Research Article
Abstract
Tumors adapt to an unfavorable microenvironment by controlling the balance between cell proliferation and cell motility, but the regulators of this process are largely unknown. Here, we show that an alternatively spliced isoform of syntaphilin (SNPH), a cytoskeletal regulator of mitochondrial movements in neurons, is directed to mitochondria of tumor cells. Mitochondrial SNPH buffers oxidative stress and maintains complex II–dependent bioenergetics, sustaining local tumor growth while restricting mitochondrial redistribution to the cortical cytoskeleton and tumor cell motility. Conversely, introduction of stress stimuli to the microenvironment, including hypoxia, acutely lowered SNPH levels, resulting in bioenergetics defects and increased superoxide production. In turn, this suppressed tumor cell proliferation but increased tumor cell invasion via greater mitochondrial trafficking to the cortical cytoskeleton. Loss of SNPH or expression of an SNPH mutant lacking the mitochondrial localization sequence resulted in increased metastatic dissemination in xenograft or syngeneic tumor models in vivo. Accordingly, tumor cells that acquired the ability to metastasize in vivo constitutively downregulated SNPH and exhibited higher oxidative stress, reduced cell proliferation, and increased cell motility. Therefore, SNPH is a stress-regulated mitochondrial switch of the cell proliferation-motility balance in cancer, and its pathway may represent a therapeutic target.
Authors
M. Cecilia Caino, Jae Ho Seo, Yuan Wang, Dayana B. Rivadeneira, Dmitry I. Gabrilovich, Eui Tae Kim, Ashani T. Weeraratna, Lucia R. Languino, Dario C. Altieri
Abstract
The kidney glomerular capillaries are frequent sites of immune complex deposition and subsequent neutrophil accumulation in post-infectious and rapidly progressive glomerulonephritis. However, the mechanisms of neutrophil recruitment remain enigmatic, and there is no targeted therapeutic to avert this proximal event in glomerular inflammation. The uniquely human activating Fc receptor FcγRIIA promotes glomerular neutrophil accumulation and damage in anti–glomerular basement membrane–induced (anti-GBM–induced) glomerulonephritis when expressed on murine neutrophils. Here, we found that neutrophils are directly captured by immobilized IgG antibodies under physiological flow conditions in vitro through FcγRIIA-dependent, Abl/Src tyrosine kinase–mediated F-actin polymerization. Biophysical measurements showed that the lifetime of FcγRIIA-IgG bonds increased under mechanical force in an F-actin–dependent manner, which could enable the capture of neutrophils under physiological flow. Kidney intravital microscopy revealed that circulating neutrophils, which were similar in diameter to glomerular capillaries, abruptly arrested following anti-GBM antibody deposition via neutrophil FcγRIIA and Abl/Src kinases. Accordingly, inhibition of Abl/Src with bosutinib reduced FcγRIIA-mediated glomerular neutrophil accumulation and renal injury in experimental, crescentic anti-GBM nephritis. These data identify a pathway of neutrophil recruitment within glomerular capillaries following IgG deposition that may be targeted by bosutinib to avert glomerular injury.
Authors
Hiroshi Nishi, Kazuhiro Furuhashi, Xavier Cullere, Gurpanna Saggu, Mark J. Miller, Yunfeng Chen, Florencia Rosetti, Samantha L. Hamilton, Lihua Yang, Spencer P. Pittman, Jiexi Liao, Jan M. Herter, Jeffrey C. Berry, Daniel J. DeAngelo, Cheng Zhu, George C. Tsokos, Tanya N. Mayadas
Abstract
Autoreactive CD4 T cells that differentiate into pathogenic Th17 cells can trigger autoimmune diseases. Therefore, investigating the regulatory network that modulates Th17 differentiation may yield important therapeutic insights. miR-146a has emerged as a critical modulator of immune reactions, but its role in regulating autoreactive Th17 cells and organ-specific autoimmunity remains largely unknown. Here, we have reported that miR-146a–deficient mice developed more severe experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis (MS). We bred miR-146a–deficient mice with 2D2 T cell receptor–Tg mice to generate 2D2 CD4 T cells that are deficient in miR-146a and specific for myelin oligodendrocyte glycoprotein (MOG), an autoantigen in the EAE model. miR-146a–deficient 2D2 T cells induced more severe EAE and were more prone to differentiate into Th17 cells. Microarray analysis revealed enhancements in IL-6– and IL-21–induced Th17 differentiation pathways in these T cells. Further study showed that miR-146a inhibited the production of autocrine IL-6 and IL-21 in 2D2 T cells, which in turn reduced their Th17 differentiation. Thus, our study identifies miR-146a as an important molecular brake that blocks the autocrine IL-6– and IL-21–induced Th17 differentiation pathways in autoreactive CD4 T cells, highlighting its potential as a therapeutic target for treating autoimmune diseases.
Authors
Bo Li, Xi Wang, In Young Choi, Yu-Chen Wang, Siyuan Liu, Alexander T. Pham, Heesung Moon, Drake J. Smith, Dinesh S. Rao, Mark P. Boldin, Lili Yang
Abstract
Maintenance of muscle structure and function depends on the precise organization of contractile proteins into sarcomeres and coupling of the contractile apparatus to the sarcoplasmic reticulum (SR), which serves as the reservoir for calcium required for contraction. Several members of the Kelch superfamily of proteins, which modulate protein stability as substrate-specific adaptors for ubiquitination, have been implicated in sarcomere formation. The Kelch protein Klhl31 is expressed in a muscle-specific manner under control of the transcription factor MEF2. To explore its functions in vivo, we created a mouse model of Klhl31 loss of function using the CRISPR-Cas9 system. Mice lacking Klhl31 exhibited stunted postnatal skeletal muscle growth, centronuclear myopathy, central cores, Z-disc streaming, and SR dilation. We used proteomics to identify several candidate Klhl31 substrates, including Filamin-C (FlnC). In the Klhl31-knockout mice, FlnC protein levels were highly upregulated with no change in transcription, and we further demonstrated that Klhl31 targets FlnC for ubiquitination and degradation. These findings highlight a role for Klhl31 in the maintenance of skeletal muscle structure and provide insight into the mechanisms underlying congenital myopathies.
Authors
James B. Papizan, Glynnis A. Garry, Svetlana Brezprozvannaya, John R. McAnally, Rhonda Bassel-Duby, Ning Liu, Eric N. Olson
Abstract
TGF-β1 signaling is a critical driver of collagen accumulation and fibrotic disease but also a vital suppressor of inflammation and epithelial cell proliferation. The nature of this multifunctional cytokine has limited the development of global TGF-β1 signaling inhibitors as therapeutic agents. We conducted phenotypic screens for small molecules that inhibit TGF-β1–induced epithelial-mesenchymal transition without immediate TGF-β1 receptor (TβR) kinase inhibition. We identified trihydroxyphenolic compounds as potent blockers of TGF-β1 responses (IC50 ~50 nM), Snail1 expression, and collagen deposition in vivo in models of pulmonary fibrosis and collagen-dependent lung cancer metastasis. Remarkably, the functional effects of trihydroxyphenolics required the presence of active lysyl oxidase–like 2 (LOXL2), thereby limiting effects to fibroblasts or cancer cells, the major LOXL2 producers. Mechanistic studies revealed that trihydroxyphenolics induce auto-oxidation of a LOXL2/3–specific lysine (K731) in a time-dependent reaction that irreversibly inhibits LOXL2 and converts the trihydrophenolic to a previously undescribed metabolite that directly inhibits TβRI kinase. Combined inhibition of LOXL2 and TβRI activities by trihydrophenolics resulted in potent blockade of pathological collagen accumulation in vivo without the toxicities associated with global inhibitors. These findings elucidate a therapeutic approach to attenuate fibrosis and the disease-promoting effects of tissue stiffness by specifically targeting TβRI kinase in LOXL2-expressing cells.
Authors
Ying Wei, Thomas J. Kim, David H. Peng, Dana Duan, Don L. Gibbons, Mitsuo Yamauchi, Julia R. Jackson, Claude J. Le Saux, Cheresa Calhoun, Jay Peters, Rik Derynck, Bradley J. Backes, Harold A. Chapman
Abstract
The gene that encodes de novo DNA methyltransferase 3A (DNMT3A) is frequently mutated in acute myeloid leukemia genomes. Point mutations at position R882 have been shown to cause a dominant negative loss of DNMT3A methylation activity, but 15% of DNMT3A mutations are predicted to produce truncated proteins that could either have dominant negative activities or cause loss of function and haploinsufficiency. Here, we demonstrate that 3 of these mutants produce truncated, inactive proteins that do not dimerize with WT DNMT3A, strongly supporting the haploinsufficiency hypothesis. We therefore evaluated hematopoiesis in mice heterozygous for a constitutive null Dnmt3a mutation. With no other manipulations, Dnmt3a+/– mice developed myeloid skewing over time, and their hematopoietic stem/progenitor cells exhibited a long-term competitive transplantation advantage. Dnmt3a+/– mice also spontaneously developed transplantable myeloid malignancies after a long latent period, and 3 of 12 tumors tested had cooperating mutations in the Ras/MAPK pathway. The residual Dnmt3a allele was neither mutated nor downregulated in these tumors. The bone marrow cells of Dnmt3a+/– mice had a subtle but statistically significant DNA hypomethylation phenotype that was not associated with gene dysregulation. These data demonstrate that haploinsufficiency for Dnmt3a alters hematopoiesis and predisposes mice (and probably humans) to myeloid malignancies by a mechanism that is not yet clear.
Authors
Christopher B. Cole, David A. Russler-Germain, Shamika Ketkar, Angela M. Verdoni, Amanda M. Smith, Celia V. Bangert, Nichole M. Helton, Mindy Guo, Jeffery M. Klco, Shelly O’Laughlin, Catrina Fronick, Robert Fulton, Gue Su Chang, Allegra A. Petti, Christopher A. Miller, Timothy J. Ley
Abstract
The mammalian target of rapamycin complex 1 (mTORC1) kinase promotes cell growth by activating biosynthetic pathways and suppressing catabolic pathways, particularly that of macroautophagy. A prerequisite for mTORC1 activation is its translocation to the lysosomal surface. Deregulation of mTORC1 has been associated with the pathogenesis of several diseases, but its role in skeletal disorders is largely unknown. Here, we show that enhanced mTORC1 signaling arrests bone growth in lysosomal storage disorders (LSDs). We found that lysosomal dysfunction induces a constitutive lysosomal association and consequent activation of mTORC1 in chondrocytes, the cells devoted to bone elongation. mTORC1 hyperphosphorylates the protein UV radiation resistance–associated gene (UVRAG), reducing the activity of the associated Beclin 1–Vps34 complex and thereby inhibiting phosphoinositide production. Limiting phosphoinositide production leads to a blockage of the autophagy flux in LSD chondrocytes. As a consequence, LSD chondrocytes fail to properly secrete collagens, the main components of the cartilage extracellular matrix. In mouse models of LSD, normalization of mTORC1 signaling or stimulation of the Beclin 1–Vps34–UVRAG complex rescued the autophagy flux, restored collagen levels in cartilage, and ameliorated the bone phenotype. Taken together, these data unveil a role for mTORC1 and autophagy in the pathogenesis of skeletal disorders and suggest potential therapeutic approaches for the treatment of LSDs.
Authors
Rosa Bartolomeo, Laura Cinque, Chiara De Leonibus, Alison Forrester, Anna Chiara Salzano, Jlenia Monfregola, Emanuela De Gennaro, Edoardo Nusco, Isabella Azario, Carmela Lanzara, Marta Serafini, Beth Levine, Andrea Ballabio, Carmine Settembre
Abstract
Blood vessels in the tumor periphery have high pericyte coverage and are resistant to vascular disrupting agents (VDAs). VDA treatment resistance leads to a viable peripheral tumor rim that contributes to treatment failure and disease recurrence. Here, we provide evidence to support a hypothesis that shifting the target of VDAs from tumor vessel endothelial cells to pericytes disrupts tumor peripheral vessels and the viable rim, circumventing VDA treatment resistance. Through chemical engineering, we developed Z-GP-DAVLBH (from the tubulin-binding VDA desacetylvinblastine monohydrazide [DAVLBH]) as a prodrug that can be selectively activated by fibroblast activation protein α (FAPα) in tumor pericytes. Z-GP-DAVLBH selectively destroys the cytoskeleton of FAPα-expressing tumor pericytes, disrupting blood vessels both within the core and around the periphery of tumors. As a result, Z-GP-DAVLBH treatment eradicated the otherwise VDA-resistant tumor rim and led to complete regression of tumors in multiple lines of xenografts without producing the drug-related toxicity that is associated with similar doses of DAVLBH. This study demonstrates that targeting tumor pericytes with an FAPα-activated VDA prodrug represents a potential vascular disruption strategy in overcoming tumor resistance to VDA treatments.
Authors
Minfeng Chen, Xueping Lei, Changzheng Shi, Maohua Huang, Xiaobo Li, Baojian Wu, Zhengqiu Li, Weili Han, Bin Du, Jianyang Hu, Qiulin Nie, Weiqian Mai, Nan Ma, Nanhui Xu, Xinyi Zhang, Chunlin Fan, Aihua Hong, Minghan Xia, Liangping Luo, Ande Ma, Hongsheng Li, Qiang Yu, Heru Chen, Dongmei Zhang, Wencai Ye
Abstract
Hematopoietic stem cells (HSCs) remain mostly quiescent under steady-state conditions but switch to a proliferative state following hematopoietic stress, e.g., bone marrow (BM) injury, transplantation, or systemic infection and inflammation. The homeostatic balance between quiescence, self-renewal, and differentiation of HSCs is strongly dependent on their interactions with cells that constitute a specialized microanatomical environment in the BM known as the HSC niche. Here, we identified the secreted extracellular matrix protein Del-1 as a component and regulator of the HSC niche. Specifically, we found that Del-1 was expressed by several cellular components of the HSC niche, including arteriolar endothelial cells, CXCL12-abundant reticular (CAR) cells, and cells of the osteoblastic lineage. Del-1 promoted critical functions of the HSC niche, as it regulated long-term HSC (LT-HSC) proliferation and differentiation toward the myeloid lineage. Del-1 deficiency in mice resulted in reduced LT-HSC proliferation and infringed preferentially upon myelopoiesis under both steady-state and stressful conditions, such as hematopoietic cell transplantation and G-CSF– or inflammation-induced stress myelopoiesis. Del-1–induced HSC proliferation and myeloid lineage commitment were mediated by β3 integrin on hematopoietic progenitors. This hitherto unknown Del-1 function in the HSC niche represents a juxtacrine homeostatic adaptation of the hematopoietic system in stress myelopoiesis.
Authors
Ioannis Mitroulis, Lan-Sun Chen, Rashim Pal Singh, Ioannis Kourtzelis, Matina Economopoulou, Tetsuhiro Kajikawa, Maria Troullinaki, Athanasios Ziogas, Klara Ruppova, Kavita Hosur, Tomoki Maekawa, Baomei Wang, Pallavi Subramanian, Torsten Tonn, Panayotis Verginis, Malte von Bonin, Manja Wobus, Martin Bornhäuser, Tatyana Grinenko, Marianna Di Scala, Andres Hidalgo, Ben Wielockx, George Hajishengallis, Triantafyllos Chavakis
Abstract
During an immune response, CD8+ T lymphocytes can undergo asymmetric division, giving rise to daughter cells that exhibit distinct tendencies to adopt terminal effector and memory cell fates. Here we show that “pre-effector” and “pre-memory” cells resulting from the first CD8+ T cell division in vivo exhibited low and high rates of endogenous proteasome activity, respectively. Pharmacologic reduction of proteasome activity in CD8+ T cells early during differentiation resulted in acquisition of terminal effector cell characteristics, whereas enhancement of proteasome activity conferred attributes of memory lymphocytes. Transcriptomic and proteomic analyses revealed that modulating proteasome activity in CD8+ T cells affected cellular metabolism. These metabolic changes were mediated, in part, through differential expression of Myc, a transcription factor that controls glycolysis and metabolic reprogramming. Taken together, these results demonstrate that proteasome activity is an important regulator of CD8+ T cell fate and raise the possibility that increasing proteasome activity may be a useful therapeutic strategy to enhance the generation of memory lymphocytes.
Authors
Christella E. Widjaja, Jocelyn G. Olvera, Patrick J. Metz, Anthony T. Phan, Jeffrey N. Savas, Gerjan de Bruin, Yves Leestemaker, Celia R. Berkers, Annemieke de Jong, Bogdan I. Florea, Kathleen Fisch, Justine Lopez, Stephanie H. Kim, Daniel A. Garcia, Stephen Searles, Jack D. Bui, Aaron N. Chang, John R. Yates III, Ananda W. Goldrath, Hermen S. Overkleeft, Huib Ovaa, John T. Chang
Copyright © 2017 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)