IFN-α induces a preferential long-lasting expression of MHC class I in human pancreatic beta cells

Aims/hypothesis IFN-α, a cytokine expressed in human islets from individuals affected by type 1 diabetes, plays a key role in the pathogenesis of diabetes by upregulating inflammation, endoplasmic reticulum (ER) stress and MHC class I overexpression, three hallmarks of islet histology in early type 1 diabetes. We tested whether expression of these mediators of beta cell loss is reversible upon IFN-α withdrawal or IFN-α pathway inhibition. Methods IFN-α-induced MHC class I overexpression, ER stress and inflammation were evaluated by flow cytometry, immunofluorescence and real-time PCR in human EndoC-βH1 cells or human islets exposed to IFN-α with or without the presence of Janus kinase (JAK) inhibitors. Protein expression was evaluated by western blot. Results IFN-α-induced expression of inflammatory and ER stress markers returned to baseline after 24–48 h following cytokine removal. In contrast, MHC class I overexpression at the cell surface persisted for at least 7 days. Treatment with JAK inhibitors, when added with IFN-α, prevented MHC class I overexpression, but when added 24 h after IFN-α exposure these inhibitors failed to accelerate MHC class I return to baseline. Conclusions/interpretation IFN-α mediates a long-lasting and preferential MHC class I overexpression in human beta cells, which is not affected by the subsequent addition of JAK inhibitors. These observations suggest that IFN-α-stimulated long-lasting MHC class I expression may amplify beta cell antigen presentation during the early phase of type 1 diabetes and that IFN-α inhibitors might need to be used at very early stages of the disease to be effective

Gene regulation by PDGF via the PI3-kinase pathway : focus on HBP1 and stearoyl-coenzyme A desaturase

Our laboratory is interested in the study of growth factors and in particular the platelet-derived growth factor (PDGF) and its receptor (PDGFR). Many signaling pathways, including the phosphatidylinositol 3-kinase (PI3K) pathway, are activated downstream of PDGFR, leading to cell proliferation, survival, migration and differentiation. In line with this, the objective of this thesis was to characterize the regulation and the function of two mediators of the PDGFR-PI3K pathway. We focused on the HMG-box protein 1 (HBP1) and the stearoyl-coenzyme A desaturase (SCD). In our first study, we identified the transcription factor HBP1 as a negative target of the growth factor-PI3K-AKT pathway, up-regulated by FOXO via a direct transcriptional regulation. These regulations were observed in different cellular models including normal and cancer cells from human and mice origin. Using small-hairpin RNA (shRNA), we found that HBP1 knockdown increased cell proliferation and potentiated the proliferative effect of growth factors. Finally, we observed that HBP1 and FOXO1 were down-regulated in breast cancer tissues compared to normal breast tissues. In addition, breast tumors with a lower expression of FOXO also presented a lower expression of HBP1, which suggests that, in these tumors, FOXO loss could explain HBP1 down-regulation, at least partially. Our second study started from published data from our laboratory showing that PDGF strongly induces SCD expression via PI3K, mTORC1 and SREBP and stimulates lipid synthesis, in particular unsaturated fatty acids. SCD is a Δ9 desaturase that converts saturated fatty acids (SFA), such as palmitoyl-CoA and stearoyl-CoA, into mono-unsaturated fatty acids (MUFA), such as palmitoleoyl-CoA and oleyl-CoA. By increasing SCD expression, PDGF increased the MUFA / SFA ratio in the total cellular lipids. Using shRNA targeting SCD, we showed that SCD was essential for the proliferation and the survival of human fibroblasts in response to PDGF. The proliferative effect was confirmed with an inhibitor of SCD. Interestingly, the treatment of cells with palmitate, but not oleate, also blocked cell proliferation in response to PDGF, and the addition of oleate to palmitate suppressed this effect. In addition, SCD knockdown and palmitate induced endoplasmic reticulum stress (ER stress), which could explain the observed effects. Together, our results strongly suggest that SCD is necessary for PDGF-mediated cell proliferation by supplying an increased amount of MUFA. It also protects cells from SFA accumulation-induced lipotoxicity and ER stress. To conclude, PDGF activates many signaling pathways that allow the regulation of plenty of genes involved in PDGF functions. Among them, we identified HBP1 and SCD as downstream targets of the PI3K pathway, both involved in the control of cell proliferation by PDGF: HBP1 blocked cell proliferation whereas SCD favored it.(BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 201

FOXO transcription factors in cancer development and therapy.

The forkhead box O (FOXO) transcription factors are considered as tumor suppressors that limit cell proliferation and induce apoptosis. FOXO gene alterations have been described in a limited number of human cancers, such as rhabdomyosarcoma, leukemia and lymphoma. In addition, FOXO proteins are inactivated by major oncogenic signals such as the phosphatidylinositol-3 kinase pathway and MAP kinases. Their expression is also repressed by micro-RNAs in multiple cancer types. FOXOs are mediators of the tumor response to various therapies. However, paradoxical roles of FOXOs in cancer progression were recently described. FOXOs contribute to the maintenance of leukemia-initiating cells in acute and chronic myeloid leukemia. These factors may also promote invasion and metastasis of subsets of colon and breast cancers. Resistance to treatment was also ascribed to FOXO activation in multiple cases, including targeted therapies. In this review, we discuss the complex role of FOXOs in cancer development and response to therapy

Interferon-α mediates human beta cell HLA class I overexpression, endoplasmic reticulum stress and apoptosis, three hallmarks of early human type 1 diabetes

Aims/hypothesis: Three hallmarks of the pancreatic islets in early human type 1 diabetes are overexpression of HLA class I, endoplasmic reticulum (ER) stress and beta cell apoptosis. The mediators of these phenomena remain to be defined. The type I interferon IFNα is expressed in human islets from type 1 diabetes patients and mediates HLA class I overexpression. We presently evaluated the mechanisms involved in IFNα-induced HLA class I expression in human beta cells and determined whether this cytokine contributes to ER stress and apoptosis. Methods: IFNα-induced inflammation, ER stress and apoptosis were evaluated by RT-PCR, western blot, immunofluorescence and nuclear dyes, and proteins involved in type I interferon signalling were inhibited by small interfering RNAs. All experiments were performed in human islets or human EndoC-βH1 cells. Results: IFNα upregulates HLA class I, inflammation and ER stress markers in human beta cells via activation of the candidate gene TYK2, and the transcription factors signal transducer and activator of transcription 2 and IFN regulatory factor 9. Furthermore, it acts synergistically with IL-1β to induce beta cell apoptosis. Conclusions/interpretation: The innate immune effects induced by IFNα may induce and amplify the adaptive immune response against human beta cells, indicating that IFNα has a central role in the early phases of diabetes

PDGF-D expression is down-regulated by TGFβ in fibroblasts

Transforming growth factor-β (TGFβ) is a key mediator of fibrogenesis. TGFβ is overexpressed and activated in fibrotic diseases, regulates fibroblast differentiation into myofibroblasts and induces extracellular matrix deposition. Platelet-derived growth factor (PDGF) is also a regulator of fibrogenesis. Some studies showed a link between TGFβ and PDGF in certain fibrotic diseases. TGFβ induces PDGF receptor alpha expression in scleroderma fibroblasts. PDGF-C and -D are the most recently discovered ligands and also play a role in fibrosis. In this study, we report the first link between TGFβ and PDGF-D and -C ligands. In normal fibroblasts, TGFβ down-regulated PDGF-D expression and up-regulated PDGF-C expression at the mRNA and protein levels. This phenomenon is not limited to TGFβ since other growth factors implicated in fibrosis, such as FGF, EGF and PDGF-B, also regulated PDGF-D and PDGF-C expression. Among different kinase inhibitors, only TGFβ receptor inhibitors and the IκB kinase (IKK) inhibitor BMS-345541 blocked the effect of TGFβ. However, activation of the classical NF-κB pathway was not involved. Interestingly, in a model of lung fibrosis induced by either bleomycin or silica, PDGF-D was down-regulated, which correlates with the production of TGFβ and other fibrotic growth factors. In conclusion, the down-regulation of PDGF-D by TGFβ and other growth factors may serve as a negative feedback in the network of cytokines that control fibrosis

PDGF-D and PDGF-C expression in lung fibrosis induced by silica.

<p>RNA samples were harvested from lungs of three control mice and four silica-treated mice. (A) PDGF-D and (B) PDGF-C gene expression was measured by quantitative PCR and reported to the expression of actin. ANOVA analysis revealed that the effect of treatment was highly significant (p-value: <2.2e-16, data not shown).</p

Growth factors regulate PDGF-D and PDGF-C expression.

<p>MRC5 cells were treated with TGFβ 4 ng/ml, EGF 50 ng/ml, FGF-4 10 ng/ml (in the presence of heparin 10 µg/ml), PDGF-BB 20 ng/ml alone or all together for 24 h and PDGF-D (A) and PDGF-C (B) gene expression was measured by RT-qPCR. (C) AG01523 fibroblasts were treated with TGFβ 4 ng/ml, IL-1β 10 ng/ml or both for 24 h and PDGF-D gene expression was measured by q-PCR. The average of 3 experiments is shown with S.E.M, * p<0.05, ** p<0.005, *** p<0.0005.</p

PDGF-D and PDGF-C regulation by TGFβ at the mRNA and protein levels.

<p>(A) and (D), MRC5 human fibroblasts were pre-treated with TGFβ receptor inhibitor SB431542 (10 µM) 30 min before treatment with TGFβ (4 ng/ml) for 24 h. The control condition was treated with DMSO alone. PDGF-D and PDGF-C mRNA expression was measured by RT-qPCR and normalized with the housekeeping gene RPLP0. (B) and (E) MRC5 cells were treated with TGFβ (4 ng/ml) for the indicated period of time. PDGF-D and PDGF-C protein expression was analyzed by western blotting. Blots were re-hybridized with an anti-β-actin antibody as control. (C) Active PDGF-DD levels were measured by ELISA in the culture supernatant of MRC5 cells treated or untreated with TGFβ 4 ng/ml for 24 h. The average of 3 experiments is shown with S.E.M, ** p<0.005, *** p<0.0005.</p

TGFβ represses PDGF-D at the transcriptional level through an IKK-dependent pathway.

<p>We used a 1.3 kb fragment of the PDGF-D promoter cloned upstream of the luciferase reporter gene <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108656#pone.0108656-Liu1" target="_blank">[31]</a>. (A) Gamma-2A cells were co-transfected with the PDGF-D promoter construct or the CAGA12 promoter construct (positive control), with either empty vector or constitutively activated Alk5 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108656#pone.0108656-Pierreux1" target="_blank">[33]</a> and with a control β-galactosidase reporter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108656#pone.0108656-Demoulin1" target="_blank">[28]</a>. Cells were treated with TGFβ 4 ng/ml or left untreated for 24 h. (B) Gamma-2A cells were co-transfected with PDGF-D promoter construct and with β-galactosidase. They were treated with BMS-345541 (10 µM) for 30 min and then TGFβ was added for 24 h. In all experiments, luciferase and β-galactosidase activities were measured and the luciferase activity was normalized by dividing by the β-galactosidase activity. The mean of three independent experiments is shown.</p