Spleen section showing a rim of B220+ (green) B cells peripheral to the MOMA-1+ (red) macrophages. Demaria and colleagues use two new murine models to demonstrate a role for TLR8 in the regulation of TLR7 expression and prevention of spontaneous autoimmunity (page 3651).
Cathepsins were originally identified as proteases that act in the lysosome. Recent work has uncovered nontraditional roles for cathepsins in the extracellular space as well as in the cytosol and nucleus. There is strong evidence that subspecialized and compartmentalized cathepsins participate in many physiologic and pathophysiologic cellular processes, in which they can act as both digestive and regulatory proteases. In this review, we discuss the transcriptional and translational control of cathepsin expression, the regulation of intracellular sorting of cathepsins, and the structural basis of cathepsin activation and inhibition. In particular, we highlight the emerging roles of various cathepsin forms in disease, particularly those of the cardiac and renal systems.
Suppressing unwanted immune responses without compromising host immunity against pathogens is considered the holy grail of immunology. Lack of responsiveness to self-antigens is normally maintained by multiple mechanisms, including the suppressive activities of several T cell subsets. In this issue of the JCI, Jiang and colleagues define a CD8+ suppressor T cell subset in humans that recapitulates a regulatory pathway previously described in mice. These investigators further show that patients with type 1 diabetes have defects in their CD8+ suppressor T cells, thus identifying these cells as potential therapeutic targets in human disease.
Voltage-gated Na+ channels (VGSCs) are responsible for the rising phase of the action potential in excitable cells, including neurons and skeletal and cardiac myocytes. Small alterations in gating properties can lead to severe changes in cellular excitability, as evidenced by the plethora of heritable conditions attributed to mutations in VGSCs highlighting the need to better understand VGSC regulation. In this issue of the JCI, Hund et al. identify the ability of a key structural protein, βIV-spectrin, to bind and recruit Ca2+/calmodulin kinase II to the channel at a cellular location key to successful action potential initiation and propagation, where it can mediate function and excitability.
Obesity is associated with infiltration of white adipose tissue (WAT) by macrophages, which contributes to the development of insulin resistance. In this issue of the JCI, Kosteli and colleagues demonstrate that weight loss is unexpectedly also associated with rapid, albeit transient, recruitment of macrophages to WAT and that this appears to be related to lipolysis.
Osteoporosis is a common, age-related bone disease that results from an imbalance between the processes of bone formation and bone resorption, resulting in reduced bone mass and increased risk of fracture. Mesenchymal stem cells have the capacity to differentiate into osteoblastic and adipogenic lineages; recent research suggests that the switch between these two fates may be key to the decreased bone density that occurs with aging. In this issue, Nishikawa et al. demonstrate that the basic leucine-zipper transcription factor Maf (also known as c-Maf) is central to osteoblast lineage commitment. In addition, they find that increased oxidative stress — as occurs with aging — decreases Maf expression. This work advances understanding of the transcriptional regulation of cell fate decisions and may help direct the development of new therapies to fight age-related bone loss.
Excess lipid accumulation in the heart is associated with decreased cardiac function in humans and in animal models. The reasons are unclear, but this is generally believed to result from either toxic effects of intracellular lipids or excessive fatty acid oxidation (FAO). PPARγ expression is increased in the hearts of humans with metabolic syndrome, and use of PPARγ agonists is associated with heart failure. Here, mice with dilated cardiomyopathy due to cardiomyocyte PPARγ overexpression were crossed with PPARα-deficient mice. Surprisingly, this cross led to enhanced expression of several PPAR-regulated genes that mediate fatty acid (FA) uptake/oxidation and triacylglycerol (TAG) synthesis. Although FA oxidation and TAG droplet size were increased, heart function was preserved and survival improved. There was no marked decrease in cardiac levels of triglyceride or the potentially toxic lipids diacylglycerol (DAG) and ceramide. However, long-chain FA coenzyme A (LCCoA) levels were increased, and acylcarnitine content was decreased. Activation of PKCα and PKCδ, apoptosis, ROS levels, and evidence of endoplasmic reticulum stress were also reduced. Thus, partitioning of lipid to storage and oxidation can reverse cardiolipotoxicity despite increased DAG and ceramide levels, suggesting a role for other toxic intermediates such as acylcarnitines in the toxic effects of lipid accumulation in the heart.
Aging leads to the disruption of the homeostatic balance of multiple biological systems. In bone marrow multipotent mesenchymal cells undergo differentiation into various anchorage-dependent cell types, including osteoblasts and adipocytes. With age as well as with treatment of antidiabetic drugs such as thiazolidinediones, mesenchymal cells favor differentiation into adipocytes, resulting in an increased number of adipocytes and a decreased number of osteoblasts, causing osteoporosis. The mechanism behind this differentiation switch is unknown. Here we show an age-related decrease in the expression of Maf in mouse mesenchymal cells, which regulated mesenchymal cell bifurcation into osteoblasts and adipocytes by cooperating with the osteogenic transcription factor Runx2 and inhibiting the expression of the adipogenic transcription factor Pparg. The crucial role of Maf in both osteogenesis and adipogenesis was underscored by in vivo observations of delayed bone formation in perinatal Maf–/– mice and an accelerated formation of fatty marrow associated with bone loss in aged Maf+/– mice. This study identifies a transcriptional mechanism for an age-related switch in cell fate determination and may provide a molecular basis for novel therapeutic strategies against age-related bone diseases.
Obesity elicits an immune response characterized by myeloid cell recruitment to key metabolic organs, including adipose tissue. However, the response of immune cells to nonpathologic metabolic stimuli has been less well studied, and the factors that regulate the metabolic-dependent accumulation of immune cells are incompletely understood. Here we characterized the response of adipose tissue macrophages (ATMs) to weight loss and fasting in mice and identified a role for lipolysis in ATM recruitment and accumulation. We found that the immune response to weight loss was dynamic; caloric restriction of high-fat diet–fed mice led to an initial increase in ATM recruitment, whereas ATM content decreased following an extended period of weight loss. The peak in ATM number coincided with the peak in the circulating concentrations of FFA and adipose tissue lipolysis, suggesting that lipolysis drives ATM accumulation. Indeed, fasting or pharmacologically induced lipolysis rapidly increased ATM accumulation, adipose tissue chemoattractant activity, and lipid uptake by ATMs. Conversely, dietary and genetic manipulations that reduced lipolysis decreased ATM accumulation. Depletion of macrophages in adipose tissue cultures increased expression of adipose triglyceride lipase and genes regulated by FFA, and increased lipolysis. These data suggest that local lipid fluxes are central regulators of ATM recruitment and that once recruited, ATMs form lipid-laden macrophages that can buffer local increases in lipid concentration.
Brain injury after focal cerebral ischemia, the most common cause of stroke, develops from a series of pathological processes, including excitotoxicity, inflammation, and apoptosis. While NMDA receptors have been implicated in excitotoxicity, attempts to prevent ischemic brain damage by blocking NMDA receptors have been disappointing. Disruption of neuroprotective pathways may be another avenue responsible for ischemic damage, and thus preservation of neuronal survival may be important for prevention of ischemic brain injury. Here, we report that suppression of proteolytic degradation of transient receptor potential canonical 6 (TRPC6) prevented ischemic neuronal cell death in a rat model of stroke. The TRPC6 protein level in neurons was greatly reduced in ischemia via NMDA receptor–dependent calpain proteolysis of the N-terminal domain of TRPC6 at Lys16. This downregulation was specific for TRPC6 and preceded neuronal death. In a rat model of ischemia, activating TRPC6 prevented neuronal death, while blocking TRPC6 increased sensitivity to ischemia. A fusion peptide derived from the calpain cleavage site in TRPC6 inhibited degradation of TRPC6, reduced infarct size, and improved behavioral performance measures via the cAMP response element–binding protein (CREB) signaling pathway. Thus, TRPC6 proteolysis contributed to ischemic neuronal cell death, and suppression of its degradation preserved neuronal survival and prevented ischemic brain damage.
Cardiac valve formation is crucial for embryonic and adult heart function. Valve malformations constitute the most common congenital cardiac defect, but little is known about the molecular mechanisms regulating valve formation and homeostasis. Here, we show that endocardial Notch1 and myocardial Bmp2 signal integration establish a valve-forming field between 2 chamber developmental domains. Patterning occurs through the activation of endocardial epithelial-to-mesenchymal transition (EMT) exclusively in prospective valve territories. Mice with constitutive endocardial Notch1 activity ectopically express Hey1 and Heyl. They also display an activated mesenchymal gene program in ventricles and a partial (noninvasive) EMT in vitro that becomes invasive upon BMP2 treatment. Snail1, TGF-β2, or Notch1 inhibition reduces BMP2-induced ventricular transformation and invasion, whereas BMP2 treatment inhibits endothelial Gsk3β, stabilizing Snail1 and promoting invasiveness. Integration of Notch and Bmp2 signals is consistent with Notch1 signaling being attenuated after myocardial Bmp2 deletion. Notch1 activation in myocardium extends Hey1 expression to nonchamber myocardium, represses Bmp2, and impairs EMT. In contrast, Notch deletion abrogates endocardial Hey gene transcription and extends Bmp2 expression to the ventricular endocardium. This embryonic Notch1-Bmp2-Snail1 relationship may be relevant in adult valve disease, in which decreased NOTCH signaling causes valve mesenchyme cell formation, fibrosis, and calcification.
Ion channel function is fundamental to the existence of life. In metazoans, the coordinate activities of voltage-gated Na+ channels underlie cellular excitability and control neuronal communication, cardiac excitation-contraction coupling, and skeletal muscle function. However, despite decades of research and linkage of Na+ channel dysfunction with arrhythmia, epilepsy, and myotonia, little progress has been made toward understanding the fundamental processes that regulate this family of proteins. Here, we have identified βIV-spectrin as a multifunctional regulatory platform for Na+ channels in mice. We found that βIV-spectrin targeted critical structural and regulatory proteins to excitable membranes in the heart and brain. Animal models harboring mutant βIV-spectrin alleles displayed aberrant cellular excitability and whole animal physiology. Moreover, we identified a regulatory mechanism for Na+ channels, via direct phosphorylation by βIV-spectrin–targeted calcium/calmodulin-dependent kinase II (CaMKII). Collectively, our data define an unexpected but indispensable molecular platform that determines membrane excitability in the mouse heart and brain.
Mutations in sarcomere protein genes can cause hypertrophic cardiomyopathy (HCM), a disorder characterized by myocyte enlargement, fibrosis, and impaired ventricular relaxation. Here, we demonstrate that sarcomere protein gene mutations activate proliferative and profibrotic signals in non-myocyte cells to produce pathologic remodeling in HCM. Gene expression analyses of non-myocyte cells isolated from HCM mouse hearts showed increased levels of RNAs encoding cell-cycle proteins, Tgf-β, periostin, and other profibrotic proteins. Markedly increased BrdU labeling, Ki67 antigen expression, and periostin immunohistochemistry in the fibrotic regions of HCM hearts confirmed the transcriptional profiling data. Genetic ablation of periostin in HCM mice reduced but did not extinguish non-myocyte proliferation and fibrosis. In contrast, administration of Tgf-β–neutralizing antibodies abrogated non-myocyte proliferation and fibrosis. Chronic administration of the angiotensin II type 1 receptor antagonist losartan to mutation-positive, hypertrophy-negative (prehypertrophic) mice prevented the emergence of hypertrophy, non-myocyte proliferation, and fibrosis. Losartan treatment did not reverse pathologic remodeling of established HCM but did reduce non-myocyte proliferation. These data define non-myocyte activation of Tgf-β signaling as a pivotal mechanism for increased fibrosis in HCM and a potentially important factor contributing to diastolic dysfunction and heart failure. Preemptive pharmacologic inhibition of Tgf-β signals warrants study in human patients with sarcomere gene mutations.
Human parvovirus B19 (B19V) is the only human pathogenic parvovirus. It causes a wide spectrum of human diseases, including fifth disease (erythema infectiosum) in children and pure red cell aplasia in immunocompromised patients. B19V is highly erythrotropic and preferentially replicates in erythroid progenitor cells (EPCs). Current understanding of how B19V interacts with cellular factors to regulate disease progression is limited, due to a lack of permissive cell lines and animal models. Here, we employed a recently developed primary human CD36+ EPC culture system that is highly permissive for B19V infection to identify cellular factors that lead to cell cycle arrest after B19V infection. We found that B19V exploited the E2F family of transcription factors by downregulating activating E2Fs (E2F1 to E2F3a) and upregulating repressive E2Fs (E2F4 to E2F8) in the primary CD36+ EPCs. B19V nonstructural protein 1 (NS1) was a key viral factor responsible for altering E2F1–E2F5 expression, but not E2F6–E2F8 expression. Interaction between NS1 and E2F4 or E2F5 enhanced the nuclear import of these repressive E2Fs and induced stable G2 arrest. NS1-induced G2 arrest was independent of p53 activation and increased viral replication. Downstream E2F4/E2F5 targets, which are potentially involved in the progression from G2 into M phase and erythroid differentiation, were identified by microarray analysis. These findings provide new insight into the molecular pathogenesis of B19V in highly permissive erythroid progenitors.
The alternative pathway (AP) of complement activation is constitutively active and must be regulated by host proteins to prevent autologous tissue injury. Dysfunction of AP regulatory proteins has been linked to several human inflammatory disorders. Properdin is a positive regulator of AP complement activation that has been shown to extend the half-life of cell surface–bound C3 convertase C3bBb; it may also initiate AP complement activation. Here, we demonstrate a critical role for properdin in autologous tissue injury mediated by AP complement activation. We identified myeloid lineage cells as the principal source of plasma properdin by generating mice with global and tissue-specific knockout of Cfp (which encodes properdin) and by generating BM chimeric mice. Properdin deficiency rescued mice from AP complement–mediated embryonic lethality caused by deficiency of the membrane complement regulator Crry and markedly reduced disease severity in the K/BxN model of arthritis. Ab neutralization of properdin in WT mice similarly ameliorated arthritis development, whereas reconstitution of properdin-null mice with exogenous properdin restored arthritis sensitivity. These data implicate systemic properdin as a key contributor to AP complement–mediated injury and support its therapeutic targeting in complement-dependent human diseases.
Mammalian sterile 20-like kinase 1 (Mst1) is a mammalian homolog of Drosophila Hippo, the master regulator of cell death, proliferation, and organ size in flies. It is the chief component of the mammalian Hippo pathway and promotes apoptosis and inhibits compensatory cardiac hypertrophy, playing a critical role in mediating heart failure. How Mst1 is regulated, however, remains unclear. Using genetically altered mice in which expression of the tumor suppressor Ras-association domain family 1 isoform A (Rassf1A) was modulated in a cell type–specific manner, we demonstrate here that Rassf1A is an endogenous activator of Mst1 in the heart. Although the Rassf1A/Mst1 pathway promoted apoptosis in cardiomyocytes, thereby playing a detrimental role, the same pathway surprisingly inhibited fibroblast proliferation and cardiac hypertrophy through both cell-autonomous and autocrine/paracrine mechanisms, playing a protective role during pressure overload. In cardiac fibroblasts, the Rassf1A/Mst1 pathway negatively regulated TNF-α, a key mediator of hypertrophy, fibrosis, and resulting cardiac dysfunction. These results suggest that the functional consequence of activating the proapoptotic Rassf1A/Mst1 pathway during pressure overload is cell type dependent in the heart and that suppressing this mechanism in cardiac fibroblasts could be detrimental.
Retinal degeneration causes vision impairment and blindness in humans. If one day we are to harness the potential of stem cell–based cell replacement therapies to treat these conditions, it is imperative that we better understand normal retina development. Currently, the genes and mechanisms that regulate the specification of the neuroretina during vertebrate eye development remain unknown. Here, we identify sine oculis–related homeobox 3 (Six3) as a crucial player in this process in mice. In Six3 conditional–mutant mouse embryos, specification of the neuroretina was abrogated, but that of the retinal pigmented epithelium was normal. Conditional deletion of Six3 did not affect the initial development of the optic vesicle but did arrest subsequent neuroretina specification. Ectopic rostral expansion of Wnt8b expression was the major response to Six3 deletion and the leading cause for the specific lack of neuroretina, as ectopic Wnt8b expression in transgenic embryos was sufficient to suppress neuroretina specification. Using chromatin immunoprecipitation assays, we identified Six3-responsive elements in the Wnt8b locus and demonstrated that Six3 directly repressed Wnt8b expression in vivo. Our findings provide a molecular framework to the program leading to neuroretina differentiation and may be relevant for the development of novel strategies aimed at characterizing and eventually treating different abnormalities in eye formation.
JAK2 kinase inhibitors were developed for the treatment of myeloproliferative neoplasms (MPNs), following the discovery of activating JAK2 mutations in the majority of patients with MPN. However, to date JAK2 inhibitor treatment has shown limited efficacy and apparent toxicities in clinical trials. We report here that an HSP90 inhibitor, PU-H71, demonstrated efficacy in cell line and mouse models of the MPN polycythemia vera (PV) and essential thrombocytosis (ET) by disrupting JAK2 protein stability. JAK2 physically associated with both HSP90 and PU-H71 and was degraded by PU-H71 treatment in vitro and in vivo, demonstrating that JAK2 is an HSP90 chaperone client. PU-H71 treatment caused potent, dose-dependent inhibition of cell growth and signaling in JAK2 mutant cell lines and in primary MPN patient samples. PU-H71 treatment of mice resulted in JAK2 degradation, inhibition of JAK-STAT signaling, normalization of peripheral blood counts, and improved survival in MPN models at doses that did not degrade JAK2 in normal tissues or cause substantial toxicity. Importantly, PU-H71 treatment also reduced the mutant allele burden in mice. These data establish what we believe to be a novel therapeutic rationale for HSP90 inhibition in the treatment of JAK2-dependent MPN.
Platinum-based chemotherapies such as cisplatin are used as first-line treatment for many cancers. Although there is often a high initial responsiveness, the majority of patients eventually relapse with platinum-resistant disease. For example, a subset of testicular cancer patients still die even though testicular cancer is considered a paradigm of cisplatin-sensitive solid tumors, but the mechanisms of chemoresistance remain elusive. Here, we have shown that one key determinant of cisplatin-resistance in testicular embryonal carcinoma (EC) is high cytoplasmic expression of the cyclin-dependent kinase (CDK) inhibitor p21. The EC component of the majority of refractory testicular cancer patients exhibited high cytoplasmic p21 expression, which protected EC cell lines against cisplatin-induced apoptosis via CDK2 inhibition. Localization of p21 in the cytoplasm was critical for cisplatin resistance, since relocalization of p21 to the nucleus by Akt inhibition sensitized EC cell lines to cisplatin. We also demonstrated in EC cell lines and human tumor tissue that high cytoplasmic p21 expression and cisplatin resistance of EC were inversely associated with the expression of Oct4 and miR-106b seed family members. Thus, targeting cytoplasmic p21, including by modulation of the Oct4/miR-106b/p21 pathway, may offer new strategies for the treatment of chemoresistant testicular and other types of cancer.
TGF-β signaling can promote tumor formation and development or suppress it, depending on the cellular context and tumor stage. A potential target of this dual effect of TGF-β is HGF, as TGF-β can inhibit or promote its expression, although the mechanisms underlying this are largely unknown. In the present study, we found that mice with keratinocyte-specific deletion of the TGF-β signaling mediator Smad2 (referred to herein as K5.Smad2–/– mice), which have increased susceptibility to squamous cell carcinomas (SCCs), exhibited angiogenesis associated with epithelial overexpression of HGF and endothelial activation of the HGF receptor c-Met. Application of a c-Met inhibitor abrogated angiogenesis, suggesting that HGF overexpression plays a major role in angiogenesis associated with epithelial Smad2 loss. On the Hgf promoter, Smad2 was mainly associated with transcriptional corepressors, whereas Smad4 was mainly associated with the transcriptional coactivator CREB-binding protein (CBP/p300). Smad2 loss caused increased binding of Smad4 and CBP/p300 to the Hgf promoter. Consistent with this, knocking down Smad2 in human keratinocytes caused increased levels of HGF, which were abrogated by concomitant knockdown of Smad3 and Smad4. Importantly, the incidence of HGF-positive human SCC was high in cases with Smad2 loss and lower when Smad4 was also lost. We therefore conclude that Smad2 loss causes HGF upregulation via loss of Smad2-mediated transcriptional repression and enhanced Smad3/4-mediated transactivation. Since Smad2 is often downregulated in human SCCs, our data suggest a therapeutic strategy of blocking HGF/c-Met activation for Smad2-deficient SCCs.
Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disorder that is caused by mutations at two loci, polycystin 1 (PKD1) and polycystin 2 (PKD2). It is characterized by the formation of multiple cysts in the kidneys that can lead to chronic renal failure. Previous studies have suggested a role for hyperactivation of mammalian target of rapamycin (mTOR) in cystogenesis, but the etiology of mTOR hyperactivation has not been fully elucidated. In this report we have shown that mTOR is hyperactivated in Pkd1-null mouse cells due to failure of the HGF receptor c-Met to be properly ubiquitinated and subsequently degraded after stimulation by HGF. In Pkd1-null cells, Casitas B-lineage lymphoma (c-Cbl), an E3-ubiquitin ligase for c-Met, was sequestered in the Golgi apparatus with α3β1 integrin, resulting in the inability to ubiquitinate c-Met. Treatment of mouse Pkd1-null cystic kidneys in organ culture with a c-Met pharmacological inhibitor resulted in inhibition of mTOR activity and blocked cystogenesis in this mouse model of ADPKD. We therefore suggest that blockade of c-Met is a potential novel therapeutic approach to the treatment of ADPKD.
TNF receptor superfamily member 25 (TNFRSF25; also known as DR3, and referred to herein as TNFR25) is constitutively and highly expressed by CD4+FoxP3+ Tregs. However, its function on these cells has not been determined. Here we used a TNFR25-specific agonistic monoclonal antibody, 4C12, to study the effects of TNFR25 signaling on Tregs in vivo in mice. Signaling through TNFR25 induced rapid and selective expansion of preexisting Tregs in vivo such that they became 30%–35% of all CD4+ T cells in the peripheral blood within 4 days. TNFR25-induced Treg proliferation was dependent upon TCR engagement with MHC class II, IL-2 receptor, and Akt signaling, but not upon costimulation by CD80 or CD86; it was unaffected by rapamycin. TNFR25-expanded Tregs remained highly suppressive ex vivo, and Tregs expanded by TNFR25 in vivo were protective against allergic lung inflammation, a mouse model for asthma, by reversing the ratio of effector T cells to Tregs in the lung, suppressing IL-13 and Th2 cytokine production, and blocking eosinophil exudation into bronchoalveolar fluid. Our studies define what we believe to be a novel mechanism for Treg control and important functions for TNFR25 in regulating autoaggression that balance its known role in enhancing autoimmunity.
A key feature of the immune system is its ability to discriminate self from nonself. Breakdown in any of the mechanisms that maintain unresponsiveness to self (a state known as self-tolerance) contributes to the development of autoimmune conditions. Recent studies in mice show that CD8+ T cells specific for the unconventional MHC class I molecule Qa-1 bound to peptides derived from the signal sequence of Hsp60 (Hsp60sp) contribute to self/nonself discrimination. However, it is unclear whether they exist in humans and play a role in human autoimmune diseases. Here we have shown that CD8+ T cells specific for Hsp60sp bound to HLA-E (the human homolog of Qa-1) exist and play an important role in maintaining peripheral self-tolerance by discriminating self from nonself in humans. Furthermore, in the majority of type 1 diabetes (T1D) patients tested, there was a specific defect in CD8+ T cell recognition of HLA-E/Hsp60sp, which was associated with failure of self/nonself discrimination. However, the defect in the CD8+ T cells from most of the T1D patients tested could be corrected in vitro by exposure to autologous immature DCs loaded with the Hsp60sp peptide. These data suggest that HLA-E–restricted CD8+ T cells may play an important role in keeping self-reactive T cells in check. Thus, correction of this defect could be a potentially effective and safe approach in the therapy of T1D.
TLRs play an essential role in the induction of immune responses by detecting conserved molecular products of microorganisms. However, the function of TLR8 is largely unknown. In the current study, we investigated the role of TLR8 signaling in immunity in mice. We found that Tlr8–/– DCs overexpressed TLR7, were hyperresponsive to various TLR7 ligands, and showed stronger and faster NF-κB activation upon stimulation with the TLR7 ligand R848. Tlr8–/– mice showed splenomegaly, defective development of marginal zone (MZ) and B1 B cells, and increased serum levels of IgM and IgG2a. Furthermore, Tlr8–/– mice exhibited increased serum levels of autoantibodies against small nuclear ribonucleoproteins, ribonucleoprotein, and dsDNA and developed glomerulonephritis, whereas neither Tlr7–/– nor Tlr8–/–Tlr7–/– mice showed any of the phenotypes observed in Tlr8–/– mice. These data provide evidence for a pivotal role for mouse TLR8 in the regulation of mouse TLR7 expression and prevention of spontaneous autoimmunity.
Accumulating evidence points to inflammation as a promoter of carcinogenesis. MyD88 is an adaptor molecule in TLR and IL-1R signaling that was recently implicated in tumorigenesis through proinflammatory mechanisms. Here we have shown that MyD88 is also required in a cell-autonomous fashion for RAS-mediated carcinogenesis in mice in vivo and for MAPK activation and transformation in vitro. Mechanistically, MyD88 bound to the key MAPK, Erk, and prevented its inactivation by its phosphatase, MKP3, thereby amplifying the activation of the canonical RAS pathway. The relevance of this mechanism to human neoplasia was suggested by the finding that MyD88 was overexpressed and interacted with activated Erk in primary human cancer tissues. Collectively, these results show that in addition to its role in inflammation, MyD88 plays what we believe to be a crucial direct role in RAS signaling, cell-cycle control, and cell transformation.
Patients with Kallmann syndrome (KS) have hypogonadotropic hypogonadism caused by a deficiency of gonadotropin-releasing hormone (GnRH) and a defective sense of smell related to olfactory bulb aplasia. Based on the findings in a fetus affected by the X chromosome–linked form of the disease, it has been suggested that hypogonadism in KS results from the failed embryonic migration of neuroendocrine GnRH1 cells from the nasal epithelium to the forebrain. We asked whether this singular observation might extend to other developmental disorders that also include arrhinencephaly. We therefore studied the location of GnRH1 cells in fetuses affected by different arrhinencephalic disorders, specifically X-linked KS, CHARGE syndrome, trisomy 13, and trisomy 18, using immunohistochemistry. Few or no neuroendocrine GnRH1 cells were detected in the preoptic and hypothalamic regions of all arrhinencephalic fetuses, whereas large numbers of these cells were present in control fetuses. In all arrhinencephalic fetuses, many GnRH1 cells were present in the frontonasal region, the first part of their migratory path, as were interrupted olfactory nerve fibers that formed bilateral neuromas. Our findings define a pathological sequence whereby a lack of migration of neuroendocrine GnRH cells stems from the primary embryonic failure of peripheral olfactory structures. This can occur either alone, as in isolated KS, or as part of a pleiotropic disease, such as CHARGE syndrome, trisomy 13, and trisomy 18.
Apoptosis of motor neurons is a well-documented feature in amyotrophic lateral sclerosis (ALS) and related motor neuron diseases (MNDs). However, the role of apoptosis in the pathogenesis of these diseases remains unresolved. One possibility is that the affected motor neurons only succumb to apoptosis once they have exhausted functional capacity. If true, blocking apoptosis should confer no therapeutic benefit. To directly investigate this idea, we tested whether tissue-specific deletion in the mouse CNS of BCL2-associated X protein (BAX) and BCL2-homologous antagonist/killer (BAK), 2 proapoptotic BCL-2 family proteins that together represent an essential gateway to the mitochondrial apoptotic pathway, would protect against motor neuron degeneration. We found that neuronal deletion of Bax and Bak in a mouse model of familial ALS not only halted neuronal loss, but prevented axonal degeneration, symptom onset, weight loss, and paralysis and extended survival. These results show that motor neurons damaged in ALS activate the mitochondrial apoptotic pathway early in the disease process and that apoptotic signaling directly contributes to neuromuscular degeneration and neuronal dysfunction. Hence, inhibiting apoptosis upstream of mitochondrial permeabilization represents a possible therapeutic strategy for preserving functional motor neurons in ALS and other MNDs.
Cyclophilin D (which is encoded by the Ppif gene) is a mitochondrial matrix peptidyl-prolyl isomerase known to modulate opening of the mitochondrial permeability transition pore (MPTP). Apart from regulating necrotic cell death, the physiologic function of the MPTP is largely unknown. Here we have shown that Ppif–/– mice exhibit substantially greater cardiac hypertrophy, fibrosis, and reduction in myocardial function in response to pressure overload stimulation than control mice. In addition, Ppif–/– mice showed greater hypertrophy and lung edema as well as reduced survival in response to sustained exercise stimulation. Cardiomyocyte-specific transgene expression of cyclophilin D in Ppif–/– mice rescued the enhanced hypertrophy, reduction in cardiac function, and rapid onset of heart failure following pressure overload stimulation. Mechanistically, the maladaptive phenotype in the hearts of Ppif–/– mice was associated with an alteration in MPTP-mediated Ca2+ efflux resulting in elevated levels of mitochondrial matrix Ca2+ and enhanced activation of Ca2+-dependent dehydrogenases. Elevated matrix Ca2+ led to increased glucose oxidation relative to fatty acids, thereby limiting the metabolic flexibility of the heart that is critically involved in compensation during stress. These findings suggest that the MPTP maintains homeostatic mitochondrial Ca2+ levels to match metabolism with alterations in myocardial workload, thereby suggesting a physiologic function for the MPTP.
Although adenovirus (Ad) has been regarded as an excellent vaccine vector, there are 2 major drawbacks to using this platform: (a) Ad-based vaccines induce a relatively weak humoral response against encoded transgenes, and (b) preexisting immunity to Ad is highly prevalent among the general population. To overcome these obstacles, we constructed an Ad-based malaria vaccine by inserting a B cell epitope derived from a Plasmodium yoelii circumsporozoite (CS) protein (referred to as the PyCS-B epitope) into the capsid proteins of WT/CS-GFP, a recombinant Ad expressing P. yoelii CS protein and GFP as its transgene. Multiple vaccinations with the capsid-modified Ad induced a substantially increased level of protection against subsequent malaria challenge in mice when compared with that of unmodified WT/CS-GFP. Increased protection correlated with augmented antibody responses against the PyCS-B epitope expressed in the capsid. Furthermore, replacement of hypervariable region 1 (HVR1) of the Ad capsid proteins with the PyCS-B epitope circumvented neutralization of the modified Ad by preexisting Ad-specific antibody, both in vivo and in vitro. Importantly, the immunogenicity of the Ad-containing PyCS-B epitope in the HVR1 and a P. yoelii CS transgene was maintained. Overall, this study demonstrates that the HVR1-modifed Ad vastly improves upon Ad as a promising malaria vaccine platform candidate.
Dense deposit disease (DDD) is a severe renal disease characterized by accumulation of electron-dense material in the mesangium and glomerular basement membrane. Previously, DDD has been associated with deficiency of factor H (fH), a plasma regulator of the alternative pathway (AP) of complement activation, and studies in animal models have linked pathogenesis to the massive complement factor 3 (C3) activation caused by this deficiency. Here, we identified a unique DDD pedigree that associates disease with a mutation in the C3 gene. Mutant C3923ΔDG, which lacks 2 amino acids, could not be cleaved to C3b by the AP C3-convertase and was therefore the predominant circulating C3 protein in the patients. However, upon activation to C3b by proteases, or to C3(H2O) by spontaneous thioester hydrolysis, C3923ΔDG generated an active AP C3-convertase that was regulated normally by decay accelerating factor (DAF) but was resistant to decay by fH. Moreover, activated C3b923ΔDG and C3(H2O)923ΔDG were resistant to proteolysis by factor I (fI) in the presence of fH, but were efficiently inactivated in the presence of membrane cofactor protein (MCP). These characteristics cause a fluid phase–restricted AP dysregulation in the patients that continuously activated and consumed C3 produced by the normal C3 allele. These findings expose structural requirements in C3 that are critical for recognition of the substrate C3 by the AP C3-convertase and for the regulatory activities of fH, DAF, and MCP, all of which have implications for therapeutic developments.
The homeodomain transcription factor pancreatic duodenal homeobox 1 (Pdx1) is a major mediator of insulin transcription and a key regulator of the β cell phenotype. Heterozygous mutations in PDX1 are associated with the development of diabetes in humans. Understanding how Pdx1 expression levels are controlled is therefore of intense interest in the study and treatment of diabetes. Pdx1 C terminus–interacting factor-1 (Pcif1, also known as SPOP) is a nuclear protein that inhibits Pdx1 transactivation. Here, we show that Pcif1 targets Pdx1 for ubiquitination and proteasomal degradation. Silencing of Pcif1 increased Pdx1 protein levels in cultured mouse β cells, and Pcif1 heterozygosity normalized Pdx1 protein levels in Pdx1+/– mouse islets, thereby increasing expression of key Pdx1 transcriptional targets. Remarkably, Pcif1 heterozygosity improved glucose homeostasis and β cell function and normalized β cell mass in Pdx1+/– mice by modulating β cell survival. These findings indicate that in adult mouse β cells, Pcif1 limits Pdx1 protein accumulation and thus the expression of insulin and other gene targets important in the maintenance of β cell mass and function. They also provide evidence that targeting the turnover of a pancreatic transcription factor in vivo can improve glucose homeostasis.
The clinical use of adoptive immunotherapy with tumor-reactive T cells to treat established cancers is limited in part by the poor in vivo survival and function of the transferred T cells. Although administration of exogenous cytokines such as IL-2 can promote T cell survival, such strategies have many nonspecific activities and are often associated with toxicity. We show here that abrogating expression of Casitas B-lineage lymphoma b (Cbl-b), a negative regulator of lymphocyte activation, in tumor-reactive CD8+ T cells expanded ex vivo increased the efficacy of adoptive immunotherapy of disseminated leukemia in mice. Mechanistically, Cbl-b abrogation bypassed the requirement for exogenous IL-2 administration for tumor eradication in vivo. In addition, CD8+ T cells lacking Cbl-b demonstrated a lower threshold for activation, better survival following target recognition and stimulation, and enhanced proliferative responses as a result of both IL-2–dependent and –independent pathways. Importantly, siRNA knockdown of Cbl-b in human CD8+CD28– effector T cell clones similarly restored IL-2 production and proliferation following target recognition independent of exogenous IL-2, enhanced IFN-γ production, and increased target avidity. Thus, abrogating Cbl-b expression in effector T cells may improve the efficacy of adoptive therapy of some human malignancies.
Copyright © 2014 American Society for Clinical Investigation