Jun Phosphorylation Research Articles

Inductive effect of SORT1 on odontoblastic differentiation of human dental pulp-derived stem cells

This study investigated the expression of sortilin 1 (SORT1) in cultured human dental pulp-derived stem cells (hDPSCs) and its role in their odontoblastic differentiation. Permanent teeth were extracted from five patients, and the dental pulp was harvested for explant culture. Fluorescence-activated cell sorting was used to analyze the outgrowth of adherent cells and cells that had migrated from the tissue margin. SORT1 expression was detected in hDPSCs simultaneously expressing the mesenchymal stem cell markers CD44 and CD90. The odontoblastic differentiation potential of SORT1-positive hDPSCs was examined via staining for alkaline phosphatase (ALP), an early odontoblastic differentiation marker. ALP staining was more intense in SORT1-positive than in SORT1-negative hDPSCs. Consistently, the expression of mRNA encoding SORT1 and p75NTR, a binding partner of SORT1, increased in SORT1-positive hDPSCs during odontoblastic differentiation. In addition, pro-nerve growth factor (NGF), a ligand for SORT1-p75NTR co-receptor, promoted ALP expression in SORT1-positive hDPSCs, and the interaction between SORT1 and p75NTR was detected using a coimmunoprecipitation assay. The function of SORT1 in odontoblastic differentiation was examined via RNA interference using shRNA targeting SORT1. ALP staining intensity in SORT1/shRNA-transfected cells was markedly lower than in control/shRNA-transfected cells. SORT1 knockdown decreased JUN phosphorylation and recruitment of phosphorylated JUN to the ALP promoter. Collectively, these results indicate that SORT1 is involved in the odontoblastic differentiation of hDPSCs through the JUN N-terminal kinases (JNK)/JUN signaling pathway and that the binding of SORT1 and p75NTR plays an important role in this process.

JUN activation modulates chromatin accessibility to drive TNFα-induced mesenchymal transition in glioblastoma.

Chromatin dynamics as well as genetic evolution underlies the adaptability of tumour cells to environmental cues. Three subtypes of tumour cells have been identified in glioblastoma, one of the commonest malignant brain tumours in adults. During tumour progression or under therapeutic pressure, the non‐mesenchymal subtypes may progress to the mesenchymal subtype, leading to unfavourable prognosis. However, the molecular mechanisms for this transition remain poorly understood. Here taking a TNFα‐induced cellular model, we profile the chromatin accessibility dynamics during mesenchymal transition. Moreover, we identify the JUN family as one of the key driving transcription factors for the gained chromatin accessibility. Accordingly, inhibition of JUN phosphorylation and therefore its transcription activity successfully impedes TNFα‐induced chromatin remodelling and mesenchymal transition. In line with these findings based on experimental models, JUN activity is positively correlated with mesenchymal features in clinical glioblastoma specimens. Together, this study unveils a deregulated transcription regulatory network in glioblastoma progression and hopefully provides a rationale for anti‐glioblastoma therapy.

Curcumin treatment attenuates cisplatin-induced gastric mucosal inflammation and apoptosis through the NF- κ B and MAPKs signaling pathway.

To investigate the protective effects of curcumin (Cur) on gastric mucosal injury induced by cisplatin (DDP), and explore possible molecular mechanisms. A mouse of gastric mucosal injury was established by intraperitoneal injection of DDP (27mg/kg). Thirty mice were randomly divided into control group, DDP group and DDP + Cur group. Serum and gastric mucosal samples were collected on the 7th day after Cur treatment. The index of gastric mucosa injury was calculated, and the expression levels of inflammation, apoptosis and signaling pathway proteins were evaluated using hematoxylin and eosin staining, ELISA and western blotting analysis. These data showed that Cur treatment significantly attenuated DDP-induced decrease in body weight, food intake, fat and muscle ratios, and improved the gross gastric injury, scores of ulcer index, and histopathology changes triggered by DDP (p < .05). Meanwhile, Cur significantly decreased serum IL-23 and IL-17 proteins, reduced the expression levels of gastric mucosal IL-1β, TNF- α and MPO, and restored the level of IL-10 protein (p < .05). Moreover, Cur treatment significantly inhibited the expression levels of Caspase-3, PARP and Bax, and increased the expression of Bcl-2 protein. Furthermore, Cur treatment significantly decreased the expression levels of IL-1R, MyD88 and TAK1, and also repressed the activation of NF-κB and nuclear translocation of NF-κB p65. And more importantly, Cur treatment significantly inhibited DDP-induced gastric mucosal JNK1/2, ASK1, P38 and JUN phosphorylation, and promoted the phosphorylation of ERK1/2 and C-Myc proteins. Our data suggest that Cur treatment alleviates DDP-induced gastric mucosal inflammation and apoptosis, which may be mediated through the NF- κ B and MAPKs signaling pathway.

Citrus Naringenin Increases Neuron Survival in Optic Nerve Crush Injury Model by Inhibiting JNK-JUN Pathway.

Traumatic nerve injury activates cell stress pathways, resulting in neuronal death and loss of vital neural functions. To date, there are no available neuroprotectants for the treatment of traumatic neural injuries. Here, we studied three important flavanones of citrus components, in vitro and in vivo, to reveal their roles in inhibiting the JNK (c-Jun N-terminal kinase)-JUN pathway and their neuroprotective effects in the optic nerve crush injury model, a kind of traumatic nerve injury in the central nervous system. Results showed that both neural injury in vivo and cell stress in vitro activated the JNK-JUN pathway and increased JUN phosphorylation. We also demonstrated that naringenin treatment completely inhibited stress-induced JUN phosphorylation in cultured cells, whereas nobiletin and hesperidin only partially inhibited JUN phosphorylation. Neuroprotection studies in optic nerve crush injury mouse models revealed that naringenin treatment increased the survival of retinal ganglion cells after traumatic optic nerve injury, while the other two components had no neuroprotective effect. The neuroprotection effect of naringenin was due to the inhibition of JUN phosphorylation in crush-injured retinal ganglion cells. Therefore, the citrus component naringenin provides neuroprotection through the inhibition of the JNK-JUN pathway by inhibiting JUN phosphorylation, indicating the potential application of citrus chemical components in the clinical therapy of traumatic optic nerve injuries.

Based on Network Pharmacology Tools to Investigate the Mechanism of Tripterygium wilfordii Against IgA Nephropathy.

Background: Immunoglobulin A nephropathy (IgAN) is the most common primary glomerular disease and poses a global major public health burden. The preparation of Tripterygium wilfordii Hook F (TwHF) is widely applied for treating patients with Immunoglobulin A nephropathy in China, while the molecular mechanisms remain unclear. This study aimed to verify the therapeutic mechanism of TwHF on IgAN by undertaking a holistic network pharmacology strategy in combination with in vitro and in vivo experiments.Methods: TwHF active ingredients and their targets were obtained via the Traditional Chinese Medicine Systems Pharmacology Database. The collection of IgAN-related target genes was collected from GeneCards and OMIM. TwHF-IgAN common targets were integrated and visualized by Cytoscape. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to determine the predominant molecular mechanisms and pathways of TwHF on the treatment of IgAN. The protein-protein interaction network was constructed by the STRING online search tool, and hub genes were identified using R software. The expression of hub gene and related signaling were evaluated in TwHF-treated mice through immunohistochemistry and western blot and further validated in human mesangial cells (HMCs). In addition, Cell counting kit 8 (CCK8) and flow cytometry were used to detect the effects of TwHF on cell proliferation and cell cycle of mesangial cells.Results: A total of 51 active ingredients were screened from TwHF and 61 overlapping targets related to IgAN were considered potential therapeutic targets, GO functions and KEGG analyses demonstrated that these genes were primarily associated with DNA-binding transcription factor binding, lipid and atherosclerosis pathway. Genes with higher degrees including AKT1, CXCL8, MMP9, PTGS2, CASP3, JUN are hub genes of TwHF against IgAN. Verification of hub gene JUN both in vitro and in vivo showed that TwHF significantly attenuated JUN phosphorylation in the kidneys of IgAN mice and aIgA1-activated HMCs, meanwhile suppressing HMCs proliferation and arresting G1-S cell cycle progression.Conclusion: Our research strengthened the mechanisms of TwHF in treating IgAN, inhibition of JUN activation may play a pivotal role in TwHF in alleviating IgAN renal injury.

Microarry Analysis Identifies Activation Of Multiple Signalling Pathways In Primary Prostate Epithelial Cells By Low Temperature Plasma

We have previously reported the killing effects of Low Temperature Plasma (LTP) on primary prostate epithelial cells, of both normal and cancerous origin, via necrosis [1] following initial DNA damage. However it was evident that a population of cells was resistant to LTP treatment. To discern possible survival mechanisms in treated cells; the gene expression of 6 samples (4 cancers and 2 matched normal) was analysed 2 hours post-treatment, on a transcriptome wide level, by microarray. Initial analysis found alteration in the expression of 646 transcripts with significant activation of Notch, NF-kB, Nrf2 and AP-1 signalling in our primary cells. Pathway activation was subsequently validated by qRT-PCR in four different patient-derived prostate cultures to give a robust LTP-induced expression signature. Upstream effector activation was then confirmed by immunofluorescence and Western blot. We found as other groups have; [2, 3] accumulation of Nrf2 in response to LTP, alongside AP-1 activation [4, 5] – through phosphorylation of both JNK and Jun. The Notch signalling pathway is typically described in relation to stem cell maintenance and we observed LTP induced Notch activation through release of its intracellular domain (NICD) that then acts as a nuclear transcription factor. Tumours are innately heterogeneous and contain multiple cell populations, both intrinsic and recruited. We have established that our cultures contain three readily identifiable epithelial cell pools that can be separated to permit further analysis.[6] Preliminary investigation has found that the progenitor epithelial cells respond in greater measure than their more differentiated progeny, suggesting that they are more resistant to plasma. Similar observations of prostate stem cell resistance have been made with radiotherapy.[7] However, if these stem-like cells can be targeted – LTP may have a greater killing effect if utilized in prostate cancer treatments. Download : Download high-res image (151KB) Download : Download full-size image Figure – Pathway activation in primary prostate cancer epithelial culture; Nrf2 accumulation, AP-1 activation by JNK and Jun phosphorylation and Notch cleavage, with release of NICD by LTP. U – Untreated, T- Treated. Samples taken 0.5 and 2 hours following treatment with 3 minutes LTP.

Hydroxyurea-inducible SAR1 gene acts through the Giα/JNK/Jun pathway to regulate γ-globin expression

AbstractHydroxyurea (HU) is effectively used in the management of β-hemoglobinopathies by augmenting the production of fetal hemoglobin (HbF). However, the molecular mechanisms underlying HU-mediated HbF regulation remain unclear. We previously reported that overexpression of the HU-induced SAR1 gene closely mimics the known effects of HU on K562 and CD34+ cells, including γ-globin induction and cell-cycle regulation. Here, we show that HU stimulated nuclear factor-κB interaction with its cognate-binding site on the SAR1 promoter to regulate transcriptional expression of SAR1 in K562 and CD34+ cells. Silencing SAR1 expression not only significantly lowered both basal and HU-elicited HbF production in K562 and CD34+ cells, but also significantly reduced HU-mediated S-phase cell-cycle arrest and apoptosis in K562 cells. Inhibition of c-Jun N-terminal kinase (JNK)/Jun phosphorylation and silencing of Giα expression in SAR1-transfected K562 and CD34+ cells reduced both γ-globin expression and HbF level, indicating that activation of Giα/JNK/Jun proteins is required for SAR1-mediated HbF induction. Furthermore, reciprocal coimmunoprecipitation assays revealed an association between forcibly expressed SAR1 and Giα2 or Giα3 proteins in both K562 and nonerythroid cells. These results indicate that HU induces SAR1, which in turn activates γ-globin expression, predominantly through the Giα/JNK/Jun pathway. Our findings identify SAR1 as an alternative therapeutic target for β-globin disorders.

Intracochlear Electrical Stimulation Suppresses Apoptotic Signaling in Rat Spiral Ganglion Neurons after Deafening in Vivo

To establish the intracellular consequences of electrical stimulation to spiral ganglion neurons after deafferentation. Here we use a rat model to determine the effect of both low and high pulse rate acute electrical stimulation on activation of the proapoptotic transcription factor Jun in deafferented spiral ganglion neurons in vivo. Experimental animal study. Hearing research laboratories of the University of Iowa Departments of Biology and Otolaryngology. A single electrode was implanted through the round window of kanamycin-deafened rats at either postnatal day 32 (P32, n = 24) or P60 (n = 22) for 4 hours of stimulation (monopolar, biphasic pulses, amplitude twice electrically evoked auditory brainstem response [eABR] threshold) at either 100 or 5000 Hz. Jun phosphorylation was assayed by immunofluorescence to quantitatively assess the effect of electrical stimulation on proapoptotic signaling. Jun phosphorylation was reliably suppressed by 100 Hz stimuli in deafened cochleae of P32 but not P60 rats. This effect was not significant in the basal cochlear turns. Stimulation frequency may be consequential: 100 Hz was significantly more effective than was 5 kHz stimulation in suppressing phospho-Jun. Suppression of Jun phosphorylation occurs in deafferented spiral ganglion neurons after only 4 hours of electrical stimulation. This finding is consistent with the hypothesis that electrical stimulation can decrease spiral ganglion neuron death after deafferentation.

Planar Cell Polarity Controls Pancreatic Beta Cell Differentiation and Glucose Homeostasis

Planar cell polarity (PCP) refers to the collective orientation of cells within the epithelial plane. We show that progenitor cells forming the ducts of the embryonic pancreas express PCP proteins and exhibit an active PCP pathway. Planar polarity proteins are acquired at embryonic day 11.5 synchronously to apicobasal polarization of pancreas progenitors. Loss of function of the two PCP core components Celsr2 and Celsr3 shows that they control the differentiation of endocrine cells from polarized progenitors, with a prevalent effect on insulin-producing beta cells. This results in a decreased glucose clearance. Loss of Celsr2 and 3 leads to a reduction of Jun phosphorylation in progenitors, which, in turn, reduces beta cell differentiation from endocrine progenitors. These results highlight the importance of the PCP pathway in cell differentiation in vertebrates. In addition, they reveal that tridimensional organization and collective communication of cells are needed in the pancreatic epithelium in order to generate appropriate numbers of endocrine cells.

Electrical Stimulation Reduces Apoptotic Signaling in SGNs

Objective: To establish the intracellular consequences of electrical stimulation (ES) to spiral ganglion neurons (SGNs) after deafferentation. SGNs die as a result of loss of hair cells, their sole afferent input. Some histologic animal studies of chronic ES after deafening show that ES alone may improve SGN survival. Here we use a rat model to determine the effect of both low and high pulse rate ES on activation of the pro-apoptotic transcription factor Jun in deafferented SGNs in vivo. Methods: A single electrode was implanted through the round window of kanamycin-deafened rats at either postnatal day 32 (P32) or P60 for 4 hours of ES (monopolar, biphasic pulses, amplitude twice eABR threshold) at either 100 or 5000 Hz. Jun phosphorylation, a proapoptotic signaling event known to occur in apoptotic SGNs after deafening, was assayed by immunofluorescence to quantitatively assess the effect of ES on proapoptotic signaling. Results: Jun phosphorylation was reliably suppressed by 100 Hz ES in deafened cochleae of P32 rats ( P = .045) but this effect was less robust when 5000 Hz ES was used. This effect was most significant in the cochlear turn just apical to the stimulating electrode and was not significant in P60 rats. Conclusion: Suppression of phospho-Jun occurs in deafferented SGNs after only 4 hours of ES, consistent with the hypothesis that ES provides trophic support to SGNs after deafferentation. Stimulation frequency may be consequential: 100 Hz ES was significantly more effective than 5000 Hz ES in suppressing phospho-Jun.

JNK2 and JNK3 are major regulators of axonal injury-induced retinal ganglion cell death

Glaucoma is a neurodegenerative disease characterized by the apoptotic death of retinal ganglion cells (RGCs). The primary insult to RGCs in glaucoma is thought to occur to their axons as they exit the eye in the optic nerve head. However, pathological signaling pathways that exert central roles in triggering RGC death following axonal injury remain unidentified. It is likely that the first changes to occur following axonal injury are signal relay events that transduce the injury signal from the axon to the cell body. Here we focus on the c-Jun N-terminal kinase (JNK1–3) family, a signaling pathway implicated in axonal injury signaling and neurodegenerative apoptosis, and likely to function as a central node in axonal injury-induced RGC death. We show that JNK signaling is activated immediately after axonal injury in RGC axons at the site of injury. Following its early activation, sustained JNK signaling is observed in axonally-injured RGCs in the form of JUN phosphorylation and upregulation. Using mice lacking specific Jnk isoforms, we show that Jnk2 and Jnk3 are the isoforms activated in injured axons. Combined deficiency of Jnk2 and Jnk3 provides robust long-term protection against axonal injury-induced RGC death and prevents downregulation of the RGC marker, BRN3B, and phosphorylation of JUN. Finally, using Jun deficient mice, we show that JUN-dependent pathways are important for axonal injury-induced RGC death. Together these data demonstrate that JNK signaling is the major early pathway triggering RGC death after axonal injury and may directly link axon injury to transcriptional activity that controls RGC death.

2.33 Modulation of Autophagy by the UPR Results in Apoptosis in CLL Cells

Background: Macroautophagy (autophagy) has been shown to be both a pro-survival and pro-death signalling pathway. The decision between life or death is dependent on the type and developmental stage of the cell and the duration and intensity of the signal. Autophagy is used by cancer cells as a mechanism to overcome cellular stresses such as starvation, reactive oxygen species (ROS) generation and misfolded protein accumulation and can result in tumorigenesis and resistance to chemotherapy. One of the pathways which regulate responses to ROS/ER stress is the unfolded protein response (UPR). Briefly, upon ER stress, the HSP70 family member BiP is sequestered away from IRE1 , PERK and ATF6, resulting in IRE1 and PERK phosphorylation and activation of downstream signalling pathways. These signals can result in cell survival by upregulating AKT or cell death via induction of CHOP and noxa. However this pathway has not been fully investigated in chronic lymphocytic leukaemia (CLL). Interestingly the UPR is also known to regulate autophagy. The lipidation of LC3 from LC3-I to LC3-II is a surrogate marker of autophagy, since LC3-II is essential for formation of the autophagasome and the initiation of autophagy. Beclin-1 is another essential protein involved in canonical autophagy and is sequestered by Bcl-2 preventing autophagy in a number of cell types. Its release from Bcl-2 enables Beclin-1 to interact with other autophagy specific proteins and initiates autophagasome formation. Methods: Twenty CLL samples were treated with the HSP70 inhibitor 2-phenylacetylenesulphonamide (PAS; 10-20 M), an activator of the UPR, in the presence of the pan-caspase inhibitor ZVAD. In addition 4 control samples (normal B cells) were treated with PAS for the indicated times. Immunoblotting was performed for BiP, HSP70, Phosphorylated eIF2 (eIF2 -P), JNK (JNK-P), Jun (Jun-P), p38MAPK, LC3-I/II, Beclin expression and its co-immunoprecipitation with Bcl-2. Ten CLL cell isolates were treated with 2 autophagy inhibitors bafilomycin (Baf) (50-200 nM) and 3-methyl adenine (3-MA) (2.5-10 mM) and their effects on cell killing evaluated using PARP cleavage, a surrogate marker of caspase 3 activation. Results: CLL cells expressed a greater basal level of LC3-II in a proportion of samples relative to normal B cells. In addition Beclin-1 did not coimmunoprecipitate with Bcl-2 above that shown with the isotype control in CLL cells. Treatment with PAS resulted in an increase in stress which resulted in the upregulation of BiP and HSP70 protein. This subsequently led to phosphorylation of eIF2 indicative of PERK activation, coincidental with an increase in the eIF2 targets LC3-I/II and noxa expression. Treatment with PAS also resulted in an increase in JNK, Jun and p38MAPK phosphorylation, 3 proteins involved in regulating autophagy in mammalian cells. Furthermore, CLL cells treated with the autophagy inhibitors 3-MA and Baf rapidly underwent apoptosis within 24 h. Conclusion: These data suggest that autophagy is active at a basal level in a proportion of CLL cells and is greater than that seen in control B cells. Activation of the UPR with PAS led to an upregulation of proteins known to regulate autophagy and resulted in an increase in LC3-II, a protein routinely used as an indicator of autophagy. Inhibitors of autophagy also rapidly induced apoptosis in CLL cells. These data suggested that autophagy may have a pro-survival role in the biology of CLL. However pro-survival autophagy is balanced on a knife-edge, such that perturbation by inducing or inhibiting the pathway results in CLL cell death.

Role of antigen persistence and dose for CD4+ T-cell exhaustion and recovery (159.7)

Abstract It is currently not understood how some chronic infections exhaust antigen-specific T cells over time and how different pathogen components contribute to the exhausted state. Here, we dissected the behavior of primed CD4+ T cells exposed to persistent antigen using an inducible transgenic mouse system that allowed us to control antigen presentation as the only experimental variable independent of persistent inflammation and disease progression. In this system presentation of an MHC-II presented antigen is restricted to dendritic cells so that confounding B, CD8+ T, or innate cell responses are not triggered. When antigen presentation was extended beyond the expansion phase, primed CD4+ T cells survived, but exhibited reduced memory functionality in terms of their proliferative capacity and cytokine expression potential. The effect was antigen dose and time dependent, not associated with increased PD-1 expression or reduced calcium influx, but with impaired Jun phosphorylation in response to TCR engagement. Upon antigen removal, the cells regained the ability to proliferate, but remained unable to produce high levels of IL-2 and TNF-α. These data show that persistent antigen at a high dose rapidly induces a dysfunctional state in CD4+ T cells that is only partially reversible upon antigen removal. At a low dose, however, antigen has to persist longer before T cell impairment becomes detectable.

213 HEPATOCELLULAR CARCINOMA PROGRESSION IS ACCELERATED BY CANCER-ASSOCIATED FIBROBLASTS STIMULATED BY TUMOR PRODUCTION OF LYSOPHOSPHATIDIC ACID

Aim of the study: The purpose of the present work was to examine the intracellular pathway activated by SDF-1 in HuCCt cells and whether SDF-1 could act as a growth factor and/or a survival factor for HuCCt cells. Materials and Methods: HuCCt cells and primary hHSC were employed. Intracellular signaling and apoptotic pathways were investigated by Western blot analysis. Differences in the activation of different signaling proteins compared to control conditions were detected by densitometric analysis. Growth of HuCCt cells was investigated by cell count. Results: Following stimulation of HuCCt with SDF-1, the phosphorylation of Akt, Erk and JUN increased compared to control after 5 minutes and decreased at later time points. Phosphorylation of Akt and Erk was inhibited by AMD3100, a specific inhibitor of CXCR4. HSC conditioned medium (CM) induced a 30% increase of HuCCt growth compared to control, whereas stimulation with SDF-1 was without any effect. The antiapoptotic effect of SDF-1 was evaluated by measurement of cleaved-PARP and expression of BclXl in presence of an apoptotic stimulus, i.e. Arsenic trioxide (AT). AT induced apoptosis was inhibited by the coincubation of SDF-1. Conclusions: SDF-1, secreted by HSC, induces Erk, Akt and JUN phosphorylation at different time points and this coincides with a modest survival effect on HuCCt cells.

An experimental model for the study of well-differentiated and dedifferentiated liposarcoma; deregulation of targetable tyrosine kinase receptors

Therapeutic progress in well-differentiated/dedifferentiated liposarcoma (WDLPS/DDLPS) is hampered by lack of relevant experimental models, thereby limiting comprehensive molecularly based investigations. Our goal is to bridge this experimental gap by establishing and characterizing an in vitro/in vivo model useful for examining WDLPS/DDLPS molecular pathogenesis and also therapeutic screening and testing. WDLPS/DDLPS cells were isolated from freshly resected human surgical specimens and were phenotypically and molecularly characterized. MDM2 amplification was determined via FISH analysis. Adipogenic differentiation was evaluated using Oil Red O staining and western blotting (WB). Tyrosine kinase receptors' (TKRs) expression in pre-adipocytes, adipocytes, WDLPS, and DDLPS cells was determined via western blot analysis. SCID mouse xenograft growth was assessed after subcutaneous and/or intraperitoneal tumor cell injection. There was enhanced proliferation, migration, invasion, survival, and pro-angiogenic capacity in DDLPS cells vs WDLPS cells. DDLPS cells formed tumors in SCID mice whereas WDLPS did not. WDLPS/DDLPS cells, especially those that exhibited baseline PPARγ expression, partially retained terminal adipogenic differentiation capacity. MDM2 amplification was found in all WDLPS/DDLPS cell strains, CDK4 overexpression was observed in LPS cells as compared with normal adipocytes, and enhanced JUN expression and phosphorylation was seen in DDLPS cells as compared with WDLPS cells. The TKRs: MET, AXL, KIT, and IGF-1R were overexpressed in LPS cells vs normal adipocytes and pre-adipocytes. In conclusion, these newly established cellular and xenograft models can facilitate investigation of liposarcomagenesis, dedifferentiation, and tumor progression. Further studies of the molecular deregulations so identified may lead to improved therapeutic strategies for patients afflicted by these unfavorable malignancies.

Role of antigen persistence and dose for CD4+T-cell exhaustion and recovery

It is currently not understood how some chronic infections exhaust antigen-specific T cells over time and which pathogen components contribute to exhaustion. Here, we dissected the behavior of primed CD4(+) T cells exposed to persistent antigen using an inducible transgenic mouse system that allowed us to control antigen presentation as the only experimental variable, independent of the persistent inflammation and disease progression that complicate infectious models. Moreover, this system restricted antigen presentation to dendritic cells (DCs) and avoided confounding B, CD8(+) T, or innate cell responses. When antigen presentation was extended beyond the expansion phase, primed CD4(+) T cells survived, but exhibited reduced memory functionality in terms of their proliferative capacity and cytokine expression potential. The effect was antigen dose and time dependent, not associated with increased PD-1 expression or reduced calcium influx, but impaired Jun phosphorylation in response to TCR engagement. Upon antigen removal, the cells regained the ability to proliferate, but remained unable to produce high levels of IL-2 and TNF-α. These data show that persistent antigen by itself rapidly induces a dysfunctional state in CD4(+) T cells that is only partially reversible upon antigen removal. These findings have implications for vaccine optimization and for the possible reinvigoration of CD4(+) T cells during chronic infection.

Pathogen Specific, IRF3-Dependent Signaling and Innate Resistance to Human Kidney Infection

The mucosal immune system identifies and fights invading pathogens, while allowing non-pathogenic organisms to persist. Mechanisms of pathogen/non-pathogen discrimination are poorly understood, as is the contribution of human genetic variation in disease susceptibility. We describe here a new, IRF3-dependent signaling pathway that is critical for distinguishing pathogens from normal flora at the mucosal barrier. Following uropathogenic E. coli infection, Irf3−/− mice showed a pathogen-specific increase in acute mortality, bacterial burden, abscess formation and renal damage compared to wild type mice. TLR4 signaling was initiated after ceramide release from glycosphingolipid receptors, through TRAM, CREB, Fos and Jun phosphorylation and p38 MAPK-dependent mechanisms, resulting in nuclear translocation of IRF3 and activation of IRF3/IFNβ-dependent antibacterial effector mechanisms. This TLR4/IRF3 pathway of pathogen discrimination was activated by ceramide and by P-fimbriated E. coli, which use ceramide-anchored glycosphingolipid receptors. Relevance of this pathway for human disease was supported by polymorphic IRF3 promoter sequences, differing between children with severe, symptomatic kidney infection and children who were asymptomatic bacterial carriers. IRF3 promoter activity was reduced by the disease-associated genotype, consistent with the pathology in Irf3−/− mice. Host susceptibility to common infections like UTI may thus be strongly influenced by single gene modifications affecting the innate immune response.

Akt-dependent Pp2a activity is required for epidermal barrier formation during late embryonic development

Acquisition of epidermal barrier function occurs late in mouse gestation. Several days before birth a wave of barrier acquisition sweeps across murine fetal skin, converging on dorsal and ventral midlines. We investigated the molecular pathways active during epidermal barrier formation. Akt signaling increased as the barrier wave crossed epidermis and Jun was transiently dephosphorylated. Inhibitor experiments on embryonic explants showed that the dephosphorylation of Jun was dependent on both Akt and protein phosphatase 2A (Pp2a). Inhibition of Pp2a and Akt signaling also caused defects in epidermal barrier formation. These data are compatible with a model for developmental barrier acquisition mediated by Pp2a regulation of Jun dephosphorylation, downstream of Akt signaling. Support for this model was provided by siRNA-mediated knockdown of Ppp2r2a (Pr55alpha or B55alpha), a regulatory subunit of Pp2a expressed in an Akt-dependent manner in epidermis during barrier formation. Ppp2r2a reduction caused significant increase in Jun phosphorylation and interfered with the acquisition of barrier function, with barrier acquisition being restored by inhibition of Jun phosphorylation. Our data provide strong evidence that Ppp2r2a is a regulatory subunit of Pp2a that targets this phosphatase to Jun, and that Pp2a action is necessary for barrier formation. We therefore describe a novel Akt-dependent Pp2a activity that acts at least partly through Jun to affect initial barrier formation during late embryonic epidermal development.

Methylprednisolone inhibits interleukin-17 and interferon-gamma expression by both naive and primed T cells

BackgroundInterleukin-17 (IL-17)-producing cells are increasingly considered to be the major pathogenic population in various autoimmune disorders. The effects of glucocorticoids, widely used as therapeutics for inflammatory and autoimmune disorders, on IL-17 generation have not been thoroughly investigated so far. Therefore, we have explored the influence of methylprednisolone (MP) on IL-17 expression in rat lymphocytes, and compared it to the effect of the drug on interferon (IFN)-γ.ResultsProduction of IL-17 in mitogen-stimulated lymph node cells (LNC) from non-treated rats, as well as in myelin basic protein (MBP)-stimulated draining LNC from rats immunized with spinal cord homogenate and complete Freund's adjuvant was significantly reduced by MP. The reduction was dose-dependent, sustained through the follow-up period of 48 hours, and was not achieved through anti-proliferative effect. Additionally, MP inhibited IL-17 production in purified T cells as well, but to less extent than in LNC. In its influence on IL-17 production MP inhibited Ror-γT transcription factor expression, as well as Jun phosphorylation, but not ERK or p38 activation in mitogen-stimulated LNC. Importantly, MP collaborated with IFN-γ in inhibiting IL-17 generation in LNC.ConclusionThe observed difference in the effect of MP on IL-17 and IFN-γ could be important for the understanding of the variability in the efficiency of glucocorticoids in the treatment of autoimmune diseases.

TNFα and GM-CSF-induced activation of the CAEV promoter is independent of AP-1

Caprine arthritis encephalitis virus transcription is under the control of the viral promoter within the long terminal repeat. Previous studies with the closely related maedi visna lentivirus have indicated that viral transcription is dependent upon the AP-1 transcription factor. Other studies have indicated a potential role for the cytokines TNFα and GM-CSF in CAEV pathogenesis by increasing viral loads in infected tissues. The hypotheses that AP-1 transcription factors are necessary for transcriptional activation of the CAEV promoter and that CAEV transcriptional activation results from treatment with the cytokines GM-CSF and TNFα were tested with a stably transduced U937 cell line. Here, we found that TNFα and GM-CSF activated CAEV transcription in U937 cells. However, this activation effect was not blocked by SP600125, an inhibitor of Jun N-terminal kinase. SP600125 effectively prevented Jun phosphorylation in cells subsequently treated with cytokines. The cytokines TNFα and GM-CSF therefore activate CAEV transcription, and this effect occurs independently of AP-1. A set of progressive deletion mutants was utilized to show that TNFα-induced expression depends on an element or elements within the U3 70-bp repeat.