A human obesity-associated MC4R mutation with defective Gq/11α signaling leads to hyperphagia in mice

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Abstract

Melanocortin 4 receptor (MC4R) mutations are the most common cause of human monogenic obesity and are associated with hyperphagia and increased linear growth. While MC4R is known to activate Gsα/cAMP signaling, a significant proportion of obesity-associated MC4R mutations do not affect MC4R/Gsα signaling. To further explore the role of specific MC4R signaling pathways in regulation of energy balance, we examined the signaling properties of one such mutant MC4R (F51L), as well as the metabolic consequences of MC4RF51L mutation in mice. The MC4RF51L mutation produced a specific defect in MC4R/Gq/11α signaling and led to obesity, hyperphagia and increased linear growth in mice. The ability of a melanocortin agonist to acutely inhibit food intake when delivered to the paraventricular nucleus (PVN) was lost in MC4RF51L mice, as well as in wild-type mice in which a specific Gq/11α inhibitor was delivered to the PVN, providing evidence that a Gsα-independent signaling pathway, namely Gq/11α, significantly contributes to the actions of MC4R on food intake and linear growth. These results suggest that a biased MC4R agonist that primarily activates Gq/11α may be a potential agent to treat obesity with less untoward cardiovascular and other side effects.

Authors

Peter J. Metzger, Aileen Zhang, Bradley A. Carlson, Hui Sun, Zhenzhong Cui, Yongqi Li, Marshal T. Jahnke, Daniel R. Layton, Meenakshi B. Gupta, Naili Liu, Evi Kostenis, Oksana Gavrilova, Min Chen, Lee S. Weinstein

Single-cell deconvolution reveals high lineage- and location-dependent heterogeneity in mesenchymal multivisceral stage IV colorectal cancer

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Abstract

Metastasized colorectal cancer (CRC) is associated with a poor prognosis and rapid disease progression. Besides hepatic metastasis, peritoneal carcinomatosis is the major cause of death in UICC (Union for International Cancer Control) stage IV CRC patients. Insights into differential site-specific reconstitution of tumour cells and the corresponding tumour microenvironment are still missing. Here, we analysed the transcriptome of single cells derived from murine multivisceral CRC and delineated the inter-metastatic cellular heterogeneity regarding tumour epithelium, stroma and immune cells. Interestingly, we found an intercellular site-specific network of cancer associated fibroblasts and tumour epithelium during peritoneal metastasis as well as an autologous feed-forward loop in cancer stem cells. We furthermore deciphered a metastatic dysfunctional adaptive immunity by a loss of B cell dependent antigen presentation and consecutive effector T cell exhaustion. Furthermore, we demonstrated major similarities of this murine metastatic CRC model with human disease and -based on the results of our analysis- provided an auspicious site-specific immune modulatory treatment approach for stage IV CRC by intraperitoneal checkpoint inhibition.

Authors

Christopher Berlin, Bernhard Mauerer, Pierre Cauchy, Jost Luenstedt, Roman Sankowski, Lisa Marx, Reinhild Feuerstein, Luisa Schäfer, Florian R. Greten, Marina Pesic, Olaf Groß, Marco Prinz, Naomi Rühl, Laura Miketiuk, Dominik Jauch, Claudia Laessle, Andreas Jud, Esther A. Biesel, Hannes P. Neeff, Stefan Fichtner-Feigl, Philipp A. Holzner, Rebecca Kesselring

Hyperactive mTORC1 in lung mesenchyme induces endothelial cell dysfunction and pulmonary vascular remodeling

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Abstract

Lymphangioleiomyomatosis (LAM) is a progressive cystic lung disease caused by tuberous sclerosis complex 1/2 (TSC1/2) gene mutations in pulmonary mesenchymal cells resulting in activation of the mechanistic target of rapamycin complex 1 (mTORC1). A subset of LAM patients develops pulmonary vascular remodeling and pulmonary hypertension. Little, however, is known regarding how LAM cells communicate with endothelial cells (ECs) to trigger vascular remodeling. In end-stage LAM lung explants, we identified endothelial cell dysfunction characterized by increased proliferation, migration, defective angiogenesis, and dysmorphic endothelial tube network formation. To model LAM disease, we utilized an mTORC1 gain-of-function mouse model with a Tsc2 knock-out (Tsc2KO) specific to lung mesenchyme (Tbx4LME-CreTsc2fl/fl), similar to the mesenchyme specific genetic alterations seen in human disease. As early as 8 weeks of age, ECs from Tbx4LME-CreTsc2fl/fl mice exhibited marked transcriptomic changes despite absence of morphological changes to the distal lung microvasculature. In contrast, 1 year old Tbx4LME-CreTsc2fl/fl mice spontaneously developed pulmonary vascular remodeling with increased medial thickness. Single cell RNA-sequencing of 1 year old mouse lung identified paracrine ligands originating from Tsc2KO mesenchyme which can signal through receptors in arterial ECs. These ECs had transcriptionally altered genes including those in pathways associated with blood vessel remodeling. The proposed pathophysiologic mesenchymal ligand/ EC receptor crosstalk highlights the importance of an altered mesenchymal-EC axis in LAM and other hyperactive mTORC1-driven diseases. Since ECs in LAM patients and in Tbx4LME-CreTsc2fl/fl mice do not harbor TSC2 mutations, our study demonstrates that constitutively active mTORC1 lung mesenchymal cells orchestrate dysfunctional EC responses which contribute to pulmonary vascular remodeling.

Authors

Susan M. Lin, Ryan Rue, Alexander R. Mukhitov, Akansha Goel, Maria C. Basil, Kseniya Obraztsova, Apoorva Babu, Slaven Crnkovic, Owen Ledwell, Laura T. Ferguson, Joseph D. Planer, Ana N. Nottingham, Kanth Swaroop Vanka, Carly J. Smith, Edward Cantu III, Grazyna Kwapiszewska, Edward E. Morrisey, Jillian F. Evans, Vera P. Krymskaya

Mechanosensitive channels TMEM63A and TMEM63B mediate lung inflation-induced surfactant secretion

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Abstract

Pulmonary surfactant is a lipoprotein complex lining the alveolar surface to decrease the surface tension and facilitate inspiration. Surfactant deficiency is often seen in premature infants and also children and adults with respiratory distress syndrome. Mechanical stretch of alveolar type 2 epithelial (AT2) cells during lung expansion is the primary physiological factor that stimulates surfactant secretion; however, it is unclear whether there is a mechanosensor dedicated for this process. Here we show that loss of mechanosensitive channels TMEM63A and TMEM63B resulted in atelectasis and respiratory failure in mice due to deficit of surfactant secretion. TMEM63A/B were predominantly localized at the limiting membrane of lamellar body, a lysosome-related organelle that stores pulmonary surfactant and ATP in AT2 cells. Activation of TMEM63A/B channels during cell stretch facilitated release of surfactant and ATP from lamellar bodies fused with the plasma membrane. The released ATP evoked Ca2+ signaling in AT2 cells and potentiated exocytic fusion of more lamellar bodies. Our study uncovered a vital physiological function of TMEM63 mechanosensitive channels, which makes the lung ready for the first breath at birth and maintains respiration through the life.

Authors

Gui-Lan Chen, Jing-Yi Li, Xin Chen, Jia-Wei Liu, Qian Zhang, Jie-Yu Liu, Jing Wen, Na Wang, Ming Lei, Jun-Peng Wei, Li Yi, Jia-Jia Li, Yu-Peng Ling, He-Qiang Yi, Zhenying Hu, Jingjing Duan, Jin Zhang, Bo Zeng

Lysosomal processing of sulfatide analogues alters target NKT cell specificity and immune responses in cancer

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Abstract

In a structure-function study of sulfatides, that typically stimulate type II NKT cells, we made an unexpected discovery. We compared analogues with sphingosine or phytosphingosine chains and 24-carbon acyl chains with 0-1-2 double bonds (C or pC24:0, 24:1, or 24:2). C24:1 and C24:2 sulfatide presented by CD1d monomer on plastic stimulated type II, not type I, NKT-cell hybridomas as expected. Unexpectedly, when presented by bone-marrow-derived DCs (BMDCs), C24:2 reversed specificity to stimulate type I, not type II, NKT-cell hybridomas, mimicking the corresponding βGalCer without sulfate. It induced IFNγ-dependent immunoprotection against CT26 colon-cancer lung metastases, skewed the cytokine profile, and activated cDC1s. This was abrogated by blocking lysosomal processing with bafilomycin A1, or sulfite-blocking or deletion of arylsulfatase A that cleaves off sulfate. Thus, C24:2 is unexpectedly processed in BMDCs from a type II to a type I NKT cell-stimulating ligand, promoting tumor immunity. We believe this is the first discovery of antigen processing of glycosylceramides altering the specificity for the target cell that reverses its function from stimulating type II to stimulating type I NKT cells, introducing protective functional activity in cancer. It also uncovers a new role for antigen processing, not to allow MHC loading but to alter the cell responding.

Authors

Kumiko Nishio, Lise Pasquet, Kaddy Camara, Julia DiSapio, Shingo Kato, Kevin S. Hsu, Anja Bloom, Stewart K. Richardson, Joshua A. Welsh, Tianbo Jiang, Jennifer C. Jones, Susanna Cardell, Hiroshi Watarai, Masaki Terabe, Purevdorj B. Olkhanud, Amy R. Howell, Jay A. Berzofsky

Bik promotes proteasomal degradation to control low grade inflammation

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Abstract

Although chronic low-grade inflammation does not cause immediate clinical symptoms, over longer term can enhance other insults or of age-dependent damage to organ systems and thereby contribute to age-related disorders, such as respiratory disorders, heart disease, metabolic disorders, autoimmunity, and cancer. However, the molecular mechanisms governing low-level inflammation are largely unknown. We discovered that Bik-deficiency causes low level inflammation even at baseline and the development of spontaneous emphysema in female but not male mice. Similarly, a single nucleotide polymorphism that reduced Bik levels was associated with increased inflammation and enhanced decline in lung function in humans. Transgenic expression of Bik in the airways of Bik-deficient mice inhibited allergen- or LPS-induced lung inflammation and reversed emphysema in female mice. Bik-deficiency increased nuclear but not cytosolic p65 levels, because Bik by modifying the BH4 domain of Bcl-2 interacted with Rpn1 and Rpn2 and enhanced proteasomal degradation of nuclear proteins. Bik-deficiency increased inflammation primarily in females because Bcl-2 and Bik levels were reduced in lung tissues and airway cells of female compared with male mice. Therefore, controlling low-grade inflammation by modifying the unappreciated role of Bik and Bcl-2 in facilitating proteasomal degradation of nuclear proteins may be crucial in treating chronic age-related diseases.

Authors

Yohannes A. Mebratu, Jane T. Jones, Congjian Liu, Zerihun H. Negasi, Mizanur Rahman, Joselyn Rojas-Quintero, George T. O'Connor, Wei Gao, Josee Dupuis, Michael H. Cho, Augusto A. Litonjua, Scott Randell, Yohannes Tesfaigzi

Microbiota-dependent indole production stimulates the development of collagen-induced arthritis in mice

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Abstract

Altered tryptophan catabolism has been identified in inflammatory diseases like rheumatoid arthritis (RA) and spondyloarthritis (SpA), but the causal mechanisms linking tryptophan metabolites to disease are unknown. Using the collagen-induced arthritis (CIA) model we identified alterations in tryptophan metabolism, and specifically indole, that correlated with disease. We demonstrated that both bacteria and dietary tryptophan were required for disease, and indole supplementation was sufficient to induce disease in their absence. When mice with CIA on a low-tryptophan diet were supplemented with indole, we observed significant increases in serum IL-6, TNF, and IL-1β; splenic RORγt+CD4+ T cells and ex vivo collagen-stimulated IL-17 production; and a pattern of anti-collagen antibody isotype switching and glycosylation that corresponded with increased complement fixation. IL-23 neutralization reduced disease severity in indole-induced CIA. Finally, exposure of human colon lymphocytes to indole increased expression of genes involved in IL-17 signaling and plasma cell activation. Altogether, we propose a mechanism by which intestinal dysbiosis during inflammatory arthritis results in altered tryptophan catabolism, leading to indole stimulation of arthritis development. Blockade of indole generation may present a unique therapeutic pathway for RA and SpA.

Authors

Brenda J. Seymour, Brandon Trent, Brendan E. Allen, Adam J. Berlinberg, Jimmy Tangchittsumran, Widian K. Jubair, Meagan E. Chriswell, Sucai Liu, Alfredo Ornelas, Andrew Stahly, Erica E. Alexeev, Alexander S. Dowdell, Sunny L. Sneed, Sabrina Fechtner, Jennifer M. Kofonow, Charles E. Robertson, Stephanie M. Dillon, Cara C. Wilson, Robert M. Anthony, Daniel N. Frank, Sean P. Colgan, Kristine A. Kuhn

KCTD1/KCTD15 complexes control ectodermal and neural crest cell functions and their impairment causes aplasia cutis

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Abstract

Aplasia cutis congenita (ACC) is a congenital epidermal defect of the midline scalp and has been proposed to be due to a primary keratinocyte abnormality. Why it forms mainly at this anatomic site has remained a longstanding enigma. KCTD1 mutations cause ACC, ectodermal abnormalities, and kidney fibrosis, whereas KCTD15 mutations cause ACC and cardiac outflow tract abnormalities. Here, we find that KCTD1 and KCTD15 can form multimeric complexes and can compensate for each other's loss, and that disease mutations are dominant-negative, resulting in lack of KCTD1/KCTD15 function. We demonstrate that KCTD15 is critical for cardiac outflow tract development, whereas KCTD1 regulates distal nephron function. Combined inactivation of KCTD1/KCTD15 in keratinocytes results in abnormal skin appendages, but not in ACC. Instead, KCTD1/KCTD15 inactivation in neural crest cells results in ACC linked to midline skull defects, demonstrating that ACC is not caused by a primary defect in keratinocytes but is a secondary consequence of impaired cranial neural crest cells giving rise to midline cranial suture cells that express keratinocyte-promoting growth factors. Our findings explain the clinical observations in patients with KCTD1 versus KCTD15 mutations, establish KCTD1/KCTD15 as critical regulators of ectodermal and neural crest cell functions, and define ACC as a neurocristopathy.

Authors

Jackelyn R. Raymundo, Hui Zhang, Giovanni Smaldone, Wenjuan Zhu, Kathleen E. Daly, Benjamin J. Glennon, Giovanni Pecoraro, Marco Salvatore, William A. Devine, Cecilia W. Lo, Luigi Vitagliano, Alexander G. Marneros

Itaconate suppresses atherosclerosis by activating a Nrf2-dependent anti-inflammatory response in macrophages in mice

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Abstract

Itaconate has emerged as a critical immunoregulatory metabolite. Here, we examined the therapeutic potential of itaconate in atherosclerosis. We found that both itaconate and the enzyme that synthesizes it, aconitate decarboxylase 1 (Acod1, also known as “immune-responsive gene 1”/IRG1) are upregulated during atherogenesis in mice. Deletion of Acod1 in myeloid cells exacerbated inflammation and atherosclerosis in vivo and resulted in an elevated frequency of a specific subset of M1-polarized proinflammatory macrophages in the atherosclerotic aorta. Importantly, Acod1 levels were inversely correlated with clinical occlusion in atherosclerotic human aorta specimens. Treating mice with the itaconate derivative 4-ocytyl itaconate attenuated inflammation and atherosclerosis induced by high cholesterol. Mechanistically, we found that the antioxidant transcription factor, Nuclear factor erythroid-2 Related Factor 2 (Nrf2) was required for itaconate to suppress macrophage activation induced by oxidized lipids in vitro and to decrease atherosclerotic lesion areas in vivo. Overall, our work shows that itaconate suppresses atherogenesis by inducing Nrf2-dependent inhibition of proinflammatory responses in macrophages. Activation of the itaconate pathway may represent an important approach to treat atherosclerosis.

Authors

Jianrui Song, Yanling Zhang, Ryan A. Frieler, Anthony Andren, Sherri C. Wood, Daniel J. Tyrrell, Peter Sajjakulnukit, Jane C. Deng, Costas A. Lyssiotis, Richard M. Mortensen, Morgan Salmon, Daniel R. Goldstein

Proteogenomic analysis reveals cytoplasmic sequestration of RUNX1 by the acute myeloid leukemia-initiating CBFB::MYH11 oncofusion protein

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Abstract

Several canonical translocations produce oncofusion genes that can initiate Acute Myeloid Leukemia (AML). Although each translocation is associated with unique features, the mechanisms responsible remain unclear. While proteins interacting with each oncofusion are known to be relevant for how they act, these interactions have not yet been systematically defined. To address this issue in an unbiased fashion, we fused a promiscuous biotin ligase ("TurboID") in-frame with three favorable-risk acute myeloid leukemia (AML) oncofusion cDNAs (PML::RARA, RUNX1::RUNX1T1, and CBFB::MYH11), and identified their interacting proteins in primary murine hematopoietic cells. The PML::RARA- and RUNX1::RUNX1T1-TurboID fusion proteins labeled common and unique nuclear repressor complexes, implying their nuclear localization. However, CBFB::MYH11-TurboID interacting proteins were largely cytoplasmic, probably due to an interaction of the MYH11 domain with several cytoplasmic myosin-related proteins. Using a variety of methods, we showed that the CBFB domain of CBFB::MYH11 sequesters RUNX1 in cytoplasmic aggregates; these findings were confirmed in primary human AML cells. Paradoxically, CBFB::MYH11 expression was associated with increased RUNX1/2 expression, suggesting the presence of a sensor for reduced functional RUNX1 protein, and a feedback loop that that may attempt to compensate by increasing RUNX1/2 transcription. These findings may have broad implications for AML pathogenesis.

Authors

Ryan B. Day, Julia A. Hickman, Ziheng Xu, Casey D.S. Katerndahl, Francesca Ferraro, Sai Mukund Ramakrishnan, Petra Erdmann-Gilmore, Robert W. Sprung, Yiling Mi, R. Reid Townsend, Christopher A. Miller, Timothy J. Ley