Satomi Niwa

Involvement of dominant-negative, spliced variants of the intermediate-conductance CA2+-activated K+ channel, KCA3.1 in immune function of lymphoid cells

The intermediate conductance Ca2+-activated K+ channel (IKCa channel) encoded by KCa3.1 is responsible for the control of proliferation and differentiation in various types of cells. We identified novel spliced variants of KCa3.1 (human (h) KCa3.1b) from the human thymus, which were lacking the N-terminal domains of the original hKCa3.1a as a result of alternative splicing events. hKCa3.1b was significantly expressed in human lymphoid tissues. Western blot analysis showed that hKCa3.1a proteins were mainly expressed in the plasma membrane fraction, whereas hKCa3.1b was in the cytoplasmic fraction. We also identified a similar N terminus lacking KCa3.1 variants from mice and rat lymphoid tissues (mKCa3.1b and rKCa3.1b). In the HEK293 heterologous expression system, the cellular distribution of cyan fluorescent protein-tagged hKCa3.1a and/or YFP-tagged hKCa3.1b isoforms showed that hKCa3.1b suppressed the localization of hKCa3.1a to the plasma membrane. In the Xenopus oocyte translation system, co-expression of hKCa3.1b with hKCa3.1a suppressed IKCa channel activity of hKCa3.1a in a dominant-negative manner. In addition, this study indicated that up-regulation of mKCa3.1b in mouse thymocytes differentiated CD4(+)CD8(+) phenotype thymocytes into CD4(−)CD8(−) ones and suppressed concanavalin-A-stimulated thymocyte growth by down-regulation of mIL-2 transcripts. Anti-proliferative effects and down-regulation of mIL-2 transcripts were also observed in mKCa3.1b-overexpressing mouse thymocytes. These suggest that the N-terminal domain of KCa3.1 is critical for channel trafficking to the plasma membrane and that the fine-tuning of IKCa channel activity modulated through alternative splicing events may be related to the control in physiological and pathophysiological conditions in T-lymphocytes.

Intermediate-Conductance Ca2+-Activated K+ Channel, KCa3.1, as a Novel Therapeutic Target for Benign Prostatic Hyperplasia

Recently, a new experimental stromal hyperplasia animal model corresponding to clinical benign prostatic hyperplasia (BPH) was established. The main objective of this study was to elucidate the roles of the intermediate-conductance Ca2+-activated K+ channel (KCa3.1) in the implanted urogenital sinus (UGS) of stromal hyperplasia BPH model rats. Using DNA microarray, real-time polymerase chain reaction, Western blot, and/or immunohistochemical analyses, we identified the expression of KCa3.1 and its transcriptional regulators in implanted UGS of BPH model rats and prostate needle-biopsy samples and surgical prostate specimens of BPH patients. We also examined the in vivo effects of a KCa3.1 blocker, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34), on the proliferation index of implanted UGS by measurement of UGS weights and proliferating cell nuclear antigen immunostaining. KCa3.1 genes and proteins were highly expressed in implanted UGS rather than in the normal host prostate. In the implanted UGS, the gene expressions of two transcriptional regulators of KCa3.1, repressor element 1-silencing transcription factor and c-Jun, were significantly down- and up-regulated, and the regulations were correlated negatively or positively with KCa3.1 expression, respectively. Positive signals of KCa3.1 proteins were detected exclusively in stromal cells, whereas they were scarcely immunolocalized to basal cells of the epithelium in implanted UGS. In vivo treatment with TRAM-34 significantly suppressed the increase in implanted UGS weights compared with the decrease in stromal cell components. Moreover, significant levels of KCa3.1 expression were observed in human BPH samples. KCa3.1 blockers may be a novel treatment option for patients suffering from BPH.

Downregulation of Ca2+-Activated Cl− Channel TMEM16A by the Inhibition of Histone Deacetylase in TMEM16A-Expressing Cancer Cells

The Ca2+-activated Cl− channel transmembrane proteins with unknown function 16 A (TMEM16A; also known as anoctamin 1 or discovered on gastrointestinal stromal tumor 1) plays an important role in facilitating the cell growth and metastasis of TMEM16A-expressing cancer cells. Histone deacetylase (HDAC) inhibitors (HDACi) are useful agents for cancer therapy, but it remains unclear whether ion channels are epigenetically regulated by them. Using real-time polymerase chain reaction, Western blot analysis, and whole-cell patch-clamp assays, we found a significant decrease in TMEM16A expression and its functional activity was induced by the vorinostat, a pan-HDACi in TMEM16A-expressing human cancer cell lines, the prostatic cancer cell line PC-3, and the breast cancer cell line YMB-1. TMEM16A downregulation was not induced by the chemotherapy drug paclitaxel in either cell type. Pharmacologic blockade of HDAC3 by 1 μM T247 [N-(2-aminophenyl)-4-[1-(2-thiophen-3-ylethyl)-1H-[1],[2],[3]triazol-4-yl]benzamide], a HDAC3-selective HDACi, elicited a large decrease in TMEM16A expression and functional activity in both cell types, and pharmacologic blockade of HDAC2 by AATB [4-(acetylamino)-N-[2-amino-5-(2-thienyl)phenyl]-benzamide; 300 nM] elicited partial inhibition of TMEM16A expression (∼40%) in both. Pharmacologic blockade of HDAC1 or HDAC6 did not elicit any significant change in TMEM16A expression, respectively. In addition, inhibition of HDAC3 induced by small interfering RNA elicited a large decrease in TMEM16A transcripts in both cell types. Taken together, in malignancies with a frequent gene amplification of TMEM16A, HDAC3 inhibition may exert suppressive effects on cancer cell viability via downregulation of TMEM16A.

Downregulation of the Ca2+-activated K+ channel KCa3.1 by histone deacetylase inhibition in human breast cancer cells

The intermediate‐conductance Ca2+‐activated K+ channel KCa3.1 is involved in the promotion of tumor growth and metastasis, and is a potential therapeutic target and biomarker for cancer. Histone deacetylase inhibitors (HDACis) have considerable potential for cancer therapy, however, the effects of HDACis on ion channel expression have not yet been investigated in detail. The results of this study showed a significant decrease in KCa3.1 transcription by HDAC inhibition in the human breast cancer cell line YMB‐1, which functionally expresses KCa3.1. A treatment with the clinically available, class I, II, and IV HDAC inhibitor, vorinostat significantly downregulated KCa3.1 transcription in a concentration‐dependent manner, and the plasmalemmal expression of the KCa3.1 protein and its functional activity were correspondingly decreased. Pharmacological and siRNA‐based HDAC inhibition both revealed the involvement of HDAC2 and HDAC3 in KCa3.1 transcription through the same mechanism. The downregulation of KCa3.1 in YMB‐1 was not due to the upregulation of the repressor element‐1 silencing transcription factor, REST and the insulin‐like growth factor‐binding protein 5, IGFBP5. The significant decrease in KCa3.1 transcription by HDAC inhibition was also observed in the KCa3.1‐expressing human prostate cancer cell line, PC‐3. These results suggest that vorinostat and the selective HDACis for HDAC2 and/or HDAC3 are effective drug candidates for KCa3.1‐overexpressing cancers.

Down-Regulation of Ca2+-Activated K+ Channel KCa1.1 in Human Breast Cancer MDA-MB-453 Cells Treated with Vitamin D Receptor Agonists

Vitamin D (VD) reduces the risk of breast cancer and improves disease prognoses. Potential VD analogs are being developed as therapeutic agents for breast cancer treatments. The large-conductance Ca2+-activated K+ channel KCa1.1 regulates intracellular Ca2+ signaling pathways and is associated with high grade tumors and poor prognoses. In the present study, we examined the effects of treatments with VD receptor (VDR) agonists on the expression and activity of KCa1.1 in human breast cancer MDA-MB-453 cells using real-time PCR, Western blotting, flow cytometry, and voltage-sensitive dye imaging. Treatments with VDR agonists for 72 h markedly decreased the expression levels of KCa1.1 transcripts and proteins in MDA-MB-453 cells, resulting in the significant inhibition of depolarization responses induced by paxilline, a specific KCa1.1 blocker. The specific proteasome inhibitor MG132 suppressed VDR agonist-induced decreases in KCa1.1 protein expression. These results suggest that KCa1.1 is a new downstream target of VDR signaling and the down-regulation of KCa1.1 through the transcriptional repression of KCa1.1 and enhancement of KCa1.1 protein degradation contribute, at least partly, to the antiproliferative effects of VDR agonists in breast cancer cells.

Transcriptional Repression and Protein Degradation of the Ca2+-Activated K+ Channel KCa1.1 by Androgen Receptor Inhibition in Human Breast Cancer Cells

The large-conductance Ca2+-activated K+ channel KCa1.1 plays an important role in the promotion of breast cancer cell proliferation and metastasis. The androgen receptor (AR) is proposed as a therapeutic target for AR-positive advanced triple-negative breast cancer. We herein investigated the effects of a treatment with antiandrogens on the functional activity, activation kinetics, transcriptional expression, and protein degradation of KCa1.1 in human breast cancer MDA-MB-453 cells using real-time PCR, Western blotting, voltage-sensitive dye imaging, and whole-cell patch clamp recording. A treatment with the antiandrogen bicalutamide or enzalutamide for 48 h significantly suppressed (1) depolarization responses induced by paxilline (PAX), a specific KCa1.1 blocker and (2) PAX-sensitive outward currents induced by the depolarizing voltage step. The expression levels of KCa1.1 transcripts and proteins were significantly decreased in MDA-MB-453 cells, and the protein degradation of KCa1.1 mainly contributed to reductions in KCa1.1 activity. Among the eight regulatory β and γ subunits, LRRC26 alone was expressed at high levels in MDA-MB-453 cells and primary and metastatic breast cancer tissues, whereas no significant changes were observed in the expression levels of LRRC26 and activation kinetics of PAX-sensitive outward currents in MDA-MB-453 cells by the treatment with antiandrogens. The treatment with antiandrogens up-regulated the expression of the ubiquitin E3 ligases, FBW7, MDM2, and MDM4 in MDA-MB-453 cells, and the protein degradation of KCa1.1 was significantly inhibited by the respective siRNA-mediated blockade of FBW7 and MDM2. Based on these results, we concluded that KCa1.1 is an androgen-responsive gene in AR-positive breast cancer cells, and its down-regulation through enhancements in its protein degradation by FBW7 and/or MDM2 may contribute, at least in part, to the antiproliferative and antimetastatic effects of antiandrogens in breast cancer cells.

Dominant-Negative, Spliced Variant of the Intermediate-Conductance Ca2+-Activated K+ Channel, KCa3.1 in Lymphoid Cells

Intermediate-conductance Ca2+-activated K+ channel (IKCa channel) encoded by KCa3.1 is responsible for the control of proliferation and differentiation in various types of cells. We identified novel spliced variants of KCa3.1 (hKCa3.1b) from the human thymus, which were lacking the N-terminal domains of the original hKCa3.1a as a result of alternative splicing. hKCa3.1b was significantly expressed in human lymphoid tissues. Western blot analysis showed that hKCa3.1a proteins were mainly expressed in plasma membrane fraction, whereas hKCa3.1b was in the cytoplasmic fraction. We also identified a similar N-terminus lacking KCa3.1 spliced variants from mice and rat lymphoid tissues (mKCa3.1b, rKCa3.1b). In the HEK293 heterologous expression system, the cellular distribution of CFP-tagged hKCa3.1a and/or YFP-tagged hKCa3.1b isoforms showed that hKCa3.1b suppressed the localization of hKCa3.1a to the plasma membrane. In the Xenopus oocyte translation system, co-expression of hKCa3.1b with hKCa3.1a suppressed IKCa channel activity of hKCa3.1a in a dominant-negative manner. In addition, the present study indicated that up-regulation of mKCa3.1b in mice thymocytes differentiated CD4+CD8+ phenotype thymocytes into CD4-CD8- ones, and suppressed concanavalin-A-stimulated thymocyte growth by down-regulation of mIL-2 transcripts. Anti-proliferative effects and down-regulation of mIL-2 transcripts were also observed in mKCa3.1b over-expressing mice thymocytes. These suggest that the N-terminal domain of KCa3.1 is critical for channel trafficking to the plasma membrane, and that the fine tuning of IKCa channel activity modulated through alternative splicing may be related to the control in physiological and pathophysiological conditions in T-lymphocytes.