Many adipose tissue-related diseases, such as obesity and type 2 diabetes, are worldwide epidemics. For studying these diseases, relevant human cell models are needed. In this study, we developed a vascularized adipose tissue model where human adipose stromal cells and human umbilical cord vein endothelial cells were cocultured with natural adipogenic and defined serum-free angiogenic media for 14 days. Several different protocols were compared to each other. The protocols varied in cell numbers and plating sequences. Lipid accumulation was studied with AdipoRed reagent, relative cell number with WST-1 reagent, gene expression of glut4, leptin, aP2, adiponectin, PPARγ and PPARγ2 with RT-qPCR. Secretion of adiponectin, leptin and aP2 was analysed with ELISA. The immunostained vascular network was imaged with Cell-IQ and area quantified using ImageJ. In this study, both angiogenesis and adipogenesis were successfully induced. Protocols produced strong lipid accumulation, good vascular network formation and induced adipocyte-specific protein secretion and expression of studied adipocyte genes. Results showed that cell numbers and cell plating sequences are important factors when aiming at in vitro standardized tissue model. Presence of mature vasculature appeared leads to faster the maturation of adipocytes judged by the lipid accumulation and gene expression results. The developed vascularized adipose tissue model is simple to use, easily modifiable to suit various applications and as such, a promising new tool for adipose tissue research when, for example, studying the effect of different cell types on adipose tissue function or for mechanistic studies.

BackgroundBone morphogenetic protein 4 (BMP4) plays an important role in cancer pathogenesis. In breast cancer, it reduces proliferation and increases migration in a cell line-dependent manner. To characterize the transcriptional mediators of these phenotypes, we performed RNA-seq and DNase-seq analyses after BMP4 treatment in MDA-MB-231 and T-47D breast cancer cells that respond to BMP4 with enhanced migration and decreased cell growth, respectively.ResultsThe RNA-seq data revealed gene expression changes that were consistent with the in vitro phenotypes of the cell lines, particularly in MDA-MB-231, where migration-related processes were enriched. These results were confirmed when enrichment of BMP4-induced open chromatin regions was analyzed. Interestingly, the chromatin in transcription start sites of differentially expressed genes was already open in unstimulated cells, thus enabling rapid recruitment of transcription factors to the promoters as a response to stimulation. Further analysis and functional validation identified MBD2, CBFB, and HIF1A as downstream regulators of BMP4 signaling. Silencing of these transcription factors revealed that MBD2 was a consistent activator of target genes in both cell lines, CBFB an activator in cells with reduced proliferation phenotype, and HIF1A a repressor in cells with induced migration phenotype.ConclusionsIntegrating RNA-seq and DNase-seq data showed that the phenotypic responses to BMP4 in breast cancer cell lines are reflected in transcriptomic and chromatin levels. We identified and experimentally validated downstream regulators of BMP4 signaling that relate to the different in vitro phenotypes and thus demonstrate that the downstream BMP4 response is regulated in a cell type-specific manner.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3428-1) contains supplementary material, which is available to authorized users.

ObjectiveCurrent knowledge on the actions of tofacitinib on cytokine signaling pathways in rheumatoid arthritis (RA) is based on in vitro studies. Our study is the first to examine the effects of tofacitinib treatment on Janus kinase (JAK) - signal transducer and activator of transcription (STAT) pathways in vivo in patients with RA.MethodsSixteen patients with active RA, despite treatment with conventional synthetic disease-modifying antirheumatic drugs (csDMARDs), received tofacitinib 5 mg twice daily for three months. Levels of constitutive and cytokine-induced phosphorylated STATs in peripheral blood monocytes, T cells and B cells were measured by flow cytometry at baseline and three-month visits. mRNA expression of JAKs, STATs and suppressors of cytokine signaling (SOCS) were measured from peripheral blood mononuclear cells (PBMCs) by quantitative PCR. Association of baseline signaling profile with treatment response was also investigated.ResultsTofacitinib, in csDMARDs background, decreased median disease activity score (DAS28) from 4.4 to 2.6 (p < 0.001). Tofacitinib treatment significantly decreased cytokine-induced phosphorylation of all JAK-STAT pathways studied. However, the magnitude of the inhibitory effect depended on the cytokine and cell type studied, varying from 10% to 73% inhibition following 3-month treatment with tofacitinib. In general, strongest inhibition by tofacitinib was observed with STAT phosphorylations induced by cytokines signaling through the common-γ-chain cytokine receptor in T cells, while lowest inhibition was demonstrated for IL-10 -induced STAT3 phosphorylation in monocytes. Constitutive STAT1, STAT3, STAT4 and STAT5 phosphorylation in monocytes and/or T cells was also downregulated by tofacitinib. Tofacitinib treatment downregulated the expression of several JAK-STAT pathway components in PBMCs, SOCSs showing the strongest downregulation. Baseline STAT phosphorylation levels in T cells and monocytes and SOCS3 expression in PBMCs correlated with treatment response.ConclusionsTofacitinib suppresses multiple JAK-STAT pathways in cytokine and cell population specific manner in RA patients in vivo. Besides directly inhibiting JAK activation, tofacitinib downregulates the expression of JAK-STAT pathway components. This may modulate the effects of tofacitinib on JAK-STAT pathway activation in vivo and explain some of the differential findings between the current study and previous in vitro studies. Finally, baseline immunological markers associate with the treatment response to tofacitinib.

Interferon regulatory factor 2 binding protein 2 (IRF2BP2) is a transcriptional coregulator that has an important role in the regulation of the immune response. IRF2BP2 has been associated with the Janus kinase (JAK)—signal transducers and activators of transcription (STAT) pathway, but its exact role remains elusive. Here, we identified a novel clinical variant, IRF2BP2 c.625_665del, from two members of a family with inflammatory conditions and investigated the function of IRF2BP2 and c.625_665del mutation in JAK–STAT pathway activation and inflammatory signaling. The levels of constitutive and cytokine-induced phosphorylation of STATs and total STAT1 in peripheral blood monocytes, T cells, and B cells from the patients and four healthy controls were measured by flow cytometry. Inflammation-related gene expression was studied in peripheral blood mononuclear cells using direct digital detection of mRNA (NanoString). Finally, we studied the relationship between IRF2BP2 and STAT1 activation using a luciferase reporter system in a cell model. Our results show that patients having the IRF2BP2 c.625_665del mutation presented overexpression of STAT1 protein and increased constitutive activation of STAT1. In addition, interferon-induced JAK–STAT signaling was upregulated, and several interferon-inducible genes were overexpressed. Constitutive phosphorylation of STAT5 was also found to be upregulated in CD4+ T cells from the patients. Using a cell model, we show that IRF2BP2 was needed to attenuate STAT1 transcriptional activity and that IRF2BP2 c.625_665del mutation failed in this. We conclude that IRF2BP2 has an important role in suppressing immune responses elicited by STAT1 and STAT5 and suggest that aberrations in IRF2BP2 can lead to abnormal function of intrinsic immunity.

The data on effects of tofacitinib on soluble proteins in patients with rheumatoid arthritis (RA) is currently very limited. We analysed how tofacitinib treatment and thus inhibition of the Janus kinase – signal transducer and activation on transcription pathway affects the in vivo levels of inflammation-related plasma proteins in RA patients. In this study, sixteen patients with active RA [28-joint Disease Activity Score (DAS28) >3.2] despite treatment with conventional synthetic disease-modifying antirheumatic drugs (csDMARDs) started tofacitinib treatment 5mg twice daily. Levels of 92 inflammation-related plasma proteins were determined by proximity extension assay at baseline and at three months. Tofacitinib treatment for three months, in csDMARD background, decreased mean DAS28 from 4.4 to 2.6 (p<0.001). Marked (>20%) and statistically significant (p<0.05) changes were found in the levels of 21 proteins, 18 of which decreased and three increased. Of these proteins 17 are directly involved in inflammatory responses or in the cellular response to cytokines. Highest (>50%) decrease was observed for interleukin-6 (IL-6), C-X-C motif chemokine ligand 1, matrix metalloproteinase-1 and AXIN1. Higher baseline level of IL-6, and lower levels of C-C motif chemokine 11 and Delta and Notch-like epidermal growth factor-related receptor were associated with DAS28 improvement. Our results indicate that tofacitinib downregulates several proinflammatory plasma proteins which may contribute to the clinical efficacy of tofacitinib. In addition, soluble biomarkers may predict the treatment response to tofacitinib.