Elevated blood glucose (hyperglycemia) is a hallmark metabolic abnormality in diabetes. Hyperglycemia is associated with protein kinase A (PKA)-mediated stimulation of L-type Ca2+ channels in arterial myocytes resulting in increased vasoconstriction. However, the mechanisms by which glucose activates PKA remain unclear. Here, we showed that elevating extracellular glucose stimulates cAMP production in arterial myocytes, and that this was specifically dependent on adenylyl cyclase 5 (AC5) activity. Super-resolution imaging suggested nanometer proximity between subpopulations of AC5 and the L-type Ca2+ channel pore-forming subunit CaV1.2. In vitro, in silico, ex vivo and in vivo experiments revealed that this close association is critical for stimulation of L-type Ca2+ channels in arterial myocytes and increased myogenic tone upon acute hyperglycemia. This pathway supported the increase in L-type Ca2+ channel activity and myogenic tone in two animal models of diabetes. Our collective findings demonstrate a unique role for AC5 in PKA-dependent modulation of L-type Ca2+ channel activity and vascular reactivity during acute hyperglycemia and diabetes.
Arsalan U. Syed, Gopireddy R. Reddy, Debapriya Ghosh, Maria Paz Prada, Matthew A. Nystoriak, Stefano Morotti, Eleonora Grandi, Padmini Sirish, Nipavan Chiamvimonvat, Johannes W. Hell, Luis F. Santana, Yang K. Xiang, Madeline Nieves-Cintrón, Manuel F. Navedo
Physiological effects of cellular hypoxia are sensed by prolyl hydroxylase (PHD) enzymes which regulate HIFs. Genetic interventions on HIF/PHD pathways reveal multiple phenotypes that extend the known biology of hypoxia. Recent studies unexpectedly implicate HIF in aspects of multiple immune and inflammatory pathways. However such studies are often limited by systemic lethal effects and/or use tissue-specific recombination systems, which are inherently irreversible, un-physiologically restricted and difficult to time. To study these processes better we developed recombinant mice which express tetracycline-regulated shRNAs broadly targeting the main components of the HIF/PHD pathway, permitting timed bi-directional intervention. We have shown that stabilization of HIF levels in adult mice through PHD2 enzyme silencing by RNA interference, or inducible recombination of floxed alleles, results in multi-lineage leukocytosis and features of autoimmunity. This phenotype was rapidly normalized on re-establishment of the hypoxia-sensing machinery when shRNA expression was discontinued. In both situations these effects were mediated principally through the Hif2a isoform. Assessment of cells bearing regulatory T cell markers from these mice revealed defective function and pro-inflammatory effects in vivo. We believe our findings have shown a new role for the PHD2/Hif2a couple in the reversible regulation of T cell and immune activity.
Atsushi Yamamoto, Joanna Hester, Philip S. Macklin, Kento Kawai, Masateru Uchiyama, Daniel Biggs, Tammie Bishop, Katherine Bull, Xiaotong Cheng, Eleanor Cawthorne, Mathew L. Coleman, Tanya L. Crockford, Ben Davies, Lukas E. Dow, Rob Goldin, Kamil Kranc, Hiromi Kudo, Hannah Lawson, James McAuliffe, Kate Milward, Cheryl L. Scudamore, Elizabeth Soilleux, Fadi Issa, Peter J. Ratcliffe, Chris W. Pugh
Resistance to immunotherapy is one of the biggest problems of current oncotherapeutics. WhileT cell abundance is essential for tumor responsiveness to immunotherapy, factors that define the T cell inflamed tumor microenvironment are not fully understood. We conducted an unbiased approach to identify tumor-intrinsic mechanisms shaping the immune tumor microenvironment(TME), focusing on pancreatic adenocarcinoma because it is refractory to immunotherapy and excludes T cells from the TME. From human tumors, we identified EPHA2 as a candidate tumor intrinsic driver of immunosuppression. Epha2 deletion reversed T cell exclusion and sensitized tumors to immunotherapy. We found that PTGS2, the gene encoding cyclooxygenase-2, lies downstream of EPHA2 signaling through TGFβ and is associated with poor patient survival. Ptgs2 deletion reversed T cell exclusion and sensitized tumors to immunotherapy; pharmacological inhibition of PTGS2 was similarly effective. Thus, EPHA2-PTGS2 signaling in tumor cells regulates tumor immune phenotypes; blockade may represent a novel therapeutic avenue for immunotherapy-refractory cancers. Our findings warrant clinical trials testing the effectiveness of therapies combining EPHA2-TGFβ-PTGS2 pathway inhibitors with anti-tumor immunotherapy, and may change the treatment of notoriously therapy-resistant pancreatic adenocarcinoma.
Nune Markosyan, Jinyang Li, Yu H. Sun, Lee P. Richman, Jeffrey H. Lin, Fangxue Yan, Liz Quinones, Yogev Sela, Taiji Yamazoe, Naomi Gordon, John W. Tobias, Katelyn T. Byrne, Andrew J. Rech, Garret A. FitzGerald, Ben Z. Stanger, Robert H. Vonderheide
Cystic Fibrosis (CF) is a multi-organ progressive genetic disease caused by loss of functional cystic fibrosis transmembrane conductance regulator (CFTR) channel. Previously, we identified a significant dysfunction in CF cells and model mice of the transcription factor nuclear-factor-E2-related factor-2 (Nrf2), a major regulator of redox balance and inflammatory signaling. Here we report that approved F508del CFTR correctors VX809/VX661 recover diminished Nrf2 function and colocalization with CFTR in CF human primary bronchial epithelia by proximity ligation assay, immunoprecipitation, and immunofluorescence, concordant with CFTR correction. F508del CFTR correctors induced Nrf2 nuclear translocation, Nrf2-dependent luciferase activity, and transcriptional activation of target genes. Rescue of Nrf2 function by VX809/VX661 was dependent on significant correction of F508del and was blocked by inhibition of corrected channel function, or high-level shRNA knockdown of CFTR or F508del-CFTR. Mechanistically, F508del-CFTR modulation restored Nrf2 phosphorylation and its interaction with the coactivator CBP. Our findings demonstrate that sufficient modulation of F508del CFTR function corrects Nrf2 dysfunction in CF.
Dana C. Borcherding, Matthew E. Siefert, Songbai Lin, John Brewington, Hesham Sadek, John P. Clancy, Scott M. Plafker, Assem G. Ziady
Idiopathic Pulmonary Fibrosis (IPF) is a deadly disease with limited therapies. Tissue fibrosis is associated with Type 2 immune response, although the causal contribution of immune cells is not defined. The AP-1 transcription factor Fra-2 is upregulated in IPF lung sections and Fra-2 transgenic mice (Fra-2tg) exhibit spontaneous lung fibrosis. Here we show that Bleomycin-induced lung fibrosis is attenuated upon myeloid-inactivation of Fra-2 and aggravated in Fra-2tg bone marrow chimeras. Type VI collagen (ColVI), a Fra-2 transcriptional target, is up-regulated in three lung fibrosis models, and macrophages promote myofibroblast activation in vitro in a ColVI- and Fra-2-dependent manner. Fra-2 or ColVI inactivation does not affect macrophage recruitment and alternative activation, suggesting that Fra-2/ColVI specifically controls the paracrine pro-fibrotic activity of macrophages. Importantly, ColVI knock-out mice (KO) and ColVI-KO bone marrow chimeras are protected from Bleomycin-induced lung fibrosis. Therapeutic administration of a Fra-2/AP-1 inhibitor reduces ColVI expression and ameliorates fibrosis in Fra-2tg mice and in the Bleomycin model. Finally, Fra-2 and ColVI positively correlate in IPF patient samples and co-localize in lung macrophages. Therefore, the Fra-2/ColVI pro-fibrotic axis is a promising biomarker and therapeutic target for lung fibrosis, and possibly other fibrotic diseases.
Alvaro C. Ucero, Latifa Bakiri, Ben Roediger, Masakatsu Suzuki, Maria Jimenez, Pratyusha Mandal, Paola Braghetta, Paolo Bonaldo, Luis Paz-Ares, Coral Fustero-Torre, Pilar Ximenez-Embun, Ana Isabel Hernandez, Diego Megias, Erwin F. Wagner
Receptor activator of Nfkb ligand (RANKL) activates, while osteoprotegerin (OPG) inhibits, osteoclastogenesis. In turn a neutralizing Ab against RANKL, denosumab improves bone strength in osteoporosis. OPG also improves muscle strength in mouse models of Duchenne’s muscular dystrophy (mdx) and denervation-induce atrophy, but its role and mechanisms of action on muscle weakness in other conditions remains to be investigated. We investigated the effects of RANKL inhibitors on muscle in osteoporotic women and mice that either overexpress RANKL (HuRANKL-Tg+), or lack Pparb and concomitantly develop sarcopenia (Pparb-/-). In women, denosumab over 3 years improved appendicular lean mass and handgrip strength compared to no treatment, whereas bisphosphonate did not. HuRANKL-Tg+ mice displayed lower limb force and maximal speed, while their leg muscle mass was diminished, with a lower number of type I and II fibers. Both OPG and denosumab increased limb force proportionally to the increase in muscle mass. They markedly improved muscle insulin sensitivity and glucose uptake, and decrease anti-myogenic and inflammatory gene expression in muscle, such as myostatin and protein tyrosine phosphatase receptor-γ. Similarly, in Pparb-/-, OPG increased muscle volume and force, while also normalizing their insulin signaling and higher expression of inflammatory genes in skeletal muscle. In conclusions, RANKL deteriorates, while its inhibitor improves, muscle strength and insulin sensitivity in osteoporotic mice and humans. Hence denosumab could represent a novel therapeutic approach for sarcopenia.
Nicolas Bonnet, Lucie Bourgoin, Emmanuel Biver, Eleni Douni, Serge Ferrari
Lactation induces bone loss to provide sufficient calcium in the milk, a process that involves osteoclastic bone resorption but also osteocytes and perilacunar resorption. The exact mechanisms by which osteocytes contribute to bone loss remain elusive. Osteocytes express genes required in osteoclasts for bone resorption, including cathepsin K (Ctsk), and lactation elevates their expression. We show that Ctsk deletion in osteocytes prevented the increase in osteocyte lacunar area seen during lactation, as well as the effects of lactation to increase osteoclast numbers and decrease trabecular bone volume, cortical thickness and mechanical properties. In addition, Ctsk deletion in osteocytes increased bone Parathyroid Hormone related Peptide (PTHrP), prevented the decrease in serum Parathyroid Hormone (PTH) induced by lactation, but amplified the increase in serum 1,25(OH)2D. The net result of these changes is to maintain serum and milk calcium levels in the normal range, ensuring normal offspring skeletal development. Our studies confirm the fundamental role of osteocytic perilacunar remodeling in physiological states of lactation and provides genetic evidence that osteocyte-derived Ctsk contributes not only to osteocyte perilacunar remodeling, but also to the regulation of PTH, PTHrP, 1,25-Dyhydroxyvitamin D (1,25(OH)2D), osteoclastogenesis and bone loss in response to the high calcium demand associated with lactation.
Sutada Lotinun, Yoshihito Ishihara, Kenichi Nagano, Riku Kiviranta, Vincent Carpentier, Lynn Neff, Virginia Parkman, Noriko Ide, Dorothy Hu, Pamela Dann, Daniel Brooks, Mary L. Bouxsein, John Wysolmerski, Francesca Gori, Roland Baron
Mechanisms underlying motor neuron degeneration in amyotrophic lateral sclerosis (ALS) are yet unclear. Specific deletion of the ER-component membralin in astrocytes manifested postnatal motor defects and lethality in mice, causing the accumulation of extracellular glutamate through reducing the glutamate transporter EAAT2. Restoring EAAT2 levels in membralin KO astrocytes limited astrocyte-dependent excitotoxicity in motor neurons. Transcriptomic profiles from mouse astrocytic membralin KO motor cortex indicated significant perturbation in KEGG pathway components related to ALS, including downregulation of Eaat2 and upregulation of Tnfrsf1a. Changes in gene expression with membralin deletion also overlapped with mouse ALS models and reactive astrocytes. Our results shown that activation of TNF receptor (TNFR1)-NFκB pathway known to suppress Eaat2 transcription was upregulated with membralin deletion. Further, reduced membralin and EAAT2 levels correlated with disease progression in spinal cord from SOD1-mutant mouse models, and reductions in membralin/EAAT2 were observed in human ALS spinal cord. Importantly, overexpression of membralin in SOD1G93A astrocytes decreased TNFR1 levels and increased EAAT2 expression, and improved motor neuron survival. Importantly, upregulation of membralin in SOD1G93A mice significantly prolonged mouse survival. Together, our study provided a mechanism for ALS pathogenesis where membralin limited glutamatergic neurotoxicity, suggesting that modulating membralin had potentials in ALS therapy.
Lu-Lin Jiang, Bing Zhu, Yingjun Zhao, Xiaoguang Li, Tongfei Liu, Juan Pina-Crespo, Lisa Zhou, Wenxi Xu, Maria J. Rodriguez, Haiyang Yu, Don W. Cleveland, John Ravits, Sandrine Da Cruz, Tao Long, Timothy Y. Huang, Huaxi Xu
Gliomas account for approximately 80% of primary malignant tumors in the central nervous system. Despite aggressive therapy, the prognosis of patients remains extremely poor. Glioma stem cells (GSCs) which considered as the potential target of therapy for their crucial role in therapeutic resistance and tumor recurrence, are believed to be key factors for the disappointing outcome. Here, we took advantage of GSCs as the cell model to perform high-throughput drug screening and the old antibiotic, clofoctol, was identified as the most effective compound, showing reduction of colony-formation and induction of apoptosis of GSCs. Moreover, growth of tumors was inhibited obviously in vivo after clofoctol treatment especially in primary patient-derived xenografts (PDXs) and transgenic xenografts. The anticancer mechanisms demonstrated by analyzing related downstream genes and discovering the targeted binding protein revealed that clofoctol exhibited the inhibition of GSCs by upregulation of Kruppel-like factor 13 (KLF13), a tumor suppressor gene, through clofoctol’s targeted binding protein, Upstream of N-ras (UNR). Collectively, these data demonstrated that induction of KLF13 expression suppressed growth of gliomas and provided a potential therapy for gliomas targeting GSCs. Importantly, our results also identified the RNA-binding protein UNR as a drug target.
Yan Hu, Meilian Zhang, Ningyu Tian, Dengke Li, Fan Wu, Peishan Hu, Zhixing Wang, Liping Wang, Wei Hao, Jingting Kang, Bin Yin, Zhi Zheng, Tao Jiang, Jiangang Yuan, Boqin Qiang, Wei Han, Xiaozhong Peng
The migration of leukocytes into the CNS drives the neuropathology of multiple sclerosis (MS). This penetration likely utilizes energy resources that remain to be defined. Using the experimental autoimmune encephalomyelitis (EAE) model of MS, we determined that macrophages within the perivascular cuff of post-capillary venules are highly glycolytic as manifested by strong expression of lactate dehydrogenase A (LDHA) that converts pyruvate to lactate. These macrophages expressed prominent levels of monocarboxylate transporter-4 (MCT-4) specialized in secreting lactate from glycolytic cells. The functional relevance of glycolysis was confirmed by siRNA-mediated knockdown of LDHA and MCT-4, which decreased lactate secretion and macrophage transmigration. MCT-4 was in turn regulated by EMMPRIN (CD147) as determined through co-expression/co-immunoprecipitation studies, and siRNA-mediated EMMPRIN silencing. The functional relevance of MCT-4/EMMPRIN interaction was affirmed by lower macrophage transmigration in culture using the MCT-4 inhibitor, α-cyano-4-hydroxy-cinnamic acid (CHCA), a cinnamon derivative. CHCA also reduced leukocyte infiltration and the clinical severity of EAE. Relevance to MS was corroborated by the strong expression of MCT-4, EMMPRIN and LDHA in perivascular macrophages in MS brains. These results detail the metabolism of macrophages for transmigration from perivascular cuffs into the CNS parenchyma and identifies CHCA and diet as potential modulators of neuro-inflammation in MS.
Deepak Kumar Kaushik, Anindita Bhattacharya, Reza Mirzaei, Khalil S. Rawji, Younghee Ahn, Jong M. Rho, V. Wee Yong
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