Estrogen Withdrawal from Osteoblasts and Osteocytes Causes Increased Mineralization and Apoptosis

M. Á. Brennan; M. G. Haugh; F. J. O’Brien; L. M. McNamara

doi:10.1055/s-0033-1363265

Hormone and Metabolic Research, Table of Contents

Horm Metab Res 2014; 46(08): 537-545
DOI: 10.1055/s-0033-1363265

Endocrine Research

Estrogen Withdrawal from Osteoblasts and Osteocytes Causes Increased Mineralization and Apoptosis

M. Á. Brennan

¹Biomechanics Research Centre (BMEC), Department of Mechanical and Biomedical Engineering, National University of Ireland, Galway, Ireland

²National Centre for Biomedical Engineering Sciences, NCBES, National University of Ireland, Galway, Ireland

³Bioengineering Sciences Research Group, Faculty of Engineering and the Environment, University of Southampton, UK

,

M. G. Haugh

¹Biomechanics Research Centre (BMEC), Department of Mechanical and Biomedical Engineering, National University of Ireland, Galway, Ireland

²National Centre for Biomedical Engineering Sciences, NCBES, National University of Ireland, Galway, Ireland

,

F. J. O’Brien

⁴Department of Anatomy, Royal College of Surgeons in Ireland, 123 St. Stephens Green, Dublin 2, Ireland

⁵Trinity Centre for Bioengineering, Trinity College, Dublin 2, Ireland

,

L. M. McNamara

¹Biomechanics Research Centre (BMEC), Department of Mechanical and Biomedical Engineering, National University of Ireland, Galway, Ireland

²National Centre for Biomedical Engineering Sciences, NCBES, National University of Ireland, Galway, Ireland

› Author Affiliations

Abstract

Recent studies have demonstrated increased bone mineral heterogeneity following estrogen withdrawal in vivo. Such changes likely contribute to fracture risk during post-menopausal osteoporosis since tissue mineralization is correlated with bone strength and stiffness. However, the cellular mechanisms responsible for increased mineral variability have not yet been distinguished. The objective of this study is to elucidate how alterations in mineral distribution are initiated during estrogen depletion. Specifically, we tested two separate hypotheses; (1) estrogen deficiency directly alters osteoblast mineralization and (2) estrogen deficiency increases bone cell apoptosis. Osteoblast-like cells (MC3T3-E1) and osteocyte-like cells (MLO-Y4) were pretreated with or without estrogen (17β-estradiol) for 14 days. Estrogen deficiency was subsequently induced by either withdrawing estrogen from cells or blocking estrogen receptors using an estrogen antagonist, fulvestrant (ICI 182,780). Cell number (Hoechst DNA), alkaline phosphatase activity (p-NPP), mineralization (alizarin red) and apoptosis (Caspase 3/7) were evaluated. Whether estrogen withdrawal altered apoptosis rates in the presence of an apoptosis promoting agent (etoposide) was also determined. Interestingly, estrogen withdrawal from cells accustomed to estrogen exposure caused significantly increased osteoblast mineralization and osteocyte apoptosis compared with continued estrogen treatment. In contrast, blocking estrogen receptors with fulvestrant abrogated the mineralization induced by estrogen treatment. When apoptosis was induced using etoposide, cells undergoing estrogen withdrawal increased apoptosis compared to cells with continued estrogen treatment. Recognizing the underlying mechanisms regulating bone cell mineralization and apoptosis during estrogen deficiency and their consequences is necessary to further our knowledge of osteoporosis.

Key words

estrogen deficiency - osteoblasts - osteocytes - mineralization - apoptosis

Full Text

References

References
1 Balena R, Toolan BC, Shea M, Markatos A, Myers ER, Lee SC, Opas EE, Seedor JG, Klein H, Frankenfield D. The effects of 2-year treatment with the aminobisphosphonate alendronate on bone metabolism, bone histomorphometry, and bone strength in ovariectomized nonhuman primates. J Clin Invest 1993; 92: 2577-2586
2 Cummings SR, Melton LJ. Epidemiology and outcomes of osteoporotic fractures. Lancet 2002; 359: 1761-1767
3 Windahl SH, Börjesson AE, Farman HH, Engdahl C, Movérare-Skrtic S, Sjögren K, Lagerquist MK, Kindblom JM, Koskela A, Tuukkanen J, Divieti Pajevic P, Feng JQ, Dahlman-Wright K, Antonson P, Gustafsson J-Å, Ohlsson C. Estrogen receptor-α in osteocytes is important for trabecular bone formation in male mice. Proc Natl Acad Sci USA 2013; 110: 2294-2299
4 Almeida M, Iyer S, Martin-Millan M, Bartell SM, Han L, Ambrogini E, Onal M, Xiong J, Weinstein RS, Jilka RL, O’Brien CA, Manolagas SC. Estrogen receptor-α signaling in osteoblast progenitors stimulates cortical bone accrual. J Clin Invest 2013; 123: 394-404
5 Almeida M, Han L, Martin-Millan M, Plotkin LI, Stewart SA, Roberson PK, Kousteni S, O’Brien CA, Bellido T, Parfitt AM, Weinstein RS, Jilka RL, Manolagas SC. Skeletal involution by age-associated oxidative stress and its acceleration by loss of sex steroids. J Biol Chem 2007; 282: 27285-27297
6 Currey JD. Effects of differences in mineralization on the mechanical properties of bone. Philos Trans R Soc Lond B Biol Sci 1984; 304: 509-518
7 Follet H, Boivin G, Rumelhart C, Meunier PJ. The degree of mineralization is a determinant of bone strength: a study on human calcanei. Bone 2004; 34: 783-789
8 Brennan MA, Gleeson JP, Browne M, O’Brien FJ, Thurner PJ, McNamara LM. Site specific increase in heterogeneity of trabecular bone tissue mineral during oestrogen deficiency. Eur Cell Mater 2011; 21: 396-406
9 Mulvihill BM, McNamara LM, Prendergast PJ. Loss of trabeculae by mechano-biological means may explain rapid bone loss in osteoporosis. J R Soc Interface 2008; 5: 1243-1253
10 Van der Linden JC, Birkenhager-Frenkel DH, Verhaar JA, Weinans H. Trabecular bone’s mechanical properties are affected by its non-uniform mineral distribution. J Biomech 2001; 34: 1573-1580
11 Krum SA, Miranda-Carboni GA, Hauschka PV, Carroll JS, Lane TF, Freedman LP, Brown M. Estrogen protects bone by inducing Fas ligand in osteoblasts to regulate osteoclast survival. EMBO J 2008; 27: 535-545
12 Manolagas SC, Kousteni S, Jilka RL. Sex Steroids and Bone. Recent Prog Horm Res 2002; 57: 385-409
13 Robinson J, Harris S, Riggs L, Spelsberg T. Estrogen Regulation of Human Osteoblastic Cell Proliferation and Differentiation. Endocrinology 1997; 138: 2919-2927
14 Chen F, Lee N, Wang K, Soong Y, Huang K. Effect of Estrogen and 1α, 25(OH)2 – Vitamin D3 on the Activity and Growth of Human Primary Osteoblast-like Cells in Vitro. Fertil Steril 2002; 77: 1038-1043
15 Rao L, Liu L, Murray T, McDermot E, Zhang X. Estrogen Added Intermittently, but not Continuously, Stimulates Differentiation and Bone Formation in SaS-2 Cells. Biol Pharm Bull 2003; 26: 936-945
16 Harris J. Estrogens and Progestins. New York: Academic Press; 1996
17 Patlas N, Zadik Y, Yaffe P, Patlas M, Schwartz Z, Ornoy A. The response to sex steroid hormones and vitamin D of cultured osteoblasts derived from ovariectomized mice with and without 17beta-estradiol pretreatment. Odontology 2005; 93: 16-23
18 Park J-B. Effects of low doses of estrone on the proliferation, differentiation and mineralization of osteoprecursor cells. Exp Ther Med 2012; 4: 681-684
19 Park J-B. The effects of fulvestrant, an estrogen receptor antagonist, on the proliferation, differentiation and mineralization of osteoprecursor cells. Mol Med Rep 2013; 7: 555-558
20 Passeri G, Girasole G, Jilka RL, Manolagas SC. Increased interleukin-6 production by murine bone marrow and bone cells after estrogen withdrawal. Endocrinology 1993; 133: 822-828
21 Baud C. Morphology and inframicroscopic structure of osteocytes. Acta Anat 1962; 209-225
22 Bélanger LF. Resorption without osteoclasts (osteolysis). In: Mechanisms of Hard Tissue Destruction. New York: American Association for the Advancement of Science; 1963
23 Baud C. Submicroscopic Structure and Functional aspects of the osteocyte. Clin Orthop Relat Res 1968; 227-236
24 Baylink D. Bone Formation by Osteocytes. Am J Physiol 1971; 221: 669-678
26 Delgado-Calle J, Arozamena J, Garcia-Renedo R, Garcia-Ibarbia C, Pascual-Carra MA, Gonzalez-Macias J, Riancho JA. Osteocyte deficiency in hip fractures. Calcif Tissue Int 2011; 89: 327-334
27 Tomkinson A, Reeve J, Shaw RW, Noble BS. The death of osteocytes via apoptosis accompanies estrogen withdrawal in human bone. J Clin Endocrinol Metab 1997; 82: 3128-3135
28 Kousteni S, Bellido T, Plotkin LI, O’Brien CA, Bodenner DL, Han L, Han K, DiGregorio GB, Katzenellenbogen JA, Katzenellenbogen BS, Roberson PK, Weinstein RS, Jilka RL, Manolagas SC. Nongenotropic, Sex-Nonspecific Signaling through the Estrogen or Androgen Receptors: Dissociation from Transcriptional Activity. Cell 2001; 104: 719-730
25 Frost HM. Micropetrosis. J Bone Joint Surg Am 1960; 42-A: 144-150
29 Kingsmill VJ, Boyde A. Mineralisation density of human mandibular bone: quantitative backscattered electron image analysis. J Anat 1998; 192 (Pt 2) 245-256
30 Pantschenko AG, Zhang W, Nahounou M, McCarthy MB, Stover ML, Lichtler AC, Clark SH, Gronowicz GA. Effect of osteoblast-targeted expression of bcl-2 in bone: differential response in male and female mice. J Bone Miner Res 2005; 20: 1414-1429
31 Sudo H, Kodama HA, Amagai Y, Yamamoto S, Kasai S. In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria. J Cell Biol 1983; 96: 191-198
32 Kato Y, Windle JJ, Koop BA, Mundy GR, Bonewald LF. Establishment of an osteocyte-like cell line, MLO-Y4. J Bone Miner Res 1997; 12: 2014-2023
33 Haisenleder DJ, Schoenfelder AH, Marcinko ES, Geddis LM, Marshall JC. Estimation of Estradiol in Mouse Serum Samples: Evaluation of Commercial Estradiol Immunoassays. Endocrinology 2011; 152: 4443-4447
34 Porter KL, Chanda S, Wang HQ, Gaido KW, Smart RC, Robinette CL. 17β-Estradiol Is a Hormonal Regulator of Mirex Tumor Promotion Sensitivity in Mice. Toxicol Sci 2002; 69: 42-48
35 Wood GA, Fata JE, Watson KLM, Khokha R. Circulating hormones and estrous stage predict cellular and stromal remodeling in murine uterus. Reproduction 2007; 133: 1035-1044
36 Wakeling AE, Bowler J. Steroidal pure antioestrogens. J Endocrinol 1987; 112: 7-10
37 Brennan O, O’Brien FJ, McNamara LM, McNamara L. Estrogen plus estrogen receptor antagonists alter mineral production by osteoblasts in vitro. Horm Metab Res 2012; 44: 47-53
38 Golub EE, Boesze-Battaglia K. The role of alkaline phosphatase in mineralization. Curr Opin Orthop 2007; 18: 444-448
39 Quarles LD, Yohay DA, Lever LW, Caton R, Wenstrup RJ. Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: An in vitro model of osteoblast development. J Bone Miner Res 1992; 7: 683-692
40 Mikunitakagaki Y, Kakai Y, Satoyoshi M, Kawano E, Suzuki Y, Kawase T, Saito S. Matrix mineralization and the differentiation of osteocyte-like cells in culture. J Bone Miner Res 1995; 10: 231-242
41 Norgaard R, Kassem M, Rattan SI. Heat shock-induced enhancement of osteoblastic differentiation of hTERT-immortalized mesenchymal stem cells. Ann N Y Acad Sci 2006; 1067: 443-447
42 Stern AR, Stern MM, Van Dyke ME, Jähn K, Prideaux M, Bonewald LF. Isolation and culture of primary osteocytes from the long bones of skeletally mature and aged mice. BioTechniques 2012; 52: 361-373
43 Balkan W, Burnstein KL, Schiller PC, Perez-Stable C, D’Ippolito G, Howard GA, Roos BA. Androgen-induced mineralization by MC3T3-E1 osteoblastic cells reveals a critical window of hormone responsiveness. Biochem Biophys Res Commun 2005; 328: 783-789
44 Hoac B, Kiffer-Moreira T, Millán JL, McKee MD. Polyphosphates inhibit extracellular matrix mineralization in MC3T3-E1 osteoblast cultures. Bone 2013; 53: 478-486
45 Luppen CA, Smith E, Spevak L, Boskey AL, Frenkel B. Bone morphogenetic protein-2 restores mineralization in glucocorticoid-inhibited MC3T3-E1 osteoblast cultures. J Bone Miner Res 2003; 18: 1186-1197
46 Nakano Y, Addison WN, Kaartinen MT. ATP-mediated mineralization of MC3T3-E1 osteoblast cultures. Bone 2007; 41: 549-561
47 Wang W, Li F, Wang K, Cheng B, Guo X. PAPSS2 Promotes Alkaline Phosphates Activity and Mineralization of Osteoblastic MC3T3-E1 Cells by Crosstalk and Smads Signal Pathways. PLoS One 2012; 7: e43475
48 Birmingham E, Niebur GL, McHugh PE, Shaw G, Barry FP, McNamara LM. Osteogenic differentiation of mesenchymal stem cells is regulated by osteocyte and osteoblast cells in a simplified bone niche. Eur Cell Mater 2012; 23: 13-27
49 Kyono A, Avishai N, Ouyang Z, Landreth GE, Murakami S. FGF and ERK signaling coordinately regulate mineralization-related genes and play essential roles in osteocyte differentiation. J Bone Miner Metab 2012; 30: 19-30
50 Kennedy OD, Brennan O, Rackard SM, Staines A, O’Brien FJ, Taylor D, Lee TC. Effects of Ovariectomy on Bone Turnover, Porosity and Biomechanical Properties in Ovine Compact Bone 12-Months Post-Surgery. J Orthop Res 2009; 27: 303-309
51 Brennan O, Kennedy OD, Lee TC, Rackard SM, O’Brien FJ. Biomechanical properties across trabeculae from the proximal femur of normal and ovariectomised sheep. J Biomech 2009; 42: 498-503
52 Chavassieux P, Garnero P, Duboeuf F, Vergnaud P, Brunner-Ferber F, Delmas PD, Meunier PJ. Effects of a new selective estrogen receptor modulator (MDL 103,323) on cancellous and cortical bone in ovariectomized ewes: a biochemical, histomorphometric, and densitometric study. J Bone Miner Res 2001; 16: 89-96
53 Yokose S, Ishizuya T, Ikeda T, Nakamura T, Tsurukami H, Kawasaki K, Suda T, Yoshiki S, Yamaguchi A. An estrogen deficiency caused by ovariectomy increases plasma levels of systemic factors that stimulate proliferation and differentiation of osteoblasts in rats. Endocrinology 1996; 137: 469-478
54 Dickenson RP, Hutton WC, Stott JR. The mechanical properties of bone in osteoporosis. J Bone Joint Surg Br 1981; 63-B: 233-238
55 Boyde A, Compston JE, Reeve J, Bell KL, Noble BS, Jones SJ, Loveridge N. Effect of estrogen suppression on the mineralization density of iliac crest biopsies in young women as assessed by backscattered electron imaging. Bone 1998; 22: 241-250
56 Jilka RL, Weinstein RS, Takahashi K, Parfitt AM, Manolagas SC. Linkage of decreased bone mass with impaired osteoblastogenesis in a murine model of accelerated senescence. J Clin Invest 1996; 97: 1732-1740
57 Bradford PG, Gerace KV, Roland RL, Chrzan BG. Estrogen regulation of apoptosis in osteoblasts. Physiol Behav 2010; 99: 181-185
58 Bronckers AL, Goei W, Luo G, Karsenty G, D’Souza RN, Lyaruu DM, Burger EH. DNA fragmentation during bone formation in neonatal rodents assessed by transferase-mediated end labeling. J Bone Miner Res 1996; 11: 1281-1291
59 Noble BS, Stevens H, Loveridge N, Reeve J. Identification of apoptotic changes in osteocytes in normal and pathological human bone. Bone 1997; 20: 273-282
60 Bonewald L. Osteocyte: A Proposed Multifunctional Bone Cell. J Musculos Neur Inter 2002; 2: 239-241
61 Carpentier VT, Wong J, Yeap Y, Gan C, Sutton-Smith P, Badiei A, Fazzalari NL, Kuliwaba JS. Increased proportion of hypermineralized osteocyte lacunae in osteoporotic and osteoarthritic human trabecular bone: implications for bone remodeling. Bone 2012; 50: 688-694