Research Article
Abstract
Glucocorticoids (GCs) are administered to human fetuses at risk of premature delivery and to infants with life-threatening respiratory and cardiac conditions. However, there are ongoing concerns about adverse effects of GC treatment on the developing human brain, although the precise molecular mechanisms underlying GC-induced brain injury are unclear. Here, we identified what we believe to be novel cross-antagonistic interactions of Sonic hedgehog (Shh) and GC signaling in proliferating mouse cerebellar granule neuron precursors (CGNPs). Chronic GC treatment (from P0 through P7) in mouse pups inhibited Shh-induced proliferation and upregulation of expression of N-myc, Gli1, and D-type cyclin protein in CGNPs. Conversely, acute GC treatment (on P7 only) caused transient apoptosis. Shh signaling antagonized these effects of GCs, in part by induction of 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2). Importantly, 11βHSD2 antagonized the effects of the GCs corticosterone, hydrocortisone, and prednisolone, but not the synthetic GC dexamethasone. Our findings indicate that Shh signaling is protective in the setting of GC-induced mouse neonatal brain injury. Furthermore, they led us to propose that 11βHSD2-sensitive GCs (e.g., hydrocortisone) should be used in preference to dexamethasone in neonatal human infants because of the potential for reduced neurotoxicity.
Authors
Vivi M. Heine, David H. Rowitch
Abstract
The bone marrow compartment is enriched in stem and progenitor cells, and an unidentified subpopulation of these cells can contribute to lung epithelial repair. Here we identify this subpopulation and quantitate its relative contribution to injured airway epithelium. A subpopulation of adherent human and murine bone marrow cells that expresses Clara cell secretory protein (CCSP) was identified using flow cytometry. When cultured at the air-liquid interface in ex vivo cultures, Ccsp+ cells expressed type I and type II alveolar markers as well as basal cell markers and active epithelial sodium channels. Ccsp+ cells preferentially homed to naphthalene-damaged airways when delivered transtracheally or intravenously, with the former being more efficient than the latter. Interestingly, naphthalene-induced lung damage transiently increased Ccsp expression in bone marrow and peripheral circulation. Furthermore, lethally irradiated Ccsp-null mice that received tagged wild-type bone marrow contained donor-derived epithelium in both normal and naphthalene-damaged airways. This study therefore identifies what we believe to be a newly discovered cell in the bone marrow that might have airway reconstitution potential in the context of cell-based therapies for lung disease. Additionally, these data could reconcile previous controversies regarding the contribution of bone marrow to lung regeneration.
Authors
Amy P. Wong, Armand Keating, Wei-Yang Lu, Pascal Duchesneau, Xinghua Wang, Adrian Sacher, Jim Hu, Thomas K. Waddell
Abstract
Platelets are central players in atherothrombosis development in coronary artery disease. The PKC family provides important intracellular mechanisms for regulating platelet activity, and platelets express several members of this family, including the classical isoforms PKCα and PKCβ and novel isoforms PKCδ and PKCθ. Here, we used a genetic approach to definitively demonstrate the role played by PKCα in regulating thrombus formation and platelet function. Thrombus formation in vivo was attenuated in Prkca–/– mice, and PKCα was required for thrombus formation in vitro, although this PKC isoform did not regulate platelet adhesion to collagen. The ablation of in vitro thrombus formation in Prkca–/– platelets was rescued by the addition of ADP, consistent with the key mechanistic finding that dense-granule biogenesis and secretion depend upon PKCα expression. Furthermore, defective platelet aggregation in response to either collagen-related peptide or thrombin could be overcome by an increase in agonist concentration. Evidence of overt bleeding, including gastrointestinal and tail bleeding, was not seen in Prkca–/– mice. In summary, the effects of PKCα ablation on thrombus formation and granule secretion may implicate PKCα as a drug target for antithrombotic therapy.
Authors
Olga Konopatskaya, Karen Gilio, Matthew T. Harper, Yan Zhao, Judith M.E.M. Cosemans, Zubair A. Karim, Sidney W. Whiteheart, Jeffery D. Molkentin, Paul Verkade, Steve P. Watson, Johan W.M. Heemskerk, Alastair W. Poole
Abstract
Cancer cells require sustained oncogenic signaling in order to maintain their malignant properties. It is, however, unclear whether they possess other dependencies that can be exploited therapeutically. We report here that in a large fraction of human breast cancers, the gene encoding focal adhesion kinase (FAK), a core component of integrin signaling, was amplified and FAK mRNA was overexpressed. A mammary gland–specific deletion of Fak in mice did not seem to affect normal mammary epithelial cells, and these mice were protected from tumors initiated by the polyoma middle T oncoprotein (PyMT), which activates Ras and PI3K. FAK-deficient PyMT-transformed cells displayed both growth arrest and apoptosis, as well as diminished invasive and metastatic capacity. Upon silencing of Fak, mouse mammary tumor cells transformed by activated Ras became senescent and lost their invasive ability. Further, Neu-transformed cells also underwent growth arrest and apoptosis if integrin β4–dependent signaling was simultaneously inactivated. Human breast cancer cells carrying oncogenic mutations that activate Ras or PI3K signaling displayed similar responses upon silencing of FAK. Mechanistic studies indicated that FAK sustains tumorigenesis by promoting Src-mediated phosphorylation of p130Cas. These results suggest that FAK supports Ras- and PI3K-dependent mammary tumor initiation, maintenance, and progression to metastasis by orchestrating multiple core cellular functions, including proliferation, survival, and avoidance of senescence.
Authors
Yuliya Pylayeva, Kelly M. Gillen, William Gerald, Hilary E. Beggs, Louis F. Reichardt, Filippo G. Giancotti
Abstract
Maternal obesity is thought to increase the offspring’s risk of juvenile obesity and metabolic diseases; however, the mechanism(s) whereby excess maternal nutrition affects fetal development remain poorly understood. Here, we investigated in nonhuman primates the effect of chronic high-fat diet (HFD) on the development of fetal metabolic systems. We found that fetal offspring from both lean and obese mothers chronically consuming a HFD had a 3-fold increase in liver triglycerides (TGs). In addition, fetal offspring from HFD-fed mothers (O-HFD) showed increased evidence of hepatic oxidative stress early in the third trimester, consistent with the development of nonalcoholic fatty liver disease (NAFLD). O-HFD animals also exhibited elevated hepatic expression of gluconeogenic enzymes and transcription factors. Furthermore, fetal glycerol levels were 2-fold higher in O-HFD animals than in control fetal offspring and correlated with maternal levels. The increased fetal hepatic TG levels persisted at P180, concurrent with a 2-fold increase in percent body fat. Importantly, reversing the maternal HFD to a low-fat diet during a subsequent pregnancy improved fetal hepatic TG levels and partially normalized gluconeogenic enzyme expression, without changing maternal body weight. These results suggest that a developing fetus is highly vulnerable to excess lipids, independent of maternal diabetes and/or obesity, and that exposure to this may increase the risk of pediatric NAFLD.
Authors
Carrie E. McCurdy, Jacalyn M. Bishop, Sarah M. Williams, Bernadette E. Grayson, M. Susan Smith, Jacob E. Friedman, Kevin L. Grove
Abstract
Provirus integration site for Moloney murine leukemia virus (PIM1) is a proto-oncogene that encodes a serine/threonine kinase with multiple cellular functions. Overexpression of PIM-1 plays a critical role in progression of prostatic and hematopoietic malignancies. Here we describe the generation of a mAb specific for GST–PIM-1, which reacted strongly with most human and mouse cancer tissues and cell lines of prostate, breast, and colon origin but only weakly (if at all) with normal tissues. The mAb binds to PIM-1 in the cytosol and nucleus as well as to PIM-1 on the surface of human and murine cancer cells. Treatment of human and mouse prostate cancer cell lines with the PIM-1–specific mAb resulted in disruption of PIM-1/Hsp90 complexes, decreased PIM-1 and Hsp90 levels, reduced Akt phosphorylation at Ser473, reduced phosphorylation of Bad at Ser112 and Ser136, and increased cleavage of caspase-9, an indicator of activation of the mitochondrial cell death pathway. The mAb induced cancer cell apoptosis and synergistically enhanced antitumor activity when used in combination with cisplatin and epirubicin. In tumor models, the PIM-1–specific mAb substantially inhibited growth of the human prostate cancer cell line DU145 in SCID mice and the mouse prostate cancer cell TRAMP-C1 in C57BL/6 mice. These findings are important because they provide what we believe to be the first in vivo evidence that treatment of prostate cancer may be possible by targeting PIM-1 using an Ab-based therapy.
Authors
Xiu Feng Hu, Jie Li, Scott Vandervalk, Zeping Wang, Nancy S. Magnuson, Pei Xiang Xing
Abstract
There is an association between expression of the MHC class I molecule HLA-B27 and protection following human infection with either HIV or HCV. In both cases, protection has been linked to HLA-B27 presentation of a single immunodominant viral peptide epitope to CD8+ T cells. If HIV mutates the HLA-B27–binding anchor of this epitope to escape the protective immune response, the result is a less-fit virus that requires additional compensatory clustered mutations. Here, we sought to determine whether the immunodominant HLA-B27–restricted HCV epitope was similarly constrained by analyzing the replication competence and immunogenicity of different escape mutants. Interestingly, in most HLA-B27–positive patients chronically infected with HCV, the escape mutations spared the HLA-B27–binding anchor. Instead, the escape mutations were clustered at other sites within the epitope and had only a modest impact on replication competence. Further analysis revealed that the cluster of mutations is required for efficient escape because a combination of mutations is needed to impair T cell recognition of the epitope. Artificially introduced mutations at the HLA-B27–binding anchors were found to be either completely cross-reactive or to lead to substantial loss of fitness. These results suggest that protection by HLA-B27 in HCV infection can be explained by the requirement to accumulate a cluster of mutations within the immunodominant epitope to escape T cell recognition.
Authors
Eva Dazert, Christoph Neumann-Haefelin, Stéphane Bressanelli, Karen Fitzmaurice, Julia Kort, Jörg Timm, Susan McKiernan, Dermot Kelleher, Norbert Gruener, John E. Tavis, Hugo R. Rosen, Jaqueline Shaw, Paul Bowness, Hubert E. Blum, Paul Klenerman, Ralf Bartenschlager, Robert Thimme
Abstract
Signaling through the TLR family of molecular pattern recognition receptors has been implicated in the induction of innate and adaptive immune responses. A role for TLR signaling in the maintenance and/or regulation of Treg function has been proposed, however its functional relevance remains unclear. Here we have shown that TLR9 is highly expressed by human Treg secreting the antiinflammatory cytokine IL-10 induced following stimulation of blood and tissue CD3+ T cells in the presence of 1α,25-dihydroxyvitamin D3 (1α25VitD3), the active form of Vitamin D, with or without the glucocorticoid dexamethasone. By contrast, TLR9 was not highly expressed by naturally occurring CD4+CD25+ Treg or by Th1 and Th2 effector cells. Induction of TLR9, but not other TLRs, was IL-10 dependent and primarily regulated by 1α25VitD3 in vitro. Furthermore, ingestion of calcitriol (1α25VitD3) by human volunteers led to an increase of both IL-10 and TLR9 expression by CD3+CD4+ T cells analyzed directly ex vivo. Stimulation of 1α25VitD3-induced IL-10–secreting Treg with TLR9 agonists, CpG oligonucleotides, resulted in decreased IL-10 and IFN-γ synthesis and a concurrent loss of regulatory function, but, unexpectedly, increased IL-4 synthesis. We therefore suggest that TLR9 could be used to monitor and potentially modulate the function of 1α25VitD3-induced IL-10–secreting Treg in vivo, and that this has implications in cancer therapy and vaccine design.
Authors
Zoë Urry, Emmanuel Xystrakis, David F. Richards, Joanne McDonald, Zahid Sattar, David J. Cousins, Christopher J. Corrigan, Emma Hickman, Zarin Brown, Catherine M. Hawrylowicz
Abstract
Pain after nerve injury, a phenomenon referred to as neuropathic pain, is a debilitating clinical condition, but the underlying mechanisms remain unclear. As leptin, an adipocytokine produced mainly by nonneuronal tissue, has been implicated in the regulation of neuronal functions, we examined the role of leptin in neuropathic pain using a rat model of the condition chronic constriction sciatic nerve injury (CCI). We report that leptin critically contributed to pain behaviors following CCI. Specifically, spinal administration of a leptin antagonist prevented and reversed neuropathic pain behaviors in rats. Further examination revealed that levels of both leptin and the long form of the leptin receptor (Ob-Rb) were substantially increased within the ipsilateral spinal cord dorsal horn after peripheral nerve injury. Mechanistic studies showed that leptin upregulated the expression of both the spinal NMDA receptor and IL-1β through the JAK/STAT pathway. Furthermore, these CCI-induced behavioral and cellular responses were diminished in leptin-deficient mice and mimicked by spinal administration of exogenous leptin in naive rats. Our findings reveal a critical role for spinal leptin in the pathogenesis of neuropathic pain and suggest what we believe to be a novel form of nonneuronal and neuronal interactions in the mechanisms of pathological pain.
Authors
Grewo Lim, Shuxing Wang, Yi Zhang, Yinghong Tian, Jianren Mao
Abstract
The analgesic effects of leukocyte-derived opioids have been exclusively demonstrated for somatic inflammatory pain, for example, the pain associated with surgery and arthritis. Neuropathic pain results from injury to nerves, is often resistant to current treatments, and can seriously impair a patient’s quality of life. Although it has been recognized that neuronal damage can involve inflammation, it is generally assumed that immune cells act predominately as generators of neuropathic pain. However, in this study we have demonstrated that leukocytes containing opioids are essential regulators of pain in a mouse model of neuropathy. About 30%–40% of immune cells that accumulated at injured nerves expressed opioid peptides such as β-endorphin, Met-enkephalin, and dynorphin A. Selective stimulation of these cells by local application of corticotropin-releasing factor led to opioid peptide–mediated activation of opioid receptors in damaged nerves. This ultimately abolished tactile allodynia, a highly debilitating heightened response to normally innocuous mechanical stimuli, which is symptomatic of neuropathy. Our findings suggest that selective targeting of opioid-containing immune cells promotes endogenous pain control and offers novel opportunities for management of painful neuropathies.
Authors
Dominika Labuz, Yvonne Schmidt, Anja Schreiter, Heike L. Rittner, Shaaban A. Mousa, Halina Machelska
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