Igbp1 is part of a positive feedback loop in stem cell factor–dependent, selective mRNAtranslation initiation inhibiting erythroid differentiation

The authors thank Dr Victor de Jager for assistance with the Rosetta Resolver software; Dr Ivo Touw for many fruitful discussions and critical reading of the manuscript; Liu Wing for technical assistance; Drs Peter Seither, Andreas Weith (Boehringer Ingelheim, Biberach, Germany), Helmuth Dolznig, Thomas Waerner, and Sandra Pilat (IMP, Vienna, Austria) for mRNA profiling of erythroblasts, of which the complete data will be published elsewhere; Dr Bart Aarts (Erasmus MC, Rotterdam, The Netherlands) for assistance in confocal scanning microscopy; Dr David Brautigan (University of Virginia, Charlottesville) for anti-Igbp1 antibodies; Dr Manfred Boehm (National Institutes of Health/National Heart, Lung, and Blood Institute, Bethesda, MD) for anti-Uhmk1 antibodies; and Ortho-Biotech (Tilburg, The Netherlands) for their kind gift of Eprex (erythropoietin).Stem cell factor (SCF)–induced activation of phosphoinositide-3-kinase (PI3K) is required for transient amplification of the erythroblast compartment. PI3K stimulates the activation of mTOR (target of rapamycin) and subsequent release of the cap-binding translation initiation factor 4E (eIF4E) from the 4E-binding protein 4EBP, which controls the recruitment of structured mRNAs to polysomes. Enhanced expression of eIF4E renders proliferation of erythroblasts independent of PI3K. To investigate which mRNAs are selectively recruited to polysomes, we compared SCF-dependent gene expression between total and polysome-bound mRNA. This identified 111 genes primarily subject to translational regulation. For 8 of 9 genes studied in more detail, the SCF-induced polysome recruitment of transcripts exceeded 5-fold regulation and was PI3K-dependent and eIF4E-sensitive, whereas total mRNA was not affected by signal transduction. One of the targets, Immunoglobulin binding protein 1 (Igbp1), is a regulatory subunit of protein phosphatase 2A (Pp2a) sustaining mTOR signaling. Constitutive expression of Igbp1 impaired erythroid differentiation, maintained 4EBP and p70S6k phosphorylation, and enhanced polysome recruitment of multiple eIF4E-sensitive mRNAs. Thus, PI3K-dependent polysome recruitment of Igbp1 acts as a positive feedback mechanism on translation initiation underscoring the important regulatory role of selectivemRNArecruitment to polysomes in the balance between proliferation and maturation of erythroblasts. (Blood. 2008; 112:2750-2760)peer-reviewe

FoxO3a regulates erythroid differentiation and induces BTG1, an activator of protein arginine methyl transferase 1

Erythropoiesis requires tight control of expansion, maturation, and survival of erythroid progenitors. Because activation of phosphatidylinositol-3-kinase (PI3K) is required for erythropoietin/stem cell factor–induced expansion of erythroid progenitors, we examined the role of the PI3K-controlled Forkhead box, class O (FoxO) subfamily of Forkhead transcription factors. FoxO3a expression and nuclear accumulation increased during erythroid differentiation, whereas untimely induction of FoxO3a activity accelerated differentiation of erythroid progenitors to erythrocytes. We identified B cell translocation gene 1 (BTG1)/antiproliferative protein 2 as a FoxO3a target gene in erythroid progenitors. Promoter studies indicated BTG1 as a direct target of FoxO3a. Expression of BTG1 in primary mouse bone marrow cells blocked the outgrowth of erythroid colonies, which required a domain of BTG1 that binds protein arginine methyl transferase 1. During erythroid differentiation, increased arginine methylation coincided with BTG1 expression. Concordantly, inhibition of methyl transferase activity blocked erythroid maturation without affecting expansion of progenitor cells. We propose FoxO3a-controlled expression of BTG1 and subsequent regulation of protein arginine methyl transferase activity as a novel mechanism controlling erythroid expansion and differentiation

A secondary role for hypoxia and HIF1 in the regulation of (IFNγ-induced) PD-L1 expression in melanoma

Cancer cells are able to escape immune surveillance by upregulating programmed death ligand 1 (PD-L1). A key regulator of PD-L1 expression is transcriptional stimulation by the IFNγ/JAK/STAT pathway. Recent studies suggest that hypoxia can induce PD-L1 expression. As hypoxia presents a hallmark of solid tumor development, hypoxic control of PD-L1 expression may affect the efficacy of cancer immunotherapy. This study aims to explore the hypoxic regulation of PD-L1 expression in human melanoma, and its interaction with IFNγ-induced PD-L1 expression. Analysis of the cutaneous melanoma dataset from the cancer genome atlas revealed a significant correlation of the HIF1-signaling geneset signature with PD-L1 mRNA expression. However, this correlation is less pronounced than other key pathways known to control PD-L1 expression, including the IFNγ/JAK/STAT pathway. This secondary role of HIF1 in PD-L1 regulation was confirmed by analyzing single-cell RNA-sequencing data of 33 human melanoma tissues. Interestingly, PD-L1 expression in these melanoma tissues was primarily found in macrophages. However, also in these cells STAT1, and not HIF1, displayed the most pronounced correlation with PD-L1 expression. Moreover, we observed that hypoxia differentially affects PD-L1 expression in human melanoma cell lines. Knockdown of HIF1 expression indicated a minor role for HIF1 in regulating PD-L1 expression. A more pronounced influence of hypoxia was found on IFNγ-induced PD-L1 mRNA expression, which is controlled at a 952 bp PD-L1 promoter fragment. These findings, showing the influence of hypoxia on IFNγ-induced PD-L1 expression, are relevant for immunotherapy, as both IFNγ and hypoxia are frequently present in the tumor microenvironment

Presence of Skin Tissue-Resident Memory T Cells in Human Nonmalignant and Premalignant Melanocytic Skin Lesions and in Melanoma

ABSTRACT: The infiltration of tissue-resident memory (TRM) cells in melanoma correlates with improved survival, suggesting an important role for TRM cells in immunity against melanoma. However, little is known about the presence of TRM cells in nonmalignant and premalignant melanocytic lesions. This study aimed to evaluate the presence of TRM cells in human skin melanocytic lesions, representing the spectrum from healthy skin to metastatic melanoma. FFPE sections from healthy skin, sun-exposed skin, benign nevi, lentigo maligna (LM), primary LM melanoma, and primary cutaneous and metastatic melanoma were analyzed by immunohistochemistry. The number of infiltrating cells expressing TRM-associated markers, CD3, CD4, CD8, CD69, CD103, and CD49a, was quantified by digital analyses. Multiplex immunofluorescence was performed to analyze coexpression of TRM cell markers. More T cells and CD69+ cells were found in melanoma lesions, as compared with healthy skin and nevi. CD103+ and CD49a+ cell numbers did not significantly differ. More importantly, no differences were seen in expression of all markers between healthy skin and benign nevi. Similar results, except for CD69, were observed in LM melanoma, as compared with LM and sun-exposed skin. Interestingly, multiplex immunofluorescence showed that nevi tissues have comparable CD103+ T cell numbers with healthy skin but comprise more CD103+ CD8+ cells. Expression of TRM cell markers is significantly increased in melanoma, as compared with nonmalignant skin. Our data also show that TRM cells are not abundantly present already in premalignant tissues. Further studies on the specificity of TRM cells for melanocyte/melanoma antigens may reveal their significance in cancer immunosurveillance

IFN-γ-induced PD-L1 expression on human melanocytes is impaired in vitiligo

Mounting evidence shows that the PD-1/PD-L1 axis is involved in tumor immune evasion. This is demonstrated by anti-PD-1 antibodies that can reverse tumor-associated PD-L1 to functionally suppress anti-tumor T-cell responses. Since type I and II interferons are key regulators of PD-L1 expression in melanoma cells and IFN-γ-producing CD8+ T cells and IFN-α-producing dendritic cells are abundant in vitiligo skin, we aimed to study the role of PD-1/PD-L1 signalling in melanocyte destruction in vitiligo. Moreover, impaired PD-1/PD-L1 function is observed in a variety of autoimmune diseases. It is, therefore, hypothesized that manipulating PD-1/PD-L1 signalling might have therapeutic potential in vitiligo. The PD-1+ T cells were abundantly present in situ in perilesional vitiligo skin, but expression of PD-L1 was limited and confined exclusively to dermal T cells. More specifically, neither melanocytes nor other epidermal skin cells expressed PD-L1. Exposure to IFN-γ, but also type I interferons, increased PD-L1 expression in primary melanocytes and fibroblasts, derived from healthy donors. Primary human keratinocytes only showed increased PD-L1 expression upon stimulation with IFN-γ. More interestingly, melanocytes derived from non-lesional vitiligo skin showed no PD-L1 upregulation upon IFN-γ exposure, while other skin cells displayed significant PD-L1 expression after exposure. In a vitiligo skin explant model, incubation of non-lesional vitiligo skin with activated (IFN-γ-producing) T cells from vitiligo lesions was previously described to induce melanocyte apoptosis. Although PD-L1 expression was induced in epidermal cells in these explants, this induction was completely absent in melanocytes. The lack of PD-L1 upregulation by melanocytes in the presence of IFN-γ-producing T cells shows that melanocytes lack protection against T-cell attack during vitiligo pathogenesis. Manipulating PD-1/PD-L1 signalling may, therefore, be a therapeutic option for vitiligo patients

Differential Regulation of Foxo3a Target Genes in Erythropoiesis▿ †

The cooperation of stem cell factor (SCF) and erythropoietin (Epo) is required to induce renewal divisions in erythroid progenitors, whereas differentiation to mature erythrocytes requires the presence of Epo only. Epo and SCF activate common signaling pathways such as the activation of protein kinase B (PKB) and the subsequent phosphorylation and inactivation of Foxo3a. In contrast, only Epo activates Stat5. Both Foxo3a and Stat5 promote erythroid differentiation. To understand the interplay of SCF and Epo in maintaining the balance between renewal and differentiation during erythroid development, we investigated differential Foxo3a target regulation by Epo and SCF. Expression profiling revealed that a subset of Foxo3a targets was not inhibited but was activated by Epo. One of these genes was Cited2. Transcriptional control of Epo/Foxo3a-induced Cited2 was studied and compared with that of the Epo-repressed Foxo3a target Btg1. We show that in response to Epo, the allegedly growth-inhibitory factor Foxo3a associates with the allegedly growth-stimulatory factor Stat5 in the nucleus, which is required for Epo-induced Cited2 expression. In contrast, Btg1 expression is controlled by the cooperation of Foxo3a with cyclic AMP- and Jun kinase-dependent Creb family members. Thus, Foxo3a not only is an effector of PKB but also integrates distinct signals to regulate gene expression in erythropoiesis

Genome-wide analysis reveals NRP1 as a direct HIF1α-E2F7 target in the regulation of motorneuron guidance in vivo

In this study, we explored the existence of a transcriptional network co-regulated by E2F7 and HIF1α, as we show that expression of E2F7, like HIF1α, is induced in hypoxia, and because of the previously reported ability of E2F7 to interact with HIF1α. Our genome-wide analysis uncovers a transcriptional network that is directly controlled by HIF1α and E2F7, and demonstrates both stimulatory and repressive functions of the HIF1α -E2F7 complex. Among this network we reveal Neuropilin 1 (NRP1) as a HIF1α-E2F7 repressed gene. By performing in vitro and in vivo reporter assays we demonstrate that the HIF1α-E2F7 mediated NRP1 repression depends on a 41 base pairs 'E2F-binding site hub', providing a molecular mechanism for a previously unanticipated role for HIF1α in transcriptional repression. To explore the biological significance of this regulation we performed in situ hybridizations and observed enhanced nrp1a expression in spinal motorneurons (MN) of zebrafish embryos, upon morpholino-inhibition of e2f7/8 or hif1α. Consistent with the chemo-repellent role of nrp1a, morpholino-inhibition of e2f7/8 or hif1α caused MN truncations, which was rescued in TALEN-induced nrp1a(hu10012) mutants, and phenocopied in e2f7/8 mutant zebrafish. Therefore, we conclude that repression of NRP1 by the HIF1α-E2F7 complex regulates MN axon guidance in vivo