Discovery of Novel Human Gene Regulatory Modules from Gene Co-expression and Promoter Motif Analysis.

Deciphering gene regulatory networks requires identification of gene expression modules. We describe a novel bottom-up approach to identify gene modules regulated by cis-regulatory motifs from a human gene co-expression network. Target genes of a cis-regulatory motif were identified from the network via the motif's enrichment or biased distribution towards transcription start sites in the promoters of co-expressed genes. A gene sub-network containing the target genes was extracted and used to derive gene modules. The analysis revealed known and novel gene modules regulated by the NF-Y motif. The binding of NF-Y proteins to these modules' gene promoters were verified using ENCODE ChIP-Seq data. The analyses also identified 8,048 Sp1 motif target genes, interestingly many of which were not detected by ENCODE ChIP-Seq. These target genes assemble into house-keeping, tissues-specific developmental, and immune response modules. Integration of Sp1 modules with genomic and epigenomic data indicates epigenetic control of Sp1 targets' expression in a cell/tissue specific manner. Finally, known and novel target genes and modules regulated by the YY1, RFX1, IRF1, and 34 other motifs were also identified. The study described here provides a valuable resource to understand transcriptional regulation of various human developmental, disease, or immunity pathways.

Inhibitory effect of trans-caryophyllene (TC) on leukocyte-endothelial attachment.

trans-Caryophyllene (TC) is a major component found in the essential oils of many spices and foods/medicinal plants. It is a natural sesquiterpene and has been the subject of numerous studies. However, the effects of TC on vascular inflammation remain unknown. In this study, we reported that TC treatment in human umbilical vein endothelial cells (HUVECs) prevented attachment of monocytic leukemia cell line THP-1 cells to endothelial cells. In addition, in vivo results indicate that TC inhibited macrophage infiltration to the aortic surface and reduced total serum levels of cholesterol and triglycerides. Importantly, administration of TC could inhibit the induction of vascular cell adhesion molecule-1 (VCAM-1) both in vitro and in vivo. Notably, our data indicate that the inhibitory effects of TC on the expression of VCAM-1 are mediated by the JAK2/STAT1/IRF-1 pathway. TC is a specific agonist of the type 2 cannabinoid receptor (CB2R). Importantly, we further verified that the inhibitory effects of TC on the expression of IRF-1 and VCAM-1 are dependent on activation of CB2R. Inhibition of CB2R by either specific inhibitors or RNA interference abolished the inhibitory effects of TC on the expression of IRF-1 and VCAM-1. Our results suggest that TC might have a capacity to suppress the development of atherosclerosis.

Zinc supplementation induces CD4+CD25+Foxp3+ antigen-specific regulatory T cells and suppresses IFN-γ production by upregulation of Foxp3 and KLF-10 and downregulation of IRF-1.

PURPOSE: The essential trace element zinc plays a fundamental role in immune function and regulation since its deficiency is associated with autoimmunity, allergies, and transplant rejection. Thus, we investigated the influence of zinc supplementation on the Th1-driven alloreaction in mixed lymphocyte cultures (MLC), on generation of antigen-specific T cells, and analyzed underlying molecular mechanisms. METHODS: Cell proliferation and pro-inflammatory cytokine production were monitored by [3H]-thymidine proliferation assay and ELISA, respectively. Analysis of surface and intracellular T cell marker was performed by flow cytometry. Western blotting and mRNA analysis were used for Foxp3, KLF-10, and IRF-1 expression. RESULTS: Zinc supplementation on antigen-specific T cells in physiological doses (50 µM) provokes a significant amelioration of cell proliferation and pro-inflammatory cytokine production after reactivation compared to untreated controls. Zinc administration on MLC results in an increased induction and stabilization of CD4+CD25+Foxp3+ and CD4+CD25+CTLA-4+ T cells (p < 0.05). The effect is based on zinc-induced upregulation of Foxp3 and KLF-10 and downregulation of IRF-1. However, in resting lymphocytes zinc increases IRF-1. CONCLUSION: In summary, zinc is capable of ameliorating the allogeneic immune reaction by enhancement of antigen-specific iTreg cells due to modulation of essential molecular targets: Foxp3, KLF-10, and IRF-1. Thus, zinc can be seen as an auspicious tool for inducing tolerance in adverse immune reactions.

Phytoestrogens mediated anti-inflammatory effect through suppression of IRF-1 and pSTAT1 expressions in lipopolysaccharide-activated microglia.

Microglial activation has been implicated in various neurological disorders, including Alzheimer's disease, Parkinson's disease, multiple sclerosis, and HIV encephalopathy. Phytoestrogens have been shown to be neuroprotective in neurotoxicity models; however, their effect on microglia has not been well established. In the current study, we report that the soy phytoestrogens, genistein, daidzein, and coumestrol, decreased nitric oxide (NO) production induced by lipopolysaccharide (LPS) in the rat microglial cell line (HAPI). The levels of inducible NO synthase (iNOS) mRNA and protein expression were also reduced. Transcription factors known to govern iNOS expression including interferon regulatory factor-1 (IRF-1) and phosphorylated STAT1 were down regulated. These observations explain, at least in part, the inhibitory effect of phytoestrogens on NO production. The levels of monocyte chemoattractant protein-1 and interleukin-6 mRNA, proinflammatory chemokine and cytokine associated with various neurological disorders, were also reduced following LPS stimulation when HAPI cells were pretreated with phytoestrogens. Hence, genistein, daidzein, and coumestrol could serve as anti-inflammatory agents and may have beneficial effects in the treatment of neurodegenerative diseases.

Early angiogenic response to shock waves in a three-dimensional model of human microvascular endothelial cell culture (HMEC-1).

The exact nature of shock wave (SW) action is not, as yet, fully understood, although a possible hypothesis may be that shock waves induce neoangiogenesis. To test this hypothesis, a three-dimensional (3D) culture model on Matrigel was developed employing a human microvascular endothelial cell line (HMEC-1) which was stimulated with low energy soft- focused SW generated by an SW lithotripter. After 12 hours we observed a statistically significant increase in capillary connections subsequent to shock-wave treatment in respect to the control group and a marked 3-hour down-regulation in genes involved in the apoptotic processes (BAX, BCL2LI, GADD45A, PRKCA), in cell cycle (CDKN2C, CEBPB, HK2, IRF1, PRKCA), oncogenes (JUN, WNT1), cell adhesion (ICAM-1), and proteolytic systems (CTSD, KLK2, MMP10). Our preliminary results indicate that microvascular endothelial cells in vitro quickly respond to SW, proliferating and forming vessel-like structures, depending on the energy level employed and the number of shocks released. The early decreased expression in the analysed genes could be interpreted as the first reactive response of the endothelial cells to the external stimuli and the prelude to the events characterizing the neo-angiogenic sequence.

New mimetic peptides of the kinase-inhibitory region (KIR) of SOCS1 through focused peptide libraries.

SOCS (suppressor of cytokine signalling) proteins are negative-feedback regulators of the JAK (Janus kinase)/STAT (signal transducer and activator of transcription) pathway. Their expression levels are low under physiological conditions, but they are up-regulated in response to cytokine stimulation in many immune and inflammatory processes. Overexpression of SOCS1 in keratinocyte clones abrogates the IFNγ (interferon γ)-induced expression of many pro-inflammatory genes and the release of related chemokines by blocking the JAK/STAT pathway. SOCS1 inhibits JAK2 kinase activity by binding the catalytic site of JAK2, with its KIR (kinase-inhibitory region) acting as a pseudo-substrate of the enzyme. In the present study, we screened a focused combinatorial peptide library of KIR to identify new peptides able to mimic its function with an improved affinity towards the JAK2 catalytic site. Using an alanine-scanning method, KIR residues that are crucial for the interaction with JAK2 were unveiled. In this way, the KIR sequence was restricted to a shorter segment and 'non-essential' residues were replaced by different amino acids following a simplified combinatorial approach. We selected a new unnatural sequence able to bind to JAK2 with Kd values in the nanomolar range. This peptide was tested in human keratinocyte cultures and reduced the phosphorylation of STAT1 and the expression levels of IRF-1 (interferon regulatory factor-1).

IFNgamma restores breast cancer sensitivity to fulvestrant by regulating STAT1, IFN regulatory factor 1, NF-kappaB, BCL2 family members, and signaling to caspase-dependent apoptosis.

Antiestrogens are effective therapies for the management of many estrogen receptor-alpha (ER)-positive breast cancers. Nonetheless, both de novo and acquired resistance occur and remain major problems in the clinical setting. IFNgamma is an inflammatory cytokine that induces the expression and function of IFN regulatory factor 1 (IRF1), a tumor suppressor gene that can increase antiestrogen responsiveness. We show that IFNgamma, but not IFNalpha, IFNbeta, or fulvestrant (ICI; ICI 182,780; Faslodex), induces IRF1 expression in antiestrogen-resistant MCF7/LCC9 and LY2 cells. Moreover, IFNgamma restores the responsiveness of these cells to fulvestrant. Increased IRF1 activation suppresses NF-kappaB p65 (RELA) activity, inhibits the expression of prosurvival (BCL2, BCL-W), and induces the expression of proapoptotic members (BAK, mitochondrial BAX) of the BCL2 family. This molecular signaling is associated with the activation of signal transducer and activator of transcription 1 and leads to increased mitochondrial membrane permeability; activation of caspase-7 (CASP7), CASP8, and CASP9; and induction of apoptosis but not autophagy. Whereas antiestrogen-resistant cells are capable of inducing autophagy through IFN-mediated signaling, their ability to do so through antiestrogen-regulated signaling is lost. The abilities of IFNgamma to activate CASP8, induce apoptosis, and restore antiestrogen sensitivity are prevented by siRNA targeting IRF1, whereas transient overexpression of IRF1 mimics the effects of IFNgamma treatment. These observations support the exploration of clinical trials combining antiestrogens and compounds that can induce IRF1, such as IFNgamma, for the treatment of some ER-positive breast cancers.

Anthocyanins from black soybean seed coats preferentially inhibit TNF-alpha-mediated induction of VCAM-1 over ICAM-1 through the regulation of GATAs and IRF-1.

Adhesion molecules have a key role in pathological inflammation. Thus, we investigated the effect of anthocyanins on the induction of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) by TNF-alpha and the possible molecular mechanisms by which anthocyanins differentially regulate ICAM-1 and VCAM-1 expression. Stimulation of cells with TNF-alpha increased ICAM-1 and VCAM-1 expression, and pretreatment with anthocyanins inhibited VCAM-1 expression, but not ICAM-1 expression. We found that IRF-1 and GATAs, especially GATA-4 and -6, were involved in the TNF-alpha-mediated expression of VCAM-1 but not ICAM-1, and anthocyanins decreased nuclear levels of GATA-4 and GATA-6 as well as IRF-1. Moreover, pretreatment with a Jak/STAT inhibitor decreased TNF-alpha-induced VCAM-1 expression and nuclear GATA-4, GATA-6, and IRF-1 levels. Furthermore, anthocyanins efficiently inhibited the phosphorylation of STAT-3. This suggests that anthocyanins differentially regulate TNF-alpha-mediated expression of VCAM-1 and ICAM-1 through modulation of the GATA and IRF-1 binding activity via Jak/STAT-3 activation.