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Tsugumichi Saito - One of the best experts on this subject based on the ideXlab platform.
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regulation of the kcnj5 gene by sf 1 in the adrenal cortex complete genomic organization and promoter function
Molecular and Cellular Endocrinology, 2020Co-Authors: Ayaka Nishikido, Shunichi Matsumoto, Kazuhiko Horiguchi, Tomoko Miyamoto, Akikokatano Toki, Satoshi Yoshino, Takashi Okamura, Yasuyo Nakajima, Emi Ishida, Tsugumichi SaitoAbstract:Abstract Activating mutations in the KCNJ5 gene are responsible for the significant number of aldosterone-producing adenomas. To elucidate the molecular mechanisms underlying KCNJ5 expression, we characterized the entire human KCNJ5 gene. The gene spanned approximately 29.8 kb and contained three exons and two introns. The strongest expression of KCNJ5 mRNA was observed in the adrenal gland. The promoter region contained a putative binding site for SF-1 at −1782 bp. A construct containing −2444 bp of the promoter region exhibited the strongest promoter activity in adrenal H295R cells, and the introduction of a mutation in the SF-1 binding site almost completely abolished promoter activity. Furthermore, deletion mutation, EMSA, and knockdown analyses revealed that SF-1 bound to this element and was functional. Immunochemistry showed that KCNJ5 was predominantly expressed in the zona glomerulosa, while SF-1 was ubiquitously expressed in the adrenal cortex. These results demonstrated that SF-1 mediates the expression of human KCNJ5 in the adrenal cortex.
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regulation of the kcnj5 gene by sf 1 in the adrenal cortex complete genomic organization and promoter function
Molecular and Cellular Endocrinology, 2020Co-Authors: Ayaka Nishikido, Shunichi Matsumoto, Kazuhiko Horiguchi, Tomoko Miyamoto, Akikokatano Toki, Satoshi Yoshino, Takashi Okamura, Yasuyo Nakajima, Emi Ishida, Tsugumichi SaitoAbstract:Abstract Activating mutations in the KCNJ5 gene are responsible for the significant number of aldosterone-producing adenomas. To elucidate the molecular mechanisms underlying KCNJ5 expression, we characterized the entire human KCNJ5 gene. The gene spanned approximately 29.8 kb and contained three exons and two introns. The strongest expression of KCNJ5 mRNA was observed in the adrenal gland. The promoter region contained a putative binding site for SF-1 at −1782 bp. A construct containing −2444 bp of the promoter region exhibited the strongest promoter activity in adrenal H295R cells, and the introduction of a mutation in the SF-1 binding site almost completely abolished promoter activity. Furthermore, deletion mutation, EMSA, and knockdown analyses revealed that SF-1 bound to this element and was functional. Immunochemistry showed that KCNJ5 was predominantly expressed in the zona glomerulosa, while SF-1 was ubiquitously expressed in the adrenal cortex. These results demonstrated that SF-1 mediates the expression of human KCNJ5 in the adrenal cortex.
Felix Beuschlein - One of the best experts on this subject based on the ideXlab platform.
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primary aldosteronism kcnj5 mutations and adrenocortical cell growth
Hypertension, 2019Co-Authors: Yuhong Yang, Felix Beuschlein, Celso E Gomezsanchez, Diana Jaquin, Elke Tatjana Aristizabal Prada, Lucie S Meyer, Thomas Knosel, Holger Schneider, Martin ReinckeAbstract:Aldosterone-producing adenomas with somatic mutations in the KCNJ5 G-protein-coupled inwardly rectifying potassium channel are a cause of primary aldosteronism. These mutations drive aldosterone excess, but their role in cell growth is undefined. Our objective was to determine the role of KCNJ5 mutations in adrenal cell proliferation and apoptosis. The Ki67 proliferative index was positively correlated with adenoma diameter in aldosterone-producing adenomas with a KCNJ5 mutation (r=0.435, P=0.007), a negative correlation was noted in adenomas with no mutation detected (r=-0.548, P=0.023). Human adrenocortical cell lines were established with stable expression of cumate-inducible wild-type or mutated KCNJ5. Increased cell proliferation was induced by low-level induction of KCNJ5-T158A expression compared with control cells (P=0.009), but increased induction ablated this difference. KCNJ5-G151R displayed no apparent proliferative effect, but KCNJ5-G151E and L168R mutations each resulted in decreased cell proliferation (difference P<0.0001 from control cells, both comparisons). Under conditions tested, T158A had no effect on apoptosis, but apoptosis increased with expression of G151R (P<0.0001), G151E (P=0.008), and L168R (P<0.0001). We generated a specific KCNJ5 monoclonal antibody which was used in immunohistochemistry to demonstrate strong KCNJ5 expression in adenomas without a KCNJ5 mutation and in the zona glomerulosa adjacent to adenomas irrespective of genotype as well as in aldosterone-producing cell clusters. Double immunofluorescence staining for KCNJ5 and CYP11B2 (aldosterone synthase) showed markedly decreased KCNJ5 immunostaining in CYP11B2-positive cells compared with CYP11B2-negative cells in aldosterone-producing adenomas with a KCNJ5 mutation. Together, these findings support the concept that cell growth effects of KCNJ5 mutations are determined by the expression level of the mutated channel.
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Pharmacology and pathophysiology of mutated KCNJ5 found in adrenal aldosterone-producing adenomas
Endocrinology, 2014Co-Authors: Philipp Tauber, Julia Stindl, Felix Beuschlein, David Penton, Evelyn Humberg, Ines Tegtmeier, Christina Sterner, Martin Reincke, Jacques Barhanin, Sascha BandulikAbstract:Somatic mutations of the potassium channel KCNJ5 are found in 40% of aldosterone producing adenomas (APAs). APA-related mutations of KCNJ5 lead to a pathological Na(+) permeability and a rise in cytosolic Ca(2+), the latter presumably by depolarizing the membrane and activating voltage-gated Ca(2+) channels. The aim of this study was to further investigate the effects of mutated KCNJ5 channels on intracellular Na(+) and Ca(2+) homeostasis in human adrenocortical NCI-H295R cells. Expression of mutant KCNJ5 led to a 2-fold increase in intracellular Na(+) and, in parallel, to a substantial rise in intracellular Ca(2+). The increase in Ca(2+) appeared to be caused by activation of voltage-gated Ca(2+) channels and by an impairment of Ca(2+) extrusion by Na(+)/Ca(2+) exchangers. The mutated KCNJ5 exhibited a pharmacological profile that differed from the one of wild-type channels. Mutated KCNJ5 was less Ba(2+) and tertiapin-Q sensitive but was inhibited by blockers of Na(+) and Ca(2+)-transporting proteins, such as verapamil and amiloride. The clinical use of these drugs might influence aldosterone levels in APA patients with KCNJ5 mutations. This might implicate diagnostic testing of APAs and could offer new therapeutic strategies.
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lack of influence of somatic mutations on steroid gradients during adrenal vein sampling in aldosterone producing adenoma patients
European Journal of Endocrinology, 2013Co-Authors: Andrea Oswald, Evelyn Fischer, Christoph Degenhart, Marcus Quinkler, Martin Bidlingmaier, Anna Pallauf, Katharina Lang, Thomas Mussack, K Hallfeldt, Felix BeuschleinAbstract:Objective: Adrenal vein sampling (AVS) is a technically demanding procedure required for the identification of suitable candidates for unilateral adrenalectomy in primary aldosteronism. Recently, somatic KCNJ5 K C -channel mutations in aldosterone-producing adenoma (APA) patients have been shown to influence steroid gradients during AVS. These and other recently identified genetic modifiers (ATP1A1 and ATP2B3) might affect the final diagnosis and treatment of the affected patients. Design: Fifty-nine patients with APAs who had undergone successful AVS (adrenal vein cortisol:peripheral cortisol ratio R2) and had undergone a mutation analysis of their tumor tissue were studied. The mutation status of the APAs was as follows: 19 KCNJ5 mutations, eight ATPase mutations (five ATP1A1 and three ATP2B3), and 32 patients with none of these mutations. Methods: The lateralization index (ratio of aldosterone:cortisol on the side of the adenoma to aldosterone to cortisol on the contralateral side) and the contralateral suppression index (ratio of aldosterone:cortisol on the contralateral side to aldosterone to cortisol in the periphery) were calculated for the KCNJ5-mutated, ATPase-mutated, and the KCNJ5/ATPase mutation-negative APA patients. Results: The lateralization indices of the ATPase mutation carriers had a median of 19.9 compared with a median of 16.0 in the KCNJ5 mutation carriers and that of 20.5 in the KCNJ5/ATPase mutation-negative patients. The contralateral suppression indices of the ATPase-mutated patients had a median of 0.1 compared with a median of 0.4 in the KCNJ5 mutation carriers and that of 0.2 in the KCNJ5/ATPase mutation-negative patients. The differences between the genetic groups were not statistically significant. Conclusions: We did not find evidence for a clinically important impact of mutation status on steroid gradients during AVS.
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prevalence clinical and molecular correlates of kcnj5 mutations in primary aldosteronism
Hypertension, 2012Co-Authors: Sheerazed Boulkroun, Laurence Amar, Gian Paolo Rossi, Benoit Samsoncouterie, Evelyn Fischer, Felix Beuschlein, Paolo Mulatero, Josefelipe Golibdzib, Stefanie Hahner, Marcus QuinklerAbstract:Primary aldosteronism is the most common form of secondary hypertension. Mutations in the KCNJ5 gene have been described recently in aldosterone-producing adenomas (APAs). The aim of this study was to investigate the prevalence of KCNJ5 mutations in unselected patients with primary aldosteronism and their clinical, biological and molecular correlates. KCNJ5 sequencing was performed on somatic (APA, n=380) and peripheral (APA, n=344; bilateral adrenal hyperplasia, n=174) DNA of patients with primary aldosteronism, collected through the European Network for the Study of Adrenal Tumors. Transcriptome analysis was performed in 102 tumors. Somatic KCNJ5 mutations (p.Gly151Arg or p.Leu168Arg) were found in 34% (129 of 380) of APA. They were significantly more prevalent in females (49%) than males (19%; P<10(-3)) and in younger patients (42.1±1.0 versus 47.6±0.7 years; P<10(-3)) and were associated with higher preoperative aldosterone levels (455±26 versus 376±17 ng/L; P=0.012) but not with therapeutic outcome after surgery. Germline KCNJ5 mutations were found neither in patients with APA nor those with bilateral adrenal hyperplasia. Somatic KCNJ5 mutations were specific for APA, because they were not identified in 25 peritumoral adrenal tissues or 16 cortisol-producing adenomas. Hierarchical clustering of transcriptome profiles showed that APAs with p.Gly151Arg or p.Leu168Arg mutations were indistinguishable from tumors without KCNJ5 mutations. In conclusion, although a large proportion of sporadic APAs harbors somatic KCNJ5 mutations, germline mutations are not similarly causative for bilateral adrenal hyperplasia. KCNJ5 mutation carriers are more likely to be females; younger age and higher aldosterone levels at diagnosis suggest that KCNJ5 mutations may be associated with a more florid phenotype of primary aldosteronism.
Felipe A. - One of the best experts on this subject based on the ideXlab platform.
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KCNE4-dependent functional consequences of Kv1.3-related leukocyte physiology
Springer Nature, 2021Co-Authors: Capera Aragones Jca, Sole L., Sastre D., Vallejo-gracia A., Felipe A., Colomer-molera M, Navarro-pérez M, Hajdú P, Pedrós-gámez OAbstract:The voltage-dependent potassium channel Kv1.3 plays essential roles in the immune system, participating in leukocyte activation, proliferation and apoptosis. The regulatory subunit KCNE4 acts as an ancillary peptide of Kv1.3, modulates K+ currents and controls channel abundance at the cell surface. KCNE4-dependent regulation of the oligomeric complex fine-tunes the physiological role of Kv1.3. Thus, KCNE4 is crucial for Ca2+-dependent Kv1.3-related leukocyte functions. To better understand the role of KCNE4 in the regulation of the immune system, we manipulated its expression in various leukocyte cell lines. Jurkat T lymphocytes exhibit low KCNE4 levels, whereas CY15 dendritic cells, a model of professional antigen-presenting cells, robustly express KCNE4. When the cellular KCNE4 abundance was increased in T cells, the interaction between KCNE4 and Kv1.3 affected important T cell physiological features, such as channel rearrangement in the immunological synapse, cell growth, apoptosis and activation, as indicated by decreased IL-2 production. Conversely, ablation of KCNE4 in dendritic cells augmented proliferation. Furthermore, the LPS-dependent activation of CY15 cells, which induced Kv1.3 but not KCNE4, increased the Kv1.3-KCNE4 ratio and increased the expression of free Kv1.3 without KCNE4 interaction. Our results demonstrate that KCNE4 is a pivotal regulator of the Kv1.3 channelosome, which fine-tunes immune system physiology by modulating Kv1.3-associated leukocyte functions
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The calmodulin-binding tetraleucine motif of KCNE4 is responsible for association with Kv1.3.
'FASEB', 2019Co-Authors: Sole L., Sastre D., Vallejo-gracia A., Serrano-albarras A., Ferrer-montiel A., Fernandez-ballester G., Tamkun M.m., Felipe A.Abstract:The voltage-dependent potassium (Kv) channel Kv1.3 regulates leukocyte proliferation, activation, and apoptosis, and altered expression of this channel is linked to autoimmune diseases. Thus, the fine-tuning of Kv1.3 function is crucial for the immune system response. The Kv1.3 accessory protein, potassium voltage-gated channel subfamily E (KCNE) subunit 4, acts as a dominant negative regulatory subunit to both enhance inactivation and induce intracellular retention of Kv1.3. Mutations in KCNE4 also cause immune system dysfunction. Although the formation of Kv1.3-KCNE4 complexes has profound consequences for leukocyte physiology, the molecular determinants involved in the Kv1.3-KCNE4 association are unknown. We now show that KCNE4 associates with Kv1.3 via a tetraleucine motif situated within the carboxy-terminal domain of this accessory protein. This motif would function as an interaction platform, in which Kv1.3 and Ca(2+)/calmodulin compete for the KCNE4 interaction. Finally, we propose a structural model of the Kv1.3-KCNE4 complex. Our experimental data and the in silico structure suggest that the KCNE4 interaction hides a forward-trafficking motif within Kv1.3 in addition to adding a strong endoplasmic reticulum retention signature to the Kv1.3-KCNE4 complex. Thus, the oligomeric composition of the Kv1.3 channelosome fine-tunes the precise balance between anterograde and intracellular retention elements that control the cell surface expression of Kv1.3 and immune system physiology.-Sole, L., Roig, S. R., Sastre, D., Vallejo-Gracia, A., Serrano-Albarras, A., Ferrer-Montiel, A., Fernandez-Ballester, G., Tamkun, M. M., Felipe, A. The calmodulin-binding tetraleucine motif of KCNE4 is responsible for association with Kv1.3
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The calmodulin-binding tetraleucine motif of KCNE4 is responsible for association with Kv1.3.
'FASEB', 2019Co-Authors: Sole L., Sastre D., Vallejo-gracia A., Serrano-albarras A., Ferrer-montiel A., Fernandez-ballester G., Tamkun M.m., Felipe A.Abstract:Item does not contain fulltextThe voltage-dependent potassium (Kv) channel Kv1.3 regulates leukocyte proliferation, activation, and apoptosis, and altered expression of this channel is linked to autoimmune diseases. Thus, the fine-tuning of Kv1.3 function is crucial for the immune system response. The Kv1.3 accessory protein, potassium voltage-gated channel subfamily E (KCNE) subunit 4, acts as a dominant negative regulatory subunit to both enhance inactivation and induce intracellular retention of Kv1.3. Mutations in KCNE4 also cause immune system dysfunction. Although the formation of Kv1.3-KCNE4 complexes has profound consequences for leukocyte physiology, the molecular determinants involved in the Kv1.3-KCNE4 association are unknown. We now show that KCNE4 associates with Kv1.3 via a tetraleucine motif situated within the carboxy-terminal domain of this accessory protein. This motif would function as an interaction platform, in which Kv1.3 and Ca(2+)/calmodulin compete for the KCNE4 interaction. Finally, we propose a structural model of the Kv1.3-KCNE4 complex. Our experimental data and the in silico structure suggest that the KCNE4 interaction hides a forward-trafficking motif within Kv1.3 in addition to adding a strong endoplasmic reticulum retention signature to the Kv1.3-KCNE4 complex. Thus, the oligomeric composition of the Kv1.3 channelosome fine-tunes the precise balance between anterograde and intracellular retention elements that control the cell surface expression of Kv1.3 and immune system physiology.-Sole, L., Roig, S. R., Sastre, D., Vallejo-Gracia, A., Serrano-Albarras, A., Ferrer-Montiel, A., Fernandez-Ballester, G., Tamkun, M. M., Felipe, A. The calmodulin-binding tetraleucine motif of KCNE4 is responsible for association with Kv1.3.1 juli 201
Ayaka Nishikido - One of the best experts on this subject based on the ideXlab platform.
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regulation of the kcnj5 gene by sf 1 in the adrenal cortex complete genomic organization and promoter function
Molecular and Cellular Endocrinology, 2020Co-Authors: Ayaka Nishikido, Shunichi Matsumoto, Kazuhiko Horiguchi, Tomoko Miyamoto, Akikokatano Toki, Satoshi Yoshino, Takashi Okamura, Yasuyo Nakajima, Emi Ishida, Tsugumichi SaitoAbstract:Abstract Activating mutations in the KCNJ5 gene are responsible for the significant number of aldosterone-producing adenomas. To elucidate the molecular mechanisms underlying KCNJ5 expression, we characterized the entire human KCNJ5 gene. The gene spanned approximately 29.8 kb and contained three exons and two introns. The strongest expression of KCNJ5 mRNA was observed in the adrenal gland. The promoter region contained a putative binding site for SF-1 at −1782 bp. A construct containing −2444 bp of the promoter region exhibited the strongest promoter activity in adrenal H295R cells, and the introduction of a mutation in the SF-1 binding site almost completely abolished promoter activity. Furthermore, deletion mutation, EMSA, and knockdown analyses revealed that SF-1 bound to this element and was functional. Immunochemistry showed that KCNJ5 was predominantly expressed in the zona glomerulosa, while SF-1 was ubiquitously expressed in the adrenal cortex. These results demonstrated that SF-1 mediates the expression of human KCNJ5 in the adrenal cortex.
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regulation of the kcnj5 gene by sf 1 in the adrenal cortex complete genomic organization and promoter function
Molecular and Cellular Endocrinology, 2020Co-Authors: Ayaka Nishikido, Shunichi Matsumoto, Kazuhiko Horiguchi, Tomoko Miyamoto, Akikokatano Toki, Satoshi Yoshino, Takashi Okamura, Yasuyo Nakajima, Emi Ishida, Tsugumichi SaitoAbstract:Abstract Activating mutations in the KCNJ5 gene are responsible for the significant number of aldosterone-producing adenomas. To elucidate the molecular mechanisms underlying KCNJ5 expression, we characterized the entire human KCNJ5 gene. The gene spanned approximately 29.8 kb and contained three exons and two introns. The strongest expression of KCNJ5 mRNA was observed in the adrenal gland. The promoter region contained a putative binding site for SF-1 at −1782 bp. A construct containing −2444 bp of the promoter region exhibited the strongest promoter activity in adrenal H295R cells, and the introduction of a mutation in the SF-1 binding site almost completely abolished promoter activity. Furthermore, deletion mutation, EMSA, and knockdown analyses revealed that SF-1 bound to this element and was functional. Immunochemistry showed that KCNJ5 was predominantly expressed in the zona glomerulosa, while SF-1 was ubiquitously expressed in the adrenal cortex. These results demonstrated that SF-1 mediates the expression of human KCNJ5 in the adrenal cortex.
Yasuyo Nakajima - One of the best experts on this subject based on the ideXlab platform.
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regulation of the kcnj5 gene by sf 1 in the adrenal cortex complete genomic organization and promoter function
Molecular and Cellular Endocrinology, 2020Co-Authors: Ayaka Nishikido, Shunichi Matsumoto, Kazuhiko Horiguchi, Tomoko Miyamoto, Akikokatano Toki, Satoshi Yoshino, Takashi Okamura, Yasuyo Nakajima, Emi Ishida, Tsugumichi SaitoAbstract:Abstract Activating mutations in the KCNJ5 gene are responsible for the significant number of aldosterone-producing adenomas. To elucidate the molecular mechanisms underlying KCNJ5 expression, we characterized the entire human KCNJ5 gene. The gene spanned approximately 29.8 kb and contained three exons and two introns. The strongest expression of KCNJ5 mRNA was observed in the adrenal gland. The promoter region contained a putative binding site for SF-1 at −1782 bp. A construct containing −2444 bp of the promoter region exhibited the strongest promoter activity in adrenal H295R cells, and the introduction of a mutation in the SF-1 binding site almost completely abolished promoter activity. Furthermore, deletion mutation, EMSA, and knockdown analyses revealed that SF-1 bound to this element and was functional. Immunochemistry showed that KCNJ5 was predominantly expressed in the zona glomerulosa, while SF-1 was ubiquitously expressed in the adrenal cortex. These results demonstrated that SF-1 mediates the expression of human KCNJ5 in the adrenal cortex.
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regulation of the kcnj5 gene by sf 1 in the adrenal cortex complete genomic organization and promoter function
Molecular and Cellular Endocrinology, 2020Co-Authors: Ayaka Nishikido, Shunichi Matsumoto, Kazuhiko Horiguchi, Tomoko Miyamoto, Akikokatano Toki, Satoshi Yoshino, Takashi Okamura, Yasuyo Nakajima, Emi Ishida, Tsugumichi SaitoAbstract:Abstract Activating mutations in the KCNJ5 gene are responsible for the significant number of aldosterone-producing adenomas. To elucidate the molecular mechanisms underlying KCNJ5 expression, we characterized the entire human KCNJ5 gene. The gene spanned approximately 29.8 kb and contained three exons and two introns. The strongest expression of KCNJ5 mRNA was observed in the adrenal gland. The promoter region contained a putative binding site for SF-1 at −1782 bp. A construct containing −2444 bp of the promoter region exhibited the strongest promoter activity in adrenal H295R cells, and the introduction of a mutation in the SF-1 binding site almost completely abolished promoter activity. Furthermore, deletion mutation, EMSA, and knockdown analyses revealed that SF-1 bound to this element and was functional. Immunochemistry showed that KCNJ5 was predominantly expressed in the zona glomerulosa, while SF-1 was ubiquitously expressed in the adrenal cortex. These results demonstrated that SF-1 mediates the expression of human KCNJ5 in the adrenal cortex.
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expression and mutations of kcnj5 mrna in japanese patients with aldosterone producing adenomas
The Journal of Clinical Endocrinology and Metabolism, 2012Co-Authors: Ryo Taguchi, Yasuyo Nakajima, Masanobu Yamada, Tetsurou Satoh, Koshi Hashimoto, Nobuyuki Shibusawa, Atsushi Ozawa, Shuichi Okada, Nana Rokutanda, Daisuke TakataAbstract:Context: Mutations of the KCNJ5 gene have recently been identified in patients with aldosterone-producing adenomas (APA). Objective: Our objective was to investigate the expression and mutations of the KCNJ5 gene in Japanese patients with APA. Design and Patients: We sequenced KCNJ5 cDNA and measured KCNJ5 mRNA levels in 23 patients with APA operated on at Gunma University Hospital. Main Outcome Measures: Mutations and mRNA levels of the KCNJ5 gene were examined and compared to those in cortisol-producing adenomas (Cushing's syndrome) and pheochromocytomas. Results: Of the 23 patients with APA, 15 (65.2%) had two somatic mutations of the KCNJ5 gene: 12 cases of p.G151R (eight with c.451G>A, and four with c.451G>C) and three cases of p.L168R (c.503T>G). Levels of KCNJ5 mRNA were significantly higher in the APA with mutations than those without. Immunohistochemistry also showed a stronger staining of KCNJ5 on the cell membrane in the tumor with a mutation. Furthermore, a PCR-restriction fragment length poly...