MiR221 promotes precursor B-cell retention in the bone marrow by amplifying the PI3K-signaling pathway in mice.

Hematopoietic stem cells and lineage-uncommitted progenitors are able to home to the bone marrow upon transplantation and reconstitute the host with hematopoietic progeny. Expression of miR221 in B-lineage committed preBI-cells induces their capacity to home to the bone marrow. However, the molecular mechanisms underlying miR221-controlled bone marrow homing and retention remain poorly understood. Here, we demonstrate, that miR221 regulates bone marrow retention of such B-cell precursors by targeting PTEN, thus enhancing PI3K signaling in response to the chemokine CXCL12. MiR221-enhanced PI3K signaling leads to increased expression of the anti-apoptotic protein Bcl2 and VLA4 integrin-mediated adhesion to VCAM1 in response to CXCL12 in vitro. Ablation of elevated PI3K activity abolishes the retention of miR221 expressing preBI-cells in the bone marrow. These results suggest that amplification of PI3K signaling by miR221 could be a general mechanism for bone marrow residence, shared by miR221-expressing hematopoietic cells.

A respiratory chain controlled signal transduction cascade in the mitochondrial intermembrane space mediates hydrogen peroxide signaling.

Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) govern cellular homeostasis by inducing signaling. H2O2 modulates the activity of phosphatases and many other signaling molecules through oxidation of critical cysteine residues, which led to the notion that initiation of ROS signaling is broad and nonspecific, and thus fundamentally distinct from other signaling pathways. Here, we report that H2O2 signaling bears hallmarks of a regular signal transduction cascade. It is controlled by hierarchical signaling events resulting in a focused response as the results place the mitochondrial respiratory chain upstream of tyrosine-protein kinase Lyn, Lyn upstream of tyrosine-protein kinase SYK (Syk), and Syk upstream of numerous targets involved in signaling, transcription, translation, metabolism, and cell cycle regulation. The active mediators of H2O2 signaling colocalize as H2O2 induces mitochondria-associated Lyn and Syk phosphorylation, and a pool of Lyn and Syk reside in the mitochondrial intermembrane space. Finally, the same intermediaries control the signaling response in tissues and species responsive to H2O2 as the respiratory chain, Lyn, and Syk were similarly required for H2O2 signaling in mouse B cells, fibroblasts, and chicken DT40 B cells. Consistent with a broad role, the Syk pathway is coexpressed across tissues, is of early metazoan origin, and displays evidence of evolutionary constraint in the human. These results suggest that H2O2 signaling is under control of a signal transduction pathway that links the respiratory chain to the mitochondrial intermembrane space-localized, ubiquitous, and ancient Syk pathway in hematopoietic and nonhematopoietic cells.

miR-221 redirects precursor B cells to the BM and regulates their residence.

Pluripotent hematopoietic stem cells and multipotent myeloid/lymphoid progenitors express miR-221 and miR-222. When Pax5 expression commits these progenitors to monopotent pre-B lymphocytes the two microRNAs (miRNAs) are downregulated. Upon transplantation, stem cells and progenitors can reside in the BM, while pre-B cells, after their commitment, no longer do so. Retrovirally transduced, doxycycline-induced overexpression of either miR-221 or miR-222 in pre-B-I cells does not revert their monopotency to multipotency. However, upon transplantation miR-221, but not miR-222, transduced pre-B-I cells regain the capacity to reside in the BM. Upon subsequent termination of miR-221-expression by removal of doxycycline, the transplanted cells leave the BM again. Microarray analyses identified 25 downregulated miR-221-target genes, which could function to localize phases of B-lymphocyte development in BM before and after commitment.

Biphenotypic B-lymphoid/myeloid cells expressing low levels of Pax5: potential targets of BAL development.

The expression of Pax5 commits common lymphoid progenitor cells to B-lymphoid lineage differentiation. Little is known of possible variations in the levels of Pax5 expression and their influences on hematopoietic development. We have developed a retroviral transduction system that allows for the study of possible intermediate stages of this commitment by controlling the levels of Pax5 expressed in Pax5-deficient progenitors in vitro and in vivo. Retroviral transduction of Pax5-deficient pro-/pre-B cell lines with a doxycycline-inducible (TetON) form of the human Pax5 (huPax5) gene yielded cell clones that could be induced to different levels of huPax5 expression. Clones inducible to high levels developed B220(+)/CD19(+)/IgM(+) B cells, while clones with low levels differentiated to B220(+)/CD19(-)/CD11b(+)/Gr-1(-) B-lymphoid/myeloid biphenotypic cells in vitro and in vivo. Microarray analyses of genes expressed at these lower levels of huPax5 identified C/ebpα, C/ebpδ, Pu.1, Csf1r, Csf2r, and Gata-3 as myeloid-related genes selectively expressed in the pro-/pre-B cells that can develop under myeloid/lymphoid conditions to biphenotypic cells. Therefore, reduced expression of huPax5 during the induction of early lymphoid progenitors to B-lineage-committed cells can fix this cellular development at a stage that has previously been seen during embryonic development and in acute lymphoblastic lymphoma-like biphenotypic acute leukemias.

The non-Ig parts of the VpreB and λ5 proteins of the surrogate light chain play opposite roles in the surface representation of the precursor B cell receptor.

The VpreB and λ5 proteins, together with Igµ-H chains, form precursor BCRs (preBCRs). We established λ5(-/-)/VpreB1(-/-)/VpreB2(-/-) Abelson virus-transformed cell lines and reconstituted these cells with λ5 and VpreB in wild-type form or with a deleted non-Ig part. Whenever preBCRs had the non-Ig part of λ5 deleted, surface deposition was increased, whereas deletion of VpreB non-Ig part decreased it. The levels of phosphorylation of Syk, SLP65, or PLC-γ2, and of Ca(2+) mobilization from intracellular stores, stimulated by µH chain crosslinking Ab were dependent on the levels of surface-bound preBCRs. It appears that VpreB probes the fitness of newly generated VH domains of IgH chains for later pairing with IgL chains, and its non-Ig part fixes the preBCRs on the surface. By contrast, the non-Ig part of λ5 crosslinks preBCRs for downregulation and stimulation.

An adipose lncRAP2-Igf2bp2 complex enhances adipogenesis and energy expenditure by stabilizing target mRNAs.

lncRAP2 is a conserved cytoplasmic lncRNA enriched in adipose tissue and required for adipogenesis. Using purification and in vivo interactome analyses, we show that lncRAP2 forms complexes with proteins that stabilize mRNAs and modulate translation, among them Igf2bp2. Surveying transcriptome-wide Igf2bp2 client mRNAs in white adipocytes reveals selective binding to mRNAs encoding adipogenic regulators and energy expenditure effectors, including adiponectin. These same target proteins are downregulated when either Igf2bp2 or lncRAP2 is downregulated, hindering adipocyte lipolysis. Proteomics and ribosome profiling show this occurs predominantly through mRNA accumulation, as lncRAP2-Igf2bp2 complex binding does not impact translation efficiency. Phenome-wide association studies reveal specific associations of genetic variants within both lncRAP2 and Igf2bp2 with body mass and type 2 diabetes, and both lncRAP2 and Igf2bp2 are suppressed in adipose depots of obese and diabetic individuals. Thus, the lncRAP2-Igf2bp2 complex potentiates adipose development and energy expenditure and is associated with susceptibility to obesity-linked diabetes.

The Super-Enhancer-Derived alncRNA-EC7/Bloodlinc Potentiates Red Blood Cell Development in trans.

Enhancer-derived RNAs are thought to act locally by contributing to their parent enhancer function. Whether large domains of clustered enhancers (super-enhancers) also produce cis-acting RNAs, however, remains unclear. Unlike typical enhancers, super-enhancers form large spans of robustly transcribed chromatin, amassing capped and polyadenylated RNAs that are sufficiently abundant to sustain trans functions. Here, we show that one such RNA, alncRNA-EC7/Bloodlinc, is transcribed from a super-enhancer of the erythroid membrane transporter SLC4A1/BAND3 but diffuses beyond this site. Bloodlinc localizes to trans-chromosomal loci encoding critical regulators and effectors of terminal erythropoiesis and directly binds chromatin-organizing and transcription factors, including the chromatin attachment factor HNRNPU. Inhibiting Bloodlinc or Hnrnpu compromises the terminal erythropoiesis gene program, blocking red cell production, whereas expressing Bloodlinc ectopically stimulates this program and can promote erythroblast proliferation and enucleation in the absence of differentiation stimuli. Thus, Bloodlinc is a trans-acting super-enhancer RNA that potentiates red blood cell development.

SYK kinase mediates brown fat differentiation and activation.

Brown adipose tissue (BAT) metabolism influences glucose homeostasis and metabolic health in mice and humans. Sympathetic stimulation of ß-adrenergic receptors in response to cold induces proliferation, differentiation, and UCP1 expression in pre-adipocytes and mature brown adipocytes. Here we show that spleen tyrosine kinase (SYK) is upregulated during brown adipocyte differentiation and activated by ß-adrenergic stimulation. Deletion or inhibition of SYK, a kinase known for its essential roles in the immune system, blocks brown and white pre-adipocyte proliferation and differentiation in vitro, and results in diminished expression of Ucp1 and other genes regulating brown adipocyte function in response to ß-adrenergic stimulation. Adipocyte-specific SYK deletion in mice reduces BAT mass and BAT that developed consisted of SYK-expressing brown adipocytes that had escaped homozygous Syk deletion. SYK inhibition in vivo represses ß-agonist-induced thermogenesis and oxygen consumption. These results establish SYK as an essential mediator of brown fat formation and function.

PD-L1 is an activation-independent marker of brown adipocytes.

Programmed death ligand 1 (PD-L1) is expressed on a number of immune and cancer cells, where it can downregulate antitumor immune responses. Its expression has been linked to metabolic changes in these cells. Here we develop a radiolabeled camelid single-domain antibody (anti-PD-L1 VHH) to track PD-L1 expression by immuno-positron emission tomography (PET). PET-CT imaging shows a robust and specific PD-L1 signal in brown adipose tissue (BAT). We confirm expression of PD-L1 on brown adipocytes and demonstrate that signal intensity does not change in response to cold exposure or ß-adrenergic activation. This is the first robust method of visualizing murine brown fat independent of its activation state.Current approaches to visualise brown adipose tissue (BAT) rely primarily on markers that reflect its metabolic activity. Here, the authors show that PD-L1 is expressed on brown adipocytes, does not change upon BAT activation, and that BAT volume in mice can be measured by PET-CT with a radiolabeled anti-PD-L1 antibody.

Long non-coding RNAs as regulators of the endocrine system.

Long non-coding RNAs (lncRNAs) are a large and diverse group of RNAs that are often lineage-specific and that regulate multiple biological functions. Many are nuclear and are essential parts of ribonucleoprotein complexes that modify chromatin segments and establish active or repressive chromatin states; others are cytosolic and regulate the stability of mRNA or act as microRNA sponges. This Review summarizes the current knowledge of lncRNAs as regulators of the endocrine system, with a focus on the identification and mode of action of several endocrine-important lncRNAs. We highlight lncRNAs that have a role in the development and function of pancreatic ß cells, white and brown adipose tissue, and other endocrine organs, and discuss the involvement of these molecules in endocrine dysfunction (for example, diabetes mellitus). We also address the associations of lncRNAs with nuclear receptors involved in major hormonal signalling pathways, such as estrogen and androgen receptors, and the relevance of these associations in certain endocrine cancers.

De Novo Reconstruction of Adipose Tissue Transcriptomes Reveals Long Non-coding RNA Regulators of Brown Adipocyte Development.

Brown adipose tissue (BAT) protects against obesity by promoting energy expenditure via uncoupled respiration. To uncover BAT-specific long non-coding RNAs (lncRNAs), we used RNA-seq to reconstruct de novo transcriptomes of mouse brown, inguinal white, and epididymal white fat and identified ∼1,500 lncRNAs, including 127 BAT-restricted loci induced during differentiation and often targeted by key regulators PPARγ, C/EBPα, and C/EBPß. One of them, lnc-BATE1, is required for establishment and maintenance of BAT identity and thermogenic capacity. lnc-BATE1 inhibition impairs concurrent activation of brown fat and repression of white fat genes and is partially rescued by exogenous lnc-BATE1 with mutated siRNA-targeting sites, demonstrating a function in trans. We show that lnc-BATE1 binds heterogeneous nuclear ribonucleoprotein U and that both are required for brown adipogenesis. Our work provides an annotated catalog for the study of fat depot-selective lncRNAs and establishes lnc-BATE1 as a regulator of BAT development and physiology.

Environments of B cell development.

B lymphocyte development in the mouse begins with the generation of long-term reconstituting, pluripotent hematopoietic stem cells, over multipotent myeloid/lymphoid progenitors and common lymphoid progenitors to B-lineage committed pro/pre B and pre B cells, which first express pre B cell receptors and then immunoglobulins, B cell receptors, to generate the repertoires of peripheral B cells. This development is influenced and guided by cells of non-hematopoietic and hematopoietic origins. We review here some of the recent developments, and our contributions in this fascinating field of developmental immunology.

Reprint of: Environments of B cell development.

B lymphocyte development in the mouse begins with the generation of long-term reconstituting, pluripotent hematopoietic stem cells, over multipotent myeloid/lymphoid progenitors and common lymphoid progenitors to B-lineage committed pro/pre B and pre B cells, which first express pre B cell receptors and then immunoglobulins, B cell receptors, to generate the repertoires of peripheral B cells. This development is influenced and guided by cells of non-hematopoietic and hematopoietic origins. We review here some of the recent developments, and our contributions in this fascinating field of developmental immunology.