BK Knockout by TALEN-Mediated Gene Targeting in Osteoblasts: KCNMA1 Determines the Proliferation and Differentiation of Osteoblasts

Hei, Hongya; Gao, Jianjun; Dong, Jianyu; Tao, Jie; Tian, Lulu; Pan, Wanma; Wang, Hongyu; Zhang, Xuemei

doi:10.14348/molcells.2016.0033

Cited by 16 publications

(13 citation statements)

References 28 publications

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“…Stable suppression of BKs in hMSCs with shRNA significantly decreased both mRNA and protein levels of PPARγ and osteocalcin (intermediate and early biomarkers of adipogenesis and osteogenesis, respectively) (Figure ) (Zhang et al, ). A recent study also showed that BK channel knockout in MSC‐derived osteoblasts largely reduced osteocalcin, osteopontin and Runx2 expression (Hei et al, ). Similarly, K Ca channels have been found to be involved in the initiation of other SCs.…”

Section: Kca's In Mesenchymal Stem Cells and The Differentiation Processmentioning

confidence: 95%

Mesenchymal stem cell differentiation: Control by calcium‐activated potassium channels

Pchelintseva

Djamgoz

2017

Journal Cellular Physiology

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Mesenchymal stem cells (MSCs) are widely used in modern medicine for which understanding the mechanisms controlling their differentiation is fundamental. Ion channels offer novel insights to this process because of their role in modulating membrane potential and intracellular milieu. Here, we evaluate the contribution of calcium-activated potassium (K ) channels to the three main components of MSC differentiation: initiation, proliferation, and migration. First, we demonstrate the importance of the membrane potential (V ) and the apparent association of hyperpolarization with differentiation. Of K subtypes, most evidence points to activity of big-conductance channels in inducing initiation. On the other hand, intermediate-conductance currents have been shown to promote progression through the cell cycle. While there is no information on the role of K channels in migration of MSCs, work from other stem cells and cancer cells suggest that intermediate-conductance and to a lesser extent big-conductance channels drive migration. In all cases, these effects depend on species, tissue origin and lineage. Finally, we present a conceptual model that demonstrates how K activity could influence differentiation by regulating V and intracellular Ca oscillations. We conclude that K channels have significant involvement in MSC differentiation and could potentially enable novel tissue engineering approaches and therapies.

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Section: Kca's In Mesenchymal Stem Cells and The Differentiation Processmentioning

confidence: 95%

Mesenchymal stem cell differentiation: Control by calcium‐activated potassium channels

Pchelintseva

Djamgoz

2017

Journal Cellular Physiology

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“…Immunocytochemistry analysis was performed according to a previous report 21 . The cells were seeded onto slides.…”

Section: Methodsmentioning

confidence: 99%

“…Total proteins from the long bones or cells were analyzed by western blotting using a modified method 21 . Briefly, the bone powder and cells were lysed with RIPA buffer supplemented with 1% protease inhibitor cocktail (P0013B; Beyotime) for 30 min on ice.…”

Section: Methodsmentioning

confidence: 99%

“…BK channels are expressed in primary osteoblasts 14–16 , osteoblast cell lines (e.g., MG63, SaOS2, and ROS17/2, 8) 14,16–21 and progenitors (e.g., mesenchymal stem cells) 22–24 . Emerging evidence indicate that the BK channels in osteoblasts may act as mechanosensors to transduce extracellular mechanical signals into cell; Davidson et al was the first to report activation of BK channels in human G292 osteoblast-like osteosarcoma cells by the membrane stretch; 19,25 in human ondonosteoblasts, BK channels also exhibit mechanosensitivity 26 .…”

Section: Introductionmentioning

confidence: 99%

“…Similarly, silencing BK in human BMSCs decrease osteogenic differentiation by reducing mineral precipitation and OCN 24 . We have previously observed that BK knockout in ROS17/2.8 osteoblast cells posses a lower expression of runt-related transcription factor 2 (Runx2, the core transcription factor in osteoblast differentiation) associated with a reduction in osteoblast markers OPN and OCN 21 . Activity of the BK channels can be regulated by prostaglandin E2 (PGE2) 18,25,36 , via the recruitment and activation of tyrosine phosphorylated protein Syk.…”

Section: Introductionmentioning

confidence: 99%

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BK ablation attenuates osteoblast bone formation via integrin pathway

et al. 2019

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Impaired bone formation is one of the major causes of low bone mass and skeletal fragility that occurs in osteoporosis. However, the mechanisms underlying the defects in bone formation are not well understood. Here, we report that big conductance calcium-activated potassium channels (BKs) are required for bone formation and osteoblast function both in vivo and in vitro. By 15 weeks of age, BK knockout (BKO) mice exhibited a decline in bone mineral density and trabecular bone volume of the tibiae and lumbar vertebrae, which were associated with impaired bone formation and osteoblast activity. Mechanistically, BK ablation in bone and bone marrow mesenchymal stem cells (BMSCs) of BKO mice inhibited integrin signaling. Furthermore, the binding of α subunit of BK with integrin β1 protein in osteoblasts was confirmed, and FAK-ERK1/2 signaling was proved to be involved by genetic modification of KCNMA1 (which encodes the α subunit of BK) in ROS17/2.8 osteoblast cells. These findings indicated that BK regulates bone formation by promoting osteoblast differentiation via integrin pathway, which provided novel insight into ion transporter crosstalk with the extracellular matrix in osteoblast regulation and revealed a new potential strategy for intervention in correcting bone formation defects.

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Loss of the large conductance calcium-activated potassium channel causes an increase in mitochondrial reactive oxygen species in glioblastoma cells

Kulawiak

Żochowska

Bednarczyk

et al. 2023

Pflugers Arch - Eur J Physiol

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Mitochondrial potassium (mitoK) channels play an important role in cellular physiology. These channels are expressed in healthy tissues and cancer cells. Activation of mitoK channels can protect neurons and cardiac tissue against injury induced by ischemia–reperfusion. In cancer cells, inhibition of mitoK channels leads to an increase in mitochondrial reactive oxygen species, which leads to cell death. In glioma cell activity of the mitochondrial, large conductance calcium-activated potassium (mitoBKCa) channel is regulated by the mitochondrial respiratory chain. In our project, we used CRISPR/Cas9 technology in human glioblastoma U-87 MG cells to generate knockout cell lines lacking the α-subunit of the BKCa channel encoded by the KCNMA1 gene, which also encodes cardiac mitoBKCa. Mitochondrial patch-clamp experiments showed the absence of an active mitoBKCa channel in knockout cells. Additionally, the absence of this channel resulted in increased levels of mitochondrial reactive oxygen species. However, analysis of the mitochondrial respiration rate did not show significant changes in oxygen consumption in the cell lines lacking BKCa channels compared to the wild-type U-87 MG cell line. These observations were reflected in the expression levels of selected mitochondrial genes, organization of the respiratory chain, and mitochondrial morphology, which did not show significant differences between the analyzed cell lines. In conclusion, we show that in U-87 MG cells, the pore-forming subunit of the mitoBKCa channel is encoded by the KCNMA1 gene. Additionally, the presence of this channel is important for the regulation of reactive oxygen species levels in mitochondria.

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BK Knockout by TALEN-Mediated Gene Targeting in Osteoblasts: KCNMA1 Determines the Proliferation and Differentiation of Osteoblasts

Cited by 16 publications

References 28 publications

Mesenchymal stem cell differentiation: Control by calcium‐activated potassium channels

Mesenchymal stem cell differentiation: Control by calcium‐activated potassium channels

BK ablation attenuates osteoblast bone formation via integrin pathway

Loss of the large conductance calcium-activated potassium channel causes an increase in mitochondrial reactive oxygen species in glioblastoma cells

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