Discovery of agonist-antagonist pairs for the modulation of Ca [2]+ and voltage-gated K+ channels of large conductance that contain beta1 subunits.

Large conductance, calcium/voltage-gated potassium channels (BK) regulate critical body processes, including neuronal, secretory and smooth muscle (SM) function. While BK-forming alpha subunits are ubiquitous, accessory beta1 subunits are highly expressed in SM. This makes beta1 an attractive target for pharmaceutical development to treat SM disorders, such as hypertension or cerebrovascular spasm. Compounds activating BK via beta1 have been identified, yet they exhibit low potency and off-target effects while antagonists that limit agonist activity via beta 1 remain unexplored. Beta1-dependent BK ligand-based pharmacophore modeling and ZINC database searches identified 15 commercially available hits. Concentration-response curves on BK alpha + beta1 subunit-mediated currents were obtained in CHO cells. One potent (EC50 = 20 nM) and highly efficacious activator (maximal activation = ×10.3 of control) was identified along with a potent antagonist (KB = 3.02 nM), both of which were dependent on beta1. Our study provides the first proof-of-principle that an agonist/antagonist pair can be used to control beta1-containing BK activity.

Cholesterol Inhibition of Slo1 Channels Is Calcium-Dependent and Can Be Mediated by Either High-Affinity Calcium-Sensing Site in the Slo1 Cytosolic Tail.

Calcium- and voltage-gated K+ channels of large conductance (BKs) are expressed in the cell membranes of all excitable tissues. Currents mediated by BK channel-forming slo1 homotetramers are consistently inhibited by increases in membrane cholesterol (CLR). The molecular mechanisms leading to this CLR action, however, remain unknown. Slo1 channels are activated by increases in calcium (Ca2+) nearby Ca2+-recognition sites in the slo1 cytosolic tail: one high-affinity and one low-affinity site locate to the regulator of conductance for K+ (RCK) 1 domain, whereas another high-affinity site locates within the RCK2 domain. Here, we first evaluated the crosstalking between Ca2+ and CLR on the function of slo1 (cbv1 isoform) channels reconstituted into planar lipid bilayers. CLR robustly reduced channel open probability while barely decreasing unitary current amplitude, with CLR maximal effects being observed at 10-30 µM internal Ca2+ CLR actions were not only modulated by internal Ca2+ levels but also disappeared in absence of this divalent. Moreover, in absence of Ca2+, BK channel-activating concentrations of magnesium (10 mM) did not support CLR action. Next, we evaluated CLR actions on channels where the different Ca2+-sensing sites present in the slo1 cytosolic domain became nonfunctional via mutagenesis. CLR still reduced the activity of low-affinity Ca2+ (RCK1:E379A, E404A) mutants. In contrast, CLR became inefficacious when both high-affinity Ca2+ sites were mutated (RCK1:D367A,D372A and RCK2:D899N,D900N,D901N,D902N,D903N), yet still was able to decrease the activity of each high-affinity site mutant. Therefore, BK channel inhibition by CLR selectively requires optimal levels of Ca2+ being recognized by either of the slo1 high-affinity Ca2+-sensing sites. SIGNIFICANCE STATEMENT: Results reveal that inhibition of calcium/voltage-gated K+ channel of large conductance (BK) (slo1) channels by membrane cholesterol requires a physiologically range of internal calcium (Ca2+) and is selectively linked to the two high-affinity Ca2+-sensing sites located in the cytosolic tail domain, which underscores that Ca2+ and cholesterol actions are allosterically coupled to the channel gate. Cholesterol modification of BK channel activity likely contributes to disruption of normal physiology by common health conditions that are triggered by disruption of cholesterol homeostasis.

Activation of Estrogen Receptor Alpha by Decitabine Inhibits Osteosarcoma Growth and Metastasis.

Osteosarcoma is a malignant tumor in the bone, which originates from normal osteoblasts or osteoblast precursors. Normal osteoblasts express estrogen receptor alpha (ERα); however, osteosarcomas do not express ERα due to promoter DNA methylation. Here we show that treatment of 143B osteosarcoma cells with decitabine (DAC, 5-Aza-2'-deoxycytidine) induces expression of ERα and leads to decreased proliferation and concurrent induction of osteoblast differentiation. DAC exposure reduced protein expression of metastasis-associated markers VIMENTIN, SLUG, ZEB1, and MMP9, with a concurrent decrease in mRNA expression of known stem cell markers SOX2, OCT4, and NANOG. Treatment with 17ß-estradiol (E2) synergized with DAC to reduce proliferation. Overexpression of ERα inhibited proliferation and induced osteoblast differentiation, whereas knockout of ERα by CRISPR/Cas9 prevented the effects of DAC. In an orthotopic model of osteosarcoma, DAC inhibited tumor growth and metastasis of 143B cells injected into the tibia of NOD SCID gamma mice. Furthermore, ERα overexpression reduced tumor growth and metastasis, and ERα knockout prevented the effects of DAC in vivo. Together, these experiments provide preclinical evidence that the FDA-approved DNA methylation inhibitor DAC may be repurposed to treat patients with osteosarcoma based on its efficacy to decrease proliferation, to induce osteoblast differentiation, and to reduce metastasis to visceral organs.Significance: These findings describe the effects of DNA methyltransferase inhibition on ERα and its potential role as a tumor suppressor in osteosarcoma.See related commentary by Roberts, p. 1034 See related article by El Ayachi and colleagues; Cancer Res 79(5);982-93.

GATA4 represses RANKL in osteoblasts via multiple long-range enhancers to regulate osteoclast differentiation.

GATA4 is a transcription factor that is responsible for tissue-specific gene regulation in many tissues, and more recent studies showed that it is necessary for osteoblast differentiation. Previously, we showed that in vivo deletion of Gata4 using Cre-recombinase under the control of the Col1a1 2.3 kb promoter, showed significantly reduced trabecular bone properties. To understand the role of GATA4 in more differentiated cells, GATA4fl/fl mice were crossed with mice expressing Cre-recombinase under the control of the osteocalcin promoter. MicroCT analysis of trabecular bone properties of the femur and tibia from 14-week-old female osteocalcin-Cre/GATA4fl/fl (OCN-cKO) mice showed a significant reduction in percentage bone volume, a decrease in trabecular number and an increase in trabecular spacing. In vivo, histomorphometric analysis revealed a decrease in the number of osteoblasts and an increase in the number of osteoclasts in the tibiae of OCN-cKO mice. In vivo and in vitro systems correlated a decrease in Gata4 mRNA with increased RANKL gene expression. To determine if RANKL is a direct target of GATA4, chromatin immunoprecipitation (ChIP)-sequencing was performed, and it demonstrated that GATA4 is recruited to seven enhancers near RANKL. Furthermore, when Gata4 is knocked down, the chromatin at the RANKL region is further opened, as detected by a reduction in histone 3 lysine 27 trimethylation (H3K27me3) and an increase in histone 3 lysine 4 dimethylation (H3K4me2) in the RANKL locus. In vitro, TRAP staining of cells from bone marrow cultures from Gata4 knockout cells show that the increased levels of RANKL are sufficient for osteoclast formation. Together, the data suggest that GATA4 directly represses RANKL expression via seven cis-regulatory regions and plays an important role in maintaining proper bone development and osteoclast formation.

GATA4 Directly Regulates Runx2 Expression and Osteoblast Differentiation.

GATA4 is a zinc-finger transcription factor that is a pioneer factor in various tissues and regulates tissue-specific gene regulation. In vivo deletion of Gata4 using Cre-recombinase under the control of the Col1a1 2.3 kb promoter showed significantly reduced values for trabecular bone properties by microCT analysis of femur and tibia of 14-week-old male and female mice, suggesting GATA4 is necessary for maintaining normal adult bone phenotype. Quantitative PCR analysis revealed higher expression of Gata4 in trabecular bone compared with cortical bone, suggesting a role for GATA4 in maintaining normal trabecular bone mass. In vivo and in vitro, reduction of Gata4 correlates with reduced Runx2 gene expression, along with reduced osteoblast mineralization. To determine if Runx2 is a direct target of GATA4, chromatin immunoprecipitation (ChIP) was performed, and it demonstrated that GATA4 is recruited to the two Runx2 promoters and an enhancer region. Furthermore, when Gata4 is knocked down, the chromatin at the Runx2 region is not open, as detected by DNase assays and ChIP with antibodies to the open chromatin marks H3K4me2 (histone 3 lysine 4 dimethylation) and H3K27ac (histone 3 lysine 27 acetylation) and the closed chromatin mark H3K27me2 (histone 3 lysine 27 trimethylation). Together, the data suggest that GATA4 binds near the Runx2 promoter and enhancer and helps maintain open chromatin to regulate Runx2 expression leading to bone mineralization.