Tob proteins suppress steroid hormone receptor-mediated transcriptional activation

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Abstract

Although sex steroid hormones have significant effects on bone metabolism, the molecular mechanisms of these actions have not been fully elucidated yet. We examined the functional relationship between steroid hormone receptors and Tob, a member of an anti-prolifelative protein family and a negative regulator of osteoblast proliferation and differentiation. Luciferase assay using promoters carrying hormone-responsive elements revealed that both Tob1 and Tob2 proteins but not PC3 suppressed steroid hormone receptor-dependent transcriptional activation in MC3T3-E1 osteoblastic cells. Mutated Tob proteins carrying amino acid substitutions at an LXXLL motif also showed the same degree of inhibition of the transcriptional activation as the wild type. By observation of androgen receptor (AR)-tagged with green fluorescent protein under a confocal laser scanning microscope, we found that Tob1 inhibits the nuclear foci formation of dihydrotestosterone-bound AR. These results indicate that Tob family proteins may negatively regulate sex steroid hormone action in bone formation.

Introduction

Sex steroid hormones play important roles not only in the reproductive system but also in bone metabolism. It is well known that estrogen deficiency after menopause is the most important factor in osteoporosis development among women. Estrogen replacement is one of the major therapies for female postmenoposal osteoporosis. On the other hand, loss of androgen function also causes various abnormalities in bone metabolism (Hofbauer and Khosla, 1999, Manolagas et al., 2002). As well as ovariectomy, orchidectomized mice exhibited marked bone loss due to excessive bone resorption (Onoe et al., 2000). More recently, androgen receptor-deficient mice were generated and showed decreased bone volume with increased bone resorption (Yeh et al., 2002, Kawano et al., 2003).

In the last few years, the mechanism of steroid hormone action in a cell has been extensively studied at molecular level. After steroid hormones enter their target cells, these hormones bind to their specific receptors. Ligand-bound receptors change their conformation and regulate transcription by binding to hormone responsive elements located in regulatory regions of target genes. The effects of nuclear receptors on transcription are modulated by coregulator proteins, which enhance (coactivators) or reduce (corepressors) transactivation of target genes. Many of these coactivators possess histone acetylase activity, whereas corepressor complexes often contain histone deacetylase activity. Reversible acetylation of core histones modulates chromatin structure and regulates transcription. These coactivators commonly possess an LXXLL motif in their amino acid sequences through which they can interact with steroid hormone receptors. Introducing mutations in this motif abolished the function of these coactivators (Leo and Chen, 2000, Aranda and Pascual, 2001, Cheng et al., 2002, Heinlein and Chang, 2002).

Tob, which was originally discovered as a protein interacting with a membrane receptor c-erbB2, is a member of a functionally and structurally related protein family involved in negative control of the cell cycle (Matsuda et al., 1996, Ikematsu et al., 1999, Matsuda et al., 2001, Tirone, 2001). This family comprises six proteins; Tob1, Tob2, BTG1, BTG2/PC3/TIS21, ANA/BTG3 and PC3B. These proteins share a homology at N-terminal 120 amino acid residues. Anti-proliferating activity of Tob protein is regulated by phosphorylation of three serine residues (Ser152, 154 and 164) that are modified by Erk MAP Kinase. In quiescent cells, Tob1 blocks their proliferation by suppression of cyclin D1 expression. Growth signals activate Erk that phosphorylates Tob protein to lose its anti-proliferating activity, resulting in continuous expression of cyclin D1 and cell cycle progression (Maekawa et al., 2002, Suzuki et al., 2002, Yoshida et al., 2003).

It has been indicated that Tob member proteins would mainly act on transcription. These proteins have been shown to physically interact with Caf1, whose yeast homologue is involved in a transcription complex (Rouault et al., 1998, Ikematsu et al., 1999, Prevot et al., 2001). Furthermore, four of the Tob family proteins carry an LXXLL motif being common in coactivators for nuclear receptors. In fact, BTG1 and BTG2 modulate the estrogen receptor (ER)-mediated transcriptional activation through this LXXLL motif (Prevot et al., 2001).

Mice carrying a targeted deletion of the Tob gene have a greater bone mass resulting from increased numbers of osteoblasts. Bone morphogenetic protein-2 (BMP-2), a member of the transforming growth factor-beta (TGF-β) superfamily, has been shown to control osteoblast proliferation and differentiation. BMP signals are mainly mediated by Smad proteins. In these Tob-deficient mice, BMP-2-induced osteoblast proliferation and differentiation was enhanced. Tob was shown to associate with receptor-regulated Smads (Smad1, 5 and 8), which are the mediators of BMP signals, and inhibit the signal transduction. These results indicate that Tob protein negatively regulates bone formation in vivo by repressing BMP-2-induced, Smad-mediated transcriptional activation (Yoshida et al., 2000).

In the present study, we investigated whether Tob expression influences steroid hormone receptor-dependent transcriptional activation in osteoblasts. Tob proteins suppressed the ligand-dependent transcriptional activation of steroid hormone receptors such as androgen receptor (AR) and estrogen receptor (ER) in osteoblastic cells. These results indicate that Tob proteins could regulate the action of steroid hormone in bone formation.

Section snippets

Cells

MC3T3-E1 osteoblastic cells were obtained from Riken Cell Bank (Tokyo, Japan). Cells were maintained in α-MEM (Invitrogen Corp., Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Cansera International Inc., Canada). COS-7 monkey kidney cells were maintained in DMEM (Invitrogen) with 10% FBS and PC3 prostatic carcinoma cells were maintained in RPMI-160 medium (Sigma-Aldrich Co., St. Louis, MO).

Plasmid constructs

The firefly luciferase reporter plasmid, pGL3-MMTV (Tomura et al., 2001) and the expression

Suppression of steroid hormone receptor-mediated transcriptional activation by Tob protein

We investigated whether Tob family proteins modulate the function of steroid hormone receptors in osteoblasts because some of Tob family proteins have one or two copies of an LXXLL motif that is commonly found in interaction domains of coactivators for nuclear receptors (Fig. 1). To examine whether expression of the Tob family proteins influences the transcriptional activation mediated by steroid hormone receptors, cotransfection of AR and Tob expression plasmids was performed with the

Discussion

In the present study, we demonstrated that expression of Tob protein in MC3T3-C1 osteoblastic cells suppressed the ligand-dependent transactivation function of steroid hormone receptors, AR and ERα, and inhibited the ligand-dependent nuclear foci formation of AR.

Some of the Tob family proteins have an LXXLL motif that has been identified in many nuclear receptor coregulators. Through this motif, these coregulators can directly interact with nuclear receptors to modulate the transcriptional

Acknowledgements

This work was supported in part by grants-in-aid for Scientific Research (B) and Exploratory Research and a grant for the 21st Century Center of Excellence (COE) Program (Kyushu University) from the Japanese Ministry of Education, Culture, Sports, Science and Technology.

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