Miyuki Suzawa

A histone lysine methyltransferase activated by non-canonical Wnt signalling suppresses PPAR-gamma transactivation.

Histone modifications induced by activated signalling cascades are crucial to cell-lineage decisions. Osteoblast and adipocyte differentiation from common mesenchymal stem cells is under transcriptional control by numerous factors. Although PPAR-γ (peroxisome proliferator activated receptor-γ) has been established as a prime inducer of adipogenesis, cellular signalling factors that determine cell lineage in bone marrow remain generally unknown. Here, we show that the non-canonical Wnt pathway through CaMKII–TAK1–TAB2–NLK transcriptionally represses PPAR-γ transactivation and induces Runx2 expression, promoting osteoblastogenesis in preference to adipogenesis in bone marrow mesenchymal progenitors. Wnt-5a activates NLK (Nemo-like kinase), which in turn phosphorylates a histone methyltransferase, SETDB1 (SET domain bifurcated 1), leading to the formation of a co-repressor complex that inactivates PPAR-γ function through histone H3-K9 methylation. These findings suggest that the non-canonical Wnt signalling pathway suppresses PPAR-γ function through chromatin inactivation triggered by recruitment of a repressing histone methyltransferase, thus leading to an osteoblastic cell lineage from mesenchymal stem cells.

Structural analyses reveal phosphatidyl inositols as ligands for the NR5 orphan receptors SF-1 and LRH-1

Vertebrate members of the nuclear receptor NR5A subfamily, which includes steroidogenic factor 1 (SF-1) and liver receptor homolog 1 (LRH-1), regulate crucial aspects of development, endocrine homeostasis, and metabolism. Mouse LRH-1 is believed to be a ligand-independent transcription factor with a large and empty hydrophobic pocket. Here we present structural and biochemical data for three other NR5A members-mouse and human SF-1 and human LRH-1-which reveal that these receptors bind phosphatidyl inositol second messengers and that ligand binding is required for maximal activity. Evolutionary analysis of structure-function relationships across the SF-1/LRH-1 subfamily indicates that ligand binding is the ancestral state of NR5A receptors and was uniquely diminished or altered in the rodent LRH-1 lineage. We propose that phospholipids regulate gene expression by directly binding to NR5A nuclear receptors.

Cytokines suppress adipogenesis and PPAR-gamma function through the TAK1/TAB1/NIK cascade.

Pluripotent mesenchymal stem cells in bone marrow differentiate into adipocytes, osteoblasts and other cells. Balanced cytodifferentiation of stem cells is essential for the formation and maintenance of bone marrow; however, the mechanisms that control this balance remain largely unknown. Whereas cytokines such as interleukin-1 (IL-1) and tumour-necrosis factor-α (TNF-α) inhibit adipogenesis, the ligand-induced transcription factor peroxisome proliferator-activated receptor-γ (PPAR-γ), is a key inducer of adipogenesis. Therefore, regulatory coupling between cytokine- and PPAR-γ-mediated signals might occur during adipogenesis. Here we show that the ligand-induced transactivation function of PPAR-γ is suppressed by IL-1 and TNF-α, and that this suppression is mediated through NF-κB activated by the TAK1/TAB1/NF-κB-inducing kinase (NIK) cascade, a downstream cascade associated with IL-1 and TNF-α signalling. Unlike suppression of the PPAR-γ transactivation function by mitogen-activated protein kinase-induced growth factor signalling through phosphorylation of the A/B domain, NF-κB blocks PPAR-γ binding to DNA by forming a complex with PPAR-γ and its AF-1-specific co-activator PGC-2. Our results suggest that expression of IL-1 and TNF-α in bone marrow may alter the fate of pluripotent mesenchymal stem cells, directing cellular differentiation towards osteoblasts rather than adipocytes by suppressing PPAR-γ function through NF-κB activated by the TAK1/TAB1/NIK cascade.

Retracted: A subfamily of RNA‐binding DEAD‐box proteins acts as an estrogen receptor α coactivator through the N‐terminal activation domain (AF‐1) with an RNA coactivator, SRA

One class of the nuclear receptor AF‐2 coactivator complexes contains the SRC‐1/TIF2 family, CBP/p300 and an RNA coactivator, SRA. We identified a subfamily of RNA‐binding DEAD‐box proteins (p72/p68) as a human estrogen receptor α (hERα) coactivator in the complex containing these factors. p72/p68 interacted with both the AD2 of any SRC‐1/TIF2 family protein and the hERα A/B domain, but not with any other nuclear receptor tested. p72/p68, TIF2 (SRC‐1) and SRA were co‐immunoprecipitated with estrogen‐bound hERα in MCF7 cells and in partially purified complexes associated with hERα from HeLa nuclear extracts. Estrogen induced co‐localization of p72 with hERα and TIF2 in the nucleus. The presence of p72/p68 potentiated the estrogen‐induced expression of the endogenous pS2 gene in MCF7 cells. In a transient expression assay, a combination of p72/p68 with SRA and one TIF2 brought an ultimate synergism to the estrogen‐induced transactivation of hERα. These findings indicate that p72/p68 acts as an ER subtype‐selective coactivator through ERα AF‐1 by associating with the coactivator complex to bind its AF‐2 through direct binding with SRA and the SRC‐1/TIF2 family proteins.

Differentiation and Transforming Growth Factor-β Receptor Down-regulation by Collagen-α2β1 Integrin Interaction Is Mediated by Focal Adhesion Kinase and Its Downstream Signals in Murine Osteoblastic Cells

Interaction of type I collagen (COL(I)) with α2β1 integrin causes differentiation and transforming growth factor (TGF)-β receptor down-regulation in osteoblastic cells (Takeuchi, Y., Nakayama, K., and Matsumoto, T. (1996) J. Biol. Chem.271, 3938–3644). The TGF-β receptor down-regulation enables cells to escape from the inhibition of differentiation by TGF-β. To clarify how the cell-matrix interaction regulates these phenotypic changes, signaling pathways were examined in murine MC3T3-E1 cells. Attachment of cells to COL(I) stimulated tyrosine phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK), a mitogen-activated protein kinase (MAPK), and enhanced MAPK activity. Inhibition of tyrosine kinase by herbimycin A, destruction of focal adhesion by cytochalasin D, or overexpression of antisense FAK mRNA prevented the activation of ERK/MAPK and the increase in alkaline phosphatase (ALP) activity. Transient expression of a MAPK-specific phosphatase, CL100, also suppressed the elevation of ALP activity. In addition, introduction of a constitutively active MAPK kinase enhanced ALP activity in the absence of collagen production. TGF-β receptor down-regulation was abrogated by treatments that inactivate FAK, whereas the expression of CL100 had no effect. These results demonstrate that COL(I)-α2β1 integrin interaction facilitates differentiation and down-regulates TGF-β receptors via the activation of FAK and its diverse downstream signals. These signaling pathways may play an important role in the sequential differentiation of osteoblasts during bone formation.

Selective Interaction of Vitamin D Receptor with Transcriptional Coactivators by a Vitamin D Analog

ABSTRACT The nuclear vitamin D receptor (VDR) is a member of a nuclear receptor superfamily and acts as a ligand-dependent transcription factor. A family of cotranscriptional activators (SRC-1, TIF2, and AIB-1) interacts with and activates the transactivation function of nuclear receptors in a ligand-dependent way. We examined interaction of VDR with these coactivators that was induced by several vitamin D analogs, since they exert differential subsets of the biological action of vitamin D through unknown mechanisms. Unlike other vitamin D analogs tested, OCT (22-oxa-1α,25-dihydroxyvitamin D3) induced interaction of VDR with TIF2 but not with SRC-1 or AIB-1. Consistent with these interactions, only TIF2 was able to potentiate the transactivation function of VDR bound to OCT. Thus, the present findings suggest that the structure of VDR is altered in a vitamin D analog-specific way, resulting in selective interactions of VDR with coactivators. Such selective interaction of coactivators with VDR may specify the array of biological actions of a vitamin D analog like OCT, possibly through activating a particular set of target gene promoters.

Ligand type-specific Interactions of Peroxisome Proliferator-activated Receptor γ with Transcriptional Coactivators

Ligand type-specific interactions of peroxisome proliferator-activated receptor with transcriptional coactivators. Yasuo Kodera, Ken-ichi Takeyama, Akiko Murayama, Miyuki Suzawa, Yoshikazu Masuhiro, and Shigeaki Kato This article has been withdrawn by the authors. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 42, p. 30505, October 18, 2013

The Herbicide Atrazine Activates Endocrine Gene Networks via Non-Steroidal NR5A Nuclear Receptors in Fish and Mammalian Cells

Atrazine (ATR) remains a widely used broadleaf herbicide in the United States despite the fact that this s-chlorotriazine has been linked to reproductive abnormalities in fish and amphibians. Here, using zebrafish we report that environmentally relevant ATR concentrations elevated zcyp19a1 expression encoding aromatase (2.2 µg/L), and increased the ratio of female to male fish (22 µg/L). ATR selectively increased zcyp19a1, a known gene target of the nuclear receptor SF-1 (NR5A1), whereas zcyp19a2, which is estrogen responsive, remained unchanged. Remarkably, in mammalian cells ATR functions in a cell-specific manner to upregulate SF-1 targets and other genes critical for steroid synthesis and reproduction, including Cyp19A1, StAR, Cyp11A1, hCG, FSTL3, LHß, INHα, αGSU, and 11ß-HSD2. Our data appear to eliminate the possibility that ATR directly affects SF-1 DNA- or ligand-binding. Instead, we suggest that the stimulatory effects of ATR on the NR5A receptor subfamily (SF-1, LRH-1, and zff1d) are likely mediated by receptor phosphorylation, amplification of cAMP and PI3K signaling, and possibly an increase in the cAMP-responsive cellular kinase SGK-1, which is known to be upregulated in infertile women. Taken together, we propose that this pervasive and persistent environmental chemical alters hormone networks via convergence of NR5A activity and cAMP signaling, to potentially disrupt normal endocrine development and function in lower and higher vertebrates.

Suppression of PPAR transactivation switches cell fate of bone marrow stem cells from adipocytes into osteoblasts.

Abstract:  Osteoblasts and adipocytes differentiate from common pleiotropic mesenchymal stem cells under transcriptional controls by numerous factors and multiple intracellular signalings. However, cellular signaling factors that determine cell fates of mensenchymal stem cells in bone marrow remain to be largely uncovered, though peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) is well established as a prime inducer of adipogenesis. Here, we describe two signaling pathways that induce the cell fate decision into osteoblasts from adipocytes. One signaling is a TAK1/TAB1/NIK cascade activated by TNF‐α and IL‐1, and the activated NF‐κB blocked the DNA binding of PPAR‐γ, attenuating the activated PPAR‐mediated adipogenesis. The second signaling is the noncanonical Wnt pathway through CaMKII‐TAK1/TAB2‐NLK. Activated NLK by a noncanonical Wnt ligand (Wnt‐5a) transrepresses PPAR transactivation through a histone methyltransferase, SETDB1. Wnt‐5a induces phosphorylation of NLK, leading to the formation of a corepressor complex that inactivates PPAR function through histone H3‐K9 methylation. Thus, two signaling pathways lead to an osteoblastic cell lineage decision from mesenchymal stem cells through two distinct modes of PPAR transrepression.

The DEAD-Box Protein DP103 (Ddx20 or Gemin-3) Represses Orphan Nuclear Receptor Activity via SUMO Modification

ABSTRACT Structural analysis of nuclear receptor subfamily V orphan nuclear receptors suggests that ligand-independent mechanisms must regulate this subclass of receptors. Here, we report that steroidogenic factor 1 (SF-1) and liver receptor homolog 1 are repressed via posttranslational SUMO modification at conserved lysines within the hinge domain. Indeed, mutating these lysines or adding the SUMO isopeptidase SENP1 dramatically increased both native and Gal4-chimera receptor activities. The mechanism by which SUMO conjugation attenuates SF-1 activity was found to be largely histone deacetylase independent and was unaffected by the AF2 corepressor Dax1. Instead, our data suggest that SUMO-mediated repression involves direct interaction of the DEAD-box protein DP103 with sumoylated SF-1. Of potential E3-SUMO ligase candidates, PIASy and PIASxα strongly promoted SF-1 sumoylation, and addition of DP103 enhanced both PIAS-dependent receptor sumoylation and SF-1 relocalization to discrete nuclear bodies. Taken together, we propose that DEAD-box RNA helicases are directly coupled to transcriptional repression by protein sumoylation.