Psychiatric patients treated with lithium (Li+) may develop nephrogenic diabetes insipidus (NDI). Although the etiology of Li+-induced NDI (Li-NDI) is poorly understood, it occurs partially due to reduced aquaporin-2 (AQP2) expression in the kidney collecting ducts. A mechanism postulated for this is that Li+ inhibits adenylyl cyclase (AC) activity, leading to decreased cAMP, reduced AQP2 abundance, and less membrane targeting. We hypothesized that Li-NDI would not develop in mice lacking AC6. Whole-body AC6 knockout (AC6–/–) mice and potentially novel connecting tubule/principal cell–specific AC6 knockout (AC6loxloxCre) mice had approximately 50% lower urine osmolality and doubled water intake under baseline conditions compared with controls. Dietary Li+ administration increased water intake and reduced urine osmolality in control, AC6–/–, and AC6loxloxCre mice. Consistent with AC6–/– mice, medullary AQP2 and pS256-AQP2 abundances were lower in AC6loxloxCre mice compared with controls under standard conditions, and levels were further reduced after Li+ administration. AC6loxloxCre and control mice had a similar increase in the numbers of proliferating cell nuclear antigen–positive cells in response to Li+. However, AC6loxloxCre mice had a higher number of H+-ATPase B1 subunit–positive cells under standard conditions and after Li+ administration. Collectively, AC6 has a minor role in Li-NDI development but may be important for determining the intercalated cell–to–principal cell ratio.
Søren Brandt Poulsen, Tina Bøgelund Kristensen, Heddwen L. Brooks, Donald E. Kohan, Timo Rieg, Robert A. Fenton
Loss-of-function mutations in stromal interaction molecule 1 (STIM1) impair the activation of Ca2+ release–activated Ca2+ (CRAC) channels and store-operated Ca2+ entry (SOCE), resulting in a disease syndrome called CRAC channelopathy that is characterized by severe dental enamel defects. The cause of these enamel defects has remained unclear given a lack of animal models. We generated
Miriam Eckstein, Martin Vaeth, Cinzia Fornai, Manikandan Vinu, Timothy G. Bromage, Meerim K. Nurbaeva, Jessica L. Sorge, Paulo G. Coelho, Youssef Idaghdour, Stefan Feske, Rodrigo S. Lacruz
Loss of LKB1 activity is prevalent in
Melissa Gilbert-Ross, Jessica Konen, Junghui Koo, John Shupe, Brian S. Robinson, Walter Guy Wiles IV, Chunzi Huang, W. David Martin, Madhusmita Behera, Geoffrey H. Smith, Charles E. Hill, Michael R. Rossi, Gabriel L. Sica, Manali Rupji, Zhengjia Chen, Jeanne Kowalski, Andrea L. Kasinski, Suresh S. Ramalingam, Haian Fu, Fadlo R. Khuri, Wei Zhou, Adam I. Marcus
The leucine rich repeat containing protein 8A (LRRC8A), or SWELL1, is an essential component of the volume-regulated anion channel (VRAC) that is activated by cell swelling and ionic strength. We report here for the first time to our knowledge its expression in a primary cell culture of nodose ganglia neurons and its localization in the soma, neurites, and neuronal membrane. We show that this neuronal VRAC/SWELL1 senses low external pH (pHo) in addition to hypoosmolarity. A robust sustained chloride current is seen in 77% of isolated nodose neurons following brief exposures to extracellular acid pH. Its activation involves proton efflux, intracellular alkalinity, and an increase in NOX-derived H2O2. The molecular identity of both the hypoosmolarity-induced and acid pHo–conditioned VRAC as LRRC8A (SWELL1) was confirmed by Cre-flox–mediated KO, shRNA-mediated knockdown, and CRISPR/Cas9-mediated LRRC8A deletion in HEK cells and in primary nodose neuronal cultures. Activation of VRAC by low pHo reduces neuronal injury during simulated ischemia and N-methyl-D-aspartate–induced (NMDA-induced) apoptosis. These results identify the VRAC (LRRC8A) as a dual sensor of hypoosmolarity and low pHo in vagal afferent neurons and define the mechanisms of its activation and its neuroprotective potential.
Runping Wang, Yongjun Lu, Susheel Gunasekar, Yanhui Zhang, Christopher J. Benson, Mark W. Chapleau, Rajan Sah, François M. Abboud
ice and humans lacking the caveolae component polymerase I transcription release factor (PTRF, also known as cavin-1) exhibit lipo- and muscular dystrophy. Here we describe the molecular features underlying the muscle phenotype for PTRF/cavin-1 null mice. These animals had a decreased ability to exercise, and exhibited muscle hypertrophy with increased muscle fiber size and muscle mass due, in part, to constitutive activation of the Akt pathway. Their muscles were fibrotic and exhibited impaired membrane integrity accompanied by an apparent compensatory activation of the dystrophin-glycoprotein complex along with elevated expression of proteins involved in muscle repair function.
Shi-Ying Ding, Libin Liu, Paul F. Pilch
Mitophagy occurs during ischemia/reperfusion (I/R) and limits oxidative stress and injury. Mitochondrial turnover was assessed in patients undergoing cardiac surgery involving cardiopulmonary bypass (CPB). Paired biopsies of right atrial appendage before initiation and after weaning from CPB were processed for protein analysis, mitochondrial DNA/nuclear DNA ratio (mtDNA:nucDNA ratio), mtDNA damage, mRNA, and polysome profiling. Mitophagy in the post-CPB samples was evidenced by decreased levels of mitophagy adapters NDP52 and optineurin in whole tissue lysate, decreased Opa1 long form, and translocation of Parkin to the mitochondrial fraction. PCR analysis of mtDNA comparing amplification of short vs. long segments of mtDNA revealed increased damage following cardiac surgery. Surprisingly, a marked increase in several mitochondria-specific protein markers and mtDNA:nucDNA ratio was observed, consistent with increased mitochondrial biogenesis. mRNA analysis suggested that mitochondrial biogenesis was traniscription independent and likely driven by increased translation of existing mRNAs. These findings demonstrate in humans that both mitophagy and mitochondrial biogenesis occur during cardiac surgery involving CPB. We suggest that mitophagy is balanced by mitochondrial biogenesis during I/R stress experienced during surgery. Mitigating mtDNA damage and elucidating mechanisms regulating mitochondrial turnover will lead to interventions to improve outcome after I/R in the setting of heart disease.
Allen M. Andres, Kyle C. Tucker, Amandine Thomas, David J.R. Taylor, David Sengstock, Salik M. Jahania, Reza Dabir, Somayeh Pourpirali, Jamelle A. Brown, David G. Westbrook, Scott W. Ballinger, Robert M. Mentzer Jr., Roberta A. Gottlieb
Pathogen-specific polyfunctional T cell responses have been associated with favorable clinical outcomes, but it is not known whether molecular differences exist between polyfunctional and monofunctional cytokine-producing T cells. Here, we report that polyfunctional CD4+ T cells induced during
Julie G. Burel, Simon H. Apte, Penny L. Groves, James S. McCarthy, Denise L. Doolan
Amphetamine (AMPH) or methamphetamine (METH) abuse can cause oxidative damage and is a risk factor for diseases including pulmonary arterial hypertension (PAH). Pulmonary artery endothelial cells (PAECs) from AMPH-associated-PAH patients show DNA damage as judged by γH2AX foci and DNA comet tails. We therefore hypothesized that AMPH induces DNA damage and vascular pathology by interfering with normal adaptation to an environmental perturbation causing oxidative stress. Consistent with this, we found that AMPH alone does not cause DNA damage in normoxic PAECs, but greatly amplifies DNA damage in hypoxic PAECs. The mechanism involves AMPH activation of protein phosphatase 2A, which potentiates inhibition of Akt. This increases sirtuin 1, causing deacetylation and degradation of HIF1α, thereby impairing its transcriptional activity, resulting in a reduction in pyruvate dehydrogenase kinase 1 and impaired cytochrome
Pin-I Chen, Aiqin Cao, Kazuya Miyagawa, Nancy F. Tojais, Jan K. Hennigs, Caiyun G. Li, Nathaly M. Sweeney, Audrey S. Inglis, Lingli Wang, Dan Li, Matthew Ye, Brian J. Feldman, Marlene Rabinovitch
Hypertrophic cardiomyopathy (HCM) is a common heart disease with a prevalence of 1 in 500 in the general population. Several mutations in genes encoding cardiac proteins have been found in HCM patients, but these changes do not predict occurrence or prognosis and the molecular mechanisms underlying HCM remain largely elusive. Here we show that cardiac expression of vacuolar protein sorting 34 (Vps34) is reduced in a subset of HCM patients. In a mouse model, muscle-specific loss of Vps34 led to HCM-like manifestations and sudden death. Vps34-deficient hearts exhibited abnormal histopathologies, including myofibrillar disarray and aggregates containing αB-crystallin (CryAB). These features result from a block in the ESCRT-mediated proteolysis that normally degrades K63-polyubiquitinated CryAB. CryAB deposition was also found in myocardial specimens from a subset of HCM patients whose hearts showed decreased Vps34. Our results identify disruption of the previously unknown Vps34-CryAB axis as a potentially novel etiology of HCM.
Hirotaka Kimura, Satoshi Eguchi, Junko Sasaki, Keiji Kuba, Hiroki Nakanishi, Shunsuke Takasuga, Masakazu Yamazaki, Akiteru Goto, Hiroyuki Watanabe, Hiroshi Itoh, Yumiko Imai, Akira Suzuki, Noboru Mizushima, Takehiko Sasaki
Current therapies to treat non–small cell lung carcinoma (NSCLC) have proven ineffective owing to transient, variable, and incomplete responses. Here we show that ABL kinases, ABL1 and ABL2, promote metastasis of lung cancer cells harboring EGFR or KRAS mutations. Inactivation of ABL kinases suppresses NSCLC metastasis to brain and bone, and other organs. ABL kinases are required for expression of prometastasis genes. Notably, ABL1 and ABL2 depletion impairs extravasation of lung adenocarcinoma cells into the lung parenchyma. We found that ABL-mediated activation of the TAZ and β-catenin transcriptional coactivators is required for NSCLC metastasis. ABL kinases activate TAZ and β-catenin by decreasing their interaction with the β-TrCP ubiquitin ligase, leading to increased protein stability. High-level expression of
Jing Jin Gu, Clay Rouse, Xia Xu, Jun Wang, Mark W. Onaitis, Ann Marie Pendergast
Lymphangioleiomyomatosis (LAM) is a progressive lung disease that primarily affects young women. Genetic evidence suggests that LAM cells bearing
Chenggang Li, Na Li, Xiaolei Liu, Erik Y. Zhang, Yang Sun, Kouhei Masuda, Jing Li, Julia Sun, Tasha Morrison, Xiangke Li, Yuanguang Chen, Jiang Wang, Nagla A. Karim, Yi Zhang, John Blenis, Mauricio J. Reginato, Elizabeth P. Henske, Jane J. Yu
Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease and one of the leading inherited causes of infant mortality. SMA results from insufficient levels of the survival motor neuron (SMN) protein, and studies in animal models of the disease have shown that increasing SMN protein levels ameliorates the disease phenotype. Our group previously identified and optimized a new series of small molecules, with good potency and toxicity profiles and reasonable pharmacokinetics, that were able to increase SMN protein levels in SMA patient–derived cells. We show here that ML372, a representative of this series, almost doubles the half-life of residual SMN protein expressed from the SMN2 locus by blocking its ubiquitination and subsequent degradation by the proteasome. ML372 increased SMN protein levels in muscle, spinal cord, and brain tissue of SMA mice. Importantly, ML372 treatment improved the righting reflex and extended survival of a severe mouse model of SMA. These results demonstrate that slowing SMN degradation by selectively inhibiting its ubiquitination can improve the motor phenotype and lifespan of SMA model mice.
Mahlet B. Abera, Jingbo Xiao, Jonathan Nofziger, Steve Titus, Noel Southall, Wei Zheng, Kasey E. Moritz, Marc Ferrer, Jonathan J. Cherry, Elliot J. Androphy, Amy Wang, Xin Xu, Christopher Austin, Kenneth H. Fischbeck, Juan J. Marugan, Barrington G. Burnett
Bcl-2–associated athanogene 3 (BAG3) is an evolutionarily conserved protein expressed at high levels in the heart and the vasculature and in many cancers. While altered BAG3 expression has been associated with cardiac dysfunction, its role in ischemia/reperfusion (I/R) is unknown. To test the hypothesis that BAG3 protects the heart from reperfusion injury, in vivo cardiac function was measured in hearts infected with either recombinant adeno-associated virus serotype 9–expressing (rAAV9-expressing) BAG3 or GFP and subjected to I/R. To elucidate molecular mechanisms by which BAG3 protects against I/R injury, neonatal mouse ventricular cardiomyocytes (NMVCs) in which BAG3 levels were modified by adenovirus expressing (Ad-expressing) BAG3 or siBAG3 were exposed to hypoxia/reoxygenation (H/R). H/R significantly reduced NMVC BAG3 levels, which were associated with enhanced expression of apoptosis markers, decreased expression of autophagy markers, and reduced autophagy flux. The deleterious effects of H/R on apoptosis and autophagy were recapitulated by knockdown of BAG3 with Ad-siBAG3 and were rescued by Ad-BAG3. In vivo, treatment of mice with rAAV9-BAG3 prior to I/R significantly decreased infarct size and improved left ventricular function when compared with mice receiving rAAV9-GFP and improved markers of autophagy and apoptosis. These findings suggest that BAG3 may provide a therapeutic target in patients undergoing reperfusion after myocardial infarction.
Feifei Su, Valerie D. Myers, Tijana Knezevic, JuFang Wang, Erhe Gao, Muniswamy Madesh, Farzaneh G. Tahrir, Manish K. Gupta, Jennifer Gordon, Joseph Rabinowitz, Frederick V. Ramsey, Douglas G. Tilley, Kamel Khalili, Joseph Y. Cheung, Arthur M. Feldman
Loss of functional pancreatic β cells is a hallmark of both type 1 and 2 diabetes. Identifying the pathways that promote β cell proliferation and/or block β cell apoptosis is a potential strategy for diabetes therapy. The transcriptional coactivator Yes-associated protein (YAP), a major downstream effector of the Hippo signaling pathway, is a key regulator of organ size and tissue homeostasis by modulating cell proliferation and apoptosis. YAP is not expressed in mature primary human and mouse β cells. We aimed to identify whether reexpression of a constitutively active form of YAP promotes β cell proliferation/survival. Overexpression of YAP remarkably induced β cell proliferation in isolated human islets, while β cell function and functional identity genes were fully preserved. The transcription factor forkhead box M1 (FOXM1) was upregulated upon YAP overexpression and necessary for YAP-dependent β cell proliferation. YAP overexpression protected β cells from apoptosis triggered by multiple diabetic conditions. The small redox proteins thioredoxin-1 and thioredoxin-2 (Trx1/2) were upregulated by YAP; disruption of the Trx system revealed that Trx1/2 was required for the antiapoptotic action of YAP in insulin-producing β cells. Our data show the robust proproliferative and antiapoptotic function of YAP in pancreatic β cells. YAP reconstitution may represent a disease-modifying approach to restore a functional β cell mass in diabetes.
Ting Yuan, Sahar Rafizadeh, Zahra Azizi, Blaz Lupse, Kanaka Durga Devi Gorrepati, Sushil Awal, Jose Oberholzer, Kathrin Maedler, Amin Ardestani
The small intestine has an underappreciated role as a lipid storage organ. Under conditions of high dietary fat intake, enterocytes can minimize the extent of postprandial lipemia by storing newly absorbed dietary fat in cytoplasmic lipid droplets. Lipid droplets can be subsequently mobilized for the production of chylomicrons. The mechanisms that regulate this process are poorly understood. We report here that the milk protein Mfge8 regulates hydrolysis of cytoplasmic lipid droplets in enterocytes after interacting with the αvβ3 and αvβ5 integrins. Mice deficient in Mfge8 or the αvβ3 and αvβ5 integrins accumulate excess cytoplasmic lipid droplets after a fat challenge. Mechanistically, interruption of the Mfge8-integrin axis leads to impaired enterocyte intracellular triglyceride hydrolase activity in vitro and in vivo. Furthermore, Mfge8 increases triglyceride hydrolase activity through a PI3 kinase/mTORC2–dependent signaling pathway. These data identify a key role for Mfge8 and the αvβ3 and αvβ5 integrins in regulating enterocyte lipid processing.
Amin Khalifeh-Soltani, Deepti Gupta, Arnold Ha, Jahangir Iqbal, Mahmood Hussain, Michael J. Podolsky, Kamran Atabai
Adipose tissue is a key endocrine organ that governs systemic homeostasis. PPARγ is a master regulator of adipose tissue signaling that plays an essential role in insulin sensitivity, making it an important therapeutic target. The selective PPARγ agonist rosiglitazone (RSG) has been used to treat diabetes. However, adverse cardiovascular effects have seriously hindered its clinical application. Experimental models have revealed that PPARγ activation increases cardiac hypertrophy. RSG stimulates cardiac hypertrophy and oxidative stress in cardiomyocyte-specific PPARγ knockout mice, implying that RSG might stimulate cardiac hypertrophy independently of cardiomyocyte PPARγ. However, candidate cell types responsible for RSG-induced cardiomyocyte hypertrophy remain unexplored. Utilizing cocultures of adipocytes and cardiomyocytes, we found that stimulation of PPARγ signaling in adipocytes increased miR-200a expression and secretion. Delivery of miR-200a in adipocyte-derived exosomes to cardiomyocytes resulted in decreased TSC1 and subsequent mTOR activation, leading to cardiomyocyte hypertrophy. Treatment with an antagomir to miR-200a blunted this hypertrophic response in cardiomyocytes. In vivo, specific ablation of PPARγ in adipocytes was sufficient to blunt hypertrophy induced by RSG treatment. By delineating mechanisms by which RSG elicits cardiac hypertrophy, we have identified pathways that mediate the crosstalk between adipocytes and cardiomyocytes to regulate cardiac remodeling.
Xi Fang, Matthew J. Stroud, Kunfu Ouyang, Li Fang, Jianlin Zhang, Nancy D. Dalton, Yusu Gu, Tongbin Wu, Kirk L. Peterson, Hsien-Da Huang, Ju Chen, Nanping Wang
Emerging knowledge indicates the difficulty in categorizing unusual cystic fibrosis (CF) mutations, with regard to both pathogenic mechanism and theratype. As case in point, we present data concerning P67L mutation of the cystic fibrosis transmembrane conductance regulator (CFTR), a defect carried by a small number of individuals with CF and sometimes attributed to a channel conductance abnormality. Findings from our laboratory and others establish that P67L causes protein misfolding, disrupts maturation, confers gating defects, is thermally stable, and exhibits near normal conductance. These results provide one framework by which rare CF alleles such as P67L can be more comprehensively profiled vis-à-vis molecular pathogenesis. We also demonstrate that emerging CF treatments — ivacaftor and lumacaftor — can mediate pronounced pharmacologic activation of P67L CFTR. Infrequent CF alleles are often improperly characterized, in part, due to the small numbers of patients involved. Moreover, access to new personalized treatments among patients with ultra-orphan genotypes has been limited by difficulty arranging phase III clinical trials, and off-label prescribing has been impaired by high drug cost and difficulty arranging third party reimbursement. Rare CFTR mutations such as P67L are emblematic of the challenges to “precision” medicine, including use of the best available mechanistic knowledge to treat patients with unusual forms of disease.
Carleen M. Sabusap, Wei Wang, Carmel M. McNicholas, W. Joon Chung, Lianwu Fu, Hui Wen, Marina Mazur, Kevin L. Kirk, James F. Collawn, Jeong S. Hong, Eric J. Sorscher
Kidney fibrosis following kidney injury is an unresolved health problem and causes significant morbidity and mortality worldwide. In a study into its molecular mechanism, we identified essential causative features. Acute or chronic kidney injury causes sustained elevation of a disintegrin and metalloprotease 17 (ADAM17); of its cleavage-activated proligand substrates, in particular of pro-TNFα and the EGFR ligand amphiregulin (pro-AREG); and of the substrates’ receptors. As a consequence, EGFR is persistently activated and triggers the synthesis and release of proinflammatory and profibrotic factors, resulting in macrophage/neutrophil ingress and fibrosis. ADAM17 hypomorphic mice, specific ADAM17 inhibitor–treated WT mice, or mice with inducible KO of ADAM17 in proximal tubule (Slc34a1-Cre) were significantly protected against these effects. In vitro, in proximal tubule cells, we show that AREG has unique profibrotic actions that are potentiated by TNFα-induced AREG cleavage. In vivo, in acute kidney injury (AKI) and chronic kidney disease (CKD, fibrosis) patients, soluble AREG is indeed highly upregulated in human urine, and both ADAM17 and AREG expression show strong positive correlation with fibrosis markers in related kidney biopsies. Our results indicate that targeting of the ADAM17 pathway represents a therapeutic target for human kidney fibrosis.
Eirini Kefaloyianni, Muthu Lakshmi Muthu, Jakob Kaeppler, Xiaoming Sun, Venkata Sabbisetti, Athena Chalaris, Stefan Rose-John, Eitan Wong, Irit Sagi, Sushrut S. Waikar, Helmut Rennke, Benjamin D. Humphreys, Joseph V. Bonventre, Andreas Herrlich
Motile airway cilia that propel contaminants out of the lung are oriented in a common direction by planar cell polarity (PCP) signaling, which localizes PCP protein complexes to opposite cell sides throughout the epithelium to orient cytoskeletal remodeling. In airway epithelia, PCP is determined in a 2-phase process. First, cell-cell communication via PCP complexes polarizes all cells with respect to the proximal-distal tissue axis. Second, during ciliogenesis, multiciliated cells (MCCs) undergo cytoskeletal remodeling to orient their cilia in the proximal direction. The second phase not only directs cilium polarization, but also consolidates polarization across the epithelium. Here, we demonstrate that in airway epithelia, PCP depends on MCC differentiation. PCP mutant epithelia have misaligned cilia, and also display defective barrier function and regeneration, indicating that PCP regulates multiple aspects of airway epithelial homeostasis. In humans, MCCs are often sparse in chronic inflammatory diseases, and these airways exhibit PCP dysfunction. The presence of insufficient MCCs impairs mucociliary clearance in part by disrupting PCP-driven polarization of the epithelium. Consistent with defective PCP, barrier function and regeneration are also disrupted. Pharmacological stimulation of MCC differentiation restores PCP and reverses these defects, suggesting its potential for broad therapeutic benefit in chronic inflammatory disease.
Eszter K. Vladar, Jayakar V. Nayak, Carlos E. Milla, Jeffrey D. Axelrod
Osteoarthritis (OA) of spine (facet joints [FJs]) is one of the major causes of severe low back pain and disability worldwide. The degeneration of facet cartilage is a hallmark of FJ OA. However, endogenous mechanisms that initiate degeneration of facet cartilage are unknown, and there are no disease-modifying therapies to stop FJ OA. In this study, we have identified microRNAs (small noncoding RNAs) as mediators of FJ cartilage degeneration. We first established a cohort of patients with varying degrees of facet cartilage degeneration (control group: normal or mild facet cartilage degeneration; FJ OA group: moderate to severe facet cartilage degeneration) and then screened 2,100 miRNAs and identified 2 miRNAs (miR-181a-5p and miR-4454) that were significantly elevated in FJ OA cartilage compared with control facet cartilage. We further explored their role, function, and signaling mechanisms using computational, in vitro functional, and in vivo studies. We specifically indicate that miR-181a-5p and miR-4454 are involved in promoting inflammatory, catabolic, and cell death activity in FJ chondrocytes. This is the first report to our knowledge that identifies miR-181a-5p and miR-4454 as mediators of cartilage degeneration in FJs and potential therapeutic targets for stopping cartilage degeneration.
Akihiro Nakamura, Y. Raja Rampersaud, Anirudh Sharma, Stephen J. Lewis, Brian Wu, Poulami Datta, Kala Sundararajan, Helal Endisha, Evgeny Rossomacha, Jason S. Rockel, Igor Jurisica, Mohit Kapoor
No posts were found with this tag.