Vionnie W.C. Yu

FIAT represses ATF4-mediated transcription to regulate bone mass in transgenic mice

We report the characterization of factor inhibiting activating transcription factor 4 (ATF4)–mediated transcription (FIAT), a leucine zipper nuclear protein. FIAT interacted with ATF4 to inhibit binding of ATF4 to DNA and block ATF4-mediated transcription of the osteocalcin gene in vitro. Transgenic mice overexpressing FIAT in osteoblasts also had reduced osteocalcin gene expression and decreased bone mineral density, bone volume, mineralized volume, trabecular thickness, trabecular number, and decreased rigidity of long bones. Mineral homeostasis, osteoclast number and activity, and osteoblast proliferation and apoptosis were unchanged in transgenics. Expression of osteoblastic differentiation markers was largely unaffected and type I collagen synthesis was unchanged. Mineral apposition rate was reduced in transgenic mice, suggesting that the lowered bone mass was due to a decline in osteoblast activity. This cell-autonomous decrease in osteoblast activity was confirmed by measuring reduced alkaline phosphatase activity and mineralization in primary osteoblast cultures. These results show that FIAT regulates bone mass accrual and establish FIAT as a novel transcriptional regulator of osteoblastic function.

Sex steroid blockade enhances thymopoiesis by modulating Notch signaling

Velardi et al. show that sex steroids regulate thymopoiesis by directly modulating Notch signaling, and provide a novel clinical strategy to boost immune regeneration.

Specific bone cells produce DLL4 to generate thymus-seeding progenitors from bone marrow

Osteocalcin (Ocn)-expressing bone marrow cells produce the Notch ligand DLL4, and this is required for lymphoid progenitor cells to seed the thymus.

Inhibition of ATF4 Transcriptional Activity by FIAT/γ‐Taxilin Modulates Bone Mass Accrual

Abstract:  The basic domain‐leucine zipper protein, activating transcription factor 4 (ATF4), was recently shown to control key aspects of osteoblast biology. ATF4 regulates the timely onset of osteoblast differentiation, the synthesis of type I collagen, and the transcription of the osteocalcin and RANKL (receptor activator of NFκ‐B ligand) genes. Accordingly, the levels and activity of ATF4 are under tight control through mechanisms that include protein stability and phosphorylation. We have uncovered yet another mode of inhibition of ATF4 through its interaction with the leucine zipper protein FIAT (Factor Inhibiting ATF4‐mediated Transcription, also described as γ‐taxilin). FIAT/γ‐taxilin localizes to the nucleus in osteoblasts and dimerizes with ATF4 to form inactive dimers, because it does not contain a DNA‐binding basic domain moiety. The interaction of FIAT/γ‐taxilin with ATF4 thus inhibits ATF4‐mediated transcription. Transgenic mice overexpressing FIAT/γ‐taxilin show osteopenia and reduced expression of the ATF4 target gene, osteocalcin. Interestingly, FIAT/γ‐taxilin also interacts with the transcriptional co‐activator αNAC (Nascent polypeptide associated complex And Coactivator alpha), suggesting alternative, non‐mutually exclusive mechanisms contributing to the inhibition of ATF4‐dependent osteocalcin gene transcription by FIAT/γ‐taxilin.

Inhibiting stromal cell heparan sulfate synthesis improves stem cell mobilization and enables engraftment without cytotoxic conditioning.

The glycosyltransferase gene, Ext1, is essential for heparan sulfate production. Induced deletion of Ext1 selectively in Mx1-expressing bone marrow (BM) stromal cells, a known population of skeletal stem/progenitor cells, in adult mice resulted in marked changes in hematopoietic stem and progenitor cell (HSPC) localization. HSPC egressed from BM to spleen after Ext1 deletion. This was associated with altered signaling in the stromal cells and with reduced vascular cell adhesion molecule 1 production by them. Further, pharmacologic inhibition of heparan sulfate mobilized qualitatively more potent and quantitatively more HSPC from the BM than granulocyte colony-stimulating factor alone, including in a setting of granulocyte colony-stimulating factor resistance. The reduced presence of endogenous HSPC after Ext1 deletion was associated with engraftment of transfused HSPC without any toxic conditioning of the host. Therefore, inhibiting heparan sulfate production may provide a means for avoiding the toxicities of radiation or chemotherapy in HSPC transplantation for nonmalignant conditions.

Heterogeneity of the bone marrow niche.

Purpose of reviewThe bone marrow niche is increasingly recognized as heterogeneous with specific subtypes of mesenchymal niche cells governing the development or homeostasis of selective parenchymal hematopoietic subsets. The present review outlines recent efforts in dissecting these microniches regulated by unique cell pairings within the bone marrow and provides an overview of how the bone marrow orchestrates multiple facets of hematopoiesis. Recent findingsRecent advancement in technologies has significantly improved our understanding of the cellular and molecular constituents that contribute to regulation of hematopoiesis and to maintenance of the hematopoietic stem cells (HSCs). Transgenic mouse models that enable endogenous cell deletion or lineage tracing, coupled with advanced intravital microscopy has identified several mesenchymal cell types, including the osteolineage cells, megakaryocytes, macrophages, perivascular cells, and Schwann cells, to be indispensible regulators of hematopoiesis. These niche cells, when perturbed, each caused very specific hematopoietic consequences including impairment in B-cell maturation, T lineage development, erythropoiesis, and impact different aspects of HSC behavior such as quiescence, mobilization, and response to acute stress signals. SummaryThe emerging concept is that the bone marrow environment is composed of multiple microniches, each consisting of unique pairing of distinct supportive stromal cells with distinct hematopoietic subtypes to regulate a particular branch of hematopoietic cell process. The bone marrow can be viewed as a carrier with subcompartments tailored to support different hematopoietic activities.

Notch Receptor‐Ligand Engagement Maintains Hematopoietic Stem Cell Quiescence and Niche Retention

Notch is long recognized as a signaling molecule important for stem cell self‐renewal and fate determination. Here, we reveal a novel adhesive role of Notch‐ligand engagement in hematopoietic stem and progenitor cells (HSPCs). Using mice with conditional loss of O‐fucosylglycans on Notch EGF‐like repeats important for the binding of Notch ligands, we report that HSPCs with faulty ligand binding ability display enhanced cycling accompanied by increased egress from the marrow, a phenotype mainly attributed to their reduced adhesion to Notch ligand‐expressing stromal cells and osteoblastic cells and their altered occupation in osteoblastic niches. Adhesion to Notch ligand‐bearing osteoblastic or stromal cells inhibits wild type but not O‐fucosylglycan‐deficient HSPC cycling, independent of RBP‐JK‐mediated canonical Notch signaling. Furthermore, Notch‐ligand neutralizing antibodies induce RBP‐JK‐independent HSPC egress and enhanced HSPC mobilization. We, therefore, conclude that Notch receptor–ligand engagement controls HSPC quiescence and retention in the marrow niche that is dependent on O‐fucosylglycans on Notch. Stem Cells 2015;33:2280–2293

FIAT is co-expressed with its dimerization target ATF4 in early osteoblasts, but not in osteocytes.

FIAT represses osteocalcin gene transcription by heterodimerizing with ATF4 and preventing it from binding to DNA. We report here the expression profiles of FIAT and ATF4 during osteoblastogenesis. Messenger RNA levels for the osteoblast transcriptional regulators Satb2, Runx2, Fiat, and Atf4 were quantified using real-time reverse-transcription PCR (RT-qPCR) and respective protein levels monitored by immunodetection in differentiating primary osteoblast cultures. Satb2, Fiat, and Atf4 mRNA levels remained constant throughout the differentiation sequence, whereas Runx2 transcript levels were significantly increased by 12 days post-confluency. Using immunofluorescence, the SATB2, RUNX2, and ATF4 signals appeared to increase as a function of time in culture. FIAT protein expression was readily detected in early cultures, but signal intensity decreased thereafter. When immunoblotting was used to quantify the relative amounts of FIAT and ATF4 proteins, the expression levels of the two proteins were found to be inversely correlated. The decrease in FIAT protein levels coincided with increased binding of ATF4 to the osteocalcin gene promoter, and with increased osteocalcin expression measured by RT-qPCR or immunoblotting. Immunohistochemistry of long bones from mice at E16.5 and 2 days post-natal revealed that both proteins are initially expressed in osteoblasts. In adult bone, FIAT was detected in osteocytes, while ATF4 expression was observed in active osteoblasts and lining cells, but not in osteocytes. Taken together, these data support the idea that a stoichiometric excess of ATF4 over FIAT in mature osteoblasts releases ATF4 from sequestration by FIAT, thereby allowing ATF4 homodimerization and subsequent transactivation of the osteocalcin gene.

Hematopoietic Stem Cell and Its Bone Marrow Niche

Stem cells do not thrive without their niche. The bone marrow microenvironment is where hematopoietic stem cells maintain their cell state while receiving physiological input to modify their activity in response to changing physiological demands. The complexity of the bone marrow microenvironment is being unraveled and indicates that multiple different cell types contribute to the regulation of stem and progenitor cells. Further, it is becoming evident that the bone marrow represents a composite of niches with different components and different functional roles in hematopoiesis. It is now evident that alterations in specific stromal cells that comprise the bone marrow microenvironment can contribute to hematologic pathology. In this chapter, we will review the history of the niche concept, evolving information about its components and how niche dysfunction may contribute to disease.

FIAT Represses Bone Matrix Mineralization by Interacting With ATF4 Through Its Second Leucine Zipper

We have characterized FIAT, a 66 kDa leucine zipper (LZ) protein that dimerizes with activating transcription factor 4 (ATF4) to form inactive dimers that cannot bind DNA. Computer analysis identifies three putative LZ motifs within the FIAT amino acid sequence. We have used deletion‐ and/or site‐specific mutagenesis to individually inactivate these motifs in order to identify the functional LZ that mediates the FIAT–ATF4 interaction. Amino acids 194–222 that encode the FIAT LZ2 were deleted (mutant FIAT ZIP2 DEL). We inactivated each zipper individually by replacing two or three leucine residues within each zipper by alanine residues. The engineered mutations were L142A/L149A (mutant M1, first zipper), L208A/L215A/L222A (mutant M2, second zipper), and L441A/L448A (mutant M3, third zipper). MC3T3‐E1 osteoblastic cells with an integrated 1.3 kb mouse osteocalcin gene promoter fragment driving expression of luciferase were transfected with expression vectors for ATF4 and the various FIAT deletion‐ or site‐specific mutants. Inhibition of ATF4‐mediated transcription was compared between wild‐type (WT) and LZ FIAT mutants. The deletion mutant FIAT ZIP2 DEL and the sequence‐specific M2 mutant did not interact with ATF4 and were unable to inhibit ATF4‐mediated transcription. The M1 or M3 mutations did not affect the ability of FIAT to contact ATF4 or to inhibit its transcriptional activity. Stable expression of WT FIAT in osteoblastic cells inhibited mineralization, but not expression of the FIAT ZIP2 DEL and M2 mutants. This structure–function analysis reveals that FIAT interacts with ATF4 and modulates its activity through its second leucine zipper motif. J. Cell. Biochem. 105: 859–865, 2008.