Themis2/ICB1 Is a Signaling Scaffold That Selectively Regulates Macrophage Toll-Like Receptor Signaling and Cytokine Production

BACKGROUND: Thymocyte expressed molecule involved in selection 1 (Themis1, SwissProt accession number Q8BGW0) is the recently characterised founder member of a novel family of proteins. A second member of this family, Themis2 (Q91YX0), also known as ICB1 (Induced on contact with basement membrane 1), remains unreported at the protein level despite microarray and EST databases reporting Themis2 mRNA expression in B cells and macrophages. METHODOLOGY/PRINCIPAL FINDINGS: Here we characterise Themis2 protein for the first time and show that it acts as a macrophage signalling scaffold, exerting a receptor-, mediator- and signalling pathway-specific effect on TLR responses in RAW 264.7 macrophages. Themis2 over-expression enhanced the LPS-induced production of TNF but not IL-6 or Cox-2, nor TNF production induced by ligands for TLR2 (PAM3) or TLR3 (poly IratioC). Moreover, LPS-induced activation of the MAP kinases ERK and p38 was enhanced in cells over-expressing Themis2 whereas the activation of JNK, IRF3 or NF-kappaB p65, was unaffected. Depletion of Themis2 protein by RNA inteference inhibited LPS-induced TNF production in primary human macrophages demonstrating a requirement for Themis2 in this event. Themis2 was inducibly tyrosine phosphorylated upon LPS challenge and interacted with Lyn kinase (P25911), the Rho guanine nucleotide exchange factor, Vav (P27870), and the adaptor protein Grb2 (Q60631). Mutation of either tyrosine 660 or a proline-rich sequence (PPPRPPK) simultaneously interrupted this complex and reduced by approximately 50% the capacity of Themis2 to promote LPS-induced TNF production. Finally, Themis2 protein expression was induced during macrophage development from murine bone marrow precursors and was regulated by inflammatory stimuli both in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: We hypothesise that Themis2 may constitute a novel, physiological control point in macrophage inflammatory responses

SOX2 Co-Occupies Distal Enhancer Elements with Distinct POU Factors in ESCs and NPCs to Specify Cell State

SOX2 is a master regulator of both pluripotent embryonic stem cells (ESCs) and multipotent neural progenitor cells (NPCs); however, we currently lack a detailed understanding of how SOX2 controls these distinct stem cell populations. Here we show by genome-wide analysis that, while SOX2 bound to a distinct set of gene promoters in ESCs and NPCs, the majority of regions coincided with unique distal enhancer elements, important cis-acting regulators of tissue-specific gene expression programs. Notably, SOX2 bound the same consensus DNA motif in both cell types, suggesting that additional factors contribute to target specificity. We found that, similar to its association with OCT4 (Pou5f1) in ESCs, the related POU family member BRN2 (Pou3f2) co-occupied a large set of putative distal enhancers with SOX2 in NPCs. Forced expression of BRN2 in ESCs led to functional recruitment of SOX2 to a subset of NPC-specific targets and to precocious differentiation toward a neural-like state. Further analysis of the bound sequences revealed differences in the distances of SOX and POU peaks in the two cell types and identified motifs for additional transcription factors. Together, these data suggest that SOX2 controls a larger network of genes than previously anticipated through binding of distal enhancers and that transitions in POU partner factors may control tissue-specific transcriptional programs. Our findings have important implications for understanding lineage specification and somatic cell reprogramming, where SOX2, OCT4, and BRN2 have been shown to be key factors

Serotonin and the renal blood supply: Role of prostaglandins and the 5HT-2 receptor

Serotonin and the renal blood supply: role of prostaglandins and the 5HT-2 receptor. Serotonin (5HT) has been reported to increase, to decrease, or not to change renal blood flow. We postulated that prostaglandin release in response to 5HT acted as a confusing variable, and tested the hypothesis by comparing infusions of serotonin and angiotensin into one renal artery of 14 anesthetized dogs before and after indomethacin administration. Renal blood flow (Q, by EM flowmeter) responses to 5HT were routinely biphasic, an initial sharp decrease followed by a gradual increase which stabilized well above baseline at three to six minutes. Indomethacin, 1 to 2 mg/kg, did not alter the acute Q decrement induced by 5HT but abolished the increase in Q at three to six minutes (P < 0.001). Instead, sustained vasoconstriction became apparent. Ketanserin reversed the sustained vasoconstrictor effect of 5HT in indomethacin–treated dogs, leading to striking, serotonin–induced vasodilatation. The latter vasodilatation in turn was inhibited by methysergide. Four independent elements, two promoting vasoconstriction and two vasodilatation, are suggested. One vasodilator response is abolished by prostaglandin synthetase inhibition and the other by methysergide, a complex 5HT receptor blocker. The sustained vasoconstrictor response is blocked by ketanserin, suggesting an action on the 5HT-2 receptor. The initial, transient vasoconstrictor response is resistant to the blockers employed. These complex interactions may account for the variability in reported responses of the renal blood supply to serotonin