Int J. Morphol, 25(2):385-392, 2007.

LITERATURE REVIEW

Regulation of the Bone Healing Process by Hormones

Regulación del Proceso de Reparación Ósea por Hormonas

*Joáo Paulo Mardegan Issa; *Rodrigo Tiossi; *Amaro Sergio da Silva Mello & **Mamie Mizusaki Iyomasa

* Graduate student, Faculty of Dentistry of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Sao Paulo, Brazil.
** Professor, Faculty of Dentistry of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Sao Paulo, Brazil.

Dirección para correspondencia

SUMMARY: The skeleton has several important functions, such as structural functions that provide mobility, support, and protection for the body. It also has an important function as a reservoir for calcium and phosphorus. The aim of this article is to show the principal hormones that are involved with the bone healing process. In this article, it will be pointed the mineral homeostatic mechanisms in the skeleton, controlled by the calcium-regulating hormones: parathyroid hormone (PTH) that maintains the normal extracellular calcium levels by enhancing osteoclastic bone resorption and liberating calcium from the adult skeleton; estrogen, considering that both osteoblasts and osteoclasts express estrogen receptors, it is reasonable to assume that the effects of estrogen on skeletal remodeling could be caused, at least partly, by a direct effect on bone cells; androgen with important functions on the adult male phenotype, anabolic actions on skeleton, muscle and bone, including the effects of their metabolism in diverse tissues; and steroids that inhibit the synthesis of proteins, collagen, and proteoglycans in articular cartilage, and inactivate vitamin D, limiting calcium absorption by the gastrointestinal tract, and increasing the urinary excretion of calcium, they also inhibit the release of growth hormone, which further decreases soft tissue and bone repair and lead to a decrease in bone, ligament, and tendon strength.

KEYWORDS: Bone; Repair; Hormones

RESUMEN: El esqueleto humano tiene varias importantes funciones, tales como las estructurales que proporcionan la movilidad, soporte y protección del cuerpo. Además, tiene función de reserva de calcio y fósforo. El objetivo de este artículo es mostrar las principales hormonas envueltas en los procesos de reparación ósea. Se muestran los mecanismos de homeostasis mineral en el esqueleto, controlados por las hormonas reguladoras del calcio: hormona de la paratiroide, que mantiene normales los niveles de calcio extracelular aumentando la reabsorción ósea por osteoclastos y liberando el calcio del esqueleto humano; estrógeno, considerando que tanto los oesteoblastos como los osteoclastos presentan receptores para estrógeno, es razonable presumir que los efectos del estrógeno en la remodelación ósea puedan ser causados, por lo menos parcialmente, por un diferente efecto en las células óseas; andrógeno con importantes funciones en el fenotipo masculino, acciones anabólicas en el esqueleto, músculos y huesos, incluyendo los efectos de su metabolismo en diversos tejidos; y esferoides que inhiben la síntesis de proteínas, colágeno y proteoglicanos en el cartílago articular e inactivan la vitamina D, limitando la absorción de calcio por el tracto gastrointestinal y aumentando la excreción urinaria de calcio, ellos también inhiben la liberación de hormona del crecimiento, lo que llevará a una disminución de tejido blando y de la reparación ósea y en consecuencia a una disminución en la fuerza de los huesos, ligamentos y tendones.

PALABRAS CLAVE: Hueso; Reparación, Hormonas.

INTRODUCTION

The skeleton has several important functions, such as structural functions that provide mobility, support, and protection for the body. It also has an important function as a reservoir for calcium and phosphorus. To fulfill the structural functions, it is not only the amount of bone tissue present in the skeleton that is important, but also the architecture and shapes of bones. One important structural function of the skeleton is the anchoring of the teeth to the jawbones, a function that involves bone tissue, periodontal ligament, and cementum on the roots of the teeth. The reservoir role played by the skeleton is important for regulation of blood levels of calcium and phosphorus, which, in turn, are influenced by mineral uptake in the intestine and mineral excretion in the urine. The mineral homeostatic mechanisms in the skeleton are controlled by the calcium-regulating hormones: parathyroid hormone (PTH), steroids, estrogen, androgen and l,25(OH)2-vitaminD3 (D3), which regulate the activity of the bone-resorbing cells. Thus, the objective of this article is to show by a literature review the principal hormones associated with the bone healing process.

DISCUSSION

Physiologic bone remodeling. Remodeling of the skeleton is crucial for maintaining its quality, and it is estimated that approximately 10% of the skeleton is renewed each year by this process. Trabecular rather than cortical bone is more frequently remodeled, which explains why metabolic bone diseases such as osteoporosis are mainly, but not exclusively, observed in bones with comparatively large amounts of trabecular bone, e.g., the distal forearm, spine, and hip.

Bone resorption and bone formation do not occur randomly in the skeleton, but take place at so-called bone multi-cellular units. It is estimated that the human skeleton has 1 x 106 such units (Riggs & Parfitt, 2005). The remodeling process in bone multi-cellular units is initiated by osteoclastic resorption. However, since osteoclast formation and activation are controlled by osteoblasts (covering the bone surfaces), the most initial phase consists of the catabolic activation of osteoblasts. It is not likely that actively bone-forming osteoblasts are the cells that activate osteoclasts. Rather, inactive osteoblasts, either the so-called lining cells or the pre-osteoblast, are responsible, although this has not been definitively shown. It is completely unknown which molecules activate this change in the phenotype of osteoblasts/lining cells during the physiological remodeling process, with the exception of the remodeling that is part of the hormonal regulation of calcium homeostasis. It is well-known that loading plays an important role: a low amount of loading leads to bone loss, due to decreased anabolic activity of osteoblasts and increased osteoclastic resorption, and high loading causes increased bone mineral density, due to the anabolic activation of osteoblasts. Two commonly cited examples are the decreased bone mineral density that can be observed during space flights (up to 2% loss per month) and the increased bone mineral density (up to 35% more) in the racket arms of tennis players.

The surfaces of all bone tissues are covered by a single cell layer of osteoblasts, which means that these cells cover all trabecular bone and are present as the innermost cell layer in the endosteum and periosteum of cortical bone. Activation of a remodeling cycle initially leads to osteoblastic degradation of the unmineralized osteoid that exists between the osteoblastic cell layer and the mineralized bone. This is necessary, since the osteoclast cannot adhere to unmineralized bone and is capable of only resorbing mineralized bone. Next, the osteoblasts increase their expressions of receptor activator of nuclear factor B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). In addition, the expression of osteoprotegerin (OPG; an inhibitor of RANK activation due to its function as a decoy receptor binding to RANKL) is decreased. This will allow more of the RANKL molecules to activate the receptor RANK. By a process that requires cell-to-cell contact, RANKL will activate its cognate receptors, RANK, on osteoclast progenitor cells. Together with the activation of the receptor c-Fms by M-CSF, this will lead to an expansion of the osteoclast progenitor pool, increased survival of these cells, and the initiation of a differentiation program that terminates in fusion of the mononucleated progenitor cells and the development of latent multi-nucleated osteoclasts. Finally, these latent osteoclasts become activated to bone-resorbing osteoclasts.

Recently, it has been shown that activation of two adapter proteins, DNAX-activating protein 12 (DAP 12) and Fc receptor common subunit (FcR), is also crucial for osteoclast differentiation (reviewed by Takayanagi, 2005). DAP 12 associates with a variety of ligand-recognizing receptors, so-called DAP12-associated receptors (DARs), such as triggering receptor expressed in myeloid cells 2 (TREM2), TREM3, myeloid DAP12-associated lectin-1 (MDL-1), natural killer cells group 2D (NKG2D), and signal- regulatory protein 131 (SIRP1B), whereas FcR associates with two receptors: osteoclast-associated receptor (OSCAR) and paired immunoglobulin-like receptor A (PIR-A). Activation of FcR and DAP 12 leads to recruitment and activation of the tyrosine kinases Syk and Zap70, and to the activation of immunoreceptor tyrosine-based activation motifs (ITAM), which are present in the cytoplasmic tails of both FcR and DAP12, and it seems as if there is a redundancy between the two activation pathways. Very little is known about the ligands for the receptors associated with FcR and DAP 12, but circumstantial evidence indicates that DARs are activated by an unknown ligand expressed by osteoclast progenitor cells, and that the ligand for OSCAR is expressed on osteoblasts.

The final step in the activation of the remodeling process is the retraction of the osteoblasts from the bone surface, so that the multi-nucleated osteoclasts can gain access to mineralized bone. The giant cells attach to bone by vitronection receptors (vB3), expressed preferentially in the sealing zone. Importantly, this integrin has binding sites for Arg-Gly-Asp (RGD) sequences in osteopontin and bone sialoprotein, present on the surface of the exposed mineralized bone. When bound to bone extracellular matrix, osteoclasts develop a ruffled border, and by means of a proton pump and a chloride channel (C1C-7) in the ruffled border membrane, an acidic milieu is created in Howship's resorption lacunae, and the hydroxyapatite crystals will be dissolved. The demineralized organic matrix of bone will subsequently be degraded by proteolytic enzymes, including highly collagenolytic cathepsin K.

When remodeling has been initiated by osteoblast-dependent stimulation of osteoclast formation and activity, the osteoclasts create resorption lacunae. Then, the osteoclasts leave the lacunae, and a less-well-characterized mononuclear cell appears in the lacunae, "cleaning up" the organic matrix left behind by the osteoclasts, and possibly also forming the more intensively stained cementum line in the bottom of the lacunae (Everts etal., 2002). Subsequently, osteoblast precursor cells are recruited to the lacunae, where they differentiate into fully active osteoblasts that will fill the resorption lacunae with new bone. It has been suggested that insulin growth factor-I (IGF-I) and transforming growth factor-B (TGF-B), both of which are abundant in the extracellular matrix of bone and are released during the resorption process, play important roles in the recruitment and activation of the osteoblasts in the bone multicellular units. In this context, these growth factors are referred to as "coupling factors", linking bone formation to bone resorption.

A crucial event in the initiation of bone resorption is the activation of the receptors RANK and c-Fms on osteoclast progenitor cells. Although much is known about the regulation of RANKL, OPG, RANK, and M-CSF expression by hormones and cytokines, it is not known which molecules regulate the expression of these cytokines during physiological remodeling.

Parathyroid hormone. Endogenous parathyroid hormone (PTH) functions to maintain normal extracellular calcium levels by enhancing osteoclastic bone resorption and liberating calcium from the adult skeleton. In contrast, when administered intermittently as a pharmacologic agent, exogenous PTH has been shown to exert significant anabolic effects (Baldassare etal, 2001; Ciármela etal., 2003; Croy et al, 2003). Indeed, in experimental models of osteoporosis in which bone loss was induced by ovariectomy or orchiodectomy, intermittent treatment with rhPTH(l-34) leads to increased osteoblastic activity and recovery of bone mass (Beppu et al, 2000; Casagrandi et al, 2003; Debieve etal., 2006; Dono etal., 1993).

Recent reports have demonstrated that intermittent treatment with PTH promotes osteogenesis in experimental fracture healing (Aitkene? al, 1996; Alexander et al, 1996; Ando etal, 1998; Bersinger etal, 2003; Chang etal, 2002). We have already shown that intermittent low-dose treatment with rhPTH( 1-34) can increase hard callus formation (callus formed by intramembranous bone formation in periosteal osteoblasts and osteoprogenitor cells) and its mechanical strength without apparent systemic side-effects (Chang et al, 2002). In that report, we also demonstrated that treatment with PTH stimulates proliferation and differentiation of osteoprogenitor cells and production of bone matrix proteins early in the fracture healing process. Thus, based on its osteogenic effects, treatment of fractures with intermittent low-dose rhPTH(l-34) may represent an effective strategy for the enhancement of fracture healing and could be the first systemic intervention for the repair of skeletal injuries.

Chondrogenesis, an essential component of endochondral ossification in long bones, is a key component of fracture healing. Growth factors and cytokines that are produced by the inflammatory response to skeletal injury support the chemotaxis of immature mesenchymal cells to the site of the fracture and promote their differentiation into chondrocytes. A well-organized development and maturation process progresses, leading to the formation of calcified cartilage that is subsequently replaced by bone (Chang et al, 1999; Chow et al, 2001). Thus, in addition to the knowledge of the osteogenic response to PTH in the treatment of fractures, a detailed understanding of the mechanism by which PTH regulates chondrogenesis may also have important clinical implications. Because previous studies on developing embryos and long bones have shown that PTH/PTH-related peptide (PTHrP) is a negative regulator of chondrocyte hypertrophy (Albano et al, 1993; Bulmer et al, 1988; Caniggia et al, 1999; Daluiski et al, 2001; Debieve et al, 2000; Dong et al, 1996), the possibility that exogenous PTH could suppress the hypertrophy of chondrocytes and delay the replacement of cartilage with bone during fracture healing requires investigation. To the best of our knowledge, there have been no reports that specifically describe the effects of exogenous PTH on chondrogenesis in fracture healing.

Estrogen hormone. Since both osteoblasts and osteoclasts express estrogen receptors, it is reasonable to assume that the effects of estrogen on skeletal remodeling could be caused, at least partly, by a direct effect on bone cells. It has in fact been shown that estrogen receptors in differentiated osteoclasts are functional and cause decreased bone-resorbing activity (Oursler et al, 1991 a; Taranta et al, 2002) and enhanced apoptosis (Hughes et al, 1996; Chen et al, 2005). However, ERmRNAis substantially down-regulated during osteoclastic differentiation (Garcia Palacios et al, 2005); therefore, estrogen receptors present in osteoclast progenitor cells are probably more important than those in the terminally differentiated osteoclasts. Activation of these estrogen receptors leads to the inhibition of osteoclast formation, as shown with use of the monocytic RAW 264.7 cells stimulated by RANKL (Shevde etal, 2000; Srivastava etal, 2001; Garcia Palacios etal.) and mouse bone marrow macrophages stimulated by both M-CSF and RANKL (Shevde et al; Srivastava et al.).

The mechanism has been attributed to effects by estrogen on signaling pathways downstream of RANK, including inhibition of c-Jun amino terminal kinases, resulting in decreased activation of AP-1 (Shevde et al; Srivastava etal. 2001), NF-B and ERK1,2 (Garcia Palacios etal.). These mechanisms of action by estrogen make sense, since the crucial roles of AP-1 and NF-B pathways in osteoclasto-genesis have been demonstrated in mice deficient in c-Fos, or in both the NF-B subunits p50 and p52. Thus, c-Fos-7- and p50-7- /p52-/- mice exhibit osteopetrosis due to lack of osteoclasts (Grigoriadis et al. 1994; Iotsova et al. 1997). The inhibitory effects of estrogen are mediated by ER, since osteoclast progenitor cells are devoid of ERJ3. The inhibitory effects on osteoclast formation and c-Jun amino terminal kinase are also obtained with the selective estrogen-receptor modulators raloxifene and tamoxifen (Shevde et al.).

Since osteoblasts/stromal cells are crucial for osteoclast formation, due to the expression of M-CSF, RANKL, and OPG, activation of the estrogen receptors in these cells may also play a role in the regulation of osteoclastogenesis. Consistent with this view, it has been demonstrated that estrogen increases OPG mRNA and protein expression in human osteoblasts (Hofbauer et al, 1999) and murine stromal cells (Saika et al., 2001), an effect most likely mediated by ER. Similarly, the SERM compound raloxifene stimulates OPG formation at both protein and mRNA levels in human osteoblasts that predominantly express ER (Viereck et al., 2003). Furthermore, OPG levels in blood are higher in post-menopausal women on hormone replacement therapy, compared with those in women without treatment (Browner et al, 2001). Although serum analyses of OPG are complicated by the fact that OPG is ubiquitously expressed, another study showed that OPG serum levels were positively correlated with 17B-estradiol and bone mineral density (Rogers et al. 2002). No convincing evidence for a direct effect of estrogen on RANKL expression in osteoblasts, or stromal cells, has been reported so far, although the RANKL levels are likely to be influenced secondarily to effects by estrogen on RANKL-stimulating cytokines (see below). The fact that no estrogen-responsive elements seem to exist in the RANKL promoter (Kitazawa et al., 1999) also argues for the notion that estrogen does not directly regulate RANKL transcription. The studies by Eghbali-Fatourechi et al., 2003, showing that surface-expressed RANKL protein on human bone marrow stromal cells, isolated by flow cytometry, was substantially increased in cells from post-menopausal women, as compared with pre-menopausal women, as well as with estrogen-treated post-menopausal women—demonstrate that estrogen can regulate RANKL expression on bone marrow stromal cells, although the studies do not demonstrate if this is a direct or indirect effect.

Estrogen may also influence osteoclast formation by decreasing the expression ofM-CSF (Sarma etal., 1998; Lea et al., 1999). Thus, it is possible that estrogen may control bone resorption by several mechanisms crucial for osteoclast differentiation, via receptors in both, osteoblasts/ stromal cells and osteoclast progenitor cells. Although most of the loss in bone mass caused by estrogen deficiency is primarily due to enhanced bone resorption, decreased bone formation is also a contributing factor (Chow et al, 1992; Qu et al., 1998). It is less-well-understood, however, how estrogen controls the anabolic activities of osteoblasts. It has been suggested that decreased expression of TGF-B (Oursler et al, 1991b) and IGF-I (Ernst et al, 1989) in osteoblasts leads to a decreased stimulation of osteoblast proliferation and differentiation during estrogen deficiency. Estrogen also stimulates the expression of type I collagen (Ernst etal, 1989), and decreased levels of estrogen would then result in osteoblasts less active in producing an extracellular matrix. The observation that estrogen can decrease osteoblast apoptosis and thereby increase the lifespan of these cells has been put forward as a mechanism by which estrogen could control bone formation (Manolagas, 2000). The opposite effects of estrogen on osteoblast and osteoclast apoptosis have recently been attributed to different kinetics ofErkphosphorylation, since estrogen causes a transient phosphorylation of Erk in osteoblasts/osteocytes and a sustained phosphorylation in osteoclasts (Chen et al., 2005). There is also direct in vivo evidence that estrogen is important for osteoblastic function (Tobias & Compston, 1999).

Androgens. The biological activities of androgens are universal, affecting most tissues. Their important functions include the development of the adult male phenotype, anabolic actions on skeleton, muscle and bone, including the effects of their metabolism in diverse tissues. Metabolic studies have shown that androgens, estrogens and progesterone are actively metabolized in gingival tissues of humans and animals; the presence of inflammation can increase the level of activity of the enzymes which metabolize these hormones. When testosterone was incubated with homogenate, mitochondrial, microsomal and soluble fractions of healthy and inflamed gingivae from both sexes, the metabolic activity was higher in inflamed than in healthy tissue. In both types of tissue, testosterone was converted to DHT, suggesting that gingivae are target tissue for androgen action. Expression of the androgen receptor in periodontal and gingival tissue, suggest sensitivity to the anabolic effects of androgens. The anabolic effects of the androgen testosterone and its matrix stimulatory metabolite DHT on bone and matrix tissue have been well documented (Colvard et al, 1989; Dassouli et al., 1994). The conversion of testosterone to DHT is enhanced in cells from an inflamed source. The presence of inflammation can alter the expression of steroid hormone receptors, partly mediated by inflammatory cytokines, which can modulate the metabolism of circulating steroids and their effects on target tissues. An increase in the number of DHT receptors of about 2-3-fold has been observed in inflamed and hyperplasic tissue, implying enhanced synthetic activity characteristic of androgen target tissue under these conditions.

Cellular behavior and phenotype are controlled in part by the extracellular materials in the microenvironment. In addition to growth factors, extracellular matrix molecules which interact with each other and with receptors on the cell surface play an important part in the regulation of functional differentiation. Steroid hormones associated with matrix repair are activated by these substances by ligand-dependent and independent mechanisms.

Steroids. Corticosteroids, such as cortisone and prednisone, have an adverse effect on bone and soft tissue healing, they inactivate vitamin D, limiting calcium absorption by the gastrointestinal tract, and increasing the urinary excretion of calcium. Bone shows a decrease in calcium uptake with cortisone use, ultimately leading to weakness at the fibro-osseous junction. Corticosteroids also inhibit the release of growth hormone, which further decreases soft tissue and bone repair. Ultimately, corticosteroids lead to a decrease in bone, ligament, and tendon strength.

Corticosteroids inhibit the synthesis of proteins, collagen, and proteoglycans in articular cartilage, by inhibiting chondrocyte production, the cells that comprise and produce the articular cartilage. The net catabolic effect (weakening) of corticosteroids is inhibition of fibroblast production of collagen, ground substance, and angiogenesis (new blood vessel formation). The result is weakened synovial joints, supporting structures, articular cartilage, ligaments, and tendons. This weakness increases the pain and the increased pain leads to more steroid injections. Cortisone injections should play almost no role in sports injury care. Although anti-inflammatory medications and steroid injections reduce pain, they do so at the cost of destroying tissue.

REFERENCES

Aitken, D. A.; Wallace, E. M.; Crossley, J. A.; Swanston, I. A.; Van Pareren, Y.; Van Maarle, M.; Groóme, N. P.; Macri, J. N. & Connor, J. M. Dimeric inhibin a as a marker for down's syndrome in early pregnancy. New England Journal of Medicine., 334:1231-6, 1996.

Albano, R. M.; Groóme, N. & Smith, J. C. Activins are expressed in preimplantation mouse embryos and in ES and EC cells and are regulated on their differentiation. Development, 117:111-23, 1993.

Alexander, C. M.; Hansell, E. J.; Behrendtsen, O.; Flannery, M. L.; Kishnani, N. S.; Hawkes, S. P. & Werb, Z. Expression and function of matrix metalloproteinases and their inhibitors at the maternal-embryonic boundary during mouse embryo implantation. Development., 122:1123-36, 1996.

Ando, N.; Hirahara, E; Fukushima, J.; Kawamoto, S.; Okuda, K.; Funabashi, T.; Gorai, I. & Minaguchi, H. Differential gene expression of TGF-beta isoforms and TGF-beta receptors during the first trimester of pregnancy at the human matemal-fetal interface. Am. J. of Reproduction Immunology, 40:48-56, 1998.

Baldassarre, G.; Tucci, M.; Lembo, G.; Pacifico, F.M.; Dono, R.; Lago, C.T.; Barra, A.; Bianco, C; Viglietto, G.; Salomon, D.; et al. Atruncated form of teratocarcinoma-derived growth factor-1 (cripto-1) mRNA expressed in human colon carcinoma cell lines and tumors. Tumour Biology, 22:286-93,2001.

Beppu, FL; Kawabata, M.; Hamamoto, T.; Chytil, A.; Minowa, O.; Noda, T.; Miyazono, K. BMP type II receptor is required for gastrulation and early development of mouse embryos. Developmental Biology., 221:249-58, 2000.

Bersinger, N. A.; Smarason, A. K.; Muttukrishna, S.; Groóme, N. P.; Redman, C. W. Women withpreeclampsia have increased serum levels of pregnancy-associated plasma protein A (PAPP-A), inhibin A, activin A and soluble E-selectin. Hypertension in Pregnancy, 22:45-55,2003.

Browner, W. S.; Lui, L. Y.; Cummings, S. R. Associations of serum osteoprotegerin levels with diabetes, stroke, bone density, fractures, and mortality in elderly women. J. Clin. Endocrinol. Metab., 86:631-7, 2001.

Bulmer, J. N.; Pace, D. & Ritson, A. Immunoregulatory cells in human decidua: morphology, immunohistochemistry and function. Reproduction, Nutrition, Development, 25:1599-613, 1988.

Caniggia, I.; Grisaru-Gravnosky, S.; Kuliszewsky, M; Post, M. & Lye, S. J. Inhibition of TGF-beta 3 restores the invasive capability of extravillous trophoblasts in preeclamptic pregnancies. Eur J. of Clinical Investigation, 705:1641-650, 1999.

Casagrandi, D.; Bearfield, C; Geary, J.; Redman, C.W. & Muttukrishna, S. Inhibin, activin, follistatin, activin receptors and beta-glycan gene expression in the placental tissue of patients with pre-eclampsia. Molecular Human Reproduction, 9:199-203, 2003.

Chang, FL; Huylebroeck, D.; Verschueren, K.; Guo, Q.; Matzuk, M. M. & Zwijsen, A. Smad5 knockout mice die at mid-gestation due to multiple embryonic and extraembryonic defects. Development, 126:1631-42, 1999.

Chang, FL; Brown, C.W. & Matzuk, M. M. Genetic analysis of the mammalian transforming growth factor-beta superfamily. Endocrine Reviews, 25:787-823, 2002.

Chen, J. R.; Plotkin, L. I.; Aguirre, J. I.; Han, L.; Jilka, R. L.; Kousteni, S.; et al. Transient versus sustained phosphorylation and nuclear accumulation of ERKs underlie anti- versus pro-apoptotic effects of estrogens. J. Biol. Chem., 280:4632-8, 2005.

Chow, J.; Tobias, J. FL; Colston, K.W.; Chambers, T. J. Estrogen maintains trabecular bone volume in rats not only by suppression of bone resorption but also by stimulation of bone formation. J. Clin. Invest., 89:14-8, 1992.

Chow, J. F.; Lee, K. F.; Chan, S.T. & Yeung, W.S. Quantification of transforming growth factor betal (TGFbetal) mRNA expression in mouse preimplantation embryos and determination of TGFbeta receptor (type I and type II) expression in mouse embryos and reproductive tract. Molecular Human Reproduction., 7:1047-56, 2001.

Ciármela, P.; Florio, P.; Toti, P.; Grasso, D.; Santopietro, R.; Tosi, P. & Petraglia, F. Expression of betaglycan in pregnant tissues throughout gestation. Eur. J. of Endocrinology, 149:433-7,2003.

Colvard, D. S.; Eriksen, E. E; Keeting, P. E.; Wilson, E. M.; Lubahn, D. B.; French, F. S. et al. Proc Natl Acad Sci., 86: 854-57, 1989.

Croy, B. A.; Esadeg, S.; Chantakru, S.; Van den Heuvel, M.; Paffaro, V. A.; He, H.; Black, G. P.; Ashkar, A. A.; Kiso, Y. & Zhang, J. Update on pathways regulating the activation of uterine Natural Killer cells, their interactions with decidual spiral arteries and homing of their precursors to the uterus. J. of Reproductive Immunology, 59:175-91,2003.

Daluiski, A.; Engstrand, T.; Bahamonde, M. E.; Gamer, L. W.; Agius, E.; Stevenson, S. L.; Cox, K.; Rosen, V. & Lyons, L. M. Bone morphogenetic protein-3 is a negative regulator of bone density. Nature Genetics., 27:84-8,2001.

Dassouli, A.; Manin, M.; Veyssiere, G. & Jean, C. J. Steroid Biochem Mol Biol, 48:121-8, 1994.

Debieve, E; Pampfer, S.; Thomas, K. Inhibin and activin production and subunit expression in human placental cells cultured in vitro. Molecular Human Reproduction., 6:743-49, 2000.

Debieve, F.; Hinck, L.; Biard, J. M.; Bernard, P. & Hubinont, C. Activin receptor expression and induction of apoptosis in rat blastocysts in vitro. Human Reproduction., 21:618-23, 2006.

Dong, J.; Albertini, D. E; Nishimori, K.; Kumar, T. R.; Lu, N. & Matzuk, M. M. Growth differentiation fac-tor-9 is required during early ovarian folliculogenesis. Nature, 383: 531-5, 1996.

Dono, R.; Scalera, L.; Pacifico, F.; Acampora, D.; Pérsico, M. G. & Simeone, A. The murine cripto gene: expression during mesoderm induction and early heart morphogenesis. Development, 118:1157-68, 1993.

Eghbali-Fatourechi, G.; Khosla, S.; Sanyal, A.; Boyle, W. J.; Lacey, D. L. & Riggs, B. L. Role of RANK ligand in mediating increased bone resorption in early postmenopausal women. J. Clin. Invest., 111: 1221 -30, 2003.

Ernst, M.; Heath, J. K. & Rodan, G. A. Estradiol effects on proliferation, messenger ribonucleic acid for collagen and insulin-like growth factorl, and parathyroid hormone-stimulated adenylate cyclase activity in osteoblastic cells from calvariae and long bones. Endocrinology, 125:825-33, 1989.

Everts, V.; Delaisse, J. M; Korper, W.; Jansen, D. C; Tigchelaar-Gutter, W.; Saftig, R; et al. The bone lining cell: its role in cleaning Howship's lacunae and initiating bone formation. J. Bone Miner. Res., 17:77-90, 2002.

Garcia Palacios, V.; Robinson, L. J.; Borysenko, C.W.; Lehmann, T.; Kalla, S. E. & Blair, H. C. Negative regulation of RANKL-induced osteoclastic differentiation in RAW264.7 cells by estrogen and phytoestrogens. J. Biol. Chem., 280:13720-7, 2005.

Grigoriadis, A. E.; Wang, Z. Q.; Cecchini, M. G.; Hofstetter, W.; Felix, R.; Fleisch, H. A.; et al. C-Fos: a key regulator of osteoclast-macrophage lineage determination and bone remodeling. Science, 266:443-8, 1994.

Hofbauer, L. C.; Khosla, S.; Dunstan, C. R.; Lacey, D. L.; Spelsberg, T. C; Riggs, B. L. Estrogen stimulates gene expression and protein production of osteoprotegerin in human osteoblastic cells. Endocrinology., 140:4367-70, 1999.

Hughes, D. E.; Dai, A.; Tiffee, J. C; Li, H. H.; Mundy, G. R. & Boyce, B.F. Estrogen promotes apoptosis of murine osteoclasts mediated by TGF-beta. Nat. Med., 2:1132-6,1996.

Iotsova, V.; Caamano, J.; Loy, J.; Yang, Y.; Lewin, A. & Bravo, R. Osteopetrosis in mice lacking NF-kappaBl andNF-kappaB2. Nat. Med, 5:1285-9, 1997.

Kitazawa, R.; Kitazawa, S. & Maeda, S. Promoter structure of mouse RANKL/TRANCE/OPGL/ODF gene. Biochim. Biophys Acta., 1445:134-41, 1999.

Lea, C. K.; Sarma, U. & Flanagan, A. M. Macrophage colony-stimulating factor transcripts are differentially regulated in rat bone-marrow by gender hormones. Endocrinology, 140:273-9, 1999.

Manolagas, S. C. Birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis. Endocr. Rev., 27:115-37,2000.

Oursler, M. J.; Osdoby, P.; Pyfferoen, J.; Riggs, B. L.; Spelsberg, T. C. Avian osteoclasts as estrogen target cells. Proc. Natl.Acad. Sci. USA., 88:6613-7, 1991a.

Oursler, M. J.; Córtese, C.; Keeting, P.; Anderson, M. A.; Bonde, S. K.; Riggs, B. L.; et al. Modulation of transforming growth factor-beta production in normal human osteoblast-like cells by 17 beta-estradiol and parathyroid hormone. Endocrinology, 129:3313-20, 1991b.

Qu, Q.; Perala-Heape, M.; Kapanen, A.; Dahllund, J.; Salo, J.; Váánanen, H. K.; et al. Estrogen enhances differentiation of osteoblasts in mouse bone marrow culture. Bone, 22:201-9, 1998.

Riggs, B. L. & Parfitt, A. M. Drugs used to treat osteoporosis: the critical need for a uniform nomenclature based on their action on bone remodeling. J. Bone Miner. Res., 20:177-84, 2005.

Rogers, A.; Saleh, G.; Harmon, R. A.; Greenfield, D. & Eastell, R. Circulating estradiol and osteoprotegerin as determinants of bone turnover and bone density in postmenopausal women. J. Clin. Endocrinol. Metab., 87: 4470-5, 2002.

Saika, M.; Inoue, D.; Kido, S. & Matsumoto, T. 17beta-estradiol stimulates expression of osteoprotegerin by a mouse stromal cell line, ST-2, via estrogen receptor-alpha. Endocrinology, 142:2205-12, 2001.

Sarma, U.; Edwards, M.; Motoyoshi, K. & Flanagan, A. M. Inhibition of bone resorption by 17beta-estradiol in human bone marrow cultures. J. Cell Physiol, 775:99-108, 1998.

Shevde, N. K.; Bendixen, A. C; Dienger, K. M. & Pike, J.W. Estrogens suppress RANK ligand-induced osteoclast differentiation via a stromal cell independent mechanism involving c-Jun repression. Proc. Nati. Acad. Sci. USA., 97: 7829-834, 2000.

Srivastava, S.; Toraldo, G.; Weitzmann, M. N.; Cenci, S.; Ross, F. P. & Pacifici, R. Estrogen decreases oseoclast formation by down-regulating receptor activator of NF-kappa B ligand (RANKL)-induced JNK activation. J. Biol. Chem., 276:8836-40, 2001.

Takayanagi, H. Mechanistic insight into osteoclast differentiation in osteoimmunology. J. Mol. Med., 55:170-9,2005.

Taranta, A.; Brama, M.; Teti, A.; De Luca, V.; Scandurra, R.; Spera, G.; et al. The selective estrogen receptor modulator raloxifene regulates osteoclast and osteoblast activity in vitro. Bone, 50:368-76, 2002.

Tobias, J. H. & Compston, J. E. Does estrogen stimulate osteoblast function in postmenopausal women? Bone, 24:121-4, 1999.

Viereck, V.; Grundker, C; Blaschke, S.; Niederkleine, B.; Siggelkow, H.; Frosch, K.H.; et al. Raloxifene concurrently stimulates osteoprotegerin and inhibits interleukin-6 production by human trabecular osteoblasts. J. Clin. Endocrinol. Metab., 88: 4206-13, 2003.

Received: 17-10-2006 Accepted: 27-12-2006

Correspondence to:

Dr. Joao Paulo Mardegan Issa
Rua Garibaldi, 806, ap-601
CEP: 14010-170
Bairro: Centro
Ribeirao Preto- SP
BRASIL

Email: jpmissa@forp.usp.br