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Vitamin D analogs modulate the action of gonadal steroids in human vascular cells in vitro
AJH
2000;13:396 – 403
Vitamin D Analogs Modulate the Action of Gonadal Steroids in Human Vascular Cells In Vitro D. Somjen, F. Kohen, Y. Amir-Zaltsman, E. Knoll, and N. Stern
We have previously reported that estradiol (E2) and dihydrotestosterone (DHT) regulate cell growth in human umbilical arterial smooth muscle cells (SMC) and in an endothelial cell line (E304). In SMC both gonadal steroids stimulated DNA synthesis at low concentrations and suppressed 3 [H] thymidine incorporation at high concentrations, whereas in E304 cells E2 and DHT dose dependently enhanced DNA synthesis. In both cell types gonadal steroids also induced the specific activity of creatine kinase BB (CK). Previous evidence suggets that the in vitro and in vivo CK responses to gonadal steroids in bone cells are upregulated by pretreatment with vitamin D analogs due to increased level of cellular estrogen receptors (ER). Here we analyzed the interaction of the vitamin D analogs hexafluorovitamin D (FL), JK-1624 F2-2 (JKF), and CB 1093 (CB) with gonadal steroids in regulating DNA synthesis and CK activity in human vascular cells in vitro. In E304 cells, daily treatment with FL, JKF, or CB (1 nmol/L for 3 days) increased DNA synthesis by 110 ⴞ 11%, 65 ⴞ 16%, and 88 ⴞ 23% respectively. In contrast, the same analogs inhibited 3[H] thymidine incorporation by 52 ⴞ 21%, 46 ⴞ 19%, and 50 ⴞ 10%, respectively, in SMC. In both cell types all three analogs increased CK by 25% to 75% and amplified the CK response
to E2 and to DHT by twofold to threefold. In E304 cells the vitamin D analogs also increased DNA response to gonadal steroids from 50% to 60% to 200% to 280%. In SMC these analogs did not modify the DNA synthetic response to a low E2 concentration, but prevented the suppression of DNA synthesis exerted by high concentrations of E2 and DHT. Vitamin D inhibitors known to block cellular calcium mobilization, had no effect on the proliferative activity induced by vitamin D analogs. However, the inhibitor of the nuclear effects of vitamin D, ZK 159222, blocked the stimulatory effects of CB on DNA synthesis in E304 cells. Finally, both 1,25(OH)2 D3, and JKF decreased the expression of ER proteins in SMC and increased the ER␣ isoform in E304 cells by 40% to 75%. The results indicate that vascular cells are targets for both vitamin D and gonadal steroid action and suggest a possible interaction between these hormones in the regulation of cell proliferation via modulation of vascular ER or interaction with proteins associated with ER. Am J Hypertens 2000;13:396 – 403 © 2000 American Journal of Hypertension, Ltd.
Received May 4, 1999. Accepted August 3, 1999. From the Institute of Endocrinology Metabolism and Hypertension, Tel Aviv Sourasky Medical Center and The Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv; and Department of Biological Regulation, The Weizman Institute of Science, Rehovot, Israel.
Address correspondence and reprint requests to N. Stern, MD, Institute of Endocrinology, Metabolism and Hypertension, Tel Aviv-Sourasky Medical Center, 6 Weizman Street, Tel Aviv 64239, Israel.
© 2000 by the American Journal of Hypertension, Ltd. Published by Elsevier Science, Inc.
KEY WORDS:
Vitamin D, estradiol, androgens, vascular smooth muscle cells, endothelial cells.
0895-7061/00/$20.00 PII S0895-7061(99)00203-4
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e have previously reported that 17 estradiol (E2) and dihydrotestosterone (DHT) regulate cell growth in human umbilical arterial smooth muscle cells (SMC) and in a human endothelial cell line (E304).1 In SMC both gonadal steroids had a biphasic effect, such that DNA synthesis was stimulated at low concentrations and inhibited at high concentrations.1 In E304 cells E2 and DHT dose dependently enhanced DNA synthesis.1 Additionally, in both cell types E2 and DHT stimulated, in a dose-related manner, the activity of the brain isozyme of creatine kinase (CK), an enzyme linked to cell metabolism and steroid hormone action in many cell types.2,3 Earlier reports indicated that the response of bone and cartilage cells to gonadal steroids is upregulated by pretreatment with vitamin D metabolites and their nonhypercalcemic analogs,4 –7 presumably secondary to increased expression of estrogen receptors (ER).7,8 In contrast, in the uterus cellular CK responses to E2 were downregulated by vitamin D9 in association with a slight decrease in ER␣. These observations lend support to the concept that vitamin D may be a modulator of gonadal steroid action in several sex-steroid–responsive tissues. Relative or frank sex hormone deficiency is a hallmark of human aging.10 Likewise, a low vitamin D status is quite common in older subjects.11 Indeed, vitamin D supplementation in the form of multivitamins, vitamin-D– enriched dairy products, or prescribed medications is widespread in Western countries. Thus, combined sex hormone and vitamin D replacement therapy is a common practice in older patients. The cardiovascular implications of this combination in human subjects have not been explored. In the present study we examined the effects of several nonhypercalcemic vitamin D analogs on [3H] thymidine incorporation and CK activity in human SMC and E304 cells in the presence and absence of gonadal steroids. The results indicate that vitamin D analogs are, by themselves, modulators of DNA sythesis in human vascular cells and suggest that they modify the effects of gonadal steroids on [3H] thymidine incorporation in both SMC and endothelial cells. MATERIALS AND METHODS Experimental Procedures Reagents All reagents used were of analytical grade. Chemicals, steroids, and the CK assay kit were purchased from Sigma Chemical Co. (St. Louis, MO). Anti-ER␣ antibodies (clone 13H2) were kindly provided by Dr. H. Thole, Germany. Polyclonal anti-ER antibodies were the kind gift of Dr. N. Ben-Jonathan from the University of Cincinnati. 1Chloro 25 (OH) vitamin D3 (HL) was a gift of GBF, Braunschweig Germany, and ZK 159222 was kindly provided by the Schering Co., Berlin, Germany. The secondary anti-
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bodies were rabbit antimouse IgG1 peroxidase (Zymed, San Francisco, CA), goat antimouse peroxidase, and goat antirabbit peroxidase (Sigma, Rehovot, Israel). The enhanced chemilluminescence reagents were from Amersham (Amersham, Buckinghamshire, UK). Cell Cultures Umbilical Artery Smooth Muscle Cells (SMC) Umbilical SMC were prepared as previously described, with minor modifications.1,12 In brief, umbilical cords were collected shortly after delivery, cleaned of blood and adventitia, and cut into tiny slices (1–3 mm). The segments were kept in culture in medium 199 containing 20% fetal calf serum (FCS), glutamine, and antibiotics. Cell migration was detected within 5 to 7 days. Cells were fed twice a week and, upon confluence, trypsinyzed and transferred to 24-well dishes. Cells were used only at passages 1 to 3, when expression of smooth muscle action was clearly demonstrable.1,12 Endothelial Cells (E304) E304 cells, an endothelial cell line derived from a human umbilical vein, were purchased from ATCC and grown in medium 199 containing 20% FCS, glutamine, and antibiotics.1 Pretreatment With Vitamin D Analogs and Inhibitors Cells were seeded as described and treated daily for 3 days with 1 nmol/L of 1,25 (OH)2 D3 hexafluoro vitamin D (FL),6,7 JK-1264 F2-2 (JKF)13 or CB 1093 (CB).4 In other experiments, either the inhibitor of the membranous effects of vitamin D, HL, or the inhibitor of its nuclear action, ZK 159222 (ZK), were used. On day 4 the cells were treated with E2, DHT, or 3000 nmol/L raloxifene (RAL) for 24 h for the assessment of DNA synthesis or 4 h for the measurement of CK activity.7 Assessment of DNA Synthesis Cells were grown until subconfluence and then treated with various hormones or agents as indicated. Twenty-two hours later, [3H] thymidine was added for 2 h. Cells were then treated with 10% ice-cold trichloro acetic acid (TCA) for 5 min and washed twice with 5% TCA and then with cold ethanol. The cellular layer was dissolved in 0.3 mL of 0.3 mmol/L NaOH, samples were aspirated, and 3[H] thymidine incorporation into DNA was determined.1 Creatine Kinase Extraction and Assay Cells were treated for 4 h with the various hormones and agents, as specified, and were then scraped off the culture dishes and homgenized by freezing and thawing three times in an extraction buffer, as previously described.1 Supernatant extracts were obtained by centrifugation of homogenates at 14,000 ⫻ g for 5 min at 4°C in an Eppendorf microcentrifuge. CK was determined by a coupled spectrophotometric assay described previ-
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ously.7 Protein was determined by Coomasie blue dye binding using bovine serum albumin as the standard.14 Statistical Analysis Comparisons between the control and vaious treatments were made by analysis of variance. Western Blot Analysis of ER Western blot analysis of ER was performed using specific antibodies against the two forms of the receptors.9 Cells from treated and control incubates were homogenized in a homogenization buffer consisting of 50 mmol/L -glycerophosphate (pH 7.3), 1.5 mmol/L EGTA, 1 mmol/L EDTA, 1 mmol/L dithiotreitol, 0.1 mmol/L sodium orthovanadate, 1 mmol/L benzamidine, aprotinin (10 g/gL), and leupeptin (10 g/mL), and the protein content of the homogenate was determined. For Western blot analysis equal amounts (30 g) of homogenates and molecular-weight markers were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to nitrocellulose membranes and stained with Ponceau Red to verify equal protein loading and transfer. After blocking with 5% milk or 2% bovine serum albumin (BSA) membranes were incubated overnight with the antibodies diluted in TBS buffer (20 mM Tris and 0.14 M NaCl pH, 7.6.) containing 1% milk or 1% BSA, anti-ER␣ (0.5 g/mL), and anti-ER (at 1:3000 dilution). Membranes were washed and processed for enhanced chemilluminscence using the ECL reagents. Signals were then quantified by densitometry. RESULTS Effects of Vitamin D Analogs on [3H] Thymidine Incorporation and CK Activity Daily treatment with FL, JKF, or CB (1 nmol/L) for 3 days increased DNA synthesis by 110%, 65%, and 88%, respectively, in E304 cells, whereas in SMC cells, cell proliferation was inhibited by ⬃45% to 55% by these agents (Figure 1). In both cell types the specific activity of CK increased by 75% to 120% after daily treatment with FL, JKF, or CB (Figure 2). Interaction Between Vitamin D Analogs and Gonadal Steroids in E304 Cells Hexafluoro vitamin D, JKF, and CB increased basal [3H] thymidine incorporation and further augmented the response to E2 (30 nmol/L) and DHT (300 nmol/L) by approximately twofold to sixfold, (from ⬃⫹50% to 60% to 200% to 400% in the absence and presence of vitamin D analogs, respectively (Figure 3). A parallel upregulation of the response of E304 cells to gonadal steroids by vitamin D analogs was observed with respect to CK activity (Figure 4): the E2- or DHT-induced increase in CK, on the order of 60% to 80%, was further enhanced to 300% to 320% in the presence of FL, JKF, or CB (Figure 4).
FIGURE 1. Effect of daily treatment with vitamin D analogs on DNA synthesis in E304 and SMC cells. The vitamin D analogs FL, JKF, and CB were added daily for 3 days (1 nmol/L each). DNA synthesis was assessed by [3H] thymidine incorporation. The results are means ⫾ SEM of eight to 12 incubates from at least two different cultures and are expressed as the % change induced by the vitamin D analog compared with control incubates. **P ⬍ .05 for the difference between treated and untreated cells.
Interaction Between Vitamin D Analogs and Gonadal Steroids in SMC Pretreatment with vitamin D analogs affected the response of the SMC to gonadal steroids in a complex manner. FL, JKF, and CB reduced basal [3H] thymidine incorporation but did not alter the increase induced by a low concentration of E2 (Figure 3). High concentrations of E2 (30 nmol/L) or DHT (300 nmol/L) decreased DNA synthesis by ⬃50%. However, in the presence of vitamin D analogs these inhibitory effect of E2 and DHT were no longer seen (Figure 3). FL, JKF, and CB also amplified the CK response to gonadal steroids (Figure 4) from ⬃30% to 170% to 390% (with and without pretreatment with vitamin D analogs, respectively). Effect of the Vitamin D Analog CB on RAL-Mediated DNA Synthesis Because our previous studies showed that estrogen receptor antagonists mimic the effect of E2 on DNA synthesis in SMC and E304 cells,1
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FIGURE 2. Effect of daily treatment with vitamin D analogs on CK-specific activity in E304 and SMC cells. The vitamin D analogs FL, JKF, and CB were added daily for 3 days (1 nmol/L each). CK was assayed as described in Materials and Methods. The results are means ⫾ SEM of eight to 12 incubates from at least two different cutures and are expressed as the change induced by the vitamin D analog compared with control incubates. **P ⬍ .05 for the difference between treated and untreated cells.
we examined the effect of the vitamin D analog CB on DNA synthesis in the presence of RAL. As depicted in Figure 5, in SMC a low concentration of RAL (30 nmol/L) increased [3H] thymidine incorporation, whereas a high concentration (3000 nmol/L) decreased DNA synthesis. CB augmented the proliferative response to a low concentration of RAL and prevented the suppression of [3H] thymidine incorporation induced by a high concentration of RAL. In E304 cells, CB amplified the stimulatory effect of RAL (3000 nmol/L) on DNA synthesis (Figure 5). Effect of Vitamin D Analogs on Receptor Proteins for E2 (ER) in Vascular Cells Western immunoblotting of SMC extracts detected the presence of two isoforms (32 k and 63 k) of ER␣ protein and a single 53-k band of ER protein. After 72 h of incubation with 1,25 (OH)2 vitamin D3 (added daily at 1 nmol/L), the expression of the 32-k and 63-k ER␣ peptides was
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FIGURE 3. Effect of daily treatment with vitamin D analogs on the DNA synthesis response to gonadal steroids in E304 and SMC cells. The vitamin D analogs FL, JKF, or CB were added daily for 3 days (at a concentration of 1 nmol/L) followed by 1 day of treatment with E2. In SMC, low (0.3 nmol/L) and high (30 nmol/L) concentrations of E2 were used. In E304 cells, only high concentrations of E2 (30 nmol/L) or DHT (300 nmol/L) were used (as in these cells gonadal steroids stimulated DNA synthesis dose dependently). DNA synthesis was assessed by [3H] thymidine incorporation. The results are means ⫾ SEM of eight to 12 incubates from at least two different cultures and are expressed as % change from basal [3H] thymidine uptake compared with untreated cells. **P ⬍ .05, ***P ⬍ .001.
unchanged, whereas ER levels declined by 70% to 90%; (Figure 6). Similarly, incubation with the vitamin D analog JKF (daily addition, 1 nmol/L) resulted in a 25% to 50% increase in the 63-k ER␣ associated with a 25% to 60% reduction in ER protein. In E304 cells ER protein was not clearly affected by 1,25 (OH)2 D3 or JKF (⫺15% to ⫹25%), whereas the 32-k isoform of ER␣ increased by 25% to 100% (Figure 6). Effect of Vitamin D Inhibitors on Vitamin D Analog Action We used two chemically and biologically distinct inhibitors of vitamin D action, 1 chloro 25 (OH)2 D3 (HL), a blocker of transmembranous vitamin-D–
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FIGURE 4. Effect of daily treatment with vitamin D analogs on CK-specific activity induced by gonadal steroids in E304 and SMC cells. The vitamin D analogs FL, JKF, or CB (1 nmol/L each) were added daily for 3 days, followed by 4 h of incubation with E2 (30 nmol/L) or DHT (300 nmol/L). CK activity was subsequently assayed as described in Materials and Methods. The results are means ⫾ SEM of eight to 12 incubates from at least two different cultures and are expressed as % change in CK activity compared with untreated cells. *P ⬍ .01, **P ⬍ .05, ***P ⬍ .001.
dependent calcium influx,7 and ZK 159222, an inhibitor of the nuclear action of vitamin D.7 Neither had an independent effect on DNA synthesis in either SMC or E304 cells and, similarly, these two inhibitors did not modify the effect of estradiol on DNA synthesis. In E304 cells, only the nuclear blocker of vitamin D effects, ZK 159222, (but not the membranous inhibitor HL; data not shown) prevented the amplification of E2-dependent DNA synthesis induced by the vitamin D analog CB (Figure 7). In SMC, after pretreatment with CB, high E2 concentrations no longer prevented DNA synthesis in SMC (Figure 7). When the vitamin D nuclear antagonist ZK 159222 was also added—so that cells were pretreated with both the vitamin D analog and its nuclear antagonist, followed by high
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FIGURE 5. The effect of daily treatment with vitamin D analog CB on the response of DNA synthesis to raloxifene (RAL) in E304 and SMC cells. CB (1 nmol/L) was added daily for 3 days, followed by 24 h incubation in the presence of RAL (30 nmol/L or 3000 nmol/L). DNA synthesis was determined following the experimental protocol detailed in the legend to Figure 1. The results are means ⫾ SEM of eight to 12 incubates from two different cultures and are expressed as the change from basal [3H] thimidine incorporation. *P ⬍ .01, **P ⬍ .05, ***P ⬍ .001.
concentration of E2—DNA synthesis was actually stimulated, an effect seen with low E2 concentrations alone. In contrast, the membranous antagonist of vitamin D action HL did not modify the interaction between the vitamin D analog and high concentrations of E2 (data not shown). DISCUSSION Smooth muscle cells express high-affinity receptors for 1,25(OH)2 vitamin D3, which suggests that the arterial wall may be a target for vitamin D action.13,15 Classic nuclear receptors of vitamin D could mediate several reported vascular effects of this hormone, such as increased expression of Ca-ATPase,13 induction of contractile protein synthesis,16 and hence, increased arterial resistance.17 That vitamin D may also be a modulator of vascular wall growth has been suggested by several reports that 1,25(OH)2 vitamin D3 either suppresses15,18 or stimulates15,17–19 [3H] thymi-
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FIGURE 6. The effect of 1,25(OH)2 vitamin D3 (solid symbols) and JKF (open symbols) on ER␣ and ER proteins in SMC and E304 cells. Cells were treated daily for 3 days with 1 nmol/L of either 1,25(OH)2D3 or JKF and Western immunoblotting with specific anti-ER␣ and ER antibodies was performed as described in Materials and Methods. The results represent two separate experiments and are presented as % change in protein densities.
dine uptake in SMC. To our knowledge, this is the first study addressing the effect of vitamin D on growth in human vascular cells. A key finding in this study is that vitamin D analogs exert opposite effects on human SMC and endothelial cells, ie, they inhibit growth in SMC while stimulating [3H] thymidine incorporation in E304 cells (Figure 1). The observation that the analogs are inhibitors of cell growth in human SMC is in agreement with a previous report that 1,25(OH)2 D3 inhibits growth in human mesangial cells,20 presumably a uniquely differentiated form of SMC. Extrapolation of these dual endothelial-SMC effects to the in vivo situation would suggest that vitamin D analogs may be candidate enhancers of vascular protection; by stimulating endothelial cell replication, they could promote reendothelialization and by inhibiting SMC poliferation, they might minimize postinjury myointimal proliferation. Although previous reports have indicated that high doses of vitamin D3 exert deleterious effects on the vascular wall in several animal models,21,22 the role of severe hypercalcemia and increased calcium load in these models has not been discerned. In this sense
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FIGURE 7. The effect of vitamin D inhibitors on the interaction between E2 and the vitamin D analog CB on DNA synthesis. The vitamin D inhibitor ZK 159222 (1 nmol/L) alone, CB (1 nmol/L) alone, or both were added daily for 3 days. On day 4 the cells were treated with E2 (0.3 or 30 nmol/L) for 24 h. DNA synthesis was then assessed by [3H] thymidine incorporation. The results are means ⫾ SEM of eight to 12 incubates from two different cultures and are expressed as change in [3H] thymidine incorporation into DNA with the various treatments, compared with that seen in untreated cells. *P ⬍ .01, **P ⬍ .05, ***P ⬍ .001.
vitamin D analogs provide a substantially different research opportunity in vivo, because their calcemic effect is negligible.23 The major finding in this study is that vitamin D analogs modulate the effects of gonadal steroids on vascular cell growth in a complex and cell-specific manner. In E304 cells, vitamin D analogs enhanced E2and DHT-induced [3H] thymidine incorporation. Because pretreatment with each of the analogs tested alone increased [3H] thymidine incorporation by 60% to 110%, and the response to estrogens seen in analogtreated cells rose, in relative terms, by 100% to 180% compared with ⬃50% in cells treated with E2 alone, the combined three- to sixfold increase induced by FL/E2, JKF/E2, or CB/E2 represented a synergistic rather than a simple additive effect, as shown in Fig-
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ure 3 for DNA synthesis and in Figure 4 for CK activity. To the extent that enhanced endothelial cell replication favors vascular protection, the combination of vitamin D analogs and E2 may offer an advantage over E2 alone. In SMC, FL, JKF, and CB did not affect the stimulatory influence seen with low concentrations of gonadal steroids, yet they prevented the suppression of DNA synthesis exerted by high doses of E2 and DHT. This disinhibition of SMC growth was, in essence, duplicated when RAL (an estrogen agonist in this system) rather then E2 was present, thus further confirming that vitamin D analogs block the inhibitory effects on SMC growth exerted by high concentrations of estrogenic agents (Figures 3, 5). Of note is the observation that a nuclear inhibitor of vitamin D action (but not an inhibitor of its membranous effects) prevented the analog-induced enhancement of the stimulatory effect of E2 in E304 cells (Figure 7). This suggests that the analog-dependent augmentation of [3H] thymidine incorporation and CK activity induced by E2 in endothelial cells was mediated by classic nuclear routes of vitamin D action. It is not clear why the disinhibition of the suppressive effect of E2 on DNA synthesis exerted by vitamin D analogs in SMC was not reversed by the inhibitor, such that the combination of the vitamin D agonist and its nuclear antagonist resulted in stimulation of growth after high concentrations of E2 (rather than the restoration of the inhibitory effect of high concentrations of E2; Figure 7). In fact, this effect resembled the induction of growth seen in SMC with low concentrations of E2. One potential mechanism by which vitamin D analogs may modify the vascular actions of estrogens is via modulation of ER expression. Indeed, there are precedents for such interactions in bone-marrow– derived stromal cells,8 as well as in bone and cartilage cells.7 Results depicted in Figure 6 appear consistent with this possibility; 1,25(OH)2 vitamin D3, as well as its analog JKF, reduced the expression of ER protein in SMC, but not in E304 cells. Observations that the inhibitory effect of estrogen on SMC proliferation is preserved in ER␣ knockout mice suggest that the ER isoform is involved is E2-mediated suppression of SMC growth.24 If the ER isoform is also involved in the inhibitory effect of E2 on DNA synthesis in human SMC, its downregulation by vitamin D analogs (Figure 6) could negate this effect of E2. How the observed increase in the ER␣ isoforms induced by 1,25(OH)2 vitamin D3 and its analog in E304 cells relates to the enhancement of E2-dependent growth attained by these agents cannot be determined based on these preliminary observations. In summary, vitamin D analogs and gonadal steroids have independent effects on DNA synthesis and CK activity in human SMC and E304 cells. Moreover,
vitamin D analogs interact with gonadal steroids in a dose- and cell-specific manner. Vitamin D analogs modulate the effects of E2 on DNA synthesis, apparently via classic nuclear routes of vitamin D action. Downregulation of ER expression and upregulation of ER␣ protein by vitamin D analogs may contribute to the interaction between these agents and estrogens in human vascular cells. ACKNOWLEDGMENTS FL was donated by Sumitomo Pharmaceutical Co. Ltd., Osaka, Japan. JKF was synthesized and donated by Dr. G.H. Posner from the Department of Chemistry, Johns Hopkins University, Baltimore, MD. CB 1093 was a gift of Leo Pharmaceutial Co., Ballerup, Denmark. Raloxifene was the gift of Novartis, Basel, Switzerland. HL was a gift of GBF, Braunschweig Germany, and ZK 159222 was kindly provided by Schering Co., Berlin, Germany. Polyclonal anti-ER␣ was the gift of Dr. H. Thole, Germany, and polyclonal anti-ER was kindly provided by Dr. N. Ben-Jonathan, University of Cincinnati, Cincinnati, OH. We also thank Mrs. Rivka Morris for her expert assistance in preparing the manuscript and Prof. A.M. Kaye and Mrs. Anna Waisman for their assistance in the CK assays and fruitful discussion of the data.
REFERENCES 1.
Somjen D, Kohen F, Jaffe A, Amir-Zaltsman Y, Knoll E, Stern N: Effects of gonadal steroids and their antagonists on DNA synthesis in human vascular cells. Hypertension 1998;32:39 – 45.
2.
Kaye AM, Sheratzky D, Lindner HR: Kinetics of DNA synthesis in immature rat uterus: age dependence and estradiol stimulation. Biochimica Biophysica Acta 1971; 261:475– 486.
3.
Fournier B, Haring S, Kaye AM, Somjen D: Stimulation of creatine kinase specific activity in human osteoblast and endometrial cells by estrogens and anti-estrogens and its modulation by calciotropic hormones. J Endocrinol 1996;150:275–285.
4.
James SY, Mackay AB, Binderup L, Colston K: Effects of a new synthetic vitamin D analogue, EB 1089, on estrogen-responsive growth of human breast cancer cells. J Endocrinol 1994;141:555–563.
5.
Posner GH, Lee JK, Wang O, Pelleg S, Bunke M, Brom H, Dolan P, Kensler T: Noncalcemic, antiproliferative, transeriptionally active, 24-fluorinated hybrid analogues of the hormone 1␣,25-dihydroxyvitamin D3. Synthesis and preliminary biological evaluation. J Med Chem 1998;41:3008 –3014.
6.
Somjen D, Weisman Y, Kaye AM: Increased sensitivity to sex steroids after pretreatment of skeletal derived cells with vitamin D metabolites, in Norman AW, Bouillon R, Thomasset M (eds): Proceedings of the 9th Vitamin D Workshop, 1994, Orlando, FL, pp 737–738.
7.
Somjen D, Waisman A, Weisman Y, Kaye AM: Nonhypercalcemic analogs of vitamin D stimulate creatine kinase B activity in osteoblast-like ROS 17/2.8 cells and up-regulate their responsiveness to estrogens. Steroids 1998;63:340 –343.
8.
Bellido T, Girasole G, Paseri G, Yu XP, Mocharia H, Jilka RL, Notides A, Manolagas SC: Demonstration of estrogen and vitamin D receptors in bone marrow-
AJH–APRIL 2000 –VOL. 13, NO. 4, PART 1
derived stromal cells: upregulation of the estrogen receptor by 1,25-dihydroxy vitamin D3. Endocrinology 1993;133:553–562. 9.
Somjen D, Waisman A, Kaye AM, Posner GH, Lee JK, Weisman Y: Non-hypercalcemic Vitamin D analogs upregulate female rat responses and sensitivity to estrogen. The American Endocrine Society’s 80th Annual Meeting, 1998; June 24 –27th New Orleans, p. 242 (abstract).
VITAMIN D, ESTROGENS IN VASCULAR SMC
17.
18.
10.
Ferrini RL, Barret-Connor E: Sex hormone and age: a cross sectional study of testosterone and estradiol and their bioavailable fractions in community-dwelling men. Am J Epidemiol 1998;147:750 –754.
19.
11.
Bouilion R, Carmeliet G, Boonen S: Ageing and calcium metabolism. Baillier’s Clin Endocrinol Metab 1997;11: 341–365.
20.
12.
Kisch E, Jaffe A, Knoll E, Stern N: Role of lipoxygenase pathway in angiotensin II - induced vasoconstriction in the human placenta. Hypertension 1997;29:796 – 801.
13.
Kwashima H: 1,25 dihydroxyvitamin D3 stimulates CaATPase in VSMC line. Biochem Biophys Res Commun 1988;150:1138 –1143.
22.
Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt Biochem 1976;72:248 –254.
23.
14.
15.
Mitsuhashi T, Morris RC Jr, Ives HE: 1,25 dihydroxyvitamin D3 modulates growth of vascular smooth muscle cells. J Clin Invest 1991;87:1889 –1895.
16.
Xue H, McCarron DA, Bukoski RD: 1,25(OH)2 vitamin D3 attenuates the loss of resistance artery contractile
21.
24.
403
function associated with incubation in culture media. Biochem Biophys Res Commun 1991;174:11–17. Bukoski RD, DeWan P, McCarron DA: 1,25 (OH)2 vitamin D3 modifies growth and contractile function of vascular smooth muscle of spontaneously hypertensive rats. Am J Hypertens 1989;2:553–556. Carthy EP, Yamashita W, Hsu A, Ooi BS: 1,25 dihydroxyvitamin D3 and rat vascular smooth muscle cell growth. Hypertension 1989;13:954 –959. Koh E, Morimoto S, Nobata T, Takamoto S, Kitano S, Ogihara T: Effects of 1,25 dihydroxyvitamin D3 on the synthesis of DNA and glycosaminoglycans by rat aortic smooth muscle cells in vitro. Life Sci 1990;46:1545–1551. Weinreich T, Merke J, Schonermark M, Reichel H, Diebold M, Hansch GM, Ritz E: Action of 1,25 dihydroxyvitamin D3 on human mesangial cells. Am J Kid Dis 1991;17:359 –366. Taura S, Taura M, Kamio A, Kummerow FA. Vitamin D-induced coronary atherosclerosis in normolipeuic swine: comparison with human disease. Tohoku J Exper Med 1979;129:9 –16. Takeo T, Anan M, Fujioka, Kajihara T, Hiraga S, Miyake K, Tanonaka K, Minematsu R, Mori H, Taniguchi Y: Functional changes of aorta with massive accumulation of calcium. Atherosclerosis 1989;77:175–181. Brown AJ: Vitamin D analogues. Am J Kid Dis 1988; 32(2 suppl 2):S25–S39. Iafrati MD, Karas RII, Aronovitz M, Kim S, Sullivan TR Jr, Lubahn DB, O’Donnell TF Jr, Korach KS, Mendelson ME: Estrogen inhibits the vascular injury response in estrogen receptor alpha-deficient mice. Nature Med 1997;3:545–548.
2000;13:396 – 403
Vitamin D Analogs Modulate the Action of Gonadal Steroids in Human Vascular Cells In Vitro D. Somjen, F. Kohen, Y. Amir-Zaltsman, E. Knoll, and N. Stern
We have previously reported that estradiol (E2) and dihydrotestosterone (DHT) regulate cell growth in human umbilical arterial smooth muscle cells (SMC) and in an endothelial cell line (E304). In SMC both gonadal steroids stimulated DNA synthesis at low concentrations and suppressed 3 [H] thymidine incorporation at high concentrations, whereas in E304 cells E2 and DHT dose dependently enhanced DNA synthesis. In both cell types gonadal steroids also induced the specific activity of creatine kinase BB (CK). Previous evidence suggets that the in vitro and in vivo CK responses to gonadal steroids in bone cells are upregulated by pretreatment with vitamin D analogs due to increased level of cellular estrogen receptors (ER). Here we analyzed the interaction of the vitamin D analogs hexafluorovitamin D (FL), JK-1624 F2-2 (JKF), and CB 1093 (CB) with gonadal steroids in regulating DNA synthesis and CK activity in human vascular cells in vitro. In E304 cells, daily treatment with FL, JKF, or CB (1 nmol/L for 3 days) increased DNA synthesis by 110 ⴞ 11%, 65 ⴞ 16%, and 88 ⴞ 23% respectively. In contrast, the same analogs inhibited 3[H] thymidine incorporation by 52 ⴞ 21%, 46 ⴞ 19%, and 50 ⴞ 10%, respectively, in SMC. In both cell types all three analogs increased CK by 25% to 75% and amplified the CK response
to E2 and to DHT by twofold to threefold. In E304 cells the vitamin D analogs also increased DNA response to gonadal steroids from 50% to 60% to 200% to 280%. In SMC these analogs did not modify the DNA synthetic response to a low E2 concentration, but prevented the suppression of DNA synthesis exerted by high concentrations of E2 and DHT. Vitamin D inhibitors known to block cellular calcium mobilization, had no effect on the proliferative activity induced by vitamin D analogs. However, the inhibitor of the nuclear effects of vitamin D, ZK 159222, blocked the stimulatory effects of CB on DNA synthesis in E304 cells. Finally, both 1,25(OH)2 D3, and JKF decreased the expression of ER proteins in SMC and increased the ER␣ isoform in E304 cells by 40% to 75%. The results indicate that vascular cells are targets for both vitamin D and gonadal steroid action and suggest a possible interaction between these hormones in the regulation of cell proliferation via modulation of vascular ER or interaction with proteins associated with ER. Am J Hypertens 2000;13:396 – 403 © 2000 American Journal of Hypertension, Ltd.
Received May 4, 1999. Accepted August 3, 1999. From the Institute of Endocrinology Metabolism and Hypertension, Tel Aviv Sourasky Medical Center and The Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv; and Department of Biological Regulation, The Weizman Institute of Science, Rehovot, Israel.
Address correspondence and reprint requests to N. Stern, MD, Institute of Endocrinology, Metabolism and Hypertension, Tel Aviv-Sourasky Medical Center, 6 Weizman Street, Tel Aviv 64239, Israel.
© 2000 by the American Journal of Hypertension, Ltd. Published by Elsevier Science, Inc.
KEY WORDS:
Vitamin D, estradiol, androgens, vascular smooth muscle cells, endothelial cells.
0895-7061/00/$20.00 PII S0895-7061(99)00203-4
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e have previously reported that 17 estradiol (E2) and dihydrotestosterone (DHT) regulate cell growth in human umbilical arterial smooth muscle cells (SMC) and in a human endothelial cell line (E304).1 In SMC both gonadal steroids had a biphasic effect, such that DNA synthesis was stimulated at low concentrations and inhibited at high concentrations.1 In E304 cells E2 and DHT dose dependently enhanced DNA synthesis.1 Additionally, in both cell types E2 and DHT stimulated, in a dose-related manner, the activity of the brain isozyme of creatine kinase (CK), an enzyme linked to cell metabolism and steroid hormone action in many cell types.2,3 Earlier reports indicated that the response of bone and cartilage cells to gonadal steroids is upregulated by pretreatment with vitamin D metabolites and their nonhypercalcemic analogs,4 –7 presumably secondary to increased expression of estrogen receptors (ER).7,8 In contrast, in the uterus cellular CK responses to E2 were downregulated by vitamin D9 in association with a slight decrease in ER␣. These observations lend support to the concept that vitamin D may be a modulator of gonadal steroid action in several sex-steroid–responsive tissues. Relative or frank sex hormone deficiency is a hallmark of human aging.10 Likewise, a low vitamin D status is quite common in older subjects.11 Indeed, vitamin D supplementation in the form of multivitamins, vitamin-D– enriched dairy products, or prescribed medications is widespread in Western countries. Thus, combined sex hormone and vitamin D replacement therapy is a common practice in older patients. The cardiovascular implications of this combination in human subjects have not been explored. In the present study we examined the effects of several nonhypercalcemic vitamin D analogs on [3H] thymidine incorporation and CK activity in human SMC and E304 cells in the presence and absence of gonadal steroids. The results indicate that vitamin D analogs are, by themselves, modulators of DNA sythesis in human vascular cells and suggest that they modify the effects of gonadal steroids on [3H] thymidine incorporation in both SMC and endothelial cells. MATERIALS AND METHODS Experimental Procedures Reagents All reagents used were of analytical grade. Chemicals, steroids, and the CK assay kit were purchased from Sigma Chemical Co. (St. Louis, MO). Anti-ER␣ antibodies (clone 13H2) were kindly provided by Dr. H. Thole, Germany. Polyclonal anti-ER antibodies were the kind gift of Dr. N. Ben-Jonathan from the University of Cincinnati. 1Chloro 25 (OH) vitamin D3 (HL) was a gift of GBF, Braunschweig Germany, and ZK 159222 was kindly provided by the Schering Co., Berlin, Germany. The secondary anti-
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bodies were rabbit antimouse IgG1 peroxidase (Zymed, San Francisco, CA), goat antimouse peroxidase, and goat antirabbit peroxidase (Sigma, Rehovot, Israel). The enhanced chemilluminescence reagents were from Amersham (Amersham, Buckinghamshire, UK). Cell Cultures Umbilical Artery Smooth Muscle Cells (SMC) Umbilical SMC were prepared as previously described, with minor modifications.1,12 In brief, umbilical cords were collected shortly after delivery, cleaned of blood and adventitia, and cut into tiny slices (1–3 mm). The segments were kept in culture in medium 199 containing 20% fetal calf serum (FCS), glutamine, and antibiotics. Cell migration was detected within 5 to 7 days. Cells were fed twice a week and, upon confluence, trypsinyzed and transferred to 24-well dishes. Cells were used only at passages 1 to 3, when expression of smooth muscle action was clearly demonstrable.1,12 Endothelial Cells (E304) E304 cells, an endothelial cell line derived from a human umbilical vein, were purchased from ATCC and grown in medium 199 containing 20% FCS, glutamine, and antibiotics.1 Pretreatment With Vitamin D Analogs and Inhibitors Cells were seeded as described and treated daily for 3 days with 1 nmol/L of 1,25 (OH)2 D3 hexafluoro vitamin D (FL),6,7 JK-1264 F2-2 (JKF)13 or CB 1093 (CB).4 In other experiments, either the inhibitor of the membranous effects of vitamin D, HL, or the inhibitor of its nuclear action, ZK 159222 (ZK), were used. On day 4 the cells were treated with E2, DHT, or 3000 nmol/L raloxifene (RAL) for 24 h for the assessment of DNA synthesis or 4 h for the measurement of CK activity.7 Assessment of DNA Synthesis Cells were grown until subconfluence and then treated with various hormones or agents as indicated. Twenty-two hours later, [3H] thymidine was added for 2 h. Cells were then treated with 10% ice-cold trichloro acetic acid (TCA) for 5 min and washed twice with 5% TCA and then with cold ethanol. The cellular layer was dissolved in 0.3 mL of 0.3 mmol/L NaOH, samples were aspirated, and 3[H] thymidine incorporation into DNA was determined.1 Creatine Kinase Extraction and Assay Cells were treated for 4 h with the various hormones and agents, as specified, and were then scraped off the culture dishes and homgenized by freezing and thawing three times in an extraction buffer, as previously described.1 Supernatant extracts were obtained by centrifugation of homogenates at 14,000 ⫻ g for 5 min at 4°C in an Eppendorf microcentrifuge. CK was determined by a coupled spectrophotometric assay described previ-
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ously.7 Protein was determined by Coomasie blue dye binding using bovine serum albumin as the standard.14 Statistical Analysis Comparisons between the control and vaious treatments were made by analysis of variance. Western Blot Analysis of ER Western blot analysis of ER was performed using specific antibodies against the two forms of the receptors.9 Cells from treated and control incubates were homogenized in a homogenization buffer consisting of 50 mmol/L -glycerophosphate (pH 7.3), 1.5 mmol/L EGTA, 1 mmol/L EDTA, 1 mmol/L dithiotreitol, 0.1 mmol/L sodium orthovanadate, 1 mmol/L benzamidine, aprotinin (10 g/gL), and leupeptin (10 g/mL), and the protein content of the homogenate was determined. For Western blot analysis equal amounts (30 g) of homogenates and molecular-weight markers were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to nitrocellulose membranes and stained with Ponceau Red to verify equal protein loading and transfer. After blocking with 5% milk or 2% bovine serum albumin (BSA) membranes were incubated overnight with the antibodies diluted in TBS buffer (20 mM Tris and 0.14 M NaCl pH, 7.6.) containing 1% milk or 1% BSA, anti-ER␣ (0.5 g/mL), and anti-ER (at 1:3000 dilution). Membranes were washed and processed for enhanced chemilluminscence using the ECL reagents. Signals were then quantified by densitometry. RESULTS Effects of Vitamin D Analogs on [3H] Thymidine Incorporation and CK Activity Daily treatment with FL, JKF, or CB (1 nmol/L) for 3 days increased DNA synthesis by 110%, 65%, and 88%, respectively, in E304 cells, whereas in SMC cells, cell proliferation was inhibited by ⬃45% to 55% by these agents (Figure 1). In both cell types the specific activity of CK increased by 75% to 120% after daily treatment with FL, JKF, or CB (Figure 2). Interaction Between Vitamin D Analogs and Gonadal Steroids in E304 Cells Hexafluoro vitamin D, JKF, and CB increased basal [3H] thymidine incorporation and further augmented the response to E2 (30 nmol/L) and DHT (300 nmol/L) by approximately twofold to sixfold, (from ⬃⫹50% to 60% to 200% to 400% in the absence and presence of vitamin D analogs, respectively (Figure 3). A parallel upregulation of the response of E304 cells to gonadal steroids by vitamin D analogs was observed with respect to CK activity (Figure 4): the E2- or DHT-induced increase in CK, on the order of 60% to 80%, was further enhanced to 300% to 320% in the presence of FL, JKF, or CB (Figure 4).
FIGURE 1. Effect of daily treatment with vitamin D analogs on DNA synthesis in E304 and SMC cells. The vitamin D analogs FL, JKF, and CB were added daily for 3 days (1 nmol/L each). DNA synthesis was assessed by [3H] thymidine incorporation. The results are means ⫾ SEM of eight to 12 incubates from at least two different cultures and are expressed as the % change induced by the vitamin D analog compared with control incubates. **P ⬍ .05 for the difference between treated and untreated cells.
Interaction Between Vitamin D Analogs and Gonadal Steroids in SMC Pretreatment with vitamin D analogs affected the response of the SMC to gonadal steroids in a complex manner. FL, JKF, and CB reduced basal [3H] thymidine incorporation but did not alter the increase induced by a low concentration of E2 (Figure 3). High concentrations of E2 (30 nmol/L) or DHT (300 nmol/L) decreased DNA synthesis by ⬃50%. However, in the presence of vitamin D analogs these inhibitory effect of E2 and DHT were no longer seen (Figure 3). FL, JKF, and CB also amplified the CK response to gonadal steroids (Figure 4) from ⬃30% to 170% to 390% (with and without pretreatment with vitamin D analogs, respectively). Effect of the Vitamin D Analog CB on RAL-Mediated DNA Synthesis Because our previous studies showed that estrogen receptor antagonists mimic the effect of E2 on DNA synthesis in SMC and E304 cells,1
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FIGURE 2. Effect of daily treatment with vitamin D analogs on CK-specific activity in E304 and SMC cells. The vitamin D analogs FL, JKF, and CB were added daily for 3 days (1 nmol/L each). CK was assayed as described in Materials and Methods. The results are means ⫾ SEM of eight to 12 incubates from at least two different cutures and are expressed as the change induced by the vitamin D analog compared with control incubates. **P ⬍ .05 for the difference between treated and untreated cells.
we examined the effect of the vitamin D analog CB on DNA synthesis in the presence of RAL. As depicted in Figure 5, in SMC a low concentration of RAL (30 nmol/L) increased [3H] thymidine incorporation, whereas a high concentration (3000 nmol/L) decreased DNA synthesis. CB augmented the proliferative response to a low concentration of RAL and prevented the suppression of [3H] thymidine incorporation induced by a high concentration of RAL. In E304 cells, CB amplified the stimulatory effect of RAL (3000 nmol/L) on DNA synthesis (Figure 5). Effect of Vitamin D Analogs on Receptor Proteins for E2 (ER) in Vascular Cells Western immunoblotting of SMC extracts detected the presence of two isoforms (32 k and 63 k) of ER␣ protein and a single 53-k band of ER protein. After 72 h of incubation with 1,25 (OH)2 vitamin D3 (added daily at 1 nmol/L), the expression of the 32-k and 63-k ER␣ peptides was
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FIGURE 3. Effect of daily treatment with vitamin D analogs on the DNA synthesis response to gonadal steroids in E304 and SMC cells. The vitamin D analogs FL, JKF, or CB were added daily for 3 days (at a concentration of 1 nmol/L) followed by 1 day of treatment with E2. In SMC, low (0.3 nmol/L) and high (30 nmol/L) concentrations of E2 were used. In E304 cells, only high concentrations of E2 (30 nmol/L) or DHT (300 nmol/L) were used (as in these cells gonadal steroids stimulated DNA synthesis dose dependently). DNA synthesis was assessed by [3H] thymidine incorporation. The results are means ⫾ SEM of eight to 12 incubates from at least two different cultures and are expressed as % change from basal [3H] thymidine uptake compared with untreated cells. **P ⬍ .05, ***P ⬍ .001.
unchanged, whereas ER levels declined by 70% to 90%; (Figure 6). Similarly, incubation with the vitamin D analog JKF (daily addition, 1 nmol/L) resulted in a 25% to 50% increase in the 63-k ER␣ associated with a 25% to 60% reduction in ER protein. In E304 cells ER protein was not clearly affected by 1,25 (OH)2 D3 or JKF (⫺15% to ⫹25%), whereas the 32-k isoform of ER␣ increased by 25% to 100% (Figure 6). Effect of Vitamin D Inhibitors on Vitamin D Analog Action We used two chemically and biologically distinct inhibitors of vitamin D action, 1 chloro 25 (OH)2 D3 (HL), a blocker of transmembranous vitamin-D–
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FIGURE 4. Effect of daily treatment with vitamin D analogs on CK-specific activity induced by gonadal steroids in E304 and SMC cells. The vitamin D analogs FL, JKF, or CB (1 nmol/L each) were added daily for 3 days, followed by 4 h of incubation with E2 (30 nmol/L) or DHT (300 nmol/L). CK activity was subsequently assayed as described in Materials and Methods. The results are means ⫾ SEM of eight to 12 incubates from at least two different cultures and are expressed as % change in CK activity compared with untreated cells. *P ⬍ .01, **P ⬍ .05, ***P ⬍ .001.
dependent calcium influx,7 and ZK 159222, an inhibitor of the nuclear action of vitamin D.7 Neither had an independent effect on DNA synthesis in either SMC or E304 cells and, similarly, these two inhibitors did not modify the effect of estradiol on DNA synthesis. In E304 cells, only the nuclear blocker of vitamin D effects, ZK 159222, (but not the membranous inhibitor HL; data not shown) prevented the amplification of E2-dependent DNA synthesis induced by the vitamin D analog CB (Figure 7). In SMC, after pretreatment with CB, high E2 concentrations no longer prevented DNA synthesis in SMC (Figure 7). When the vitamin D nuclear antagonist ZK 159222 was also added—so that cells were pretreated with both the vitamin D analog and its nuclear antagonist, followed by high
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FIGURE 5. The effect of daily treatment with vitamin D analog CB on the response of DNA synthesis to raloxifene (RAL) in E304 and SMC cells. CB (1 nmol/L) was added daily for 3 days, followed by 24 h incubation in the presence of RAL (30 nmol/L or 3000 nmol/L). DNA synthesis was determined following the experimental protocol detailed in the legend to Figure 1. The results are means ⫾ SEM of eight to 12 incubates from two different cultures and are expressed as the change from basal [3H] thimidine incorporation. *P ⬍ .01, **P ⬍ .05, ***P ⬍ .001.
concentration of E2—DNA synthesis was actually stimulated, an effect seen with low E2 concentrations alone. In contrast, the membranous antagonist of vitamin D action HL did not modify the interaction between the vitamin D analog and high concentrations of E2 (data not shown). DISCUSSION Smooth muscle cells express high-affinity receptors for 1,25(OH)2 vitamin D3, which suggests that the arterial wall may be a target for vitamin D action.13,15 Classic nuclear receptors of vitamin D could mediate several reported vascular effects of this hormone, such as increased expression of Ca-ATPase,13 induction of contractile protein synthesis,16 and hence, increased arterial resistance.17 That vitamin D may also be a modulator of vascular wall growth has been suggested by several reports that 1,25(OH)2 vitamin D3 either suppresses15,18 or stimulates15,17–19 [3H] thymi-
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FIGURE 6. The effect of 1,25(OH)2 vitamin D3 (solid symbols) and JKF (open symbols) on ER␣ and ER proteins in SMC and E304 cells. Cells were treated daily for 3 days with 1 nmol/L of either 1,25(OH)2D3 or JKF and Western immunoblotting with specific anti-ER␣ and ER antibodies was performed as described in Materials and Methods. The results represent two separate experiments and are presented as % change in protein densities.
dine uptake in SMC. To our knowledge, this is the first study addressing the effect of vitamin D on growth in human vascular cells. A key finding in this study is that vitamin D analogs exert opposite effects on human SMC and endothelial cells, ie, they inhibit growth in SMC while stimulating [3H] thymidine incorporation in E304 cells (Figure 1). The observation that the analogs are inhibitors of cell growth in human SMC is in agreement with a previous report that 1,25(OH)2 D3 inhibits growth in human mesangial cells,20 presumably a uniquely differentiated form of SMC. Extrapolation of these dual endothelial-SMC effects to the in vivo situation would suggest that vitamin D analogs may be candidate enhancers of vascular protection; by stimulating endothelial cell replication, they could promote reendothelialization and by inhibiting SMC poliferation, they might minimize postinjury myointimal proliferation. Although previous reports have indicated that high doses of vitamin D3 exert deleterious effects on the vascular wall in several animal models,21,22 the role of severe hypercalcemia and increased calcium load in these models has not been discerned. In this sense
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FIGURE 7. The effect of vitamin D inhibitors on the interaction between E2 and the vitamin D analog CB on DNA synthesis. The vitamin D inhibitor ZK 159222 (1 nmol/L) alone, CB (1 nmol/L) alone, or both were added daily for 3 days. On day 4 the cells were treated with E2 (0.3 or 30 nmol/L) for 24 h. DNA synthesis was then assessed by [3H] thymidine incorporation. The results are means ⫾ SEM of eight to 12 incubates from two different cultures and are expressed as change in [3H] thymidine incorporation into DNA with the various treatments, compared with that seen in untreated cells. *P ⬍ .01, **P ⬍ .05, ***P ⬍ .001.
vitamin D analogs provide a substantially different research opportunity in vivo, because their calcemic effect is negligible.23 The major finding in this study is that vitamin D analogs modulate the effects of gonadal steroids on vascular cell growth in a complex and cell-specific manner. In E304 cells, vitamin D analogs enhanced E2and DHT-induced [3H] thymidine incorporation. Because pretreatment with each of the analogs tested alone increased [3H] thymidine incorporation by 60% to 110%, and the response to estrogens seen in analogtreated cells rose, in relative terms, by 100% to 180% compared with ⬃50% in cells treated with E2 alone, the combined three- to sixfold increase induced by FL/E2, JKF/E2, or CB/E2 represented a synergistic rather than a simple additive effect, as shown in Fig-
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ure 3 for DNA synthesis and in Figure 4 for CK activity. To the extent that enhanced endothelial cell replication favors vascular protection, the combination of vitamin D analogs and E2 may offer an advantage over E2 alone. In SMC, FL, JKF, and CB did not affect the stimulatory influence seen with low concentrations of gonadal steroids, yet they prevented the suppression of DNA synthesis exerted by high doses of E2 and DHT. This disinhibition of SMC growth was, in essence, duplicated when RAL (an estrogen agonist in this system) rather then E2 was present, thus further confirming that vitamin D analogs block the inhibitory effects on SMC growth exerted by high concentrations of estrogenic agents (Figures 3, 5). Of note is the observation that a nuclear inhibitor of vitamin D action (but not an inhibitor of its membranous effects) prevented the analog-induced enhancement of the stimulatory effect of E2 in E304 cells (Figure 7). This suggests that the analog-dependent augmentation of [3H] thymidine incorporation and CK activity induced by E2 in endothelial cells was mediated by classic nuclear routes of vitamin D action. It is not clear why the disinhibition of the suppressive effect of E2 on DNA synthesis exerted by vitamin D analogs in SMC was not reversed by the inhibitor, such that the combination of the vitamin D agonist and its nuclear antagonist resulted in stimulation of growth after high concentrations of E2 (rather than the restoration of the inhibitory effect of high concentrations of E2; Figure 7). In fact, this effect resembled the induction of growth seen in SMC with low concentrations of E2. One potential mechanism by which vitamin D analogs may modify the vascular actions of estrogens is via modulation of ER expression. Indeed, there are precedents for such interactions in bone-marrow– derived stromal cells,8 as well as in bone and cartilage cells.7 Results depicted in Figure 6 appear consistent with this possibility; 1,25(OH)2 vitamin D3, as well as its analog JKF, reduced the expression of ER protein in SMC, but not in E304 cells. Observations that the inhibitory effect of estrogen on SMC proliferation is preserved in ER␣ knockout mice suggest that the ER isoform is involved is E2-mediated suppression of SMC growth.24 If the ER isoform is also involved in the inhibitory effect of E2 on DNA synthesis in human SMC, its downregulation by vitamin D analogs (Figure 6) could negate this effect of E2. How the observed increase in the ER␣ isoforms induced by 1,25(OH)2 vitamin D3 and its analog in E304 cells relates to the enhancement of E2-dependent growth attained by these agents cannot be determined based on these preliminary observations. In summary, vitamin D analogs and gonadal steroids have independent effects on DNA synthesis and CK activity in human SMC and E304 cells. Moreover,
vitamin D analogs interact with gonadal steroids in a dose- and cell-specific manner. Vitamin D analogs modulate the effects of E2 on DNA synthesis, apparently via classic nuclear routes of vitamin D action. Downregulation of ER expression and upregulation of ER␣ protein by vitamin D analogs may contribute to the interaction between these agents and estrogens in human vascular cells. ACKNOWLEDGMENTS FL was donated by Sumitomo Pharmaceutical Co. Ltd., Osaka, Japan. JKF was synthesized and donated by Dr. G.H. Posner from the Department of Chemistry, Johns Hopkins University, Baltimore, MD. CB 1093 was a gift of Leo Pharmaceutial Co., Ballerup, Denmark. Raloxifene was the gift of Novartis, Basel, Switzerland. HL was a gift of GBF, Braunschweig Germany, and ZK 159222 was kindly provided by Schering Co., Berlin, Germany. Polyclonal anti-ER␣ was the gift of Dr. H. Thole, Germany, and polyclonal anti-ER was kindly provided by Dr. N. Ben-Jonathan, University of Cincinnati, Cincinnati, OH. We also thank Mrs. Rivka Morris for her expert assistance in preparing the manuscript and Prof. A.M. Kaye and Mrs. Anna Waisman for their assistance in the CK assays and fruitful discussion of the data.
REFERENCES 1.
Somjen D, Kohen F, Jaffe A, Amir-Zaltsman Y, Knoll E, Stern N: Effects of gonadal steroids and their antagonists on DNA synthesis in human vascular cells. Hypertension 1998;32:39 – 45.
2.
Kaye AM, Sheratzky D, Lindner HR: Kinetics of DNA synthesis in immature rat uterus: age dependence and estradiol stimulation. Biochimica Biophysica Acta 1971; 261:475– 486.
3.
Fournier B, Haring S, Kaye AM, Somjen D: Stimulation of creatine kinase specific activity in human osteoblast and endometrial cells by estrogens and anti-estrogens and its modulation by calciotropic hormones. J Endocrinol 1996;150:275–285.
4.
James SY, Mackay AB, Binderup L, Colston K: Effects of a new synthetic vitamin D analogue, EB 1089, on estrogen-responsive growth of human breast cancer cells. J Endocrinol 1994;141:555–563.
5.
Posner GH, Lee JK, Wang O, Pelleg S, Bunke M, Brom H, Dolan P, Kensler T: Noncalcemic, antiproliferative, transeriptionally active, 24-fluorinated hybrid analogues of the hormone 1␣,25-dihydroxyvitamin D3. Synthesis and preliminary biological evaluation. J Med Chem 1998;41:3008 –3014.
6.
Somjen D, Weisman Y, Kaye AM: Increased sensitivity to sex steroids after pretreatment of skeletal derived cells with vitamin D metabolites, in Norman AW, Bouillon R, Thomasset M (eds): Proceedings of the 9th Vitamin D Workshop, 1994, Orlando, FL, pp 737–738.
7.
Somjen D, Waisman A, Weisman Y, Kaye AM: Nonhypercalcemic analogs of vitamin D stimulate creatine kinase B activity in osteoblast-like ROS 17/2.8 cells and up-regulate their responsiveness to estrogens. Steroids 1998;63:340 –343.
8.
Bellido T, Girasole G, Paseri G, Yu XP, Mocharia H, Jilka RL, Notides A, Manolagas SC: Demonstration of estrogen and vitamin D receptors in bone marrow-
AJH–APRIL 2000 –VOL. 13, NO. 4, PART 1
derived stromal cells: upregulation of the estrogen receptor by 1,25-dihydroxy vitamin D3. Endocrinology 1993;133:553–562. 9.
Somjen D, Waisman A, Kaye AM, Posner GH, Lee JK, Weisman Y: Non-hypercalcemic Vitamin D analogs upregulate female rat responses and sensitivity to estrogen. The American Endocrine Society’s 80th Annual Meeting, 1998; June 24 –27th New Orleans, p. 242 (abstract).
VITAMIN D, ESTROGENS IN VASCULAR SMC
17.
18.
10.
Ferrini RL, Barret-Connor E: Sex hormone and age: a cross sectional study of testosterone and estradiol and their bioavailable fractions in community-dwelling men. Am J Epidemiol 1998;147:750 –754.
19.
11.
Bouilion R, Carmeliet G, Boonen S: Ageing and calcium metabolism. Baillier’s Clin Endocrinol Metab 1997;11: 341–365.
20.
12.
Kisch E, Jaffe A, Knoll E, Stern N: Role of lipoxygenase pathway in angiotensin II - induced vasoconstriction in the human placenta. Hypertension 1997;29:796 – 801.
13.
Kwashima H: 1,25 dihydroxyvitamin D3 stimulates CaATPase in VSMC line. Biochem Biophys Res Commun 1988;150:1138 –1143.
22.
Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt Biochem 1976;72:248 –254.
23.
14.
15.
Mitsuhashi T, Morris RC Jr, Ives HE: 1,25 dihydroxyvitamin D3 modulates growth of vascular smooth muscle cells. J Clin Invest 1991;87:1889 –1895.
16.
Xue H, McCarron DA, Bukoski RD: 1,25(OH)2 vitamin D3 attenuates the loss of resistance artery contractile
21.
24.
403
function associated with incubation in culture media. Biochem Biophys Res Commun 1991;174:11–17. Bukoski RD, DeWan P, McCarron DA: 1,25 (OH)2 vitamin D3 modifies growth and contractile function of vascular smooth muscle of spontaneously hypertensive rats. Am J Hypertens 1989;2:553–556. Carthy EP, Yamashita W, Hsu A, Ooi BS: 1,25 dihydroxyvitamin D3 and rat vascular smooth muscle cell growth. Hypertension 1989;13:954 –959. Koh E, Morimoto S, Nobata T, Takamoto S, Kitano S, Ogihara T: Effects of 1,25 dihydroxyvitamin D3 on the synthesis of DNA and glycosaminoglycans by rat aortic smooth muscle cells in vitro. Life Sci 1990;46:1545–1551. Weinreich T, Merke J, Schonermark M, Reichel H, Diebold M, Hansch GM, Ritz E: Action of 1,25 dihydroxyvitamin D3 on human mesangial cells. Am J Kid Dis 1991;17:359 –366. Taura S, Taura M, Kamio A, Kummerow FA. Vitamin D-induced coronary atherosclerosis in normolipeuic swine: comparison with human disease. Tohoku J Exper Med 1979;129:9 –16. Takeo T, Anan M, Fujioka, Kajihara T, Hiraga S, Miyake K, Tanonaka K, Minematsu R, Mori H, Taniguchi Y: Functional changes of aorta with massive accumulation of calcium. Atherosclerosis 1989;77:175–181. Brown AJ: Vitamin D analogues. Am J Kid Dis 1988; 32(2 suppl 2):S25–S39. Iafrati MD, Karas RII, Aronovitz M, Kim S, Sullivan TR Jr, Lubahn DB, O’Donnell TF Jr, Korach KS, Mendelson ME: Estrogen inhibits the vascular injury response in estrogen receptor alpha-deficient mice. Nature Med 1997;3:545–548.