Charles River Laboratories Research Articles

An Artificial Antigen-presenting Cell with Paracrine Delivery of IL-2 Impacts the Magnitude and Direction of the T Cell Response

Artificial antigen-presenting cells (aAPCs) are an emerging technology to induce therapeutic cellular immunity without the need for autologous antigen-presenting cells (APCs). To fully replace natural APCs, an optimized aAPC must present antigen (signal 1), provide costimulation (signal 2), and release cytokine (signal 3). Here we demonstrate that the spatial and temporal characteristics of paracrine release of IL-2 from biodegradable polymer aAPCs (now termed paAPCs) can significantly alter the balance in the activation and proliferation of CD8+ and CD4+ T cells. Paracrine delivery of IL-2 upon T cell contact with paAPCs induces significant IL-2 accumulation in the synaptic contact region. This accumulation increases CD25 (the inducible IL-2 Rα chain) on responding T cells and increases proliferation of CD8+ T cells in vitro to levels 10 times that observed with equivalent amounts of bulk IL-2. These CD8+ T cell responses critically depend upon close contact of T cells and the paAPCs and require sustained release of low levels of IL-2. The same conditions promote activation-induced cell death in CD4+ T cells. These findings provide insight into the response of T cell subsets to paracrine IL-2.

Phosphorylation of Podocalyxin (Ser415) Prevents RhoA and Ezrin Activation and Disrupts Its Interaction with the Actin Cytoskeleton

Podocalyxin (PC) is a polysialylated, anti-adhesin that is essential for maintaining foot process architecture and the integrity of the glomerular filtration barrier. We showed previously that PC is firmly attached to the actin cytoskeleton through ezrin, that in puromycin aminonucleoside (PAN)-mediated nephrosis the PC-ezrin-actin complex is disrupted, and that PC is uncoupled from actin. However, the precise mechanisms involved remained unknown. Here we show that detachment of PC from actin is regulated by phosphorylation of PC. PC is hyperphosphorylated at serines in PAN- and protamine sulfate (PS)-treated rat glomeruli. We determined that PC is a substrate of PKC and that the site of phosphorylation is Ser415, located within the juxtamembrane, ezrin-binding domain of the cytoplasmic tail of PC. Mutation of Ser415 to the phosphomimetic residues Glu (S415E) or Asp (S415D) interfered with direct binding of the PC cytoplasmic tail to ezrin in vitro. Moreover, stable expression of a phosphomimetic (S415E) PC mutant but not the WT or the phosphorylation-deficient (S415A) PC mutant, disrupted PC-ezrin-actin interaction, failed to activate RhoA, and the cytoskeletal linker, ezrin, remained inactive. Our data indicate that phosphorylation of PC at Ser415 prevents attachment of PC and ezrin to actin and highlights the strategic position of Ser415 and direct binding of PC to ezrin in regulating podocyte foot process architecture.

Arrested Oocyst Maturation in Plasmodium Parasites Lacking Type II NADH:Ubiquinone Dehydrogenase

The Plasmodium mitochondrial electron transport chain has received considerable attention as a potential target for new antimalarial drugs. Atovaquone, a potent inhibitor of Plasmodium cytochrome bc(1), in combination with proguanil is recommended for chemoprophylaxis and treatment of malaria. The type II NADH:ubiquinone oxidoreductase (NDH2) is considered an attractive drug target, as its inhibition is thought to lead to the arrest of the mitochondrial electron transport chain and, as a consequence, pyrimidine biosynthesis, an essential pathway for the parasite. Using the rodent malaria parasite Plasmodium berghei as an in vivo infection model, we studied the role of NDH2 during Plasmodium life cycle progression. NDH2 can be deleted by targeted gene disruption and, thus, is dispensable for the pathogenic asexual blood stages, disproving the candidacy for an anti-malarial drug target. After transmission to the insect vector, NDH2-deficient ookinetes display an intact mitochondrial membrane potential. However, ndh2(-) parasites fail to develop into mature oocysts in the mosquito midgut. We propose that Plasmodium blood stage parasites rely on glycolysis as the main ATP generating process, whereas in the invertebrate vector, a glucose-deprived environment, the malaria parasite is dependent on an intact mitochondrial respiratory chain.

Targeted Training of Ultrasonic Vocalizations in Aged and Parkinsonian Rats

Voice deficits are a common complication of both Parkinson disease (PD) and aging; they can significantly diminish quality of life by impacting communication abilities. 1, 2 Targeted training (speech/voice therapy) can improve specific voice deficits,3, 4 although the underlying mechanisms of behavioral interventions are not well understood. Systematic investigation of voice deficits and therapy should consider many factors that are difficult to control in humans, such as age, home environment, age post-onset of disease, severity of disease, and medications. The method presented here uses an animal model of vocalization that allows for systematic study of how underlying sensorimotor mechanisms change with targeted voice training. The ultrasonic recording and analysis procedures outlined in this protocol are applicable to any investigation of rodent ultrasonic vocalizations. The ultrasonic vocalizations of rodents are emerging as a valuable model to investigate the neural substrates of behavior.5-8 Both rodent and human vocalizations carry semiotic value and are produced by modifying an egressive airflow with a laryngeal constriction.9, 10 Thus, rodent vocalizations may be a useful model to study voice deficits in a sensorimotor context. Further, rat models allow us to study the neurobiological underpinnings of recovery from deficits with targeted training. To model PD we use Long-Evans rats (Charles River Laboratories International, Inc.) and induce parkinsonism by a unilateral infusion of 7 μg of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle which causes moderate to severe degeneration of presynaptic striatal neurons (for details see Ciucci, 2010).11, 12 For our aging model we use the Fischer 344/Brown Norway F1 (National Institute on Aging). Our primary method for eliciting vocalizations is to expose sexually-experienced male rats to sexually receptive female rats. When the male becomes interested in the female, the female is removed and the male continues to vocalize. By rewarding complex vocalizations with food or water, both the number of complex vocalizations and the rate of vocalizations can be increased (Figure 1). An ultrasonic microphone mounted above the male's home cage records the vocalizations. Recording begins after the female rat is removed to isolate the male calls. Vocalizations can be viewed in real time for training or recorded and analyzed offline. By recording and acoustically analyzing vocalizations before and after vocal training, the effects of disease and restoration of normal function with training can be assessed. This model also allows us to relate the observed behavioral (vocal) improvements to changes in the brain and neuromuscular system.

Targeted Training of Ultrasonic Vocalizations in Aged and Parkinsonian Rats

Voice deficits are a common complication of both Parkinson disease (PD) and aging; they can significantly diminish quality of life by impacting communication abilities. 1, 2 Targeted training (speech/voice therapy) can improve specific voice deficits,3, 4 although the underlying mechanisms of behavioral interventions are not well understood. Systematic investigation of voice deficits and therapy should consider many factors that are difficult to control in humans, such as age, home environment, age post-onset of disease, severity of disease, and medications. The method presented here uses an animal model of vocalization that allows for systematic study of how underlying sensorimotor mechanisms change with targeted voice training. The ultrasonic recording and analysis procedures outlined in this protocol are applicable to any investigation of rodent ultrasonic vocalizations.The ultrasonic vocalizations of rodents are emerging as a valuable model to investigate the neural substrates of behavior.5-8 Both rodent and human vocalizations carry semiotic value and are produced by modifying an egressive airflow with a laryngeal constriction.9, 10 Thus, rodent vocalizations may be a useful model to study voice deficits in a sensorimotor context. Further, rat models allow us to study the neurobiological underpinnings of recovery from deficits with targeted training.To model PD we use Long-Evans rats (Charles River Laboratories International, Inc.) and induce parkinsonism by a unilateral infusion of 7 μg of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle which causes moderate to severe degeneration of presynaptic striatal neurons (for details see Ciucci, 2010).11, 12 For our aging model we use the Fischer 344/Brown Norway F1 (National Institute on Aging).Our primary method for eliciting vocalizations is to expose sexually-experienced male rats to sexually receptive female rats. When the male becomes interested in the female, the female is removed and the male continues to vocalize. By rewarding complex vocalizations with food or water, both the number of complex vocalizations and the rate of vocalizations can be increased (Figure 1).An ultrasonic microphone mounted above the male's home cage records the vocalizations. Recording begins after the female rat is removed to isolate the male calls. Vocalizations can be viewed in real time for training or recorded and analyzed offline. By recording and acoustically analyzing vocalizations before and after vocal training, the effects of disease and restoration of normal function with training can be assessed. This model also allows us to relate the observed behavioral (vocal) improvements to changes in the brain and neuromuscular system.

Image-based Screening Identifies Novel Roles for IκB Kinase and Glycogen Synthase Kinase 3 in Axonal Degeneration

Axon degeneration is an active, evolutionarily conserved self-destruction program by which compromised axons fragment in response to varied insults. Unlike programmed cell death, axon degeneration is poorly understood. We have combined robotic liquid handling with automated microscopy and image analysis to create a robust screening platform to measure axon degeneration in mammalian primary neuronal cultures. Using this assay, we performed an unbiased screen of 480 bioactive compounds, identifying 11 that reproducibly delay fragmentation of severed axons in vitro, including two inhibitors of glycogen synthase kinase 3 and two inhibitors of IκB kinase. Knockdown of each of these targets by shRNA lentivirus also delays axon degeneration in vitro, further supporting their role in the axon degeneration program.

Minocycline Attenuates Photoreceptor Degeneration in a Mouse Model of Subretinal Hemorrhage: Microglial Inhibition as a Potential Therapeutic Strategy

Minocycline Attenuates Photoreceptor Degeneration in a Mouse Model of Subretinal Hemorrhage: Microglial Inhibition as a Potential Therapeutic Strategy

Functional Specializations of Intestinal Dendritic Cell and Macrophage Subsets That Control Th17 and Regulatory T Cell Responses Are Dependent on the T Cell/APC Ratio, Source of Mouse Strain, and Regional Localization

Although several subsets of intestinal APCs have been described, there has been no systematic evaluation of their phenotypes, functions, and regional localization to date. In this article, we used 10-color flow cytometry to define the major APC subsets in the small and large intestine lamina propria. Lamina propria APCs could be subdivided into CD11c(+)CD11b(-), CD11c(+)CD11b(+), and CD11c(dull)CD11b(+) subsets. CD11c(+)CD11b(-) cells were largely CD103(+)F4/80(-) dendritic cells (DCs), whereas the CD11c(+)CD11b(+) subset comprised CD11c(+)CD11b(+)CD103(+)F4/80(-) DCs and CD11c(+)CD11b(+)CD103(-)F4/80(+) macrophage-like cells. The majority of CD11c(dull)CD11b(+) cells were CD103(-)F4/80(+) macrophages. Although macrophages were more efficient at inducing Foxp3(+) regulatory T (T(reg)) cells than DCs, at higher T cell/APC ratios, all of the DC subsets efficiently induced Foxp3(+) T(reg) cells. In contrast, only CD11c(+)CD11b(+)CD103(+) DCs efficiently induced Th17 cells. Consistent with this, the regional distribution of CD11c(+)CD11b(+)CD103(+) DCs correlated with that of Th17 cells, with duodenum > jejunum > ileum > colon. Conversely, CD11c(+)CD11b(-)CD103(+) DCs, macrophages, and Foxp3(+) T(reg) cells were most abundant in the colon and scarce in the duodenum. Importantly, however, the ability of DC and macrophage subsets to induce Foxp3(+) T(reg) cells versus Th17 cells was strikingly dependent on the source of the mouse strain. Thus, DCs from C57BL/6 mice from Charles River Laboratories (that have segmented filamentous bacteria, which induce robust levels of Th17 cells in situ) were more efficient at inducing Th17 cells and less efficient at inducing Foxp3(+) T(reg) cells than DCs from B6 mice from The Jackson Laboratory. Thus, the functional specializations of APC subsets in the intestine are dependent on the T cell/APC ratio, regional localization, and source of the mouse strain.

Disruption of a Ciliary B9 Protein Complex Causes Meckel Syndrome

Nearly every ciliated organism possesses three B9 domain-containing proteins: MKS1, B9D1, and B9D2. Mutations in human MKS1 cause Meckel syndrome (MKS), a severe ciliopathy characterized by occipital encephalocele, liver ductal plate malformations, polydactyly, and kidney cysts. Mouse mutations in either Mks1 or B9d2 compromise ciliogenesis and result in phenotypes similar to those of MKS. Given the importance of these two B9 proteins to ciliogenesis, we examined the role of the third B9 protein, B9d1. Mice lacking B9d1 displayed polydactyly, kidney cysts, ductal plate malformations, and abnormal patterning of the neural tube, concomitant with compromised ciliogenesis, ciliary protein localization, and Hedgehog (Hh) signal transduction. These data prompted us to screen MKS patients for mutations in B9D1 and B9D2. We identified a homozygous c.301A>C (p.Ser101Arg) B9D2 mutation that segregates with MKS, affects an evolutionarily conserved residue, and is absent from controls. Unlike wild-type B9D2 mRNA, the p.Ser101Arg mutation failed to rescue zebrafish phenotypes induced by the suppression of b9d2. With coimmunoprecipitation and mass spectrometric analyses, we found that Mks1, B9d1, and B9d2 interact physically, but that the p.Ser101Arg mutation abrogates the ability of B9d2 to interact with Mks1, further suggesting that the mutation compromises B9d2 function. Our data indicate that B9d1 is required for normal Hh signaling, ciliogenesis, and ciliary protein localization and that B9d1 and B9d2 are essential components of a B9 protein complex, disruption of which causes MKS.

Studies in mice, hamsters, and rats demonstrate that repression of hepatic apoA-I expression by taurocholic acid in mice is not mediated by the farnesoid-X-receptor

It is claimed that apoA-I expression is repressed in mice by cholic acid (CA) and its taurine conjugate, taurocholic acid (TCA) via farnesoid X receptor (FXR) activation. We measured apoA-I expression in mice, hamsters, and rats treated with highly potent and selective synthetic FXR agonists or with TCA. All of the synthetic agonists bound to FXR with high affinity in a scintillation proximity assay. However, TCA did not compete with the radioligand up to the highest concentration used (100 μM). The C-site regulatory region of apoA-I, through which FXR has been reported to regulate its expression, is completely conserved across the species investigated. In both male and female human apoA-I-transgenic mice, we reproduced the previously reported strong inhibition of human apoA-I expression upon treatment with the typical supraphysiological dose of TCA used in such studies. However, in contrast to some previous reports, TCA did not repress murine apoA-I expression in the same mice. Also, more-potent and -selective FXR agonists did not affect human or murine apoA-I expression in this model. In LDL receptor-deficient mice and Golden Syrian hamsters, selective FXR agonists did not affect apoA-I expression, whereas in Wistar rats, some even increased apoA-I expression. In conclusion, selective FXR agonists do not repress apoA-I expression in rodents. Repression of human apoA-I expression by TCA in transgenic mice is probably mediated through FXR-independent mechanisms.

Hydrogen Rich Water Prevents Progression of NASH and Accompanying Hepatic Tumorigenesis in Mice

(Background/Aims) Non-alcoholic fatty liver disease (NAFLD) is one of the common causes of chronic liver disease and a major sign of metabolic syndrome. Non-alcoholic steatohepatitis(NASH) is a more severe form of NAFLD and is broadly defined by the presence of steatosis with inflammation and progressive fibrosis, and can ultimately lead to cirrhosis and hepatocellular carcinoma (HCC). The prevalence of NAFLD is gradually increasing. It is known that the presence of reactive oxygen stress (ROS) plays important role for the onset of NASH. Molecular hydrogen has recently been shown to have therapeutic value as an antioxidant through its ability to reduce cytotoxic ROS. Therefore, it may be of potential therapeutic in the treatment of ROS-induced pathologies. However, there is no report that demonstrates the effects of hydrogen for chronic liver disease such as NASH. In this study, we evaluated the effects of hydrogen rich water in the NASH model mouse. (Method) A methionine-choline-deficient (MCD) diet mouse NASH model was introduced. Eight-weekold C57BL/6 mice were divided into 3 experimental groups and fed for 8 weeks as follows : (1) MCD diet + normal water (NW group) (2) MCD diet + hydrogen rich water (HW group) (3) MCD diet mixed with 0.01% of the PPAR-agonist pioglitazone + normal water (PGZ group). Hepatic inflammatory grade and fibrosis stage of each group was compared histologically. Hepatic 8-OHdG concentrationwasmeasured by commercially available ELISA kit. Real time PCR was administered for quantifying the expression of hepatic inflammation genes such as TNF-α and IL-6, and lipogenic genes such as fatty acid synthase (FAS), fatty acid translocase (FAT), and fatty acid transport protein (FATP). Next, a STAM mouse NASH hepatocellular carcinoma model (purchased from Charles River Laboratories Japan Inc.) was introduced. This model mice were divided into 2 experimental groups and fed for 8 weeks as follows: (1) High fat ( HF ) diet + normal water (NW-STAM group) (2) HF diet + hydrogen rich water (HW-STAM group). After 8 weeks, mice were sacrificed and counted for hepatic tumor number. (Results) HW group and PGZ group showed low inflammation and fibrosis compared with NW group histologically. The concentration of 8-OHdG was reduced in HW and PGZ group compared with NW group. The hepatic mRNA expression of TNF-α, IL-6, FAS, FAT, and FATP were also downregulated in HW and PGZ groups. Hepatic tumor number was reduced in HW-STAM group compared with NW-STAM group. (Conclusion) Hydrogen rich water reduces hepatic lipogenesis, oxidative stress and results in improvement of NASH and accompanying hepatic tumors.

Simultaneous Measurement Of Blood Pressure, Electrocardiogram And Temperature In A Single Mouse Now Possible For Chronic Cardiovascular Applications

Until recently, researchers collecting chronic cardiovascular (CV) data from mice have had to choose between blood pressure (BP) and electrocardiogram (ECG) transmitters; prioritizing the signal of most interest or choosing to implant parallel animal groups to capture both signals. A new device enabling simultaneous collection of BP, ECG, temperature (temp) and locomotor activity in mice eliminates the need for separate groups and allows for a more complete CV assessment in mice. Six male mice (C57BL6/J) were implanted with the HD-X11 (DSI, St. Paul, MN). The transmitter was implanted in the peritoneal cavity to record core body temp and the pressure-sensing catheter was subcutaneously routed to the throat area where an incision was made to access the left carotid artery. Sensing leads were externalized from the peritoneal cavity and placed in a lead II configuration. Chronic data collection over a one-month period demonstrated that the HD-X11 provides a more refined approach to mouse CV studies by eliminating variability associated with collecting from two separate groups of mice. Furthermore, it reduces the number of animals, transmitters and supporting materials used and provides a more complete CV assessment for applications such as heart failure or myocardial infarction. DSI provided all supplies and monitoring equipment required for the study; mice were obtained from Charles River Laboratory.

Involvement of Autophagy in Oncogenic K-Ras-induced Malignant Cell Transformation

Autophagy has recently been implicated in both the prevention and progression of cancer. However, the molecular basis for the relationship between autophagy induction and the initial acquisition of malignancy is currently unknown. Here, we provide the first evidence that autophagy is essential for oncogenic K-Ras (K-RasV12)-induced malignant cell transformation. Retroviral expression of K-RasV12 induced autophagic vacuole formation and malignant transformation in human breast epithelial cells. Interestingly, pharmacological inhibition of autophagy completely blocked K-RasV12-induced, anchorage-independent cell growth on soft agar. Both mRNA and protein levels of ATG5 and ATG7 (autophagy-specific genes 5 and 7, respectively) were increased in cells overexpressing K-RasV12. Targeted suppression of ATG5 or ATG7 expression by short hairpin (sh) RNA inhibited cell growth on soft agar and tumor formation in nude mice. Moreover, inhibition of reactive oxygen species (ROS) with antioxidants clearly attenuated K-RasV12-induced ATG5 and ATG7 induction, autophagy, and malignant cell transformation. MAPK pathway components were activated in cells overexpressing K-RasV12, and inhibition of JNK blunted induction of ATG5 and ATG7 and subsequent autophagy. In addition, pretreatment with antioxidants completely inhibited K-RasV12-induced JNK activation. Our results provide novel evidence that autophagy is critically involved in malignant transformation by oncogenic K-Ras and show that reactive oxygen species-mediated JNK activation plays a causal role in autophagy induction through up-regulation of ATG5 and ATG7.

Aβ42 Neurotoxicity Is Mediated by Ongoing Nucleated Polymerization Process Rather than by Discrete Aβ42 Species

The identification of toxic Aβ species and/or the process of their formation is crucial for understanding the mechanism(s) of Aβ neurotoxicity in Alzheimer disease and also for the development of effective diagnostic and therapeutic interventions. To elucidate the structural basis of Aβ toxicity, we developed different procedures to isolate Aβ species of defined size and morphology distribution, and we investigated their toxicity in different cell lines and primary neurons. We observed that crude Aβ42 preparations, containing a monomeric and heterogeneous mixture of Aβ42 oligomers, were more toxic than purified monomeric, protofibrillar fractions, or fibrils. The toxicity of protofibrils was directly linked to their interactions with monomeric Aβ42 and strongly dependent on their ability to convert into amyloid fibrils. Subfractionation of protofibrils diminished their fibrillization and toxicity, whereas reintroduction of monomeric Aβ42 into purified protofibril fractions restored amyloid formation and enhanced their toxicity. Selective removal of monomeric Aβ42 from these preparations, using insulin-degrading enzyme, reversed the toxicity of Aβ42 protofibrils. Together, our findings demonstrate that Aβ42 toxicity is not linked to specific prefibrillar aggregate(s) but rather to the ability of these species to grow and undergo fibril formation, which depends on the presence of monomeric Aβ42. These findings contribute significantly to the understanding of amyloid formation and toxicity in Alzheimer disease, provide novel insight into mechanisms of Aβ protofibril toxicity, and important implications for designing anti-amyloid therapies.

Myo1c Regulates Glucose Uptake in Mouse Skeletal Muscle

Contraction and insulin promote glucose uptake in skeletal muscle through GLUT4 translocation to cell surface membranes. Although the signaling mechanisms leading to GLUT4 translocation have been extensively studied in muscle, the cellular transport machinery is poorly understood. Myo1c is an actin-based motor protein implicated in GLUT4 translocation in adipocytes; however, the expression profile and role of Myo1c in skeletal muscle have not been investigated. Myo1c protein abundance was higher in more oxidative skeletal muscles and heart. Voluntary wheel exercise (4 weeks, 8.2 ± 0.8 km/day), which increased the oxidative profile of the triceps muscle, significantly increased Myo1c protein levels by ∼2-fold versus sedentary controls. In contrast, high fat feeding (9 weeks, 60% fat) significantly reduced Myo1c by 17% in tibialis anterior muscle. To study Myo1c regulation of glucose uptake, we expressed wild-type Myo1c or Myo1c mutated at the ATPase catalytic site (K111A-Myo1c) in mouse tibialis anterior muscles in vivo and assessed glucose uptake in vivo in the basal state, in response to 15 min of in situ contraction, and 15 min following maximal insulin injection (16.6 units/kg of body weight). Expression of wild-type Myo1c or K111A-Myo1c had no effect on basal glucose uptake. However, expression of wild-type Myo1c significantly increased contraction- and insulin-stimulated glucose uptake, whereas expression of K111A-Myo1c decreased both contraction-stimulated and insulin-stimulated glucose uptake. Neither wild-type nor K111A-Myo1c expression altered GLUT4 expression, and neither affected contraction- or insulin-stimulated signaling proteins. Myo1c is a novel mediator of both insulin-stimulated and contraction-stimulated glucose uptake in skeletal muscle.

Palmitoylation Controls Dopamine Transporter Kinetics, Degradation, and Protein Kinase C-dependent Regulation

Palmitoylation is a lipid modification that confers diverse functions to target proteins and is a contributing factor for many neuronal diseases. In this study, we demonstrate using [(3)H]palmitic acid labeling and acyl-biotinyl exchange that native and expressed dopamine transporters (DATs) are palmitoylated, and using the palmitoyl acyltransferase inhibitor 2-bromopalmitate (2BP), we identify several associated functions. Treatment of rat striatal synaptosomes with 2BP using lower doses or shorter times caused robust inhibition of transport V(max) that occurred with no losses of DAT protein or changes in DAT surface levels, indicating that acute loss of palmitoylation leads to reduction of transport kinetics. Treatment of synaptosomes or cells with 2BP using higher doses or longer times resulted in DAT protein losses and production of transporter fragments, implicating palmitoylation in regulation of transporter degradation. Site-directed mutagenesis indicated that palmitoylation of rat DAT occurs at Cys-580 at the intracellular end of transmembrane domain 12 and at one or more additional unidentified site(s). Cys-580 mutation also led to production of transporter degradation fragments and to increased phorbol ester-induced down-regulation, further supporting palmitoylation in opposing DAT turnover and in opposing protein kinase C-mediated regulation. These results identify S-palmitoylation as a major regulator of DAT properties that could significantly impact acute and long term dopamine transport capacity.

Increased Expression of P-Glycoprotein Is Associated with Doxorubicin Chemoresistance in the Metastatic 4T1 Breast Cancer Model

Development of drug resistance is one of the major causes of breast cancer treatment failure. The goal of this study was to understand the chemoresistance mechanism using the highly metastatic 4T1 breast cancer model, which emulates stage IV breast cancer in humans. The metastatic 4T1 breast cancer cell line treated with either doxorubicin or 5-FU showed a concentration-dependent reduced cell proliferation, with induced G2-phase growth arrest (doxorubicin) or G1-phase growth arrest (5-FU). Doxorubicin treatment partially suppressed the multiorgan metastasis of 4T1 breast cancer cells in the lung, heart, liver, and bone, compared with either 5-FU or cyclophosphamide. We isolated and characterized 4T1 breast cancer cells from doxorubicin-resistant metastatic tumors (cell line 4T1-R). Multiorgan metastasis of drug-resistant 4T1 breast tumors was totally resistant to doxorubicin treatment. Our results indicate that doxorubicin is localized exclusively in the cytoplasm of resistant 4T1 breast cancer cells and that it cannot reach the nucleus because of increased nuclear expression of P-glycoprotein. Pretreatment of doxorubicin-resistant 4T1-R breast cancer cells with verapamil, a general inhibitor of P-glycoprotein, increased nuclear translocation of doxorubicin and cellular cytotoxicity. Thus, impaired nuclear translocation of doxorubicin due to increased expression of P-glycoprotein is associated with doxorubicin resistance of highly metastatic 4T1 breast cancer.

Mast Cells Are an Essential Component of Human Radiation Proctitis and Contribute to Experimental Colorectal Damage in Mice

Radiation proctitis is characterized by mucosal inflammation followed by adverse chronic tissue remodeling and is associated with substantial morbidity and mortality. Mast cell hyperplasia has been associated with diseases characterized by pathological tissue remodeling and fibrosis. Rectal tissue from patients treated with radiotherapy shows mast cell hyperplasia and activation, suggesting that these cells play a role in the development of radiation-induced sequelae. To investigate the role of mast cells in radiation damage, experimental radiation proctitis was induced in a mast cell-deficient (W(sh)/W(sh)) mouse model. The colon and rectum of W(sh)/W(sh) and wild-type mice were exposed to 27-Gy single-dose irradiation and studied after 2 and 14 weeks. Irradiated rodent rectum showed mast cell hyperplasia. W(sh)/W(sh) mice developed less acute and chronic rectal radiation damage than their control littermates. Tissue protection was associated with increased tissue neutrophil influx and expression of several inflammatory mediators immediately after radiation exposure. It was further demonstrated that mast cell chymase, tryptase, and histamine could change human muscularis propria smooth muscle cells into a migrating/proliferating and proinflammatory phenotype. These data show that mast cells have deleterious effects on both acute and chronic radiation proctitis, possibly by limiting acute tissue neutrophil influx and by favoring phenotypic orientation of smooth muscle cells, thus making them active participants in the radiation-induced inflammatory process and dystrophy of the rectal wall.

STAT1 Is a Master Regulator of Pancreatic β-Cell Apoptosis and Islet Inflammation

Cytokines produced by islet-infiltrating immune cells induce β-cell apoptosis in type 1 diabetes. The IFN-γ-regulated transcription factors STAT1/IRF-1 have apparently divergent effects on β-cells. Thus, STAT1 promotes apoptosis and inflammation, whereas IRF-1 down-regulates inflammatory mediators. To understand the molecular basis for these differential outcomes within a single signal transduction pathway, we presently characterized the gene networks regulated by STAT1 and IRF-1 in β-cells. This was done by using siRNA approaches coupled to microarray analysis of insulin-producing cells exposed or not to IL-1β and IFN-γ. Relevant microarray findings were further studied in INS-1E cells and primary rat β-cells. STAT1, but not IRF-1, mediates the cytokine-induced loss of the differentiated β-cell phenotype, as indicated by decreased insulin, Pdx1, MafA, and Glut2. Furthermore, STAT1 regulates cytokine-induced apoptosis via up-regulation of the proapoptotic protein DP5. STAT1 and IRF-1 have opposite effects on cytokine-induced chemokine production, with IRF-1 exerting negative feedback inhibition on STAT1 and downstream chemokine expression. The present study elucidates the transcriptional networks through which the IFN-γ/STAT1/IRF-1 axis controls β-cell function/differentiation, demise, and islet inflammation.

Peroxisome Proliferator-activated Receptor α Is Responsible for the Up-regulation of Hepatic Glucose-6-phosphatase Gene Expression in Fasting and db/db Mice

Glucose-6-phosphatase (G6Pase) is a key enzyme that is responsible for the production of glucose in the liver during fasting or in type 2 diabetes mellitus (T2DM). During fasting or in T2DM, peroxisome proliferator-activated receptor α (PPARα) is activated, which may contribute to increased hepatic glucose output. However, the mechanism by which PPARα up-regulates hepatic G6Pase gene expression in these states is not well understood. We evaluated the mechanism by which PPARα up-regulates hepatic G6Pase gene expression in fasting and T2DM states. In PPARα-null mice, both hepatic G6Pase and phosphoenolpyruvate carboxykinase levels were not increased in the fasting state. Moreover, treatment of primary cultured hepatocytes with Wy14,643 or fenofibrate increased the G6Pase mRNA level. In addition, we have localized and characterized a PPAR-responsive element in the promoter region of the G6Pase gene. Chromatin immunoprecipitation (ChIP) assay revealed that PPARα binding to the putative PPAR-responsive element of the G6Pase promoter was increased in fasted wild-type mice and db/db mice. These results indicate that PPARα is responsible for glucose production through the up-regulation of hepatic G6Pase gene expression during fasting or T2DM animal models.