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J Biol Chem, Vol. 275, Issue 12, 8711-8718, March 24, 2000

Ribozyme Ablation Demonstrates That the Cardiac Subtype of the Voltage-sensitive Calcium Channel Is the Molecular Transducer of 1,25-Dihydroxyvitamin D₃-stimulated Calcium Influx in Osteoblastic Cells^*

Riting Liu^§, Wei Li^¶, Norman J. Karin, Joel J. Bergh^¶, Karen Adler-Storthz, and Mary C. Farach-Carson^¶

From the  Department of Basic Sciences, University of Texas-Houston, Dental Branch, Houston, Texas 77030 and the ^¶ Department of Biological Sciences, University of Delaware, Newark, Delaware 19716

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

1,25-Dihydroxyvitamin D₃ (1,25(OH)₂D₃) stimulates transmembrane influx of Ca²⁺ through L-type voltage-sensitive Ca²⁺ channels (VSCCs) in ROS 17/2.8 osteoblastic cells. Ca²⁺ influx modulates osteoblastic activities including matrix deposition, hormone responsiveness, and Ca²⁺-dependent signaling. 1,25(OH)₂D₃ also regulates transcript levels encoding VSCCs. L-type VSCCs are multisubunit complexes composed of a central pore-forming ₁ subunit and four additional subunits. The ₁ subunit is encoded by one gene in a multimember family, defining tissue-specific subtypes. Osteoblasts synthesize two splice variants of the _1C cardiac VSCC subtype; however, the molecular identity of the 1,25(OH)₂D₃-regulated VSCC remained unknown. We created a ribozyme specifically cleaving _1C mRNA. To increase target ablation efficiency, the ribozyme was inserted into U1 small nuclear RNA (snRNA) by engineering the U1 snRNA expression cassette, conferring the ribozyme transcript with stabilizing stem-loops at both sides and the Sm binding site that facilitates localization into nucleoplasm. After transfection of ROS 17/2.8 cells with U1 ribozyme-encoding vector, stable clonal cells were selected in which the expression of _1C transcript and protein were strikingly reduced. Ca²⁺ influx assays in ribozyme transfectants showed selective attenuation of depolarization and 1,25(OH)₂D₃-regulated Ca²⁺ responses. We conclude that the cardiac subtype of the L-type VSCC is the transducer of stimulated Ca²⁺ influx in ROS 17/2.8 osteoblastic cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Various Ca²⁺ channels transduce signals that control the physiological activities of target cells. Voltage-sensitive calcium channels (VSCCs)¹ are classified as L, T, N, P/Q, and R type according to their voltage sensitivity, single-channel properties, ion selectivity, and pharmacological properties (1, 2). A high threshold for voltage-dependent activation and sensitivity to dihydropyridine type organic channel blockers characterize L-type VSCCs that consist of five distinct subunits (₁, ₂, , , and ) encoded by four genes (3). The ion translocating function of the L-type channel is conferred by its pore forming ₁ subunit that contains four homologous repeats, each having six putative transmembrane segments (S1-S6) (1-4). At least four subtypes have been identified and ascribed to coding gene differences; _1C, _1D, _1F, and _1S correspond to cardiac muscle, neuroendocrine tissue, retina, and skeletal muscle, respectively. The diversity of the channel is further generated by alternative splicing into various isoforms, as is the case for the _1C subunit (5).

L-type VSCCs have been identified in osteoblasts of various origins (6-9), where they are thought to play important roles in regulating osteoblast responses to external stimuli. In particular, L-type VSCCs transduce the rapid effects of 1,25(OH)₂D₃ and depolarization, leading to Ca²⁺ influx in various osteoblastic cells (6, 7, 10). In the longer term, 1,25(OH)₂D₃ treatment alters gene expression in osteoblastic cells, resulting in increased expression of matrix proteins including osteopontin and osteocalcin and reduced expression of the VSCC _1C subunit (11, 12). However, the molecular identity of the VSCC involved in Ca²⁺ flux induced by 1,25(OH)₂D₃ and depolarization has never been demonstrated conclusively.

Recent developments in ribozyme technology provide powerful means to selectively inhibit expression of specific target genes. Ribozymes are a class of small catalytic RNA molecules that recognize specific substrate RNA molecules by their complementary nucleotide sequence, cleaving the substrate RNA as an endoribonuclease at enzymatic rates (13). Ribozyme expression in transfected target cells thus can inhibit the functional expression of a specific gene. Factors that contribute to ribozyme efficiency in transfected cells are expression levels, stability against rapid degradation, correct folding for exposure to the target, and co-localization and processing at sites where target transcripts are produced and accumulated. By all of these criteria, the U1 small nuclear RNA (snRNA) expression cassette provides an excellent vehicle for ribozyme delivery and expression (14-16). This cassette has been successfully used to introduce a ribozyme to inhibit fibrillin 1 expression in a human osteosarcoma cell line (16).

U1 snRNA is a highly expressed, stable small RNA (164 nucleotides) involved in both spliceosome and catalytic processing during pre-mRNA splicing (14). The U1 snRNA expression cassette has potent, constitutive upstream elements comparable in strength with the SV40 early promoter (15). At the 5'-end, there is a trimethylguanosine 5' cap. At both ends, there are stable stem-loop structures, with the 3' loop having a high GC content. These structures confer resistance to exonucleases (14). U1 snRNA has a conserved binding site, referred to as the Sm binding site, for the small nuclear ribonucleoproteins. The consensus sequence is RAU_3-4NUGR. The hypermethylation of the 5' cap structure and the binding to the small nuclear ribonucleoprotein enable U1 snRNA to accumulate in the nucleoplasm, where the newly transcribed _1C mRNA molecules are most abundant (14). In consideration of these advantages of the U1 snRNA expression cassette, the ribozyme vehicle used in these studies was derived from an engineered U1 snRNA expression vector (16), maintaining the conserved Sm binding sequence AAUUUGUGG.

This study was designed with the aim of producing a hammerhead ribozyme specifically targeting the _1C subtype of the L-type VSCC, transfecting it into ROS 17/2.8 cells in the snRNA expression cassette, and determining whether efficient ablation of the _1C transcript would reduce or eliminate Ca²⁺ influx stimulated by 1,25(OH)₂D₃ or depolarization. Our results provide conclusive evidence that the cardiac subtype of the L-type VSCC present in osteoblastic cells, including ROS 17/2.8 cells and primary cultures, can serve as the major plasma membrane transducer of external stimuli including both 1,25(OH)₂D₃-stimulated and depolarization-induced Ca²⁺ influx.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Generation of Partial cDNA Encoding _1C mRNA Target-- For selecting and testing ribozyme activity in vitro, a cDNA copy of partial _1C mRNA was ligated into pGEM^®-3Z vector (Promega, Madison, WI). Total RNA was extracted from cultured ROS 17/2.8 cells with RNA STAT-60^TM (Tel-Test, Inc., Friendswood, TX). The upstream primer CCC1 (5'-GGTAATCACCCGAAGGAGCAAG-3') in the untranslated region and the downstream primer CCC3 (5'-ATGAATTCGCGTTGGGGTGGAAGAGTAGTC-3'; ATGAATTC represents an extra modified sequence) in the open reading frame were selected to amplify a 734-base pair fragment corresponding to bases 160 to 566 of the rat _1C subtype. cDNA was synthesized using reverse transcriptase (RT) and random hexamer priming (Advantage^TM RT for PCR Kit, CLONTECH, Palo Alto, CA).

Polymerase chain reactions (PCRs) were performed as follows: 30 cycles of 1 min at 95 °C, 1 min at 55 or 65 °C, and 2 min at 72 °C. The PCR products were subjected to electrophoresis in 1.2% (w/v) agarose gels and visualized with ethidium bromide. The 734-base pair amplimer was excised, extracted with the QIAEX II gel extraction kit (Qiagen, Valencia, CA), and ligated into a pGEM^®-3Z vector. Subsequently, the ligated product was transformed into JM109 competent cells (Promega) for amplification. At least 10 colonies were selected, and the cloned DNA and ligation junctions were sequenced to verify the identity, sequence, and orientation of the insert. A ligated plasmid, termed pGEM_1C7, was utilized to perform in vitro transcription. pGEM_1C7 contains an SP6 promoter upstream from the cloned DNA corresponding to _1C mRNA of the L-type VSCC.

Identification of a Specific Ribozyme Target Site in the _1C mRNA-- The DNA sequence encoding _1C transcript between primers CCC1 and CCC3 in rat ROS 17/2.8 cells was aligned and compared with other rat subtypes of VSCC ₁ subunits, including _1S (GenBank^TM accession no. X05921), _1D (GenBank^TM accession no. M57682), and reported sequence for _1C (GenBank^TM accession no. M67516). The computer program Align Plus (Scientific and Educational Software, State Line, PA) was used for sequence analysis and identification of subtype-specific sequences (see Fig. 1). A search for the nucleotide triplet NUX was made (where N is any nucleotide, and X is A, U, or C). NUX represents the cleavage triplet of hammerhead ribozymes (17, 18). The secondary RNA structure was then analyzed using the RNA MFold program (by Mike Zuker; available on the World Wide Web). Sites buried within complex predicted secondary structures were eliminated from consideration (19), and a site at nt 6 was selected for this study. The target sequence (25 nt) of ribozyme 6 was compared with available sequences in GenBank^TM and found to be unique to _1C.

Ribozyme Design-- A hammerhead ribozyme targeting _1C mRNA was designed according to published procedures (13). The ribozyme contained a 24-nt hammerhead domain flanked by 10-12 nt complementary to the targeted region of the _1C mRNA. To construct the ribozymes in vitro, two strands of DNA template containing a SP6 RNA polymerase promoter sequence and ribozyme sequence were synthesized. The ribozyme was targeted to a GUC at nt 6 of _1C mRNA, relative to the translational site, and was thus named ribozyme 6.

In Vitro Testing of Ribozyme Activity-- The ribozyme-encoding oligonucleotides were incubated in 10 mM Tris (pH 8.0) and 50 mM NaCl at 94 °C for 5 min and then cooled gradually to room temperature to allow annealing into double-stranded DNA. Substrate RNA was synthesized from pGEM_1C7 that contains a SP6 promoter upstream from the cloned DNA corresponding to partial _1C mRNA. The plasmid pGEM_1C7 was linearized with SacI and then isolated from an agarose gel and extracted using the QIAEX II gel extraction kit. Transcription of substrate and ribozyme RNA was performed with the RiboMAX^TM large scale RNA production system (Promega). The transcription reaction mixture contained 2.5 µg of linearized plasmid or 1.5 µg of ribozyme DNA templates and 25 mM each ATP, CTP, GTP, and UTP. In addition, a prescribed amount of SP6 RNA polymerase and buffers was added as suggested by the manufacturer's instructions in a total volume of 100 µl. Following transcription, the DNA templates were removed from the reaction mixture with RQ1-RNase-Free DNase. Unincorporated nucleotides were removed from the RNA transcript by size exclusion chromatography through a small prepacked MicroSpin^TM G-50 column (Amersham Pharmacia Biotech).

The target RNA and ribozyme 6 were mixed, and the in vitro cleavage reaction was performed in 50 mM Tris (pH 8.0), 20 mM MgCl₂ at 37 °C for various times. Prior to the addition of MgCl₂, the mixture was heated at 95 °C for 2 min and then quick chilled on ice. The cleavage reaction was terminated by the addition of a 3-fold (v/v) excess of Ambion (Austin, TX) loading buffer containing formaldehyde, formamide, and 6 mM EDTA. Cleavage products were resolved by electrophoresis in 1.9% (w/v) agarose/formaldehyde gels and then stained with ethidium bromide and visualized after destaining with RNase-free water.

Construction of the Eukaryotic Expression Vector Encoding Ribozyme-- The _1C ribozyme used for transfection was designed and modified from the ribozyme tested in vitro. The vector for expressing this ribozyme in transfected cells was constructed from the plasmid pZeoU1EcoSpe (16). pZeoU1EcoSpe contains the pZeoSV plasmid DNA modified by excising the SV40 promoter, SV40 polyadenylation site, and polylinker at the BamHI sites. In constructing the pZeoU1EcoSpe, a U1 snRNA expression cassette in pUC13 (20, 21) was excised with BamHI digestion and ligated into the BamHI sites of the modified pZeoSV. Two rounds of site-directed mutagenesis were then performed to change 4 nt flanking the Sm protein binding site of U1 snRNA, creating unique EcoRI and SpeI restriction sites. The 5'-flanking region of the inserted U1 snRNA expression cassette possesses a promoter/enhancer comparable in strength with the SV40 early promoter (15).

The transfection vector expressing ribozyme 6 was termed pU1RB_1C6, and the vector expressing the control RNA was designated pU1control. In construction of pU1RB_1C6 and pU1control, the Sm binding sequence AATTTGTGG (AAUUUGUGG in mRNA) was inserted prior to the template DNA sequence. The DNA sequence of pU1control differs from that of pU1RB_1C6 only in that the nucleotides corresponding to those in ribozyme 6 were randomly selected. Oligonucleotides were synthesized and chemically phosphorylated at the 5'-end (Integrated DNA Technologies, Inc., Coralville, IA). The single-stranded oligonucleotides were annealed and ligated into the EcoRI and SpeI sites of pZeoU1EcoSpe to create pU1RB_1C6 and pU1control. The sequence and orientation of the inserts as well as the U1 snRNA expression cassette were confirmed by DNA sequencing.

Cell Culture-- ROS 17/2.8 rat osteoblastic sarcoma cells were cultured in Ham's F-12 medium/Dulbecco's modified Eagle's medium-high glucose (1:1) containing 10% fetal calf serum at 37 °C in a humidified incubator containing 5% CO₂ as described previously (11). Zeocin-resistant clones, into which an anti-_1C ribozyme construct or control plasmid had been transfected (see below) were maintained in the same medium supplemented with 50-150 µg/ml Zeocin (Invitrogen Inc., Carlsbad, CA). The cell lines were weaned from an initial selection concentration of 225 µg/ml (see below).

Transfection of ROS 17/2.8 Cells-- pU1RB_1C6 and pU1control were linearized with ApaI and cleaned with Wizard DNA Clean-Up Resin (Promega). ROS 17/2.8 cells were grown to 50-80% confluency and transfected with the linearized plasmids. Transfections were performed with LipofectAMINE^TM (Life Technologies, Inc.) according to the manufacturer's protocol. At 72 h post-transfection, the cells were passaged to less than 25% confluency in medium containing 225 µg/ml Zeocin. After 14 days, widely spaced clonal colonies were scraped and aspirated into a micropipettor as described (22). The cell clumps were added to wells containing 0.15 ml of trypsin EDTA (22). After 15 min, the cell clumps were dispersed by pipetting, and the trypsin reaction was terminated with 1 ml of medium containing 10% (v/v) serum and 225 µg/ml Zeocin. The medium was replaced with fresh medium after the cells had attached firmly. To ensure plasmid retention, the Zeocin concentration was maintained at 50-150 µg/ml throughout all remaining phases of experimentation.

RT-PCR Analysis of Ribozyme 6 and _1C Expression-- Total RNA extraction and reverse transcription with random hexamer were performed as described above. For detection of ribozyme expression, PCR was performed with primers PR_1C6 (5'-CATTCTGATGAGTCCGTGAGG-3') and PRU1 (5'-GGAAAGCGCGAACGCAGT-3'). For detection of control plasmid expression, a nucleotide sequence termed PRcntr (5'-GCAGTTGACCGAAGAGTTGGA-3') was used with PRU1. The amplification conditions were as follows: 45 s at 95 °C, 45 s at 58 °C, and 45 s at 72 °C for 30-35 cycles. PCR products were electrophoresed in 1.4% agarose gels and visualized with ethidium bromide.

For detection of the mRNA encoding _1C, PCR was performed with primers CCC1 and CCC3 as described above. PCR also was performed with a downstream primer pair near the 3'-end used previously in our laboratory (23) and termed PR11 (5'-AGAACCAGAGACAATGTGTGGAATA-3') and PR12 (5'-TACAGGCTAGCATGATATCTTGCCA-3').

Confocal, Immunofluorescent Analysis of _1C Expression-- Cells were grown overnight, rinsed with Hanks' balanced salt solution three times, and fixed with 2.5% paraformaldehyde in phosphate-buffered saline for 10 min. Fixed cells were blocked for 20 min with blocking solution (1% gelatin, 0.05% casein, and 0.2% donkey serum) and then rinsed three times with phosphate-buffered saline. Commercial rabbit anti-_1C primary antibodies that do not react with either _1S or _1D subtypes (Alomone Laboratories, Jerusalem, Israel) were applied at a dilution of 1:500 in blocking solution for 1 h. A second antibody solution containing donkey anti-rabbit Ig-Texas Red (Amersham Pharmacia Biotech) at a dilution of 1:10 in blocking solution was applied for 1 h. Prior to visualization, the cells were rinsed three times in buffer containing 0.1% (w/v) gelatin and 0.05% (w/v) casein for 10 min each. Finally, the cells were prepared with Fluoromount G (Fisher) and observed with a Zeiss 510 LSM confocal fluorescence microscope (Carl Zeiss, Inc., Thornwood, NY) as described previously (24). It should be noted that Western blot analyses using a variety of osteoblastic cells confirm the specificity of the _1C antibody.²

Calcium Uptake Assays-- Cultured cells were assayed for Ca²⁺ uptake using previously published procedures (25). Cells were seeded at a density of 50,000 cells/ml onto 3.5-cm dishes and grown to approximately 50% confluency. Culture medium was aspirated, and the cells were washed with resting buffer (132 mM NaCl, 5 mM KCl, 1.3 mM MgCl₂, 1.2 mM CaCl₂, 10 mM D-glucose, and 25 mM Tris-HCl, pH 7.4). Then the cells were incubated for 2 min with either 37 °C or room temperature resting buffer, resting buffer plus 1 nM 1,25(OH)₂D₃, or depolarizing buffer (5 mM NaCl, 132 mM KCl, 1.3 mM MgCl₂, 1.2 mM CaCl₂, 10 mM D-glucose, and 25 mM Tris-HCl, pH 7.4). 1,25(OH)₂D₃ from stock solution (1 µM) in absolute ethanol was added into the resting buffer, and the same amount of ethanol was added into all other test tubes. All uptake solutions contained 12.5 µCi/ml ⁴⁵Ca²⁺ (NEN Life Science Products). Uptake was terminated by aspiration of the labeling solution followed by three washes with ice-cold resting buffer. Cell-associated ⁴⁵Ca²⁺ was extracted by a 2-h incubation with 0.5 N NaOH and measured by liquid scintillation counting. Two-tailed t tests were performed to assess the significance of the difference between each stimulated group compared with its control in resting buffer.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Identification of Subtype-specific Ribozyme Cleavage Sites in the Osteoblastic _1C Transcript-- The first objective was to verify the sequence of _1C transcript produced in ROS 17/2.8 cells for identification of ribozyme cleavage sites. The cDNA corresponding to the 5' region of _1C in ROS 17/2.8 cells that encompasses the initiating AUG codon was cloned and sequenced. This region was chosen for ribozyme targeting because the mRNA near the translational start site is more likely to be exposed on the outer surface of the three-dimensional structure of the folded transcript. Only a few nucleotide discrepancies upstream of the translational start site were found in a comparison of the _1C mRNA from ROS 17/2.8 osteoblastic sarcoma cells with the _1C mRNA sequence in GenBank^TM (26); the coding sequences were identical. For selection of subtype-specific ribozyme cleavage sites in the _1C mRNA in ROS 17/2.8 cells, the mRNA sequence of _1C mRNA was compared with those of _1D from rat brain mRNA (27) and _1S from rabbit skeletal muscle mRNA (28) obtained from GenBank^TM. No corresponding rat sequence of _1S was found in the GenBank^TM data base. Both K_cat and K_cat/K_m for GUC and CUC cleavage sites are comparatively higher than for other potential sites. Because _1C mRNA is not an abundant transcript in ROS 17/2.8 cells, both K_cat and K_m were considered (29). GUC at nt 6, relative to the translational site of _1C, was selected as the best potential ribozyme target site. Fig. 1 shows the design and specificity of the ribozyme that was termed ribozyme 6. MFold program analysis of the _1C transcript further showed that the ribozyme 6 cleavage site was likely to be in a region of the predicted folded structure accessible for efficient ribozyme cleavage (Fig. 2A).

View larger version (8K): [in this window] [in a new window]   Fig. 1.   Ribozyme 6 is designed to specifically cleave the 1C subtype of the VSCC. Sequences encoding 1C, 1D, and 1S subtypes of the L-type VSCC were obtained from GenBankTM (accession numbers M67516, M57682, and X05921, respectively). The rat osteoblastic 1C sequence present in ROS 17/2.8 cells was deduced from a cloned cDNA produced using primers CCC1 and CCC3 (see "Experimental Procedures") and found to match the published sequence for 1C in the complementary region to ribozyme 6. The GUC site chosen for ribozyme targeting is located at nt 6 (relative to start codon AUG designated nt 1-3) and contains a sequence unique to the 1C VSCC subtype. Because this sequence is not present in either 1S or 1D subtypes, the ribozyme will not hybridize with or cleave these transcripts if present.

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Ribozyme 6 cleaves a synthetic 1C target in vitro. A, a synthetic target mRNA corresponding to nt 160 to 566 of the 1C sequence was produced as described under "Experimental Procedures." The site for cleavage by ribozyme 6 (Ribo-6) in the predicted secondary structure of the synthetic target is shown. The smaller nt numbers indicate the position of the nt as input for the RNA MFold program (see "Experimental Procedures"). B, 1C mRNA transcripts (783 nt including flanking sequences) were incubated alone or with ribozyme 6 (3:1, w/w) at 37 °C for various periods of time in 20 mM MgCl2, 50 mM Tris-HCl, pH 8.0. Cleavage products were resolved by electrophoresis using 1.9% agarose/formaldehyde gels and visualized by ethidium bromide staining. Shown are the lanes corresponding to uncleaved 1C substrate (lane 2), 1C mRNA and ribozyme 6 co-incubated for 1.5 h (lane 3), and 1C mRNA and ribozyme 6 co-incubated for 3.0 h (lane 4), 4.0 h (lane 5), or 6.0 h (lane 6).

In Vitro Testing of Ribozyme Cleavage of the Substrate _1C mRNA-- To assess the ability of ribozyme 6 to cleave the _1C transcript, reactions were performed in vitro with cloned products. To create a synthetic _1C mRNA substrate, a region of _1C mRNA that encompasses the selected subtype-specific target sites was inserted into the pGEM3 vector. This vector contains a SP6 promoter directly upstream of the cloned _1C cDNA that corresponds to nt 160 to 566 relative to the translational start site. Transcription of the sense RNA construct by SP6 RNA polymerase yielded a 783-nt RNA substrate that contains 726 nt from the _1C mRNA, plus 57 nt at the ends (46 plus 11 nts) contributed by the pGEM3 vector. Ribozyme 6 was synthesized from double-stranded synthetic DNA oligonucleotide as described (30).

The synthetic _1C mRNA substrate and ribozyme were mixed as described in the legend to Fig. 2B. Cleavage was assessed after various incubation times in the cell-free system. As shown in Fig. 2B, cleavage of the _1C target by ribozyme 6 occurred efficiently within the first 1.5 h (lane 3). The 783-base substrate was cleaved by ribozyme 6 into limit 571-base and 212-base fragments that appeared to be fairly stable for at least 6 h. Two other ribozymes targeting _1C sites were also designed and cloned (data not shown). Ribozyme 6 was the most efficient, consistent with its target being located in the proximity of the start site.

Expression of Ribozyme 6 mRNA in Transfected Cells-- Ribozyme 6 and control double-stranded DNA oligonucleotides were inserted separately into a modified U1 snRNA expression vector as described under "Experimental Procedures." The predicted secondary structures of both transcripts of U1-ribozyme and U1-control chimeric RNA were compared with the parent U1 snRNA using the RNA MFold program. As shown in Fig. 3, the U1-ribozyme 6 chimeric transcript was predicted to preserve the 5' and 3' stem-loop structures of U1 snRNA. The Sm binding site (AAUUUGUGG) of U1-ribozyme also is predicted to maintain its single-stranded character seen in U1 snRNA. While these models do not necessarily reflect the actual structure of the RNA molecules under study, they provide useful models for the design of active ribozymes.

View larger version (10K): [in this window] [in a new window]   Fig. 3.   Sequence and predicted secondary structure of chimeric ribozyme transcript of pU1RB1C6. Ribozyme 6 DNA was inserted into a modified U1 snRNA expression vector to produce a modified anti-1C ribozyme-producing vector termed pU1RB1C6. The hammerhead ribozyme is located at the center of the transcript flanked by stem structures at both ends. The 5' cap structure was methylated by 1 or 3 methyl (m) groups. A Sm binding site sequence (AAUUUGUGG) was included. Primers PR1C6 (5'-CATTCTGATGAGTCCGTGAGG-3) (sense) and PRU1 (5'-GGAAAGCGCGAACGCAGT-3) (antisense) were designed to amplify the ribozyme transcripts. The number at 1C mRNA indicates the position relative to the translational start site. An arrow indicates the cleavage site.

The ribozyme-encoding and control plasmids were transfected into ROS17/2.8 cells using LipofectAMINE^TM and subcloned as described under "Experimental Procedures." Multiple, stable, and Zeocin-resistant clones transfected with ribozyme 6 were screened for _1C mRNA expression by RT-PCR analysis. The clone that demonstrated the most efficient ablation of _1C mRNA as revealed by PCR was selected for further study.

For detecting the transcripts of U1-ribozyme and U1-control chimeric genes, specific primers were designed as described under "Experimental Procedures." The RT-PCR analysis shown in Fig. 4 demonstrates that ribozyme 6 was expressed only in the selected clonal cells transfected with ribozyme 6-encoding plasmids, while control ribozyme transcripts were found only in clonal cells transfected with control plasmids. As expected, both ribozyme and control transcripts were absent in the parental ROS 17/2.8 cells. The cDNA bands migrate at a size corresponding to the expected 66 base pairs, demonstrating that the ribozyme and control RNA constructs were expressed in the respective clonal cells.

View larger version (79K): [in this window] [in a new window]   Fig. 4.   Ribozyme 6 and control chimeric RNA are expressed in transfected ROS 17/2.8 cells as detected by RT-PCR. Total RNA was extracted from ROS 17/2.8 parental cells (lanes 1 and 1'), and subclones were transfected with ribozyme encoding pU1RB1C6 (lanes 2 and 2') or with pU1control (lanes 3 and 3'). Preparation of the ribozyme and control constructs and transfection and subcloning were as described under "Experimental Procedures." Reverse transcription reactions were performed with random hexamer primers. PCR was performed using ribozyme-specific primers (lanes 1, 2, and 3) as shown in Fig. 3. The control chimeric RNA was detected using control chimeric RNA-specific 5' primer (lanes 1', 2', and 3') and the same 3' primer as that used to detect the ribozyme.

Inhibition of _1C Expression in ROS 17/2.8 Cells-- Selected clonal cells were analyzed by RT-PCR and immunofluorescence microscopy for _1C transcript and protein expression. As shown in Fig. 5, the _1C mRNA levels were strikingly reduced in cell lines transfected with ribozyme-encoding plasmids, while the _1C mRNA levels in cell lines transfected with control plasmids were comparable with those found in the original ROS 17/2.8 cells. The primers (CCC1 and CCC3) employed in Fig. 5, A and B, amplified _1C transcripts that spanned the predicted ribozyme cleavage site shown in Fig. 1. It was considered that short versions of _1C transcripts containing 3' sequences might be present in cells transfected with active ribozyme. To address this, a downstream primer pair (PR11 and PR12) that amplifies region IV of _1C transcripts corresponding to bases 3551-4276 relative to the translational start site of rat _1C was used in RT-PCRs. No products were detected, suggesting that 5'-targeted, ribozyme ablation of _1C transcript leads to complete degradation. The amplimer size of the PR11/PR12 product has been revised from earlier estimates (11) of 740 nt to a more precise determination of 726 nt, shown in Fig. 5C, lanes 1 and 3. Similar amounts of glyceraldehyde-3'-phosphate dehydrogenase were amplified in reactions from all three cell lines (Fig. 5C, lanes 1'-3'), indicating that _1C targets were specifically affected. In Fig. 5B, additional nonspecific DNA bands were observed when the annealing temperature was decreased from 65 to 55 °C during the PCR process, but _1C transcripts remained undetectable. The inability to detect _1C transcript in ribozyme 6-transfected cells, even under lower stringency PCRs, points to the efficiency of the pU1RB_1C6 construct.

View larger version (46K): [in this window] [in a new window]   Fig. 5.   Ribozyme 6 inhibits the expression of 1C-encoding mRNA as detected by RT-PCR. Total RNA was extracted from nontransfected ROS 17/2.8 cells (lanes 1 and 1') or subclones transfected with ribozyme encoding pU1RB1C6 (lanes 2 and 2') or with control pU1control (lanes 3 and 3'). Reverse transcriptase reactions were performed with random hexamer primers. PCR reactions shown in lanes 1, 2, and 3 were performed using primers CCC1 and CCC3 (see "Experimental Procedures") located near the 5'-end or subtype-nonspecific primers PR11 and PR12 (23) located closer to the 3'-end of the 1C transcript. The annealing temperature of the PCR shown in A was 10 °C higher than that used in the reaction shown in B and C. Only 1C subtype products were detected in all cases. The PCRs shown in lanes 1', 2', and 3' were performed using glyceraldehyde-3'-phosphate dehydrogenase (G3PDH)-specific primers and provided a loading assessment.

The selected clonal cells were further characterized by immunofluorescence using specific antibodies to _1C protein and confocal microscopy. The antibodies are specific to the _1C subtype of the VSCC as determined by the manufacturer. As shown in Fig. 6, A-C, the number of detectable immunoreactive cell surface _1C VSCCs was substantially reduced in clonal cells harboring ribozyme-coding pU1RB_1C6 (Fig. 6B). In contrast, clonal cells transfected with pU1control (Fig. 6C) showed a pattern and density of _1C protein expression similar to that found in the parental ROS 17/2.8 cells (Fig. 6A). This finding demonstrated that ribozyme 6 successfully inhibited cell surface expression of _1C protein as well as transcript in transfected cells.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Confocal immunocytochemistry demonstrates blockade of 1C protein expression in ROS 17/2.8 cells expressing ribozyme 6. Antibodies specific to 1C were used to assess cardiac VSCC protein expression in parental cells or pU1RB1C6- or pU1control-transfected ROS 17/2.8 subclones. A, parental ROS 17/2.8 cells; B, subclone transfected with pU1RB1C6; C, subclone transfected with pU1control. Methods were as described under "Experimental Procedures." Magnification was × 1000. Note that the micrograph in A was taken in an area of the plate where cells were at higher density.

ROS 17/2.8 Cells Ablated of _1C Transcript Do Not Respond to 1,25(OH)₂D₃ and Depolarization-- ⁴⁵Ca²⁺ uptake assays were performed using subconfluent cell monolayers as described under "Experimental Procedures." As shown in Fig. 7, cells in which _1C of VSCC was ablated by active ribozyme 6 lost the ability to respond to either depolarization or 1,25(OH)₂D₃. In contrast, cells transfected with control plasmids continued to express _1C and maintained their sensitivity to 1,25(OH)₂D₃ and high K⁺-stimulated depolarization reflected by a statistically significant 2-3-fold increase in ⁴⁵Ca²⁺ influx. As seen previously (6), slightly higher levels of stimulated ⁴⁵Ca²⁺ influx were seen when cells were depolarized. Overall, treatment with 1,25(OH)₂D₃ increased ⁴⁵Ca²⁺ influx 2.2-fold, compared with 2.8-fold after depolarization. Together, these results revealed that the _1C subtype of VSCC is responsible for 1,25(OH)₂D₃-stimulated influx in ROS 17/2.8 cells.

View larger version (65K): [in this window] [in a new window]   Fig. 7.   ROS 17/2.8 cells ablated of 1C do not demonstrate stimulated 45Ca2+ influx in response to depolarization or 1,25(OH)2D3. 45Ca2+ influx assays were performed as described under "Experimental Procedures" using subclones demonstrating efficient ribozyme-mediated ablation of the 1C transcript assessed by RT-PCR and immunohistochemistry as shown in Figs. 5 and 6. Depolarizing buffer differs from resting buffer in that it contains high KCl (see "Experimental Procedures"). 1,25(OH)2D3 was used at a concentration of 1 nM. Values for influx within each experiment were normalized to values of cells in resting buffer that were assigned a normalized value of 1.0. Data represent the mean and S.D. in five experiments. The p values indicated over the bar graphs for these experiments were as follows: p < 0.01 (**) and p > 0.05 (). Only those groups marked with a double asterisk demonstrated influx values significantly different from controls receiving resting buffer.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ca²⁺ influx induced by various stimuli, including depolarization and treatment with calcitropic hormones such as 1,25(OH)₂D₃, plays an important role in signaling processes related to bone growth, differentiation, and remodeling. For example, recent work demonstrated that 1,25(OH)₂D₃-dependent Ca²⁺ influx modulates the post-translational state of the extracellular bone matrix phosphoprotein, osteopontin (31). The VSCC represents a major pathway for Ca²⁺ entry into bone cells (6, 32). Several laboratories, including our own, have demonstrated the presence and activation of VSCCs in osteoblasts treated with 1,25(OH)₂D₃ (6), parathyroid hormone (8), and epidermal growth factor (32) and during depolarization (6). The development of 1,25(OH)₂D₃ analogs with differential actions on nuclear receptor-mediated or membrane-initiated pathways provided a powerful approach to dissect the dual effects of 1,25(OH)₂D₃, demonstrating conclusively that the rapid activation of cell surface VSCCs is independent of the classical nuclear receptor (12, 33). Vitamin D₃ analogs that bind to the nuclear receptor but fail to stimulate Ca²⁺ influx have clinical utility because they stimulate bone formation with a lesser tendency to produce hypercalcemia compared with 1,25(OH)₂D₃ (34). This implies that activation of VSCCs may be of pathophysiological importance in the development of hypercalcemia.

Early studies established that depolarization and hormone-stimulated Ca²⁺ influx into osteoblasts could be inhibited by dihydropyridines (6), indicating that the VSCCs involved in this influx are of the high threshold, or L-type. Since the first cDNA encoding the _1S skeletal muscle Ca²⁺ channel subunit was cloned (28), two major additional subtypes of L-type VSCCs were discovered: the cardiac muscle subtype, _1C, and the neuroendocrine subtype, _1D. Recently, a new L-type subtype (_1F) from retina also was reported (35). Major subtypes of VSCCs are frequently found outside the excitable tissue where they were first discovered; for example, the expression of the _1C subtype was detected in rat brain as well as cardiac tissue (26).

Since the initial discovery that Ca²⁺ influx by 1,25(OH)₂D₃ was transduced by L-type VSCCs in ROS 17/2.8 osteoblastic cells (6), many efforts have been made to discern the exact molecular nature of the Ca²⁺ channel(s) present in osteoblastic cells. L-type VSCCs are the best studied Ca²⁺ channel type and appear to be the major VSCC in osteoblasts, although T-type channels may be expressed transiently during development (36, 37). Three types of cDNA sequences corresponding to _1S, _1C, and _1D were detected by Barry et al. (8) in the rat UMR-106 osteosarcoma cell line. Two alternate splice isoforms of the _1C subtype were identified in rat ROS 17/2.8 osteoblastic sarcoma cells and primary osteoblasts in our laboratory, but the other two subtypes were undetected (11). The molecular identification of the cardiac subtype was confirmed recently in primary cultures of rat calvarial osteoblasts, where it was proposed that the _1C is involved in development of epidermal growth factor responsiveness of these cells (32). This finding was consistent with our previous report (11) that the _1C subtype was present in primary cultures of cells grown from neonatal rat calvaria. Taken together, these studies indicate that the _1C subtype of the VSCC is the major subtype expressed both in primary cultures and cell lines of osteoblastic phenotype. Nonetheless, a collective limitation of all prior studies is that they rely upon circumstantial evidence to predict relationships between structure and function. No evidence has been presented to definitively link the presence of _1C VSCC subtype to a demonstrated function.

The approach taken in this study involved ribozyme-mediated ablation of the _1C transcript in transfected osteoblastic cells, followed by functional assays using stable transfectants. For the study to be definitive, it was essential that _1C transcript levels be reduced to negligible levels. Antisense approaches have been used previously in this system (38), but we believed more efficient ablation would be achieved using a ribozyme approach with catalytic efficiency. Considering that targeting the initiating AUG codon or adjacent sequences may increase the antisense effect of ribozymes (39, 40), ribozyme-6 was chosen for transfection and expression in ROS 17/2.8 cells. Because the alternative splice that occurs in the osteoblast _1C mRNA is downstream in the coding region at transmembrane segments IVS3-IVS4 (11), ribozyme-6 was expected to cleave transcripts encoding both of these isoforms. We also engineered a ribozyme construct in which ribozyme 6 was inserted into a U1 snRNA expression cassette (16). This cassette offered the advantage of producing a ribozyme that should be highly expressed, resistant to exonucleases, and capable of concentrating in the nucleoplasm, where newly transcribed _1C transcripts should be most abundant.

In this study, we prepared stable sublines of ROS 17/2.8 cells in which _1C expression is highly inhibited by expression of active ribozyme 6. Control cell lines expressing inactive ribozyme constructs also were prepared. The selected clonal cells showing most efficient ribozyme-mediated ablation of _1C transcripts, as well as clonal control transfectants, were employed to investigate the relationship between _1C and Ca²⁺ influx modulated by 1,25(OH)₂D₃ and depolarization. ⁴⁵Ca²⁺ uptake assays were performed in subconfluent cell monolayers of sublines expressing active and inactive ribozymes, as well as in the parental line. As presented under "Results," cells expressing active ribozyme 6 lost their rapid Ca²⁺ response to both 1,25(OH)₂D₃ and depolarizing high K⁺ buffer. In contrast, cells transfected with control plasmids maintained their sensitivity to 1,25(OH)₂D₃ and high K⁺ buffer, demonstrated by rapid ⁴⁵Ca²⁺ influx at levels comparable with nontransfected cells. Taken together, these data establish that the _1C subtype of the L-type channel is the depolarization and 1,25(OH)₂D₃-responsive VSCC in osteoblasts.

The results presented here integrate and extend previous findings regarding the nature of the osteoblast VSCC. For the first time, we definitively demonstrated that the rapid membrane-initiated effects of 1,25(OH)₂D₃ on Ca²⁺ influx in osteoblasts are transduced by the _1C subtype of the VSCC. By defining the cardiac subtype to be the molecular form of VSCCs responsive to 1,25(OH)₂D₃ and depolarization, we are now in a position to further explore the physiological importance of the VSCC in osteoblast function and to begin to define the signaling pathways activated directly by the VSCC.

	ACKNOWLEDGEMENTS

We thank Drs. William T. Butler, Zhou (Georgia) Chen, and Dan Carson for many wonderful ideas and enthusiastic support of this project. We are grateful to Dr. Harry C. Dietz at The Johns Hopkins University for providing the plasmid used to create the ribozyme vector and Dr. Kirk Czymmek for assisting with the confocal microscopy. We thank all of the members of the Farach-Carson laboratory and Dr. Gary Meszaros for many useful discussions. We especially thank Dr. Kamil Akanbi for assistance with sequence analysis. Finally, we are grateful to Sharron Kingston and Margie Barrett for assistance in the preparation of the manuscript.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grant AR39273 (to William T. Butler and M. C. F.-C.) and a grant from NASA/Texas Medical Center (to M. C. F.-C. and N. J. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Present address: Dept. of Biological Sciences, University of Delaware, Newark, DE 19716.

To whom correspondence should be addressed: Dept. of Biological Sciences, University of Delaware, 304 Wolf Hall, Newark, DE 19716. Tel.: 302-831-2277; Fax: 302-831-2281; E-mail: farachca@udel.edu.

² K. Brubaker and R. Duncan, personal communication.

	ABBREVIATIONS

The abbreviations used are: VSCC, voltage-sensitive calcium channel; nt, nucleotide(s); PCR, polymerase chain reaction; RT, reverse transcriptase; snRNA, small nuclear RNA; 1, 25(OH)₂D₃, 1,25-dihydroxyvitamin D₃.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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