BK ablation attenuates osteoblast bone formation via integrin pathway.

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 ß1 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.

Large-Conductance Calcium-Activated Potassium Channels in Glomerulus: From Cell Signal Integration to Disease.

Large-conductance calcium-activated potassium (BK) channels are currently considered as vital players in a variety of renal physiological processes. In podocytes, BK channels become active in response to stimuli that increase local cytosolic Ca(2+), possibly secondary to activation of slit diaphragm TRPC6 channels by chemical or mechanical stimuli. Insulin increases filtration barrier permeability through mobilization of BK channels. In mesangial cells, BK channels co-expressed with ß1 subunits act as a major component of the counteractive response to contraction in order to regulate glomerular filtration. This review aims to highlight recent discoveries on the localization, physiological and pathological roles of BK channels in glomerulus.

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

Large conductance calcium-activated potassium (BK) channels participate in many important physiological functions in excitable tissues such as neurons, cardiac and smooth muscles, whereas the knowledge of BK channels in bone tissues and osteoblasts remains elusive. To investigate the role of BK channels in osteoblasts, we used transcription activator-like effector nuclease (TALEN) to establish a BK knockout cell line on rat ROS17/2.8 osteoblast, and detected the proliferation and mineralization of the BK-knockout cells. Our study found that the BK-knockout cells significantly decreased the ability of proliferation and mineralization as osteoblasts, compared to the wild type cells. The overall expression of osteoblast differentiation marker genes in the BK-knockout cells was significantly lower than that in wild type osteoblast cells. The BK-knockout osteoblast cell line in our study displays a phenotype decrease in osteoblast function which can mimic the pathological state of osteoblast and thus provide a working cell line as a tool for study of osteoblast function and bone related diseases.

Hydrogen Sulfide Inhibits Transforming Growth Factor-ß1-Induced EMT via Wnt/Catenin Pathway.

Hydrogen sulfide (H2S) has anti-fibrotic potential in lung, kidney and other organs. The exogenous H2S is released from sodium hydrosulfide (NaHS) and can influence the renal fibrosis by blocking the differentiation of quiescent renal fibroblasts to myofibroblasts. But whether H2S affects renal epithelial-to-mesenchymal transition (EMT) and the underlying mechanisms remain unknown. Our study is aimed at investigating the in vitro effects of H2S on transforming growth factor-ß1 (TGF-ß1)-induced EMT in renal tubular epithelial cells (HK-2 cells) and the associated mechanisms. The induced EMT is assessed by Western blotting analysis on the expressions of α-SMA, E-cadherin and fibronectin. HK-2 cells were treated with NaHS before incubating with TGF-ß1 to investigate its effect on EMT and the related molecular mechanism. Results demonstrated that NaHS decreased the expression of α-SMA and fibronectin, and increased the expression of E-cadherin. NaHS reduced the expression of TGF-ß receptor type I (TßR I) and TGF-ß receptor type II (TßR II). In addition, NaHS attenuated TGF-ß1-induced increase of ß-catenin expression and ERK phosphorylation. Moreover, it inhibited the TGF-ß1-induced nuclear translocation of ßß-catenin. These effects of NaHS on fibronectin, E-cadherin and TßR I were abolished by the ERK inhibitor U0126 or ß-catenin inhibitor XAV939, or ß-catenin siRNA interference. We get the conclusion that NaHS attenuated TGF-ß1-induced EMT in HK-2 cells through both ERK-dependent and ß-catenin-dependent pathways.

Up-Regulatory Effects of Curcumin on Large Conductance Ca2+-Activated K+ Channels.

Large conductance Ca2+-activated potassium channels (BK) are targets for research that explores therapeutic means to various diseases, owing to the roles of the channels in mediating multiple physiological processes in various cells and tissues. We investigated the pharmacological effects of curcumin, a compound isolated from the herb Curcuma longa, on BK channels. As recorded by whole-cell patch-clamp, curcumin increased BK (α) and BK (α+ß1) currents in transfected HEK293 cells as well as the current density of BK in A7r5 smooth muscle cells in a dose-dependent manner. By incubating with curcumin for 24 hours, the current density of exogenous BK (α) in HEK293 cells and the endogenous BK in A7r5 cells were both enhanced notably, though the steady-state activation of the channels did not shift significantly, except for BK (α+ß1). Curcumin up-regulated the BK protein expression without changing its mRNA level in A7r5 cells. The surface expression and the half-life of BK channels were also increased by curcumin in HEK293 cells. These effects of curcumin were abolished by MG-132, a proteasome inhibitor. Curcumin also increased ERK 1/2 phosphorylation, while inhibiting ERK by U0126 attenuated the curcumin-induced up-regulation of BK protein expression. We also observed that the curcumin-induced relaxation in the isolated rat aortic rings was significantly attenuated by paxilline, a BK channel specific blocker. These results show that curcumin enhances the activity of the BK channels by interacting with BK directly as well as enhancing BK protein expression through inhibiting proteasomal degradation and activating ERK signaling pathway. The findings suggest that curcumin is a potential BK channel activator and provide novel insight into its complicated pharmacological effects and the underlying mechanisms.

Forskolin Regulates L-Type Calcium Channel through Interaction between Actinin 4 and ß3 Subunit in Osteoblasts.

Voltage-dependent L-type calcium channels that permit cellular calcium influx are essential in calcium-mediated modulation of cellular signaling. Although the regulation of voltage-dependent L-type calcium channels is linked to many factors including cAMP-dependent protein kinase A (PKA) activity and actin cytoskeleton, little is known about the detailed mechanisms underlying the regulation in osteoblasts. Our present study investigated the modulation of L-type calcium channel activities through the effects of forskolin on actin reorganization and on its functional interaction with actin binding protein actinin 4. The results showed that forskolin did not significantly affect the trafficking of pore forming α1c subunit and its interaction with actin binding protein actinin 4, whereas it significantly increased the expression of ß3 subunit and its interaction with actinin 4 in osteoblast cells as assessed by co-immunoprecipitation, pull-down assay, and immunostaining. Further mapping showed that the ABD and EF domains of actinin 4 were interaction sites. This interaction is independent of PKA phosphorylation. Knockdown of actinin 4 significantly decreased the activities of L-type calcium channels. Our study revealed a new aspect of the mechanisms by which the forskolin activation of adenylyl cyclase - cAMP cascade regulates the L-type calcium channel in osteoblast cells, besides the PKA mediated phosphorylation of the channel subunits. These data provide insight into the important role of interconnection among adenylyl cyclase, cAMP, PKA, the actin cytoskeleton, and the channel proteins in the regulation of voltage-dependent L-type calcium channels in osteoblast cells.