While cancer is commonly perceived as a disease of dedifferentiation, the hallmark of early stage prostate cancer is paradoxically the loss of more plastic basal cells and the abnormal proliferation of more differentiated secretory luminal cells. However, the mechanism of prostate cancer pro-luminal differentiation is largely unknown. Through integrating analysis of the transcription factors (TFs) from 806 human prostate cancers, we have identified that ERG highly correlated with prostate cancer luminal subtyping. ERG overexpression in luminal epithelial cells inhibits its normal plasticity to transdifferentiate into basal lineage and ERG supersedes PTEN-loss which favors basal differentiation. ERG knock-out disrupted prostate cell luminal differentiation, whereas AR knock-out had no such effects. Trp63 is a known master regulator of prostate basal lineage. Through analysis of 3D chromatin architecture, we found that ERG binds and inhibits the enhancer activity and chromatin looping of a Trp63 distal enhancer, thereby silencing its gene expression. Specific deletion of the distal ERG binding site resulted in the loss of ERG-mediated inhibition of basal differentiation. Thus, ERG orchestrates chromatin interactions and regulates prostate cell lineage toward pro-luminal program, as its fundamental role on lineage differentiation in prostate cancer initiation.
Fei Li, Qiuyue Yuan, Wei Di, Xinyi Xia, Zhuang Liu, Ninghui Mao, Lin Li, Chunfeng Li, Juan He, Yunguang Li, Wangxin Guo, Xiaoyu Zhang, Yiqin Zhu, Rebiguli Aji, Shangqian Wang, Xinyuan Tong, Hongbin Ji, Ping Chi, Brett Carver, Yong Wang, Yu Chen, Dong Gao
Utilizing the Nephrotic Syndrome Study Network Consortium and other publicly available transcriptomic datasets, we identified Retinoic acid receptor responder protein 1 (RARRES1) as a gene whose expression positively correlated with renal function decline in human glomerular disease. The glomerular expression of RARRES1, which is largely restricted to podocytes, increased in focal segmental glomerulosclerosis (FSGS) and diabetic kidney disease (DKD). Tumor necrosis factor-α (TNF-α) was a potent inducer of RARRES1 expression in cultured podocytes, and transcriptomic analysis showed the enrichment of cell death pathway genes with RARRES1 overexpression. The overexpression of RARRES1 indeed induced podocyte apoptosis in vitro. Notably, this effect was dependent on its cleavage in the extracellular domain, as the mutation of its cleavage site abolished the apoptotic effect. Mechanistically, the soluble RARRES1 is endocytosed and interacts with and inhibits RIO kinase 1 (RIOK1), resulting in p53 activation and podocyte apoptosis. In mice, podocyte-specific overexpression of RARRES1 resulted in marked glomerular injury and albuminuria, while the overexpression of RARRES1 cleavage mutant had no effect. Conversely, podocyte-specific knockdown of Rarres1 in mice ameliorated glomerular injury in the setting of Adriamycin-induced nephropathy. Together, our study demonstrates an important role and the mechanism of RARRES1 in podocyte injury in glomerular disease.
Anqun Chen, Ye Feng, Han Lai, Wenjun Ju, Zhengzhe Li, Yu Li, Andrew Wang, Quan Hong, Fang Zhong, Chengguo Wei, Jia Fu, Tian-Jun Guan, Bi-Cheng Liu, Matthias Kretzler, Kyung Lee, John Cijiang He
TGFβ is a master regulator of fibrosis, driving the differentiation of fibroblasts into apoptosis resistant myofibroblasts and sustaining the production of extracellular matrix (ECM) components. Here, we identify the nuclear lncRNA H19X as a master regulator of TGFβ-driven tissue fibrosis. H19X was consistently upregulated in a wide variety of human fibrotic tissues and diseases and was strongly induced by TGFβ, particularly in fibroblasts and fibroblast-related cells. Functional experiments following H19X silencing revealed that H19X is an obligatory factor for the TGFβ-induced ECM synthesis as well as differentiation and survival of ECM-producing myofibroblasts. We showed that H19X regulates DDIT4L gene expression, specifically interacting with a region upstream of DDIT4L gene and changing the chromatin accessibility of a DDIT4L enhancer. These events resulted in transcriptional repression of DDIT4L and, in turn, in increased collagen expression and fibrosis. Our results shed light on key effectors of the TGFβ-induced ECM remodeling and fibrosis.
Elena Pachera, Shervin Assassi, Gloria A. Salazar, Mara Stellato, Florian Renoux, Adam Wunderlin, Przemyslaw Blyszczuk, Robert Lafyatis, Fina Kurreeman, Jeska de Vries-Bouwstra, Tobias Messemaker, Carol A. Feghali-Bostwick, Gerhard Rogler, Wouter T. van Haaften, Gerard Dijkstra, Fiona Oakley, Maurizio Calcagni, Janine Schniering, Britta Maurer, Jörg H.W. Distler, Gabriela Kania, Mojca Frank-Bertoncelj, Oliver Distler
Lysosomal enzymes are synthesized in the endoplasmic reticulum (ER) and transferred to the Golgi complex by interaction with the Batten disease protein CLN8 (ceroid lipofuscinosis, neuronal, 8). Here we investigated the relationship of this pathway with CLN6, an ER-associated protein of unknown function that is defective in a different Batten disease subtype. Experiments focused on protein interaction and trafficking identified CLN6 as an obligate component of a CLN6-CLN8 complex (herein referred to as EGRESS: ER-to-Golgi relaying of enzymes of the lysosomal system), which recruits lysosomal enzymes at the ER to promote their Golgi transfer. Mutagenesis experiments showed that the second luminal loop of CLN6 is required for the interaction of CLN6 with the enzymes but dispensable for interaction with CLN8. In vitro and in vivo studies showed that CLN6 deficiency results in inefficient ER export of lysosomal enzymes and diminished levels of the enzymes at the lysosome. Mice lacking both CLN6 and CLN8 did not display aggravated pathology compared with the single deficiencies, indicating that the EGRESS complex works as a functional unit. These results identify CLN6 and the EGRESS complex as key players in lysosome biogenesis and shed light on the molecular etiology of Batten disease caused by defects in CLN6.
Lakshya Bajaj, Jaiprakash Sharma, Alberto di Ronza, Pengcheng Zhang, Aiden Eblimit, Rituraj Pal, Dany Roman, John R. Collette, Clarissa Booth, Kevin T. Chang, Richard N. Sifers, Sung Y. Jung, Jill M. Weimer, Rui Chen, Randy W. Schekman, Marco Sardiello
Ligand-dependent activation of Hedgehog (Hh) signaling in cancer occurs without mutations in canonical pathway genes. Consequently, the genetic basis of Hh pathway activation in adult solid tumors, such as small-cell lung cancer (SCLC), is unknown. Here we show that combined inactivation of Trp53 and Rb1, a defining genetic feature of SCLC, leads to hypersensitivity to Hh ligand in vitro, and during neural tube development in vivo. This response is associated with the aberrant formation of primary cilia, an organelle essential for canonical Hh signaling through smoothened, a transmembrane protein targeted by small-molecule Hh inhibitors. We further show that loss of both Trp53 and Rb1 disables transcription of genes in the autophagic machinery necessary for the degradation of primary cilia. In turn, we also demonstrate a requirement for Kif3a, a gene essential for the formation of primary cilia, in a mouse model of SCLC induced by conditional deletion of both Trp53 and Rb1 in the adult airway. Our results provide a mechanistic framework for therapeutic targeting of ligand-dependent Hh signaling in human cancers with somatic mutations in both TP53 and RB1.
Catherine R. Cochrane, Vijesh Vaghjiani, Anette Szczepny, W. Samantha N. Jayasekara, Alvaro Gonzalez-Rajal, Kazu Kikuchi, Geoffrey W. McCaughan, Andrew Burgess, Daniel J. Gough, D. Neil Watkins, Jason E. Cain
Connexin 43 (Cx43) gap junctions provide intercellular coupling which ensures rapid action potential propagation and synchronized heart contraction. Altered Cx43 localization and reduced gap junction coupling occur in failing hearts, contributing to ventricular arrhythmias and sudden cardiac death. Recent reports have found that an internally translated Cx43 isoform, GJA1-20k, is an auxiliary subunit for the trafficking of Cx43 in heterologous expression systems. Here, we have created a mouse model by using CRISPR technology to mutate a single internal translation initiation site in Cx43 (M213L mutation), which generates full length Cx43 but not GJA1-20k. We find that GJA1M213L/M213L mice have severely abnormal electrocardiograms despite preserved contractile function, reduced total Cx43, reduced gap junctions, and die suddenly at two to four weeks of age. Heterozygous GJA1M213L/WT mice survive to adulthood with increased ventricular ectopy. Biochemical experiments indicate that cytoplasmic Cx43 has a half life that is 50% shorter than membrane associated Cx43. Without GJA1-20k, poorly trafficked Cx43 is degraded. The data support that GJA1-20k, an endogenous entity translated independently of Cx43, is critical for Cx43 gap junction trafficking, maintenance of Cx43 protein, and normal electrical function of the mammalian heart.
Shaohua Xiao, Daisuke Shimura, Rachel Baum, Diana M. Hernandez, Sosse Agvanian, Yoshiko Nagaoka, Makoto Katsumata, Paul D. Lampe, Andre G. Kleber, TingTing Hong, Robin M. Shaw
The tight junction protein claudin-2 is upregulated in disease. Although many studies have linked intestinal barrier loss to local and systemic disease, these have relied on macromolecular probes. In vitro analyses show however that these probes cannot be accommodated by size- and charge-selective claudin-2 channels. We sought to define the impact of claudin-2 channels on disease. Transgenic claudin-2 overexpression or IL-13-induced claudin-2 upregulation increased intestinal small cation permeability in vivo. IL-13 did not however affect permeability in claudin-2-knockout mice. Claudin-2 is therefore necessary and sufficient to effect size- and charge-selective permeability increases in vivo. In chronic disease, T-cell transfer colitis severity was augmented or diminished in claudin-2 transgenic or knockout mice, respectively. We translated in vitro data suggesting that casein kinase-2 (CK2) inhibition blocks claudin-2 channel function and found that CK2 inhibition prevented IL-13-induced, claudin-2-mediated permeability increases in vivo. In chronic immune-mediated colitis, CK2 inhibition attenuated progression in claudin-2-sufficient, but not claudin-2-knockout, mice, i.e., the effect was claudin-2-dependent. Paracellular flux mediated by claudin-2 channels can therefore promote immune-mediated colitis progression. Although the mechanisms by which claudin-2 channels intensify disease remain to be defined, these data suggest that claudin-2 may be an accessible target in immune-mediated disorders, including inflammatory bowel disease.
Preeti Raju, Nitesh Shashikanth, Pei-Yun Tsai, Pawin Pongkorpsakol, Sandra Chanez-Parades, Peter R. Steinhagen, Wei-Ting Kuo, Gurminder Singh, Sachiko Tsukita, Jerrold R. Turner
Emerging immune therapy, such as with the anti–programmed cell death–1 (anti–PD-1) monoclonal antibody nivolumab, has shown efficacy in tumor suppression. Patients with terminal cancer suffer from cancer pain as a result of bone metastasis and bone destruction, but how PD-1 blockade affects bone cancer pain remains unknown. Here, we report that mice lacking Pdcd1 (Pd1−/−) demonstrated remarkable protection against bone destruction induced by femoral inoculation of Lewis lung cancer cells. Compared with WT mice, Pd1−/− mice exhibited increased baseline pain sensitivity, but the development of bone cancer pain was compromised in Pd1−/− mice. Consistently, these beneficial effects in Pd1−/− mice were recapitulated by repeated i.v. applications of nivolumab in WT mice, even though nivolumab initially increased mechanical and thermal pain. Notably, PD-1 deficiency or nivolumab treatment inhibited osteoclastogenesis without altering tumor burden. PD-L1 and CCL2 are upregulated within the local tumor microenvironment, and PD-L1 promoted RANKL-induced osteoclastogenesis through JNK activation and CCL2 secretion. Bone cancer upregulated CCR2 in primary sensory neurons, and CCR2 antagonism effectively reduced bone cancer pain. Our findings suggest that, despite a transient increase in pain sensitivity following each treatment, anti–PD-1 immunotherapy could produce long-term benefits in preventing bone destruction and alleviating bone cancer pain by suppressing osteoclastogenesis.
Kaiyuan Wang, Yun Gu, Yihan Liao, Sangsu Bang, Christopher R. Donnelly, Ouyang Chen, Xueshu Tao, Anthony J. Mirando, Matthew J. Hilton, Ru-Rong Ji
Dominant mutations in the HSP70 co-chaperone DNAJB6 cause a late onset muscle disease termed limb girdle muscular dystrophy type D1 (LGMDD1), which is characterized by protein aggregation and vacuolar myopathology. Disease mutations reside within the G/F domain of DNAJB6, but the molecular mechanisms underlying dysfunction are not well understood. Using yeast, cell culture, and mouse models of LGMDD1, we found that the toxicity associated with disease-associated DNAJB6 required its interaction with HSP70, and that abrogating this interaction genetically or with small molecules was protective. In skeletal muscle, DNAJB6 localizes to the Z-disc with HSP70. Whereas HSP70 normally diffused rapidly between the Z-disc and sarcoplasm, the rate of HSP70’s diffusion in LGMDD1 mouse muscle was diminished likely because it has an unusual affinity for the Z-disc and mutant DNAJB6. Treating LGMDD1 mice with a small molecule inhibitor of the DNAJ-HSP70 complex re-mobilized HSP70, improved strength and corrected myopathology. These data support a model in which LGMDD1 mutations in DNAJB6 are a gain-of-function disease that is, counter-intuitively, mediated via HSP70 binding. Thus, therapeutic approaches targeting HSP70:DNAJB6 may be effective in treating this inherited muscular dystrophy.
Rocio Bengoechea, Andrew R. Findlay, Ankan K. Bhadra, Hao Shao, Kevin C. Stein, Sara K. Pittman, Jill Daw, Jason E. Gestwicki, Heather L. True, Conrad C. Weihl
While Canakinumab Anti-Inflammatory Thrombosis Outcomes Study (CANTOS) established the role of treating inflammation in atherosclerosis, our understanding of endothelial activation at atherosclerosis-prone sites remains limited. Disturbed flow at atheroprone regions primes plaque inflammation by enhancing endothelial NF-κB signaling. Herein, we demonstrate a role for the Nck adaptor proteins in disturbed flow-induced endothelial activation. Although highly similar, only Nck1 deletion, but not Nck2 deletion, limited flow-induced NF-κB activation and proinflammatory gene expression. Nck1 knockout mice showed reduced endothelial activation and inflammation in both models of disturbed flow and high fat diet-induced atherosclerosis, whereas Nck2 deletion did not. Bone marrow chimeras confirmed that vascular Nck1, but not hematopoietic Nck1, mediated this effect. Domain swap experiments and point mutations identified the Nck1 SH2 domain and the first SH3 domain as critical for flow-induced endothelial activation. We further characterized Nck1’s proinflammatory role by identifying interleukin-1 type I receptor kinase-1 (IRAK-1) as a Nck1-selective binding partner, demonstrating IRAK-1 activation by disturbed flow required Nck1 in vitro and in vivo, showing endothelial Nck1 and IRAK-1 staining in early human atherosclerosis, and demonstrating that disturbed flow-induced endothelial activation required IRAK-1. Taken together, our data reveal a hitherto unknown link between Nck1 and IRAK-1 in atherogenic inflammation.
Mabruka Alfaidi, Christina H. Acosta, Dongdong Wang, James G. Traylor, A. Wayne Orr
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