Identification of a highly specific and versatile vitamin D receptor antibody

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Abstract

The active form of vitamin D, 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) is critical for regulation of serum calcium and phosphorus levels and for proper maintenance of bone mineralization and neuromuscular function. Biological effects of 1,25(OH)2D3 are mediated through a nuclear steroid hormone receptor, known as the vitamin D receptor (VDR). The discovery of VDR in a number of different cell and tissue types, suggests that the physiological role of vitamin D may extend beyond the regulation of calcium homeostasis and bone function. Unfortunately, identification of tissues expressing VDR has been controversial due to low abundance of the receptor and quality of the antibodies used. Therefore, we elected to characterize a panel of commercially available VDR antibodies in order to identify antibodies with high specificity and sensitivity. To address these objectives, we have used multiple immunoassays to determine VDR expression in tissues from several organs from multiple species employing tissues from VDR knockout mice as critical negative controls. Many of the antibodies tested showed nonspecific binding that can account for divergent reports. However, one antibody, identified as D-6, is highly specific and extremely sensitive. The specificity, sensitivity, and versatility of this antibody make it the preferred antibody for identifying VDR expression in target tissues using immunological methods.

Introduction

Vitamin D mediates many biological functions, including intestinal calcium and phosphate absorption, calcium reabsorption in the kidney, and calcium mobilization in bone [1]. Vitamin D also regulates parathyroid growth and parathyroid hormone production. The parathyroid hormone plays a critical role in the calcium homeostasis and calcium mobilization from bone [1]. Vitamin D also performs a number of functions outside of calcium homeostasis including promoting differentiation and inhibiting proliferation of certain cell types. These functions suggest that vitamin D may have a role in cancer chemoprevention [2]. It is also a potent immunomodulator and has been shown to suppress disease in several animal models of autoimmunity [1], [3]. Most if not all the functions of vitamin D are believed to be mediated by the vitamin D receptor (VDR)1[4], [5], [6]. Thus, accurate identification of VDR in tissues is critical to understanding the biological functions of Vitamin D and could be key to the development of novel therapeutic modalities targeting the VDR.

The VDR is a transcriptional factor which regulates gene expression in a ligand-dependent manner. It is classified as a member of the steroid hormone receptor superfamily [4]. The human VDR gene located on chromosome 12q is composed of promoter and regulatory regions (exons 1a–f) and the coding sequence (exons 2–9) for the full length VDR protein. The VDR protein contains several structurally and functionally important domains, including nuclear localization signal, hormone ligand-binding, DNA-binding, dimerization, and activation function 2 (AF2) transactivation domains [7]. The active form of vitamin D, 1,25(OH)2D3, exerts transcriptional activation of target genes by binding to the VDR. 1,25(OH)2D3–VDR-dependent transcriptional activity is modulated through synergistic ligand-binding and dimerization with retinoic X receptor (RXR). The 1,25(OH)2D3–VDR–RXR complex binds to the vitamin D response elements (VDREs) through the DNA-binding domain in the promoters of target genes. Conformational changes in the VDR results in the dissociation of the co-repressor, known as the silencing mediator for retinoid and thyroid hormone receptors (SMRT). This allows interaction of the VDR activation function 2 (AF2) transactivation domain with stimulatory coactivators, such as steroid receptor coactivators (SRCs), vitamin D receptor-interacting protein complex and nuclear coactivator-62 kDa–Ski-interacting protein (NCoA62–SKIP), and thereby mediates transcriptional activation [2].

The VDR appears not only in the target cells of enterocytes, osteoblasts, and renal distal tubule cells but also in parathyroid gland cells, skin keratinocytes, promyelocytes, lymphocytes, colon epithelium, islet cells of the pancreas, pituitary gland cells, and ovarian cells [1]. Some of these tissues have no direct impact on calcium or phosphorus homeostasis and thus, the functions of VDR within many of these cells remain uncertain [1]. Recent studies have shown that the VDR is also present in several tumors such as prostate, colon, breast, leukemia, and lung cancers [8]. Patients with breast cancer whose tumors contain immunochemically detectable VDR have a longer disease-free interval following treatment than those patients with VDR negative tumors [9]. Such clinical studies suggest that the expression of VDR may be a useful prognostic indicator [8]. Consequently, treatment with vitamin D and/or its analogs, may have a role in the inhibition of cancer cell growth and metastasis [2]. The VDR may also constitute an important prerequisite for using vitamin D and/or its analogs in treatment of selected cancers [8].

Early studies to determine VDR distribution among tissues used an autoradiographic technique based on radio-labeled ligand-receptor binding [10], [11]. This assay required an injection of radio-labeled ligand into an animal followed by detection of the cellular distribution of the radiation signal. Although this technique is highly sensitive, errors in VDR detection can occur as a result of the receptor stability, ligand/receptor dissociation, or presence of the endogenous ligand [12]. Utility of vitamin D deficient animals instead of the vitamin D sufficient animals as recipients of the radiolabelled ligand can increase the exogenous ligand-binding to the receptor. However, vitamin D deficiency results in downregulation of VDR expression because of the autoregulation of VDR by its ligand [13]. Therefore, VDR determination in the deficient animal may not represent the normal physiological expression of VDR in target tissues.

Other assays, such as in situ hybridization, PCR/qPCR, immunoblotting, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry (IHC), have also been used to determine VDR transcript or protein expression. Only in situ hybridization and immunohistochemistry can spatiotemporally determine VDR mRNA and protein in target tissues.

The VDR antibody is a key component for immunoassays. Several VDR antibodies, such as rat monoclonal antibody 9A7, mouse monoclonal antibody IVG8C11, or rabbit polyclonal antibody C-20, have been developed for this purpose [14], [15], [16], [17], [18], [19], [20], [21], [22], [23]. The application of these antibody-based methods has provided important information regarding tissue-specific expression of VDR. However, VDR determinations using different antibodies have produced variable and contradictory results. For example, immunohistochemistry of VDR using antibody 9A7, the first VDR antibody developed in 1980s and now commercially available from Affinity BioReagents, demonstrated VDR expression in human renal proximal tubules [24]. However, a similar assay using C-20, an antibody recently developed by Santa Cruz Biotechnology and commonly used in VDR detection, did not identify VDR in these same tissues [14]. It has been noted that the widely-used rat monoclonal antibody 9A7 cross-reacts with other proteins, especially in rat tissues [25]. In this investigation, we address these issues by testing the characteristics of 9 VDR antibodies, which recognize distinct epitopes located in different regions of the VDR protein.

More than 10 VDR antibodies are commercially available, but none has been systematically characterized in terms of their specificity and immunosensitivity. Therefore, we used multiple immunoassays for antibody characterization and included negative control samples from Demay VDR knockout mice for each immunoassay. We demonstrated that the mouse monoclonal VDR antibody against the C-terminus of human VDR, D-6 (Santa Cruz Biotechnology) possesses high specificity, high sensitivity, and versatility.

Section snippets

Antibodies against VDR

VDR antibodies raised against distinct epitopes are listed in Table 1. Mouse monoclonal antibodies (MoAb) IVG8C11 and XVIE6E6 were raised against the porcine intestinal VDR [20], [22], [26] and made in the DeLuca laboratory (University of Wisconsin, Madison). The rat MoAb 9A7 and rabbit polyclonal antibody 711 was purchased from Affinity BioReagents. The antibodies D-6 (Lot # B0806, J3108, and E2809), C-20, H-81, and N-20 were purchased from Santa Cruz Biotechnology; antibody 39069 was

Specie specificity of VDR antibodies

First, we sought to characterize the specie specificity of nine VDR antibodies (Table 1) using immunoblotting analysis of the VDR proteins overexpressed in human epithelial kidney cells for human, rat, mouse, and chicken VDRs or isolated from porcine and monkey kidney tissues. The antibodies D-6, 9A7, IVG8C11, C-20, N-20, and H-81 all reacted with the human, rat, mouse, chicken and porcine VDRs (Fig. 1). The polyclonal antibody 711 did not react with any of the VDR proteins that were analyzed.

Discussion

Approximately 3% of the mouse or human genome is regulated, directly or indirectly, by the vitamin D endocrine system, suggesting that vitamin D has more widespread functions than calcium and phosphorus homeostasis [34]. Since vitamin D exerts its biological functions through ligand-binding of the VDR, determination of VDR levels in tissues and cells is critical information. The lack of a well-characterized VDR antibody, proper controls, and standardized protocols have led to contradictory

Acknowledgments

We are thankful to Dr. Daniel D. Bikle, Department of Medicine, Veterans Affairs Medical Center, University of California, San Francisco, California, USA, for his gift of the VDR adenoviral vector; and to Dr. Sachiko Yamada, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan, for her gift of the human VDR expression plasmid vector. We also thank Lance Rodenkirch, Laboratory Manager of the W.M.Keck

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