| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Department of Pharmacology and Toxicology and Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555
Address all correspondence and requests for reprints to: Miriam Falzon, Ph.D., Department of Pharmacology and Toxicology, 10th and Market Streets, University of Texas Medical Branch, Galveston, Texas 77555-1031. E-mail: mfalzon{at}utmb.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
PTHrP was originally purified from a human cancer cell line (BEN) isolated from a patient with the HHM syndrome (6). In fact, HHM is a very common complication in patients with squamous cell carcinomata of the lung and is a major contributor to the morbidity of such patients (7, 8, 9, 10). Cell lines derived from squamous cell carcinomas of the lung secrete high levels of PTHrP; one such example is the human cell line, NCI H520 (11). As the molecular mechanism(s) through which the PTHrP gene is up-regulated in certain malignancies has not yet been identified, there has been no effective approach to control increased production of PTHrP in cancer cells.
PTHrP gene transcription in various cell types is repressed by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], the hormonally active form of vitamin D3 (11, 12, 13, 14). The hypercalcemic activity of 1,25-(OH)2D3 has prevented its clinical application, so various vitamin D3 analogs with preferential effects on cellular differentiation and proliferation but little or no calcemic activity have been synthesized. Although these compounds are not absolutely noncalcemic, for simplicity they are referred to as nonhypercalcemic in the text because their effects on calcium levels are much less pronounced than those of 1,25-(OH)2D3. Two of these compounds, EB1089 and 22-oxacalcitriol (OCT), were used in the present study. OCT has an oxygen atom at the C22 position of the side-chain skeleton (15). The side chain in EB1089 has been elongated by introduction of terminal ethyl groups, and double bonds have been introduced at positions 22 and 24 (16). We and others have shown that EB1089 and OCT decrease PTHrP messenger RNA (mRNA) and secreted peptide levels through a transcriptional mechanism in NCI H520 cells (11) and other cells lines (15, 16, 17, 18, 19, 20, 21, 22). Here, we extend these findings to address a potential role for PTHrP as an autocrine regulator of NCI H520 cell proliferation.
PTHrP gene expression is positively regulated at both the transcriptional and posttranscriptional level by serum-derived growth factors (23, 24, 25, 26, 27). Cancer cells produce a number of growth factors. It is likely that these growth factors play an important role in increasing PTHrP production by these cells, resulting in the development of hypercalcemia. The present study was undertaken to evaluate whether 1,25-(OH)2D3 and the two nonhypercalcemic analogs reverse the stimulatory effects of the serum-derived growth factors on PTHrP mRNA levels and peptide secretion.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Plasmids and oligonucleotides
The DNA fragment used as a probe to detect PTHrP by Northern
blot analysis was generated by RT-PCR using two oligonucleotides
(upstream, 5'-CTGGTTCAGCAGTGGAGCGTC-3'; downstream,
5'-GTTAGGGGACACCTCCGAGGT-3') spanning exons 3 and 4 of the human PTHrP
gene. Complementary DNA obtained by RT of total RNA from NCI H520 cells
was used as the template for PCR. The resulting 231-bp fragment was
cloned into the vector pCR II (Invitrogen, San Diego, CA), and its
identity was verified by dideoxy sequencing. The recombinant plasmid
was cut with EcoRI. For Northern blot analysis, the isolated
fragment was labeled by asymmetric PCR (28), using the downstream
primer and [
-32P]deoxy-CTP (6000 Ci/mmol; Amersham,
Arlington Heights, IL). A DNA fragment containing cyclophilin sequences
(29) was labeled by the random primer extension reaction with a
multiprime labeling kit (Amersham) and [-32P]deoxy-CTP
(6000 Ci/mmol) and was used to control for equal RNA loading and
transfer.
In nuclear run-on experiments to determine promoter usage, the following promoter-specific probes were used: for promoter 1 usage, an XbaI fragment extending approximately 5.7 kb upstream of exon 1a to the XbaI site situated within the intron between exons 1b and 1c (nomenclature as in Ref.30), cloned in pUC18 (Life Technologies, Gaithersburg, MD); for promoter 2 usage, a BamHI/AccI fragment (extending from the BamHI site located within the intron separating exons 1b and 1c to the AccI site located just downstream of exon 1c) cloned in pCAT basic (Promega, Madison, WI); and for promoter 3 usage, a fragment generated by PCR, including the last 25 bp upstream of exon 1c and extending to 42 bp within exon 2, cloned in pCAT basic.
Cell culture
To determine promoter utilization by nuclear run-on analysis,
cells were grown to 
80% confluence in 10% FBS. The vitamin D
analogs 1,25-(OH)2D3 (obtained from Dr. Milan
Uskokovic, Hoffmann La Roche, Nutley, NJ), EB1089 (from Dr. Lise
Binderup, Leo Pharmaceuticals, Ballerup, Denmark), and OCT (from Dr.
Noboru Kubodera, Chugai Pharmaceutical Co., Tokyo, Japan) were then
added at the indicated concentrations. Ethanol was used as the vehicle
control. In experiments in which the inductive effects of serum or EGF
were studied, cells were plated in 10% FBS. After 12 h to allow
for the cells to attach, they were transferred to medium containing
0.5% FBS. At approximately 80% confluence (after 48–72 h in 0.5%
FBS), the cells were exposed to various concentrations of either FBS
(range, 2–20%, vol/vol) or EGF (1–100 ng/ml; Clonetics, San Diego,
CA) for the indicated time intervals. Control cells were kept in 0.5%
FBS. In experiments in which the combined effects of FBS or EGF and
vitamin D3 analog were studied, cells were plated in 10%
FBS. After 12 h to allow the cells to attach, they were
transferred to 0.5% FBS. When the cells had reached approximately 80%
confluence, they were treated with the vitamin D analogs at
10-7 M; after 24 h, they were treated
with 20% FBS or 50 ng/ml EGF for 2 h (for Northern blot analysis
or nuclear run-on assays) or for 24 h (for the radioimmunometric
assay to measure secreted PTHrP levels).
Northern blot analysis
Total RNA was isolated at the indicated time points using RNA
STAT-60 (Tel-Test "B", Friendswood, TX). RNA gel electrophoresis
was performed under standard conditions (31), using 15 µg RNA. The
RNA was then blotted onto nitrocellulose (Schleicher and Schuell,
Keene, NH) by capillary action and fixed by baking at 80 C under vacuum
for 2 h. Prehybridization and hybridization were carried out as
previously described (12). The blots were then washed twice in 2
x SSC (1 x SSC is 0.15 M NaCl plus 0.15
M sodium citrate)-0.1% SDS for 15 min at room temperature,
then in 0.2 x SSC-0.1% SDS at 60 C for 45 min. The washed
membranes were exposed to Kodak X-Omat film (Eastman Kodak, Rochester,
NY) at -70 C with intensifying screens. After autoradiography, the
intensities of the bands representing PTHrP and cyclophilin were
evaluated using the Sigmagel program (Jandel Scientific, San Rafael,
CA). Quantitation was always performed using signal intensities within
the linear range of film sensitivity, as evaluated by scanning an
autoradiograph with multiple signal intensities (multiple sample
dilutions) and ascertaining that the experimental signals were always
within the linear range. This allowed calculation of the ratio of the
PTHrP/cyclophilin band intensities for each individual treatment.
Nuclear run-on assays
For nuclear run-on assays of PTHrP gene transcription, cells
were treated with FBS and/or vitamin D3 analog as described
above. The cells were then harvested, and nuclei were prepared as
previously described (11, 12, 32, 33). The following DNAs were used as
membrane-attached probes: the promoter-specific probes described above,
a rat cyclophilin probe (29), and, as a negative control, pCAT basic
DNA linearized with EcoRI. Promoter-specific probes were
linearized as follows: promoter 1-specific probe with SalI,
and promoter 2- and 3-specific probes with XhoI. Each probe
DNA (4 µg) was applied to a nitrocellulose membrane (Schleicher and
Schuell) under vacuum and fixed by baking at 80 C under vacuum for
2 h. After hybridization for 40–48 h, the membranes were washed
for 1 h at 65 C in 2 x SSC, for 30 min at 37 C in 2 x
SSC containing 10 µg/ml ribonuclease A, and for 1 h at 37 C in
2 x SSC, then exposed to film at -70 C with intensifying screens
for 3–7 days. The intensities of the bands were quantitated using
Sigmagel (Jandel Scientific) as described above, and the ratio of the
PTHrP/cyclophilin band intensities was calculated for each individual
treatment.
Immunoassay for secreted PTHrP
The amount of PTHrP secreted into the culture medium was
measured using an immunoradiometric sandwich assay (Nichols Institute,
San Juan Capistrano, CA) employing two affinity-purified antisera to
human PTHrP. One antiserum, labeled with 125I, recognizes
amino acid residues 1–40, whereas the second antiserum, labeled with
biotin, recognizes residues 60–72. This kit provides a standard, human
PTHrP-(1–86), as a positive control. The detection limit of this kit
is 0.7 pmol/liter (34). The NCI H520 cells were grown to approximately
80% confluence and then treated with the indicated concentrations of
FBS, EGF, 1,25-(OH)2D3, EB1089, or OCT or
combinations of vitamin D3 compounds and FBS or EGF, as
specified in the text. After 24 h (or 48 h for combination
treatments), the conditioned medium was collected and frozen at -80 C
for future use, and the cell number was determined using a Coulter
counter (Hialeah, FL). Before assay, aliquots of the conditioned medium
(range, 0.4–1 ml, calculated to represent the same number of cells)
were concentrated to 0.2 ml using acetone precipitation. Unconditioned
medium (never exposed to cells) similarly concentrated served as the
negative control. The assay was carried out according to the
manufacturer’s specifications.
Cell proliferation studies
For experiments to measure the effects of
1,25-(OH)2D3 and analogs on cell proliferation,
NCI H520 cells were plated at a density of 5 x 105
cells in six-well dishes in RPMI 1640 containing 10% Nu-serum. (The
vitamin D3 compounds produced the largest effects on cell
growth when cells where grown in Nu-serum as opposed to 0.5% or 10%
FBS). After 12 h to allow the cells to attach, they were treated
with the various vitamin D3 compounds
(10-7–10-10 M) for periods
ranging from 24–72 h, as specified in the text. The cells were then
treated with trypsin. After the trypsin was inactivated with medium
containing 10% FBS, cell number was determined with a Coulter
counter.
Thymidine incorporation assay
For these experiments, NCI H520 cells were plated in 48-well
plates in RPMI 1640 medium supplemented with 10% Nu-serum. Cells were
allowed to attach for 12 h. They were then treated with various
concentrations of 1,25-(OH)2D3, EB1089, or OCT
for 24 h. After 12 h of treatment, 0.5 µCi/well
[3H]thymidine (DuPont-New England Nuclear, Boston, MA)
was added, and treatment was continued for an additional 12 h. To
determine [3H]thymidine incorporation, the cell monolayer
was washed twice with PBS, and a fraction of the cells were counted
with a Coulter counter. The nucleic acids in the rest of the cell
fraction were precipitated with trichloroacetic acid and solubilized
with sodium hydroxide for scintillation counting (35). The results are
expressed as counts per number of cells.
Statistics
Numerical data are presented as the mean ±
SEM. Data were analyzed by ANOVA with t tests to
determine the statistical significance of differences.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). The PTHrP mRNA levels peaked at
2 h (4-fold increase) and had started to decline by 4 h. This
increase in PTHrP mRNA levels was concentration dependent at 2 h
and was already evident with 2% FBS. Higher serum concentrations
produced proportionately larger increases in PTHrP mRNA levels (Fig. 1B). The maximum serum effect was not determined, because a plateau was
not demonstrated with 20% FBS, the highest concentration used (Fig. 1B).
|
). When NCI H520 cells
maintained in 0.5% FBS for 48 h were treated with EGF (50 ng/ml),
there was an increase in PTHrP mRNA levels within 30 min. Peak
induction occurred at 2 h, and levels declined back to the control
value by 4 h. This increase in PTHrP mRNA levels was also
concentration dependent at 2 h and reached a peak with 50 ng/ml
EGF (Fig. 2B). There did not seem to be a further increase with 100
ng/ml EGF, indicating that the effect reached a plateau with the 50
ng/ml concentration.
|
). When maintained in 0.5% FBS, NCI
H520 cells produced 1.2 pmol PTHrP/liter·24 h. These levels rose to a
maximum of 5.1 and 4.2 pmol/liter after treatment for 24 h with
20% FBS or 50 ng/ml EGF, respectively (the highest concentrations
tested; Fig. 3). A minimum of 5% FBS or 5 ng/ml EGF was required to
produce an increase in secreted PTHrP levels (Fig. 3). Induction times
shorter than 24 h could not be measured, because there was not
enough accumulation of PTHrP in the culture medium.
|
). Transcription from promoter 2 was
much weaker, whereas transcription from promoter 3 was not detected,
even after prolonged exposure of the autoradiographs. We also
determined the effect of serum and EGF on transcription from each of
the three promoters. For these experiments, nuclei were prepared from
NCI H520 cells maintained in 0.5% serum for 72 h and then treated
with either 20% FBS or 50 ng/ml EGF for 2 h. Control cells were
kept in 0.5% FBS. The data in Fig. 4 show that both serum and FBS
exerted their effects through sequences within promoter 1; the
transcription rate from this promoter was increased approximately
4-fold in the presence of serum and 3-fold in the presence of EGF (Fig. 4). There was no change in the transcription rate from promoters 2 and
3 in the presence of serum or EGF (Fig. 4).
|
, the effects of these compounds
on PTHrP gene transcription were also mediated via promoter 1. There
was an approximately 3-fold decrease in the transcription rate from
this promoter after treatment with each of the three vitamin
D3 compounds, a value that matches the decreased mRNA
levels observed by Northern blot analysis (11). These data suggest that
the negative effects of the three vitamin D3 compounds on
steady state PTHrP mRNA levels are mediated primarily at the
transcriptional level, via promoter 1. The promoter usage of cells
grown in 10% FBS was identical to that in cells grown in 0.5% FBS
(shown in Fig. 4
).
|
, A and B,
shows that this is indeed the case, in that after cotreatment with 20%
FBS (Fig. 6A) or 50 ng/ml EGF (Fig. 6B) and OCT (10-7
M), PTHrP mRNA levels were significantly lower than those
in cells treated with FBS or EGF alone. OCT was more effective in
preventing the EGF-induced than the FBS-induced up-regulation,
presumably because serum is a complex mixture of factors working
through different mechanisms, not all of which may interact with the
OCT pathway. OCT also partially prevented the serum (Fig. 7)- and EGF (data not shown)-induced
increase in the PTHrP run-on transcription rate mediated via sequences
within promoter 1. The transcription rates from promoters 2 and 3 were
unchanged (Fig. 7). Similar effects were produced when
1,25-(OH)2D3 or EB1089 was used in conjunction
with serum or EGF (data not shown). We also measured the effect of the
serum or EGF/vitamin D3 combination on PTHrP secretion. As
shown in Fig. 8, all three vitamin
D3 compounds produced a significant decrease in the serum-
and EGF-stimulated secretion of peptide into the medium.
|
|
|
, at 10-8 M,
all three compounds inhibited cell growth in a time-dependent manner.
OCT produced the greatest effect, reducing cell number to approximately
60% that of the control (vehicle-treated) after 72 h. EB1089 and
1,25-(OH)2D3 reduced the growth by 30% and
25%, respectively, at the same time point. A smaller effect on cell
growth was evident at 24 and 48 h.
|
). The effect of OCT was again larger than that of the other two
compounds, especially at the lower concentration range
(10-10 and 10-9 M).
25-Hydroxyvitamin D3 was used as a negative control. This
compound had no effect on NCI H520 cell proliferation at the
concentrations tested (Fig. 9B). The three compounds also decreased [3H]thymidine incorporation. Thus, a 24-h exposure to 10-7 M OCT, EB1089, and 1,25-(OH)2D3 decreased [3H]thymidine incorporation by 24%, 18%, and 15%, respectively (data not shown). Lower concentrations (down to 10-10 M) produced progressively smaller effects (data not shown).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
One such growth factor is EGF, a component of serum (37, 38). Squamous cell lung carcinomas express the EGF receptor (39, 40, 41, 42), and these cells are induced to proliferate in response to EGF (41, 42). In fact, anti-EGF receptor monoclonal antibodies are being investigated as potential anticancer agents in patients with squamous cell lung carcinomas (39, 40, 41, 42). It has been shown that EGF induces PTHrP mRNA levels in rat osteosarcoma (ROS 17/2.8) (23) cells and in an immortalized human keratinocyte cell line (27). EGF also induces c-fos gene expression; the primary mediator of c-fos responsiveness to EGF in HeLa cells is the serum response element (43). However, EGF also induces a second pathway that involves rapid activation of latent cytoplasmic transcription factors called STATs (signal transducers and activators of transcription) (44, 45). Six distinct mammalian STAT family members have been cloned to date. At least two of these, STAT1 and STAT3, are activated by EGF (45). Once phosphorylated, STATs dimerize and bind to a number of DNA elements, for example, the c-sis-inducible element (46, 47), resulting in activated gene transcription. EGF-dependent activation of PTHrP gene expression in NCI H520 cells may involve one of these pathways.
Lung cancer patients have a very poor prognosis. It is therefore important to develop a therapeutic agent with antiproliferative effects as well as the potential to control PTHrP gene transcription, but which itself has minimal side-effects.
As these cancer cells produce many growth factors, such a therapeutic agent must suppress not only basal production of PTHrP, but also secretion stimulated by these growth factors. 1,25-(OH)2D3 fits these criteria. This compound is a potent antiproliferative agent and induces differentiation in several systems (48, 49). Here, we have shown that 1,25-(OH)2D3 both suppresses proliferation and down-regulates PTHrP gene transcription in NCI H520. Similar results have been found with other cells, including ROS 17/2.8 (rat osteosarcoma) cells (12), human TT cells (a C cell line derived from a medullary thyroid carcinoma) (14), MT-2 cells (a cell line derived from human T cell leukemia virus I-infected T cells) (16), and cultured normal keratinocytes (13). These observations in conjunction with the fact that VDR is widely expressed not only in the classic target organs involved in calcium homeostasis but also in a number of cancer cells, including NCI H520 cells (11, 50, 51), suggest that 1,25-(OH)2D3 might be a useful agent in the treatment of cancer.
However, as high doses of 1,25-(OH)2D3 cause hypercalcemia, it cannot be used therapeutically. Structural changes in the 1,25-(OH)2D3 molecule have yielded analogs that retain the beneficial effects of 1,25-(OH)2D3 but lack a hypercalcemic ability. We have previously reported that two such analogs, OCT and EB1089, suppress basal PTHrP gene transcription and secreted peptide levels in NCI H520 cells (11). Here we have shown that both analogs have the potential to suppress cell proliferation as well as serum- and EGF-induced PTHrP transcription and secretion. OCT has also been shown to decrease the interleukin-2- and cAMP-induced stimulated secretion of PTHrP in MT-2 cells, which are derived from a human T cell lymphotropic virus type I-infected T cell line (16). These properties make nonhypercalcemic vitamin D3 analogs of potential value therapeutically in squamous cell carcinoma of the lung and other carcinomas by suppressing PTHrP secretion and the accompanying hypercalcemia as well as cell proliferation.
The human PTHrP gene is a highly complex transcriptional unit, with nine exons spanning more than 15 kb of genomic DNA (30). The gene uses at least three different promoters, located upstream of exons 1a, 1c, and 2. Promoters 1 and 3 are TATA containing, whereas promoter 2 is GC rich (Ref. 30 and references quoted therein). We have shown here by nuclear run-on assays that PTHrP gene transcription in NCI H520 cells is mediated primarily by sequences within the upstream promoter 1 under both basal and serum- or EGF-stimulated conditions. Down-regulation by 1,25-(OH)2D3, EB1089, and OCT is also mediated via promoter 1, suggesting that this promoter region contains a negative vitamin D response element (nVDRE).
The human PTH gene is also down-regulated by 1,25-(OH)2D3. Studies of the 5'-flanking sequence of the human PTH gene have identified a nVDRE on the sense strand (sequence AAACTTGGATATC), which includes a half-site of the consensus VDRE sequence (underlined). This sequence interacts with the VDR and confers negative 1,25-(OH)2D3 responsiveness on the PTH gene, as has been shown in transient transfection and electrophoretic mobility shift assays (52, 53). A sequence with significant homology (11 of 13 bp) to this nVDRE (sequence CTATAGATTCATA) is located on the antisense strand approximately -540 bp upstream of exon 1a, that is within promoter 1, in the human PTHrP gene. We are currently investigating whether this sequence does confer negative 1,25-(OH)2D3 responsiveness on the human PTHrP gene in NCI H520 cells.
In conclusion, our present results indicate that both basal and serum- and EGF-induced PTHrP gene transcription and peptide secretion are down-regulated by 1,25-(OH)2D3 and two nonhypercalcemic analogs, EB1089 and OCT, in NCI H520 cells. These compounds also suppress proliferation of this cell line. Therefore, these compounds may provide a new strategy to treat squamous cell carcinoma of the lung as well as other malignancies, both because of their potent antiproliferative effects and their ability to prevent PTHrP-induced HHM.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received August 4, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, 25-dihydroxyvitamin
D3. Proc Natl Acad Sci USA 78:4990–4994This article has been cited by other articles:
 |
|
 |
|
  J. L. Gilmore, R. M. Gonterman, K. Menon, G. Lorch, D. J. Riese II, A. Robling, and J. Foley Reconstitution of Amphiregulin-Epidermal Growth Factor Receptor Signaling in Lung Squamous Cell Carcinomas Activates PTHrP Gene Expression and Contributes to Cancer-Mediated Diseases of the Bone Mol. Cancer Res., October 1, 2009; 7(10): 1714 - 1728. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. F. McCarty and K. I. Block Toward a Core Nutraceutical Program for Cancer Management Integr Cancer Ther, June 1, 2006; 5(2): 150 - 171. [Abstract] [PDF]
|
|
|
|
 |
|
 K. Nakagawa, Y. Sasaki, S. Kato, N. Kubodera, and T. Okano 22-Oxa-1{alpha},25-dihydroxyvitamin D3 inhibits metastasis and angiogenesis in lung cancer Carcinogenesis, June 1, 2005; 26(6): 1044 - 1054. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Nakagawa, A. Kawaura, S. Kato, E. Takeda, and T. Okano 1{alpha},25-Dihydroxyvitamin D3 is a preventive factor in the metastasis of lung cancer Carcinogenesis, February 1, 2005; 26(2): 429 - 440. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. A. Tovar Sepulveda, X. Shen, and M. Falzon Intracrine PTHrP Protects against Serum Starvation-Induced Apoptosis and Regulates the Cell Cycle in MCF-7 Breast Cancer Cells Endocrinology, February 1, 2002; 143(2): 596 - 606. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Falzon and P. Du Enhanced Growth of MCF-7 Breast Cancer Cells Overexpressing Parathyroid Hormone-Related Peptide Endocrinology, May 1, 2000; 141(5): 1882 - 1892. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
0.05.
, Untreated (A) or
25-hydroxyvitamin D3 (B); 
,
1,25-(OH)2D3; 
, EB1089; •, OCT.