Research Article
Abstract
The B cell lymphoma 2 (Bcl-2) family member Bcl-xL has a well-characterized antiapoptotic function in lymphoid cells. However, its functions in other cells — including osteoclasts, which are of hematopoietic origin — and other cellular processes remain unknown. Here we report an unexpected function of Bcl-xL in attenuating the bone-resorbing activity of osteoclasts in mice. To investigate the role of Bcl-xL in osteoclasts, we generated mice with osteoclast-specific conditional deletion of Bcl-x (referred to herein as Bcl-x cKO mice) by mating Bcl-xfl/fl mice with mice in which the gene encoding the Cre recombinase has been knocked into the cathepsin K locus and specifically expressed in mature osteoclasts. Although the Bcl-x cKO mice grew normally with no apparent morphological abnormalities, they developed substantial osteopenia at 1 year of age, which was caused by increased bone resorption. Bcl-x deficiency increased the bone-resorbing activity of osteoclasts despite their high susceptibility to apoptosis, whereas Bcl-xL overexpression produced the opposite effect. In addition, Bcl-x cKO osteoclasts displayed increased c-Src activity, which was linked to increased levels of vitronectin and fibronectin expression. These results suggest that Bcl-xL attenuates osteoclastic bone-resorbing activity through the decreased production of ECM proteins, such as vitronectin and fibronectin, and thus provide evidence for what we believe to be a novel cellular function of Bcl-xL.
Authors
Mitsuyasu Iwasawa, Tsuyoshi Miyazaki, Yuichi Nagase, Toru Akiyama, Yuho Kadono, Masaki Nakamura, Yasushi Oshima, Tetsuro Yasui, Takumi Matsumoto, Takashi Nakamura, Shigeaki Kato, Lothar Hennighausen, Kozo Nakamura, Sakae Tanaka
Abstract
Viruses that infect T cells, including those of the lentivirus genus, such as HIV-1, modulate the responsiveness of infected T cells to stimulation by interacting APCs in a manner that renders the T cells more permissive for viral replication. HIV-1 and other primate lentiviruses use their Nef proteins to manipulate the T cell/APC contact zone, the immunological synapse (IS). It is known that primate lentiviral Nef proteins differ substantially in their ability to modulate cell surface expression of the TCR-CD3 and CD28 receptors critical for the formation and function of the IS. However, the impact of these differences in Nef function on the interaction and communication between virally infected T cells and primary APCs has not been investigated. Here we have used primary human cells to show that Nef proteins encoded by HIV-2 and most SIVs, which downmodulate cell surface expression of TCR-CD3, disrupt formation of the IS between infected T cells and Ag-presenting macrophages or DCs. In contrast, nef alleles from HIV-1 and its simian precursor SIVcpz failed to suppress synapse formation and events downstream of TCR signaling. Our data suggest that most primate lentiviruses disrupt communication between virally infected CD4+ Th cells and APCs, whereas HIV-1 and its SIV precursor have largely lost this capability. The resulting differences in the levels of T cell activation and apoptosis may play a role in the pathogenesis of AIDS.
Authors
Nathalie Arhel, Martin Lehmann, Karen Clauß, G. Ulrich Nienhaus, Vincent Piguet, Frank Kirchhoff
Abstract
X-linked lymphoproliferative disease (XLP) is a rare congenital immunodeficiency that leads to an extreme, usually fatal increase in the number of lymphocytes upon infection with EBV. It is most commonly defined molecularly by loss of expression of SLAM-associated protein (SAP). Despite this, there is little understanding of how SAP deficiency causes lymphocytosis following EBV infection. Here we show that T cells from individuals with XLP are specifically resistant to apoptosis mediated by TCR restimulation, a process that normally constrains T cell expansion during immune responses. Expression of SAP and the SLAM family receptor NK, T, and B cell antigen (NTB-A) were required for TCR-induced upregulation of key pro-apoptotic molecules and subsequent apoptosis. Further, SAP/NTB-A signaling augmented the strength of the proximal TCR signal to achieve the threshold required for restimulation-induced cell death (RICD). Strikingly, TCR ligation in activated T cells triggered increased recruitment of SAP to NTB-A, dissociation of the phosphatase SHP-1, and colocalization of NTB-A with CD3 aggregates. In contrast, NTB-A and SHP-1 contributed to RICD resistance in XLP T cells. Our results reveal what we believe to be novel roles for NTB-A and SAP in regulating T cell homeostasis through apoptosis and provide mechanistic insight into the pathogenesis of lymphoproliferative disease in XLP.
Authors
Andrew L. Snow, Rebecca A. Marsh, Scott M. Krummey, Philip Roehrs, Lisa R. Young, Kejian Zhang, Jack van Hoff, Deepali Dhar, Kim E. Nichols, Alexandra H. Filipovich, Helen C. Su, Jack J. Bleesing, Michael J. Lenardo
Abstract
Various acute and chronic inflammatory stimuli increase the number and activity of pulmonary mucus-producing goblet cells, and goblet cell hyperplasia and excess mucus production are central to the pathogenesis of chronic pulmonary diseases. However, little is known about the transcriptional programs that regulate goblet cell differentiation. Here, we show that SAM-pointed domain–containing Ets-like factor (SPDEF) controls a transcriptional program critical for pulmonary goblet cell differentiation in mice. Initial cell-lineage–tracing analysis identified nonciliated secretory epithelial cells, known as Clara cells, as the progenitors of goblet cells induced by pulmonary allergen exposure in vivo. Furthermore, in vivo expression of SPDEF in Clara cells caused rapid and reversible goblet cell differentiation in the absence of cell proliferation. This was associated with enhanced expression of genes regulating goblet cell differentiation and protein glycosylation, including forkhead box A3 (Foxa3), anterior gradient 2 (Agr2), and glucosaminyl (N-acetyl) transferase 3, mucin type (Gcnt3). Consistent with these findings, levels of SPDEF and FOXA3 were increased in mouse goblet cells after sensitization with pulmonary allergen, and the proteins were colocalized in goblet cells lining the airways of patients with chronic lung diseases. Deletion of the mouse Spdef gene resulted in the absence of goblet cells in tracheal/laryngeal submucosal glands and in the conducting airway epithelium after pulmonary allergen exposure in vivo. These data show that SPDEF plays a critical role in regulating a transcriptional network mediating the goblet cell differentiation and mucus hyperproduction associated with chronic pulmonary disorders.
Authors
Gang Chen, Thomas R. Korfhagen, Yan Xu, Joseph Kitzmiller, Susan E. Wert, Yutaka Maeda, Alexander Gregorieff, Hans Clevers, Jeffrey A. Whitsett
Abstract
Muscle wasting is associated with a number of pathophysiologic conditions, including metabolic acidosis, diabetes, sepsis, and high angiotensin II levels. Under these conditions, activation of muscle protein degradation requires endogenous glucocorticoids. As the mechanism(s) underlying this dependence on glucocorticoids have not been identified, we analyzed the effects of glucocorticoids on muscle wasting in a mouse model of acute diabetes. Adrenalectomized, acutely diabetic mice given a physiologic dose of glucocorticoids exhibited decreased IRS-1–associated PI3K activity in muscle and progressive muscle atrophy. These responses were related to increased association of PI3K with the glucocorticoid receptor (GR). In mice with muscle-specific GR deletion (referred to as MGRKO mice), acute diabetes minimally suppressed IRS-1–associated PI3K activity in muscle and did not cause muscle atrophy. However, when a physiologic dose of glucocorticoids was given to mice with muscle-specific IR deletion, muscle protein degradation was accelerated. Fluorescence resonance energy transfer and an in vitro competition assay revealed that activated GRs competed for PI3K, reducing its association with IRS-1. Reexpression of WT GRs or those with a mutation in the nuclear localization signal in the muscle of MGRKO mice indicated that competition for PI3K was a prominent mechanism underlying reduced IRS-1–associated PI3K activity. This nongenomic influence of the GR contributes to activation of muscle protein degradation. We therefore conclude that stimulation of muscle proteolysis requires 2 events, increased glucocorticoid levels and impaired insulin signaling.
Authors
Zhaoyong Hu, Huiling Wang, In Hee Lee, Jie Du, William E. Mitch
Abstract
Inflammation is associated with blood vessel and lymphatic vessel proliferation and remodeling. The microvasculature of the mouse trachea provides an ideal opportunity to study this process, as Mycoplasma pulmonis infection of mouse airways induces widespread and sustained vessel remodeling, including enlargement of capillaries into venules and lymphangiogenesis. Although the mediators responsible for these vascular changes in mice have not been identified, VEGF-A is known not to be involved. Here, we sought to determine whether TNF-α drives the changes in blood vessels and lymphatics in M. pulmonis–infected mice. The endothelial cells, but not pericytes, of blood vessels, but not lymphatics, were immunoreactive for TNF receptor 1 (TNF-R1) and lymphotoxin B receptors. Most TNF-R2 immunoreactivity was on leukocytes. Infection resulted in a large and sustained increase in TNF-α expression, as measured by real-time quantitative RT-PCR, and smaller increases in lymphotoxins and TNF receptors that preceded vessel remodeling. Substantially less vessel remodeling and lymphangiogenesis occurred when TNF-α signaling was inhibited by a blocking antibody or was silenced in Tnfr1–/– mice. When administered after infection was established, the TNF-α–specific antibody slowed but did not reverse blood vessel remodeling and lymphangiogenesis. The action of TNF-α on blood vessels is probably mediated through direct effects on endothelial cells, but its effects on lymphangiogenesis may require inflammatory mediators from recruited leukocytes. We conclude that TNF-α is a strong candidate for a mediator that drives blood vessel remodeling and lymphangiogenesis in inflammation.
Authors
Peter Baluk, Li-Chin Yao, Jennifer Feng, Talia Romano, Sonia S. Jung, Jessica L. Schreiter, Li Yan, David J. Shealy, Donald M. McDonald
Abstract
New World monkeys of the genus Aotus synthesize a fusion protein (AoT5Cyp) containing tripartite motif-containing 5 (TRIM5) and cyclophilin A (CypA) that potently blocks HIV-1 infection. We attempted to generate a human HIV-1 inhibitor modeled after AoT5Cyp, by fusing human CypA to human TRIM5 (hT5Cyp). Of 13 constructs, 3 showed substantial HIV-1–inhibitory activity when expressed in human cell lines. This activity required capsid binding by CypA and correlated with CypA linkage to the TRIM5a capsid-specificity determinant and the ability to form cytoplasmic bodies. CXCR4- and CCR5-tropic HIV-1 clones and primary isolates were inhibited from infecting multiple human macrophage and T cell lines and primary cells by hT5Cyp, as were HIV-2ROD, SIVAGMtan, FIVPET, and a circulating HIV-1 isolate previously reported to be AoT5Cyp resistant. The anti–HIV-1 activity of hT5Cyp was surprisingly more effective than that of the well-characterized rhesus TRIM5α, especially in T cells. hT5Cyp also blocked HIV-1 infection of primary CD4+ T cells and macrophages and conferred a survival advantage to these cells without disrupting their function. Extensive attempts to elicit HIV-1 resistance to hT5Cyp were unsuccessful. Finally, Rag2–/–γc–/– mice were engrafted with human CD4+ T cells that had been transduced by optimized lentiviral vectors bearing hT5Cyp. Upon challenge with HIV-1, these mice showed decreased viremia and productive infection in lymphoid organs and preserved numbers of human CD4+ T cells. We conclude that hT5Cyp is an extraordinarily robust inhibitor of HIV-1 replication and a promising anti–HIV-1 gene therapy candidate.
Authors
Martha R. Neagu, Patrick Ziegler, Thomas Pertel, Caterina Strambio-De-Castillia, Christian Grütter, Gladys Martinetti, Luca Mazzucchelli, Markus Grütter, Markus G. Manz, Jeremy Luban
Abstract
To improve contractile function, the myocardium undergoes hypertrophic growth without myocyte proliferation in response to both pathologic and physiologic stimulation. Various membrane-bound receptors and intermediate signal transduction pathways regulate the induction of cardiac hypertrophy, but the cardioprotective regulatory pathways or effectors that antagonize cardiac hypertrophy remain poorly understood. Here we identify the small GTPase Cdc42 as a signaling intermediate that restrained the cardiac growth response to physiologic and pathologic stimuli. Cdc42 was specifically activated in the heart after pressure overload and in cultured cardiomyocytes by multiple agonists. Mice with a heart-specific deletion of Cdc42 developed greater cardiac hypertrophy at 2 and 8 weeks of stimulation and transitioned more quickly into heart failure than did wild-type controls. These mice also displayed greater cardiac hypertrophy in response to neuroendocrine agonist infusion for 2 weeks and, more remarkably, enhanced exercise-induced hypertrophy and sudden death. These pathologies were associated with an inability to activate JNK following stimulation through a MEKK1/MKK4/MKK7 pathway, resulting in greater cardiac nuclear factor of activated T cells (NFAT) activity. Restoration of cardiac JNK signaling with an Mkk7 heart-specific transgene reversed the enhanced growth effect. These results identify what we believe to be a novel antihypertrophic and protective cardiac signaling pathway, whereby Cdc42-dependent JNK activation antagonizes calcineurin-NFAT activity to reduce hypertrophy and prevent transition to heart failure.
Authors
Marjorie Maillet, Jeffrey M. Lynch, Bastiano Sanna, Allen J. York, Yi Zheng, Jeffery D. Molkentin
Abstract
Cytokines orchestrate the tumor-promoting interplay between malignant cells and the immune system. In many experimental and human cancers, the cytokine TNF-α is an important component of this interplay, but its effects are pleiotropic and therefore remain to be completely defined. Using a mouse model of ovarian cancer in which either TNF receptor 1 (TNFR1) signaling was manipulated in different leukocyte populations or TNF-α was neutralized by antibody treatment, we found that this inflammatory cytokine maintained TNFR1-dependent IL-17 production by CD4+ cells and that this led to myeloid cell recruitment into the tumor microenvironment and enhanced tumor growth. Consistent with this, in patients with advanced cancer, treatment with the TNF-α–specific antibody infliximab substantially reduced plasma IL-17 levels. Furthermore, expression of IL-1R and IL-23R was downregulated in CD4+CD25– cells isolated from ascites of ovarian cancer patients treated with infliximab. We have also shown that genes ascribed to the Th17 pathway map closely with the TNF-α signaling pathway in ovarian cancer biopsy samples, showing particularly high levels of expression of genes encoding IL-23, components of the NF-κB system, TGF-β1, and proteins involved in neutrophil activation. We conclude that chronic production of TNF-α in the tumor microenvironment increases myeloid cell recruitment in an IL-17–dependent manner that contributes to the tumor-promoting action of this proinflammatory cytokine.
Authors
Kellie A. Charles, Hagen Kulbe, Robin Soper, Monica Escorcio-Correia, Toby Lawrence, Anne Schultheis, Probir Chakravarty, Richard G. Thompson, George Kollias, John F. Smyth, Frances R. Balkwill, Thorsten Hagemann
Abstract
ER stress–induced apoptosis is implicated in various pathological conditions, but the mechanisms linking ER stress–mediated signaling to downstream apoptotic pathways remain unclear. Using human and mouse cell culture and in vivo mouse models of ER stress–induced apoptosis, we have shown that cytosolic calcium resulting from ER stress induces expression of the Fas death receptor through a pathway involving calcium/calmodulin-dependent protein kinase IIγ (CaMKIIγ) and JNK. Remarkably, CaMKIIγ was also responsible for processes involved in mitochondrial-dependent apoptosis, including release of mitochondrial cytochrome c and loss of mitochondrial membrane potential. CaMKII-dependent apoptosis was also observed in a number of cultured human and mouse cells relevant to ER stress–induced pathology, including cultured macrophages, endothelial cells, and neuronal cells subjected to proapoptotic ER stress. Moreover, WT mice subjected to systemic ER stress showed evidence of macrophage mitochondrial dysfunction and apoptosis, renal epithelial cell apoptosis, and renal dysfunction, and these effects were markedly reduced in CaMKIIγ-deficient mice. These data support an integrated model in which CaMKII serves as a unifying link between ER stress and the Fas and mitochondrial apoptotic pathways. Our study also revealed what we believe to be a novel proapoptotic function for CaMKII, namely, promotion of mitochondrial calcium uptake. These findings raise the possibility that CaMKII inhibitors could be useful in preventing apoptosis in pathological settings involving ER stress–induced apoptosis.
Authors
Jenelle M. Timmins, Lale Ozcan, Tracie A. Seimon, Gang Li, Cristina Malagelada, Johannes Backs, Thea Backs, Rhonda Bassel-Duby, Eric N. Olson, Mark E. Anderson, Ira Tabas
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