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Am J Physiol Cell Physiol 290: C822-C831, 2006. First published October 12, 2005; doi:10.1152/ajpcell.00403.2005
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Osteoblast Ca2+ permeability and voltage-sensitive Ca2+ channel expression is temporally regulated by 1,25-dihydroxyvitamin D3

Joel J. Bergh,1 Ying Shao,1 Erwin Puente,1 Randall L. Duncan,2 and Mary C. Farach-Carson1

1Department of Biological Sciences, University of Delaware, Newark, Delaware; and 2Department of Orthopaedics, Indiana University College of Medicine, Indianapolis, Indiana

Submitted 10 August 2005 ; accepted in final form 5 October 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The cardiac subtype of the L-type voltage-sensitive Ca2+ channel (VSCC) Cav1.2 ({alpha}1C) is the primary voltage-sensitive channel responsible for Ca2+ influx into actively proliferating osteoblasts. This channel also serves as the major transducer of Ca2+ signals in growth-phase osteoblasts in response to hormone treatment. In this study, we have demonstrated that 24-h treatment of MC3T3-E1 preosteoblasts with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], a coupling factor for bone resorption, coordinately downregulates Cav1.2 ({alpha}1C) and uniquely upregulates T-type channel Cav3.2 ({alpha}1H). No other voltage-sensitive channel {alpha}-subunit of the 10 that were surveyed was upregulated by 1,25(OH)2D3. The shift from predominantly L-type to T-type channel expression has been demonstrated to occur at both mRNA and protein levels detected using quantitative PCR and immunohistochemistry with antibodies specific for each channel type. Functional and pharmacological studies using specific inhibitors have revealed that treatment with 1,25(OH)2D3 also alters the Ca2+ permeability properties of the osteoblast membrane from a state of primarily L-current sensitivity to T-current sensitivity. We conclude that the L-type channel is likely to support proliferation of osteoblast cells, whereas T-type channels are more likely to be involved in supporting differentiated functions after 1,25(OH)2D3-mediated reversal of remodeling has occurred. This latter observation is consistent with the unique expression of the T-type VSCC Cav3.2 ({alpha}1H) in terminally differentiated osteocytes as we recently reported.

calcium influx; bone

1,25-DIHYDROXYVITAMIN D3 [1,25(OH)2D3] is a critical hormonal modulator of Ca2+ homeostasis and of osteoblast function during bone remodeling (5, 14). Regulation of bone resorption by osteoclasts in response to 1,25(OH)2D3 occurs through coupling factors generated by osteoblastic cells. Multiple interacting 1,25(OH)2D3 response pathways in osteoblasts coordinate physiological events (31). These events include rapid responses that involve activation of voltage-sensitive Ca2+ channels (VSCCs) (18) and genomic changes in gene transcription that may involve the nuclear vitamin D receptor (nVDR) or other transcription factors such as the cAMP response element-binding protein (CREB) (5, 19). Acute exposure to 1,25(OH)2D3 increases plasma membrane Ca2+ permeability by increasing the mean open time of the L-type VSCC and shifting the threshold of activation toward the resting potential, thereby easing the requirement for Ca2+ influx (9, 25, 43).

VSCCs are present in all excitable tissues and at lower levels in most nonexcitable cell types, in which they mediate the influx of Ca2+ in response to membrane depolarization and regulate intracellular functions, including excitation-secretion, gene transcription, neurotransmitter release, and cell differentiation. These responses are fine-tuned to a specific temporospatial pattern of Ca2+ entry (17, 20) that is accomplished by expression of unique subtypes of VSCCs, each of which differs regarding the kinetics of activation and inactivation, pharmacology, and tissue distribution. In osteoblasts, L-type VSCCs consist of four discrete protein subunits ({alpha}1, {alpha}2{delta}, and beta) (4). The {alpha}1-subunit provides the pore through which Ca2+ ions enter the cell and can generate a Ca2+ current in the absence of the other subunits (35). At present, at least 10 distinct {alpha}1-subunits have been identified (37). Physiological and pharmacological studies have demonstrated functional similarities among various {alpha}1-subunits that allow VSCCs to be classified into high-voltage activated (L-, P/Q-, N, and R-type) and low-voltage activated (T-type) types (39). L-type Ca2+ currents are mediated by VSCCs containing Cav1.1 {alpha}1S-, Cav1.2 {alpha}1C-, Cav1.3 {alpha}1D-, and Cav1.4 {alpha}1F-subunits, while Cav2.1 ({alpha}1A), Cav2.2 ({alpha}1B), and Cav2.3 ({alpha}1E) (P/Q-, N-, and R-type VSCCs, respectively) compose the remainder of the high-voltage activated VSCC family. The low-voltage activated T-type VSCCs include Cav3.1 ({alpha}1G), Cav3.2 ({alpha}1H), and Cav3.3 ({alpha}1I). Low- and high-voltage activated channels share <25% sequence similarity at the protein level and display remarkably different functional properties.

Previous studies conducted at our laboratory have definitively established that the L-type VSCC Cav1.2 {alpha}1C-subunit is the primary site for Ca2+ influx into the proliferating osteoblast (9, 27) and also have shown that application of 1,25(OH)2D3 increases plasma membrane permeability to Ca2+ within milliseconds by shifting the threshold of activation toward the resting potential and increasing the mean open time of the L-type VSCC (9, 25). Interestingly, rat osteoblastic cells (ROS17/2.8) treated for 24–48 h with 1,25(OH)2D3 reduced L-type VSCC transcription levels while they increased expression of differentiation markers, including osteopontin and osteocalcin (30). Prior observations also have shown that terminally differentiated osteocytes express Cav3.2 ({alpha}1H), but not Cav1.2 ({alpha}1C) (41), although the effects of 1,25(OH)2D3 on the expression of this channel have not been examined.

In this study, we used quantitative real-time PCR (QPCR) and specific immunostaining approaches to identify the spectrum of VSCC {alpha}1-subunits expressed in murine MC3T3-E1 osteoblastic cells. In addition, we examined the effects of 1,25(OH)2D3 on VSCC {alpha}1-subunit expression. Finally, 45Ca2+ influx assays were performed in the presence of various pharmacological inhibitors specific for VSCC classes to determine how treatment with 1,25(OH)2D3 altered the properties of Ca2+ permeability.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. MC3T3-E1 cells, a gift from Dr. Renny T. Franceschi (Department of Periodontics/Prevention/Geriatrics, University of Michigan School of Dentistry, Ann Arbor, MI), were plated at 5,000 cells/cm2 and maintained in {alpha}-MEM containing ribonucleosides and deoxyribonucleosides supplemented with 10% (vol/vol) heat-inactivated FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10 mM HEPES buffer. All culture reagents were purchased from Invitrogen (Carlsbad, CA). Cultures were maintained in a 37°C humidified chamber with 5% CO2. The medium was changed every 3 days, and the cell lines were passaged at 80% confluence using trypsin-EDTA.

For 1,25(OH)2D3 treatment, cells were plated into T-75 culture flasks (Corning, Corning, NY) and cultured for 72 h in serum-containing medium. The cells were then rinsed twice with PBS and fed serum-free {alpha}-MEM supplemented with penicillin, streptomycin, and HEPES. After 24-h incubation in serum-free medium, the cells were rinsed with PBS and fresh serum-free medium was added that contained either 1,25(OH)2D3 or an equal amount of vehicle 0.01% ethanol (vol/vol).

RNA isolation and RT-PCR. Total RNA was extracted from MC3T3-E1 cell cultures at 80% confluence using the RNeasy kit, which we obtained from Qiagen (Valencia, CA), according to the manufacturer's instructions. First Choice total RNA from normal mouse tissue was purchased from Ambion (Austin, TX) and contained RNA from mouse liver, brain, thymus, heart, lung, spleen, testis, ovary, kidney, and embryonic day 10 tissue. Total RNA was reverse transcribed using the Advantage for RT-PCR kit available from BD Biosciences Clontech (Palo Alto, CA). Total RNA (1 µg) was reverse transcribed for 45 min at 40°C. The enzyme then was heat inactivated at 95°C for 15 min.

QPCR was performed using the QuantiTect SYBR Green PCR kit (Qiagen) according to the manufacturer's instructions, except that fluorescein reference dye (1 nM) was added to each reaction to normalize the instrument's optics and to compensate for variations in fluorescence among wells. The primer sequences used, which are listed in Table 1, were designed on the basis of published species-specific sequences. Standards were generated by cloning the PCR product into the pCR2.1 vector using the TOPO T/A cloning kit (Invitrogen). Isolated plasmid was linearized using EcoRV, quantitated, and diluted for use as standards in QPCR. QPCR was performed using the iCycler iQ real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA), and true mRNA levels were calculated exactly as recommended by the manufacturer. Data were analyzed using Prism 3.0 software (GraphPad, San Diego, CA). For confirmation of specificity, nonlinear RT-PCR also was performed with the same primer sets. After 10-min incubation at 95°C, the cycling conditions were as follows: denaturation at 94°C for 45 s, annealing at 58°C for 45 s, and extension for 60 s at 72°C for 45 cycles, well beyond the linear range for most channel subunits. All PCR products were sequence verified using the BioResource Center at Cornell University (available online at http://www.brc.cornell.edu/brcinfo/index.php).


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  		Table 1. Oligonucleotide sequences used for QPCR

 
Immunodetection of {alpha}1-subunit expression using confocal microscopy. Detection of {alpha}1-subunits was performed using affinity-purified rabbit PAbs directed against the various {alpha}1-subunits. Primary antibodies and blocking peptides specific for Cav2.1 ({alpha}1A), Cav2.2 ({alpha}1B), Cav1.2 ({alpha}1C), Cav1.3 ({alpha}1D), Cav2.3 ({alpha}1E), and Cav1.4 ({alpha}1F) were purchased from Alomone Laboratories (Jerusalem, Israel). The rabbit anti-mouse PAb to Cav3.2 ({alpha}1H) was designed as part of this work and was generated against the peptide sequence [C]HLEEDFDKLRDVRATE located in the intracellular loop between transmembrane domains II and III. The affinity-purified antibody was obtained from a commercially available source (Invitrogen Life Technologies).

MC3T3-E1 (5,000 cells/well) were plated onto eight-well Lab-Tek chamber slides (Nalg Nunc International, Naperville, IL). The cells were grown overnight in a 37°C incubator containing 5% CO2. After 24 h, the cells were washed three times with PBS and then fixed in 4% (vol/vol) methanol-free paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA) in 0.1% (vol/vol) PBS for 30 min on ice. After fixation, the cells were rinsed with 0.01% (wt/vol) sodium azide in PBS for 5 min on ice. To permeabilize and block nonspecific binding, the cells were incubated in blocking buffer containing 5% (vol/vol) normal donkey serum, 0.3% (vol/vol) Triton X-100, and PBS for 30 min at room temperature. The primary antibody was diluted 1:50 with 1% (vol/vol) normal donkey serum, 0.1% (wt/vol) BSA, and 0.01% (wt/vol) sodium azide in PBS and then applied to the fixed cells and incubated for 1 h at room temperature. After being washed three times for 10 min each with 1% (vol/vol) normal donkey serum and PBS, the cells were incubated in the dark for 1 h at room temperature using FITC-conjugated donkey anti-rabbit IgG (1:100 dilution; Jackson ImmunoResearch, West Grove, PA). The cells were counterstained for 10 min in the dark with the nuclear dye ToPro3 (Molecular Probes, Eugene, OR) diluted 1:4,000 in PBS. The cells were washed three times for 5 min each in PBS and stored at 4°C until being studied. The fluorescence was analyzed using an inverted microscope linked to a confocal scanning unit (LSM 510; Carl Zeiss, Oberkochen, Germany). To determine the specificity of staining, images were compared with cells that had been incubated with FITC-conjugated donkey anti-rabbit IgG in the absence of primary antibody. Immunostained cells also were compared using slides stained with primary antibody that had been preincubated for 1 h with 1 µg of antigenic peptide per 1 µg of primary antibody (peptide block).

45Ca2+ flux assays. MC3T3-E1 cells were grown to 80% confluence in 35-mm-diameter tissue culture dishes and switched to serum-free medium for 24 h with 1,25(OH)2D3 or vehicle as described above. After 24 h in serum-free medium, the cells were incubated with VSCC inhibitors for 15 min. Ca2+ channel inhibitors (Alomone Laboratories) were diluted according to the manufacturer's instructions and added at the following concentrations: 1 µM L-type inhibitor nifedipine, 200 nM T-type inhibitor sFTX-3.3, 1 µM P/Q-type inhibitor {omega}-agatoxin IVA, and 1 µM N-type inhibitor {omega}-conotoxin GVIA. After 15-min application of VSCC inhibitors, the cells were rinsed twice with HBSS and placed in either resting buffer (in mM: 132 NaCl, 5 KCl, 1.3 MgCl2, 1.2 CaCl2, 10 D-glucose, and 25 Tris) or depolarizing buffer (in mM: 5 NaCl, 132 KCl, 1.3 MgCl2, 1.2 CaCl2, 10 D-glucose, and 25 Tris) containing 12.5 µCi/ml 45Ca2+ (as described in Ref. 9; NEN Life Sciences, Boston, MA) and fresh VSCC inhibitors. Cells were incubated for 2 min and then rinsed three times with ice-cold nonlabeled resting buffer. The cells were lysed for 4 h in 0.5 M NaOH, and then the lysate was placed into a scintillation counter and the amount of radioactivity in the sample was measured.

Statistical analysis. All values are expressed as means ± SD. QPCR assays were performed in triplicate, and the entire experiment was repeated three times using two independent RNA isolates. 45Ca2+ flux assays were performed in triplicate and repeated twice. Statistical comparisons were made between treatment and control groups using Student's t-test with Dunn's post hoc test. Relative changes referred to in the text were calculated by dividing the value of the treatment group by the value of the control sample at the same time point. For each experiment, data were normalized by arbitrarily setting the value of the vehicle-treated MC3T3-E1 cells to 1.0 at time 0, essentially the basal level.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Detection of {alpha}1-subunit mRNA in osteoblastic cells. To identify the VSCC {alpha}1-subunits expressed in osteoblastic cells, QPCR was performed on RNA isolated from 80% confluent growth-phase MC3T3-E1 cells. The primer sequences used are listed in Table 1 and were chosen on the basis of sequence information available in the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/). Sequence analysis of PCR amplimers revealed that each shared at least 98% sequence identity with the published sequence for that subunit. The amplification products from each {alpha}1-subunit primer produced from MC3T3-E1 cells are shown in Fig. 1A. Note that these amplification products are based not on quantitative gels but on extended cycles (45) optimized to produce a roughly identical amplimer product for sequence analysis. As shown, growth-phase MC3T3-E1 cells contained at least minimal levels of transcripts encoding all of the known {alpha}1-subunits except for Cav1.4 ({alpha}1F), which to date has been found only in RNA isolated from retinal cells. To ensure that each primer set was capable of generating a specific product, RT-PCR also was performed using an RNA mixture containing RNA from mouse liver, brain, thymus, heart, lung, spleen, testis, ovary, kidney, and embryonic day 10 tissue (Fig. 1B). Each primer set generated product bands identical in size to the bands detected in MC3T3-E1 cells, and sequence analysis confirmed that positive control RNA generated PCR products that were the same as those from MC3T3-E1 cells. RT-PCR analysis performed on the positive control RNA confirmed that the primer set specific for the Cav1.4 {alpha}1F-subunit produced a 121-bp product that had the proper sequence, confirming that MC3T3-E1 cells do not produce transcripts encoding the Cav1.4 {alpha}1F-subunit.


Figure 1
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  		Fig. 1. Standard RT-PCR of cardiac subtype of the L-type voltage-sensitive Ca2+ channel (VSCC), Cav1.2 ({alpha}1C subunit), in MC3T3-E1 cells. Nonquantitative RT-PCR was performed to identify the various pore-forming {alpha}1-subunits present in proliferating osteoblasts. A: RT-PCR performed on RNA isolated from 80% confluent MC3T3-E1 cells using specific primer sets (see Table 1) for transcripts encoding the indicated {alpha}1-subunits. B: RT-PCR was performed on a control RNA mix using primer sets and cycle conditions described previously. Control total RNA was purchased from Ambion and contained liver, brain, thymus, heart, lung, spleen, testicle, ovary, kidney, and embryonic day 10 RNA. All RT-PCR results were sequenced to confirm product identity.

 
Effects of 24-h exposure of 1,25(OH)2D3 on VSCC {alpha}1-subunit MRNA expression. QPCR was performed to quantify mRNA levels encoding various {alpha}1-subunits after 24-h treatment with 1,25(OH)2D3 or vehicle (0.01% ethanol). After 24-h incubation in serum-free medium, 80% confluent MC3T3-E1 cell cultures were treated with 0.1, 1, 10, or 100 nM 1,25(OH)2D3 or vehicle (0.01% ethanol), or they were left untreated. After 24 h, total RNA was isolated and QPCR was performed. To detect changes in expression levels, the QPCR results for the VSCCs expressed in MC3T3-E1 cells were normalized to untreated control values for each primer set. The relative L-type VSCC Cav1.2 {alpha}1C-subunit transcript levels are shown in Fig. 2. There was no difference in Cav1.2 {alpha}1C-subunit transcript levels in MC3T3-E1 cells treated for 24 h with 0.1 nM 1,25(OH)2D3, cells treated with vehicle (ethanol), or cells that were left untreated. When treated for 24 h with 1, 10, or 100 nM 1,25(OH)2D3, Cav1.2 {alpha}1C-subunit transcription levels were reduced to approximately one-half the levels found in untreated MC3T3-E1 cells, all of which were statistically significant. Cav1.3 {alpha}1D-subunit transcription levels in MC3T3-E1 cells treated with 0.1, 1, 10, or 100 nM 1,25(OH)2D3 did not differ from transcription levels in vehicle-treated and untreated cells (Fig. 2). Similarly, the other L-type VSCC present in osteoblastic cells, the Cav1.1 {alpha}1S-subunit, also was unaffected by 24-h 1,25(OH)2D3 treatment (Fig. 2). These data suggest that the Cav1.2 {alpha}1C-subunit is the only L-type VSCC present in MC3T3-E1 osteoblastic cells, the expression of which is modulated by 1,25(OH)2D3 treatment.


Figure 2
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  		Fig. 2. Changes in relative L-type VSCC {alpha}1-subunit mRNA levels in MC3T3-E1 cells treated with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. After treatment for 24 h with different doses of 1,25(OH)2D3, RNA was extracted for QPCR analysis. The black bars represent the levels of Cav1.2 ({alpha}1C) mRNA levels after treatment with various concentrations of 1,25(OH)2D3. Treatment with 1,25(OH)2D3 did not change levels of Cav1.3 ({alpha}1D) transcript (gray bars) or the levels of Cav1.1 ({alpha}1S) mRNA (open bars). All data were normalized as described in text. Data shown are means ± SD. ***P < 0.001, 1,25(OH)2D3-treated vs. vehicle-treated cultures.

 
Analysis of T-type VSCC {alpha}1-subunit transcription levels are displayed in Fig. 3. Expression of Cav3.1 {alpha}1G-subunit mRNA was not significantly different in untreated, vehicle-treated, or 0.1 nM 1,25(OH)2D3-treated cells (Fig. 3). When the concentration of 1,25(OH)2D3 was increased to 1 nM, transcription levels were reduced to 48% of the levels measured in vehicle-treated cell cultures. This suppression also was observed when secosteroid levels were increased to 10 nM, and expression was further reduced to 34% of the levels in vehicle-treated cells when the 1,25(OH)2D3 dose was raised to 100 nM. A second T-type VSCC {alpha}1-subunit found to be expressed in osteoblastic cells is Cav3.2 ({alpha}1H). The expression of Cav3.2 ({alpha}1H) transcription levels in ethanol-treated MC3T3-E1 cells was not different from the levels observed in untreated cells (Fig. 3); however, the application of 0.1 nM 1,25(OH)2D3 led to Cav3.2 {alpha}1H-subunit transcription levels that were 3.6-fold greater than those found in vehicle-treated MC3T3-E1 cells. This response was maintained when the hormone levels were elevated to 1, 10, and 100 nM 1,25(OH)2D3. The expression of the third T-type VSCC {alpha}1-subunit, which we found to be present in osteoblastic cells (Cav3.3 {alpha}1I-subunit), was not significantly affected by 1,25(OH)2D3 treatment (Fig. 3).


Figure 3
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  		Fig. 3. Changes in relative T-type VSCC {alpha}1-subunit mRNA levels in MC3T3-E1 cells treated with 1,25(OH)2D3. After treatment for 24 h with different doses of 1,25(OH)2D3, RNA was extracted for QPCR analysis. Black bars show reduction in levels of Cav3.1 ({alpha}1G) mRNA after treatment with various concentrations of 1,25(OH)2D3. Gray bars show the upregulation of transcript levels for Cav3.2 ({alpha}1H) after treatment with increasing concentrations of 1,25(OH)2D3. Exposure to 1,25(OH)2D3 had no significant effect on the expression of the Cav3.3 {alpha}1I-subunit (open bars). All data were normalized as described in text. Data are means ± SD. ***P < 0.001, 1,25(OH)2D3-treated vs. vehicle-treated cultures.

 
In addition to L- and T-type VSCCs, MC3T3-E1 cells expressed low levels of mRNA encoding P/Q-, N-, and R-type VSCCs (Fig. 4 and Table 2). The P/Q-type VSCC transcription level encoding Cav2.1 ({alpha}1A) in vehicle-treated MC3T3-E1 cells was not significantly different from that found in untreated cells (Fig. 4). Application of 1,25(OH)2D3 for 24 h did not alter Cav2.1 ({alpha}1A) mRNA levels when applied at doses of 0.1, 1, or 10 nM (Table 2); however, treatment of MC3T3-E1 cells with a high dose of 1,25(OH)2D3 (100 nM) increased Cav2.1 ({alpha}1A) transcription levels 2.2-fold compared with levels detected in vehicle-treated MC3T3-E1 cells. The expression of N-type VSCC Cav2.2 ({alpha}1B) mRNA showed a similar pattern in transcript downregulation by 1,25(OH)2D3, as did the L-type VSCC Cav1.2 ({alpha}1C) (Fig. 4 and Table 2). Untreated, vehicle-treated, and 0.1 nM 1,25(OH)2D3-treated MC3T3-E1 cells did not express significantly different transcription levels. When the 1,25(OH)2D3 hormone dose was increased to 1 nM, Cav2.2 ({alpha}1B) mRNA expression was decreased to 54% of the levels in vehicle-treated cells. Cav2.2 ({alpha}1B) transcription levels were reduced further in vehicle-treated cells when cultures were treated with 10 nM 1,25(OH)2D3 and to 19% of the vehicle-treated cells when the 1,25(OH)2D3 dose was increased to 100 nM. The levels of RNA encoding the osteoblastic VSCC Cav2.3 {alpha}1E-subunit were low and were not regulated by any 1,25(OH)2D3 treatment (Fig. 4 and Table 2).


Figure 4
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  		Fig. 4. Changes in relative P/Q-, N-, and R-type VSCC {alpha}1-subunit mRNA levels in MC3T3-E1 cells treated with 1,25(OH)2D3. After treatment for 24 h with various doses of 1,25(OH)2D3, RNA was extracted for QPCR analysis. Black bars show increase in transcript levels for Cav2.1 {alpha}1A-subunit in response to treatment with 100 nM 1,25(OH)2D3. Gray bars show downregulation of transcript levels for Cav2.2 ({alpha}1B) after exposure to different concentrations of 1,25(OH)2D3. Exposure to 1,25(OH)2D3 had no significant effect on levels of Cav2.3 {alpha}1E-subunit (open bars). All data were normalized as described in text. Data are means ± SD. ***P < 0.001 and **P < 0.01, 1,25(OH)2D3-treated vs. vehicle-treated cultures.

 

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  		Table 2. Transcript levels of VSCC {alpha}-subunits in MC3T3-E1 cells treated with 1,25(OH)2D3 (10 nM) or vehicle for 24 h

 
On the basis of mRNA expression levels, the most abundant VSCCs in untreated osteoblastic MC3T3-E1 cells were found to be the L-type Cav1.2 {alpha}1C-subunit and the T-type Cav3.1 {alpha}1G-subunit (Table 2). The next highest transcription levels encoded Cav2.2 ({alpha}1B), Cav2.1 ({alpha}1A), Cav1.1 ({alpha}1S), and Cav1.3 ({alpha}1D), followed by Cav2.3 ({alpha}1E), Cav3.3 ({alpha}1I), and Cav3.2 ({alpha}1H) as the least abundant messages. Table 2 lists the amount of each transcript, calculated as attomolar concentration per 5 ng of reverse-transcribed RNA found to be present in vehicle- and 10 nM 1,25(OH)2D3-treated MC3T3-E1 cells. Application of physiological levels (1 nM) of 1,25(OH)2D3 for 24 h decreased mRNA transcription levels of the L-type VSCC Cav1.2 {alpha}1C-, T-type Cav3.1 {alpha}1G-, and the N-type Cav2.2 {alpha}1B- subunits. The only VSCC subunit that significantly increased expression after the addition of 1,25(OH)2D3 was the T-type VSCC Cav3.2 {alpha}1H-subunit. In separate experiments, this increase varied from approximately four- to eightfold. The sum of the transcription levels encoding all three L-type channels was reduced twofold (from 2,887 to 1,427 aM) after 24-h treatment with 10 nM 1,25(OH)2D3.

Immunostaining of VSCC {alpha}1-subunits in MC3T3-E1 cells. Immunostaining of the VSCC {alpha}1-subunits in MC3T3-E1 cells was performed to validate the results of the mRNA studies with regard to protein expression and also to study cellular localization in cells treated for 24 h with 10 or 20 nM 1,25(OH)2D3. Detection of three VSCC {alpha}1-subunits is shown in Fig. 5. Immunostaining of Cav1.2 {alpha}1C-, Cav1.3 {alpha}1D-, and Cav3.2 {alpha}1H-subunits are shown before and after treatment of MC3T3-E1 preosteoblasts with 1,25(OH)2D3. Cav1.3 ({alpha}1D) showed little detectable fluorescent signal in MC3T3-E1 cells, regardless of treatment condition, consistent with the low transcription levels that we had detected earlier (Fig. 5, C and D). Most of the staining that was observed appeared to be intracellular. Similar immunostaining patterns were observed in the osteosarcoma cell line ROS17/2.8 when stained for Cav1.3 ({alpha}1D) in 1,25(OH)2D3- and vehicle-treated cells (data not shown). A similar pattern of diffuse intracellular staining was observed for all of the other poorly expressed {alpha}-subunits found in MC3T3-E1 cells, including Cav2.1, Cav2.2, Cav2.3, and Cav3.1, regardless of whether they had been treated with 1,25(OH)2D3 (data not shown). In contrast, vehicle-treated cells stained for Cav1.2 ({alpha}1C) showed abundant plasma membrane and intracellular immunostaining (Fig. 5A), particularly in regions of the cell in close proximity to the nucleus, presumably the rough endoplasmic reticulum. Treatment for 24 h with 1,25(OH)2D3 decreased immunostaining for Cav1.2 ({alpha}1C) (Fig. 5B). The staining of regions of the cell close to the nucleus persisted but was less intense than that observed in vehicle-treated cells. Most of the staining in the osteoblastic processes had disappeared. MC3T3-E1 cells immunostained with our anti-Cav3.2 ({alpha}1H) antibody produced intense plasma membrane signal (Fig. 5E). The fluorescent signal in the cytoplasm was dramatically increased after 24-h treatment with 1,25(OH)2D3 (Fig. 5F). The increase in Cav3.2 ({alpha}1H) staining was consistent with the increase in mRNA transcription levels that we observed after hormone treatment (Fig. 3 and Table 2). Despite high levels of Cav3.1 ({alpha}1G) transcripts, immunostaining using the commercially available antibody was poor (data not shown).


Figure 5
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  		Fig. 5. Immunohistochemistry of VSCC {alpha}1-subunits in MC3T3-E1 cells in response to 24-h treatment with 1,25(OH)2D3. Cells were fixed in 4% (vol/vol) paraformaldehyde and stained with rabbit anti-{alpha}1 antibodies. Green staining shows {alpha}1-subunits, and cells were counterstained with the nucleic acid dye ToPro-3 (red). A, C, and E: staining of MC3T3-E1 cells with rabbit anti-Cav1.2 ({alpha}1C) (A), anti-Cav1.3 ({alpha}1D) (C), anti-Cav3.2 ({alpha}1H) (E), and FITC-conjugated donkey anti-rabbit IgG as secondary antibody. B, D, and F: 24-h 1,25(OH)2D3-treated MC3T3-E1 cells stained with rabbit anti-Cav1.2 ({alpha}1C) (B), anti-Cav1.3 ({alpha}1D) (D), anti-Cav3.2 ({alpha}1H) (F), and FITC-conjugated donkey anti-rabbit IgG as secondary antibody. G and H: MC3T3-E1 cells incubated with competing peptides for anti-Cav1.2 ({alpha}1C) (G) and anti-Cav3.2 ({alpha}1H) (H) antibodies. All cultures incubated with the competing peptide or normal rabbit serum showed little green staining and comparable levels of nuclear staining. Note the decrease in Cav1.2 ({alpha}1C) and the increase in Cav3.2 ({alpha}1H) immunostaining in cells treated for 24 h with 1,25(OH)2D3.

 
Signal specificity for each antibody was determined by incubating MC3T3-E1 cells with competing peptide and anti-{alpha}1 antibody (Fig. 5). Additional specificity was demonstrated using preimmune antibodies and, in the case of Cav3.2 ({alpha}1H), by demonstrating negative immunostaining in tissues from mice null for this subunit (41).

1,25(OH)2D3 alters the sensitivity of plasma membrane Ca2+ permeability to pharmacological inhibitors. 45Ca2+ influx assays were performed to determine which families of VSCCs contribute to osteoblastic cell plasma membrane Ca2+ permeability and to determine whether 24-h exposure to 1,25(OH)2D3 modulates influx through different VSCC families. MC3T3-E1 cells were pretreated for 15 min with specific VSCC inhibitors, the L-type inhibitor nifedipine (1 µM), the T-type inhibitor sFTX-3.3 (200 nM), the P/Q-type inhibitor {omega}-agatoxin IVA (1 µM), the N-type inhibitor {omega}-conotoxin GVIA (1 µM), or vehicle. The cells then were rinsed in HBSS and immediately placed in either resting buffer or depolarizing buffer containing fresh VSCC inhibitors or vehicle. As shown in Fig. 6A, vehicle-treated cells depolarized with the high-K+ stimulating buffer demonstrated 3.8-fold greater 45Ca2+ influx than cells placed in the nondepolarizing resting buffer. Cells treated with nifedipine placed in stimulating buffer displayed a reduction of 45Ca2+ influx to 37% of vehicle-treated cells in the same buffer, consistent with a major role for this channel previously demonstrated in preosteoblasts (9). Cells treated with inhibitors of T-, P/Q-, and N-type VSCCs had a reduction in 45Ca2+ influx to 47%, 49%, and 67%, respectively.


Figure 6
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  		Fig. 6. 45Ca2+ influx assay of control and 1,25(OH)2D3-treated (24 h) MC3T3-E1 cells treated with VSCC inhibitors. A: growth phase, vehicle-treated MC3T3-E1 cells were incubated for 2 min in either low-K+ resting buffer or high-K+ depolarizing buffer. An ~4-fold increase in influx was observed with depolarization. Pretreatment (15 min) with various channel blockers was performed before addition of high-K+ buffer (see text). y-Axis is normalized to 1.0 as the level of Ca2+ influx in vehicle-treated cells incubated in resting buffer in the absence of VSCC inhibitors. ***P < 0.001, Ca2+ influx in vehicle-treated cells placed in depolarizing buffer vs. vehicle-treated cultures incubated in resting buffer. bulletbulletP < 0.01, Ca2+ influx in MC3T3-E1 cells placed in depolarizing buffer in presence of nifedipine (L-type blocker) vs. vehicle-treated cultures incubated in depolarizing buffer. bulletP < 0.05, Ca2+ influx in MC3T3-E1 cells placed in depolarizing buffer in presence of T-type inhibitor (sFTX-3.3) or P/Q inhibitor ({omega}-agatoxin IVA) vs. vehicle-treated culture incubated in depolarizing buffer. Effect of N-type blocker ({omega}-conotoxin GVIA) was not significant. B: growth phase MC3T3-E1 cells were cultured for 24 h in 20 nM 1,25(OH)2D3 before influx assays were performed as in A. An ~2.5-fold increase in influx was observed with depolarization. Inhibitors and statistical analysis were as described in A, with the exception of bulletbulletbulletP < 0.001, which was observed only for T-type inhibitor.

 
MC3T3-E1 cells next were treated for 24 h with 1,25(OH)2D3 to determine whether hormone treatment affected cell Ca2+ permeability assessed on the basis of sensitivity to class-specific pharmacological inhibition. MC3T3-E1 cells were transferred to serum-free medium for 24 h before being treated with 1,25(OH)2D3 or vehicle. Vehicle-treated cells depolarized with high-K+ buffer demonstrated a 2.4-fold increase in 45Ca2+ influx compared with vehicle-treated cells incubated in nondepolarizing resting buffer (Fig. 6B). Total stimulated influx was reduced (from 3.8- to 2.4-fold) in hormone-treated compared with untreated cells, suggesting a slight reduction in total VSCC function after 1,25(OH)2D3 treatment. This occurs during the same period and under the same conditions in which we and others have shown that expression of well-established osteoblast differentiation markers, including alkaline phosphatase, osteopontin, and osteocalcin, occur. In MC3T3-E1 cells treated for 24 h with 1,25(OH)2D3, blocking the T-type VSCCs reduced 45Ca2+ influx to 24% of the levels of cells that were not exposed to VSCC inhibitors. Inhibition of P/Q- and L-type VSCCs modestly reduced 45Ca2+ influx in depolarized 24-h 1,25(OH)2D3-treated MC3T3-E1 cells, whereas inhibition of N-type VSCCs had an insignificant effect. Thus treatment of MC3T3-E1 cells with 1,25(OH)2D3 for 24 h changes both the pattern of VSCC {alpha}1-subunit transcript expression and the pharmacological sensitivity of the cells to class-specific VSCC inhibitors.


   DISCUSSION
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 ABSTRACT
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1,25(OH)2D3 is a calcitropic hormone that serves as a key regulator of bone remodeling, and it is well accepted that vitamin D status plays a major role in bone homeostasis. Rapid actions of 1,25(OH)2D3 on cells of the osteoblastic lineage include modulation of cAMP (16), phosphatidylinositol 3-kinase (38), activation of Ca2+ channels (9), and, along with parathyroid hormone, elevation in the levels of intracellular Ca2+ (7, 14, 26). In the longer term, 1,25(OH)2D3 also alters rates of gene transcription of target molecules, including markers of osteoblast differentiation (22, 32, 33). This study examined the effect of 24-h 1,25(OH)2D3 treatment on patterns of VSCC expression and function in osteoblasts, with the aim of linking rapid and long-term calcemic actions of 1,25(OH)2D3 into a novel mechanism describing Ca2+ permeability and Ca2+-driven cross talk.

It has been established that depolarization and hormone-stimulated Ca2+ influx into osteoblastic cells is inhibited by L-type VSCC inhibitors, including the dihydropyridines (9), and by a ribozyme specifically targeting the L-type VSCC Cav1.2 ({alpha}1c) (27). The identification of VSCC transcripts that are typically observed in excitable tissues, including neurons (36) and skeletal muscle (44), in osteoblastic cells is not particularly surprising, given that VSCCs have been found in a variety of nonexcitable tissues and cells, including lung (6), kidney (46), pancreas (40), and fibroblasts (42). In this systematic study of 10 VSCC types, we have shown that although three of the four known L-type VSCCs, Cav1.1 ({alpha}1S), Cav1.2 ({alpha}1C), and Cav1.3 ({alpha}1D), were detected using QPCR in preosteoblastic MC3T3-E1 cells, only Cav1.2 ({alpha}1C) was expressed at significant levels. In a previous study, it was shown that application of 1,25(OH)2D3 for a duration of seconds to minutes increased the mean open time and Ca2+ permeability of the L-type VSCC in the short term (9, 45). In primary osteoblast cultures, 1,25(OH)2D3 treatment produces an increase in Ca2+ entry that, along with subsequent release of Ca2+ from intracellular stores (14, 26), elevates cytoplasmic Ca2+ levels. This phenomenon supports signal propagation and stimulates Ca2+-dependent signaling pathways. In addition, Ca2+ influx through L-type VSCCs in response to membrane depolarization activates the cAMP- and Ca2+-dependent transcription factor CREB (2). This method of transcriptional activation is most efficient when the Ca2+ signal is generated through the L-type VSCC rather than by other methods of Ca2+ entry (10). It is thus of critical importance to the function of a cell to modulate both the type and the density of VSCCs expressed on the cell surface.

Using an analog of 1,25(OH)2D3 that binds the nuclear vitamin D receptor and does not elicit a plasma membrane-initiated response, we previously demonstrated that the downregulation of the Cav1.2 {alpha}1C-subunit involves transcriptional changes that require the presence of the nVDR (30). Consistent with previous studies conducted at our laboratory in which another cell line was used (30), we found in the present study that long-term exposure of MC3T3-E1 cells to nanomolar concentrations of 1,25(OH)2D3 decreased Cav1.2 ({alpha}1C) mRNA transcription and protein levels. Because mRNA levels for the two other L-type VSCCs detected in osteoblasts, Cav1.3 ({alpha}1D) and Cav1.1 ({alpha}1S), remain unchanged, it is likely that the Cav1.2 {alpha}1C-subunit is the only pore-forming subunit in the L-type VSCC family whose transcriptional expression is modulated by 1,25(OH)2D3 treatment. Radioactive Ca2+ influx assays revealed that prolonged exposure to 1,25(OH)2D3 decreased Ca2+ entry through the L-type VSCC, presumably because of decreased expression of the Cav1.2 {alpha}1C-subunit. A potential role for downregulation of the Cav1.2 {alpha}1C-subunit in response to long-term exposure to 1,25(OH)2D3 is to protect the cell from chronic elevation in intracellular Ca2+ levels that could lead to cell apoptosis. To that end, it has been demonstrated that neuronal vulnerability to excitotoxicity in hippocampal neurons is mediated by Ca2+ influx through the L-type VSCC and that downregulation of these channels with long-term exposure to 1,25(OH)2D3 increases neuroprotection (8). Together, these results suggest that the initial 1,25(OH)2D3 exposure elicits a rapid cellular response, including the activation of various protein kinases, protein lipases, and cAMP, by increasing the short-term ability of Ca2+ to enter the osteoblast through the Cav1.2 {alpha}1C-subunit of the L-type VSCC. Long-term exposure to the steroid subsequently downregulates the Cav1.2 {alpha}1C-subunit in a nuclear receptor-dependent pathway that then diminishes Ca2+ influx, preventing Ca2+ toxicity.

Although the L-type VSCCs are the best studied Ca2+ channel type and the most highly expressed VSCC in osteoblasts, it has been reported that T-type VSCCs are expressed in osteoblasts during development (13, 29, 41). T-type channels are expressed throughout the body, including in various parts of the nervous system, heart, kidney, smooth muscle, and sperm. These channels have been implicated in a range of physiological events, including cardiac pacemaker activity, neuronal firing, smooth muscle contraction, fertilization, and hormone secretion (34). Electrophysiological properties of T-type currents are similar in many tissues, but differences in inactivation kinetics and pharmacology have been identified and are due to the existence of three distinct T-type VSCCs (15, 24). We report that all three of the T-type VSCC isoforms, Cav3.1 ({alpha}1G), Cav3.2 ({alpha}1H), and Cav3.3 ({alpha}1I), are detectable by performing QPCR in the preosteoblast, but that Cav3.1 ({alpha}1G) is the major transcript (Table 2). At the protein level, Cav3.2 ({alpha}1H) was prominent at the plasma membrane (Fig. 5), unlike Cav3.1 ({alpha}1G), which appeared to be largely intracellular (data not shown). Upon application of 1,25(OH)2D3, Cav3.1 ({alpha}1G) transcription levels are reduced by 24 h, whereas the expression of Cav3.2 ({alpha}1H) increases almost threefold during the same period. The levels of Cav3.3 ({alpha}1I) transcription remain low and unchanged. Immunostaining for Cav3.2 ({alpha}1H) revealed a significant increase in the amount of Cav3.2 ({alpha}1H) protein in the osteoblastic cells after 24-h treatment with 1,25(OH)2D3. Little staining of Cav3.1 ({alpha}1G) protein was observed under any conditions, indicating that the levels of transcription vastly exceeded the amount of protein for this channel class in MC3T3-E1 cells (data not shown), which may be due to a failure to be translated or to assemble. Long-term treatment with 1,25(OH)2D3 concomitantly produces a shift in the proportion of Ca2+ influx into the cells that occurs through the class of T-type VSCCs assessed by susceptibility to pharmacological class-specific blockers. Together, these data suggest that the increases in the Cav3.2 {alpha}1H-subunit transcription and protein expression also increase T-type plasma membrane VSCC activity.

Much of the current understanding of the physiological roles of the T-type currents comes from work performed in neuronal (28) and cardiac cells (3), in which the T-type VSCCs generate low-threshold Ca2+ spikes. These Ca2+ spikes are associated with burst firing and oscillatory behavior (23), which produce pacemaker currents and rapid neuronal depolarization. Mice null for the T-type VSCC Cav3.2 ({alpha}1H) are viable but show defects in the relaxation of coronary arterioles and focal coronary fibrosis as well as skeletal defects (Ref. 12 and Shao Y, Chen CC, Campbell K, and Farach-Carson MC, unpublished data). The role of the T-type VSCC in the osteoblast has not been studied extensively, although it has been known for some time that MC3T3-E1 cells have T-type currents (1). After treatment with ATP, osteocytic MLO-Y4 cells express T-type currents and Cav3.1 ({alpha}1G) transcripts, but Cav3.2 ({alpha}1H) levels have not been investigated (21). We recently reported that Cav3.2 {alpha}1H-subunits were present in, but Cav1.2 {alpha}1C-subunits were absent from, osteocytes in long bone (41). A potential reason for the upregulation of the T-type VSCC Cav3.2 {alpha}1H-subunit after treatment with 1,25(OH)2D3 is to compensate for the loss of the L-type VSCC-mediated Ca2+ influx. With the decrease in Cav1.2 ({alpha}1C) levels, the ability of the cell to maintain Ca2+-dependent cellular processes would be compromised if a parallel increase in another {alpha}1-subunit did not occur. For the terminally differentiated osteocyte, loss of Cav1.2 ({alpha}1C) without upregulation of another subunit would render the cell unable to respond to Ca2+-mobilizing stimuli, including mechanical load and shear stress.

N-, P/Q-, and R-type VSCCs typically are found in neuronal cells and are key regulators of neurotransmitter release (11). In osteoblasts, mRNA encoding for Cav2.1 ({alpha}1A), Cav2.2 ({alpha}1B), and Cav2.3 ({alpha}1E) all were detected at low levels. However, application of 1,25(OH)2D3 at physiologically relevant concentrations did not affect mRNA or protein levels for these subunits, except for Cav2.2 ({alpha}1B). The downregulation of Cav2.2 ({alpha}1B) mRNA observed after 24-h exposure to the steroid did not result in a significant change in the diffuse immunofluorescent staining pattern for the protein (data not shown). Ca2+ influx experiments showed that the N-type VSCC contribution to plasma membrane Ca2+ permeability was not affected by 1,25(OH)2D3 treatment. Together, these data suggest that the P/Q-, N-, and R-type VSCCs are not major contributors to the modulation of osteoblast Ca2+ permeability after 1,25(OH)2D3 treatment.

This study was undertaken to identify the role of VSCCs in regulating Ca2+ permeability in osteoblastic cells under various conditions of vitamin D hormone status. To identify the potential role of the VSCC in Ca2+ permeability, we first had to systematically determine which VSCC {alpha}1-subunits are present under various conditions. Although researchers at several laboratories, including our own, have examined the ability of 1,25(OH)2D3 to modulate VSCC activity, a complete explanation of the link between 1,25(OH)2D3 treatment, total VSCC expression, and Ca2+ permeability in the osteoblast has never been reported. A novel aspect of the quantitative approach presented herein is that it allowed us to predict the total potential contribution of L-type and T-type Ca2+ channels to overall Ca2+ permeability under both 1,25(OH)2D3 replete and depleted conditions. The L-type VSCC has a conductance of ~25 pS, whereas the T-type VSCC has a conductance of ~8 pS; thus most Ca2+ entry is due to the amount of functional L-type channel present. The expression levels thus predict that in the absence of vitamin D hormone, cells have approximately twice the L-type current potential of 1,25(OH)2D3-treated cells. This prediction was borne out in the responses to high K+ concentration in vehicle- and 1,25(OH)2D3-treated cells as shown in Fig. 6. In hormone-treated cells, expression levels of total L-type and T-type VSCC transcripts were reduced to one-half and one-third, respectively (Table 2). However, the increase in the T-type channel Cav3.2 ({alpha}1H) expression after hormone treatment was dramatic, especially at the protein level, and likely contributed to the shift to a T-type sensitivity shown in Fig. 6B. In a physiological context, we would suggest that the high Ca2+ permeability of the preosteoblast supports cell proliferation and growth, whereas the loss of Ca2+ permeability during 1,25(OH)2D3-stimulated differentiation is likely to turn off Ca2+-dependent proliferation signals and associated gene expression. Such modulation may occur in parallel with the loss of L-type Cav1.2 ({alpha}1c) expression that we recently reported in terminally differentiated osteocytes in intact bone (41).


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Dental and Craniofacial Research Grant DE-12641 (to M. C. Farach-Carson) and National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant P01 AR-45218 (to R. L. Duncan).


   ACKNOWLEDGMENTS
 
We thank Dr. Kamil Akanbi (formerly of the University of Delaware, Newark, DE) for many helpful discussions and Dr. J. Gary Meszaros (Northeastern Ohio Universities College of Medicine, Rootstown, OH) for suggestions regarding the necessity of this work in the discussion of his doctoral thesis. We acknowledge a former graduate student, Yihuan (Catherine) Xu, for assistance with data analysis. We thank Sharron Kingston for assistance with preparation of the manuscript.

Present address of J. J. Bergh: Ordway Research Institute, Albany, NY 12208.

Present address of R. L. Duncan: Department of Biological Sciences, University of Delaware, Newark, DE 19716.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. C. Farach-Carson, Dept. of Biological Sciences, Univ. of Delaware, 326 Wolf Hall, Newark, DE 19716 (e-mail: farachca{at}udel.edu)

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.


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