Ca2+-Dependent Regulation of NFATc1 via KCa3.1 in Inflammatory Osteoclastogenesis

Eva M. Grössinger, Mincheol Kang, Laura Bouchareychas, Ritu Sarin, Dominik R Haudenschild, Laura N Borodinsky, Iannis Adamopoulos

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

In inflammatory arthritis, the dysregulation of osteoclast activity by proinflammatory cytokines, including TNF, interferes with bone remodeling during inflammation through Ca2+-dependent mechanisms causing pathological bone loss. Ca2+-dependent CREB/c-fos activation via Ca2+-calmodulin kinase IV (CaMKIV) induces transcriptional regulation of osteoclast-specific genes via NFATc1, which facilitate bone resorption. In leukocytes, Ca2+ regulation of NFAT-dependent gene expression oftentimes involves the activity of the Ca2+-activated K+ channel KCa3.1. In this study, we evaluate KCa3.1 as a modulator of Ca2+-induced NFAT-dependent osteoclast differentiation in inflammatory bone loss. Microarray analysis of receptor activator of NF-kB ligand (RANKL)-activated murine bone marrow macrophage (BMM) cultures revealed unique upregulation of KCa3.1 during osteo-clastogenesis. The expression of KCa3.1 in vivo was confirmed by immunofluorescence staining on multinucleated cells at the bone surface of inflamed mouse joints. Experiments on in vitro BMM cultures revealed that KCa3.12/2 and TRAM-34 treatment significantly reduced the expression of osteoclast-specific genes (p < 0.05) alongside decreased osteoclast formation (p < 0.0001) in inflammatory (RANKL+TNF) and noninflammatory (RANKL) conditions. In particular, live cell Ca2+ imaging and Western blot analysis showed that TRAM-34 pretreatment decreased transient RANKL-induced Ca2+ amplitudes in BMMs by ∼50% (p < 0.0001) and prevented phosphorylation of CaMKIV. KCa3.12/2 reduced RANKL+/2TNF-stimulated phosphorylation of CREB and expression of c-fos in BMMs (p < 0.01), culminating in decreased NFATc1 protein expression and transcriptional activity (p < 0.01). These data indicate that KCa3.1 regulates Ca2+-dependent NFATc1 expression via CaMKIV/CREB during inflammatory osteoclastogenesis in the presence of TNF, corroborating its role as a target candidate for the treatment of bone erosion in inflammatory arthritis.

Original languageEnglish (US)
Pages (from-to)749-757
Number of pages9
JournalJournal of Immunology
Volume200
Issue number2
DOIs
StatePublished - Jan 15 2018

Fingerprint

NF-kappa B
Osteoclasts
Osteogenesis
Calcium-Calmodulin-Dependent Protein Kinases
Ligands
Bone and Bones
Arthritis
Tumor Necrosis Factors
Bone Marrow
Macrophages
Phosphorylation
Calcium-Activated Potassium Channels
Bone Remodeling
Microarray Analysis
Bone Resorption
Genes
Fluorescent Antibody Technique
Leukocytes
Up-Regulation
Joints

ASJC Scopus subject areas

  • Immunology

Cite this

Ca2+-Dependent Regulation of NFATc1 via KCa3.1 in Inflammatory Osteoclastogenesis. / Grössinger, Eva M.; Kang, Mincheol; Bouchareychas, Laura; Sarin, Ritu; Haudenschild, Dominik R; Borodinsky, Laura N; Adamopoulos, Iannis.

In: Journal of Immunology, Vol. 200, No. 2, 15.01.2018, p. 749-757.

Research output: Contribution to journalArticle

Grössinger, Eva M. ; Kang, Mincheol ; Bouchareychas, Laura ; Sarin, Ritu ; Haudenschild, Dominik R ; Borodinsky, Laura N ; Adamopoulos, Iannis. / Ca2+-Dependent Regulation of NFATc1 via KCa3.1 in Inflammatory Osteoclastogenesis. In: Journal of Immunology. 2018 ; Vol. 200, No. 2. pp. 749-757.
@article{5604a3e2909a4eda8dcbc3ec91939b34,
title = "Ca2+-Dependent Regulation of NFATc1 via KCa3.1 in Inflammatory Osteoclastogenesis",
abstract = "In inflammatory arthritis, the dysregulation of osteoclast activity by proinflammatory cytokines, including TNF, interferes with bone remodeling during inflammation through Ca2+-dependent mechanisms causing pathological bone loss. Ca2+-dependent CREB/c-fos activation via Ca2+-calmodulin kinase IV (CaMKIV) induces transcriptional regulation of osteoclast-specific genes via NFATc1, which facilitate bone resorption. In leukocytes, Ca2+ regulation of NFAT-dependent gene expression oftentimes involves the activity of the Ca2+-activated K+ channel KCa3.1. In this study, we evaluate KCa3.1 as a modulator of Ca2+-induced NFAT-dependent osteoclast differentiation in inflammatory bone loss. Microarray analysis of receptor activator of NF-kB ligand (RANKL)-activated murine bone marrow macrophage (BMM) cultures revealed unique upregulation of KCa3.1 during osteo-clastogenesis. The expression of KCa3.1 in vivo was confirmed by immunofluorescence staining on multinucleated cells at the bone surface of inflamed mouse joints. Experiments on in vitro BMM cultures revealed that KCa3.12/2 and TRAM-34 treatment significantly reduced the expression of osteoclast-specific genes (p < 0.05) alongside decreased osteoclast formation (p < 0.0001) in inflammatory (RANKL+TNF) and noninflammatory (RANKL) conditions. In particular, live cell Ca2+ imaging and Western blot analysis showed that TRAM-34 pretreatment decreased transient RANKL-induced Ca2+ amplitudes in BMMs by ∼50{\%} (p < 0.0001) and prevented phosphorylation of CaMKIV. KCa3.12/2 reduced RANKL+/2TNF-stimulated phosphorylation of CREB and expression of c-fos in BMMs (p < 0.01), culminating in decreased NFATc1 protein expression and transcriptional activity (p < 0.01). These data indicate that KCa3.1 regulates Ca2+-dependent NFATc1 expression via CaMKIV/CREB during inflammatory osteoclastogenesis in the presence of TNF, corroborating its role as a target candidate for the treatment of bone erosion in inflammatory arthritis.",
author = "Gr{\"o}ssinger, {Eva M.} and Mincheol Kang and Laura Bouchareychas and Ritu Sarin and Haudenschild, {Dominik R} and Borodinsky, {Laura N} and Iannis Adamopoulos",
year = "2018",
month = "1",
day = "15",
doi = "10.4049/jimmunol.1701170",
language = "English (US)",
volume = "200",
pages = "749--757",
journal = "Journal of Immunology",
issn = "0022-1767",
publisher = "American Association of Immunologists",
number = "2",

}

TY - JOUR

T1 - Ca2+-Dependent Regulation of NFATc1 via KCa3.1 in Inflammatory Osteoclastogenesis

AU - Grössinger, Eva M.

AU - Kang, Mincheol

AU - Bouchareychas, Laura

AU - Sarin, Ritu

AU - Haudenschild, Dominik R

AU - Borodinsky, Laura N

AU - Adamopoulos, Iannis

PY - 2018/1/15

Y1 - 2018/1/15

N2 - In inflammatory arthritis, the dysregulation of osteoclast activity by proinflammatory cytokines, including TNF, interferes with bone remodeling during inflammation through Ca2+-dependent mechanisms causing pathological bone loss. Ca2+-dependent CREB/c-fos activation via Ca2+-calmodulin kinase IV (CaMKIV) induces transcriptional regulation of osteoclast-specific genes via NFATc1, which facilitate bone resorption. In leukocytes, Ca2+ regulation of NFAT-dependent gene expression oftentimes involves the activity of the Ca2+-activated K+ channel KCa3.1. In this study, we evaluate KCa3.1 as a modulator of Ca2+-induced NFAT-dependent osteoclast differentiation in inflammatory bone loss. Microarray analysis of receptor activator of NF-kB ligand (RANKL)-activated murine bone marrow macrophage (BMM) cultures revealed unique upregulation of KCa3.1 during osteo-clastogenesis. The expression of KCa3.1 in vivo was confirmed by immunofluorescence staining on multinucleated cells at the bone surface of inflamed mouse joints. Experiments on in vitro BMM cultures revealed that KCa3.12/2 and TRAM-34 treatment significantly reduced the expression of osteoclast-specific genes (p < 0.05) alongside decreased osteoclast formation (p < 0.0001) in inflammatory (RANKL+TNF) and noninflammatory (RANKL) conditions. In particular, live cell Ca2+ imaging and Western blot analysis showed that TRAM-34 pretreatment decreased transient RANKL-induced Ca2+ amplitudes in BMMs by ∼50% (p < 0.0001) and prevented phosphorylation of CaMKIV. KCa3.12/2 reduced RANKL+/2TNF-stimulated phosphorylation of CREB and expression of c-fos in BMMs (p < 0.01), culminating in decreased NFATc1 protein expression and transcriptional activity (p < 0.01). These data indicate that KCa3.1 regulates Ca2+-dependent NFATc1 expression via CaMKIV/CREB during inflammatory osteoclastogenesis in the presence of TNF, corroborating its role as a target candidate for the treatment of bone erosion in inflammatory arthritis.

AB - In inflammatory arthritis, the dysregulation of osteoclast activity by proinflammatory cytokines, including TNF, interferes with bone remodeling during inflammation through Ca2+-dependent mechanisms causing pathological bone loss. Ca2+-dependent CREB/c-fos activation via Ca2+-calmodulin kinase IV (CaMKIV) induces transcriptional regulation of osteoclast-specific genes via NFATc1, which facilitate bone resorption. In leukocytes, Ca2+ regulation of NFAT-dependent gene expression oftentimes involves the activity of the Ca2+-activated K+ channel KCa3.1. In this study, we evaluate KCa3.1 as a modulator of Ca2+-induced NFAT-dependent osteoclast differentiation in inflammatory bone loss. Microarray analysis of receptor activator of NF-kB ligand (RANKL)-activated murine bone marrow macrophage (BMM) cultures revealed unique upregulation of KCa3.1 during osteo-clastogenesis. The expression of KCa3.1 in vivo was confirmed by immunofluorescence staining on multinucleated cells at the bone surface of inflamed mouse joints. Experiments on in vitro BMM cultures revealed that KCa3.12/2 and TRAM-34 treatment significantly reduced the expression of osteoclast-specific genes (p < 0.05) alongside decreased osteoclast formation (p < 0.0001) in inflammatory (RANKL+TNF) and noninflammatory (RANKL) conditions. In particular, live cell Ca2+ imaging and Western blot analysis showed that TRAM-34 pretreatment decreased transient RANKL-induced Ca2+ amplitudes in BMMs by ∼50% (p < 0.0001) and prevented phosphorylation of CaMKIV. KCa3.12/2 reduced RANKL+/2TNF-stimulated phosphorylation of CREB and expression of c-fos in BMMs (p < 0.01), culminating in decreased NFATc1 protein expression and transcriptional activity (p < 0.01). These data indicate that KCa3.1 regulates Ca2+-dependent NFATc1 expression via CaMKIV/CREB during inflammatory osteoclastogenesis in the presence of TNF, corroborating its role as a target candidate for the treatment of bone erosion in inflammatory arthritis.

UR - http://www.scopus.com/inward/record.url?scp=85044742330&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85044742330&partnerID=8YFLogxK

U2 - 10.4049/jimmunol.1701170

DO - 10.4049/jimmunol.1701170

M3 - Article

VL - 200

SP - 749

EP - 757

JO - Journal of Immunology

JF - Journal of Immunology

SN - 0022-1767

IS - 2

ER -