Molecular and Cellular Endocrinology

Acetylation modulates thyroid hormone receptor intracellular localization and intranuclear mobility Publication date: 15 September 2019 Source: Molecular and Cellular Endocrinology, Volume 495 Author(s): Cyril S. Anyetei-Anum, Rochelle M. Evans, Amanda M. Back, Vincent R. Roggero, Lizabeth A. Allison Abstract The thyroid hormone receptor (TR) undergoes nucleocytoplasmic shuttling, but is primarily nuclear-localized and mediates expression of genes involved in development and homeostasis. Given the proximity of TR acetylation and sumoylation sites to nuclear localization (NLS) and nuclear export signals, we investigated their role in regulating intracellular localization. The nuclear/cytosolic fluorescence ratio (N/C) of fluorescent protein-tagged acetylation mimic, nonacetylation mimic, and sumoylation-deficient TR was quantified in transfected mammalian cells. While nonacetylation mimic and sumoylation-deficient TRs displayed wild-type N/C, the acetylation mimic's N/C was significantly lower. Importins that interact with wild-type TR also interact with acetylation and nonacetylation mimics, suggesting factors other than reduced importin binding alter nuclear localization. FRAP analysis showed wild-type intranuclear dynamics of acetylation mimic and sumoylation-deficient TRs, whereas the nonacetylation mimic had significantly reduced mobility and transcriptional activity. Acetyltransferase CBP/p300 inhibition enhanced TR's nuclear localization, further suggesting that nonacetylation correlates with nuclear retention, while acetylation promotes cytosolic localization.

Nicotine induces insulin resistance via downregulation of Nrf2 in cardiomyocyte Publication date: 15 September 2019 Source: Molecular and Cellular Endocrinology, Volume 495 Author(s): Zhi Li, Wang Xu, Yiwan Su, Kai Gao, Yuqiang Chen, Lian Ma, Yang Xie Abstract Clinical studies have demonstrated that cigarette smoking is strongly associated with insulin resistance and heart disease. Nicotine is considered the primary toxin constituent associated with smoking. However, the distinct molecular mechanism of nicotine-induced cardiac dysfunction remains unclear. Cardiomyocytes with nicotine-induced insulin resistance are characterized by decreased glucose uptake, as measured by 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG), a fluorescent derivative of glucose, and reactive oxygen species (ROS) generation. Immunoblotting was used to evaluate the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), extracellular signal-related kinase (ERK) and phosphoinositide 3-kinase (PI3K, p85, Y607). We determined the impact of nicotine on insulin resistance and Nrf2, phospho-ERK and phospho-PI3K expression in the myocardial tissue of a mouse model. Nicotine increased ROS production and depressed insulin-induced glucose uptake in cardiomyocytes. Pretreatment with N-acetyl-L-cysteine (NAC), an antioxidant, reversed nicotine-inhibited glucose uptake induced by insulin. Nicotine exposure directly inhibited Nrf2 and increased ERK phosphorylation in cardiomyocytes, which were obstructed by NAC. Further exploration of signaling cascades revealed nicotine-induced ROS involved in inhibiting PI3K/Nrf2 and activating ERK in cardiomyocytes. Moreover, the mouse model treated with nicotine showed glucose intolerance and impaired insulin tolerance accompanied by inhibited PI3K/Nrf2 and increased ERK in myocardial tissues. Thus, nicotine induces insulin resistance via the downregulation of Nrf2 activity in cardiomyocytes, which is a potential mechanism of the pharmacological effects of nicotine. This study identified potential therapeutic targets against nicotine-related cardiovascular diseases.

Kynurenic acid attenuates pro-inflammatory reactions in lipopolysaccharide-stimulated endothelial cells through the PPARδ/HO-1-dependent pathway Publication date: 15 September 2019 Source: Molecular and Cellular Endocrinology, Volume 495 Author(s): Taeseung Lee, Hyung Sub Park, Ji Hoon Jeong, Tae Woo Jung Abstract Kynurenic acid (KA) regulates energy homeostasis and alleviates inflammation in adipose tissue but how KA affects the atherosclerotic response in HUVECs remains unclear. We evaluated the effects of KA on lipopolysaccharide (LPS)-induced inflammation and apoptosis in HUVECs. KA enhanced peroxisome proliferator-activated receptor delta (PPARδ) expression in HUVECs and THP-1 cells and suppressed LPS-induced atherosclerotic responses through PPARδ-mediated signaling. Moreover, KA treatment mitigated LPS-induced phosphorylation of nuclear factor kappa B and pro-inflammatory cytokine release in HUVECs and THP-1 cells, and down-regulated adhesion molecules in HUVECs and adhesion of THP-1 cells to HUVECs following LPS treatment. KA treatment decreased LPS-induced inflammation and apoptosis, and also promoted heme oxygenase (HO)-1 expression, which suppresses inflammation in HUVECs. Suppression of PPARδ or HO-1 expression markedly mitigated the effects of KA on atherosclerotic responses in HUVECs. Thus, KA attenuates LPS-induced atherosclerotic responses by suppressing inflammation via the PPARδ/HO-1-dependent pathway. Graphical abstract

MicroRNA-126-5p downregulates BCAR3 expression to promote cell migration and invasion in endometriosis Publication date: 20 August 2019 Source: Molecular and Cellular Endocrinology, Volume 494 Author(s): Xiannan Meng, Jing Liu, Huimin Wang, Peng Chen, Danbo Wang Abstract Purpose Endometriosis (EMs) is an estrogen-dependent multifactorial disease. Inhibition of estrogen in endometrial cells contributes to their failure to form lesions in ectopic sites. However, whether reducing or suppressing the inhibitory effect of estrogen results in the establishment of ectopic lesions remains unclear. The BCAR3 gene induces estrogen resistance in estrogen-dependent breast cancer cells and promotes cell migration, invasion, and epithelial-mesenchymal transition (EMT). However, the expression of BCAR3 in endometriosis and its effect on endometrial cell function and the anti-estrogen effect of endometriosis have not been reported. These issues are addressed in the present study. Methods The study included 32 cases of ectopic endometrium and eutopic endometrium in patients with endometriosis and 31 cases of normal endometrium as controls. The expression of BCAR3 and microRNA (miR)-126-5p was detected by real-time PCR, immunohistochemistry, and western blotting. The effects of BCAR3 and miR-126-5p on the morphology and biological behavior of eutopic endometrial cells were verified using lentivirus overexpression and a vector knockdown model, the CCK-8 assay, Transwell experiments, and estrogen intervention experiments using primary cultures of epithelial and stromal cells. Results The BCAR3 gene was highly expressed in ectopic endometrium and the eutopic endometrium of patients with endometriosis, and the expression level was higher in stage III-IV patients than in stage I-II patients. In vitro cell experiments showed that miR-126-5p negatively regulated the expression of BCAR3 and its effect on the migration and invasion of stromal cells. Low expression of miR-126-5p and high expression of BCAR3 promoted endometriosis stromal cell migration and invasion. Assessment of EMT in endometriosis compared with eutopic endometrium showed that the expression of vimentin was significantly increased and the expression of E-cadherin was significantly decreased in ectopic endometrium. Estrogen promoted EMT in eutopic endometrial epithelial cells and this effect was reversed by estrogen inhibitors. BCAR3 had no direct effect on EMT and did not act synergistically with estrogen on promoting EMT. Conclusion miR-126-5p negatively regulated BCAR3 expression in eutopic endometriosis, enhanced the migration and invasion of endometrial cells, and promoted the occurrence of endometriosis. BCAR3 did not induce EMT and had no synergistic effect with estrogen, but its inhibition of anti-estrogen function may provide new insight into the mechanism of local estrogen action in endometriosis.

Microtubule actin crosslinking factor 1 (MACF1) knockdown inhibits RANKL-induced osteoclastogenesis via Akt/GSK3β/NFATc1 signalling pathway Publication date: 20 August 2019 Source: Molecular and Cellular Endocrinology, Volume 494 Author(s): Xiao Lin, Yunyun Xiao, Zhihao Chen, Jianhua Ma, Wuxia Qiu, Kewen Zhang, Fang Xu, Kai Dang, Airong Qian Abstract Osteoclasts are responsible for bone resorption and play essential roles in causing bone diseases such as osteoporosis. Microtubule actin crosslinking factor 1 (MACF1) is a large spectraplakin protein that has been implicated in regulating cytoskeletal distribution, cell migration, cell survival and cell differentiation. However, whether MACF1 regulates the differentiation of osteoclasts has not been elucidated. In this study, we found that the expression of MACF1 was increased in primary bone marrow-derived monocytes (BMMs) of osteoporotic mice and was downregulated during receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis of pre-osteoclast cell lines RAW264.7 cells. RAW264.7 cells were transfected with shMACF1 using a lentiviral vector to study the role of MACF1 in osteoclastogenic differentiation. Knockdown of MACF1 in RAW264.7 cells inhibited the formation of multinucleated osteoclasts and decreased the expression of osteoclast-marker genes (Ctsk, Acp5, Mmp9 and Oscar) during RANKL-induced osteoclastogenesis. Additionally, knockdown of MACF1 disrupted actin ring formation in osteoclasts and further blocked the bone resorption activity of osteoclasts by reducing the area and depth of pits. Knockdown of MACF1 had no effect on the survival of pre-osteoclasts and mature osteoclasts. We further established that knockdown of MACF1 attenuated the phosphorylation of Akt and GSK3β and inhibited the expression of its downstream target NFATc1. Akt activator rescued the inhibition of osteoclast differentiation by MACF1 knockdown. These data demonstrate that MACF1 positively regulates osteoclast differentiation via the Akt/GSK3β/NFATc1 signalling pathway, suggesting that targeting MACF1 may be a novel therapeutic approach against osteoporosis.

Compositional analysis and biological characterization of Cornus officinalis on human 1.1B4 pancreatic β cells Publication date: 20 August 2019 Source: Molecular and Cellular Endocrinology, Volume 494 Author(s): Arielle E. Sharp-Tawfik, Alexis M. Coiner, Catherine B. MarElia, Melissa Kazantzis, Clare Zhang, Brant R. Burkhardt Abstract Type 1 diabetes (T1D) is an autoimmune disease resulting from the loss of pancreatic β cells and subsequent insulin production. Novel interventional therapies are urgently needed that can protect existing β cells from cytokine-induced death and enhance their function before symptomatic onset. Our initial evidence is suggesting that bioactive ingredients within Cornus officinalis (CO) may be able to serve in this function. CO has been extensively used in Traditional Chinese Medicine (TCM) and reported to possess both anti-inflammatory and pro-metabolic effects. We hypothesize that CO treatment may provide a future potential candidate for interventional therapy for early stage T1D prior to significant β cell loss. Our data demonstrated that CO can inhibit cytokine-mediated β cell death, increase cell viability and oxidative capacity, and increase expression of NFATC2 (Nuclear Factor of Activated T Cells, Cytoplasmic 2). We have also profiled the bioactive components in CO from multiple sources by HPLC/MS (High Performance Liquid Chromatography/Mass Spectrometry) analysis. Altogether, CO significantly increases the energy metabolism of β cells while inducing the NFAT pathway to signal for increased proliferation and endocrine function.

Natriuretic peptide signaling is involved in the expression of oxidative metabolism-related and muscle fiber constitutive genes in the gastrocnemius muscle Publication date: 20 August 2019 Source: Molecular and Cellular Endocrinology, Volume 494 Author(s): Kiyoshi Ishikawa, Taiki Hara, Mao Mizukawa, Yasufumi Fukano, Takeshi Shimomura Abstract Natriuretic peptides regulate cyclic guanosine monophosphate (cGMP) levels via their receptors and have various physiological effects. Natriuretic peptide receptor C (NPR-C) increases cGMP signaling by functioning as a clearance receptor. We analyzed the role of natriuretic peptides in the skeletal muscle, which increases in mass with bone elongation, of NPR-C− mice. High-fat diet (HFD)-fed NPR-C− mice exhibited obesity resistance and higher oxygen consumption. PGC1α gene expression was upregulated in the gastrocnemius muscle of HFD-fed NPR-C− mice compared with HFD-fed NPR-C+ (wild-type) mice. Gene expression of proliferator-activated receptor delta and estrogen-related receptor α, which upregulate oxidative metabolism, was increased in the gastrocnemius muscle of NPR-C− mice, irrespective of diet. Expression of myosin heavy chain 7, a component of type I slow-twitch fiber, was enhanced. Natriuretic peptide signaling may influence oxidative metabolism-related and slow-twitch fiber constitutive gene expression in the fast-twitch gastrocnemius muscle but not in slow-twitch muscles such as the soleus.

Uterus globulin associated protein 1 (UGRP1) is a potential marker of progression of Graves' disease into hypothyroidism Publication date: 20 August 2019 Source: Molecular and Cellular Endocrinology, Volume 494 Author(s): Zheng Zhou, Chun-Lin Zuo, Xue-Song Li, Xiao-Ping Ye, Qian-Yue Zhang, Ping Wang, Rong-Xin Zhang, Gang Chen, Jia-Lin Yang, Yue Chen, Qin-Yun Ma, Huai-Dong Song Abstract Approximately 20% of Graves' disease (GD) patients may result eventually in hypothyroidism in their natural course. Uterus globulin-associated protein 1 (UGRP1) was associated with GD in our previous study. Here we investigated the role of UGRP1 in the development of autoimmune thyroid disease (AITD). The results showed that UGRP1 was expressed in the thyrocytes of most Hashimoto's thyroiditis (HT) patients and a proportion of GD patients (293 HT and 198 GD). The pathologic features of UGRP1-positive thyrocytes resembled “Hürthle cells”, and were surrounded by infiltrated leukocytes. The positivity rate of TPOAb in UGRP1-positive GD patients was much higher than that in -negative GD patients. Moreover, UGRP1 was co-expressed with Fas and HLA-DR in the thyrocytes of AITD patients. We also found IL-1β but not Th1 or Th2 cytokines was able to upregulate the expression of UGRP1. Our findings indicated that UGRP1 may be a novel marker in thyrocytes to predict GD patients who develop hypothyroidism.

The acute effects of hydrocortisone on cardiac electrocardiography, action potentials, intracellular calcium, and contraction: The role of protein kinase C Publication date: 20 August 2019 Source: Molecular and Cellular Endocrinology, Volume 494 Author(s): Mi-Hyeong Park, Seo-In Park, Jong-Hui Kim, Jing Yu, Eun Hye Lee, Su Ryeon Seo, Su-Hyun Jo Abstract Hydrocortisone exerts adverse effects on various organs, including the heart. This study investigated the still unclear effects of hydrocortisone on electrophysiological and biochemical aspects of cardiac excitation–contraction coupling. In guinea pigs’ hearts, hydrocortisone administration reduced the QT interval of ECG and the action potential duration (APD). In guinea pig ventricular myocytes, hydrocortisone reduced contraction and Ca2+ transient amplitudes. These reductions and the effects on APD were prevented by pretreatment with the protein kinase C (PKC) inhibitor staurosporine. In an overexpression system of Xenopus oocytes, hydrocortisone increased hERG K+ currents and reduced Kv1.5 K+ currents; these effects were negated by pretreatment with staurosporine. Western blot analysis revealed dose- and time-dependent changes in PKCα/βII, PKCε, and PKCγ phosphorylation by hydrocortisone in guinea pig ventricular myocytes. Therefore, hydrocortisone can acutely affect cardiac excitation–contraction coupling, including ion channel activity, APD, ECG, Ca2+ transients, and contraction, possibly via biochemical changes in PKC.

Klotho inhibits PKCα/p66SHC-mediated podocyte injury in diabetic nephropathy Publication date: 20 August 2019 Source: Molecular and Cellular Endocrinology, Volume 494 Author(s): Wei Jiang, Tangli Xiao, Wenhao Han, Jiachuan Xiong, Ting He, Yong Liu, Yinghui Huang, Ke Yang, Xianjin Bi, Xinli Xu, Yanlin Yu, Yan Li, Jun Gu, Jingbo Zhang, Yunjian Huang, Bo Zhang, Jinghong Zhao Abstract Diabetic nephropathy (DN) is a progressive disease, the main pathogeny of which is podocyte injury. As a calcium-dependent serine/threonine protein kinase involved in podocyte injury, protein kinase C isoform α (PKCα) was reported to regulate the phosphorylation of p66SHC. However, the role of PKCα/p66SHC in DN remains unknown. Klotho, an anti-aging protein with critical roles in protecting kidney, is expressed predominantly in the kidney and secreted in the blood. Nonetheless, the mechanism underlying amelioration of podocyte injury by Klotho in DN remains unclear. Our data showed that Klotho was decreased in STZ-treated mice and was further declined in diabetic KL ± mice. As expected, Klotho deficiency aggravated diabetes-induced proteinuria and podocyte injury, accompanied by the activation of PKCα and p66SHC. In contrast, overexpression of Klotho partially ameliorated PKCα/p66SHC-mediated podocyte injury and proteinuria. In addition, in vitro experiments showed that activation of PKCα and subsequently increased intracellular reactive oxygen species (ROS) was involved in podocytic apoptosis induced by high glucose (HG), which could be partially reversed by Klotho. Hence, we conclude that Klotho might inhibit PKCα/p66SHC-mediated podocyte injury in diabetic nephropathy. Graphical abstract Schematic diagram of the proposed mechanism. HG promotes calcium influx by activating calcium channel (e.g. TRCPC6) in podocytes. Excess intracellular calcium activates PKCα which then contributes to the phosphorylation of p66SHC. Phosphorylated p66SHC then enters mitochondria by altering its configuration. Thereafter, the altered configuration of p66SHC oxidizes cytochrome c (Cytc) releasing a large amount of reactive oxygen species (ROS). Continuous generation of ROS eventually leads to cellular injury and apoptosis of podocytes. However, Klotho (sKL) can function by inhibiting calcium influx induced by HG. SKL reduces intracellular calcium to inhibit PKCα/p66SHC. Therefore, Klotho inhibits continuous generation of ROS eventually ameliorating injury and apoptosis of podocytes.