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Supplementary Fig. S6. Gross anatomical features of mice treated with MN-anti-miR10b and doxorubicin.

supplemental figure legends

Supplementary Video S8. Physical appearance of mice treated with MN-anti-miR10b/MN-scr-miR and doxorubicin.

Although metabolic syndrome (MS) is a significant risk of cardiovascular disease (CVD), the cardiac response (MR) to MS remains unclear due to traditional MS models’ narrow scope around a limited number of cell-cycle regulation biomarkers and drawbacks of limited human tissue samples. To date, we developed the most comprehensive platform studying MR to MS in a pig model tightly related to human MS criteria. By incorporating comparative metabolomic, transcriptomic, functional analyses, and unsupervised machine learning (UML), we can discover unknown metabolic pathways connections and links on numerous biomarkers across the MS-associated issues in the heart. For the first time, we show severely diminished availability of glycolytic and citric acid cycle (CAC) pathways metabolites, altered expression, GlcNAcylation, and activity of involved enzymes. A notable exception, however, is the excessive succinate accumulation despite reduced succinate dehydrogenase complex iron-sulfur subunit b (SDHB) expression and decreased content of precursor metabolites. Finally, the expression of metabolites and enzymes from the GABA-glutamate, GABA-putrescine, and the glyoxylate pathways significantly increase, suggesting an alternative cardiac means to replenish succinate and malate in MS. Our platform discovers potential therapeutic targets for MS-associated CVD within pathways that were previously unknown to corelate with the disease.

The underglycosylated mucin 1 tumor antigen (uMUC1) is a biomarker that forecasts the progression of adenocarcinomas. In this study, we evaluated the utility of a dual-modality molecular imaging approach based on targeting uMUC1 for monitoring chemotherapeutic response in a transgenic murine model of pancreatic cancer (KCM triple transgenic mice). An uMUC1-specific contrast agent (MN-EPPT) was synthesized for use with magnetic resonance imaging (MRI) and fluorescence optical imaging. It consisted of dextran-coated iron oxide nanoparticles conjugated to the near infrared fluorescent dye Cy5.5 and to a uMUC1-specific peptide (EPPT). KCM triple transgenic mice were given gemcitabine as chemotherapy while control animals received saline injections following the same schedule. Changes in uMUC1 levels following chemotherapy were monitored using T2-weighted MRI and optical imaging before and 24 hr after injection of the MN-EPPT. uMUC1 expression in tumors from both groups was evaluated by histology and qRT-PCR. We observed that the average delta-T2 in the gemcitabine-treated group was significantly reduced compared to the control group indicating lower accumulation of MN-EPPT, and correspondingly, a lower level of uMUC1 expression. In vivo optical imaging confirmed the MRI findings. Fluorescence microscopy of pancreatic tumor sections showed a lower level of uMUC1 expression in the gemcitabine-treated group compared to the control, which was confirmed by qRT-PCR. Our data proved that changes in uMUC1 expression after gemcitabine chemotherapy could be evaluated using MN-EPPT-enhanced in vivo MR and optical imaging. These results suggest that the uMUC1-targeted imaging approach could provide a useful tool for the predictive assessment of therapeutic response.

Background T-helper 2 (Th2) inflammation is a hallmark of chronic rhinosinusitis with nasal polyps (CRSwNP) although the pathogenesis is poorly understood. P-glycoprotein (permeability glycoprotein, P-gp) is an efflux pump that is capable of regulating cytokine transport and is expressed within sinonasal mucosa. The purpose of this study was to examine if the oversecretion of interleukin 5 (IL-5) and thymic stromal lymphopoietin (TSLP) in CRSwNP could be explained through P-gp–mediated secretory pathways. Methods Fifteen ethmoid mucosal explants were harvested from patients with CRS (n = 10) and CRSwNP (n = 10) and stimulated with Staphylococcus aureus enterotoxin B (SEB). P-gp was inhibited using zosuquidar trihydrochloride (herein Zosuquidar). P-gp expression was measured using real-time polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). IL-5, IL-8, and TSLP secretion were quantified using ELISA. Results P-gp protein was overexpressed in CRSwNP (28.32 ± 25.94 ng/mL per mg explant) as compared to CRS (10.74 ± 8.61; p = 0.01, 2-tailed Mann-Whitney U test). There was no difference in messenger RNA (mRNA) expression. SEB induced a significant increase in IL-5 and TSLP but not IL-8 secretion relative to control in the CRSwNP explants only. Subsequent P-gp inhibition significantly reduced IL-5 and TSLP secretion (p = 0.04 for both, 2-tailed Student t test) to control levels. The concentration of IL-5 and TSLP secretion were strongly and significantly correlated to the concentration of P-gp within the same explant (IL-5: r = 0.791, p = 0.001; TSLP: r = 0.687, p = 0.003; 2-tailed Spearman's rank-order correlation). Conclusion P-gp protein is expressed at higher concentrations in CRSwNP as compared to CRS. This overexpression directly contributes to the relative hypersecretion of IL-5 and TSLP. These findings suggest a novel mechanism for Th2 skewing in CRSwNP.

The therapeutic promise of microRNA (miRNA) in cancer has yet to be realized. In this study, we identified and therapeutically exploited a new role for miR-10b at the metastatic site, which links its overexpression to tumor cell viability and proliferation. In the protocol developed, we combined a miR-10b–inhibitory nanodrug with low-dose anthracycline to achieve complete durable regressions of metastatic disease in a murine model of metastatic breast cancer. Mechanistic investigations suggested a potent antiproliferative, proapoptotic effect of the nanodrug in the metastatic cells, potentiated by a cell-cycle arrest produced by administration of the low-dose anthracycline. miR-10b was overexpressed specifically in cells with high metastatic potential, suggesting a role for this miRNA as a metastasis-specific therapeutic target. Taken together, our results implied the existence of pathways that regulate the viability and proliferation of tumor cells only after they have acquired the ability to grow at distant metastatic sites. As illustrated by miR-10b targeting, such metastasis-dependent apoptotic pathways would offer attractive targets for further therapeutic exploration. Cancer Res; 75(20); 4407–15. ©2015 AACR.

Metastases, and not the primary tumor from which they originate, are the main reason for mortality from carcinoma. Although the molecular mechanisms behind metastasis are poorly understood, it is clear that epigenetic dysregulation at the level of microRNA expression is a key characteristic of the metastatic process that can be exploited for therapy. Here, we describe an miRNA-targeted therapeutic approach for the prevention and arrest of lymph node metastasis. Therapy relies on the inhibition of the pro-metastatic microRNA-10b. It is delivered to primary and lymph node metastatic tumor cells using an imaging-capable nanodrug that is designed to specifically home to these tissues. Treatment of invasive human breast tumor cells (MDA-MB-231) with the nanodrug in vitro downregulates miR-10b and abolishes the invasion and migration of the tumor cells. After intravenous delivery to mice bearing orthotopic MDA-MB-231-luc-D3H2LN tumors, the nanodrug accumulates in the primary tumor and lymph nodes. When treatment is initiated before metastasis to lymph nodes, metastasis is prevented. Treatment after the formation of lymph node metastases arrests the metastatic process without a concomitant effect on primary tumor growth raising the possibility of a context-dependent variation in miR-10b breast oncogenesis.

Resveratrol (3,5,4’-trihydroxystilbene) has been ascribed multiple beneficial biological effects but the influence of resveratrol on glucocorticoid-induced muscle atrophy is not known. We examined the effects of resveratrol on dexamethasone-induced atrogin-1 and MuRF1 expression, FOXO1 acetylation, protein degradation and atrophy in cultured L6 myotubes. In addition, the role of the deacetylase SIRT1 in the effects of resveratrol was determined by transfecting myotubes with SIRT1 siRNA. The catabolic effects of dexamethasone were prevented by resveratrol and the protective effects of resveratrol on dexamethasone-induced atrogin-1 and MuRF1 expression were abolished in myotubes transfected with SIRT1 siRNA. Results suggest that resveratrol can prevent glucocorticoid-induced muscle wasting and that this effect is at least in part SIRT1-dependent.

Myostatin is a negative regulator of muscle mass and has been reported to be upregulated in several conditions characterized by muscle atrophy. The influence of sepsis on myostatin expression and activity is poorly understood. Here, we tested the hypothesis that sepsis upregulates the expression and downstream signaling of myostatin in skeletal muscle. Because sepsis-induced muscle wasting is at least in part regulated by glucocorticoids, we also determined the influence of glucocorticoids on myostatin expression. Sepsis was induced in rats by cecal ligation and puncture and control rats were sham-operated. In other experiments, rats were injected intraperitoneally with dexamethasone (10 mg/kg) or corresponding volume of vehicle. Surprisingly, myostatin mRNA levels were reduced and myostatin protein levels were unchanged in muscles from septic rats. Muscle levels of activin A, follistatin, and total and phosphorylated Smad2 (p-Smad2) were not influenced by sepsis, suggesting that myostatin downstream signaling was not altered during sepsis. Interestingly, total and p-Smad3 levels were increased in septic muscle, possibly reflecting altered signaling through pathways other than myostatin. Similar to sepsis, treatment of rats with dexamethasone reduced myostatin mRNA levels and did not alter myostatin protein levels. Fasting, an additional condition characterized by muscle wasting, reduced myostatin mRNA and activin A protein levels, increased myostatin protein, and did not influence follistatin and p-Smad2 levels. Of note, total and p-Smad3 levels were reduced in muscle during fasting. The results suggest that sepsis and glucocorticoids do not upregulate the expression and activity of myostatin in skeletal muscle. The role of myostatin may vary between different conditions characterized by muscle wasting. Downstream signaling through Smad2 and 3 is probably regulated not only by myostatin but by other mechanisms as well.

The inflammatory response that accompanies central nervous system (CNS) injury can affect neurological outcome in both positive and negative ways. In the optic nerve, a CNS pathway that normally fails to regenerate when damaged, intraocular inflammation causes retinal ganglion cells (RGCs) to switch into an active growth state and extend lengthy axons down the nerve. The molecular basis of this phenomenon is uncertain. A prior study showed that oncomodulin (Ocm), a Ca 2+ -binding protein secreted by a macrophage cell line, is a potent axon-promoting factor for RGCs. However, it is not known whether Ocm contributes to the physiological effects of intraocular inflammation in vivo, and there are conflicting reports in the literature regarding its expression and significance. We show here that intraocular inflammation causes infiltrative cells of the innate immune system to secrete high levels of Ocm, and that agents that prevent Ocm from binding to its receptor suppress axon regeneration. These results were verified in different strains, species, and experimental models, and establish Ocm as a potent growth-promoting signal between the innate immune system and neurons in vivo.

The transcription factor PU.1 is an important regulator of hematopoiesis; precise expression levels are critical for normal hematopoietic development and suppression of leukemia. We show here that noncoding antisense RNAs are important modulators of proper dosages of PU.1. Antisense and sense RNAs are regulated by shared evolutionarily conserved cis-regulatory elements, and we can show that antisense RNAs inhibit PU.1 expression by modulating mRNA translation. We propose that such antisense RNAs will likely be important in the regulation of many genes and may be the reason for the large number of overlapping complementary transcripts with so far unknown function.

T-helper 2 (Th2) inflammation is a hallmark of chronic rhinosinusitis with nasal polyps (CRSwNP) although the pathogenesis is poorly understood. P-glycoprotein (permeability glycoprotein, P-gp) is an efflux pump that is capable of regulating cytokine transport and is expressed within sinonasal mucosa. The purpose of this study was to examine if the oversecretion of interleukin 5 (IL-5) and thymic stromal lymphopoietin (TSLP) in CRSwNP could be explained through P-gp-mediated secretory pathways.Fifteen ethmoid mucosal explants were harvested from patients with CRS (n = 10) and CRSwNP (n = 10) and stimulated with Staphylococcus aureus enterotoxin B (SEB). P-gp was inhibited using zosuquidar trihydrochloride (herein Zosuquidar). P-gp expression was measured using real-time polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). IL-5, IL-8, and TSLP secretion were quantified using ELISA.P-gp protein was overexpressed in CRSwNP (28.32 ± 25.94 ng/mL per mg explant) as compared to CRS (10.74 ± 8.61; p = 0.01, 2-tailed Mann-Whitney U test). There was no difference in messenger RNA (mRNA) expression. SEB induced a significant increase in IL-5 and TSLP but not IL-8 secretion relative to control in the CRSwNP explants only. Subsequent P-gp inhibition significantly reduced IL-5 and TSLP secretion (p = 0.04 for both, 2-tailed Student t test) to control levels. The concentration of IL-5 and TSLP secretion were strongly and significantly correlated to the concentration of P-gp within the same explant (IL-5: r = 0.791, p = 0.001; TSLP: r = 0.687, p = 0.003; 2-tailed Spearman's rank-order correlation).P-gp protein is expressed at higher concentrations in CRSwNP as compared to CRS. This overexpression directly contributes to the relative hypersecretion of IL-5 and TSLP. These findings suggest a novel mechanism for Th2 skewing in CRSwNP.

We examined the influence of sepsis on the expression and activity of the calpain and caspase systems in skeletal muscle. Sepsis was induced in rats by cecal ligation and puncture (CLP). Control rats were sham operated. Calpain activity was determined by measuring the calcium-dependent hydrolysis of casein and by casein zymography. The activity of the endogenous calpain inhibitor calpastatin was measured by determining the inhibitory effect on calpain activity in muscle extracts. Protein levels of μ- and m-calpain and calpastatin were determined by Western blotting, and calpastatin mRNA was measured by real-time PCR. Caspase-3 activity was determined by measuring the hydrolysis of the fluorogenic caspase-3 substrate Ac-DEVD-AMC and by determining protein and mRNA expression for caspase-3 by Western blotting and real-time PCR, respectively. In addition, the role of calpains and caspase-3 in sepsis-induced muscle protein breakdown was determined by measuring protein breakdown rates in the presence of specific inhibitors. Sepsis resulted in increased muscle calpain activity caused by reduced calpastatin activity. In contrast, caspase-3 activity, mRNA levels, and activated caspase-3 29-kDa fragment were not altered in muscle from septic rats. Sepsis-induced muscle proteolysis was blocked by the calpain inhibitor calpeptin but was not influenced by the caspase-3 inhibitor Ac-DEVD-CHO. The results suggest that sepsis-induced muscle wasting is associated with increased calpain activity, secondary to reduced calpastatin activity, and that caspase-3 activity is not involved in the catabolic response to sepsis.

The inability of mature CNS neurons to regenerate injured axons has been attributed to a loss of inherent growth potential of cells and to inhibitory signals associated with myelin and the glial scar. The present study investigated two complementary issues: (1) whether mature CNS neurons can be stimulated to alter their gene expression profile and switch into a strong growth state; and (2) whether inactivating RhoA, a convergence point for multiple inhibitory signals, is sufficient to produce strong regeneration even without activating the growth state of neurons. In the mature rat, retinal ganglion cells (RGCs) normally fail to regenerate axons through the injured optic nerve but can be stimulated to do so by activating macrophages in the eye (e.g., by lens injury). To investigate underlying changes in gene expression, we retrogradely labeled RGCs with a fluorescent dye, performed optic nerve surgery with or without lens injury, and 4 d later, dissociated retinas, isolated RGCs by fluorescence-activated cell sorting, and examined their profiles of gene expression using microarrays. To investigate the effects of inactivating RhoA, we transfected RGCs with adeno-associated viruses carrying a gene for C3 ribosyltransferase. Our results show that, with appropriate stimulation, mature CNS neurons can undergo dramatic changes in gene expression comparable with those seen in regenerating neurons of the PNS, and that RhoA inactivation by itself results in moderate regeneration, and strongly potentiates axon regeneration through the mature optic nerve when the growth state of neurons is activated.

Myotonic dystrophy type 1 (DM1) is caused by a CUG n expansion ( n ≈ 50 to 5000) in the 3′ untranslated region of the mRNA of the DM protein kinase gene. We show that mutant RNA binds and sequesters transcription factors (TFs), with up to 90% depletion of selected TFs from active chromatin. Diverse genes are consequently reduced in expression, including the ion transporter CIC-1, which has been implicated in myotonia. When TF specificity protein 1 (Sp1) was overexpressed in DM1-affected cells, low levels of messenger RNA for CIC-1 were restored to normal. Transcription factor leaching from chromatin by mutant RNA provides a potentially unifying pathomechanistic explanation for this disease.

During activation, T cells undergo metabolic reprogramming, which imprints distinct functional fates. We determined that on PD-1 ligation, activated T cells are unable to engage in glycolysis or amino acid metabolism but have an increased rate of fatty acid β-oxidation (FAO). PD-1 promotes FAO of endogenous lipids by increasing expression of CPT1A, and inducing lipolysis as indicated by elevation of the lipase ATGL, the lipolysis marker glycerol and release of fatty acids. Conversely, CTLA-4 inhibits glycolysis without augmenting FAO, suggesting that CTLA-4 sustains the metabolic profile of non-activated cells. Because T cells utilize glycolysis during differentiation to effectors, our findings reveal a metabolic mechanism responsible for PD-1-mediated blockade of T-effector cell differentiation. The enhancement of FAO provides a mechanistic explanation for the longevity of T cells receiving PD-1 signals in patients with chronic infections and cancer, and for their capacity to be reinvigorated by PD-1 blockade., Activation of T cells results in metabolic reprogramming to favour glycolysis. Here, Patsoukis et al. show that the surface receptor PD-1 inhibits glycolysis and increases the metabolism of lipids, providing a potential mechanism for the blockade of T effector functions but also for the longevity accompanying T cell exhaustion.

The elements regulating gene expression in hematopoietic stem cells are still poorly understood. We previously reported that a 141-kilobase (kb) human CD34 transgene confers properly regulated human CD34 expression in transgenic mice. A construct with only the human CD34 promoter and 3′ enhancer region is not sufficient, suggesting that critical distal elements are necessary for expression of the human CD34 gene. To further localize such elements, we analyzed deletion constructs of the human CD34 gene and evaluated their function in transgenic mice. Constructs harboring as little as 18 kb of 5′ and 26 kb of 3′ human CD34 flanking sequence conferred human expression in tissues of transgenic mice with a pattern similar to that of the 141-kb human transgene. In contrast, a construct harboring 10 kb of 5′ and 17 kb of 3′ human CD34 flanking sequence gave no expression. These data demonstrate that regions between 10 to 18 kb upstream and/or 17 to 26 kb downstream of the human CD34 gene contain critical elements for human CD34 expression in vivo. Further functional analysis of these regions in transgenic mice will be crucial for understanding CD34 gene expression in hematopoietic stem and progenitor cells.

Phosphorylation on serines or threonines preceding proline (Ser/Thr-Pro) is a major signaling mechanism. The conformation of a subset of phosphorylated Ser/Thr-Pro motifs is regulated by the prolyl isomerase Pin1. Inhibition of Pin1 induces apoptosis and may also contribute to neuronal death in Alzheimer’s disease. However, little is known about the role of Pin1 in cancer or in modulating transcription factor activity. Here we report that Pin1 is strikingly overexpressed in human breast cancers, and that its levels correlate with cyclin D1 levels in tumors. Overexpression of Pin1 increases cellular cyclin D1 protein and activates its promoter. Furthermore, Pin1 binds c-Jun that is phosphorylated on Ser63/73-Pro motifs by activated JNK or oncogenic Ras. Moreover, Pin1 cooperates with either activated Ras or JNK to increase transcriptional activity of c-Jun towards the cyclin D1 promoter. Thus, Pin1 is up-regulated in human tumors and cooperates with Ras signaling in increasing c-Jun transcriptional activity towards cyclin D1. Given the crucial roles of Ras signaling and cyclin D1 overexpression in oncogenesis, our results suggest that overexpression of Pin1 may promote tumor growth.

The outcome of umbilical cord blood transplantation (UCBT) is compromised by low hematopoietic stem cell (HSC) doses leading to prolonged time to engraftment, delayed immunological reconstitution and late memory T-cell skewing. Exposure of UCB to dimethyl-prostaglandin E2 (dmPGE2) increases HSC in vivo. We determined that exposure of UCB T lymphocytes to dmPGE2 modified Wnt signaling resulting in T cell factor (TCF)-mediated transcription. Wnt signaling upregulated interleukin (IL)-7R and IL-2Rβ, resulting in enhanced survival mediated by the homeostatic cytokines IL-7 and IL-15. dmPGE2 also induced components of the Wnt pathway and Wnt receptors, thereby priming UCB T cells to receive signals via Wnt ligands in vivo. We observed that the Wnt transcription factor TCF7 and its target EOMES were elevated in the T cells of patients who received PGE2-treated UCBs. Consistent with the role of Wnt/β-catenin signaling to induce and maintain naive, memory precursors and long-lived central memory CD8(+) cells, these patients also had increased fractions of CD8(+)CD45RO(-)CD62L(+) plus CD8(+)CD45RO(+)CD62L(+) subsets encompassing these T-cell populations. These effects of the PGE2/Wnt/β-catenin axis may have significant implications for harnessing immunity in the context of UCBT, where impaired immune reconstitution is associated with late memory T-cell skewing.

Programmed death 1 (PD-1) is a potent inhibitor of T cell responses. PD-1 abrogates activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, but the mechanism remains unclear. We determined that during T cell receptor (TCR)/CD3- and CD28-mediated stimulation, PTEN is phosphorylated by casein kinase 2 (CK2) in the Ser380-Thr382-Thr383 cluster within the C-terminal regulatory domain, which stabilizes PTEN, resulting in increased protein abundance but suppressed PTEN phosphatase activity. PD-1 inhibited the stabilizing phosphorylation of the Ser380-Thr382-Thr383 cluster within the C-terminal domain of PTEN, thereby resulting in ubiquitin-dependent degradation and diminished abundance of PTEN protein but increased PTEN phosphatase activity. These effects on PTEN were secondary to PD-1-mediated inhibition of CK2 and were recapitulated by pharmacologic inhibition of CK2 during TCR/CD3- and CD28-mediated stimulation without PD-1. Furthermore, PD-1-mediated diminished abundance of PTEN was reversed by inhibition of ubiquitin-dependent proteasomal degradation. Our results identify CK2 as a new target of PD-1 and reveal an unexpected mechanism by which PD-1 decreases PTEN protein expression while increasing PTEN activity, thereby inhibiting the PI3K/Akt signaling axis.

The receptor programmed death 1 (PD-1) inhibits T cell proliferation and plays a critical role in suppressing self-reactive T cells, and it also compromises antiviral and antitumor responses. To determine how PD-1 signaling inhibits T cell proliferation, we used human CD4(+) T cells to examine the effects of PD-1 signaling on the molecular control of the cell cycle. The ubiquitin ligase SCF(Skp2) degrades p27(kip1), an inhibitor of cyclin-dependent kinases (Cdks), and PD-1 blocked cell cycle progression through the G(1) phase by suppressing transcription of SKP2, which encodes a component of this ubiquitin ligase. Thus, in T cells stimulated through PD-1, Cdks were not activated, and two critical Cdk substrates were not phosphorylated. Activation of PD-1 inhibited phosphorylation of the retinoblastoma gene product, which suppressed expression of E2F target genes. PD-1 also inhibited phosphorylation of the transcription factor Smad3, which increased its activity. These events induced additional inhibitory checkpoints in the cell cycle by increasing the abundance of the G(1) phase inhibitor p15(INK4) and repressing the Cdk-activating phosphatase Cdc25A. PD-1 suppressed SKP2 transcription by inhibiting phosphoinositide 3-kinase-Akt and Ras-mitogen-activated and extracellular signal-regulated kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) signaling. Exposure of cells to the proliferation-promoting cytokine interleukin-2 restored activation of MEK-ERK signaling, but not Akt signaling, and only partially restored SKP2 expression. Thus, PD-1 blocks cell cycle progression and proliferation of T lymphocytes by affecting multiple regulators of the cell cycle.

Muscle wasting in catabolic patients is in part mediated by glucocorticoids and is associated with increased expression and activity of the transcription factor C/EBPβ. It is not known, however, if C/EBPβ is causally linked to glucocorticoid-induced muscle atrophy. We used dexamethasone-treated L6 myoblasts and myotubes to test the role of C/EBPβ in glucocorticoid-induced expression of the muscle-specific ubiquitin ligases atrogin-1 and MuRF1, protein degradation, and muscle atrophy by transfecting cells with C/EBPβ siRNA. In myoblasts, silencing C/EBPβ expression with siRNA inhibited dexamethasone-induced increase in protein degradation, atrogin-1 and MuRF1 expression, and muscle cell atrophy. Similar effects of C/EBPβ siRNA were seen in myotubes except that the dexamethasone-induced increase in MuRF1 expression was not affected by C/EBPβ siRNA in myotubes. In additional experiments, overexpressing C/EBPβ did not influence atrogin-1 or MuRF1 expression in myoblasts or myotubes. Taken together, our observations suggest that glucocorticoid-induced muscle wasting is at least in part regulated by C/EBPβ. Increased C/EBPβ expression alone, however, is not sufficient to upregulate atrogin-1 and MuRF1 expression.

Background—With the ultimate hope of finding a cure for diabetes, researches are looking into altering the genetic profile of the beta cell as a way to manage metabolic dysregulation. One of the most powerful new approaches for the directed regulation of gene expression utilizes the phenomenon of RNA interference. Methods—Here, we establish the feasibility of a novel technology centered around multifunctional magnetic nanocarriers, which concurrently deliver siRNA to intact pancreatic islets and can be detected by magnetic resonance (MR) and optical imaging. Results—In the proof-of-principle studies described here, we demonstrate that, following in vitro incubation, magnetic nanoparticles carrying siRNA designed to target the model gene for enhanced green fluorescent protein, are efficiently taken up by murine pancreatic islets, derived from egfp transgenic animals. This uptake can be visualized by magnetic resonance imaging (MRI) and nearinfrared fluorescence optical imaging and results in down-regulation of the target gene. Conclusions—These results illustrate the value of our approach in overcoming the challenges associated with genetic modification of intact pancreatic islets in a clinically acceptable manner. Furthermore, an added advantage of our technology derives from the combined capability of our magnetic nanoparticles for siRNA delivery and magnetic labeling of pancreatic islets.

Dexamethasone-treated L6 (a rat cell line) and C2C12 (a mouse cell line) myotubes are frequently used as in vitro models of muscle wasting. We compared the effects of different concentrations of dexamethasone and corticosterone (the naturally occurring glucocorticoid in rodents) on protein breakdown rates, myotube size, and atrogin-1 and MuRF1 mRNA levels in the two cell lines. In addition, the expression of the glucocorticoid receptor (GR) and its role in glucocorticoid-induced metabolic changes were determined. Treatment with dexamethasone or corticosterone resulted in dose-dependent increases in protein degradation rates in both L6 and C2C12 myotubes accompanied by 25-30% reduction of myotube diameter. The same treatments increased atrogin-1 mRNA levels in L6 and C2C12 myotubes but, surprisingly, upregulated the expression of MuRF1 in L6 myotubes only. Both cell types expressed the GR and treatment with dexamethasone or corticosterone downregulated total cellular GR levels while increasing nuclear translocation of the GR in both L6 and C2C12 myotubes. The GR antagonist RU38486 inhibited the dexamethasone- and corticosterone-induced increases in atrogin-1 and MuRF1 expression in L6 myotubes but not in C2C12 myotubes. Interestingly, RU38486 exerted agonist effects in the C2C12, but not in the L6 myotubes. The present results suggest that muscle wasting-related responses to dexamethasone and corticosterone are similar, but not identical, in L6 and C2C12 myotubes. Most notably, the regulation by glucocorticoids of MuRF1 and the role of the GR may be different in the two cell lines. These differences need to be taken into account when cultured myotubes are used in future studies to further explore mechanisms of muscle wasting.

With the increased potential of RNA interference (RNAi) as a therapeutic strategy, new noninvasive methods for detection of siRNA delivery and silencing are urgently needed. Here we describe the development of dual-purpose probes for in vivo transfer of siRNA and the simultaneous imaging of its accumulation in tumors by high-resolution magnetic resonance imaging (MRI) and near-infrared in vivo optical imaging (NIRF). These probes consisted of magnetic nanoparticles labeled with a near-infrared dye and covalently linked to siRNA molecules specific for model or therapeutic targets. Additionally, these nanoparticles were modified with a membrane translocation peptide for intracellular delivery. We show the feasibility of in vivo tracking of tumor uptake of these probes by MRI and optical imaging in two separate tumor models. We also used proof-of-principle optical imaging to corroborate the efficiency of the silencing process. These studies represent the first step toward the advancement of siRNA delivery and imaging strategies, essential for cancer therapeutic product development and optimization.

Members of the C/EBP transcription factor family regulate cell differentiation and multiple other cellular functions. The cellular levels of C/EBPalpha, gamma, delta, epsilon, and Gadd153/CHOP are regulated in part by proteasome-dependent degradation. In contrast, mechanisms regulating the degradation of C/EBPbeta are poorly understood. We tested the hypothesis that the degradation of C/EBPbeta is calpain-dependent. Studies were performed in cultured L6 myotubes (a rat skeletal muscle cell line) because we have found previously that C/EBPbeta may be involved in the regulation of muscle proteolysis. Treatment of cultured L6 myotubes with the calpain inhibitors calpeptin and Calpain Inhibitor I and II resulted in increased C/EBPbeta concentrations but did not influence cellular levels of the other C/EBP transcription factor family members. Transfection of myoblasts with a plasmid expressing the endogenous calpain inhibitor calpastatin resulted in increased cellular levels of C/EBPbeta whereas the opposite result was observed in myoblasts overexpressing micro- or m-calpain. Co-immunoprecipitation provided evidence for protein-protein interaction between C/EBPbeta and micro- and m-calpain suggesting that C/EBPbeta may be a calpain substrate. This notion was supported by experiments in which immunoprecipitated C/EBPbeta was incubated with purified micro-calpain in a cell-free system. The increase in C/EBPbeta levels caused by inhibition of calpain activity was accompanied by increased C/EBPbeta DNA-binding and gene activation. The present results suggest that C/EBPbeta is degraded by a calpain-dependent mechanism in skeletal muscle cells and that the role of calpains is specific for C/EBPbeta among different members of the C/EBP transcription factor family.

Muscle wasting in sepsis is a significant clinical problem because it results in muscle weakness and fatigue that may delay ambulation and increase the risk for thromboembolic and pulmonary complications. Treatments aimed at preventing or reducing muscle wasting in sepsis, therefore, may have important clinical implications. Recent studies suggest that sepsis-induced muscle proteolysis may be initiated by calpain-dependent release of myofilaments from the sarcomere, followed by ubiquitination and degradation of the myofilaments by the 26S proteasome. In the present experiments, treatment of rats with one of the calpain inhibitors calpeptin or BN82270 inhibited protein breakdown in muscles from rats made septic by cecal ligation and puncture. The inhibition of protein breakdown was not accompanied by reduced expression of the ubiquitin ligases atrogin-1/MAFbx and MuRF1, suggesting that the ubiquitin-proteasome system is regulated independent of the calpain system in septic muscle. When incubated muscles were treated in vitro with calpain inhibitor, protein breakdown rates and calpain activity were reduced, consistent with a direct effect in skeletal muscle. Additional experiments suggested that the effects of BN82270 on muscle protein breakdown may, in part, reflect inhibited cathepsin L activity, in addition to inhibited calpain activity. When cultured myoblasts were transfected with a plasmid expressing the endogenous calpain inhibitor calpastatin, the increased protein breakdown rates in dexamethasone-treated myoblasts were reduced, supporting a role of calpain activity in atrophying muscle. The present results suggest that treatment with calpain inhibitors may prevent sepsis-induced muscle wasting.

Muscle wasting during sepsis and other catabolic conditions is, at least in part, mediated by glucocorticoids and is associated with upregulated transcription of multiple genes in the ubiquitin-proteasome proteolytic pathway. In addition to transcription factors, nuclear cofactors, including p300, regulate gene transcription. We tested the hypothesis that glucocorticoids upregulate the expression of p300 in muscle cells. Treatment of cultured L6 myotubes, a rat skeletal muscle cell line, with dexamethasone resulted in a dose- and time-dependent increase in p300 protein and mRNA levels. Surprisingly, the effect of dexamethasone on p300 levels was not inhibited by the glucocorticoid receptor (GR) antagonist RU38486 and RU38486 exerted an agonist effect on p300, increasing its expression. Co-immunoprecipitation showed that treatment of the myotubes with dexamethasone resulted in protein–protein interaction between p300 and C/EBPβ, but not C/EBPδ. The present results suggest that glucocorticoids upregulate the expression of p300 and its interaction with C/EBPβ in skeletal muscle. Increased expression and activity of p300 may be involved in the regulation of gene transcription in glucocorticoid-dependent muscle wasting. J. Cell. Biochem. 94: 1058–1067, 2005. © 2005 Wiley-Liss, Inc.

The influence of glucocorticoids on the expression and activity of the transcription factors CCAAT/enhancer binding protein (C/EBP)beta and delta in skeletal muscle was examined by treating rats or cultured L6 myotubes with dexamethasone. Treatment of rats with 10 mg/kg of dexamethasone resulted in increased C/EBPbeta and delta DNA binding activity in the extensor digitorum longus muscle as determined by electrophoretic mobility shift assay (EMSA) and supershift analysis. A similar response was noticed in dexamethasone-treated myotubes. In other experiments, myocytes were transfected with a plasmid containing a promoter construct consisting of multiple C/EBP binding elements upstream of a luciferase reporter gene. Treatment of these cells with dexamethasone resulted in a fourfold increase in luciferase activity, suggesting that glucocorticoids increase C/EBP-dependent gene activation in muscle cells. In addition, dexamethasone upregulated the protein and gene expression of C/EBPbeta and delta in the myotubes in a time- and dose-dependent fashion as determined by Western blotting and real-time PCR, respectively. The results suggest that glucocorticoids increase C/EBPbeta and delta activity and expression through a direct effect in skeletal muscle.

Programmed death (PD)-1 is a potent T cell inhibitor and can compromise anti-viral and anti-tumor T cell responses. PD-1 abrogates activation of the PI3K/Akt pathway but the mechanism remains unclear. The main negative regulator of PI3K/Akt is the lipid phosphatase PTEN, which dephosphorylates PIP3 at the 3′ position to generate PI(4,5)P2. By limiting the amount of PIP3, PTEN opposes PI3K activation and blunts the survival and proliferative signal of the PI3K/Akt pathway. A critical regulatory component of PTEN is the 50-amino-acid C-terminus domain (aa residues 354 to 403), which undergoes phopshorylation by CK2 (casein kinase 2) on three residues S380, T382 and T383, thereby decreasing PTEN phosphatase activity while stabilizing PTEN protein. To examine the effects of PD-1 on PTEN we used primary human CD4+ T cells and magnetic beads conjugated with monoclonal antibodies against TCR/CD3 and CD28 with or without PDL-1-Ig fusion protein to induce PD-1-mediated signals. During TCR/CD3/CD28-mediated stimulation, PTEN was phosphorylated in the Ser380/Thr382/Thr383 cluster within the C-terminus regulatory domain, resulting in suppressed PTEN phosphatase activity and increased protein stability. PD-1 inhibited phosphorylation of the Ser380/Thr382/Thr383 cluster resulting in decreased PTEN stability but increased phosphatase activity. These effects were secondary to inhibition of CK2 and were recapitulated by pharmacologic CK2 inhibitors upon TCR/CD3/CD28-mediated stimulation without PD-1. PI3K/Akt has a central role in regulating expression of Glut1 that is required for glucose uptake and utilization for glycolysis, which is the main metabolic pathway for energy generation upon T cell activation. Because PD-1 activated PTEN and inhibited PI3K/Akt, we examined the effects of PD-1 on cell survival and energy generation. Surprisingly, T cells receiving PD-1 signals remained viable at levels comparable to T cells stimulated by TCR/CD3 and CD28, although failed to express Glut1 and did not display uptake and incorporation of 14C(U)}-deoxy-D-glucose. Moreover, T cells receiving PD-1 signals were metabolically active, as determined by mitochondrial membrane potential. We determined that PD-1 induced a sustained activation of the key energy sensor AMP-activated protein kinase (AMPK) and phosphorylation of the AMPK substrate Ulk1, the mammalian homologue of the yeast kinase ATG, which is essential for autophagy, survival and longevity under limited nutrient supplies. Under these conditions, T cells also displayed phosphorylation of Raptor on the AMPK-specific site, leading to inactivation of mTORC1. Because Ulk1 activation is essential for autophagy and survival under limited nutrient supplies, we analyzed markers of autophagy. Compared to TCR/CD3/CD28-stimulated cells, T cells receiving PD-1 signals had higher LC3B-II/LC3B-I ratio and decreased expression of SQSTM1/p62, indicating a higher degree of autophagy. Besides autophagy, AMPK has a central role in lipid metabolism by stimulating fatty acid oxidation (FAO). In the liver, the rate of FAO is controlled by allosteric regulation of carnitine palmitoyltransferase I (CPTI), which is located on the outer mitochondrial membrane and esterifies long chain fatty acids to carnitine, thereby initiating mitochondrial import. In contrast, in hematopoietic cells, FAO is controlled by CPT1A abundance, and metabolic reprogramming is regulated by alterations of CPT1A expression. Assessment of CPT1A in T cells stimulated via TCR/CD3 and CD28 revealed decrease of CPT1A expression and parallel suppression of FAO compared to base line levels of unstimulated cells. In contrast, T cells receiving PD-1 signals displayed a dramatic upregulation of CPT1A mRNA and protein, which temporally coincided with a robust increase of FAO as determined by assessment of fatty acid β-oxidation using [9,10-3H] palmitate. Our results uncover an unexpected biochemical and metabolic mechanism coupling PD-1-mediated PI3K/Akt inhibition to AMPK activation leading to a metabolic shift from glycolysis to autophagy and lipid oxidation in T cells receiving PD-1 signals. Furthermore, our findings provide a mechanistic explanation for the survival and persistence of PD-1-exhausted T cells in patients with chronic infections and tumors and identify novel targets that can be exploited therapeutically to overcome PD-1-mediated T cell exhaustion. Disclosures: No relevant conflicts of interest to declare.

Programmed death (PD)-1 is a potent T cell inhibitor critical for peripheral tolerance. Currently, it is unclear whether PD-1 has a global or a selective inhibitory effect on TCR signaling pathways involved in T cell expansion. It is also unclear how PD-1 affects the molecular machinery of cell cycle progression. Using primary human T cells we determined that PD-1 blocked cell cycle progression in the G1 phase. PD-1 did not alter the expression of G1 phase cyclins or cyclin-dependent kinases (Cdks) but, instead, suppressed transcription of the SCFSkp2 ubiquitin ligase that leads p27kip1 to degradation and resulted in accumulation of p27kip1. T cells receiving PD-1 signals displayed impaired Cdk2 activation and failed to phosphorylate Rb and Smad3 leading to suppression of E2F target genes but enhanced Smad3 transactivation. These events resulted in abrogation of the Cdk-activating phosphatase Cdc25A and upregulation of the Cdk4/6 inhibitor p15INK4. Suppression of Skp2 by PD-1 was mediated by inhibition of both PI3K/Akt and Ras/MEK/Erk pathways and was only partially reversed by IL-2, which restored activation of MEK/Erk but not Akt. In contrast to the inhibition of Ras and MEK/Erk pathway, PD-1 did not affect activation of Rap1, p38 and JNK MAP kinases. Thus, PD-1 selectively targets Ras/MEK/Erk and PI3K/Akt signaling to inhibit transcription of Skp2 and to activate Smad3 as an integral component of a pathway regulating blockade of cell cycle progression in T lymphocytes.

Immune suppression by T regulatory (Treg) cells is required for peripheral T cell tolerance. Treg cells display plasticity and are subjected to regulation by other immune cells. We determined previously that co-culture of CD4+CD25- cells and Treg cells in the presence of antigen presenting cells induced Treg cells producing IL-17 (named Tr17 cells). In the present study, we investigated the conditions required for the generation of Tr17 cells and we analyzed their molecular and functional properties. We determined that both IL-1β and IL-2 were required for the development of Tr17 cells. Compared to IL-17- Treg cells, Tr17 cells expressed increased levels of RORγt and Runx1 while retaining expression of Foxp3. To gain insight into how IL-17 transcription is regulated in Treg cells, we performed CHIP assays and found that Runx1 was able to bind to the IL-17 promoter and enhancer. Tr17 cells displayed more potent suppressive activity compared to IL-17- Treg and responder cells co-cultured with Tr17 cells were more prone to cell death than those co-cultured with IL-17- Treg. Tr17 cells expressed increased levels of perforin and granzyme B relative to IL-17- Treg and used this pathway to induce killing of responder cells as determined by degranulation assay. Our findings suggest that Tr17 cells represent a subset of Treg cells that is generated under inflammatory conditions and may have a unique role in suppressing inflammatory responses by inducing effector cell killing.