+, and FTY720 dose dependently decreased basal [Ca2+]i. A spontaneously active inward current (Ispont) contributive to basal [Ca2+]i was identified by the nystatin-perforated whole-cell recording. Ispont permeated Ca2+ comparably to Na+, and was greatly eliminated by siRNA targeting TRPM7, a melastatin member of the transient receptor potential (TRP) superfamily. Consistent with these findings, TRPM7 immune reactivity was detected by western blotting, and immunofluorescence representing TRPM7 was found localized to the K562 cell membrane. Strikingly, all these procedures, that is, Ca2+ removal, TRPM7 blockers and siRNA-mediated TRPM7 knockdown significantly retarded the growth and suppressed hemin-induced γ-globin and hemoglobin syntheses in K562 cells, respectively, both of which appeared associated with the inhibition of ERK activation. These results collectively suggest that spontaneous Ca2+ influx through constitutively active TRPM7 channels may critically regulate both proliferative and erythroid differentiation potentials of K562 cells.",
author = "Kiriko Takahashi and Chisato Umebayashi and Tomohiro Numata and Akira Honda and Jun Ichikawa and Yaopeng Hu and Ken Yamaura and Ryuji Inoue",
year = "2018",
month = "7",
doi = "10.14814/phy2.13796",
language = "English",
volume = "6",
journal = "Physiological Reports",
issn = "2051-817X",
publisher = "John Wiley and Sons Inc.",
number = "14",

}

TY - JOUR

T1 - TRPM7-mediated spontaneous Ca2+ entry regulates the proliferation and differentiation of human leukemia cell line K562

AU - Takahashi, Kiriko

AU - Umebayashi, Chisato

AU - Numata, Tomohiro

AU - Honda, Akira

AU - Ichikawa, Jun

AU - Hu, Yaopeng

AU - Yamaura, Ken

AU - Inoue, Ryuji

PY - 2018/7

Y1 - 2018/7

N2 - Continuous Ca2+ influx is essential to maintain intracellular Ca2+ homeostasis and its dysregulation leads to a variety of cellular dysfunctions. In this study, we explored the functional roles of spontaneous Ca2+ influx for the proliferation and differentiation of a human erythromyeloid leukemia cell line K562. mRNA/protein expressions were assessed by the real-time RT-PCR, western blotting, and immunocytochemical staining. Intracellular Ca2+ concentration ([Ca2+]i) and ionic currents were measured by fluorescent imaging and patch clamping techniques, respectively. Cell counting/viability and colorimetric assays were applied to assess proliferation rate and hemoglobin synthesis, respectively. Elimination of extracellular Ca2+ decreased basal [Ca2+]i in proliferating K562 cells. Cation channel blockers such as SK&F96365, 2-APB, Gd3+, and FTY720 dose dependently decreased basal [Ca2+]i. A spontaneously active inward current (Ispont) contributive to basal [Ca2+]i was identified by the nystatin-perforated whole-cell recording. Ispont permeated Ca2+ comparably to Na+, and was greatly eliminated by siRNA targeting TRPM7, a melastatin member of the transient receptor potential (TRP) superfamily. Consistent with these findings, TRPM7 immune reactivity was detected by western blotting, and immunofluorescence representing TRPM7 was found localized to the K562 cell membrane. Strikingly, all these procedures, that is, Ca2+ removal, TRPM7 blockers and siRNA-mediated TRPM7 knockdown significantly retarded the growth and suppressed hemin-induced γ-globin and hemoglobin syntheses in K562 cells, respectively, both of which appeared associated with the inhibition of ERK activation. These results collectively suggest that spontaneous Ca2+ influx through constitutively active TRPM7 channels may critically regulate both proliferative and erythroid differentiation potentials of K562 cells.

AB - Continuous Ca2+ influx is essential to maintain intracellular Ca2+ homeostasis and its dysregulation leads to a variety of cellular dysfunctions. In this study, we explored the functional roles of spontaneous Ca2+ influx for the proliferation and differentiation of a human erythromyeloid leukemia cell line K562. mRNA/protein expressions were assessed by the real-time RT-PCR, western blotting, and immunocytochemical staining. Intracellular Ca2+ concentration ([Ca2+]i) and ionic currents were measured by fluorescent imaging and patch clamping techniques, respectively. Cell counting/viability and colorimetric assays were applied to assess proliferation rate and hemoglobin synthesis, respectively. Elimination of extracellular Ca2+ decreased basal [Ca2+]i in proliferating K562 cells. Cation channel blockers such as SK&F96365, 2-APB, Gd3+, and FTY720 dose dependently decreased basal [Ca2+]i. A spontaneously active inward current (Ispont) contributive to basal [Ca2+]i was identified by the nystatin-perforated whole-cell recording. Ispont permeated Ca2+ comparably to Na+, and was greatly eliminated by siRNA targeting TRPM7, a melastatin member of the transient receptor potential (TRP) superfamily. Consistent with these findings, TRPM7 immune reactivity was detected by western blotting, and immunofluorescence representing TRPM7 was found localized to the K562 cell membrane. Strikingly, all these procedures, that is, Ca2+ removal, TRPM7 blockers and siRNA-mediated TRPM7 knockdown significantly retarded the growth and suppressed hemin-induced γ-globin and hemoglobin syntheses in K562 cells, respectively, both of which appeared associated with the inhibition of ERK activation. These results collectively suggest that spontaneous Ca2+ influx through constitutively active TRPM7 channels may critically regulate both proliferative and erythroid differentiation potentials of K562 cells.

UR - http://www.scopus.com/inward/record.url?scp=85050815958&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85050815958&partnerID=8YFLogxK

U2 - 10.14814/phy2.13796

DO - 10.14814/phy2.13796

M3 - Article

C2 - 30033625

AN - SCOPUS:85050815958

VL - 6

JO - Physiological Reports

JF - Physiological Reports

SN - 2051-817X

IS - 14

M1 - e13796

ER -