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Department of Biochemistry, University of Wisconsin-Madison, College of Agricultural of Life Sciences, Madison, WI 53706
3To whom correspondence should be addressed. No reprints will be available from the authors. E-mail: deluca{at}biochem.wisc.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • retinoids • osteoclasts • osteoblasts • vitamin A and bone
The administration of excess vitamin A has profound effects on bone. Early in vivo studies have revealed that large amounts of vitamin A administered to rats produced rarefied bones and spontaneous fractures (1 –3 ). Later in vitro studies have demonstrated that vitamin A is capable of inducing bone resorption in neonatal rat and chick bone rudiment cultures (4 ,5 ). More recent investigations have confirmed and expanded these early observations. Excess vitamin A causes an increase in bone resorption, an increase in the number and size of osteoclasts, a decrease in osteoid surface and a deterioration of cartilage in rats in addition to the previously observed spontaneous fractures (6 ,7 ). Similar abnormalities in bone as well as cases of hypercalcemia have been reported in patients administered retinoids for the treatment of disease (8 –11 ).
The exact mechanism of vitamin A action on bone, however, remains unclear. One possible explanation is that vitamin A acts directly on bone cells. Both retinoic acid receptors and retinoid X receptors have been found in osteoblasts, osteoclasts and possibly chondrocytes (12 –15 ). In addition, all-trans retinoic acid (ATRA)4 (4 ) has been shown to stimulate bone resorption in neonatal mice calvarial cultures as well as in dentine pit assays (13 ,16 ). Furthermore, ATRA up-regulates the expression of several proteins in osteoblasts and osteoclasts, including matrix proteins and lysosomal enzymes that may play a direct role in bone resorption.
A second possible mechanism of action is that vitamin A might affect bone by influencing the actions of other bone active agents, such as parathyroid hormone (PTH) and 1,25-dihydroxycholecalciferol [1,25(OH)2D3]. Unfortunately, studies of the effect of vitamin A on PTH have yielded conflicting results (6 ,17 ,18 ). Investigations into the effect of vitamin A on vitamin D action, on the other hand, have clearly shown that vitamin A is able to antagonize the ability of vitamin D to optimally raise serum calcium (Ca) concentrations and normalize bone mineralization (19 ,20 ). It also has been shown to suppress the toxic effects of high concentrations of vitamin D (21 –25 ).
A third possibility is that vitamin A, like PTH, may require 1,25(OH)2D3 to resorb bone in vivo. Although PTH can stimulate bone resorption in vitro, as seen using neonatal rat bone cultures (5 ), even large amounts of PTH are unable to cause bone Ca mobilization in vitamin D–deficient rats (26 ). In addition, thyroparathyroidectomized, nephrectomized, vitamin D–deficient rats fed a low Ca diet require both PTH and 1,25(OH)2D3 to resorb bone and normalize serum Ca concentrations (27 ).
This third hypothesis was tested in the present study. In addition to investigating ATRA-induced bone resorption and its requirement for vitamin D under normal dietary Ca and phosphorus (P) conditions, we examined the ability of ATRA to resorb bone under conditions that cause decreased bone mineralization (high dietary Ca and low dietary P) or inhibition of bone growth (low dietary Ca and normal dietary P). We report here that ATRA-induced bone resorption is independent of vitamin D and dietary Ca and P concentrations.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In all experiments, male Holtzman rats (Harlan Sprague-Dawley, Indianapolis, IN) were used. They were maintained in individual overhanging wire cages and had access to distilled water ad libitum. All rats were fed purified diets based on those described by Suda et al. (28 ). Briefly, these diets use glucose monohydrate (67%) as the carbohydrate source, vitamin-free casein (18%) as the protein source and soybean oil (10%) as fat. Vitamins and minerals were supplied exactly as described (28 ). At the completion of each experiment, rats were anesthetized and killed by decapitation. All experimental protocols were reviewed and approved by the Research Animal Resources Center [(RARC) University of Wisconsin-Madison, Madison, WI].
Experiment 1: determination of the amount of ATRA required to affect bone mineral density.
Forty-three rats (31 ± 2 d old, 
100 g) were acclimated to the animal facility for 2 wk. During this time, rats were fed Diet 11 (4.7 g of Ca and 3 g of P per kg of diet) (28
). The diet was supplemented with soybean oil (Wesson oil; Hunt-Wesson Foods, Fullerton, CA) containing 5 g vitamin E (DL-
-tocopherol; Spectrum, Gardena, CA), 0.6 g of vitamin K (menadione; Spectrum, Gardena, CA) and 0.4 g of vitamin A (ß-carotene; Sigma, St. Louis, MO) per L. The vitamin mixture (0.1 mL/rat) was added to the diet three times weekly. In addition, rats were fed 625 ng of cholecalciferol (vitamin D3; Sigma)/d as an oral dose in 0.1 mL of soybean oil. After 2 wk, rats were divided into six groups (n = 7 or 8) and switched to diets containing varying amounts (0, 10, 25, 50, 75 and 100 µg) of ATRA (Spectrum, Gardena, CA)/g of diet. Rats were pair-fed after administration of ATRA diets to normalize body weights, which were measured every 2–3 d. The rats continued to receive the previously described amounts of vitamins A, E, K and D3. The rats were fed the diets for 16 wk. During this time, the rats were scanned at 0, 8, and 16 wk with a DPX- bone scanning system (LUNAR, Madison, WI) to determine bone mineral density. For this measurement, the rats were anesthetized by an intramuscular injection (0.5–1.0 mL/kg of body) of 0.062 mmol of fentanyl citrate and 2.2 mmol of droperidol per L to determine whole body bone mineral density. The rats were killed after the 16-wk scan, and blood and femurs were collected for analysis.
Experiment 2: effect of ATRA on bone in the presence and absence of vitamin D3.
Fifty-nine weanling (20 ± 1 d old, 50–60 g) rats were depleted of vitamin D for 6 wk. During depletion, rats were fed a purified diet, Diet 11 (4.7 g of Ca and 3 g of P per kg of diet) and supplemented with the previously described amounts of vitamins A, E and K. Additionally, rats were maintained under incandescent lighting. At the end of 6 wk, serum Ca concentrations were analyzed to confirm vitamin D deficiency. An initial group (n = 6) was used to collect blood, femurs and radius/ulna for analysis. The remaining rats were divided into four groups (n = 12–14). Two of the groups were given an oral dose of 625 ng vitamin D3 in 0.1 mL of oil daily (+D groups). The other two groups were given an oral dose of 0.1 mL of oil daily (-D groups). Two of the four groups, one +D group and one –D group, were fed Diet 11 (4.7 g of Ca and 3 g of P per kg of diet) containing no additional ATRA, and the other two groups were switched to the same diet but containing 150 µg of ATRA/g of diet. Groups were pair-fed to normalize body weights, which were measured every 2–3 d. The diets were supplemented with the previously described amounts of vitamins A, E and K. After 4 wk, half of the rats in each of the four groups were killed. Blood, femurs and radius/ulna were collected. The remaining rats were killed after an additional 4 wk, and the same tissues were collected.
Experiement 3: effect of ATRA on bone in rats fed a high Ca, low P diet.
Thirty weanling rats were depleted of vitamin D for 4 wk. During this time rats were treated as described above. At the end of 4 wk, serum Ca concentrations were analyzed to confirm vitamin D deficiency. Rats were then divided into five groups (n = 6), and an initial group was killed. Blood and femurs were collected for further analysis. The remaining four groups were treated as previously described with the exception that the basal diet used was Diet 24 (12 g of Ca and 1 g of P per kg of diet) (29 ). Rats were killed after 3 wk on the ATRA diets. Blood and femurs were collected.
Experiment 4: effect of ATRA on bone in rats fed a low Ca, normal P eiet.
Forty-three weanling rats were depleted of vitamin D for 5 wk. During this time, rats were treated as described above. At the end of 5 wk, serum Ca concentrations were analyzed to confirm vitamin D deficiency. Rats were divided into five groups, and an initial group (n = 11) was killed. Blood and femurs were collected for further analysis. The remaining four groups (n = 8) were treated as described above except that the basal diet contained 0.2 g of Ca and 3 g of P per kg of diet (28 ). After being fed the diets for 3 wk, the rats were killed. Blood and femurs were collected.
Pair-feeding protocol.
Rats were pair-fed as follows. Food consumption of each rat was monitored daily. Then, the average daily food consumption of each group was calculated. The least amount of food consumed by the groups was fed to all groups the next day.
Bone ash determination.
Femurs were cleaned of adhering tissue and placed in 100% ethanol overnight. The ethanol was removed and the bones were extracted a second time in chloroform (CHCl3) overnight. The bones were removed from the CHCl3 and allowed to air dry for 5 min. Then they were placed in crucibles that had been baked in a muffle furnace, cooled in a dessicator for 3 h and weighed. The bones were baked at 100°C for 8–12 h. On removal from the 100°C oven, the bones were placed in a dessicator to cool. After 3 h, the bones were weighed. This measurement was termed the dry weight. After weighing, the bones were placed in a muffle furnace and ashed at 649°C for 24 h. Ash weight was then determined.
Rickets Severity Score.
The radius/ulna were removed from each rat and cleaned of adherent tissue. The bones were soaked overnight in distilled water, split longitudinally with a razor blade and rinsed again with distilled water before staining. The bones were stained for 10 min with AgNO3 (15 g/L) in water and then rinsed with distilled water. This procedure reveals calcified areas as a black stain while uncalcified regions remain unstained. The bone sections were examined microscopically. Epiphyseal plate width was scored on a scale of 0 to 4 [0 = normal growth plate width (area between epiphysis and diaphysis) and 4 = severe rickets] by an investigator who was blind to treatment status (30 ).
Serum calcium measurement.
Blood samples were centrifuged at 3500 rpm (1096 x g) for 15 min at room temperature using a Microfuge R centrifuge (Beckman, Fullerton, CA). Serum Ca concentrations were determined using atomic absorption spectrophotometry (Model 3110; Perkin Elmer, Wellesley, MA) (31 ) on samples diluted with lanthanum chloride (1 g/L).
Serum phosphorus analysis.
Serum P concentrations were determined as previously described (32 ). Briefly, 25 µL of serum was added to 4 mL of color developer [42 g of NH4)6MoO24 · 4H2O/L in 5 mol of HCl:2 g of malachite green per L in dH2O (3:1)] and vortexed. Then 200 µL of Tween 20 (15 g/L) was added to the developer, and the solution was vortexed again. Samples were immediately read at 660 nm on an UV spectrophotometer (UV-2100; Shimadzu, Columbia, MD).
Statistical analysis.
SAS Version 8 (SAS Institute, Cary, NC) was used to determine statistical significance of treatment groups. Data were analyzed using a mixed procedure (Proc Mixed). Contrasts were used to identify significance differences (P < 0.05) between treatment group means.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Rats fed a diet with 
10 µg of ATRA/g had significant decreases in dried and ashed femur weights after 16 wk of being fed the ATRA diet (Table 1
). The amount of dried and ashed bone decreased with increasing amounts of ATRA in the diet, with the greatest decreases in the group fed 100 µg of ATRA/g of diet. In addition, the groups fed 10 µg of ATRA/g of diet had decreased whole body bone mineral density compared with controls after 16 wk (Fig. 1
). However, only the groups fed 25 and 100 µg of ATRA/g of diet had significantly lower bone mineral density values at 8 wk compared with the group not receiving ATRA (Fig. 1) . No differences were observed in percent ash among the groups (Table 1)
. Furthermore, despite the losses in bone mineral, no significant differences were observed in serum Ca concentrations among the groups receiving 0–75 µg of ATRA/g of diet (data not shown).
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Experiment 2: effect of ATRA on bone in the presence and absence of vitamin D3.
In this study, the effect of vitamin D on the ability of ATRA to stimulate bone resorption was investigated. In the absence of vitamin D3, ATRA caused a significant decrease in bone mineral content and overall bone growth at 4 wk (Table 2 ). The loss of mineral from bone was severe enough to produce low percent ash and a widening of the epiphyseal plate. In the presence of vitamin D3, 150 µg of ATRA/g of diet similarly decreased femur ash and dry weight (Table 2) .
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For the most part, the results observed after 4 wk of feeding with the ATRA diets were very similar to the results seen after 8 wk (Table 3 ). Vitamin D–deficient rats fed 150 µg of ATRA/g of diet had significantly decreased ashed femur weight, as was seen previously. Furthermore, these rats had decreased percent ash and a widened epiphyseal plate, as were seen at 4 wk. When rats were fed vitamin D3, 150 µg of ATRA/g of diet significantly decreased dried femur weight and ashed femur weight.
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In the absence of vitamin D3, feeding of ATRA caused a significant decrease in overall bone growth and bone mineral content after 3 wk on a rachitogenic diet, a diet that impairs bone mineralization (Table 4 ). Vitamin D–deficient rats fed 150 µg of ATRA/g of diet had significantly lower dried femur weights, ashed femur weights and mineral accumulation compared with vitamin D–deficient rats not fed ATRA. Despite the significant loss of bone in these rats compared with the control group, ATRA did not affect percent ash. ATRA also did not affect the body weight of these rats compared with controls.
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ATRA had no effect on serum Ca or P concentrations (Fig. 3 ), but as expected, vitamin D3 increased both serum Ca and P concentrations. When groups did not receive vitamin D3 supplements, serum Ca concentrations were slightly below normal, as expected for a high Ca diet that allows passive absorption to occur. When rats were fed vitamin D3, the serum Ca concentrations rose to normal. Likewise in the absence of vitamin D3, serum P concentrations remained low. However, when rats were fed vitamin D3, these concentrations rose to normal.
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In addition to investigating the effect of vitamin D on ATRA-stimulated bone resorption when mineralization is impaired, we studied this effect under conditions where new bone formation is suppressed.
ATRA further suppressed bone growth and decreased mineral accumulation in rats on the low Ca, normal P diet regardless of whether vitamin D was present (Table 5 ). However, the effect of ATRA was more severe in the presence of vitamin D3 than in its absence. Regarding dried femur weight, ashed femur weight and mineral accumulation, the difference between vitamin D–replete rats fed and those not fed ATRA was greater than the difference in vitamin D–deficient rats. Percent ash, final body weights, serum Ca concentrations and serum phosphate concentrations were not significantly different between rats fed ATRA and controls in the absence or presence of vitamin D3 (Table 5 , Fig. 4 ).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The current investigation was launched to determine whether the effect of retinoids on the skeleton involves the vitamin D endocrine system. Our results clearly indicated that ATRA fed in pharmacological doses had a major impact on the skeleton by reducing skeletal mass. This was determined by both the noninvasive technique of dual photon absorptometry and ex vivo examination of the skeleton by chemical means. Clearly retinoids do not increase bone mass but markedly diminish the amount of bone. The effect of the retinoids on diminishing bone mass is independent of vitamin D. Thus, vitamin D–deficient animals show very similar losses of skeleton, ruling out the possibility that retinoids are working through the vitamin D endocrine system.
In Expt. 2, in which Ca and P were supplied at approximately normal amounts, a decrease in the skeleton was found, but it was unclear whether the decrease was due to an inhibition of skeletal formation or the result of increased bone resorption. Subsequent experiments were carried out in which diets containing very low Ca or very low P concentrations were fed, thereby limiting either skeletal growth or skeletal mineralization. Under these conditions, retinoids nevertheless caused a decrease in bone mass. In fact, under conditions where bone mineralization or new bone formation was limited, retinoids caused a loss in bone mass from the initial values, demonstrating that existing bone was resorbed under the influence of retinoids. These results appear to us to suggest that the primary action of retinoids is to stimulate osteoclast-mediated bone resorption independent of the action of vitamin D. These results were consistent with the earlier reported finding that retinoids stimulated increases in osteoclast number and activity (6
,7
,13
). Whether this bone resorption, brought about by retinoids, is mediated by the RANKL (receptor activator of nuclear factor-
B ligand) signaling pathway, the pathway by which 1,25(OH)2D3 stimulates osteoclast differentiation, activation and survival, remains unknown.
Our results cannot totally exclude a possible block in bone formation by retinoids, nor can they exclude the idea that a small component of the impact of retinoids on bone may be the result of interference with the vitamin D endocrine system. For example, it is known that retinoids will activate the 24-hydroxylase enzyme (CYP-24), the chief enzyme responsible for destruction of the active form of vitamin D (33 ,34 ). It is also possible that the antagonism between vitamin A and vitamin D observed earlier may be involved in the interference by retinoids in the formation of new bone.
Our results were consistent with the idea that the major impact of pharmacological doses of retinoids is to cause extensive bone resorption resulting in osteopenia and a high risk of fractures. It also presents a model for study of the retinoid-induced bone loss so that the mechanism can be more clearly understood.
It is of some interest that we did not observe hypercalcemia as a result of ATRA-induced bone resorption. Ca and P remained in the normal range although bone resorption was clearly taking place. It is, indeed, likely that although bone resorption was taking place, the Ca released from bone brought about a suppression of PTH, which in turn suppressed the vitamin D endocrine sources of Ca, namely intestinal absorption and bone resorption, by suppressing the production of 1,25-(OH)2D3. In any case, we did not observe hypercalcemia as a result of retinoid administration. Although our studies exclude the vitamin D endocrine system as being involved in retinoid-induced bone resorption, additional and extensive work is needed to understand exactly how the retinoids carry out this unwanted pharmacological effect. To that end, the vitamin D–deficient rat model presents a tool in this investigation.
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FOOTNOTES |
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2 Present address: Department of Pharmacology, The University of Michigan, Ann Arbor, MI 48109.
4 Abbreviations used: ATRA, all-trans retinoic acid; Ca, calcium; P, phosphorus; 1,25(OH)2D3, 1,25-dihydroxycholecalciferol; PTH, parathyroid hormone; vitamin D3, cholecalciferol.
Manuscript received 18 July 2002. Initial review completed 3 September 2002. Revision accepted 25 November 2002.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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