We examined the role of microRNAs (miRNAs) in targeting the stromal-derived factor 1α/CXCR4 (SDF-1α/CXCR4) axis to overcome chemoresistance of AML cells. Microarray analysis of OCI-AML3 cells revealed that the miRNA let-7a was downregulated by SDF-1α-mediated CXCR4 activation and increased by CXCR4 inhibition. Overexpression of let-7a in AML cell lines was associated with decreased c-Myc and BCL-XL protein expression and enhanced chemosensitivity, both in vitro and in vivo. We identified the transcription factor Yin Yang 1 (YY1) as a link between SDF-1α/CXCR4 signaling and let-7a, as YY1 was upregulated by SDF-1α and downregulated by treatment with a CXCR4 antagonist. ChIP assay confirmed the binding of YY1 to unprocessed let-7a DNA fragments, and treatment with YY1 shRNA increased let-7a expression. In primary human AML samples, high CXCR4 expression was associated with low let-7a levels. Xenografts of primary human AML cells engineered to overexpress let-7a exhibited enhanced sensitivity to cytarabine, resulting in greatly extended survival of immunodeficient mice. Based on these data, we propose that CXCR4 induces chemoresistance by downregulating let-7a to promote YY1-mediated transcriptional activation of MYC and BCLXL in AML cells.

Cystic fibrosis (CF) pigs develop disease with features remarkably similar to those in people with CF, including exocrine pancreatic destruction, focal biliary cirrhosis, micro-gallbladder, vas deferens loss, airway disease, and meconium ileus. Whereas meconium ileus occurs in 15% of babies with CF, the penetrance is 100% in newborn CF pigs. We hypothesized that transgenic expression of porcine CF transmembrane conductance regulator (pCFTR) cDNA under control of the intestinal fatty acid-binding protein (iFABP) promoter would alleviate the meconium ileus. We produced 5 CFTR-/-;TgFABP>pCFTR lines. In 3 lines, intestinal expression of CFTR at least partially restored CFTR-mediated anion transport and improved the intestinal phenotype. In contrast, these pigs still had pancreatic destruction, liver disease, and reduced weight gain, and within weeks of birth, they developed sinus and lung disease, the severity of which varied over time. These data indicate that expressing CFTR in intestine without pancreatic or hepatic correction is sufficient to rescue meconium ileus. Comparing CFTR expression in different lines revealed that approximately 20% of wild-type CFTR mRNA largely prevented meconium ileus. This model may be of value for understanding CF pathophysiology and testing new preventions and therapies.

To explore the physiological functions of endothelin-2 (ET-2), we generated gene-targeted mouse models. Global Et2 knockout mice exhibited severe growth retardation and juvenile lethality. Despite normal milk intake, they suffered from internal starvation characterized by hypoglycemia, ketonemia, and increased levels of starvation-induced genes. Although ET-2 is abundantly expressed in the gastrointestinal tract, the intestine was morphologically and functionally normal. Moreover, intestinal epithelium-specific Et2 knockout mice showed no abnormalities in growth and survival. Global Et2 knockout mice were also profoundly hypothermic. Housing Et2 knockout mice in a warm environment significantly extended their median lifespan. However, neuron-specific Et2 knockout mice displayed a normal core body temperature. Low levels of Et2 mRNA were also detected in the lung, with transient increases soon after birth. The lungs of Et2 knockout mice showed emphysematous structural changes with an increase in total lung capacity, resulting in chronic hypoxemia, hypercapnia, and increased erythropoietin synthesis. Finally, systemically inducible ET-2 deficiency in neonatal and adult mice fully reproduced the phenotype previously observed in global Et2 knockout mice. Together, these findings reveal that ET-2 is critical for the growth and survival of postnatal mice and plays important roles in energy homeostasis, thermoregulation, and the maintenance of lung morphology and function.

JAK2 activity is tightly controlled through a self-inhibitory effect via its JAK homology domain 2 (JH2), which restricts the strength and duration of JAK2/STAT3 signaling under physiological conditions. Although multiple mutations within JAK2, which abrogate the function of JH2 and sustain JAK2 activation, are widely observed in hematological malignancies, comparable mutations have not been detected in solid tumors. How solid tumor cells override the autoinhibitory effect of the JH2 domain to maintain constitutive activation of JAK2/STAT3 signaling remains puzzling. Herein, we demonstrate that AGK directly interacted with the JH2 domain to relieve inhibition of JAK2 and activate JAK2/STAT3 signaling. Overexpression of AGK sustained constitutive JAK2/STAT3 activation, consequently promoting the cancer stem cell population and augmenting the tumorigenicity of esophageal squamous cell carcinoma (ESCC) cells both in vivo and in vitro. Furthermore, AGK levels significantly correlated with increased STAT3 phosphorylation, poorer disease-free survival, and shorter overall survival in primary ESCC. More importantly, AGK expression was significantly correlated with JAK2/STAT3 hyperactivation in ESCC, as well as in lung and breast cancer. These findings uncover a mechanism for constitutive activation of JAK2/STAT3 signaling in solid tumors and may represent a prognostic biomarker and therapeutic target.

Abnormalities in cell-cell communication and growth factor signaling pathways can lead to defects in maternal-fetal interactions during pregnancy, including immunologic rejection of the fetal/placental unit. In this study, we discovered that bone morphogenetic protein receptor type 2 (BMPR2) is essential for postimplantation physiology and fertility. Despite normal implantation and early placental/fetal development, deletion of Bmpr2 in the uterine deciduae of mice triggered midgestation abnormalities in decidualization that resulted in abnormal vascular development, trophoblast defects, and a deficiency of uterine natural killer cells. Absence of BMPR2 signaling in the uterine decidua consequently suppressed IL-15, VEGF, angiopoietin, and corin signaling. Disruption of these pathways collectively lead to placental abruption, fetal demise, and female sterility, thereby placing BMPR2 at a central point in the regulation of several physiologic signaling pathways and events at the maternal-fetal interface. Since trophoblast invasion and uterine vascular modification are implicated in normal placentation and fetal growth in humans, our findings suggest that abnormalities in uterine BMPR2-mediated signaling pathways can have catastrophic consequences in women for the maintenance of pregnancy.

A disintegrin and metalloproteinase 10 (ADAM10), a disintegrin and metalloproteinase that resides in the postsynaptic densities (PSDs) of excitatory synapses, has previously been shown to limit β-amyloid peptide (Aβ) formation in Alzheimer's disease (AD). ADAM10 also plays a critical role in regulating functional membrane proteins at the synapse. Using human hippocampal homogenates, we found that ADAM10 removal from the plasma membrane was mediated by clathrin-dependent endocytosis. Additionally, we identified the clathrin adaptor AP2 as an interacting partner of a previously uncharacterized atypical binding motif in the ADAM10 C-terminal domain. This domain was required for ADAM10 endocytosis and modulation of its plasma membrane levels. We found that the ADAM10/AP2 association was increased in the hippocampi of AD patients compared with healthy controls. Long-term potentiation (LTP) in hippocampal neuronal cultures induced ADAM10 endocytosis through AP2 association and decreased surface ADAM10 levels and activity. Conversely, long-term depression (LTD) promoted ADAM10 synaptic membrane insertion and stimulated its activity. ADAM10 interaction with the synapse-associated protein-97 (SAP97) was necessary for LTD-induced ADAM10 trafficking and required for LTD maintenance and LTD-induced changes in spine morphogenesis. These data identify and characterize a mechanism controlling ADAM10 localization and activity at excitatory synapses that is relevant to AD pathogenesis.

The mechanisms involved in the coordinate regulation of the metabolic and structural programs controlling muscle fitness and endurance are unknown. Recently, the nuclear receptor PPARβ/δ was shown to activate muscle endurance programs in transgenic mice. In contrast, muscle-specific transgenic overexpression of the related nuclear receptor, PPARα, results in reduced capacity for endurance exercise. We took advantage of the divergent actions of PPARβ/δ and PPARα to explore the downstream regulatory circuitry that orchestrates the programs linking muscle fiber type with energy metabolism. Our results indicate that, in addition to the well-established role in transcriptional control of muscle metabolic genes, PPARβ/δ and PPARα participate in programs that exert opposing actions upon the type I fiber program through a distinct muscle microRNA (miRNA) network, dependent on the actions of another nuclear receptor, estrogen-related receptor γ (ERRγ). Gain-of-function and loss-of-function strategies in mice, together with assessment of muscle biopsies from humans, demonstrated that type I muscle fiber proportion is increased via the stimulatory actions of ERRγ on the expression of miR-499 and miR-208b. This nuclear receptor/miRNA regulatory circuit shows promise for the identification of therapeutic targets aimed at maintaining muscle fitness in a variety of chronic disease states, such as obesity, skeletal myopathies, and heart failure.

The heparan sulfate proteoglycan (HSPG) syndecan-1 (SDC1) acts as a major receptor for triglyceride-rich lipoprotein (TRL) clearance in the liver. We sought to identify the relevant apolipoproteins on TRLs that mediate binding to SDC1 and determine their clinical relevance. Evidence supporting ApoE as a major determinant arose from its enrichment in TRLs from mice defective in hepatic heparan sulfate (Ndst1f/fAlbCre+ mice), decreased binding of ApoE-deficient TRLs to HSPGs on human hepatoma cells, and decreased clearance of ApoE-deficient [3H]TRLs in vivo. Evidence for a second ligand was suggested by the faster clearance of ApoE-deficient TRLs after injection into WT Ndst1f/fAlbCre- versus mutant Ndst1f/fAlbCre+ mice and elevated fasting and postprandial plasma triglycerides in compound Apoe-/-Ndst1f/fAlbCre+ mice compared with either single mutant. ApoAV emerged as a candidate based on 6-fold enrichment of ApoAV in TRLs accumulating in Ndst1f/fAlbCre+ mice, decreased binding of TRLs to proteoglycans after depletion of ApoAV or addition of anti-ApoAV mAb, and decreased heparan sulfate-dependent binding of ApoAV-deficient particles to hepatocytes. Importantly, disruption of hepatic heparan sulfate-mediated clearance increased atherosclerosis. We conclude that clearance of TRLs by hepatic HSPGs is atheroprotective and mediated by multivalent binding to ApoE and ApoAV.

TNF has remarkable antitumor activities; however, therapeutic applications have not been possible because of the systemic and lethal proinflammatory effects induced by TNF. Both the antitumor and inflammatory effects of TNF are mediated by the TNF receptor p55 (p55TNFR) (encoded by the Tnfrsf1a gene). The antitumor effect stems from an induction of cell death in tumor endothelium, but the cell type that initiates the lethal inflammatory cascade has been unclear. Using conditional Tnfrsf1a knockout or reactivation mice, we found that the expression level of p55TNFR in intestinal epithelial cells (IECs) is a crucial determinant in TNF-induced lethal inflammation. Remarkably, tumor endothelium and IECs exhibited differential sensitivities to TNF when p55TNFR levels were reduced. Tumor-bearing Tnfrsf1a+/- or IEC-specific p55TNFR-deficient mice showed resistance to TNF-induced lethality, while the tumor endothelium remained fully responsive to TNF-induced apoptosis and tumors regressed. We demonstrate proof of principle for clinical application of this approach using neutralizing anti-human p55TNFR antibodies in human TNFRSF1A knockin mice. Our results uncover an important cellular basis of TNF toxicity and reveal that IEC-specific or systemic reduction of p55TNFR mitigates TNF toxicity without loss of antitumor efficacy.

Adult-onset autosomal-dominant leukodystrophy (ADLD) is a progressive and fatal neurological disorder characterized by early autonomic dysfunction, cognitive impairment, pyramidal tract and cerebellar dysfunction, and white matter loss in the central nervous system. ADLD is caused by duplication of the LMNB1 gene, which results in increased lamin B1 transcripts and protein expression. How duplication of LMNB1 leads to myelin defects is unknown. To address this question, we developed a mouse model of ADLD that overexpresses lamin B1. These mice exhibited cognitive impairment and epilepsy, followed by age-dependent motor deficits. Selective overexpression of lamin B1 in oligodendrocytes also resulted in marked motor deficits and myelin defects, suggesting these deficits are cell autonomous. Proteomic and genome-wide transcriptome studies indicated that lamin B1 overexpression is associated with downregulation of proteolipid protein, a highly abundant myelin sheath component that was previously linked to another myelin-related disorder, Pelizaeus-Merzbacher disease. Furthermore, we found that lamin B1 overexpression leads to reduced occupancy of Yin Yang 1 transcription factor at the promoter region of proteolipid protein. These studies identify a mechanism by which lamin B1 overexpression mediates oligodendrocyte cell-autonomous neuropathology in ADLD and implicate lamin B1 as an important regulator of myelin formation and maintenance during aging.