Abstract
Estrogen plays an important role in the human growth plate by accelerating growth and promoting epiphyseal fusion in both sexes. Nevertheless, the precise mechanisms responsible for these effects are poorly understood. In the present study, we examined the role of 17β-estradiol (E2) on cell proliferation and viability, type X collagen synthesis, alkaline phosphatase activity, and matrix calcification in primary cultures of resting, proliferating, and prehypertrophic chondrocytes derived from explants of the bovine fetal epiphyseal growth plate. Growth plate chondrocytes were isolated and separated into maturationally distinct subpopulations, which were cultured for 7–21 days to high density in either (1) serum-free medium, (2) 1 nM thyroid hormone (T3), (3) E2 concentrations ranging from 10−13 M to 10−7 M, or (4) a combination of T3 and E2. To compare E2 effects in both sexes, chondrocytes were harvested from 8 fetuses of both sexes. After hormone treatment, cell cultures were analyzed for cell number and viability, collagen type X, alkaline phosphatase (ALP), and matrix calcification. Neither DNA content nor cell viability were affected by the duration or type of hormone treatment. By itself, E2 stimulated maturation of all subpopulations only in pharmacologic doses (10−7 M). Physiologic E2 concentrations were no different than negative controls treated with ITS (insulin, transferrin, and selenite). Regardless of E2 concentrations, the addition of E2 to 1 nM T3 did not appreciably affect the response to T3 alone, which stimulates maturation of the phenotype. All effects were comparable in both male and female chondrocytes, in all cell subpopulations (maturation stages) and fetuses of varying gestational age. These findings indicate that at physiologic concentrations, the effects of E2 on fetal bovine growth plate chondrocyte appear to be indirect and independent of T3, suggesting that, in vivo, E2 acts in concert with other factors or hormones to induce fusion of the growth plate.
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References
E Smith J Boyd G Frank H Takshashi RM Cohen B Specker TC Williams DB Lubahn KS Korach (1994) ArticleTitleEstrogen resistance caused by a mutation in the estrogen receptor gene in a man N Engl J Med 331 1056–1061
C Carani K Qin M Faustini–Fustini S Serpents J Boyd KS Korach ER Simpson (1997) ArticleTitleEffect of testosterone and estradiol in a man with aromatase deficiency N Engl J Med 337 91–95
F Conte M Grumbach Y Ito C Fisher E Simspon (1994) ArticleTitleA syndrome of female pseudohermaphrodism, hypergonadotropic hypogonadism, and multicystic ovaries associated with missense mutations in the gene encoding aromatase (P450arom) J Clin Endocinol 78 1287–1292
A Morishima M Grumbach E Simpson C Fisher K Qin (1995) ArticleTitleAromatase deficiency in male and female siblings caused by a novel mutation and the physiological role of estrogens J Clin Endocrinol 80 3689–3698
S Drop W Waal ParticleDe S Muinck Keizer–Schrama ParticleDe (1998) ArticleTitleSex steroid treatment of constitutionally tall stature Endocr Rev 19 540–558
AR Poole (1991) The growth plate: Cellular physiology, cartilage assembly and mineralization B Hall S Newman (Eds) Cartilage Molecular Aspects CRC Press Boco Raton, FL 179–211
R Cancedda F Cancedda ParticleDescalzi P Castagnola (1995) ArticleTitleChondrocyte diflferentiation Int Rev Cytol 159 265–353
DA Stevens GR Williams (1999) ArticleTitleHormonal regulation of chondrocyte differentiation and endochondral bone formation Mol Cell Endocrinol 151 195–204
E Hunziker R Schenk L Cruz-Orive (1987) ArticleTitleQuantitation of chondrocyte performance in growth plate cartilage during longitudinal bone growth J of Bone Joint Surg AM 69 162–172
J Buckwalter D Mower R Ungar J Schaeffer B Ginsberg (1986) ArticleTitleMorphometric analysis of chondrocyte hypertrophy J Bone Joint Surg Am 68 243–255
G Gibson S Schor M Grant (1982) ArticleTitleEffect of matrix macromolecules on chondrocyte gene expression: Synthesis of a low molecular weight collagen species of cell cultures within collagen gels J Cell Biol 93 767–774
T Schmid H Conrad (1982) ArticleTitleA unique low molecular weight collagen secreted by cultured chick embryo chondrocytes J Bio Chem 257 12444–12450
M Alini D Carey S Hirata M Grynpas I Pidoux A Poole (1994) ArticleTitleCellular and matrix changes before and at the time of calcification in the growth plate studied in vitro: Arrest of type X collagen synthesis and net loss of collagen when calcification is initiated J Bone Mine Res 9 1077–1087
F Mwale C Billinghurst W Wu M Alini C Webber M Ionescu A Reiner J Poole AR Poole (2000) ArticleTitleSelective assembly and remodeling of collagens II and IX associated with expression of the chondrocyte hypertrophic phenotype Dev Dyn 218 IssueID4 648–662
D Dean O Muniz J Woessner D Howell (1990) ArticleTitleProduction of collagenase and tissue inhibitor of metalloproteinases by rat growth plates in culture Matrix 10 320–330
M Alini A Marriott RM Chen A Suji AR Poole (1996) ArticleTitleA novel angiogenic molecule produced at the time of chondrocyte hypertrophy during endochondral bone formation Dev Biol 176 124–132
MF Carlevaro A Albini D Ribatti C Gentili R Benelli S Cermelli R Cancedda F Cancedda Descalzi (1997) ArticleTitleTransferrin promotes endothelial cell migr ation and invasion: implication in cartilage neovascularization J Cell Biol 136 1375–1384
K Oerter Klein J Baron M Colli D McDonnell G Cutler (1994) ArticleTitleEstrogen levels in childhood determined by an ultrasensitive recombinant cell bioassay J Clin Invest 94 2475–2480
E Nasatzky Z Schwartz B Boyan W Soskolne A Ornoy (1993) ArticleTitleSex dependent effects of 17 beta estradiol on chondrocyte differentiation in culture J Cell Physiol 154 359–367
E Lee Y Matsui A Poole (1990) ArticleTitleImmunochemical and immunocytochemical studies of C-propeptide of type II collagen in chondrocytes of the growth plate J Histochem Cytochem 38 659–673
M Alini Y Kofsky W Wu I Pidoux A Poole (1996) ArticleTitleIn serum-free culture thyroid hormones induce full expression of chondrocyte hypertrophy leading to matrix calcification J Bone Miner Res 11 105–113
N Gassler T Peuschel R Pankau (2000) ArticleTitlePediatric reference values of estradiol, testosterone, leutropin, follitropin and prolactin Clin Lab 46 553–560
C Labarca K Paigen (1980) ArticleTitleA single and rapid DNA assay procedure Anal Biochem 102 344–352
N Papadopoulos G Dedoussis G Spanakos A Gritzapis C Baxevanis M Papamichail (1984) ArticleTitleAn improved fluorescence assay for the determination of lymphocyte-mediated cytotoxicity using flow cytometry J Immunol Meth 177 101–111
V Sylvia I Gay R Hardin D Dean B Boyay Z Schwartz (2002) ArticleTitleRat costochondral chondrocytes produce 17β-estradiol and regulate its production by lα,25(OH)2D3 Bone 30 57–63
B Eerden Particlevan der J Ven Particlevan de C Lowik J Wit M Karperien (2002) ArticleTitleSex steroid metabolism in the tibial growth plate of the rat Endocrinology 143 4048–4055
O Blanchard L Tsagris R Rappaport G Duval–Beaupre M Corvol (1991) ArticleTitleAge-dependent responsiveness of rabbit and human cartilage cells to sex steroids in vitro J Steroid Biochem 40 711–716
M Corvol A Carrascosa L Tsagris O Blanchard R Rappaport (1987) ArticleTitleEvidence for a direct in vitro action of sex steroids on rabbit cartilage cells during skeletal growth Endocrinology 120 1422–1429
V Syvlia J Walton D Dean B Boyan Z Schwartz (2001) ArticleTitle17 beta estradiol–BSA conjugates and 17-beta estradiol regulate growth plate chondrocytes by common membrane associated mechanisms involving PKC dependent and independent signal transduction J Cell Biochem 81 413–429
V Sylvia B Boyan D Dean Z Schwartz (2000) ArticleTitleThe membrane effects of 17 beta estradiol on chondrocyte phenotypic expression are mediated by activation of protein kinase C through phospholipase C and G-proteins J Steroid Biochem Mol Biol 73 211–224
B Eerden Particlevan der J Emons S Ahmed H Essen Particlevan C Lowik J Wit M Karperien (2002) ArticleTitleEvidence for genomic and nongenomic actions of estrogen in growth plate regulation in female and male rats at the onset of sexual maturation J Endocrinol 175 277–288
O Nilsson V Abad D Chrysis E Ritzen L Savendahl J Baron (2002) ArticleTitleEstrogen receptor alpha and beta are expressed throughout postnatal development in the rat and rabbit growth plate J Endocrinol 173 407–414
M Egerbacher M Helmreich W Rossmanith G Haeusler (2002) ArticleTitleEstrogen receptor-alpha and estrogen receptor beta are present in the human growth plate in childhood and adoescence, in identical ditribution Horm Res 58 99–103
O Vidal M Lindberg K Hollberg D Baylink G Andersson D Lubahn S Mohan J-A Gustasfsson C Ohlsson (2000) ArticleTitleEstrogen receptor specificity in the regulation of skeletal growth and maturation in male mice Proc Natl Acad Sci USA 97 5474–5479
M Weise S De-Levi K Barnes R Gafni V Abad J Baron (2001) ArticleTitleEffects of estrogen on growth plate senescence and epiphyseal fusion Proc Natl Acad Sci USA 98 6871–6876
Z Schwartz E Nasatzky A Ornoy B Brooks W Soskolne D Boyan (1994) ArticleTitleGender-specific, maturation-dependent effects of testosterone on chondrocytes in culture Endocrinology 134 1640–1647
J Ross F Cassorla M Skerda I Valk D Loriaux G Cutler SuffixJr (1983) ArticleTitleA preliminary study of the effect of estrogen dose on growth in Turner’s syndrome N Engl J Med 309 1104–1106
E Nasatzky D Grinfeld B Boyan D Dean A Ornoy Z Schwartz (1999) ArticleTitleTransforming growth factor β-1 modulates chondrocyte responsiveness to 17 β-estradiol Endocrine 11 241–249
S Ren Z Huang D Sweet S Malozowski F Cassorla (1990) ArticleTitleBiphasic response of rat tibial growth to thyroxine administration Acta Endocrinol 122 336–340
D Stevens R Hasserjian H Robson T Siebler S Shalet G Williams (2000) ArticleTitleThyroid hormones regulate hypertrophic chondrocyte differentiation and expression of parathyroid-hormone-regulated peptide and its receptor during endochondral bone formation J Bone Miner Res 15 2431–2442
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The Canadian Institutes of Health Research generously funded this study.
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Rodd, C., Jourdain, N. & Alini, M. Action of Estradiol on Epiphyseal Growth Plate Chondrocytes. Calcif Tissue Int 75, 214–224 (2004). https://doi.org/10.1007/s00223-004-0197-7
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DOI: https://doi.org/10.1007/s00223-004-0197-7