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Departments of Pediatrics (B.C.J.v.d.E., M.K., J.M.W.) and Endocrinology & Metabolic Diseases (M.K., C.W.G.M.L.), Leiden University Medical Center, Leiden 2300 RC, The Netherlands; and Laboratory of Endocrinology (J.v.d.V.), University Hospital Utrecht, Utrecht 3508 GA, The Netherlands
Address all correspondence and requests for reprints to: M. Karperien, Ph.D., Department of Endocrinology & Metabolic Diseases and Department of Pediatrics, Leiden University Medical Center, Building 1, Zone C4-R86, P.O. Box 9600, 2300 RC Leiden, The Netherlands. E-mail: karperien{at}lumc.nl.
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Abstract |
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-reductase were detected in proliferating and hypertrophic chondrocytes of the growth plate. The former three were strongly up-regulated around sexual maturation (7 wk), whereas the latter two were expressed at a relatively constant level during development. These data were supported by measuring aromatase, type I 17ß-HSD, and STS enzyme activities in chondrocytes collected from tibial growth plates at 1 and 7 wk of age. Of the enzymes studied, there were minor differences between the sexes in aromatase and 5-reductase expression only. In conclusion, our findings clearly indicate the presence of various enzymes involved in sex steroid metabolism in the tibial growth plate, especially in sexually maturing rats, a timepoint at which sex steroids have major effects on longitudinal growth. Our data suggest that intracrinology in the rat growth plate can occur and may be a major source of local sex steroid delivery. |
Introduction |
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and two with an aromatase enzyme deficiency, have clearly established that estrogens are involved in the initiation of the pubertal growth spurt and in fusion of the growth plate at the end of puberty in both boys and girls. Still, androgens seem to participate in establishing sexual differences in the skeleton, at least in the rat (2). A number of studies have unequivocally demonstrated the presence of the androgen receptor (AR) and both estrogen receptors, ER and ERß, in growth plate tissue at the mRNA and protein level in several species, including rat, rabbit, and human (3, 4, 5, 6, 7), suggesting that androgens and estrogens can directly regulate processes in the growth plate.
The gonads are the major source of circulating levels of sex steroids in the body. Besides the gonads, several human peripheral tissues contain enzymes capable of local formation of active androgens and estrogens (8, 9). In this process, also called intracrinology, precursor steroids such as dihydroepiandrosterone-sulfate (DHEA-S) and estrone-sulfate (E1-S), both present in high amounts in the circulation, are locally converted into active sex steroids such as 17ß-estradiol (E2) and testosterone (T) so that they can directly exert biological actions (8). The most important enzymes involved are aromatase p450, type I and II 17ß-hydroxysteroid dehydrogenase (HSD), steroid sulfatase (STS), and 5-reductase. Aromatase mediates the conversion of the androgens androstenedione (A) and T into the estrogens estrone (E1) and E2. Type I 17ß-HSD converts A into T and E1 to E2, whereas type II 17ß HSD catalyzes the conversion in the opposite direction. STS catalyzes the formation of DHEA and E1 from their respective sulfated precursors, DHEA-S and E1-S. Finally, 5-reductase irreversibly converts T into dihydrotestosterone (DHT). Recent data have demonstrated the expression and activity of these androgen- and estrogen-synthesizing enzymes in osteoblast-like cells, suggesting that local metabolism of sex steroids may contribute to bone mass accrual and bone remodeling (10, 11).
At present it is largely unclear whether the ligands for the ERs and AR in growth plate chondrocytes are derived from the circulation or can also be locally metabolized from circulating precursors. Previous studies using cultured chondrocytes from rabbits and humans suggest the presence of aromatase in the growth plate (12). Only very recently, aromatase immunoreactivity has been demonstrated in chondrocytes from human femoral growth plates (13). However, it is unclear, whether other enzymes in sex steroid metabolism are also present. Furthermore, it remains elusive whether the expression of aromatase and possibly other enzymes in the growth plate is regulated by sexual maturation. In this study we have, therefore, surveyed the mRNA expression of various enzymes involved in the formation of androgens and estrogens in tibial growth plates of female and male rats during development, using in situ hybridization. Furthermore, the biological activity of aromatase, type I 17ß-HSD, and STS in growth plate tissue has been assessed in 1- and 7-wk-old rats during weaning and sexual maturation, respectively.
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Materials and Methods |
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Generation of cRNA probes for in situ hybridization
In situ hybridization probes for rat type I and II 17ß-HSD were kindly provided by P. Vihko (University of Oulu, Finland). A cDNA fragment of rat aromatase was obtained from C. Y. Cheng (Center for Biomedical Research, New York, NY) and a full-length cDNA for rat 5-reductase was kindly provided by D. W. Russell (University of Texas, Dallas, TX). Using RT-PCR a cDNA fragment corresponding to nucleotides 472–723 for rat STS was generated and ligated into the pBluescript SK-vector (Invitrogen, Groningen, The Netherlands). In Table 1
an overview of the various cDNA fragments used to generate cRNA probes is depicted. Vector (10 µg) was linearized with the appropriate restriction enzymes. The probes were labeled with digoxigenin (Roche, Basel, Switzerland) as described before (6) and hydrolyzed to reduce the probe size to 200 bp as described in detail by Wilkinson (15). To assess for the efficiency of probe labeling, dot-blotting was performed as set out previously (6).
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-reductase) proteinase K (Life Technologies, Inc., Breda, The Netherlands). After washing, hybridization was performed overnight at 50 C (aromatase, type I 17ß-HSD, and STS) or 60 C (type II 17ß-HSD and 5-reductase). The hybridization mixture consisted of 50% formamid, 2x SSC (0.3 M sodium chloride and 0.03 M sodium citrate), 1x Denhardt’s solution, 10% dextrane sulfate, 1 mg/ml yeast tRNA, and the probe of interest (see Table 1
). The concentration of the sense and antisense probes was adjusted for the amount of incorporated Dig-UTP as assessed by dot-blot. After hybridization, ribonuclease A (20 µg/ml; Sigma, St. Louis, MO) treatment and blocking in sheep serum, alkaline phosphatase-conjugated anti-DIG (Roche; 1:1250) was applied overnight at 4 C. Staining was performed in a NBT/BCIP solution (Roche) varying from 6 h to overnight. Finally, the sections were counterstained with 0.5% Neutral Red (Sigma), dehydrated and embedded in Euparal (Klinipath, Köngen, Germany). Representative pictures of the growth plate sections were taken with a Nikon DXM 1200 digital camera (Uvikon BV, Bunnik, The Netherlands) using the same settings.
Aromatase activity assay
Aromatase bioactivity was determined using the tritiated water release assay (16) with small modifications. In short, tibial growth plates were isolated from 1- (n = 6) and 7-wk-old (n = 3) female and male rats, using a preparation microscope. The surrounding perichondrium/periost as well as the most upper and the most lower part of the growth plate, i.e. the stem cells and late hypertrophic chondrocytes, were removed and only the middle portion of the growth plates was used for further experimentation. In this way contamination with surrounding tissue such as cells of the perichondrium and primary spongiosa was avoided. After cutting the growth plate samples in small tissue blocks, they were placed in 24-well plates and incubated with 2 µCi [1ß-3H]-androstenedione (S.A. = 25.9 Ci/mmol; NEN Life Science Products, Boston, MA) overnight in 1,5 ml of serum-free and phenol red-free -MEM supplemented with 0.1% BSA and 100 U/ml aprotinin. Ovary and brain tissue were also collected and treated in an identical fashion. A number of tissue blocks from different animals were incubated with exemestane from Aromasin tablets (Amersham Pharmacia Biotech, Uppsala, Sweden) dissolved in concentrations ranging between 10 nM and 3 µM to determine the maximum inhibitory concentration. With increasing concentrations of exemestane, the inhibition of the total amount of tritiated water release gradually decreased and reached a maximum of 72% reduction at 300 nM. This concentration of exemestane was used in the other measurements to correct for aspecific tritiated water release. The next day, an equal volume of lysis buffer (100 mM NaCl; 10 mM Tris, pH 8.0; 25 mM EDTA; 0.05% sodium dodecyl sulfate; and 50 µg/ml proteinase K) was added and samples were incubated overnight at 56 C. Part of this solution was used for total DNA determination, using the Hoechst assay (ICN Biomedicals, Inc., Zoetermeer, The Netherlands). Then three extractions with chloroform were performed and the water phase was assayed for tritium radioactivity. The chloroform fractions were pooled and counted. 3H radioactivity was measured in a Packard 1600 TR liquid scintillation analyzer (Canberra Packard, Zellik, Belgium). Results were corrected for blanks (incubation without tissue), recovery loss, and DNA content. The average activity and the SE of the mean (SEM) were calculated. The amount of tritiated water released was expressed in attomoles per microgram DNA.
Type I 17ß-HSD activity assay
The assay to measure the bioactivity of type I 17ß-HSD has previously been described (16). In short, tibial growth plate chondrocytes from 1- (n = 6) and 7-wk-old (n = 3) female and male rats were collected as described above and incubated in serum-free and phenol red-free -MEM (Life Technologies, Inc.; custom made) with 5 pmol (90 Ci/mmol) [6,7-3H]-estrone overnight (NEN Life Science Products, Boston, MA). In analogy, positive control tissues such as ovary and brain were included. The next day, 1 ml of medium was collected and supplemented with 5000 dpm of [4-14C]-estradiol (NEN Life Science Products) to monitor procedural loss. The steroids were extracted with 2 ml of diethylether by vigorously shaking during 1 min. Samples were frozen in a solution of ethanol and dry ice and the ether phase was decanted in a glass tube. Diethylether was evaporated and the remaining pellet was dissolved in 6 drops of diethylether and spotted onto thin layer chromatography plates containing a fluorescent indicator (Merck, Darmstadt, Germany) together with unlabeled estrone and estradiol (25–50 ng each). Steroids were separated on thin layer chromatography plates run in a mixture of dichloorethane and ethylacetaat (4:1 vol/vol). The products were visualized under UV light, cut out, and placed in emulsifier-safe (Canberra Packard). 3H and 14C radioactivity were measured in a Packard 1600 TR liquid scintillation analyzer (Canberra Packard).
After having lysed the tissue in lysis buffer (100 mM NaCl; 10 mM Tris, pH 8.0; 25 mM EDTA; and 0.05% sodium dodecyl sulfate) containing 15 µl of proteinase K, total DNA in the samples was determined with a Hoechst assay (ICN Biomedicals, Inc.). Results were corrected for blanks (incubation without tissue), recovery loss, and DNA content. The average activity and SEM were calculated and the amount of estradiol formed was expressed in attomoles per microgram DNA.
STS activity assay
Measurement of sulfatase activity was a modification of a method described by Purohit et al. (17). In short, tibial growth plate chondrocytes were isolated from 1- (n = 6) and 7-wk-old (n = 6) female and male rats as above, lysed with an IKA Ultra-Turrax T25 polytron (Fischer Scientific, ‘s-Hertogenbosch, The Netherlands) in 10 mM phosphate buffer pH 7.4 and frozen immediately at -80 C. An aliquot was taken for measuring total DNA content with a Hoechst assay (ICN Biomedicals, Inc.). Testis, ovary, and brain tissue were also collected and treated in an identical fashion. Cells were incubated for 15 min at 37 C with 0.5 µCi [6,7-3H]-estrone-3-sulfate and supplemented to 20 µM with unlabeled estrone-sulfate (Makor, Jerusalem, Israel) and 2500 dpm of [4-14C]-estrone recovery standard. The reaction was stopped by addition of the specific sulfatase inhibitor 100 µM 667-COUMATE (a gift of Dr. Normanton, Sterix Ltd., Oxford, UK) and placing the samples on ice for 15 min. Unconjugated estrogens were extracted from 1 ml of the culture medium with 4 ml toluene, of which 3 ml was added to 10 ml Ultima Gold scintillation fluid (Packard). 3H and 14C radioactivity were counted in a Wallac, Inc. model 1414 liquid scintillation counter. Results were corrected for blanks (incubation without tissue), recovery loss, and DNA content. The average activity and SEM were calculated and the amount of estrone formation was expressed in picomoles per microgram DNA.
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Results |
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, A–D, respectively) and in uterine luminal and glandular epithelial cells and to a lesser extent in stromal cells (data not shown). In testis, 5-reductase mRNA was expressed in Sertoli cells, peritubular myoid cells and some Leydig cells (Fig. 1E). The expression patterns were in line with literature data (18, 19, 20, 21, 22). Sense hybridizations for all enzymes revealed no staining in any of the tissues studied as exemplified for aromatase and 5-reductase (Fig. 1, F and G, respectively).
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Before sexual maturation, aromatase was barely detectable in 1-wk-old male and very weakly expressed in female rats (data not shown). Expression of aromatase increased at 4 wk and reached highest levels at 7 wk of age, being less intense in males compared with females (Fig. 2, B vs. A). At 16 wk of age, both females and males showed similar levels of aromatase mRNA expression (Fig. 2, C and D, respectively), which was similar in 40-wk-old females (Fig. 2E).
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, G and H) and moderate levels for type II at 4 wk (Fig. 2, K and L) and 7 wk of age in both sexes (Fig. 2, M and N). After sexual maturation, expression of type I was very weak in 40-wk-old females (Fig. 2) and type II was weak in 16-wk-old female and male rats (Fig. 2, O and P) as well as in 40-wk-old female rats (Fig. 2Q). When sense hybridizations were performed, no signal was detected for all enzyme probes (Fig. 2, F, J, and R, respectively).
STS mRNA was expressed in growth plates of 1- and 4-wk-old animals as exemplified in 4-wk-old females and males (Fig. 3, A and B, respectively). The staining intensity slightly increased during and after sexual maturation as exemplified at 7 (Fig. 3, C and D) and 16 wk of age (Fig. 3, E and F) and decreased in 40-wk-old females (Fig. 3G). At 7 wk of age, STS mRNA expression extended into the early proliferating zone (Fig. 3, C and D).
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-reductase mRNA was consistently detected in growth plates of all age groups in the late proliferating and early hypertrophic chondrocytes being somewhat stronger in males than in females, as exemplified in females and males at 4 (Fig. 3, I and J, respectively), 7 (Fig. 3, L and K, respectively), and 16 wk of age (Fig. 3, M and N, respectively) and in 40-wk-old females (Fig. 3O).
Sense hybridizations for the two enzymes resulted in absence of signal in any of the growth plate sections (Fig. 3, H and P, respectively).
Bioactivity of aromatase, type I 17ß-HSD, and STS in growth plates
We subsequently measured aromatase p450, type I 17ß-HSD, and STS bioactivity in chondrocytes isolated from growth plates of female and male rats at 1 wk of age and during sexual maturation at 7 wk of age. In tibial growth plate material from female and male rats before sexual maturation (1 wk of age) as shown in Fig. 4A, aromatase activity was low (59.0 ± 18.1 and 46.0 ± 11.4 attomol/µg DNA, respectively), which strongly increased in 7-wk-old female and male rats (665 ± 143 and 718 ± 82.6 attomol/µg DNA, respectively; P < 0.001). In control tissues such as ovary and brain, a similar trend was observed, i.e. low activity before and higher activity during sexual maturation (data not shown).
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; 89.8 ± 14.4; P < 0.001 and 80.2 ± 43.1 attomol/µg DNA; P = 0.01, respectively). Ovary, testis, and brain tissue of 7-wk-old rats demonstrated type I 17ß-HSD activities, whereas in 1-wk-old female rats only ovary contained low levels of activity (data not shown).
STS activity in growth plate tissue was evident both in 1-wk-old and 7-wk-old female and male rats (Fig. 4C; 1 wk: 0.44 ± 0.17 and 0.19 ± 0.03 pmol/µg DNA, respectively; 7 wk: 0.11 ± 0.19 and 0.26 ± 0.03 pmol/µg DNA, respectively) with no apparent age-related regulation. In both age groups, control tissues such as ovary, testis, and brain demonstrated enzyme activity at a similar or higher level compared with the growth plate samples (data not shown).
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Discussion |
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and ß as well as AR have been demonstrated by our group and by others in growth plate chondrocytes of several species, including the rat (3, 4, 5, 6, 7). One of the questions that remain is what the source is of the ligands for these receptors. Are these derived from the circulation or can they be locally metabolized in the growth plate? In this study, we demonstrate that various key enzymes involved in sex steroid metabolism are present in growth plate chondrocytes and are functionally active, suggesting that intracrinology can occur in the growth plate. We furthermore demonstrate that sex steroid metabolism in the growth plate is up-regulated during sexual maturation, a timepoint at which sex steroids have an important effect on longitudinal growth. Our conclusions are based on a systemic survey of mRNA expression of the enzymes involved in sex steroid metabolism using in situ hybridization and on measurements of enzyme activity in preparations of growth plate chondrocytes.
Based on the in situ hybridization data, the mRNA expression patterns in the growth plate for aromatase p450, type I and II 17ß-HSD, STS, and 5-reductase could be divided in two distinct patterns. The pattern for aromatase and type I and II 17ß-HSD shows a gradual increase from a low or undetectable mRNA expression at 1 wk to a peak at 7 wk of age, followed by a decline thereafter. The mRNAs were, however, still present at 16 and 40 wk of age. The other expression pattern is one with a relatively consistent level of mRNA expression during development, which was observed for STS and 5-reductase.
These data were supported by the enzyme bioactivity data, in which aromatase was present at low levels and type I 17ß-HSD was absent at 1 wk of age. The bioactivity of both enzymes was strongly increased at 7 wk of age. In contrast, STS bioactivity was relatively unaffected over time. We did not observe any differences in bioactivity between female and male rats at any given age, whereas the mRNA data only showed minor differences for aromatase and 5-reductase between the genders.
The results clearly indicate that sex steroid metabolism in the growth plate is up-regulated at 7 wk of age, concomitant with sexual maturation, a timepoint at which sex steroids have a major effect on longitudinal growth. Also in other tissues, steroidogenesis increases during sexual maturation, such as in chicken gonad, rat testis, and human adrenal (23, 24, 25). The observed up-regulation of sex steroid metabolism in the growth plate during sexual maturation may well be under influence of the pituitary, which seems evident in other tissues as well (26, 27). This may be a direct effect of gonadotropins, especially LH, which is up-regulated at the beginning of puberty in humans and rats (28, 29) or it may be an indirect effect, in which the increased levels of sex steroids during puberty autoregulate their own metabolism.
So far only a limited number of reports has identified aromatase in chondrocytes. Blanchard and co-workers demonstrated conversion of T to E2 in growth plate chondrocytes from a prepubertal boy and girl and only very recently, aromatase mRNA and protein expression were demonstrated in proliferating and hypertrophic chondrocytes from human femoral growth plates, which corresponded with our data (12, 13). The presence of aromatase in the growth plate opens new possibilities for the use of selective aromatase modulators to target the growth plate specifically in boys with short stature, reducing the risk of side-effects (30). To date, besides aromatase p450, no other enzymes involved in sex steroid metabolism have been identified in growth plate chondrocytes.
Although the bioactivities of aromatase and type I 17ß-HSD seem to be quite low (<1 fmol/h/µg DNA), it is difficult to compare our data with activities mentioned in other studies because the activities are often expressed per mg of protein or per million cells. Aromatase activities in mouse and rat gonads have been reported in the range of 10 fmol/h/mg protein (31, 32), but these tissues serve as the main producers of systemic 17ß-estradiol. In contrast, human osteoblasts, recognized as a cell type in which intracrinology occurs (33), contained similar amounts of enzyme activity as in our study (±2 fmol/h/mg DNA). The lower activities in chondrocytes and osteoblasts are probably sufficient to fulfill in local needs. The relatively low bioactivities may also reflect the use of tissue blocks instead of cell cultures or cell homogenates, hampering optimal exchange of substituents but perhaps better reflecting the in vivo situation.
The presence of STS in the growth plate of the rat is of particular interest because it may significantly contribute to the formation of sex steroids in the growth plate from sulfated precursors, which circulate in the body at high levels. STS has a broad tissue distribution, including ovary, breast, brain, and cultured human osteoblasts (33, 34). Interestingly, as a consequence of metabolizing sulfated precursors into active estrogens such as E1 and E2, free sulfate groups are generated, which can be used for incorporation in proteoglycans, the major matrix components in cartilage (35).
The presence of 5-reductase supports a specific role for androgens in the control of growth plate activity. The somewhat higher 5-reductase mRNA expression in males around sexual maturation combined with lower aromatase levels at this stage than in females suggests a more active androgen metabolism in males. This is in line with recent data obtained in our group that during sexual maturation in the growth plate the AR in male rats was predominantly localized in the nucleus, whereas in females the immunostaining was mainly found in the cytoplasm (7).
Based on the mRNA expression and bioactivity data, we have clearly demonstrated that in the tibial growth plate of the rat androgen and estrogen metabolism occurs within chondrocytes. This is compatible with the idea that sex steroid target organs such as the gonad, brain, breast but also bone, and now also growth plate cartilage express steroidogenic enzymes to fulfill at least in part in local needs of sex steroids. Our study, combined with studies demonstrating aromatase, as well as ERs and AR in the growth plate (3, 5, 6, 7, 12, 13), strongly suggests that intracrinology can occur in growth plate chondrocytes of various species including the rat. Moreover, our data are in contrast to recent claims that in rodents sex steroids are exclusively formed in the gonads (8).
In conclusion, our findings clearly indicate the presence of enzymes involved in sex steroid metabolism in the tibial growth plate, which reaches high levels of expression and bioactivity during sexual maturation. Hence, intracrinology may occur in the growth plate and may be a major source of ligands for sex steroid receptors in chondrocytes.
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Acknowledgments |
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-reductase probe, respectively. We like to thank G. H. Donker for performing the STS assays. We are grateful to Prof. Dr. Thijssen for critically reading the manuscript. |
Footnotes |
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Abbreviations: A, Androstenedione; AR, androgen receptor; DHEA-S, dihydroepiandrosterone-sulfate; DHT, dihydrotestosterone; E1, estrone; E1-S, estrone-sulfate; E2, 17ß-estradiol; ER, estrogen receptor; HSD, hydroxysteroid dehydrogenase; STS, steroid sulfatase; T, testosterone.
Received January 24, 2002.
Accepted for publication June 5, 2002.
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References |
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in human and rabbit skeletal tissues. J Clin Endocrinol Metab 83:2421–2428 in growth plates of rabbits, but not in rats, at sexual maturity. Bone 24:9–16[Medline]
and ß in the epiphyseal plate of the rat. Bone 30:478–485[Medline]
and ß mRNA during differentiation of human osteoblast SV-HFO cells. Endocrinology 138:5067–5070-hydroxysteroid dehydrogenase enzymes in immature, mature, and pregnant rats. Endocrinology 138:2886–2892-reductase messenger ribonucleic acid levels and immunocytochemical localization of the type 1 protein in the rat testis during postnatal development. Endocrinology 136:5409–5415[Abstract]
) hydroxylase and P450 aromatase genes in the chicken gonad before and after sexual differentiation. Gen Comp Endocrinol 102:233–240[CrossRef][Medline]
hydroxylase/lyase and 3ß-hydroxysteroid dehydrogenase/(
5-4) isomerase messenger ribonucleic acids and steroidogenesis during pubertal development in the rat testis. J Steroid Biochem Mol Biol 73:19–28[CrossRef][Medline]
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