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Endocrinology Vol. 148, No. 7 3410-3418
Copyright © 2007 by The Endocrine Society

Splice Variants of the CYP27b1 Gene and the Regulation of 1,25-Dihydroxyvitamin D3 Production

S. Wu, S. Ren, L. Nguyen, J. S. Adams and M. Hewison

Burns and Allen Research Institute, Division of Endocrinology, Diabetes and Metabolism, Cedars-Sinai Medical Center, University of California, Los Angeles, School of Medicine, Los Angeles, California 90048

Address all correspondence and requests for reprints to: Martin Hewison, Division of Endocrinology, Diabetes and Metabolism, Cedars-Sinai Medical Center, Los Angeles, California 90048. E-mail: martin.hewison{at}cshs.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The cytochrome P450 25-hydroxyvitamin D3-1{alpha}-hydroxylase (CYP27b1) plays a pivotal role in vitamin D physiology by catalyzing synthesis of active 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. In common with other P450s, CYP27b1 is known to exhibit alternative splicing. Here we have cloned and sequenced several novel intron 2-containing, noncoding splice variant mRNAs for CYP27b1 in 1,25(OH)2D3-producing HKC-8 human proximal tubule and THP-1 monocytic cells. Regulation of 1,25(OH)2D3 synthesis in these cell lines by calciotropic and noncalciotropic factors was associated with altered expression of the CYP27b1 splice variants. To assess the functional significance of this, HKC-8 cells were transfected with short hairpin RNA (shRNA) to inhibit mRNAs containing sequences from intron 2. This resulted in a significant increase in the expression of CYP27b1 protein and synthesis of 1,25(OH)2D3 by HKC-8 cells compared with control cells for two different intron 2-containing shRNAs (both P < 0.001). shRNA to intron 2 had no significant effect on the levels of wild-type CYP27b1 mRNA, suggesting a posttranscriptional mechanism of action. By contrast, shRNA to wild-type CYP27b1 suppressed transcription and activity of the enzyme by 70 and 31%, respectively (both P < 0.01). These data indicate that noncoding splice variants of CYP27b1 are functionally active and may play a significant role in the regulation of 1,25(OH)2D3 synthesis during normal physiology.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ACTIVE FORM of vitamin D, 1,25-dihydroxvitamin D3 [1,25(OH)2D3] is a pluripotent seco-steroid that has been implicated in the regulation of a diverse array of physiological functions (1, 2). However, despite its many beneficial actions, 1,25(OH)2D3 is also capable of detrimental hypercalcemic side effects when present in excess (3). This is particularly important in view of the fact that circulating levels of parental vitamin D and its major circulating metabolite 25-hydroxyvitamin D3 (25OHD3) vary considerably within individuals depending on nutritional restriction and the level of cutaneous exposure to sunlight (necessary for endogenous production of vitamin D in the skin) (4). To prevent excessive production of 1,25(OH)2D3, a complex system of feedback regulation exists involving both metabolic and catabolic pathways (5). Endocrine synthesis of 1,25(OH)2D3 occurs primarily in the proximal tubule cells of the kidney, a process that is catalyzed by the mitochondrial cytochrome P450, 25-hydroxyvitamin D3-1{alpha}-hydroxylase (CYP27b1) (6, 7). Renal metabolism of precursor 25OHD3 to 1,25(OH)2D3 is stimulated by calciotropic factors such as PTH and calcitonin (8, 9) but is also subject to feedback regulation by 1,25(OH)2D3 itself and by extracellular calcium, phosphate, and associated factors (10, 11, 12, 13). In addition to direct regulation of CYP27b1 expression, circulating levels of 1,25(OH)2D3 are also attenuated by the related mitochondrial cytochrome P450 vitamin D-24-hydroxylase (CYP24), which catalyzes synthesis of less active 24-hydroxylated vitamin D metabolites (14). In contrast to CYP27b1, transcription of CYP24 is potently induced by 1,25(OH)2D3, underlining its importance as part of the machinery for feedback regulation of 1,25(OH)2D3-directed signaling (15, 16).

Regulation of 1,25(OH)2D3 production at multiple levels plays a key role in the renal endocrinology of vitamin D so that, under normal physiology, there is no clear correlation between circulating levels of substrate 25OHD3 and product 1,25(OH)2D3 (17). However, integration of CYP27b1 and CYP24 metabolic activity is also likely to be a crucial determinant of nonclassical aspects of 1,25(OH)2D3 function. For example, increased synthesis of 1,25(OH)2D3 and concomitant hypercalciuric/hypercalcemic complications have been described for patients with granulomatous diseases (18) and some neoplasms (19, 20). In a similar manner, dysregulated expression of CYP24 has been described in breast tumors and may be a key factor in the apparent resistance of cancer cells to the antiproliferative effects of 1,25(OH)2D3 (21, 22). These alterations in CYP27b1/CYP24 metabolic activity and the highly sensitive vitamin D receptor-directed responses of many tissues to 1,25(OH)2D3 suggest that our understanding of the mechanisms by which vitamin D metabolism is regulated is far from complete.

In recent studies, we identified a splice variant of CYP24 that was abundantly expressed in macrophages and that encoded an amino-terminal-truncated but functional version of this enzyme (23). Although this variant of CYP24 was unable to associate with the mitochondrion, it was more efficient in suppressing synthesis of 1,25(OH)2D3 than its wild-type counterpart, presumably by acting as a cytosolic decoy for CYP24/CYP27b1 substrates. Other groups have reported differential expression of splice variants for CYP27b1 in normal and cancer cells (24, 25, 26, 27, 28), suggesting a role for gene splicing in the tissue-specific regulation of 1,25(OH)2D3 production. To investigate this further, we assessed the regulation and functional impact of intron-containing splice variants of CYP27b1 on the synthesis of 1,25(OH)2D3 by human renal and nonrenal cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Unlabeled 1,25(OH)2D3 and 25-OHD3 were purchased from Biomol (Plymouth Meeting, PA). [3H]25OHD3 (specific activity, 187 Ci/mmol) and [3H]1,25(OH)2D3 (179 Ci/mmol) were purchased from GE Healthcare Biosciences (Piscataway, NJ). CaCl2 and tetradecanoylphorbol 13-acetate (TPA) were obtained from Sigma (St. Louis, MO).

Cell culture
The HKC-8 human proximal kidney tubule cell line, which is known to synthesize 1,25(OH)2D3 (10), was maintained in DMEM/F-12 medium supplemented with 5% fetal calf serum (FCS). THP-1 human monocytic cells were cultured in RPMI 1640 medium supplemented with 10% FCS. Cell treatments included increased extracellular calcium concentration (2 mM CaCl2 compared with 1 mM calcium in control medium), 1,25(OH)2D3 (100 nM), 25OHD3 (200 nM), and TPA (160 nM).

Analysis of wild-type and splice variant transcripts for CYP27b1
Total RNA was extracted from HKC-8 and THP-1 cells using an RNAeasy kit (Qiagen, Valencia, CA). RT was performed using 5 µg of total RNA and PowerScript Reverse Transcriptase (Clontech, Palo Alto, CA). To ensure that only mRNA sequences were reverse transcribed, oligo-dT16 was used as RT primer. The resulting cDNA was then used as a template for subsequent PCR reactions using combinations of primers outlined in Table 1Go. Primers used for specific experiments are defined in the legend of the respective figure. PCR reaction amplification of cDNAs was performed using either a one-step or two-step protocol as indicated. The one-step protocol consisted of the following: 1 cycle of 94 C (2 min); 10 cycles of 94 C (5 sec), 62 C (10 sec), 72 C (2 min); 12 cycles of 94 C (5 sec), 68 C (10 sec with touchdown of –0.5 C each cycle), 72 C (2 min); and 25 cycles of 94 C (5 sec), 68 C (30 sec), 72 C (2 min).


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  		TABLE 1. Primer details for PCR amplification and sequencing of splice variant cDNAs to CYP27b1

 
The two-step protocol consisted of the following: 1 cycle of 94 C (2 min); and 12 cycles of 94 C (5 sec), 68 C (10 sec with touchdown of –0.5 C each cycle), 72 C (2 min). After this, an aliquot of purified PCR product (1 µl or 1/10 to 1/25 of the original reaction mix) was taken from each reaction as template for additional amplification using fresh reagents and nested PCR primers as indicated. The second round of amplification then consisted of the following: 1 cycle of 94 C (2 min); and 37 cycles of 94 C (5 sec), 68 C (30 sec), 72 C (2 min).

Cloning and sequencing of CYP27b1 intron 2-containing splice variant mRNAs
PCR products were separated on a 1% agarose gel, specific bands were excised and extracted, and the resulting DNA was cloned into the pCR4-TOPO vector using the TOPO TA Cloning kit (Invitrogen, Carlsbad, CA). Sequence analyses were performed by Sequetech (Mountain View, CA) using primers to the T3 or T7 sites within the cloning vector.

Preparation of short hairpin RNAs (shRNAs) for silencing of CYP27b1 intron-containing splice variants
shRNAs corresponding to regions of intron 2 and exon 2 of the CYP27b1 gene were synthesized using the MessageMuter shRNA Production kit (EPICENTRE, Madison, WI) and targeted at the following DNA sequences: intron 2.i, 5'-GGC AGG AAA TGA GTA AAG AGG AA(1763–1785 of CYP27b1); intron 2.ii, 5'-GGA TCT GGA TGG AAT GAA TGT (1699–1719 of CYP27b1); exon 2, 5'-GGT GTG GCT AGC CAG CTT T (1276–1294 of the CYP27b1 gene); and scrambled control, 5'-ACG AGA GAG AGA GAG AAT GAG AT.

Transient transfection of shRNAs to CYP27b1 and CYP27b1 splice variants
HKC-8 cells were placed in a 12-well plate 1 d before transfection and cultured to 60% confluence. Each shRNA was then transiently transfected into cells by lipofection (LipoTAXI; Stratagene, La Jolla, CA) at a concentration of 2 µg/well and then cultured for an additional 72 h to facilitate the maximum inhibitory effect of the shRNA.

Analysis of 25OHD3 metabolism by HKC-8 and THP-1 cells
Synthesis of 1,25(OH)2D3 by HKC-8 and THP-1 cells was assessed first by quantifying the conversion of radiolabeled 25OHD3 to 1,25(OH)2D3 in serum-free cultures of these cells. For each assay, 50 nM [3H]25OHD3 (GE Healthcare Biosciences) was added to cells in 200 µl of serum-free medium and then incubated for 5 h at 37 C, with the reaction being terminated by freezing at –20 C. Protein from these samples was initially precipitated with added acetonitrile (1:1). Vitamin D metabolites were then extracted from the reaction mixtures by elution on C18-OH columns according to the instructions of the manufacturer (DiaSorin, Stillwater, MN). The resulting eluent was resuspended in 25 µl of elution solvent hexane/methanol/isopropanol (90:5:5) and vortexed for 15 sec, and individual metabolites were separated by HPLC using a Beckman Coulter (Fullerton, CA) System Gold system with an Agilent Technologies (Palo Alto, CA) Zobax Sil normal-phase column eluted at a rate of 1.5 ml/min for 20 min. Elution profiles for standard vitamin D metabolites [25OHD3, 24,25(OH)2D3, and 1,25(OH)2D3] were determined by UV absorbance at 264 nm. Elution of metabolites of [3H]25OHD3 was assessed using a ß-Ram Model 4 in-flow detector (IN/US Systems, Tampa, FL) in conjunction with Ultima-Flo M scintillation fluid (PerkinElmer, Boston, MA) at a 2:1 ratio with a 5-sec dwell time to designate the increments for data collection. Lauralite 3 software (LabLogic, Sheffield, UK) was used to quantitate peaks of radioactivity corresponding to 25OHD3, 24,25(OH)2D3, or 1,25(OH)2D3. Data were reported as mean ± SD femtomoles metabolite synthesized per hour per milligram cellular protein following n = 3 separate incubations. Analysis of 1,25(OH)2D3 production by cells after incubation with shRNA constructs was performed using 25OHD3 (200 nM) as substrate in medium containing 2% FCS for 4 h. The reaction was terminated by addition of acetonitrile, and both cells and medium were used to extract 1,25(OH)2D3 for quantification using a 1,25(OH)2D3 IRA kit (DiaSorin). Data were reported as mean ± SD pg 1,25(OH)2D3 per 105 cells (n = 4 separate assays).

Quantitative PCR analysis of wild-type CYP27b1 gene expression
Expression of wild-type CYP27b1 mRNA was quantified using an ABI 7700 sequence detection system (Applied Biosystems, Foster City, CA) as described previously (29). Approximately 50 ng cDNA were used per reaction. All reactions were multiplexed with the housekeeping gene 18S rRNA, provided as an optimized control probe labeled with VIC fluorochrome (Applied Biosystems), enabling data to be expressed in relation to an internal reference to allow for differences in sampling. The fluorogenic probe for CYP27b1 was labeled with five-carboxy fluorescein. Data were obtained as Ct values (the cycle number at which logarithmic PCR plots cross a calculated threshold line) and used to determine {Delta}Ct values (Ct of target gene – Ct of housekeeping gene, 18S rRNA). All reactions were performed in triplicate and expressed as a mean of these values. PCR amplification of CYP27B1 cDNA was performed using Taqman gene expression assay Hs01096149 (Applied Biosystems), which uses probes and primers to the boundary of exons 3 and 4. cDNAs were amplified under the following conditions: 50 C for 2 min; 95 C for 10 min; followed by 44 cycles of 95 C for 15 sec and 60 C for 1 min.

Statistics
When indicated, experimental means were compared statistically using an unpaired Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We have shown previously that the gene for the vitamin D catabolic enzyme CYP24 is alternatively spliced, resulting in the expression of variant mRNAs including an intron 2-containing transcript that encodes a truncated CYP24 peptide (23). We hypothesized that similar splice variants may also be associated with the related vitamin D-activating enzyme CYP27b1. To test this, we used the human proximal tubule cell line HKC-8, a well-characterized renal source of CYP27b1 expression and activity. Initial RT-PCR analyses performed using oligo-dT-derived mRNA from these cells and PCR primers directed to the C terminal of the wild-type CYP27b1 gene product and sequences within intron 2 (Fig. 1Go) confirmed the presence of nonclassical PCR products. However, levels of expression for these putative novel transcripts were relatively low compared with mRNA for wild-type CYP27b1 (<10% of total transcripts in untreated HKC-8 cells when using the primer pairs and PCR conditions outlined in Fig. 1Go). To recover sufficient product for sequence analysis, we developed a two-stage PCR amplification protocol, involving nested primer pairs (Fig. 2AGo). This generated a series of PCR products, each of which contained fragments of intron 2 (Fig. 2BGo).


Figure 1
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  		FIG. 1. Expression of wild-type and intron 2-containing splice variant mRNAs for CYP27b1 in human kidney HKC-8 cells. Total RNA from HKC-8 cells was reverse transcribed using oligo-dT to isolate cDNAs corresponding to polyadenylated RNA. PCR was then performed using the one-step method outlined in Materials and Methods with primer pairs to the following: intron 2 and exon 6; intron 2 and exon 7; and exon 4 and exon 8. in, Intron; ex, exon.

 

Figure 2
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  		FIG. 2. Expression of intron 2-containing splice variant mRNAs for CYP27b1 in HKC-8 cells. Polyadenylated mRNA from HKC-8 cells was subjected to RT-PCR using the two-step method outlined in Materials and Methods with various primers within intron 2 in conjunction with primers to exon 7, 8, or 9. A, The location of each PCR primer within the CYP27b1 genomic sequence. PCR amplification was performed using the two-step protocol outlined in Materials and Methods. B, Separation of resulting PCR products by agarose gel electrophoresis.

 
Having established these protocols, our aim was then to identify and clone sequence-specific novel intron-containing mRNAs associated with the regulated synthesis of 1,25(OH)2D3 in HKC-8 cells. RNA was isolated from HKC-8 cells after 6 h treatments with high extracellular calcium (2 mM), 1,25(OH)2D3 (100 nM), or 25OHD3 (200 nM). Two stage RT-PCR using primers to intron 2(3) and exon 9(1) showed that each of the different treatments produced a distinct pattern of alternative transcripts without any significant effect on wild-type CYP27b1 expression (Fig. 3AGo). Five of the six species amplified in this experiment (referred to as SV1 to SV5) were cloned and sequenced, and their cDNA length was shown to correspond to the original PCR product. The remaining PCR product did not produce a cloned product that corresponded in length to the original PCR product, and so this cDNA was not included in additional studies (data not shown). Vitamin D metabolism analyses showed that treatments with high calcium, 1,25(OH)2D3, and 25OHD3 exerted distinct effects on 25OHD3 metabolism in HKC-8 cells (Fig. 3BGo). Specifically, high (2 mM) extracellular calcium concentrations suppressed 1,25(OH)2D3 synthesis but had no effect on CYP27b1 mRNA expression or CYP24 activity. Cells treated with 1,25(OH)2D3 also showed suppression of CYP27b1 activity, but this was associated with inhibition of CYP27b1 mRNA expression and induction of CYP24 activity. Pro-hormone 25OHD3 had no effect on either CYP27b1 or CYP24. Each of the treatments shown in Figure 3Go was associated with expression of different intron 2-containing splice variants: control, SV1 and SV2; high extracellular calcium, SV3; 1,25(OH)2D3, SV4; and 25OHD3, SV5). Sequence analysis of SV1–SV5 confirmed that the PCR products were splice variants of CYP27b1 (GenBank accession nos.: SV1, DQ925362; SV2, DQ925363; SV3, DQ925364; SV4, DQ925365; and SV5, DQ925366). Additional analysis of these cDNAs indicated that, in addition to containing a fragment of intron 2, the splice variants were also characterized by abnormal splicing of exons 3–8, and one species (SV4) also contained a fragment of intron 3 (Fig. 4Go).


Figure 3
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  		FIG. 3. Regulation of CYP27b1 splice variant expression in human kidney HKC-8 cells. A, Total RNA was isolated from HKC-8 cells treated with vehicle (control, C; 0.1% ethanol), high extracellular calcium (Ca; 2 mM CaCl2), 1,25(OH)2D3 (1,25D; 100 nM), or 25OHD3 (25D; 200 nM) for 6 h. RT was performed using oligo-dT primers and 5 µg total RNA. The resulting cDNA was then subjected to PCR amplification using the two-step protocol described in Materials and Methods with primers to intron 2 and exon 9 for the first step and primers to intron 2(3 ) and exon 9(1 ) for the second step. Top shows agarose gel electrophoresis separation of PCR products after the second step of amplification, subsequently identified as splice variant species SV1–SV5. Bottom shows the changes in wild-type CYP27b1 mRNA expression associated with treatments as assessed by real-time PCR and expressed as fold change in expression compared with vehicle-only control. B, Effects of cell treatments on CYP27b1 and CYP24 activities. Synthesis of 1,25(OH)2D3 and 24,25(OH)2D3 was assessed by incubation with 3H-25OHD3 (50 nM) for 5 h followed by separation of radiolabeled vitamin D metabolites by HPLC and expressed as [3H]1,25(OH)2D3 or [3H]24,25(OH)2D3. Data are shown as percentage CYP27b1 or CYP24 activities relative to untreated control cells. Data are the mean ± SD of three separate experiments. *, P < 0.001, statistically different from vehicle-only control.

 

Figure 4
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  		FIG. 4. Identification of CYP27b1 splice variants in human kidney HKC-8 cells. Splice variant PCR products SV1–SV5 identified in Fig. 3Go were excised, purified, and cloned. Sequence analysis was performed using primers directed to the T3 and T7 sequences within the pCR4-TOPO cloning vector, and results are shown as schematic representations of deducted transcripts.

 
The endocrine effects of vitamin D are dependent on CYP27b1-mediated activation of 25OHD3 by proximal tubule kidney cells, but the enzyme is also expressed by cells from other sites. To determine the extent to which splice variants of CYP27b1 are also a feature of 1,25(OH)2D3 production by nonrenal cells, we performed additional experiments using the monocytic cell line THP-1 (Fig. 5Go). As with the HKC-8 kidney cells, THP-1 cells showed distinct patterns of CYP27b1 splice variant expression under culture conditions associated with modulation of monocyte or vitamin D metabolism, in the absence of any apparent change in wild-type CYP27b1 expression (Fig. 5AGo). In contrast to the HKC-8 data in Fig. 3Go, response to 1,25(OH)2D3 did not appear to be associated with enhanced CYP24 activity. Indeed data showed decreased synthesis of 24,25(OH)2D3 in cells treated with TPA and/or 1,25(OH)2D3 (Fig. 5BGo). THP-1 cells cultured with 1,25(OH)2D3 showed additional expression of two splice variants not present in vehicle (0.1% ethanol)-treated control cells (SV8 and SV9). Of these, SV8 was also expressed in THP-1 cells treated with the phorbol ester TPA (160 nM, 24 h), which had no effect on synthesis of 1,25(OH)2D3 or expression of wild-type CYP27b1 mRNA. Notably, the addition of both 1,25(OH)2D3 and TPA further decreased synthesis of 1,25(OH)2D3 but without significant expression of the SV8 and SV9 observed after treatment with 1,25(OH)2D3 alone. Data in Fig. 5Go show PCR products from three separate experiments, indicating that the expression pattern of the variant CYP27b1 mRNAs was remarkably consistent. Cloning and sequence analysis of the PCR products from THP-1 cells (SV6–SV10) revealed five novel CYP27b1 mRNAs (GenBank accession nos.: SV6, DQ925367; SV7, DQ925368; SV8, DQ925369; SV9, DQ925370; and SV10, DQ925371) with distinct patterns of splicing as shown in Fig. 6Go. This included aberrant splicing of exon 4 and the inclusion of fragments of intron 5.


Figure 5
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  		FIG. 5. Regulation of CYP27b1 splice variant expression in human myelomocytic THP-1 cells. Total RNA was isolated from THP-1 cells treated with vehicle (0.1% ethanol), TPA (160 nM), 1,25(OH)2D3 (1,25D; 100 nM), or both for 24 h. RT was performed using oligo-dT primers and 5 µg total RNA. The resulting cDNA was then subjected to PCR amplification using the two-step protocol described in Materials and Methods with primers intron 2 and exon 9 for the first step and primers intron 2(3 ) and exon 7 for the second step. Top shows agarose gel electrophoresis separation of the resulting PCR products after the second step of amplification, subsequently identified as splice variant species SV6–SV10. Data are shown as amplification products for each treatment from three separate experiments. Bottom shows effects of cell treatments on CYP27b1 and CYP24 activities. Synthesis of 1,25(OH)2D3 and 24,25(OH)2D3 was assessed by incubation with [3H]25OHD3 (50 nM) for 5 h followed by separation of radiolabeled vitamin D metabolites by HPLC and expressed as [3H]1,25(OH)2D3 or [3H]24,25(OH)2D3. Data are shown as percentage CYP27b1 or CYP24 activities relative to untreated control cells. Data are the mean ± SD of three separate experiments. *, P < 0.001, statistically different from vehicle-only control. Levels of CYP27b1 mRNA were assessed by real-time PCR and expressed as fold change in expression compared with vehicle-only control. Data are the mean ± SD of three separate experiments. *, P < 0.001, statistically different from vehicle-only control.

 

Figure 6
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  		FIG. 6. Identification of CYP27b1 splice variants in human myelomonocytic THP-1 cells. Splice variant PCR products SV6–SV10, identified in Fig. 5Go, were excised, purified, and cloned. Sequence analysis was performed using primers directed to the T3 and T7 sequences within the pCR4-TOPO cloning vector, and results are shown as schematic representations of deduced transcripts.

 
To determine whether or not the intron-containing CYP27b1 splice variants identified in the study were functionally active, we used inhibitory shRNA to knockdown mRNAs containing fragments of either intron 2 or exon 2 and then assessed changes in CYP27b1 expression and activity in the recipient HKC-8 cells (Fig. 7Go). Data showed that shRNA to intron 2 abrogated expression of intron 2-containing splice variants (Fig. 7AGo) but had no significant effect on expression of wild-type CYP27b1 mRNA levels (Fig. 7BGo). However, shRNA inhibition of intron 2-containing mRNAs did result in the up-regulation of protein levels for wild-type CYP27b1 (Fig. 7CGo) and enhanced metabolism of 25OHD3 to 1,25(OH)2D3 (Fig. 7DGo). These data were consistent for more than one intron 2 shRNA. In contrast to the effects of splice variant knockdown, shRNA targeted at wild-type CYP27b1 mRNA resulted in decreased 1,25(OH)2D3 production. This effect was associated with a significant suppression of mRNA and protein for wild-type CYP27b1, as well as altered expression of intron 2-containing CYP27b1 splice variants.


Figure 7
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  		FIG. 7. Effect of inhibitor shRNAs to intron 2-containing mRNAs on expression and activity of CYP27b1. Human kidney HKC-8 cells were transfected with inhibitory shRNAs to sequences within intron 2 (int. 2.1 and int. 2.ii) or exon 2 (ex. 2) and assessed for the following: A, changes in intron 2-containing splice variants of CYP27b1 using the primers to intron 2 and exon 9(1 ); B, changes in wild-type CYP27b1 mRNA expression as determined by real-time RT-PCR; C, changes in CYP27b1 protein expression (densitometry values are shown as arbitrary density units); and D, changes in 1,25(OH)2D3 synthesis as determined by RIA after incubation with 100 nM 25OHD3 [data are shown as picograms of 1,25(OH)2D3 per 106 cells]. In each case, values were compared with data from cultures using a scrambled shRNA oligo control (Sc). All data shown are the mean ± SD of n = 3 separate experiments, apart from D in which n = 4. **, P < 0.01, statistically different from transfection control cells. ***, P < 0.001, statistically different from transfection control cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Alternative gene splicing affects up to 70% of all human genes and plays an integral role in the transcriptosome of higher organisms (30). In many instances, alternative splicing enhances genetic diversity by generating multiple proteins with distinct functions or subcellular localization from a single gene (31, 32). In other cases, splice variants are the product of aberrant splicing, leading to out-of-frame RNA isoforms with no apparent end function (33, 34). The molecular impact of the latter has yet to be fully defined, but approximately one third of splice variants are thought to alter gene transcription as a consequence of mRNA decay or other mechanisms (32, 35, 36, 37, 38). The evolutionary conservation of both frame-altering and frame-preserving RNA isoforms suggests that alternative gene splicing is biologically relevant (30, 39) and represents a potentially important target in the development of novel therapies for human disease.

In previous studies, we reported an in-frame splice variant mRNA for the vitamin D-catabolizing enzyme CYP24 in which inclusion of part of intron 2 for this gene generated an N terminally truncated peptide with altered function (23). The splice variant, termed CYP24-SV, retained capacity for binding substrate [25OHD3 or 1,25(OH)2D3], but deletion of the mitochondrial targeting sequence encoded by exons 1 and 2 meant that its presence was restricted to the cytoplasm, thereby eliminating any catalytic activity. Although CYP24-SV had no enzyme activity, it was able to suppress synthesis of 1,25(OH)2D3 presumably by acting as a "decoy" for 25OHD3 substrate. In the current study, we were unable to identify a similar in-frame splice variant for CYP27b1, but we did detect many other PCR products that were subsequently shown to be intron 2-containing splice variants of the enzyme. Although these were noncoding and were weakly expressed compared with the wild-type CYP27b1, we hypothesized that the splice variants had the potential to exert regulatory effects on expression of the enzyme at either a transcriptional or posttranscriptional level. Our postulate was based on a series of recent reports suggesting that expression of noncoding RNAs does not simply represent "transcriptional noise" but instead provides an additional layer of control for gene expression (39). For example, ectopic expression of selected intronic sequences from the cystic fibrosis transmembrane conductance regulator gene has been shown to cause substantial changes in HeLa gene expression (40). Significantly, the molecular impact of splice variants may be disproportionate to their level of expression; splice variants of the thrombopoietin gene have been shown to substantially influence protein expression despite exhibiting relatively low abundance compared with the wild-type mRNA (41).

Splice variants of CYP27b1 have been well documented in previous reports (24, 25, 26, 27, 28), but their impact on vitamin D metabolism has so far remained unclear. To address this issue, we used shRNA technology to specifically target intron-containing CYP27b1 mRNAs, thereby allowing us to selectively inhibit splice variant mRNA expression relative to wild-type transcripts; RNA interference (RNAi) via strategies such as shRNA is thought to work primarily at a cytoplasmic, posttranscriptional level by targeting mature RNA (42), although some studies have suggested potential interaction with nonspliced pre-mRNA (43). In our study, the fact that we observed enhanced production of 1,25(OH)2D3 with shRNAs that included intronic sequences suggests that the most likely target for these shRNAs is not pre-mRNA. This is in agreement with previous reports describing the RNAi knockdown of a COX-1 splice variant that retained intron 1 of this gene (44). In a similar manner to CYP27b1, RNAi targeting of intron 1 of COX-1 enhanced expression of wild-type COX-1 and COX-2, further endorsing the idea that the target for RNAi was unlikely to be wild-type pre-mRNA. A possible explanation for studies such as these is that inhibition of splice variant mRNAs results in a compensatory increase in the expression of wild-type gene products. In the case of CYP27b1, the fact that intronic shRNA enhanced CYP27b1 protein function in the absence of any changes in wild-type mRNA levels suggests that the compensatory response was at the level of translation.

Previous studies have supported the idea that there is competition among the individual ribosome binding sites for ribosomes so that this step can become a rate-limiting factor in the ratio of mRNA to protein levels (45). This has been further endorsed by other studies showing that the altered RNA metabolism, leading ultimately to the RNA decay that is associated with nonsense codons, involves the recognition of these transcripts in the cytoplasm (37). The mechanism that mediates the resulting mRNA surveillance appears to involve a "pioneer" round of translation (46), suggesting that this is a dynamic process that actively competes with wild-type mRNA for the ribosomal machinery. We can therefore speculate that the numerous splice variants of CYP27b1 detectable in both renal and extrarenal cells may play a role in the normal regulation of 1,25(OH)2D3 production by acting as competitors for a limiting number of ribosomal mRNA binding sites. The precise molecular basis for this mechanism remains unclear, but it is interesting to note recent reports showing that splice variants of CYP27b1 are detectable in some tissues in the absence of any apparent wild-type mRNA expression (27).

The data presented in this study, together with previous reports of other distinct CYP27b1 splice variants (24, 25, 26, 27, 28), indicate that there are multiple forms of CYP27b1 mRNA in different cell types. Therefore, a key remaining question is how the potential regulatory effects of these transcripts relate to more conventional mechanisms involved in the control of vitamin D metabolism. Because of its potent calcemic properties, endocrine synthesis of 1,25(OH)2D3 is tightly regulated by renal proximal tubule cells during normal vitamin D physiology. Consequently, although conversion of 25OHD3 to 1,25(OH)2D3 is upregulated by calciotropic hormones such as calcitonin and PTH, there is concomitant suppression of 1{alpha}-hydroxylation by 1,25(OH)2D3 itself and by high extracellular concentrations of calcium (5). The transcriptional induction of CYP27b1 expression by PTH has been characterized at a molecular level via the identification of protein kinase A-responsive regions of the CYP27b1 gene promoter (47), whereas attenuation of CYP27b1 transcription by 1,25(OH)2D3 appears to involve a distinct negative vitamin D response element in the proximal promoter of the CYP27b1 gene (48). However, it is uncertain whether regulation of CYP27b1 enzyme activity occurs exclusively at the level of mRNA expression. Moreover, there is no clear molecular model for the regulation of CYP27b1 activity by other factors such as extracellular calcium, although this may involve direct (i.e. non-PTH-mediated) effects (10). One possible explanation for this is that there are alternative sites for transcriptional regulation of CYP27b1 beyond the promoter regions that have currently been characterized. Another possibility, outlined above, is that there exists an additional layer of gene regulation that is provided by alternative splicing of CYP27b1 transcripts.

In contrast to the transcriptional suppression of CYP27b1, the attenuating action of the splice variant mRNAs is not necessarily dependent on actual synthesis of 1,25(OH)2D3 itself. As such, CYP27b1 splice variants have the potential to regulate 1{alpha}-hydroxylase activity at a "pre-enzyme" level, in a similar manner to that previously described for the CYP24 splice variant (23). In the case of the latter, the ability of a truncated cytosolic CYP24 protein to act as a decoy for substrate 25OHD3 may provide a more sensitive mechanism for down-regulation of 1,25(OH)2D3 production than catabolism via wild-type CYP24. It is tempting to speculate that CYP27b1 splice variants will also act as pre-enzyme regulators of 1,25(OH)2D3 production. However, unlike the CYP24 splice variant, which is translated into a functional protein, the CYP27b1 splice variants appear to act as translational competitors, thereby limiting the availability of wild-type 1{alpha}-hydroxylase protein. This may be particularly important in extrarenal sites of 1,25(OH)2D3 production such as macrophages in which CYP27b1 is not subject to classical regulation by PTH and/or 24-hydroxylase activity (49). Nevertheless, the presence of CYP27b1 splice variants in renal proximal tubule cells suggests that this alternative mode of regulation for vitamin D metabolism may also exist alongside more classical mechanisms, potentially acting as a complementary system for the fine-tuning of 1,25(OH)2D3 production by these cells. Additional analysis of the link between this and conventional transcriptional regulation of CYP27b1, particularly the molecular basis for the alternative splicing of mRNAs, will be a pivotal target for future studies of this important steroidogenic enzyme.


   Footnotes
 
This work was supported by National Institutes of Health Grant RO1AR050626 (to M.H.).

Author Disclosure Summary: The authors have nothing to declare.

First Published Online March 29, 2007

Abbreviations: CYP24, Cytochrome P450 vitamin D-24-hydroxylase; CYP27b1, cytochrome P450 25-hydroxyvitamin D3-1{alpha}-hydroxylase; FCS, fetal calf serum; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; 25OHD3, 25-hydroxyvitamin D3; RNAi, RNA interference; shRNA, short hairpin RNA; SV, splice variant; TPA, tetradecanoylphorbol 13-acetate.

Received October 12, 2006.

Accepted for publication March 19, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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