Cyclical regulation of the insulin-like growth factor binding protein 3 gene in response to 1a,25-dihydroxyvitamin D3

The nuclear receptor vitamin D receptor (VDR) is known to associate with two vitamin D response element (VDRE) containing chromatin regions of the insulin-like growth factor binding protein 3 (IGFBP3) gene. In non-malignant MCF-10A human mammary cells, we show that the natural VDR ligand 1a,25-dihydroxyvitamin D3 (1a,25(OH)2D3) causes cyclical IGFBP3 mRNA accumulation with a periodicity of 60 min, while in the presence of the potent VDR agonist Gemini the mRNA is continuously accumulated. Accordingly, VDR also showed cyclical ligand-dependent association with the chromatin regions of both VDREs. Histone deacetylases (HDACs) play an important role both in VDR signalling and in transcriptional cycling. From the 11 HDAC gene family members, only HDAC4 and HDAC6 are up-regulated in a cyclical fashion in response to 1a,25(OH)2D3, while even these two genes do not respond to Gemini. Interestingly, HDAC4 and HDAC6 proteins show cyclical VDR ligand-induced association with both VDRE regions of the IGFBP3 gene, which coincides with histone H4 deacetylation on these regions. Moreover, combined silencing of HDAC4 and HDAC6 abolishes the cycling of the IGFBP3 gene. We assume that due to more efficient VDR interaction, Gemini induces longer lasting chromatin activation and therefore no transcriptional cycling but monotonically increasing IGFBP3 mRNA. In conclusion, 1a,25(OH)2D3 regulates IGFBP3 transcription through short-term cyclical association of VDR, HDAC4 and HDAC6 to both VDRE-containing chromatin regions. INTRODUCTION The natural vitamin D receptor (VDR) ligand 1a,25dihydroxyvitamin D3 (1a,25(OH)2D3) has an important role in the regulation of calcium and phosphate homoeostasis and bone mineralization (1). In addition to this classical role, there is both epidemiological and pre-clinical evidence that 1a,25(OH)2D3 is an anti-proliferative agent (2). The anti-proliferative effects of 1a,25(OH)2D3 include induction of a G1/G0 cell cycle arrest and stimulation of apoptosis, which are mediated by the up-regulation of tumour suppressors, such as the cyclin-dependent kinase inhibitory proteins p21 and p27 (3), and the down-regulation of oncogene products including Bcl-2 (4) and Myc (5). Mitogens, such as the insulin-like growth factors (IGFs), have also been reported to be down-regulated by 1a,25(OH)2D3 (6). In addition, also the up-regulation of factors that control the actions of mitogens, such as IGF binding proteins (IGFBPs), have important anti-cancer effects (7). Three members of the IFGBP gene family, IGFBP1, IGFBP3 and IGFBP5, respond to 1a,25(OH)2D3 (8), of which the IGFBP3 gene is the most prominent (9). This increases the impact of IGF-1 and the regulation of its circulating amounts by IGFBPs in models of the anti-proliferative action of 1a,25(OH)2D3 and its synthetic analogues (10). In addition, IGFBPs mediate IGF-independent actions, including the activation of the p21 gene, causing cell cycle arrest or cell death through induction of apoptosis (11). However, bound to cellular membranes, IGFBPs can have mitogenic, IGF-dependent effects on cellular growth (12,13). As a member of the nuclear receptor superfamily, VDR acts as a transcription factor that binds to specific vitamin D response elements (VDREs) within the regulatory regions of its primary target genes (14). Most VDR target genes contain multiple VDREs (8,15–17). For example, the IGFBP3 gene has a tandem of two VDREs *To whom correspondence should be addressed. Tel: +352 4666446267; Fax: +352 4666446435; Email: [email protected] 502–512 Nucleic Acids Research, 2011, Vol. 39, No. 2 Published online 19 September 2010 doi:10.1093/nar/gkq820 The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. at position 400 and the other VDRE at position 3350 relative to the transcription start site (TSS) (8). In the absence of ligand, VDR associates via co-repressor proteins with histone deacetylases (HDACs) (18). HDACs can also inactivate directly non-histone proteins, such as p53, E2F or a-tubulin by deacetylation (19–21). Therefore, HDACs have multiple influences in cellular processes. At present 11 human HDACs are known (22). HDACs 1, 2, 3 and 8 belonging to Class I are ubiquitously expressed and seem to be involved more in general cellular processes. The Class II HDACs 4, 5, 6, 7, 9 and 10 have more tissue-specific functions and distributions, while HDAC11 forms its own class (23,24). All these HDACs are sensitive to the inhibitor trichostatin A (TSA) (25). In addition to the classical HDACs, on which we are focusing in this study, there is a family of functionally related HDACs, referred to as sirtuins (26). The seven members of this family are not sensitive to TSA but use NAD as an essential co-factor. Recently, cyclical models have been proposed for the activation of transcription by nuclear receptors, including those for oestrogen receptor a on the trefoil factor-1 gene (27), for peroxisome proliferater-activated receptor d on the pyruvate dehydrogenase kinase 4 gene (28) and for VDR on the genes 24-hydroxylase (CYP24) (29) and cyclin-dependent kinase inhibitor 1A (CDKN1A) (30). In these models, the ligand-dependent transcription is seen as a cyclical process, where alternating de-repressing, activating and initiation actions are required, providing means to stringently regulate the endurance and strength of the transcriptional response (28). More than 3000 synthetic analogues of 1a,25(OH)2D3 are presently known and majority of them carry a modification in their aliphatic side chain (31). The 1a,25(OH)2D3 analogues have been developed with a goal to improve the biological profile of the natural hormone for therapeutic application either in hyperproliferative diseases, such as psoriasis and different types of cancer, or in bone disorders, such as osteoporosis (32). Most of the analogues described to date are agonists, with a few having been identified as antagonists. An interesting exception is Gemini, which is the first 1a,25(OH)2D3 analogue that carries two side chains (33,34). Molecular dynamics simulations of the Gemini– VDR complex showed that the analogue can bind the VDR-LBD in two different conformations (35,36). In one of these conformations Gemini acts as an agonist with one side chain taking the same position as that of the natural hormone. In contrast, in its other conformation Gemini acts as an inverse agonist, since both of its side chains take alternative positions to that of 1a,25(OH)2D3 (37). In this study, we performed detailed time course experiments and observed transcriptional cycling of IGFBP3 mRNA after 1a,25(OH)2D3 stimulation, but not in response to Gemini. This is reflected by ligand-dependent VDR association with both VDREs and histone 4 acetylation on the chromatin region of the more proximal VDRE of the IGFBP3 gene. The genes HDAC4 and HDAC6 are also up-regulated in a cyclical fashion in response to 1a,25(OH)2D3, whereas they do not respond to Gemini. Both HDACs are essential for the cycling of the IGFBP3 gene. Accordingly, HDAC4 and HDAC6 proteins show VDR ligand-induced association with both IGFBP3 VDREs. In conclusion, 1a,25(OH)2D3 regulates IGFBP3 transcription through cyclical association of HDAC4 and HDAC6 to its VDRE-containing chromatin regions. EXPERIMENTAL PROCEDURES