Akt and SHP-1 are DC-intrinsic checkpoints for tumor immunity

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Abstract

BM-derived DC (BMDC) are powerful antigen-presenting cells. When loaded with immune complexes (IC), consisting of tumor antigens bound to antitumor antibody, BMDC induce powerful antitumor immunity in mice. However, attempts to employ this strategy clinically with either tumor-associated DC (TADC) or monocyte-derived DC (MoDC) have been disappointing. To investigate the basis for this phenomenon, we compared the response of BMDC, TADC, and MoDC to tumor IgG-IC. Our findings revealed, in both mice and humans, that upon exposure to IgG-IC, BMDC internalized the IC, increased costimulatory molecule expression, and stimulated autologous T cells. In contrast, TADC and, surprisingly, MoDC remained inert upon contact with IC due to dysfunctional signaling following engagement of Fcγ receptors. Such dysfunction is associated with elevated levels of the Src homology region 2 domain–containing phosphatase-1 (SHP-1) and phosphatases regulating Akt activation. Indeed, concomitant inhibition of both SHP-1 and phosphatases that regulate Akt activation conferred upon TADC and MoDC the capacity to take up and process IC and induce antitumor immunity in vivo. This work identifies the molecular checkpoints that govern activation of MoDC and TADC and their capacity to elicit T cell immunity.

Authors

Yaron Carmi, Tyler R. Prestwood, Matthew H. Spitzer, Ian L. Linde, Jonathan Chabon, Nathan E. Reticker-Flynn, Nupur Bhattacharya, Hong Zhang, Xiangyue Zhang, Pamela A. Basto, Bryan M. Burt, Michael N. Alonso, Edgar G. Engleman

Mfge8 regulates enterocyte lipid storage by promoting enterocyte triglyceride hydrolase activity

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Abstract

The small intestine has an underappreciated role as a lipid storage organ. Under conditions of high dietary fat intake, enterocytes can minimize the extent of postprandial lipemia by storing newly absorbed dietary fat in cytoplasmic lipid droplets. Lipid droplets can be subsequently mobilized for the production of chylomicrons. The mechanisms that regulate this process are poorly understood. We report here that the milk protein Mfge8 regulates hydrolysis of cytoplasmic lipid droplets in enterocytes after interacting with the αvβ3 and αvβ5 integrins. Mice deficient in Mfge8 or the αvβ3 and αvβ5 integrins accumulate excess cytoplasmic lipid droplets after a fat challenge. Mechanistically, interruption of the Mfge8-integrin axis leads to impaired enterocyte intracellular triglyceride hydrolase activity in vitro and in vivo. Furthermore, Mfge8 increases triglyceride hydrolase activity through a PI3 kinase/mTORC2–dependent signaling pathway. These data identify a key role for Mfge8 and the αvβ3 and αvβ5 integrins in regulating enterocyte lipid processing.

Authors

Amin Khalifeh-Soltani, Deepti Gupta, Arnold Ha, Jahangir Iqbal, Mahmood Hussain, Michael J. Podolsky, Kamran Atabai

Acquired platinum resistance involves epithelial to mesenchymal transition through ubiquitin ligase FBXO32 dysregulation

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Abstract

To identify the molecules involved in epithelial to mesenchymal transition (EMT) in urothelial carcinoma (UC) after acquisition of platinum resistance, here we examined the changes in global gene expression before and after platinum treatment. Four invasive UC cell lines, T24, 5637, and their corresponding sublines T24PR and 5637PR with acquired platinum resistance, were assessed by microarray, and the ubiquitin E3 ligase FBXO32 was newly identified as a negative regulator of EMT in UC tumors after acquisition of platinum resistance. In vitro and in vivo studies showed an intimate relationship between FBXO32 expression and EMT, demonstrating that FBXO32 dysregulation in T24PR cells results in elevated expression of the mesenchymal molecules SNAIL and vimentin and decreased expression of the epithelial molecule E-cadherin. The association between FBXO32 expression and EMT was further validated using clinical samples. Knockdown of MyoD expression, a specific target of FBXO32 polyubiquitination, revealed upregulation of E-cadherin expression and downregulation of SNAIL and vimentin expression in T24PR cells. Comparative genomic hybridization array analysis demonstrated loss of heterozygosity at 8q24.13 in T24PR cells, which harbors FBXO32. Our findings suggest the importance of the association between EMT and ubiquitin-proteasome regulation when tumors develop acquired platinum resistance.

Authors

Nobuyuki Tanaka, Takeo Kosaka, Yasumasa Miyazaki, Shuji Mikami, Naoya Niwa, Yutaro Otsuka, Yoji Andrew Minamishima, Ryuichi Mizuno, Eiji Kikuchi, Akira Miyajima, Hisataka Sabe, Yasunori Okada, Per Uhlén, Makoto Suematsu, Mototsugu Oya

Activating transcription factor-4 promotes mineralization in vascular smooth muscle cells

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Abstract

Emerging evidence indicates that upregulation of the ER stress–induced pro-osteogenic transcription factor ATF4 plays an important role in vascular calcification, a common complication in patients with aging, diabetes, and chronic kidney disease (CKD). In this study, we demonstrated the pathophysiological role of ATF4 in vascular calcification using global Atf4 KO, smooth muscle cell–specific (SMC-specific) Atf4 KO, and transgenic (TG) mouse models. Reduced expression of ATF4 in global ATF4-haplodeficient and SMC-specific Atf4 KO mice reduced medial and atherosclerotic calcification under normal kidney and CKD conditions. In contrast, increased expression of ATF4 in SMC-specific Atf4 TG mice caused severe medial and atherosclerotic calcification. We further demonstrated that ATF4 transcriptionally upregulates the expression of type III sodium-dependent phosphate cotransporters (PiT1 and PiT2) by interacting with C/EBPβ. These results demonstrate that the ER stress effector ATF4 plays a critical role in the pathogenesis of vascular calcification through increased phosphate uptake in vascular SMCs.

Authors

Masashi Masuda, Shinobu Miyazaki-Anzai, Audrey L. Keenan, Yuji Shiozaki, Kayo Okamura, Wallace S. Chick, Kristina Williams, Xiaoyun Zhao, Shaikh Mizanoor Rahman, Yin Tintut, Christopher M. Adams, Makoto Miyazaki

Microstructural alterations of sputum in cystic fibrosis lung disease

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Abstract

The stasis of mucus secretions in the lungs of cystic fibrosis (CF) patients leads to recurrent infections and pulmonary exacerbations, resulting in decreased survival. Prior studies have assessed the biochemical and biophysical features of airway mucus in individuals with CF. However, these measurements are unable to probe mucus structure on microscopic length scales relevant to key players in the progression of CF-related lung disease, namely, viruses, bacteria, and neutrophils. In this study, we quantitatively determined sputum microstructure based on the diffusion of muco-inert nanoparticle probes in CF sputum and found that a reduction in sputum mesh pore size is characteristic of CF patients with reduced lung function, as indicated by measured FEV₁. We also discovered that the effect of ex vivo treatment of CF sputum with rhDNase I (Pulmozyme) on microstructure is dependent upon the time interval between the most recent inhaled rhDNase I treatment and the sample collection. Microstructure of mucus may serve as a marker for the extent of CF lung disease and as a parameter for assessing the effectiveness of mucus-altering agents.

Authors

Gregg A. Duncan, James Jung, Andrea Joseph, Abigail L. Thaxton, Natalie E. West, Michael P. Boyle, Justin Hanes, Jung Soo Suk

Proproliferative and antiapoptotic action of exogenously introduced YAP in pancreatic β cells

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Abstract

Loss of functional pancreatic β cells is a hallmark of both type 1 and 2 diabetes. Identifying the pathways that promote β cell proliferation and/or block β cell apoptosis is a potential strategy for diabetes therapy. The transcriptional coactivator Yes-associated protein (YAP), a major downstream effector of the Hippo signaling pathway, is a key regulator of organ size and tissue homeostasis by modulating cell proliferation and apoptosis. YAP is not expressed in mature primary human and mouse β cells. We aimed to identify whether reexpression of a constitutively active form of YAP promotes β cell proliferation/survival. Overexpression of YAP remarkably induced β cell proliferation in isolated human islets, while β cell function and functional identity genes were fully preserved. The transcription factor forkhead box M1 (FOXM1) was upregulated upon YAP overexpression and necessary for YAP-dependent β cell proliferation. YAP overexpression protected β cells from apoptosis triggered by multiple diabetic conditions. The small redox proteins thioredoxin-1 and thioredoxin-2 (Trx1/2) were upregulated by YAP; disruption of the Trx system revealed that Trx1/2 was required for the antiapoptotic action of YAP in insulin-producing β cells. Our data show the robust proproliferative and antiapoptotic function of YAP in pancreatic β cells. YAP reconstitution may represent a disease-modifying approach to restore a functional β cell mass in diabetes.

Authors

Ting Yuan, Sahar Rafizadeh, Zahra Azizi, Blaz Lupse, Kanaka Durga Devi Gorrepati, Sushil Awal, Jose Oberholzer, Kathrin Maedler, Amin Ardestani

β Cell–specific increased expression of calpastatin prevents diabetes induced by islet amyloid polypeptide toxicity

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Abstract

The islet in type 2 diabetes (T2D) shares many features of the brain in protein misfolding diseases. There is a deficit of β cells with islet amyloid derived from islet amyloid polypeptide (IAPP), a protein coexpressed with insulin. Small intracellular membrane-permeant oligomers, the most toxic form of IAPP, are more frequent in β cells of patients with T2D and rodents expressing human IAPP. β Cells in T2D, and affected cells in neurodegenerative diseases, share a comparable pattern of molecular pathology, including endoplasmic reticulum stress, mitochondrial dysfunction, attenuation of autophagy, and calpain hyperactivation. While this adverse functional cascade in response to toxic oligomers is well described, the sequence of events and how best to intervene is unknown. We hypothesized that calpain hyperactivation is a proximal event and tested this in vivo by β cell–specific suppression of calpain hyperactivation with calpastatin overexpression in human IAPP transgenic mice. β Cell–specific calpastatin overexpression was remarkably protective against β cell dysfunction and loss and diabetes onset. The critical autophagy/lysosomal pathway for β cell viability was protected with calpain suppression, consistent with findings in models of neurodegenerative diseases. We conclude that suppression of calpain hyperactivation is a potentially beneficial disease-modifying strategy for protein misfolding diseases, including T2D.

Authors

Tatyana Gurlo, Safia Costes, Jonathan D. Hoang, Jacqueline F. Rivera, Alexandra E. Butler, Peter C. Butler

Insulin resistance uncoupled from dyslipidemia due to C-terminal PIK3R1 mutations

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Abstract

Obesity-related insulin resistance is associated with fatty liver, dyslipidemia, and low plasma adiponectin. Insulin resistance due to insulin receptor (INSR) dysfunction is associated with none of these, but when due to dysfunction of the downstream kinase AKT2 phenocopies obesity-related insulin resistance. We report 5 patients with SHORT syndrome and C-terminal mutations in PIK3R1, encoding the p85α/p55α/p50α subunits of PI3K, which act between INSR and AKT in insulin signaling. Four of 5 patients had extreme insulin resistance without dyslipidemia or hepatic steatosis. In 3 of these 4, plasma adiponectin was preserved, as in insulin receptor dysfunction. The fourth patient and her healthy mother had low plasma adiponectin associated with a potentially novel mutation, p.Asp231Ala, in adiponectin itself. Cells studied from one patient with the p.Tyr657X PIK3R1 mutation expressed abundant truncated PIK3R1 products and showed severely reduced insulin-stimulated association of mutant but not WT p85α with IRS1, but normal downstream signaling. In 3T3-L1 preadipocytes, mutant p85α overexpression attenuated insulin-induced AKT phosphorylation and adipocyte differentiation. Thus, PIK3R1 C-terminal mutations impair insulin signaling only in some cellular contexts and produce a subphenotype of insulin resistance resembling INSR dysfunction but unlike AKT2 dysfunction, implicating PI3K in the pathogenesis of key components of the metabolic syndrome.

Authors

Isabel Huang-Doran, Patsy Tomlinson, Felicity Payne, Alexandra Gast, Alison Sleigh, William Bottomley, Julie Harris, Allan Daly, Nuno Rocha, Simon Rudge, Jonathan Clark, Albert Kwok, Stefano Romeo, Emma McCann, Barbara Müksch, Mehul Dattani, Stefano Zucchini, Michael Wakelam, Lazaros C. Foukas, David B. Savage, Rinki Murphy, Stephen O’Rahilly, Inês Barroso, Robert K. Semple

Acquired resistance to innate immune clearance promotes Klebsiella pneumoniae ST258 pulmonary infection

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Abstract

Adaptive changes in the genome of a locally predominant clinical isolate of the multidrug-resistant Klebsiella pneumoniae ST258 (KP35) were identified and help to explain the selection of this strain as a successful pulmonary pathogen. The acquisition of 4 new ortholog groups, including an arginine transporter, enabled KP35 to outcompete related ST258 strains lacking these genes. KP35 infection elicited a monocytic response, dominated by Ly6C^hi monocytic myeloid-derived suppressor cells that lacked phagocytic capabilities, expressed IL-10, arginase, and antiinflammatory surface markers. In comparison with other K. pneumoniae strains, KP35 induced global changes in the phagocytic response identified with proteomics, including evasion of Ca²⁺ and calpain activation necessary for phagocytic killing, confirmed in functional studies with neutrophils. This comprehensive analysis of an ST258 K. pneumoniae isolate reveals ongoing genetic adaptation to host microenvironments and innate immune clearance mechanisms that complements its repertoire of antimicrobial resistance genes and facilitates persistence in the lung.

Authors

Danielle Ahn, Hernán Peñaloza, Zheng Wang, Matthew Wickersham, Dane Parker, Purvi Patel, Antonius Koller, Emily I. Chen, Susan M. Bueno, Anne-Catrin Uhlemann, Alice Prince

PLEKHM1/DEF8/RAB7 complex regulates lysosome positioning and bone homeostasis

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Abstract

Mutations of the Plekhm1 gene in humans and rats cause osteopetrosis, an inherited bone disease characterized by diminished bone resorption by osteoclasts. PLEKHM1 binds to RAB7 and is critical for lysosome trafficking. However, the molecular mechanisms by which PLEKHM1 regulates lysosomal pathways remain unknown. Here, we generated germline and conditional Plekhm1-deficient mice. These mice displayed no overt abnormalities in major organs, except for an increase in trabecular bone mass. Furthermore, loss of PLEKHM1 abrogated the peripheral distribution of lysosomes and bone resorption in osteoclasts. Mechanistically, we indicated that DEF8 interacts with PLEKHM1 and promotes its binding to RAB7, whereas the binding of FAM98A and NDEL1 with PLEKHM1 connects lysosomes to microtubules. Importantly, suppression of these proteins results in lysosome positioning and bone resorption defects similar to those of Plekhm1-null osteoclasts. Thus, PLHKEM1, DEF8, FAM98A, and NDEL1 constitute a molecular complex that regulates lysosome positioning and secretion through RAB7.

Authors

Toshifumi Fujiwara, Shiqiao Ye, Thiago Castro-Gomes, Caylin G. Winchell, Norma W. Andrews, Daniel E. Voth, Kottayil I. Varughese, Samuel G. Mackintosh, Yunfeng Feng, Nathan Pavlos, Takashi Nakamura, Stavros C. Manolagas, Haibo Zhao