Presence of a Truncated Form of the Vitamin D Receptor (VDR) in a Strain of VDR-Knockout Mice
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《内分泌学杂志》
Department of Biochemistry, University of California Riverside, Riverside, California 92521
Abstract
As part of our studies on the membrane-initiated actions of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and its localization in caveolae membrane fractions, we used a vitamin D receptor (VDR)-knockout (KO) mouse model to study the binding of [3H]-1,25(OH)2D3 in the presumed absence of the VDR. In this mouse model, known as the Tokyo strain, the second exon of the VDR gene, which encodes the first of the two zinc fingers responsible for DNA binding, was removed, and the resulting animals have been considered to be VDR-null mice. To our surprise, several tissues in these KO mice showed significant (5–50% of that seen in wild-type animals) specific binding of [3H]-1,25(OH)2D3 in nuclear and caveolae membrane fractions. The dissociation constants of this binding in samples from VDR-KO and wild-type mice were indistinguishable. RT-PCR analysis of intestinal mRNA from the VDR-KO animals revealed an mRNA that lacks exon 2 but contains exons 3–9 plus two 5'-untranslated exons. Western analysis of intestinal extracts from VDR-KO mice showed a protein of a size consistent with the use of Met52 as the translational start site. Transfection of a plasmid construct containing the sequence encoding the human analog of this truncated form of the receptor, VDR(52-C), into Cos-1 cells showed that this truncated form of the receptor retains full [3H]-1,25(OH)2D3 binding ability. This same construct was inactive in transactivation assays using the osteocalcin promoter in CV1 cells. Thus, we have determined that this widely used strain of the VDR-KO mouse can express a form of the VDR that can bind ligand but not activate gene transcription.
Introduction
BEFORE EXERTING BIOLOGICAL activity, vitamin D is converted by successive hydroxylations in the liver and kidney into the dihydroxylated derivative, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. This steroid hormone, which is also produced in many extrarenal sites, initiates its biological activity through specific binding to a member of the nuclear receptor family, the vitamin D receptor (VDR) (1). As is the case with the other steroid hormones such as the sex steroids, glucocorticoids, and mineralcorticoids, these hormone receptor complexes act as transcription factors and modulate the rates of transcription of specific genes, thus altering the biological activities of the target cell. They may also be involved, in either their native or modified forms, in the membrane-initiated events that are now recognized as components of steroid hormone actions in target tissues (2, 3, 4, 5).
To examine in more detail the function of steroid hormone receptors, investigators have developed strains of mice in which the gene for one or more the receptors has been altered so that the wild-type receptor is no longer expressed. In the case of the vitamin D receptor, VDR-null mice were reported in the same year by Yoshizawa et al. (6) and Li et al. (7). In the first case, the Tokyo strain, the disruption of the VDR gene was in exon 2, which encodes the first of two zinc fingers that are characteristic of nuclear receptors and required for DNA binding. The absence of exon 2 was confirmed by PCR analysis of that region, and an antibody against the N-terminal portion of the VDR failed to detect any protein. In the case of the Boston strain, the disruption of the VDR gene was achieved by the removal of exon 3, encoding the second zinc finger. These animals produced an mRNA containing a frameshift mutation after exon 2 which resulted in an early termination codon 12 bp downstream. The phenotypes of these two strains of mice were similar, with both strains exhibiting the main features of vitamin D-dependent rickets type II when raised on a normal diet containing 1.1% calcium and 0.8% phosphorus. In both strains this phenotype was rescued by feeding a diet high in calcium (2%) and phosphorus (1.25%) and including 20% lactose (8, 9).
More recently two other strains of VDR knockout (KO) mice have been reported. The strain produced in Leuven by Van Cromphaut et al. (10) is also missing exon 2, which was confirmed by PCR analysis of the segment spanning the missing region. This strain shared great phenotypic similarity with the Tokyo strain when the two were directly compared (10). Finally, a fourth strain of VDR-KO mouse, also lacking the first zinc finger but with a demonstrated intact hormone-binding domain, was produced and reported to be phenotypically indistinguishable from the previous strains, which were characterized as totally lacking the VDR protein (11).
In the past few years, an increasing amount of data has accumulated to indicate that, in addition to modulating rates of gene expression, 1,25(OH)2D3 (12, 13, 14, 15) as well as the other steroid hormones (16, 17, 18) also affect cellular function through membrane-initiated events, sometimes referred to as rapid or nongenomic effects. As part of our ongoing interest in understanding the membrane-initiated events brought about by 1,25(OH)2D3, we carried out a series of experiments in the strain of VDR-KO mice produced by Yoshizawa et al. (6). When we measured the ability of various tissues to bind 3[H]-1,25(OH)2D3 in these experiments, we found that, although binding was markedly reduced in tissues from the VDR knockout mouse, residual specific binding could be detected in some tissues (19). To determine whether this binding was due to a hitherto unrecognized truncated version of the VDR in the Tokyo strain or whether it should be attributed to a novel 1,25(OH)2D3 binding protein, we carried out the experiments described in the present manuscript. We report here that in the Tokyo strain of the VDR-KO mouse, there are both a truncated VDR mRNA and VDR protein that retains ligand binding activity.
Materials and Methods
Animals
The Tokyo strain of VDR-KO mice was generated by targeted ablation of exon 2 that encodes the first zinc finger of the DNA-binding domain (6). Our initial breeding pairs of these mice were a generous gift from Dr. S. Kato (University of Tokyo, Tokyo, Japan). VDR-KO and wild-type (WT) mice were weaned at 3 wk of age and then maintained on a normal diet (1.0% calcium, 1.0% phosphorus, 0% lactose, 4.5 IU vitamin D3 per gram; Laboratory Rodent Diet 50, PMI Nutrition International, Richmond, IN). The experimental protocols involving animals were approved by the Institutional Animal Care and Use Committee, University of California, Riverside.
Plasmid construction
cDNA encoding amino acids 4–427 of the human VDR was cloned into pcDNA 3.1 (Invitrogen, Carlsbad, CA) in which the NheI site had been inactivated by mutation. Further mutation of this plasmid using the QuikChange system (Stratagene, Palo Alto, CA) was performed to delete the N terminus in producing pcDNA3.1-VDR(52-C). The osteocalcin VDRE-driven secreted alkaline phosphatase reporter plasmid (ocVDRE-pSEAP2) is detailed by Vertino et al. (12).
Binding assays of VDR constructs
Cos-1 cells were cultured and transfected with diethylaminoethyl-dextran (Sigma, St. Louis, MO) as described by Vertino et al. (12). Saturable, specific binding of 105 Ci/mmol [3H]-1,25(OH)2D3 (Amersham Biosciences, Piscataway, NJ) was determined in cell lysates, nuclear extracts, or caveolae membrane fractions of intestine, kidney, and lung (20). After a 17-h, 4 C incubation with increasing concentrations of radiolabeled ligand in the absence or presence of excess unlabeled ligand, hydroxylapatite was used to separate the protein-bound and free ligand (21). Dissociation constants were determined by nonlinear regression analysis of the curve generated by plotting specific binding concentration vs. concentration of [3H]-1,25(OH)2D3 with GraphPad Prism (GraphPad Software, San Diego, CA) using a one-site binding curve equation.
Transcriptional activation assays
CV-1 cells were transfected with the plasmids containing the appropriate VDR construct and reporter using diethylaminoethyl-dextran (12). All experiments were carried out in quadruplicate, with the data expressed as the mean ± SEM for each 1,25(OH)2D3 concentration. The data for reporter were fit by nonlinear regression analysis to the sigmoidal dose-response equation using GraphPad Prism (GraphPad Software).
PCR
Total RNA was extracted from intestinal tissue from VDR-KO and wild-type mice using Triazol and transcribed into cDNA using oligo dT as a primer with StrataScript reverse transcriptase (Stratagene). PCR was performed with Taq DNA polymerase (Promega, Madison, WI) using a touchdown protocol. Amplicons were separated by electrophoresis in Tris/borate/EDTA buffer on 1.5% agarose at 120 V and visualized by ethidium bromide staining.
The forward primers for exons 1b, 2, 3, 4, and 8 were 5'-AGATCTGTGAGTCTTCCCAGGAGAG, 5'-GCTTCCACTTCAACGCTATGACC, 5'-TCAATGGAGATTGCCGCATCAC, 5'-GCAACAGCACATTATCGCCATC, 5'-GCGCTCCAACCAGTCTTTTACC, respectively. The reverse primer (exon 9) for all reactions was 5'-ACCAGCTTAGCATCCTGTACCC.
Western blotting
Intestinal tissue obtained from VDR-KO and kindred wild-type mice was homogenized in TED (10 mM Tris, 1.5 mM EDTA, 1 mM dithiothreitol) with 0.3 M KCl with complete EDTA-free protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Homogenates were dialyzed against TED to a final concentration of 30 mM KCl and normalized for protein concentration. After electrophoresis in 7.5% SDS-PAGE at 15 mA per minigel, proteins were transferred to a polyvinyl difluoride membrane using a semidry blotting apparatus. Membranes were blocked for 30 min with 5% nonfat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) and probed with 1:1000 VDR-C20 rabbit antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h in 1% nonfat dry milk in TBST. After washing in TBST, the membrane was incubated with 1:5000 alkaline phosphatase-conjugated antirabbit antiserum (60 min, room temperature), washed again, and incubated with 5-bromo-4-chloro-3-indoyl-phosphate/4-nitro blue tetrazolium chloride (Sigma-Aldrich, St. Louis, MO) for visualization. The probability of usage of a given methionine as a start site was determined by NetStart 1.0 prediction server (www.cbs.dtu.dk/services/NetStart/).
Results
As we reported previously (19), when kidney nuclear and caveolae membrane fractions from WT and VDR-KO mice were tested for their ability to bind [3H]-1,25(OH)2D3, there was a small but detectable level of specific binding in both fractions obtained from the VDR-KO mice. As shown in Fig. 1 for the intestinal samples, this binding was saturable and the dissociation constants were the same in WT and KO as well as in the two subcellular fractions. Similar results were obtained for the kidney and lung, for which the maximal binding capacity data are summarized in the lower portion of Fig. 1. The amount of residual binding in the VDR-KO mouse, relative to the WT (in parentheses) varied from 5% in kidney nuclei to 47% in intestinal caveolae membrane fractions. It is interesting to note that the residual binding was greater in caveolae than nuclei, especially for the intestine and lung.
To determine whether the residual binding of [3H]-1,25(OH)2D3 that we detected in tissues from VDR-KO mice was due to a truncated form of the VDR, we carried out PCR analysis of cDNA prepared using mRNA isolated from intestine from the two groups of mice as templates. We used forward primers from exons 1b, 2, 3, 4, and 8, each paired with the same reverse primer from exon 9 (Fig. 2A). As shown in Fig. 2B, the PCR product spanning exon 2 was smaller when cDNA from the VDR-KO mouse was used as the template, relative to that of the WT mouse (Fig. 2B, both WT and KO, lanes 1b and the dashed line between them). The difference is that expected from the excision of exon 2 and its replacement in the VDR-KO mouse by the Neo cassette. As expected, when the forward primer was within exon 2, which is deleted in the VDR-KO mouse, no product was obtained from the cDNA from these mice (circles in lanes 2). Forward primers from exons 3, 4, and 8 all resulted in equivalent products using cDNA from WT and KO mice. Excision of exon 2 from the longest amplicon was confirmed by DNA sequencing using the same forward primer.
The results of Fig. 2 demonstrate that the intestinal tissue from VDR-KO mice contain mRNA with the potential to code for a portion of the VDR, and we next sought to determine whether such a protein product does exist in these tissues. We considered the possibilities that translation initiation could have begun at methionine residues 52, 89/90, or 106, resulting in protein products with calculated molecular weights of 42.3, 37.9, or 35.8, respectively. Figure 3 shows the Western analysis of proteins in extracts of intestinal mucosa from WT and VDR-KO mouse. Using an antibody directed against the C-terminal region of the VDR, we detected a 43.2-kDA protein in tissue from VDR-KO mice, strongly suggesting the use of the first ATG codon in exon 3, Met 52, as the start site for production of a truncated form of the VDR in these animals.
To demonstrate that this truncated form of the receptor is indeed capable of binding [3H]-1,25(OH)2D3, we constructed an expression plasmid containing DNA encoding the truncated VDR, which begins with Met52 (VDR52-C). For these experiments, we used the human VDR sequence due to the ready availability of its cDNA in a plasmid construct. We assessed the ability of Cos-1 cell lysates bearing this truncated construct to bind [3H]-1,25(OH)2D3, compared with that of the same cells transfected with the wild-type VDR. As shown in Fig. 4A, both constructs conferred specific [3H]-1,25(OH)2D3 binding with virtually identical equilibrium dissociation constants (0.91 and 0.62 nM for WT and mutant constructs, respectively).
Finally, we tested the transactivation activity of VDR(52-C), using the osteocalcin promoter in CV1 cells. As shown in Fig. 4B, the truncated form of the VDR, unlike that of VDR WT, was completely devoid of transactivation activity.
Discussion
We have demonstrated that in one strain of mice with a targeted disruption of the VDR gene, there is a truncated message produced and that this message is translated into a protein that is capable of binding the hormone, 1,25(OH)2D3, with the same dissociation constant as the WT receptor. As expected from the absence of the first zinc finger in the DNA binding domain, this truncated form of the receptor is incapable of mediating the gene transactivation function characteristic of the WT receptor.
In the original report describing the creation of this strain of VDR-KO mice, (6), the authors confirmed the absence of mRNA representing exon 2, which encodes the first zinc finger. In addition, using an antibody directed against the N-terminal portion of the protein, they showed that no protein with this epitope was produced in the mutant mice. The presence of a truncated protein, missing the first zinc finger, was suggested to us when we studied [3H]-1,25(OH)2D3 binding in tissues from the VDR-KO mice. We now show that this binding, which ranges from 5 to 47% of WT in nuclear fractions and 15–50% in caveolae membrane fractions, is very likely due to the presence of the truncated form of the VDR.
Our Western analysis of the proteins detected by antibodies raised against the C-terminal portion of the VDR indicates that the truncated form of the receptor in these mice is produced through translation of a message using Met52 as the start site. The sequence context of this ATG has a high probability for initiating translation, 62% in the newly spliced context, compared with 67% for Met1 in exon 2 (see Materials and Methods for calculation).
Erben et al. (11) also produced a strain (Munich strain) of mice with a mutated form of the VDR that is missing the first zinc finger but retains hormone binding activity. Although the altered VDR gene in the mutant mice retained the initiating ATG in the second exon, RT-PCR analysis of RNA from these mutant mice revealed the presence of VDR mRNA in which the second exon was removed during RNA splicing. As in the present paper, when antibodies raised against the C-terminal portions of the protein were used in immunoanalysis, VDR was detected in intestinal tissue in situ as well as in extracts of several tissues (11). These authors concluded that as in the case of the protein we report here, the Met52 at the beginning of exon 3 was the initiating amino acid for the truncated form of the receptor they detected, although they could not confirm this electrophoretically. A detailed analysis of the phenotype of the Munich VDR mutant mice, designated VDR/, showed that it is virtually indistinguishable from that of the Tokyo strain (11).
The other two strains of mice with inactivated VDR are those reported by Li et al. (7) and Van Cromphaut et al. (10). In the former case, the deletion of exon 3, encoding the second zinc finger, generated a termination codon; thus, whether this mRNA was translated, it is likely that only a severely truncated protein, with no ligand binding capability would be produced. In the VDR inactivation mutation produced by Van Cromphaut et al., the deletion of exon 2 (encoding the first zinc finger) was confirmed by RT-PCR analysis of this region; whether a truncated version of the VDR containing the ligand binding domain was present in these animals was not addressed.
During the dozen or so years that gene KO mice have been widely used to study the functions of many genes including those for steroid hormone receptors, several examples of truncated gene products have occurred. Those most relevant to the present case with the VDR include the production of messages produced by splicing over a constitutive exon carrying a nonsense mutation (22) or a Neo cassette inserted by targeting (23). In the TGF-KO mouse, although such a spliced message was produced, careful examination showed that it was not translated into a protein product (23).
Turning to the steroid receptor family, in the case of the estrogen receptor (ER) in the originally reported null mouse (ERKO) , Lubahn et al. (24) showed a small amount (5%) of residual estrogen binding in the uterus but not in other estrogen-responsive tissues. At the time this was attributed to a splicing over event such as that described by Dietz et al. (22) and Luetteke et al. (23). More detailed analysis with RT-PCR and sucrose density gradient analysis showed that a truncated mRNA, missing the inserted Neo cassette and termed ER1, was produced in these animals (25). Translation of this mRNA resulted in the protein that was responsible for the residual estradiol binding and that was recognized by appropriate anti-ER antibodies. When the existence of ER was subsequently reported (26), the possibility was recognized that this isoform could be responsible for the residual binding seen in the ERKO mouse. Recently, however, the presence of the protein encoded by the ER1 transcript has been demonstrated in several tissues, including the brain, not only from ERKO mice but also from ER/ER double-KO mice (27, 28, 29). ER1 is missing the activation function-1 domain (as opposed to the DNA binding domain as in the case of the VDR-KO mouse) and retains some tissue-specific transactivation activity (27).
In the mouse with the mutated gene for the glucocorticoid receptor, GR2KO (30), the second exon containing the 1 activation domain was disrupted, leaving intact exons 3–9, encoding the two zinc fingers; the ligand binding domain; and two other regulatory domains, 2 and activation function-2. Although these mice expressed a 39-kDa ligand binding GR fragment, several target tissues were insensitive to dexamethasone treatment (31). More recently, however, it has been shown by microarray expression analysis that this GR fragment, which contains the protein encoded by virtually all of exons 3–9, is capable of both positive and negative ligand-responsive gene expression (32). Clearly the situation with the GR2KO mouse differs from that of the VDR-KO mouse described in the present paper because the former retains an intact DNA-binding domain, but it is also known that some functions of GR, such as transrepression, do not involve DNA binding but rather interaction with other DNA binding proteins.
Thus, there is evidence from studies with both the ERKO and GR2KO mice that remnant receptors retain some ligand-binding and perhaps biological activity. In recent studies, the ligand binding domain of ER has been shown to be sufficient for membrane localization and certain signal transduction functions (33) and to protect transfected HeLa cells from chemically induced apoptosis in a ligand-dependent manner, mediated through a Srk kinase/ERK pathway (34). In addition, in this latter study, the VDR ligand binding domain was also shown to be capable of this same protective effect (12).
In summary we have shown that the Tokyo strain of the VDR-KO mouse retains the capability to produce the portion of the VDR encoded by exons 3–9 and that this protein is likely responsible for the hormone binding observed in tissues from these mice. Although the phenotype of these mice is, for the characteristics thus far studied in detail, indistinguishable from other strains (10), it may be that more subtle or local effects of the hormone can be mediated by the remnant receptor. Therefore, as further studies are carried out to confirm or rule out the participation of the VDR in particular processes, whether initiated in the nucleus or the membrane, it is important for investigators to be aware of the presence of the truncated VDR in these mice.
Acknowledgments
The authors are grateful to Dr. Mathew Mizwicki for insightful discussions and suggestions throughout the course of this work.
Footnotes
This work was supported by National Institutes of Health Grant DK-09012-038 (to A.W.N.) and a Fullbright Fellowship (to J.H.).
First Published Online September 8, 2005
Abbreviations: ER, Estrogen receptor; ERKO, ER null mouse; KO, knockout; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; TBST, Tris-buffered saline with Tween 20; VDR, vitamin D receptor; WT, wild type.
Accepted for publication August 29, 2005.
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Abstract
As part of our studies on the membrane-initiated actions of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and its localization in caveolae membrane fractions, we used a vitamin D receptor (VDR)-knockout (KO) mouse model to study the binding of [3H]-1,25(OH)2D3 in the presumed absence of the VDR. In this mouse model, known as the Tokyo strain, the second exon of the VDR gene, which encodes the first of the two zinc fingers responsible for DNA binding, was removed, and the resulting animals have been considered to be VDR-null mice. To our surprise, several tissues in these KO mice showed significant (5–50% of that seen in wild-type animals) specific binding of [3H]-1,25(OH)2D3 in nuclear and caveolae membrane fractions. The dissociation constants of this binding in samples from VDR-KO and wild-type mice were indistinguishable. RT-PCR analysis of intestinal mRNA from the VDR-KO animals revealed an mRNA that lacks exon 2 but contains exons 3–9 plus two 5'-untranslated exons. Western analysis of intestinal extracts from VDR-KO mice showed a protein of a size consistent with the use of Met52 as the translational start site. Transfection of a plasmid construct containing the sequence encoding the human analog of this truncated form of the receptor, VDR(52-C), into Cos-1 cells showed that this truncated form of the receptor retains full [3H]-1,25(OH)2D3 binding ability. This same construct was inactive in transactivation assays using the osteocalcin promoter in CV1 cells. Thus, we have determined that this widely used strain of the VDR-KO mouse can express a form of the VDR that can bind ligand but not activate gene transcription.
Introduction
BEFORE EXERTING BIOLOGICAL activity, vitamin D is converted by successive hydroxylations in the liver and kidney into the dihydroxylated derivative, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. This steroid hormone, which is also produced in many extrarenal sites, initiates its biological activity through specific binding to a member of the nuclear receptor family, the vitamin D receptor (VDR) (1). As is the case with the other steroid hormones such as the sex steroids, glucocorticoids, and mineralcorticoids, these hormone receptor complexes act as transcription factors and modulate the rates of transcription of specific genes, thus altering the biological activities of the target cell. They may also be involved, in either their native or modified forms, in the membrane-initiated events that are now recognized as components of steroid hormone actions in target tissues (2, 3, 4, 5).
To examine in more detail the function of steroid hormone receptors, investigators have developed strains of mice in which the gene for one or more the receptors has been altered so that the wild-type receptor is no longer expressed. In the case of the vitamin D receptor, VDR-null mice were reported in the same year by Yoshizawa et al. (6) and Li et al. (7). In the first case, the Tokyo strain, the disruption of the VDR gene was in exon 2, which encodes the first of two zinc fingers that are characteristic of nuclear receptors and required for DNA binding. The absence of exon 2 was confirmed by PCR analysis of that region, and an antibody against the N-terminal portion of the VDR failed to detect any protein. In the case of the Boston strain, the disruption of the VDR gene was achieved by the removal of exon 3, encoding the second zinc finger. These animals produced an mRNA containing a frameshift mutation after exon 2 which resulted in an early termination codon 12 bp downstream. The phenotypes of these two strains of mice were similar, with both strains exhibiting the main features of vitamin D-dependent rickets type II when raised on a normal diet containing 1.1% calcium and 0.8% phosphorus. In both strains this phenotype was rescued by feeding a diet high in calcium (2%) and phosphorus (1.25%) and including 20% lactose (8, 9).
More recently two other strains of VDR knockout (KO) mice have been reported. The strain produced in Leuven by Van Cromphaut et al. (10) is also missing exon 2, which was confirmed by PCR analysis of the segment spanning the missing region. This strain shared great phenotypic similarity with the Tokyo strain when the two were directly compared (10). Finally, a fourth strain of VDR-KO mouse, also lacking the first zinc finger but with a demonstrated intact hormone-binding domain, was produced and reported to be phenotypically indistinguishable from the previous strains, which were characterized as totally lacking the VDR protein (11).
In the past few years, an increasing amount of data has accumulated to indicate that, in addition to modulating rates of gene expression, 1,25(OH)2D3 (12, 13, 14, 15) as well as the other steroid hormones (16, 17, 18) also affect cellular function through membrane-initiated events, sometimes referred to as rapid or nongenomic effects. As part of our ongoing interest in understanding the membrane-initiated events brought about by 1,25(OH)2D3, we carried out a series of experiments in the strain of VDR-KO mice produced by Yoshizawa et al. (6). When we measured the ability of various tissues to bind 3[H]-1,25(OH)2D3 in these experiments, we found that, although binding was markedly reduced in tissues from the VDR knockout mouse, residual specific binding could be detected in some tissues (19). To determine whether this binding was due to a hitherto unrecognized truncated version of the VDR in the Tokyo strain or whether it should be attributed to a novel 1,25(OH)2D3 binding protein, we carried out the experiments described in the present manuscript. We report here that in the Tokyo strain of the VDR-KO mouse, there are both a truncated VDR mRNA and VDR protein that retains ligand binding activity.
Materials and Methods
Animals
The Tokyo strain of VDR-KO mice was generated by targeted ablation of exon 2 that encodes the first zinc finger of the DNA-binding domain (6). Our initial breeding pairs of these mice were a generous gift from Dr. S. Kato (University of Tokyo, Tokyo, Japan). VDR-KO and wild-type (WT) mice were weaned at 3 wk of age and then maintained on a normal diet (1.0% calcium, 1.0% phosphorus, 0% lactose, 4.5 IU vitamin D3 per gram; Laboratory Rodent Diet 50, PMI Nutrition International, Richmond, IN). The experimental protocols involving animals were approved by the Institutional Animal Care and Use Committee, University of California, Riverside.
Plasmid construction
cDNA encoding amino acids 4–427 of the human VDR was cloned into pcDNA 3.1 (Invitrogen, Carlsbad, CA) in which the NheI site had been inactivated by mutation. Further mutation of this plasmid using the QuikChange system (Stratagene, Palo Alto, CA) was performed to delete the N terminus in producing pcDNA3.1-VDR(52-C). The osteocalcin VDRE-driven secreted alkaline phosphatase reporter plasmid (ocVDRE-pSEAP2) is detailed by Vertino et al. (12).
Binding assays of VDR constructs
Cos-1 cells were cultured and transfected with diethylaminoethyl-dextran (Sigma, St. Louis, MO) as described by Vertino et al. (12). Saturable, specific binding of 105 Ci/mmol [3H]-1,25(OH)2D3 (Amersham Biosciences, Piscataway, NJ) was determined in cell lysates, nuclear extracts, or caveolae membrane fractions of intestine, kidney, and lung (20). After a 17-h, 4 C incubation with increasing concentrations of radiolabeled ligand in the absence or presence of excess unlabeled ligand, hydroxylapatite was used to separate the protein-bound and free ligand (21). Dissociation constants were determined by nonlinear regression analysis of the curve generated by plotting specific binding concentration vs. concentration of [3H]-1,25(OH)2D3 with GraphPad Prism (GraphPad Software, San Diego, CA) using a one-site binding curve equation.
Transcriptional activation assays
CV-1 cells were transfected with the plasmids containing the appropriate VDR construct and reporter using diethylaminoethyl-dextran (12). All experiments were carried out in quadruplicate, with the data expressed as the mean ± SEM for each 1,25(OH)2D3 concentration. The data for reporter were fit by nonlinear regression analysis to the sigmoidal dose-response equation using GraphPad Prism (GraphPad Software).
PCR
Total RNA was extracted from intestinal tissue from VDR-KO and wild-type mice using Triazol and transcribed into cDNA using oligo dT as a primer with StrataScript reverse transcriptase (Stratagene). PCR was performed with Taq DNA polymerase (Promega, Madison, WI) using a touchdown protocol. Amplicons were separated by electrophoresis in Tris/borate/EDTA buffer on 1.5% agarose at 120 V and visualized by ethidium bromide staining.
The forward primers for exons 1b, 2, 3, 4, and 8 were 5'-AGATCTGTGAGTCTTCCCAGGAGAG, 5'-GCTTCCACTTCAACGCTATGACC, 5'-TCAATGGAGATTGCCGCATCAC, 5'-GCAACAGCACATTATCGCCATC, 5'-GCGCTCCAACCAGTCTTTTACC, respectively. The reverse primer (exon 9) for all reactions was 5'-ACCAGCTTAGCATCCTGTACCC.
Western blotting
Intestinal tissue obtained from VDR-KO and kindred wild-type mice was homogenized in TED (10 mM Tris, 1.5 mM EDTA, 1 mM dithiothreitol) with 0.3 M KCl with complete EDTA-free protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Homogenates were dialyzed against TED to a final concentration of 30 mM KCl and normalized for protein concentration. After electrophoresis in 7.5% SDS-PAGE at 15 mA per minigel, proteins were transferred to a polyvinyl difluoride membrane using a semidry blotting apparatus. Membranes were blocked for 30 min with 5% nonfat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) and probed with 1:1000 VDR-C20 rabbit antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h in 1% nonfat dry milk in TBST. After washing in TBST, the membrane was incubated with 1:5000 alkaline phosphatase-conjugated antirabbit antiserum (60 min, room temperature), washed again, and incubated with 5-bromo-4-chloro-3-indoyl-phosphate/4-nitro blue tetrazolium chloride (Sigma-Aldrich, St. Louis, MO) for visualization. The probability of usage of a given methionine as a start site was determined by NetStart 1.0 prediction server (www.cbs.dtu.dk/services/NetStart/).
Results
As we reported previously (19), when kidney nuclear and caveolae membrane fractions from WT and VDR-KO mice were tested for their ability to bind [3H]-1,25(OH)2D3, there was a small but detectable level of specific binding in both fractions obtained from the VDR-KO mice. As shown in Fig. 1 for the intestinal samples, this binding was saturable and the dissociation constants were the same in WT and KO as well as in the two subcellular fractions. Similar results were obtained for the kidney and lung, for which the maximal binding capacity data are summarized in the lower portion of Fig. 1. The amount of residual binding in the VDR-KO mouse, relative to the WT (in parentheses) varied from 5% in kidney nuclei to 47% in intestinal caveolae membrane fractions. It is interesting to note that the residual binding was greater in caveolae than nuclei, especially for the intestine and lung.
To determine whether the residual binding of [3H]-1,25(OH)2D3 that we detected in tissues from VDR-KO mice was due to a truncated form of the VDR, we carried out PCR analysis of cDNA prepared using mRNA isolated from intestine from the two groups of mice as templates. We used forward primers from exons 1b, 2, 3, 4, and 8, each paired with the same reverse primer from exon 9 (Fig. 2A). As shown in Fig. 2B, the PCR product spanning exon 2 was smaller when cDNA from the VDR-KO mouse was used as the template, relative to that of the WT mouse (Fig. 2B, both WT and KO, lanes 1b and the dashed line between them). The difference is that expected from the excision of exon 2 and its replacement in the VDR-KO mouse by the Neo cassette. As expected, when the forward primer was within exon 2, which is deleted in the VDR-KO mouse, no product was obtained from the cDNA from these mice (circles in lanes 2). Forward primers from exons 3, 4, and 8 all resulted in equivalent products using cDNA from WT and KO mice. Excision of exon 2 from the longest amplicon was confirmed by DNA sequencing using the same forward primer.
The results of Fig. 2 demonstrate that the intestinal tissue from VDR-KO mice contain mRNA with the potential to code for a portion of the VDR, and we next sought to determine whether such a protein product does exist in these tissues. We considered the possibilities that translation initiation could have begun at methionine residues 52, 89/90, or 106, resulting in protein products with calculated molecular weights of 42.3, 37.9, or 35.8, respectively. Figure 3 shows the Western analysis of proteins in extracts of intestinal mucosa from WT and VDR-KO mouse. Using an antibody directed against the C-terminal region of the VDR, we detected a 43.2-kDA protein in tissue from VDR-KO mice, strongly suggesting the use of the first ATG codon in exon 3, Met 52, as the start site for production of a truncated form of the VDR in these animals.
To demonstrate that this truncated form of the receptor is indeed capable of binding [3H]-1,25(OH)2D3, we constructed an expression plasmid containing DNA encoding the truncated VDR, which begins with Met52 (VDR52-C). For these experiments, we used the human VDR sequence due to the ready availability of its cDNA in a plasmid construct. We assessed the ability of Cos-1 cell lysates bearing this truncated construct to bind [3H]-1,25(OH)2D3, compared with that of the same cells transfected with the wild-type VDR. As shown in Fig. 4A, both constructs conferred specific [3H]-1,25(OH)2D3 binding with virtually identical equilibrium dissociation constants (0.91 and 0.62 nM for WT and mutant constructs, respectively).
Finally, we tested the transactivation activity of VDR(52-C), using the osteocalcin promoter in CV1 cells. As shown in Fig. 4B, the truncated form of the VDR, unlike that of VDR WT, was completely devoid of transactivation activity.
Discussion
We have demonstrated that in one strain of mice with a targeted disruption of the VDR gene, there is a truncated message produced and that this message is translated into a protein that is capable of binding the hormone, 1,25(OH)2D3, with the same dissociation constant as the WT receptor. As expected from the absence of the first zinc finger in the DNA binding domain, this truncated form of the receptor is incapable of mediating the gene transactivation function characteristic of the WT receptor.
In the original report describing the creation of this strain of VDR-KO mice, (6), the authors confirmed the absence of mRNA representing exon 2, which encodes the first zinc finger. In addition, using an antibody directed against the N-terminal portion of the protein, they showed that no protein with this epitope was produced in the mutant mice. The presence of a truncated protein, missing the first zinc finger, was suggested to us when we studied [3H]-1,25(OH)2D3 binding in tissues from the VDR-KO mice. We now show that this binding, which ranges from 5 to 47% of WT in nuclear fractions and 15–50% in caveolae membrane fractions, is very likely due to the presence of the truncated form of the VDR.
Our Western analysis of the proteins detected by antibodies raised against the C-terminal portion of the VDR indicates that the truncated form of the receptor in these mice is produced through translation of a message using Met52 as the start site. The sequence context of this ATG has a high probability for initiating translation, 62% in the newly spliced context, compared with 67% for Met1 in exon 2 (see Materials and Methods for calculation).
Erben et al. (11) also produced a strain (Munich strain) of mice with a mutated form of the VDR that is missing the first zinc finger but retains hormone binding activity. Although the altered VDR gene in the mutant mice retained the initiating ATG in the second exon, RT-PCR analysis of RNA from these mutant mice revealed the presence of VDR mRNA in which the second exon was removed during RNA splicing. As in the present paper, when antibodies raised against the C-terminal portions of the protein were used in immunoanalysis, VDR was detected in intestinal tissue in situ as well as in extracts of several tissues (11). These authors concluded that as in the case of the protein we report here, the Met52 at the beginning of exon 3 was the initiating amino acid for the truncated form of the receptor they detected, although they could not confirm this electrophoretically. A detailed analysis of the phenotype of the Munich VDR mutant mice, designated VDR/, showed that it is virtually indistinguishable from that of the Tokyo strain (11).
The other two strains of mice with inactivated VDR are those reported by Li et al. (7) and Van Cromphaut et al. (10). In the former case, the deletion of exon 3, encoding the second zinc finger, generated a termination codon; thus, whether this mRNA was translated, it is likely that only a severely truncated protein, with no ligand binding capability would be produced. In the VDR inactivation mutation produced by Van Cromphaut et al., the deletion of exon 2 (encoding the first zinc finger) was confirmed by RT-PCR analysis of this region; whether a truncated version of the VDR containing the ligand binding domain was present in these animals was not addressed.
During the dozen or so years that gene KO mice have been widely used to study the functions of many genes including those for steroid hormone receptors, several examples of truncated gene products have occurred. Those most relevant to the present case with the VDR include the production of messages produced by splicing over a constitutive exon carrying a nonsense mutation (22) or a Neo cassette inserted by targeting (23). In the TGF-KO mouse, although such a spliced message was produced, careful examination showed that it was not translated into a protein product (23).
Turning to the steroid receptor family, in the case of the estrogen receptor (ER) in the originally reported null mouse (ERKO) , Lubahn et al. (24) showed a small amount (5%) of residual estrogen binding in the uterus but not in other estrogen-responsive tissues. At the time this was attributed to a splicing over event such as that described by Dietz et al. (22) and Luetteke et al. (23). More detailed analysis with RT-PCR and sucrose density gradient analysis showed that a truncated mRNA, missing the inserted Neo cassette and termed ER1, was produced in these animals (25). Translation of this mRNA resulted in the protein that was responsible for the residual estradiol binding and that was recognized by appropriate anti-ER antibodies. When the existence of ER was subsequently reported (26), the possibility was recognized that this isoform could be responsible for the residual binding seen in the ERKO mouse. Recently, however, the presence of the protein encoded by the ER1 transcript has been demonstrated in several tissues, including the brain, not only from ERKO mice but also from ER/ER double-KO mice (27, 28, 29). ER1 is missing the activation function-1 domain (as opposed to the DNA binding domain as in the case of the VDR-KO mouse) and retains some tissue-specific transactivation activity (27).
In the mouse with the mutated gene for the glucocorticoid receptor, GR2KO (30), the second exon containing the 1 activation domain was disrupted, leaving intact exons 3–9, encoding the two zinc fingers; the ligand binding domain; and two other regulatory domains, 2 and activation function-2. Although these mice expressed a 39-kDa ligand binding GR fragment, several target tissues were insensitive to dexamethasone treatment (31). More recently, however, it has been shown by microarray expression analysis that this GR fragment, which contains the protein encoded by virtually all of exons 3–9, is capable of both positive and negative ligand-responsive gene expression (32). Clearly the situation with the GR2KO mouse differs from that of the VDR-KO mouse described in the present paper because the former retains an intact DNA-binding domain, but it is also known that some functions of GR, such as transrepression, do not involve DNA binding but rather interaction with other DNA binding proteins.
Thus, there is evidence from studies with both the ERKO and GR2KO mice that remnant receptors retain some ligand-binding and perhaps biological activity. In recent studies, the ligand binding domain of ER has been shown to be sufficient for membrane localization and certain signal transduction functions (33) and to protect transfected HeLa cells from chemically induced apoptosis in a ligand-dependent manner, mediated through a Srk kinase/ERK pathway (34). In addition, in this latter study, the VDR ligand binding domain was also shown to be capable of this same protective effect (12).
In summary we have shown that the Tokyo strain of the VDR-KO mouse retains the capability to produce the portion of the VDR encoded by exons 3–9 and that this protein is likely responsible for the hormone binding observed in tissues from these mice. Although the phenotype of these mice is, for the characteristics thus far studied in detail, indistinguishable from other strains (10), it may be that more subtle or local effects of the hormone can be mediated by the remnant receptor. Therefore, as further studies are carried out to confirm or rule out the participation of the VDR in particular processes, whether initiated in the nucleus or the membrane, it is important for investigators to be aware of the presence of the truncated VDR in these mice.
Acknowledgments
The authors are grateful to Dr. Mathew Mizwicki for insightful discussions and suggestions throughout the course of this work.
Footnotes
This work was supported by National Institutes of Health Grant DK-09012-038 (to A.W.N.) and a Fullbright Fellowship (to J.H.).
First Published Online September 8, 2005
Abbreviations: ER, Estrogen receptor; ERKO, ER null mouse; KO, knockout; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; TBST, Tris-buffered saline with Tween 20; VDR, vitamin D receptor; WT, wild type.
Accepted for publication August 29, 2005.
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