Western Immunoblot Analysis Research Articles

Sortilin is associated with the chlamydial inclusion and is modulated during infection.

ABSTRACTChlamydia species are obligate intracellular pathogens that have a major impact on human health. The pathogen replicates within an intracellular niche called an inclusion and is thought to rely heavily on host-derived proteins and lipids, including ceramide. Sortilin is a transmembrane receptor implicated in the trafficking of acid sphingomyelinase, which is responsible for catalysing the breakdown of sphingomyelin to ceramide. In this study, we examined the role of sortilin in Chlamydia trachomatis L2 development. Western immunoblotting and immunocytochemistry analysis revealed that endogenous sortilin is not only associated with the inclusion, but that protein levels increase in infected cells. RNAi-mediated depletion of sortilin, however, had no detectable impact on ceramide delivery to the inclusion or the production of infectious progeny. This study demonstrates that whilst Chlamydia redirects sortilin trafficking to the chlamydial inclusion, RNAi knockdown of sortilin expression is insufficient to determine if this pathway is requisite for the development of the pathogen.

144 Expression of glucocorticoid receptors, androgen receptors and its splice variants in prostate cancer: Comparison between hormone dependent and castrate-resistant prostate cancer

protein 4 (Reg IV) as a biomarker of castration-resistant prostate cancer (CRPC). METHODS: The serum Reg IV level in 39 prostate cancer (PC) patients, 36 renal cell cancer (RCC) patients, 12 urothelial cancer (UC) patients, and 14 non-cancerous subjects was measured using an enzyme-linked immunosorbent (ELISA) assay. The cell viability of PC3 and LNCaP stably transfected with Reg IV cDNA (PC3-reg and LN-reg, respectively) was examined using an MTT assay. The expression of molecules related to epithelial-mesenchymal transition (EMT), neuroendocrine differentiation, and apoptosis in these cells was examined using western immunoblot analysis. RESULTS: Serum concentration levels of Reg IV in patients with PC, RCC, or UC were significantly higher than those in noncancerous subjects (p1⁄40.0131, p1⁄40.0122 and p1⁄40.0002, respectively). The levels with metastatic PC and metastatic RCC were significantly higher than those with early stages in each of the diseases (p1⁄40.0051 and p1⁄40.0006, respectively). A significant correlation was found between the serum Reg IV and PSA level in androgen-naive prostate cancer patients (p1⁄40.0059, R1⁄40.519). The level in CRPC patients was significantly higher than that in the androgen-naive prostate cancer patients (p1⁄40.0202) and in the non-cancerous subjects (p1⁄40.001). In all CRPC patients, it was higher than 2 ng/ml, which has been defined as the cut-off value in gastrointestinal cancer in previous studies. An EMT was induced in LN-reg. The expression of synaptophysin, NSE, and chromogranin A was upregulated in both LN-reg and PC3-reg compared with in vector control cells. The cell viability under androgen deprivation was enhanced in both LN-reg and PC3-reg, and the resistance of 5-fluorouracil and docetaxel was shown in PC3-reg. CONCLUSIONS: Reg IV is a candidate for a novel biomarker of CRPC, and it might represent the effect of therapeutic agents.

CARD9 knockout ameliorates myocardial dysfunction associated with high fat diet-induced obesity

Obesity is associated with chronic inflammation which plays a critical role in the development of cardiovascular dysfunction. Because the adaptor protein caspase recruitment domain-containing protein 9 (CARD9) in macrophages regulates innate immune responses via activation of pro-inflammatory cytokines, we hypothesize that CARD9 mediates the pro-inflammatory signaling associated with obesity en route to myocardial dysfunction.C57BL/6 wild-type (WT) and CARD9−/− mice were fed normal diet (ND, 12% fat) or a high fat diet (HFD, 45% fat) for 5months. At the end of 5-month HFD feeding, cardiac function was evaluated using echocardiography. Cardiomyocytes were isolated and contractile properties were measured. Immunofluorescence was performed to detect macrophage infiltration in the heart. Heart tissue homogenates, plasma, and supernatants from isolated macrophages were collected to measure the concentrations of pro-inflammatory cytokines using ELISA kits. Western immunoblotting analyses were performed on heart tissue homogenates and isolated macrophages to explore the underlying signaling mechanism(s). CARD9 knockout alleviated HFD-induced insulin resistance and glucose intolerance, prevented myocardial dysfunction with preserved cardiac fractional shortening and cardiomyocyte contractile properties. CARD9 knockout also significantly decreased the number of infiltrated macrophages in the heart with reduced myocardium-, plasma-, and macrophage-derived cytokines including IL-6, IL-1β and TNFα. Finally, CARD9 knockout abrogated the increase of p38 MAPK phosphorylation, the decrease of LC3BII/LC3BI ratio and the up-regulation of p62 expression in the heart induced by HFD feeding and restored cardiac autophagy signaling.In conclusion, CARD9 knockout ameliorates myocardial dysfunction associated with HFD-induced obesity, potentially through reduction of macrophage infiltration, suppression of p38 MAPK phosphorylation, and preservation of autophagy in the heart.

Effect of 5-fluorouracil on membranous PD-L1 expression in colon cancer cells.

592 Background: Colorectal cancer (CRC) is a major public health problem worldwide. Chemotherapy consisting of a fluoropyrimidine backbone constitutes a major therapeutic modality used in the treatment of CRC patients. However, cancer cells often develop resistance to such cytotoxic chemotherapy and this represents a major therapeutic challenge. B7-homolog 1 (B7-H1), also known as programmed death ligand-1 (PD-L1) is an immunoregulatory protein that belongs to the B7 family of T-cell co-regulatory molecules, and is overexpressed in several human tumors. Upregulation of PD-L1 expression is an important mechanism by which tumor cells can escape host T-cell immunity. Emerging evidence suggests that chemotherapeutic agents can regulate PD-L1 expression on cancer cells, which may have an impact on anti-tumor immunity and immune evasion. We performed this study to evaluate the effect of 5-Fluorouracil (5-FU) on PD-L1 expression in colon cancer cell lines, using an in vitro approach. Methods: Flow cytometry and western immunoblot analyses for PD-L1 expression were performed on human colon cancer cell lines (HCT116-WT, HCT116-p53 KO, SW480, HT29) upon treatment with IFN-ɣ and 5-FU. Results: We found that the tested human colon cancer cell lines rarely expressed PD-L1 protein on their cell surface at baseline, but a high level of expression was induced by treatment with IFN-ɣ. More importantly, we demonstrated that the chemotherapeutic agent 5-FU induces PD-L1 expression in colon cancer cells. Conclusions: It is therefore plausible that combining 5-FU with PD-1/PD-L1 blockade might help overcome some of the chemoresistance to 5-FU and thereby enhance its anti-cancer activity. Further experiments are being planned to formally test this hypothesis.

Temporary Immersion Bioreactors for the Contained Production of Recombinant Proteins in Transplastomic Plants.

Despite the largely maternal inheritance of plastid genomes, the risk of transgene dissemination from transplastomic plants can limit the scope for field cultivation. There is a need for a cost-effective, scalable process to grow large quantities of transplastomic plant biomass for biosynthesis of biopharmaceuticals and other high-value heterologous proteins. Temporary immersion culture is a means of achieving this under fully contained conditions. This method describes the organogenesis of transplastomic Nicotiana tabacum callus in RITA(®) temporary immersion bioreactors to produce rootless leafy biomass, and subsequent total soluble protein extraction, SDS-PAGE, and Western immunoblot analysis of heterologous protein expression. This method can be used for propagation of plastid or nuclear transformants, though is especially suitable for transplastomic biomass, as organogenesis leads to greater expression and accumulation of transplastomic proteins due to increases in chloroplast number and size.

Neurotrophic effects of amyloid precursor protein peptide 165 in vitro.

Diabetic encephalopathy is one of the risk factors for Alzheimer's disease. Our previous findings indicated that animals with diabetic encephalopathy exhibit learning and memory impairment in addition to hippocampal neurodegeneration, both of which are ameliorated with amyloid precursor protein (APP) 17-mer (APP17) peptide treatment. Although APP17 is neuroprotective, it is susceptible to enzymatic degradation. Derived from the active sequence structure of APP17, we have previously structurally transformed and modified several APP5-mer peptides (APP328-332 [RERMS], APP 5). We have developed seven different derivatives of APP5, including several analogs. Results from the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay on human neuroblastoma SH-SY5Y cells in the present study showed that P165 was the most neuroprotective APP5 derivative. Furthermore, we tested the effects of APP5 and P165 on the number of cells and the release of lactate dehydrogenase. Western immunoblot analyses were also performed. The digestion rates of P165 and APP5 were determined by the pepsin digestion test. P165 resisted pepsin digestion significantly more than APP5. Therefore, P165 may be optimal for oral administration. Overall, these findings suggest that P165 may be a potential drug for the treatment of diabetic encephalopathy.

Abstract 13458: Caspase Recruitment Domain-Containing Protein 9 Deficiency Mitigates Obesity-Induced Cardiac Dysfunction

Obesity has become an epidemic and is associated with cardiovascular anomalies. Obesity-induced low-grade chronic inflammation and macrophage infiltration into the heart plays a critical role in the development of cardiac dysfunction. Because the adaptor protein caspase recruitment domain-containing protein 9 (CARD9) regulates the innate immunity in macrophages via activation of pro-inflammatory cytokines, we hypothesized that CARD9 mediates chronic inflammation and plays a detrimental role in obesity-associated cardiac dysfunction. Male C57BL/6 wild-type (WT) and CARD9 knockout (CARD9-/-) mice were fed normal chow (ND, 12% fat) or a Western diet (WD, 45% fat) for 5 months starting at 4 weeks of age. At the end of 5-month WD feeding, cardiac geometry and function were evaluated using echocardiography. Immunofluorescence assay was performed to detect macrophage infiltration in the heart. Heart tissue homogenates, plasma, and supernatants from cultured macrophages were collected to measure the activities of pro-inflammatory cytokines by ELISA kits. Western immunoblotting analyses were performed on isolated peritoneal macrophages and heart tissue to dissect the pro-inflammatory signaling pathways. WD feeding induced insulin resistance and glucose intolerance in the WT mice and the effects were attenuated in the CARD9-/- mice. Myocardial fractional shortening and ejection fraction were significantly compromised in the WD-fed WT mice and CARD9 knockout preserved cardiac function. CARD9 knockout also significantly decreased the number of infiltrated macrophages in the heart. In addition, myocardium-, plasma- and macrophage-derived cytokines IL-6, IL-1β and TNFα were attenuated in the CARD9-/- mice compared to the WT mice. WD feeding resulted in increased protein phosphorylation levels of p38 MAPK in the heart and macrophages which were suppressed in the CARD9-/- mice. Taken together, these results suggest that CARD9 knockout ameliorates obesity-induced cardiac dysfunction, potentially through reduction of macrophage infiltration and suppression of p38 MAPK phosphorylation in the heart.

Serological diagnostic potential of recombinant outer membrane proteins (rOMPs) from Brucella melitensis in mouse model using indirect enzyme-linked immunosorbent assay.

BackgroundBrucella melitensis is the most important pathogenic species of Brucella spp. which affects goats and sheep and causes caprine and ovine brucellosis, respectively. Serological tests for diagnosis of brucellosis such as Rose Bengal plate test (RBPT) and enzyme-linked immunosorbent assay (ELISA) usually utilize smooth lipopolysaccharides (S-LPS) as a diagnostic antigen which could give false positive serological reactions. Outer membrane proteins (OMP) of B. melitensis have been used as alternative diagnostic antigens rather than S-LPS for differential serological diagnosis of brucellosis, mainly in ELISA with single recombinant OMP (rOMP) as a diagnostic antigen. Nevertheless, the use of single format mainly showed lack of sensitivity against the desired rOMP. Therefore, this study aimed to determine whether a newly developed rOMPs indirect ELISA (rOMPs I-ELISA), based on combination of rOMP25, rOMP28 and rOMP31of B. melitensis, has a potential benefit for use in the serodiagnosis of brucellosis.MethodsIn this study, omp25, omp28 and omp31 of B. melitensis were cloned and expressed using prokaryotic pET-32 Ek/LIC system and their respective rOMPs were combined as one coating antigen to develop rOMPs I-ELISA. Three groups of BALB/c mice were used to elicit antibody response. Group 1, infected with B. melitensis strain 0331 field strain; group 2, injected with B. melitensis Rev.1 vaccine strain and group 3, infected with Yersinia enterocolitica O:9. Antibody responses in three groups of mice were investigated using Rose Bengal plate test (RBPT) and rOMPs I-ELISA.ResultsThe production of rOMP25, rOMP28 and rOMP31 of B. melitensis were achieved and Western immunoblotting analysis demonstrated their reactivity. The RBPT was unable to differentiate the vaccinated mice (group 2) and mice infected with Y. enterocolitica O:9 (group 3) and categorized them wrongly as positive for brucellosis. In contrast, the rOMPs I-ELISA was able to differentiate the mice infected with B. melitensis strain 0331 (group 1) from both of group 2 and group 3, and recorded 100% sensitivity and 100% specificity. ConclusionsThe results of this study suggested that rOMPs of B. melitensis has potential diagnostic ability to differentiate the FPSR in serological diagnosis of brucellosis.

An in situ hybridization study of perlecan, DMP1, and MEPE in developing condylar cartilage of the fetal mouse mandible and limb bud cartilage.

The main purpose of this in situ hybridization study was to investigate mRNA expression of three bone/cartilage matrix components (perlecan, DMP1, and MEPE) in developing primary (tibial) and secondary (condylar) cartilage. Perlecan mRNA expression was first detected in newly formed chondrocytes in tibial cartilage at E13.0, but this expression decreased in hypertrophic chondrocytes at E14.0. In contrast, at E15.0, perlecan mRNA was first detected in the newly formed chondrocytes of condylar cartilage; these chondrocytes had characteristics of hypertrophic chondrocytes, which confirmed the previous observation that progenitor cells of developing secondary cartilage rapidly differentiate into hypertrophic chondrocytes. DMP1 mRNA was detected in many chondrocytes within the lower hypertrophic cell zone in tibial cartilage at E14.0. In contrast, DMP1 mRNA expression was only transiently detected in a few chondrocytes of condylar cartilage at E15.0. Thus, DMP1 may be less important in the developing condylar cartilage than in the tibial cartilage. Another purpose of this study was to test the hypothesis that MEPE may be a useful marker molecule for cartilage. MEPE mRNA was not detected in any chondrocytes in either tibial or condylar cartilage; however, MEPE immunoreactivity was detected throughout the cartilage matrix. Western immunoblot analysis demonstrated that MEPE antibody recognized two bands, one of 67 kDa and another of 59 kDa, in cartilage-derived samples. Thus MEPE protein may gradually accumulate in the cartilage, even though mRNA expression levels were below the limits of detection of in situ hybridization. Ultimately, we could not designate MEPE as a marker molecule for cartilage, and would modify our original hypothesis.

The pathophysiology of pseudoexfoliation syndrome is affected by interaction of TGF‐β1 and LOXL1

PurposeThe cross‐linking enzyme lysyl oxidase‐like 1 (LOXL1) and profibrotic transforming growth factor (TGF)‐ß1 play key roles in pathophysiology of pseudoexfoliation (PEX) syndrome/glaucoma. The purpose of this study was to investigate the interaction between LOXL1 and TGF‐ß1 with respect to the PEX‐specific disordered matrix metabolism.MethodsPrimary human Tenon's capsule fibroblasts (hTCF) obtained from patients were treated with TGF‐ß1 (0‐10 ng/ml) for 12‐72 hours without or with preincubation with inhibitors of TGF‐β signalling pathways. Expression of LOXL1 and PEX‐specific extracellular matrix components was examined by using quantitative RT‐PCR and Western immunoblot analysis. Direct binding of LOXL1 to TGF‐ß1 was analyzed by blot overlay assay and solid phase ELISA using purified LOXL1, recombinant human TGF‐ß1, TGFß1‐LAP. The effect of LOXL1 on TGF‐ß1 signaling was analyzed using TGF‐ß receptor signaling real time PCR assays (BioRad) after transient transfection of hTCF with a full‐length pCMV6‐LOXL1 vector construct/with empty vector .ResultsTGFß1 significantly increased LOXL1 expression, secretion and enzymatic activity and correlated with enhanced expression of BMP‐1, elastin, fibrillin‐1, fibulin‐4 and fibulin‐5 with peak effects at 10 ng/ml for 48 hours. This induction was blocked by TGF‐β receptor inhibitors and inhibitors of the canonical Smad and non‐canonical signaling pathways. Direct binding between LOXL1 and TGFß1‐LAP was demonstrated by Blot overlay assays and ELISA. LOXL1 overexpression temporarily upregulates different transcriptional regulators and some protein kinases of p38‐MAPK signalling pathway after 12 to 24 hours post‐transfection.ConclusionsThe results of this study indicate that the interaction of LOXL1 and TGF‐ß1 plays an important role in the PEX‐associated abnormal matrix metabolism and fibrosis.

Family with sequence similarity member 20C is the primary but not the only kinase for the small-integrin-binding ligand N-linked glycoproteins in bone.

Recent studies have identified family with sequence similarity member 20C (FAM20C) as a kinase that phosphorylates the Ser in Ser-X-Glu/phospho-Ser (pSer) motifs in the small-integrin-binding ligand N-linked glycoproteins (SIBLINGs). There is no in vivo evidence that validates this finding, and it is unclear whether FAM20C is the only kinase for SIBLINGs. We extracted bone noncollagenous proteins (NCPs) from Fam20C-knockout (KO) mice and analyzed the phosphorylation levels. The total NCPs were separated into osteopontin-, bone sialoprotein-, and dentin matrix protein-1-enriched fractions by anion-exchange chromatography and analyzed by SDS-PAGE, native PAGE, and Western immunoblot analysis. The NCP phosphorylation level in the KO mice was lower than that in the wild-type (WT). On the native gel, the SIBLINGs from KO mice showed a lower migration rate (Mr) than those from the WT. Calf intestine phosphatase treatment shifted SIBLINGs from the WT mice to the level adjacent to the KO, but failed to shift the latter, suggesting a phosphorylation loss of SIBLINGs in the KO mice. Mass spectrometry identified less pSers in the SIBLINGs from the KO mice [including the region of the acidic Ser- and aspartate-rich motif (ASARM) peptides]. In an intriguing finding, several pSers in the Ser-X-Glu motifs in the KO mice maintained their phosphorylation, whereas several others in non-Ser-X-Glu motifs did not. Phospho-Tyrs and phospho-Thrs in the SIBLINGs did not appear to be associated with FAM20C. Our results indicate that FAM20C is the primary, but not the only, kinase for the SIBLINGs.-Yang, X., Yan, W., Tian, Y., Ma, P., Opperman, L. A., Wang, X. Family with sequence similarity member 20C is the primary but not the only kinase for the small-integrin-binding ligand N-linked glycoproteins in bone.

Therapeutic effect of tPA in ischemic stroke is enhanced by its combination with normobaric oxygen and hypothermia or ethanol

Background and Purpose: Our lab has previously elucidated the neuroprotective effects of normobaric oxygen (NBO) and ethanol (EtOH) in ischemic stroke. The present study further evaluated the effect of EtOH or hypothermia (Hypo) in the presence of low concentration of NBO and determined whether EtOH can substitute hypothermia in a more clinically relevant autologous embolus rat stroke model in which reperfusion was established by tissue-type plasminogen activator (t-PA). Methods: At 1h of middle cerebral artery occlusion (MCAO) by an autologous embolus, rats received t-PA. In addition, at the same time, ischemic animals were treated with either EtOH (1.0g/kg) or hypothermia (33°C for 3h) in combination with NBO (60% for 3h). Extent of neuroprotection was assessed by apoptotic cell death measured by ELISA and Western immunoblotting analysis for pro- (AIF, activated Caspase-3, Bax) and anti-apoptotic (Bcl-2) protein expression at 3 and 24h of reperfusion induced by t-PA administration. Results: Compared to ischemic rats treated only with t-PA, animals with NBO, hypothermia or EtOH had significantly reduced apoptotic cell death by 32.5%, 43.1% and 36.0% respectively. Furthermore, combination therapy that included NBO+EtOH or NBO+Hypo with t-PA exhibited a much larger decline (p<0.01) in the cell death by 71.1% and 73.6%, respectively. Similarly, NBO+EtOH or NBO+Hypo treatment in addition to t-PA enhanced beneficial effects on both pro- and anti-apoptotic protein expressions as compared to other options. Conclusions: Neuroprotection after stroke can be enhanced by combination treatment with either EtOH or hypothermia in the presence of t-PA and 60% NBO. Because the effects produced by EtOH and hypothermia are comparable, their mechanism of action may be not only similar but also could be interchangeable in future clinical trials.

Abstract 4639: Metformin decreases cellular ceramides in MCF-7 and MDA-MB 231 breast cancer cell lines by inhibition of ceramide synthetic enzymes

Abstract Metformin, an anti-diabetic biguanide, has been shown to have cytotoxic and chemopreventive properties in in vitro and epidemiological studies, respectively. Metformin's predominant cytotoxic functions may be through activation of 5′ adenosine monophosphate-activated protein kinase (AMPK) with subsequent cell apoptosis. Ceramides, a group of waxy lipids found in the cellular membrane, are key players in many elements of cell signaling, including the regulation of cell differentiation, proliferation, and apoptosis. While traditionally thought of as being pro-apoptotic, this notion has been questioned with the discovery of pro-survival ceramides, as well as the demonstration that certain species of ceramides are increased in breast cancer cells. Metformin reduces ceramides in insulin-resistant mouse myoblasts and may reduce the vascular complications of diabetes mellitus. MCF-7 and MDA-MB-231 breast cancer cell lines were treated with increasing concentrations of metformin and cytotoxicity was determined by MTT cell culture experiments after 72 hours of drug exposure. Metformin induces cytotoxicity in these breast cancer cells at a lowest concentration of 1.25 mM, and percentage cytotoxicity increases in a dose-dependent manner. We then determined the effect of metformin on cell ceramide synthesis by analyzing key ceramide synthetic enzymes. Using western immunoblot and polymerase chain reaction (PCR) analysis, we determined that metformin induces a reduction in serine palmitoyl transferase, ceramide synthase, dihydroceramide desaturase, and neutral sphingomyelinase in MCF-7 and MDA-MB-231 breast cancer cells. We also analyzed the distribution of ceramides in treated breast cancer cells using polyclonal antibodies against ceramides. By confocal microscopy, ceramides are globally decreased in MCF-7 and MDA-MB-231 cells treated with metformin. This data suggests that the pro-apoptotic effect of metformin may be partly mediated through its disruption of the balance of ceramides and/or through the reduction of pro-survival ceramides within breast cancer cells. It is also possible that the predominant ceramides in MCF-7 and MDA-MB-231 breast cancer cells are pro-survival and metformin suppresses these ceramides. Further work is necessary to characterize the ceramide content of MCF-7 and MDA-MB-231 cancer cells before and after metformin treatment. Citation Format: Daniel Wann, Victoria Palau, Janet Lightner, Marianne Brannon, William Stone, Koyamangalath Krishnan. Metformin decreases cellular ceramides in MCF-7 and MDA-MB 231 breast cancer cell lines by inhibition of ceramide synthetic enzymes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4639. doi:10.1158/1538-7445.AM2015-4639

A flow cytometry-based method to simplify the analysis and quantification of protein association to chromatin in mammalian cells.

Protein accumulation on chromatin has traditionally been studied using immunofluorescence microscopy or biochemical cellular fractionation followed by western immunoblot analysis. As a way to improve the reproducibility of this kind of analysis, to make it easier to quantify and to allow a streamlined application in high-throughput screens, we recently combined a classical immunofluorescence microscopy detection technique with flow cytometry. In addition to the features described above, and by combining it with detection of both DNA content and DNA replication, this method allows unequivocal and direct assignment of cell cycle distribution of protein association to chromatin without the need for cell culture synchronization. Furthermore, it is relatively quick (takes no more than a working day from sample collection to quantification), requires less starting material compared with standard biochemical fractionation methods and overcomes the need for flat, adherent cell types that are required for immunofluorescence microscopy.

Activation of Protein Kinases and Phosphatases Coupled to Glutamate Receptors Regulates the Phosphorylation State of DARPP32 at Threonine 75 After Repeated Exposure to Cocaine in the Rat Dorsal Striatum in a Ca2+-Dependent Manner.

Background:Phosphorylation state of dopamine- and cAMP-regulated phosphoprotein, molecular weight 32kDa (DARPP32) is crucial to understand drug-mediated synaptic plasticity. In this study, mechanisms underlying repeated cocaine-stimulated phosphorylation of DARPP32 at threonine 75 (pDARPP32-Thr75) were determined by investigating the hypothesis that activation of protein kinases and phosphatases coupled to glutamate signaling is necessary for the regulation of pDARPP32-Thr75 after repeated cocaine administration.Methods:Intracaudate drug infusions into the rat dorsal striatum followed by Western immunoblot analysis were mainly performed to test this hypothesis.Results:The results demonstrated that 7 repeated daily intraperitoneal injections of cocaine (20mg/kg) upregulated the expression of pDARPP32-Thr75. Increases in the cytosolic Ca2+ concentrations followed by Ca2+-dependent protein kinase activation through stimulation of Ca2+ channels in striatal neurons were necessary for the phosphorylation. Activation of protein phosphatases further regulated the phosphorylation state by deactivating pDARPP32-Thr75 and upstream protein kinases.Conclusion:These findings suggest that activation of protein kinases and phosphatases coupled to glutamate receptors controls the phosphorylation state of DARPP32-Thr75 after repeated exposure to cocaine in the dorsal striatum in a Ca2+-dependent manner.

Activation of Wnt/β-catenin signalling promotes mesenchymal stem cells to repair injured alveolar epithelium induced by lipopolysaccharide in mice.

IntroductionMesenchymal stem cells (MSCs) have potential for re-epithelization and recovery in acute respiratory distress syndrome (ARDS). In a previous in vitro study, the results showed that the canonical Wnt/β-catenin pathway promoted the differentiation of MSCs into type II alveolar epithelial cells, conferred resistance to oxidative stress, and promoted their migration, suggesting that the Wnt/β-catenin pathway might be one of the key mechanisms underling the therapeutic effect of mouse MSCs in ARDS.MethodsMouse MSCs stable transfected with β-catenin or green fluorescent protein control were transplanted intratracheally into the ARDS mice induced by lipopolysaccharide. Lung tissue injury and repair assessment were examined using haematoxylin and eosin staining, lung injury scoring, Masson’s trichrome staining and fibrosis scoring. Homing and differentiation of mouse MSCs were assayed by labelling and tracing MSCs using NIR815 dye, immunofluorescent staining, and Western immunoblot analysis. The inflammation and permeability were evaluated by detecting the cytokine and protein measurements in bronchoalveolar lavage fluid using enzyme-linked immunosorbent assay.ResultsIn this study, β-catenin-overexpressing MSC engraftment led to more significant effects than the GFP controls, including the retention of the MSCs in the lung, differentiation into type II alveolar epithelial cells, improvement in alveolar epithelial permeability, and the pathologic impairment of the lung tissue.ConclusionThese results suggest that the activation of canonical Wnt/β-catenin pathway by mouse MSCs by overexpressing β-catenin could further improve the protection of mouse MSCs against epithelial impair and the therapeutic effects of mouse MSCs in ARDS mice.

Qingnaoyizhi decoction suppresses the formation of glial fibrillary acidic protein-positive cells in cultured neural stem cells by inhibiting the Janus kinase 2 / signal transducer and activator of transcription 3 signaling pathway

ObjectiveInactivation of the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling axis plays a crucial role in determining the fate of neural stem cells (NSCs). Qingnaoyizhi decoction (QNYZD) has been used for the treatment of vascular dementia and has shown to improve synaptic remodeling. The aim of this study was to evaluate the effect of cerebrospinal fluid (CSF) containing QNYZD (CSF-QNYZD) on the differentiation of cultured NSCs and the involvement of the JAK2/STAT3 pathway. MethodsThe protein expression levels of glial fibrillary acidic protein (GFAP), tubulin, drosophila mothers against decapentaplegic protein (SMAD-1), STAT3, and phosphorylated-STAT3 were detected by western immunoblot analysis in the groups: control, CSF, JAK/STAT inhibitor (AG490), CSF-QNYZD, and CSF-XDZ (CSF-Xidezhen). The differentiation of NSCs was determined by immunofluorescence staining. The proliferation of NSCs was measured using the Cell Counting Kit-8 proliferation assay. ResultsCompared with the control group, CSF-QNYZD and AG490 significantly increased the number and expression of tubulin-positive cells, reduced the number and expression of GFAP-positive cells, and down-regulated the expression of p-STAT3. However, CSF-QNYZD also decreased the expression of SMAD-1 and STAT3. ConclusionEnhanced neuronal differentiation may be associated with the down-regulation of glial differentiation instead of promoting proliferation in treated NSCs. Furthermore, QNYZD may play a direct role in suppressing the formation of GFAP-positive cells and enhancing neuronal differentiation by inhibiting JAK2/STAT3 activation. Overall, these results provide insights into the possible mechanism underlying QNYZD-mediated neurogenesis.

The mechanism of oxythiamine-induced collagen biosynthesis in cultured fibroblasts.

The oxythiamine (OXY) is antivitamin of thiamine. The finding that OXY increases the cytoplasmic concentration of pyruvate, known to enhance collagen biosynthesis, led us to investigate the mechanism of this antivitamin action on collagen biosynthesis in cultured human skin fibroblasts. Confluent fibroblasts were treated with micromolar concentrations (30–1,000 µM) of OXY for 24 and 48 h. It was found that OXY-dependent increase in collagen biosynthesis was accompanied by parallel increase in prolidase activity and level, compared to untreated cells. Since phosphoenolpyruvate (PEP) is known as an inhibitor of prolidase—the enzyme that plays important role in collagen biosynthesis, the mechanism of pyruvate interconversion was considered as a regulatory switch in collagen biosynthesis. In fact, 3-MPA, specific inhibitor of phosphoenolpyruvate carboxykinase (PEPCK), contributed to up-regulation of prolidase activity, suggesting that down-regulation of PEP formation is an underlying mechanism. Since collagen biosynthesis and prolidase activity are regulated by signal induced by activated α2β1 integrin receptor as well as insulin-like growth factor-I receptor (IGF-IR), the expression of these receptors was measured by Western immunoblot analysis. The exposure of the cells to OXY contributed to decrease in IGF-IR, α2β1 integrin receptor, pERK1/2, and NF-κB p65 expressions. It was accompanied by increase in total ERK1/2 expression and induction of phosphorylation of Akt protein. The data suggest that OXY-dependent increase of collagen biosynthesis in cultured human skin fibroblasts results from activation of prolidase activity and level, induction in pAkt expression and down-regulation of pERK1/2 and NF-κB p65, the known inhibitor of collagen gene expression.

Mucin-1 (MUC1) Oncoprotein in Multiple Myeloma Cells Inhibits the Th1 Responses By Down Regulating the Expression of Mir-200c and up-Regulating the PDL1 Expression

Introduction: The PDL1/PD-1 pathway is a critical mediator of immune escape in patients with multiple myeloma (MM). Regulation of this pathway has not been well characterized. MicroRNAs (miRNAs) are a conserved class of small (~22 nucleotides) RNAs that post-transcriptionally regulate gene expression by interacting with the 3′ untranslated region (3′ UTR) and, in some settings, coding regions of target mRNAs. MiRNAs suppress gene expression by promoting mRNA degradation or inhibiting translation. Of note, the 3’UTR of the PDL1 gene contains putative binding sites for miR-200 family of micro-RNA’s, suggesting a possible role of miR-200’s in regulation of PDL1 expression. We have previously demonstrated that miR-200c is suppressed by the MUC1 oncoprotein, and hypothesized that MUC1 expression on myeloma cells upregulates the expression of PDL1, via suppressing miR-200c. In the present study, we investigated the relationship between MUC1, miR-200c and PDL1 in multiple myeloma.Methods and Results: Lentivirus vectors expressing miR-200c or a control vector with green fluorescence protein (GFP) were transduced in two different MM cell lines (MM-RPMI, MM-U266). Cells were harvested sorted by Fluorescence-Activated Cell Sorting (FACS) after 72 hours of transduction, using a dual fluorescence for GFP and anti-PDL1 antibody to analyze the changes in PDL1 expression. MiR-200c transduction of U266 cells resulted in a decrease in mean expression of PDL1 from 69.55% to 1.4% (n=2). Similarly, RPMI cells demonstrated a reduction in mean expression of PDL1 from 62.5% to 1.9% (n=2) following miR-200c transduction. The abrogation of PDL1 expression in MM cells by ectopic expression of miR-200c was confirmed using western immunoblot analysis. Having previously demonstrated that miR-200c is suppressed by MUC1 in a solid tumor model, we evaluated the effect of silencing MUC1 in U266 and RPMI cell lines on miR-200c and PDL1 expression. MUC1 silenced stable cell lines of RPMI and U266 cells were generated using lentivirus shRNA vectors against MUC1 or a scrambled vector control. MUC1 silenced cells demonstrated an increase in miR-200c expression (> 2 fold, p value <0.05). Notably, PDL1 expression decreased from 52% to 3.7% and from 62.5% to 6.1% following silencing of MUC1 on U266 and RPMI cells respectively.Conclusions: Ectopic expression of micro-RNA miR-200c in RPMI-MM and U266-MM cell lines results in down regulation of PDL1 expression. Silencing MUC1 in RPMI-MM and U266-MM cell lines results in both increased expression of miR-200c and downregulation of PDL1 expression. These results support the hypothesis that MUC1 expression on myeloma cells contributes to tumor mediated immunosuppression, by suppressing miR-200c thereby enhancing PDL1 expression. Interfering with MUC1 mediated signaling represents a novel approach towards augmenting immune mediated targeting of myeloma. DisclosuresNo relevant conflicts of interest to declare.

EphrinB2/EphB4 Interaction Promotes Myeloid Leukemia Cell Invasion through RhoA-Mediated Mechanism

Background and Objective: Several studies have reported the up-regulation of EphB receptor-tyrosine kinases and ephrinB ligands in a variety of tumors, suggesting a functional relation between EphB/ephrinB signaling and tumor progression. However, how they regulate the invasiveness of myeloid leukemia cells were still unknown. Our previously study suggested that EphB4 were highly expressed in patients with extramedullary leukemia compared with patients without extramedullary leukemia, which indicated that the expression of EphB4 was related with myeloid leukemia cell invasion. To address the molecular mechanism, we aimed to characterize the role of EphB4 and ephrinB2 ligands in the interaction of myeloid leukemia cells.Methods: To clarify the question, myeloid leukemia cell lines (K562 cells and THP-1 cells) treated with clustered ephrinA1–Fc proteins, ephrinB2–Fc proteins and Fc proteins were cultured in vitro, then migration and invasion were determined by transwell assay according to different time. Pulldown western immunoblot analysis were used to detect the level of GTP-RhoA and total RhoA; the phosphorylation of EphB4 and MMP9 expression were also determined by immunoblot analysis before and after the treatment of different clustered Fc proteins.Results: The results showed that after ephrinB2–Fc stimulation, the numbers of K562 cells migrating through transwell chamber were significantly enhanced compared to Fc proteins stimulation (1.85-fold, P=0.033), meanwhile, the numbers of K562 cells invading the matrigel also enhanced (1.46 -fold, P=0.025). However, the numbers of K562 cells migrating through transwell chamber after ephrinA1–Fc stimulation didn’t significantly increase compared to Fc proteins stimulation (P=0.411), and the numbers of K562 cells invading the matrigel also didn’t enhanced (P=0.072) after ephrinA1–Fc stimulation. Moreover, after ephrinB2–Fc stimulation, the numbers of THP-1 cells migrating through transwell chamber were significantly enhanced compared to Fc proteins stimulation (2.25-fold, P<0.01), meanwhile, the numbers of THP-1 cells invading the matrigel also enhanced (1.66 -fold, P<0.01). However, the numbers of THP-1 cells migrating through transwell chamber and the numbers of THP-1 cells invading the matrigel didn’t significantly enhanced (P>0.05, P>0.05) after ephrinA1–Fc stimulation. Furthermore, EphB4 immunoprecipitation followed by immunoblotting with anti-phosphotyrosine antibody revealed that EphB4 is phosphorylated on tyrosine in K562 cells after ephrinB2–Fc stimulation. Additionally, the level of active RhoA (GTP-RhoA) and MMP9 in K562 cells were both significantly increased in response to EphB4 receptor activation with its ligand ephrin-B2-Fc ( P<0.05).Conclusions: These findings suggested that EphB4/EprinB2 signaling played an important role in myeloid leukemia cells progression by promoting their migratory ability, activating RhoA activity and increasing MMP9 expression. Our findings reveal a novel regulation of this intriguing receptor/ligand family that contributes to the cell invasiveness of myeloid leukemia cells. DisclosuresNo relevant conflicts of interest to declare.