Receptor tyrosine kinases (RTKs) are important drivers of cancers. In addition to genomic alterations, aberrant activation of wild type RTKs plays an important role in driving cancer progression. However, the underlying mechanisms of how RTKs drive prostate cancer remain incompletely characterized. Here we show that non-proteolytic ubiquitination of RTK regulates its kinase activity and contributes to RTK-mediated prostate cancer metastasis. TRAF4, an E3 ubiquitin ligase, is highly expressed in metastatic prostate cancer. We demonstrated here that it is a key player in regulating RTK mediated prostate cancer metastasis. We further identified TrkA, a neurotrophin RTK, as TRAF4-targeted ubiquitination substrate that promotes cancer cell invasion and inhibition of TrkA activity abolished TRAF4-dependent cell invasion. TRAF4 promoted K27 and K29-linked ubiquitination at the TrkA kinase domain and increased its kinase activity. Mutation of TRAF4-targeted ubiquitination sites abolished TrkA tyrosine auto-phosphorylation and its interaction with downstream proteins. TRAF4 knockdown also suppressed NGF-stimulated TrkA downstream p38 MAPK activation and invasion-associated gene expression. Furthermore, elevated TRAF4 levels significantly correlated with increased NGF-stimulated invasion-associated gene expression in prostate cancer patients, indicating that this signaling axis is significantly activated during oncogenesis. Our results revealed a post-translational modification mechanism contributing to aberrant non-mutated RTK activation in cancer cells.
Ramesh Singh, Dileep Karri, Hong Shen, Jiangyong Shao, Subhamoy Dasgupta, Shixia Huang, Dean P. Edwards, Michael M. Ittmann, Bert W. O’Malley, Ping Yi
Tyro3, Axl, Mer (TAM) receptor tyrosine kinases reduce inflammatory, innate immune responses. We demonstrate that tumor-secreted protein S (Pros1), a Mer/Tyro3 ligand, decreased macrophage M1 cytokine expression in vitro and in vivo. In contrast, tumor cells with CRISPR-based deletion of Pros1 failed to inhibit M1 polarization. Tumor cell–associated Pros1 action was abrogated in macrophages from Mer- and Tyro3- but not Axl-KO mice. In addition, several other murine and human tumor cell lines suppressed macrophage M1 cytokine expression induced by IFN-γ and LPS. Investigation of the suppressive pathway demonstrated a role for PTP1b complexing with Mer. Substantiating the role of PTP1b, M1 cytokine suppression was also lost in macrophages from PTP1b-KO mice. Mice bearing Pros1-deficient tumors showed increased innate and adaptive immune infiltration, as well as increased median survival. TAM activation can also inhibit TLR-mediated M1 polarization. Treatment with resiquimod, a TLR7/8 agonist, did not improve survival in mice bearing Pros1-secreting tumors but doubled survival for Pros1-deleted tumors. The tumor-derived Pros1 immune suppressive system, like PD-L1, was cytokine responsive, with IFN-γ inducing Pros1 transcription and secretion. Inhibition of Pros1/TAM interaction represents a potential novel strategy to block tumor-derived immune suppression.
Eric Ubil, Laura Caskey, Alisha Holtzhausen, Debra Hunter, Charlotte Story, H. Shelton Earp
Fibrosis is a prevalent pathological condition arising from the chronic activation of fibroblasts. This activation results from the extensive intercellular crosstalk mediated by both soluble factors and direct cell-cell connections. Prominent among these are the interactions of fibroblasts with immune cells, in which the fibroblast–mast cell connection, although acknowledged, is relatively unexplored. We have used a Tg mouse model of skin fibrosis, based on expression of the transcription factor Snail in the epidermis, to probe the mechanisms regulating mast cell activity and the contribution of these cells to this pathology. We have discovered that Snail-expressing keratinocytes secrete plasminogen activator inhibitor type 1 (PAI1), which functions as a chemotactic factor to increase mast cell infiltration into the skin. Moreover, we have determined that PAI1 upregulates intercellular adhesion molecule type 1 (ICAM1) expression on dermal fibroblasts, rendering them competent to bind to mast cells. This heterotypic cell-cell adhesion, also observed in the skin fibrotic disorder scleroderma, culminates in the reciprocal activation of both mast cells and fibroblasts, leading to the cascade of events that promote fibrogenesis. Thus, we have identified roles for PAI1 in the multifactorial program of fibrogenesis that expand its functional repertoire beyond its canonical role in plasmin-dependent processes.
Neha Pincha, Edries Yousaf Hajam, Krithika Badarinath, Surya Prakash Rao Batta, Tafheem Masudi, Rakesh Dey, Peter Andreasen, Toshiaki Kawakami, Rekha Samuel, Renu George, Debashish Danda, Paul Mazhuvanchary Jacob, Colin Jamora
Adult vascular smooth muscle cells (VSMCs) possess the peculiar ability to de-differentiate in response to extracellular cues, such as vascular damage and inflammation. De-differentiated VSMCs are proliferative, migratory, and have decreased contractile capacity. VSMC dedifferentiation contributes not only to vascular repair, but also to cardiovascular pathologies, such as intimal hyperplasia/restenosis in coronary artery or peripheral vascular diseases and arterial aneurysm. We here demonstrate the role of ubiquitin-like, containing PHD and RING finger domains, 1 (UHRF1) as an epigenetic master regulator of VSMC plasticity. The expression of UHRF1 correlates with the development of a wide array of vascular pathologies associated also with modulation of non-coding RNAs, such as microRNAs. Importantly, miR-145, a pivotal gene regulating VSMC plasticity, which is reduced in vascular diseases, was found to control Uhrf1 mRNA translation. In turn, UHRF1 triggers VSMC proliferation by directly repressing the promoters of cell cycle inhibitor genes, such as p21 and p27, and of key pro-differentiation genes via the methylation of DNA and histones. Local vascular viral delivery of Uhrf1 shRNAs or Uhrf1 VSMC-specific deletion prevented intimal hyperplasia in mouse carotid artery and decreased vessel damage in a mouse model of aortic aneurysm.Our study demonstrates the fundamental role of Uhrf1 in regulating VSMC phenotype by promoting proliferation and de-differentiation. UHRF1 targeting may hold therapeutic potential in vascular pathologies, modulating also the VSMC component.
Leonardo Elia, Paolo Kunderfranco, Pierluigi Carullo, Marco Vacchiano, Floriana Maria Farina, Ignacio Fernando Hall, Stefano Mantero, Cristina Panico, Roberto Papait, Gianluigi Condorelli, Manuela Quintavalle
Autophagy is important for liver homeostasis and the deficiency leads to injury, inflammation, ductular reaction (DR), fibrosis, and tumorigenesis. It is not clear how these events are mechanistically linked to autophagy deficiency. Here we reveal the role of highmobility group box 1 (HMGB1) in two of these processes. First, HMGB1 was required for DR, which represents the expansion of hepatic progenitor cells (HPC) implicated in liver repair and regeneration. DR caused by hepatic toxic diets (DDC or CDE) also depended on HMGB1, indicating that HMGB1 may be generally required for DR in various injury scenarios. Second, HMGB1 promoted tumor development in autophagy deficient livers. Receptor for advanced glycation end product (RAGE), a receptor for HMGB1, was required in the same two processes, and could mediate HMGB1’s proliferative effects in isolated HPC. HMGB1 was released from autophagy-deficient hepatocytes independently of cellular injury, but depending on NRF2 and inflammasome, which was activated by NRF2. Pharmacological or genetic activation of NRF2 alone without disabling autophagy or causing injury was sufficient to cause inflammasomedependent HMGB1 release. In conclusion, HMGB1 release is a critical mechanism in hepatic pathogenesis under the autophagy deficient condition, which leads to HPC expansion but also tumor development.
Bilon Khambu, Nazmul Huda, Xiaoyun Chen, Daniel J. Antoine, Yong Li, Guoli Dai, Ulrike A. Köhler, Wei-Xing Zong, Satoshi Waguri, Sabine Werner, Tim D. Oury, Zheng Dong, Xiao-Ming Yin
Incidence of nonalcoholic steatohepatitis (NASH), which is considered a hepatic manifestation of metabolic syndrome, has been increasing worldwide with the rise in obesity; however, its pathological mechanism is poorly understood. Here, we demonstrate that the hepatic expression of aortic carboxypeptidase–like protein (ACLP), a glycosylated, secreted protein, increases in NASH in humans and mice. Furthermore, we elucidate that ACLP is a ligand, unrelated to WNT proteins, that activates the canonical WNT pathway and exacerbates NASH pathology. In the liver, ACLP is specifically expressed in hepatic stellate cells (HSCs). As fatty liver disease progresses, ACLP expression is enhanced via activation of STAT3 signaling by obesity-related factors in serum. ACLP specifically binds to frizzled-8 and low-density lipoprotein–related receptor 6 to form a ternary complex that activates canonical WNT signaling. Consequently, ACLP activates HSCs by inhibiting PPARγ signals. HSC-specific ACLP deficiency inhibits fibrosis progression in NASH by inhibiting canonical WNT signaling in HSCs. The present study elucidates the role of canonical WNT pathway activation by ACLP in NASH pathology, indicating that NASH can be treated by targeting ACLP-induced canonical WNT pathway activation in HSCs.
Toshiaki Teratani, Kengo Tomita, Takahiro Suzuki, Hirotaka Furuhashi, Rie Irie, Makoto Nishikawa, Junji Yamamoto, Toshifumi Hibi, Soichiro Miura, Tohru Minamino, Yuichi Oike, Ryota Hokari, Takanori Kanai
During development, Sox2 is indispensable for cell division and differentiation, yet its roles in regenerating tissues are less clear. Here, we used combinations of transgenic mouse models to reveal that Sox2 haploinsufficiency (Sox2haplo) increases rather than impairs cochlear regeneration in vivo. Sox2haplo cochleae had delayed terminal mitosis and ectopic sensory cells, yet normal auditory function. Sox2haplo amplified and expanded domains of damage-induced Atoh1+ transitional cell formation in neonatal cochlea. Wnt activation via β-catenin stabilization (β-cateninGOF) alone failed to induce proliferation or transitional cell formation. By contrast, β-cateninGOF caused proliferation when either Sox2haplo or damage was present, and transitional cell formation when both were present in neonatal, but not mature, cochlea. Mechanistically, Sox2haplo or damaged neonatal cochleae showed lower levels of Sox2 and Hes5, but not of Wnt target genes. Together, our study unveils an interplay between Sox2 and damage in directing tissue regeneration and Wnt responsiveness and thus provides a foundation for potential combinatorial therapies aimed at stimulating mammalian cochlear regeneration to reverse hearing loss in humans.
Patrick J. Atkinson, Yaodong Dong, Shuping Gu, Wenwen Liu, Elvis Huarcaya Najarro, Tomokatsu Udagawa, Alan G. Cheng
Pro-opiomelanocortin (POMC) neurons function as key regulators of metabolism and physiology by releasing prohormone-derived neuropeptides with distinct biological activities. However, our understanding of early events in prohormone maturation in the ER remains incomplete. Highlighting the significance of this gap in knowledge, a single POMC cysteine-to-phenylalanine mutation at position 28 (POMC-C28F) is defective for ER processing and causes early onset obesity in a dominant-negative manner in humans through an unclear mechanism. Here, we report a pathologically important role of Sel1L-Hrd1, the protein complex of ER-associated degradation (ERAD), within POMC neurons. Mice with POMC neuron–specific Sel1L deficiency developed age-associated obesity due, at least in part, to the ER retention of POMC that led to hyperphagia. The Sel1L-Hrd1 complex targets a fraction of nascent POMC molecules for ubiquitination and proteasomal degradation, preventing accumulation of misfolded and aggregated POMC, thereby ensuring that another fraction of POMC can undergo normal posttranslational processing and trafficking for secretion. Moreover, we found that the disease-associated POMC-C28F mutant evades ERAD and becomes aggregated due to the presence of a highly reactive unpaired cysteine thiol at position 50. Thus, this study not only identifies ERAD as an important mechanism regulating POMC maturation within the ER, but also provides insights into the pathogenesis of monogenic obesity associated with defective prohormone folding.
Geun Hyang Kim, Guojun Shi, Diane R.M. Somlo, Leena Haataja, Soobin Song, Qiaoming Long, Eduardo A. Nillni, Malcolm J. Low, Peter Arvan, Martin G. Myers Jr., Ling Qi
Infection by Staphylococcus aureus strain USA300 causes tissue injury, multiorgan failure, and high mortality. However, the mechanisms by which the bacteria adhere to, then stabilize on, mucosal surfaces before causing injury remain unclear. We addressed these issues through the first real-time determinations of USA300-alveolar interactions in live lungs. We found that within minutes, inhaled USA300 established stable, self-associated microaggregates in niches at curved, but not at flat, regions of the alveolar wall. The microaggregates released α-hemolysin toxin, causing localized alveolar injury, as indicated by epithelial dye loss, mitochondrial depolarization, and cytosolic Ca2+ increase. Spread of cytosolic Ca2+ through intercellular gap junctions to adjoining, uninfected alveoli caused pulmonary edema. Systemic pretreatment with vancomycin, a USA300-cidal antibiotic, failed to protect mice infected with inhaled WT USA300. However, vancomycin pretreatment markedly abrogated mortality in mice infected with mutant USA300 that lacked the aggregation-promoting factor PhnD. We interpret USA300-induced mortality as having resulted from rapid bacterial aggregation in alveolar niches. These findings indicate, for the first time to our knowledge, that alveolar microanatomy is critical in promoting the aggregation and, hence, in causing USA300-induced alveolar injury. We propose that in addition to antibiotics, strategies for bacterial disaggregation may constitute novel therapy against USA300-induced lung injury.
Jaime L. Hook, Mohammad N. Islam, Dane Parker, Alice S. Prince, Sunita Bhattacharya, Jahar Bhattacharya
Coagulation factor XII (FXII) deficiency is associated with decreased neutrophil migration, but the mechanisms remain uncharacterized. Here, we examine how FXII contributes to the inflammatory response. In 2 models of sterile inflammation, FXII-deficient mice (F12–/–) had fewer neutrophils recruited than WT mice. We discovered that neutrophils produced a pool of FXII that is functionally distinct from hepatic-derived FXII and contributes to neutrophil trafficking at sites of inflammation. FXII signals in neutrophils through urokinase plasminogen activator receptor–mediated (uPAR-mediated) Akt2 phosphorylation at S474 (pAktS474). Downstream of pAkt2S474, FXII stimulation of neutrophils upregulated surface expression of αMβ2 integrin, increased intracellular calcium, and promoted extracellular DNA release. The sum of these activities contributed to neutrophil cell adhesion, migration, and release of neutrophil extracellular traps in a process called NETosis. Decreased neutrophil signaling in F12–/– mice resulted in less inflammation and faster wound healing. Targeting hepatic F12 with siRNA did not affect neutrophil migration, whereas WT BM transplanted into F12–/– hosts was sufficient to correct the neutrophil migration defect in F12–/– mice and restore wound inflammation. Importantly, these activities were a zymogen FXII function and independent of FXIIa and contact activation, highlighting that FXII has a sophisticated role in vivo that has not been previously appreciated.
Evi X. Stavrou, Chao Fang, Kara L. Bane, Andy T. Long, Clément Naudin, Erdem Kucukal, Agharnan Gandhi, Adina Brett-Morris, Michele M. Mumaw, Sudeh Izadmehr, Alona Merkulova, Cindy C. Reynolds, Omar Alhalabi, Lalitha Nayak, Wen-Mei Yu, Cheng-Kui Qu, Howard J. Meyerson, George R. Dubyak, Umut A. Gurkan, Marvin T. Nieman, Anirban Sen Gupta, Thomas Renné, Alvin H. Schmaier
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