Succinate dehydrogenase stain of mouse gastrocnemius muscle to show oxidative muscle fibers. Frey and colleagues show that deletion of the sarcomeric Z-disc protein calsarcin-2 improved muscle performance and enhanced running distances in exercise studies in mice (page 3598).
Unfortunately, we seem to run article amendments (corrections, errata, retractions, addenda) in every issue these days. In the current issue, we have a correction and a retraction — both coming after intensive investigations and peculiar situations we hadn’t encountered before.
Ushma S. Neill, Laurence A. Turka
Monocyte-derived macrophages can determine the outcome of the immune response and whether this response contributes to tissue repair or mediates tissue destruction. In addition to their important role in immune-mediated renal disease and host defense, macrophages play a fundamental role in tissue remodeling during embryonic development, acquired kidney disease, and renal allograft responses. This review summarizes macrophage phenotype and function in the orchestration of kidney repair and replacement of specialized renal cells following injury. Recent advances in our understanding of macrophage heterogeneity in response to their microenvironment raise new and exciting therapeutic possibilities to attenuate or conceivably reverse progressive renal disease in the context of fibrosis. Furthermore, parallels with pathological processes in many other organs also exist.
Sharon D. Ricardo, Harry van Goor, Allison A. Eddy
Cytokines are a large family of more than 100 small proteins that function as short-range mediators involved in essentially all biological processes. They have been found to be important rate-limiting signals, and it is now known that blocking some cytokines, e.g., TNF-α, and cytokine receptors, such as human EGFR 1 (HER1) or HER2, yields effective therapeutics that address unmet needs. This Review Series surveys three chronic inflammatory disease areas and two forms of cancer and discusses the important role of cytokines and their receptors in these disease processes. Their role as potential therapeutic targets is also highlighted.
A large number of cytokines are active in the joints of patients with rheumatoid arthritis (RA). It is now clear that these cytokines play a fundamental role in the processes that cause inflammation, articular destruction, and the comorbidities associated with RA. Following the success of TNF-α blockade as a treatment for RA, other cytokines now offer alternative targets for therapeutic intervention or might be useful as predictive biomarkers of disease. In this Review, we discuss the biologic contribution and therapeutic potential of the major cytokine families to RA pathology, focusing on molecules contained within the TNF-α, IL-1, IL-6, IL-23, and IL-2 families.
Fionula M. Brennan, Iain B. McInnes
Asthma and chronic obstructive pulmonary disease (COPD) are very common inflammatory diseases of the airways. They both cause airway narrowing and are increasing in incidence throughout the world, imposing enormous burdens on health care. Cytokines play a key role in orchestrating the chronic inflammation and structural changes of the respiratory tract in both asthma and COPD and have become important targets for the development of new therapeutic strategies in these diseases.
Peter J. Barnes
The three most prevalent human disorders of the CNS in which immunity and inflammation are likely to have vital roles (excluding infection of the CNS) are fever, multiple sclerosis (MS), and Alzheimer disease (AD). As reviewed here, cytokines are critical in the induction of fever, the pathogenesis of MS, and the pathobiology of AD. Indeed, antibodies targeting cytokines have been used as a therapy for individuals with unusual and persistent febrile reactions not responsive to common antipyretics, while a recombinant cytokine is the most popular treatment for the relapsing-remitting form of MS. Although cytokine-modulating therapies are not currently in clinical use for the treatment of AD, cytokines can ameliorate disease pathology in certain experimental models of AD, suggesting a potential for future therapeutic opportunities.
Studies over the past 50 years revealing the molecular events that promote normal T lymphocyte cycle competence and progression led to a detailed understanding of how cytokines function to regulate normal hematopoietic cell proliferation. During that same period, the molecular and genetic changes introduced by the Philadelphia chromosome in chronic myelogenous leukemia were unraveled, and these have led to an understanding of how mutations that constitutively activate normal cytokine signaling pathways can cause unregulated cell proliferation and malignant transformation. Based on the paradigm established by these data, it is inescapable that going forward, investigators will operate under the hypothesis that transformation of additional cells and tissues will have a similar pathogenesis.
Kendall A. Smith, James D. Griffin
The human EGFR (HER) family is essential for communication between many epithelial cancer cell types and the tumor microenvironment. Therapeutics targeting the HER family have demonstrated clinical success in the treatment of diverse epithelial cancers. Here we propose that the success of HER family–targeted monoclonal antibodies in cancer results from their ability to interfere with HER family consolidation of signals initiated by a multitude of other receptor systems. Ligand/receptor systems that initiate these signals include cytokine receptors, chemokine receptors, TLRs, GPCRs, and integrins. We further extrapolate that improvements in cancer therapeutics targeting the HER family are likely to incorporate mechanisms that block or reverse stromal support of malignant progression by isolating the HER family from autocrine and stromal influences.
H. Michael Shepard, Cathleen M. Brdlik, Hans Schreiber
Even though activating mutations of B-Raf, a kinase atop the MAPK signaling cascade, reportedly sensitize tumor cells to MEK inhibitors, Raf and MEK inhibitors have exhibited limited clinical activity. In this issue of the JCI, Cragg et al. report that MEK inhibition upregulates the proapoptotic Bcl-2 family member Bim but induces little regression of human melanoma xenografts in mice unless the Bcl-2 antagonist ABT-737 is added (see the related article beginning on page 3651). These findings illustrate the potential benefit of simultaneously inhibiting oncogenic kinases and inhibiting Bcl-2 action in solid tumors.
Andrea Wahner Hendrickson, Xue Wei Meng, Scott H. Kaufmann
The polycystic liver and kidney diseases are a family of disorders with heterogeneous etiologies. Proposed mechanisms of disease include ciliary dysfunction, excess cell proliferation, and altered cell-cell or cell-matrix interactions. In this issue of the JCI, Lee and colleagues provide data to support a novel mechanism for cystogenesis involving microRNA (miRNA) (see the related article beginning on page 3714). They demonstrate that levels of the miRNA miR15a are decreased in livers of patients with autosomal recessive and autosomal dominant polycystic kidney disease (ARPKD and ADPKD, respectively) and congenital hepatic fibrosis as well as in the PKC rat model of ARPKD. This results in increased expression of the cell-cycle regulator Cdc25A, which is a direct target of miR15a, and increased cellular proliferation and cystogenesis in vitro. These findings suggest that other miRNAs may also participate in the molecular pathogenesis of cystic liver and kidney diseases.
Andrew S. Chu, Joshua R. Friedman
Histone deacetylase (HDAC) inhibitors show remarkable therapeutic potential for a variety of disorders, including cancer, neurological disease, and cardiac hypertrophy. However, the specific HDAC isoforms that mediate their actions are unclear, as are the physiological and pathological functions of individual HDACs in vivo. To explore the role of Hdac3 in the heart, we generated mice with a conditional Hdac3 null allele. Although global deletion of Hdac3 resulted in lethality by E9.5, mice with a cardiac-specific deletion of Hdac3 survived until 3–4 months of age. At this time, they showed massive cardiac hypertrophy and upregulation of genes associated with fatty acid uptake, fatty acid oxidation, and electron transport/oxidative phosphorylation accompanied by fatty acid–induced myocardial lipid accumulation and elevated triglyceride levels. These abnormalities in cardiac metabolism can be attributed to excessive activity of the nuclear receptor PPARα. The phenotype associated with cardiac-specific Hdac3 gene deletion differs from that of all other Hdac gene mutations. These findings reveal a unique role for Hdac3 in maintenance of cardiac function and regulation of myocardial energy metabolism.
Rusty L. Montgomery, Matthew J. Potthoff, Michael Haberland, Xiaoxia Qi, Satoshi Matsuzaki, Kenneth M. Humphries, James A. Richardson, Rhonda Bassel-Duby, Eric N. Olson
The composition of skeletal muscle, in terms of the relative number of slow- and fast-twitch fibers, is tightly regulated to enable an organism to respond and adapt to changing physical demands. The phosphatase calcineurin and its downstream targets, transcription factors of the nuclear factor of activated T cells (NFAT) family, play a critical role in this process by promoting the formation of slow-twitch, oxidative fibers. Calcineurin binds to calsarcins, a family of striated muscle–specific proteins of the sarcomeric Z-disc. We show here that mice deficient in calsarcin-2, which is expressed exclusively by fast-twitch muscle and encoded by the myozenin 1 (Myoz1) gene, have substantially reduced body weight and fast-twitch muscle mass in the absence of an overt myopathic phenotype. Additionally, Myoz1 KO mice displayed markedly improved performance and enhanced running distances in exercise studies. Analysis of fiber type composition of calsarcin-2–deficient skeletal muscles showed a switch toward slow-twitch, oxidative fibers. Reporter assays in cultured myoblasts indicated an inhibitory role for calsarcin-2 on calcineurin, and Myoz1 KO mice exhibited both an excess of NFAT activity and an increase in expression of regulator of calcineurin 1-4 (RCAN1-4), indicating enhanced calcineurin signaling in vivo. Taken together, these results suggest that calsarcin-2 modulates exercise performance in vivo through regulation of calcineurin/NFAT activity and subsequent alteration of the fiber type composition of skeletal muscle.
Norbert Frey, Derk Frank, Stefanie Lippl, Christian Kuhn, Harald Kögler, Tomasa Barrientos, Claudia Rohr, Rainer Will, Oliver J. Müller, Hartmut Weiler, Rhonda Bassel-Duby, Hugo A. Katus, Eric N. Olson
Based on extensive preclinical data, glycogen synthase kinase–3 (GSK-3) has been proposed to be a viable drug target for a wide variety of disease states, ranging from diabetes to bipolar disorder. Since these new drugs, which will be more powerful GSK-3 inhibitors than lithium, may potentially be given to women of childbearing potential, and since it has controversially been suggested that lithium therapy might be linked to congenital cardiac defects, we asked whether GSK-3 family members are required for normal heart development in mice. We report that terminal cardiomyocyte differentiation was substantially blunted in Gsk3b–/– embryoid bodies. While GSK-3α–deficient mice were born without a cardiac phenotype, no live-born Gsk3b–/– pups were recovered. The Gsk3b–/– embryos had a double outlet RV, ventricular septal defects, and hypertrophic myopathy, with near obliteration of the ventricular cavities. The hypertrophic myopathy was caused by cardiomyocyte hyperproliferation without hypertrophy and was associated with increased expression and nuclear localization of three regulators of proliferation — GATA4, cyclin D1, and c-Myc. These studies, which we believe are the first in mammals to examine the role of GSK-3α and GSK-3β in the heart using loss-of-function approaches, implicate GSK-3β as a central regulator of embryonic cardiomyocyte proliferation and differentiation, as well as of outflow tract development. Although controversy over the teratogenic effects of lithium remains, our studies suggest that caution should be exercised in the use of newer, more potent drugs targeting GSK-3 in women of childbearing age.
Risto Kerkela, Lisa Kockeritz, Katrina MacAulay, Jibin Zhou, Bradley W. Doble, Cara Beahm, Sarah Greytak, Kathleen Woulfe, Chinmay M. Trivedi, James R. Woodgett, Jonathan A. Epstein, Thomas Force, Gordon S. Huggins
T cell responses to MHC-mismatched transplants can be mediated via direct recognition of allogeneic MHC molecules on the cells of the transplant or via recognition of allogeneic peptides presented on the surface of recipient APCs in recipient MHC molecules — a process known as indirect recognition. As CD4+CD25+ Tregs play an important role in regulating alloresponses, we investigated whether mouse Tregs specific for allogeneic MHC molecules could be generated in vitro and could promote transplantation tolerance in immunocompetent recipient mice. Tregs able to directly recognize allogeneic MHC class II molecules (dTregs) were obtained by stimulating CD4+CD25+ cells from C57BL/6 mice (H-2b) with allogeneic DCs from BALB/c mice (H-2d). To generate Tregs that indirectly recognized allogeneic MHC class II molecules, dTregs were retrovirally transduced with TCR genes conferring specificity for H-2Kd presented by H-2Ab MHC class II molecules. The dual direct and indirect allospecificity of the TCR-transduced Tregs was confirmed in vitro. In mice, TCR-transduced Tregs, but not dTregs, induced long-term survival of partially MHC-mismatched heart grafts when combined with short-term adjunctive immunosuppression. Further, although dTregs were only slightly less effective than TCR-transduced Tregs at inducing long-term survival of fully MHC-mismatched heart grafts, histologic analysis of long-surviving hearts demonstrated marked superiority of the TCR-transduced Tregs. Thus, Tregs specific for allogeneic MHC class II molecules are effective in promoting transplantation tolerance in mice, which suggests that such cells have clinical potential.
Julia Yuen-Shan Tsang, Yakup Tanriver, Shuiping Jiang, Shao-An Xue, Kulachelvy Ratnasothy, Daxin Chen, Hans J. Stauss, R. Pat Bucy, Giovanna Lombardi, Robert Lechler
Factors that promote pancreatic β cell growth and function are potential therapeutic targets for diabetes mellitus. In mice, genetic experiments suggest that signaling cascades initiated by insulin and IGFs positively regulate β cell mass and insulin secretion. Akt and S6 kinase (S6K) family members are activated as part of these signaling cascades, but how the interplay between these proteins controls β cell growth and function has not been determined. Here, we found that although transgenic mice overexpressing the constitutively active form of Akt1 under the rat insulin promoter (RIP-MyrAkt1 mice) had enlarged β cells and high plasma insulin levels, leading to improved glucose tolerance, a substantial proportion of the mice developed insulinomas later in life, which caused decreased viability. This oncogenic transformation tightly correlated with nuclear exclusion of the tumor suppressor PTEN. To address the role of the mammalian target of rapamycin (mTOR) substrate S6K1 in the MyrAkt1-mediated phenotype, we crossed RIP-MyrAkt1 and S6K1-deficient mice. The resulting mice displayed reduced insulinemia and glycemia compared with RIP-MyrAkt1 mice due to a combined effect of improved insulin secretion and insulin sensitivity. Importantly, although the increase in β cell size in RIP-MyrAkt1 mice was not affected by S6K1 deficiency, the hyperplastic transformation required S6K1. Our results therefore identify S6K1 as a critical element for MyrAkt1-induced tumor formation and suggest that it may represent a useful target for anticancer therapy downstream of mTOR.
Samira Alliouachene, Robyn L. Tuttle, Stephanie Boumard, Thomas Lapointe, Sophie Berissi, Stephane Germain, Francis Jaubert, David Tosh, Morris J. Birnbaum, Mario Pende
Naturally occurring CD4+CD25hiFoxp3+ Tregs (nTregs) are highly proliferative in blood. However, the kinetics of their accumulation and proliferation during a localized antigen-specific T cell response is currently unknown. To explore this, we used a human experimental system whereby tuberculin purified protein derivative (PPD) was injected into the skin and the local T cell response analyzed over time. The numbers of both CD4+Foxp3– (memory) and CD4+Foxp3+ (putative nTreg) T cells increased in parallel, with the 2 populations proliferating at the same relative rate. In contrast to CD4+Foxp3– T cell populations, skin CD4+Foxp3+ T cells expressed typical Treg markers (i.e., they were CD25hi, CD127lo, CD27+, and CD39+) and did not synthesize IL-2 or IFN-γ after restimulation in vitro, indicating that they were not recently activated effector cells. To determine whether CD4+Foxp3+ T cells in skin could be induced from memory CD4+ T cells, we expanded skin-derived memory CD4+ T cells in vitro and anergized them. These cells expressed high levels of CD25 and Foxp3 and suppressed the proliferation of skin-derived responder T cells to PPD challenge. Our data therefore demonstrate that memory and CD4+ Treg populations are regulated in tandem during a secondary antigenic response. Furthermore, it is possible to isolate effector CD4+ T cell populations from inflamed tissues and manipulate them to generate Tregs with the potential to suppress inflammatory responses.
Milica Vukmanovic-Stejic, Elaine Agius, Nicola Booth, Padraic J. Dunne, Katie E. Lacy, John R. Reed, Toni O. Sobande, Steven Kissane, Mike Salmon, Malcolm H. Rustin, Arne N. Akbar
B-RAF is frequently mutated in solid tumors, resulting in activation of the MEK/ERK signaling pathway and ultimately tumor cell growth and survival. MEK inhibition in these cells results in cell cycle arrest and cytostasis. Here, we have shown that MEK inhibition also triggers limited apoptosis of human tumor cell lines with B-RAF mutations and that this effect was dependent on upregulation and dephosphorylation of the proapoptotic, Bcl-2 homology 3–only (BH3-only) Bcl-2 family member Bim. However, upregulation of Bim was insufficient for extensive apoptosis and was countered by overexpression of Bcl-2. To overcome apoptotic resistance, we treated the B-RAF mutant cells both with MEK inhibitors and with the BH3 mimetic ABT-737, resulting in profound synergism and extensive tumor cell death. This treatment was successful because of both efficient antagonism of the prosurvival Bcl-2 family member Mcl-1 by Bim and inhibition of Bcl-2 and Bcl-xL by ABT-737. Critically, addition of ABT-737 converted the predominantly cytostatic effect of MEK inhibition to a cytotoxic effect, causing long-term tumor regression in mice xenografted with human tumor cell lines. Thus, the therapeutic efficacy of MEK inhibition requires concurrent unleashing of apoptosis by a BH3 mimetic and represents a potent combination treatment for tumors harboring B-RAF mutations.
Mark S. Cragg, Elisa S. Jansen, Michele Cook, Claire Harris, Andreas Strasser, Clare L. Scott
Melanomas are highly aggressive neoplasms resistant to most conventional therapies. These tumors result from the interaction of altered intracellular tumor suppressors and oncogenes with the microenvironment in which these changes occur. We previously demonstrated that physiologic skin hypoxia contributes to melanomagenesis in conjunction with Akt activation. Here we show that Notch1 signaling is elevated in human melanoma samples and cell lines and is required for Akt and hypoxia to transform melanocytes in vitro. Notch1 facilitated melanoma development in a xenograft model by maintaining cell proliferation and by protecting cells from stress-induced cell death. Hyperactivated PI3K/Akt signaling led to upregulation of Notch1 through NF-κB activity, while the low oxygen content normally found in skin increased mRNA and protein levels of Notch1 via stabilization of HIF-1α. Taken together, these findings demonstrate that Notch1 is a key effector of both Akt and hypoxia in melanoma development and identify the Notch signaling pathway as a potential therapeutic target in melanoma treatment.
Barbara Bedogni, James A. Warneke, Brian J. Nickoloff, Amato J. Giaccia, Marianne Broome Powell
Egg activation, which is the first step in the initiation of embryo development, involves both completion of meiosis and progression into mitotic cycles. In mammals, the fertilizing sperm delivers the activating signal, which consists of oscillations in free cytosolic Ca2+ concentration ([Ca2+]i). Intracytoplasmic sperm injection (ICSI) is a technique that in vitro fertilization clinics use to treat a myriad of male factor infertility cases. Importantly, some patients who repeatedly fail ICSI also fail to induce egg activation and are, therefore, sterile. Here, we have found that sperm from patients who repeatedly failed ICSI were unable to induce [Ca2+]i oscillations in mouse eggs. We have also shown that PLC, zeta 1 (PLCZ1), the sperm protein thought to induce [Ca2+]i oscillations, was localized to the equatorial region of wild-type sperm heads but was undetectable in sperm from patients who had failed ICSI. The absence of PLCZ1 in these patients was further confirmed by Western blot, although genomic sequencing failed to reveal conclusive PLCZ1 mutations. Using mouse eggs, we reproduced the failure of sperm from these patients to induce egg activation and rescued it by injection of mouse Plcz1 mRNA. Together, our results indicate that the inability of human sperm to initiate [Ca2+]i oscillations leads to failure of egg activation and sterility and that abnormal PLCZ1 expression underlies this functional defect.
Sook-Young Yoon, Teru Jellerette, Ana Maria Salicioni, Hoi Chang Lee, Myung-sik Yoo, Kevin Coward, John Parrington, Daniel Grow, Jose B. Cibelli, Pablo E. Visconti, Jesse Mager, Rafael A. Fissore
Mucosal diseases are often characterized by an inflammatory infiltrate that includes polymorphonuclear leukocytes (PMNs), monocytes, lymphocytes, and platelets. A number of studies have suggested that the interaction of platelets with leukocytes has an essential proinflammatory role. Here, we examined whether platelets migrate across mucosal epithelium, as PMNs are known to do, and whether platelets influence epithelial cell function. Initial studies revealed that human platelets did not efficiently transmigrate across human epithelial cell monolayers. However, in the presence of human PMNs, platelet movement across the epithelium was proportional to the extent of PMN transmigration, and strategies that blocked PMN transmigration diminished platelet movement. Furthermore, platelet-PMN comigration was observed in intestinal tissue derived from human patients with inflammatory bowel disease (IBD). The translocated platelets were found to release large quantities of ATP, which was metabolized to adenosine via a 2-step enzymatic reaction mediated by ecto-nucleotidases, including CD73 and ecto–nucleoside triphosphate diphosphohydrolases (ecto-NTPDases), expressed on the apical membrane of the intestinal epithelial cells. In vitro studies and a mouse model of intestinal inflammation were employed to define a mechanism involving adenosine-mediated induction of electrogenic chloride secretion, with concomitant water movement into the intestinal lumen. These studies demonstrate that ecto-NTPDases are expressed on the apical membrane of epithelial cells and are involved in what we believe to be a previously unappreciated function for platelets in the inflamed intestine, which might promote bacterial clearance under inflammatory conditions.
Thomas Weissmüller, Eric L. Campbell, Peter Rosenberger, Melanie Scully, Paul L. Beck, Glenn T. Furuta, Sean P. Colgan
Bitter taste–sensing G protein–coupled receptors (type 2 taste receptors [T2Rs]) are expressed in taste receptor cells of the tongue, where they play an important role in limiting ingestion of bitter-tasting, potentially toxic compounds. T2Rs are also expressed in gut-derived enteroendocrine cells, where they have also been hypothesized to play a role in limiting toxin absorption. In this study, we have shown that T2R gene expression in both cultured mouse enteroendocrine cells and mouse intestine is regulated by the cholesterol-sensitive SREBP-2. In addition, T2R stimulation of cholecystokinin (CCK) secretion was enhanced directly by SREBP-2 in cultured cells and in mice fed chow supplemented with lovastatin and ezetimibe (L/E) to decrease dietary sterol absorption and increase nuclear activity of SREBP-2. Low-cholesterol diets are naturally composed of high amounts of plant matter that is likely to contain dietary toxins, and CCK is known to improve dietary absorption of fats, slow gastric emptying, and decrease food intake. Thus, these studies suggest that SREBP-2 activation of bitter signaling receptors in the intestine may sensitize the gut to a low-fat diet and to potential accompanying food-borne toxins that make it past the initial aversive response in the mouth.
Tae-Il Jeon, Bing Zhu, Jarrod L. Larson, Timothy F. Osborne
Plasma HDL levels are inversely related to the incidence of atherosclerotic disease. Some of the atheroprotective effects of HDL are likely mediated via preservation of EC function. Whether the beneficial effects of HDL on ECs depend on its involvement in cholesterol efflux via the ATP-binding cassette transporters ABCA1 and ABCG1, which promote efflux of cholesterol and oxysterols from macrophages, has not been investigated. To address this, we assessed endothelial function in Abca1–/–, Abcg1–/–, and Abca1–/–Abcg1–/– mice fed either a high-cholesterol diet (HCD) or a Western diet (WTD). Non-atherosclerotic arteries from WTD-fed Abcg1–/– and Abca1–/–Abcg1–/– mice exhibited a marked decrease in endothelium-dependent vasorelaxation, while Abca1–/– mice had a milder defect. In addition, eNOS activity was reduced in aortic homogenates generated from Abcg1–/– mice fed either a HCD or a WTD, and this correlated with decreased levels of the active dimeric form of eNOS. More detailed analysis indicated that ABCG1 was expressed primarily in ECs, and that these cells accumulated the oxysterol 7-ketocholesterol (7-KC) when Abcg1–/– mice were fed a WTD. Consistent with these data, ABCG1 had a major role in promoting efflux of cholesterol and 7-KC in cultured human aortic ECs (HAECs). Furthermore, HDL treatment of HAECs prevented 7-KC–induced ROS production and active eNOS dimer disruption in an ABCG1-dependent manner. Our data suggest that ABCG1 and HDL maintain EC function in HCD-fed mice by promoting efflux of cholesterol and 7-oxysterols and preserving active eNOS dimer levels.
Naoki Terasaka, Shuiqing Yu, Laurent Yvan-Charvet, Nan Wang, Nino Mzhavia, Read Langlois, Tamara Pagler, Rong Li, Carrie L. Welch, Ira J. Goldberg, Alan R. Tall
Hyperproliferation of bile duct epithelial cells due to cell-cycle dysregulation is a key feature of cystogenesis in polycystic liver diseases (PCLDs). Recent evidence suggests a regulatory role for microRNAs (miRNAs) in a variety of biological processes, including cell proliferation. We therefore hypothesized that miRNAs may be involved in the regulation of selected components of the cell cycle and might contribute to hepatic cystogenesis. We found that the cholangiocyte cell line PCK-CCL, which is derived from the PCK rat, a model of autosomal recessive polycystic kidney disease (ARPKD), displayed global changes in miRNA expression compared with normal rat cholangiocytes (NRCs). More specific analysis revealed decreased levels of 1 miRNA, miR15a, both in PCK-CCL cells and in liver tissue from PCK rats and patients with a PCLD. The decrease in miR15a expression was associated with upregulation of its target, the cell-cycle regulator cell division cycle 25A (Cdc25A). Overexpression of miR15a in PCK-CCL cells decreased Cdc25A levels, inhibited cell proliferation, and reduced cyst growth. In contrast, suppression of miR15a in NRCs accelerated cell proliferation, increased Cdc25A expression, and promoted cyst growth. Taken together, these results suggest that suppression of miR15a contributes to hepatic cystogenesis through dysregulation of Cdc25A.
Seung-Ok Lee, Tatyana Masyuk, Patrick Splinter, Jesús M. Banales, Anatoliy Masyuk, Angela Stroope, Nicholas LaRusso
Mucin-type O-glycans (O-glycans) are highly expressed in vascular ECs. However, it is not known whether they are important for vascular development. To investigate the roles of EC O-glycans, we generated mice lacking T-synthase, a glycosyltransferase encoded by the gene C1galt1 that is critical for the biosynthesis of core 1–derived O-glycans, in ECs and hematopoietic cells (termed here EHC T-syn–/– mice). EHC T-syn–/– mice exhibited embryonic and neonatal lethality associated with disorganized and blood-filled lymphatic vessels. Bone marrow transplantation and EC C1galt1 transgene rescue demonstrated that lymphangiogenesis specifically requires EC O-glycans, and intestinal lymphatic microvessels in EHC T-syn–/– mice expressed a mosaic of blood and lymphatic EC markers. The level of O-glycoprotein podoplanin was significantly reduced in EHC T-syn–/– lymphatics, and podoplanin-deficient mice developed blood-filled lymphatics resembling EHC T-syn–/– defects. In addition, postnatal inactivation of C1galt1 caused blood/lymphatic vessel misconnections that were similar to the vascular defects in the EHC T-syn–/– mice. One consequence of eliminating T-synthase in ECs and hematopoietic cells was that the EHC T-syn–/– pups developed fatty liver disease, because of direct chylomicron deposition via misconnected portal vein and intestinal lymphatic systems. Our studies therefore demonstrate that EC O-glycans control the separation of blood and lymphatic vessels during embryonic and postnatal development, in part by regulating podoplanin expression.
Jianxin Fu, Holger Gerhardt, J. Michael McDaniel, Baoyun Xia, Xiaowei Liu, Lacramioara Ivanciu, Annelii Ny, Karlien Hermans, Robert Silasi-Mansat, Samuel McGee, Emma Nye, Tongzhong Ju, Maria I. Ramirez, Peter Carmeliet, Richard D. Cummings, Florea Lupu, Lijun Xia
FcγRIV is a recently identified mouse activating receptor for IgG2a and IgG2b that is expressed on monocytes, macrophages, and neutrophils; herein it is referred to as mFcγRIV. Although little is known about mFcγRIV, it has been proposed to be the mouse homolog of human FcγRIIIA (hFcγRIIIA) because of high sequence homology. Our work, however, has revealed what we believe to be new properties of mFcγRIV that endow this receptor with a previously unsuspected biological significance; we have shown that it is a low-affinity IgE receptor for all IgE allotypes. Although mFcγRIV functioned as a high-affinity IgG receptor, mFcγRIV-bound monomeric IgGs were readily displaced by IgE immune complexes. Engagement of mFcγRIV by IgE immune complexes induced bronchoalveolar and peritoneal macrophages to secrete cytokines, suggesting that mFcγRIV may be an equivalent of human FceRI(αγ), which is expressed by macrophages and neutrophils and especially in atopic individuals, rather than an equivalent of hFcγRIIIA, which has no affinity for IgE. Using mice lacking 3 FcγRs and 2 FceRs and expressing mFcγRIV only, we further demonstrated that mFcγRIV promotes IgE-induced lung inflammation. These data lead us to propose a mouse model of IgE-induced lung inflammation in which cooperation exists between mast cells and mFcγRIV-expressing lung cells. We therefore suggest that a similar cooperation may occur between mast cells and hFceRI-expressing lung cells in human allergic asthma.
David A. Mancardi, Bruno Iannascoli, Sylviane Hoos, Patrick England, Marc Daëron, Pierre Bruhns
Tumors that progress do so via their ability to escape the antitumor immune response through several mechanisms, including developing ways to induce the differentiation and/or recruitment of CD4+CD25+ Tregs. The Tregs, in turn, inhibit the cytotoxic function of T cells and NK cells, but whether they have an effect on the cytotoxic function of tumor-infiltrating DCs (TIDCs) has not been determined. Here we have shown, in 2 rodent models of colon cancer, that CD4+CD25+ Tregs inhibit the ability of CD11b+ TIDCs to mediate TNF-related apoptosis-inducing ligand–induced (TRAIL-induced) tumor cell death. In both models of cancer, combination treatment with Mycobacterium bovis Bacillus Calmette-Guérin (BCG), which activates the innate immune system via TLR2, TLR4, and TLR9, and cyclophosphamide (CTX), which depletes Tregs, eradicated the tumors. Further analysis revealed that the treatment led to a marked increase in the number of CD11b+ TIDCs that killed the tumor cells via a TRAIL-dependent mechanism. Furthermore, acquisition of TRAIL expression by the CD11b+ TIDCs was induced by BCG and dependent on signaling through TLR2, TLR4, and TLR9. In vivo transfer of Tregs abrogated the ability of BCG to induce CD11b+ TIDCs to express TRAIL and thereby nullified the efficacy of the CTX-BCG treatment. Our data have therefore delineated what we believe to be a novel mechanism by which Tregs inhibit the antitumor immune response.
Stephan Roux, Lionel Apetoh, Fanny Chalmin, Sylvain Ladoire, Grégoire Mignot, Pierre-Emmanuel Puig, Gregoire Lauvau, Laurence Zitvogel, François Martin, Bruno Chauffert, Hideo Yagita, Eric Solary, François Ghiringhelli
Mutations in the phosphatase and tensin homolog (PTEN) gene leading to PTEN protein deletion and subsequent activation of the PI3K/Akt signaling pathway are common in cancer. Here we show that PTEN inactivation in human T cell acute lymphoblastic leukemia (T-ALL) cells is not always synonymous with PTEN gene lesions and diminished protein expression. Samples taken from patients with T-ALL at the time of diagnosis very frequently showed constitutive hyperactivation of the PI3K/Akt pathway. In contrast to immortalized cell lines, most primary T-ALL cells did not harbor PTEN gene alterations, displayed normal PTEN mRNA levels, and expressed higher PTEN protein levels than normal T cell precursors. However, PTEN overexpression was associated with decreased PTEN lipid phosphatase activity, resulting from casein kinase 2 (CK2) overexpression and hyperactivation. In addition, T-ALL cells had constitutively high levels of ROS, which can also downmodulate PTEN activity. Accordingly, both CK2 inhibitors and ROS scavengers restored PTEN activity and impaired PI3K/Akt signaling in T-ALL cells. Strikingly, inhibition of PI3K and/or CK2 promoted T-ALL cell death without affecting normal T cell precursors. Overall, our data indicate that T-ALL cells inactivate PTEN mostly in a nondeletional, posttranslational manner. Pharmacological manipulation of these mechanisms may open new avenues for T-ALL treatment.
Ana Silva, J. Andrés Yunes, Bruno A. Cardoso, Leila R. Martins, Patrícia Y. Jotta, Miguel Abecasis, Alexandre E. Nowill, Nick R. Leslie, Angelo A. Cardoso, Joao T. Barata
Osteoporosis results from an imbalance in skeletal remodeling that favors bone resorption over bone formation. Bone matrix is degraded by osteoclasts, which differentiate from myeloid precursors in response to the cytokine RANKL. To gain insight into the transcriptional regulation of bone resorption during growth and disease, we generated a conditional knockout of the transcription factor nuclear factor of activated T cells c1 (Nfatc1). Deletion of Nfatc1 in young mice resulted in osteopetrosis and inhibition of osteoclastogenesis in vivo and in vitro. Transcriptional profiling revealed NFATc1 as a master regulator of the osteoclast transcriptome, promoting the expression of numerous genes needed for bone resorption. In addition, NFATc1 directly repressed osteoclast progenitor expression of osteoprotegerin, a decoy receptor for RANKL previously thought to be an osteoblast-derived inhibitor of bone resorption. “Cherubism mice”, which carry a gain-of-function mutation in SH3-domain binding protein 2 (Sh3bp2), develop osteoporosis and widespread inflammation dependent on the proinflammatory cytokine, TNF-α. Interestingly, deletion of Nfatc1 protected cherubism mice from systemic bone loss but did not inhibit inflammation. Taken together, our study demonstrates that NFATc1 is required for remodeling of the growing and adult skeleton and suggests that NFATc1 may be an effective therapeutic target for osteoporosis associated with inflammatory states.
Antonios O. Aliprantis, Yasuyoshi Ueki, Rosalyn Sulyanto, Arnold Park, Kirsten S. Sigrist, Sudarshana M. Sharma, Michael C. Ostrowski, Bjorn R. Olsen, Laurie H. Glimcher
The pancreatic islets of Langerhans are highly vascularized micro-organs that play a key role in the regulation of blood glucose homeostasis. The specific arrangement of endocrine cell types in islets suggests a coupling between morphology and function within the islet. Here, we established a line-scanning confocal microscopy approach to examine the relationship between blood flow and islet cell type arrangement by real-time in vivo imaging of intra-islet blood flow in mice. These data were used to reconstruct the in vivo 3D architecture of the islet and time-resolved blood flow patterns throughout the islet vascular bed. The results revealed 2 predominant blood flow patterns in mouse islets: inner-to-outer, in which blood perfuses the core of β cells before the islet perimeter of non–β cells, and top-to-bottom, in which blood perfuses the islet from one side to the other regardless of cell type. Our approach included both millisecond temporal resolution and submicron spatial resolution, allowing for real-time imaging of islet blood flow within the living mouse, which has not to our knowledge been attainable by other methods.
Lara R. Nyman, K. Sam Wells, W. Steve Head, Michael McCaughey, Eric Ford, Marcela Brissova, David W. Piston, Alvin C. Powers
Christopher B. Little, Clare T. Meeker, Suzanne B. Golub, Kate E. Lawlor, Pamela J. Farmer, Susan M. Smith, Amanda J. Fosang
Morayma Reyes, Arkadiusz Dudek, Balkrishna Jahagirdar, Lisa Koodie, Paul H. Marker, Catherine M. Verfaillie
Mohit Kapoor, Shangxi Liu, Xu Shi-wen, Kun Huh, Matthew McCann, Christopher P. Denton, James R. Woodgett, David J. Abraham, Andrew Leask