Yoshikazu Masuhiro

Activation of the Estrogen Receptor Through Phosphorylation by Mitogen-Activated Protein Kinase

The phosphorylation of the human estrogen receptor (ER) serine residue at position 118 is required for full activity of the ER activation function 1 (AF-1). This Ser118 is phosphorylated by mitogen-activated protein kinase (MAPK) in vitro and in cells treated with epidermal growth factor (EGF) and insulin-like growth factor (IGF) in vivo. Overexpression of MAPK kinase (MAPKK) or of the guanine nucleotide binding protein Ras, both of which activate MAPK, enhanced estrogen-induced and antiestrogen (tamoxifen)-induced transcriptional activity of wild-type ER, but not that of a mutant ER with an alanine in place of Ser118. Thus, the activity of the amino-terminal AF-1 of the ER is modulated by the phosphorylation of Ser118 through the Ras-MAPK cascade of the growth factor signaling pathways.

Purification and Identification of p68 RNA Helicase Acting as a Transcriptional Coactivator Specific for the Activation Function 1 of Human Estrogen Receptor α

ABSTRACT The estrogen receptor (ER) regulates the expression of target genes in a ligand-dependent manner. The ligand-dependent activation function AF-2 of the ER is located in the ligand binding domain (LBD), while the N-terminal A/B domain (AF-1) functions in a ligand-independent manner when isolated from the LBD. AF-1 and AF-2 exhibit cell type and promoter context specificity. Furthermore, the AF-1 activity of the human ERα (hERα) is enhanced through phosphorylation of the Ser118 residue by mitogen-activated protein kinase (MAPK). From MCF-7 cells, we purified and cloned a 68-kDa protein (p68) which interacted with the A/B domain but not with the LBD of hERα. Phosphorylation of hERα Ser118 potentiated the interaction with p68. We demonstrate that p68 enhanced the activity of AF-1 but not AF-2 and the estrogen-induced as well as the anti-estrogen-induced transcriptional activity of the full-length ERα in a cell-type-specific manner. However, it did not potentiate AF-1 or AF-2 of ERβ, androgen receptor, retinoic acid receptor alpha, or mineralocorticoid receptor. We also show that the RNA helicase activity previously ascribed to p68 is dispensable for the ERα AF-1 coactivator activity and that p68 binds to CBP in vitro. Furthermore, the interaction region for p68 in the ERα A/B domain was essential for the full activity of hERα AF-1. Taken together, these findings show that p68 acts as a coactivator specific for the ERα AF-1 and strongly suggest that the interaction between p68 and the hERα A/B domain is regulated by MAPK-induced phosphorylation of Ser118.

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.

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.

Adiponectin inhibits Toll‐like receptor family‐induced signaling

Recent studies have shown that adiponectin, an adipocyte‐derived cytokine, acts as a potent inhibitor of inflammatory responses. It has been also demonstrated that bacterial and viral signalings in host cells are triggered via Toll‐like receptor (TLR) molecules. Therefore, in the present study, we investigated whether globular adiponectin (gAd) would be able to inhibit TLR‐mediated nuclear factor‐κB (NF‐κB) signaling in mouse macrophages (RAW264). gAd predominantly bound to the AdipoR1 receptor and suppressed TLR‐mediated NF‐κB signaling. gAd‐mediated inhibition of TLR‐mediated IκB phosphorylation and NF‐κB activation was eliminated by the pretreatment of cycloheximide. Also their inhibitions of gAd were blocked by preincubation of the cells with an antibody against AdipoR1, but not with an antibody against AdipoR2. Taken together, these findings indicate that adiponectin negatively regulates macrophage‐like cell response to TLR ligands via an unknown endogenous product(s).

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

p300 mediates functional synergism between AF-1 and AF-2 of estrogen receptor alpha and beta by interacting directly with the N-terminal A/B domains.

Estrogen receptor (ER) α and β mediate estrogen actions in target cells through transcriptional control of target gene expression. For 17β-estradiol-induced transactivation, the N-terminal A/B domain (AF-1) and the C-terminal E/F domain (AF-2) of ERs are required. Ligand binding is considered to induce functional synergism between AF-1 and AF-2, but the molecular mechanism remains unknown. To clarify this synergism, we studied the role of reported AF-2 coactivators, p300/CREB binding protein, steroid receptor coactivator-1/transcriptional intermediary factor-2 (SRC-1/TIF2) family proteins and thyroid hormone receptor-associated protein-220/(vitamin D3 receptor-interacting protein- 205-(TRAP220/DRIP205) on the AF-1 activity in terms of synergism with the AF-2 function. We found that neither any of the SRC-1/TIF2 family coactivators nor TRAP220/DRIP205 is potent, whereas p300 potentiates the AF-1 function of both human ERα and human ERβ. Direct interactions of p300 with the A/B domains of ERα and ERβ were observed in an in vitro glutathioneS-transferase pull-down assay in accordance with the interactions in yeast and mammalian two-hybrid assays. Furthermore, mutations in the p300 binding sites (56–72 amino acids in ERα and 62–72 amino acids in ERβ) in the A/B domains caused a reduction in ligand-induced transactivation functions of both ERα and ERβ. Thus, these findings indicate that ligand-induced functional synergism between AF-1 and AF-2 is mediated through p300 by its direct binding to the A/B regions of ERα and ERβ.

Molecular mechanism of a cross‐talk between oestrogen and growth factor signalling pathways

Oestrogen (E2) plays significant roles in variety of biological events such as the development and maintenance of female reproductive organs, bone and lipid metabolisms. More recently, from study of knock‐out mice deficient in oestrogen receptor (ER) α and ERβ it turned out that normal spermatogenesis requires the E2 actions. Furthermore, this female steroid hormone is also well known to be deeply involved in many pathophysiological events such as osteoporosis and cancer development in female reproductive organs. It is particularly well known that most breast cancer is dependent on E2 in its development. Such E2 actions are thought to be mediated through two subtypes of ERs. Growth factors have been shown to synergize in this E2 signalling pathway, although the actual molecular mechanism largely remains unknown.

Intron retention generates a novel isoform of the murine vitamin D receptor that acts in a dominant negative way on the vitamin D signaling pathway.

We identified and characterized a novel rat vitamin D receptor isoform (rVDR1), which retains intron 8 of the canonical VDR (rVDR0) during alternative splicing. In this isoform protein directed by the stop codon in this newly identified exon, a part of the ligand binding domain (86 amino acids) is truncated at the C-terminal end but contains 19 extra amino acids. The rVDR1 transcript was expressed at a level 1/15 to 1/20 of that of rVDR0 in the kidney and intestine in adult rats but not in embryos. The recombinant rVDR1 protein showed no ligand binding activity. Homo- and heterodimers of the recombinant rVDR0 and rVDR1 proteins bound to a consensus vitamin D response element (VDRE) but not to consensus response elements for thyroid hormone and retinoic acid. However, unlike rVDR0, rVDR1 did not form a heterodimeric complex with RXR on the VDRE. A transient expression assay showed that this isoform acted as a dominant negative receptor against rVDR0 transactivation. Interestingly, the dominant negative activities of rVDR1 differed among VDREs. Thus, the present study indicates that this new VDR isoform negatively modulates the vitamin D signaling pathway, through a particular set of target genes.

Molecular mechanism of a cross-talk between estrogen and growth-factor signaling pathways.

The actions of estrogen (E2) are considered to be mediated through its nuclear E2 receptor (ER). In cancer development, growth factors are shown to act synergistically with E2. Recently, we found that the mitogen-activated protein kinase, activated by growth factors, phosphorylates human ERα and this phosphorylation potentiates the transactivation function of human ERα demonstrating a novel cross-talk between E2 and growth factor-signaling pathways. In this review, the molecular mechanism of this cross-talk is discussed.