OpenAthens / Sign in
Abstract
Dehydroepiandrosterone (DHEA) is produced by the adrenal cortex and is the most abundant steroid in humans. Although in some physiological and pathological conditions the increased secretion of DHEA and its sulfated form is associated with accelerated growth rate and skeletal maturation, it is unclear whether DHEA can affect longitudinal bone growth and skeletal maturation by acting directly at the growth plate. In our study, DHEA suppressed metatarsal growth, growth plate chondrocyte proliferation, and hypertrophy/differentiation. In addition, DHEA increased the number of apoptotic chondrocytes in the growth plate. In cultured chondrocytes, DHEA reduced chondrocyte proliferation and induced apoptosis. The DHEA-induced inhibition of metatarsal growth and growth plate chondrocyte proliferation and hypertrophy/differentiation was nullified by culturing metatarsals with DHEA in the presence of ICI 182,780, an inhibitor of estrogen receptor, but not in the presence of Casodex, an inhibitor of androgen receptor. Lastly, nuclear factor-κB DNA binding activity was inhibited by the addition of DHEA in the medium of cultured chondrocyte. Our findings indicate that DHEA suppressed bone growth by acting directly at growth plate through estrogen receptor. Such growth inhibition is mediated by decreased chondrocyte proliferation and hypertrophy/differentiation and by increased chondrocyte apoptosis.
The growth plate is the final target organ for longitudinal growth and results from chondrocyte proliferation and differentiation. In mammals, linear growth occurs at the long bone growth plates by endochondral ossification, a process by which cartilage is formed and then remodeled into bone (1, 2). Growth plate chondrocyte proliferation and hypertrophy/differentiation and extracellular matrix secretion all lead to formation of new cartilage, chondrogenesis (3). Because new cartilage is continuously formed, calcified cartilage at the metaphyseal border of the growth plate is replaced by bone tissue (4). The rate of longitudinal bone growth, which is determined primarily by the rate of chondrogenesis, is regulated by many hormones and growth factors. Among these, sex steroids are of crucial importance, especially during puberty (5).
Previous studies indicated that prenatal growth restrain may be followed by exaggerated adrenarche and higher dehydroepiandrosterone (DHEA)/DHEA sulfate (DHEA-S) levels (6, 7). It has been suggested that the programming of the endocrine axes occurs during critical phases of fetal development and will be affected by intrauterine growth retardation (8). DHEA is produced by the adrenal cortex and is the most abundant steroid in humans. The secretion of DHEA and DHEA-S by the adrenals increases during the adrenarche in children at the age of 6–8 yr, and elevated values of circulating DHEA-S and DHEA are maintained throughout adult life, providing high levels of substrates for conversion into potent androgens and estrogens in peripheral tissues (9). A number of studies have unequivocally demonstrated the presence of the androgen receptor (AR) and both estrogen receptors (ERs), ERα and ERβ, in growth plate tissue at the mRNA and protein level in several species, including rat, rabbit, and human (10–14), suggesting that androgens and estrogens can directly regulate processes in the growth plate. Although both androgens and estrogens are key regulators of skeletal physiology, little is known on the role played by DHEA in the regulation of bone growth. In addition, it is unclear whether DHEA can affect longitudinal bone growth and skeletal maturation by acting directly at the growth plate.
Numerous studies of DHEA in various disease conditions, such as atherosclerosis (15), cancer (16), diabetes (17), obesity (18), aging (19), and inflammatory arthritis (such as rheumatoid arthritis) (20), have been conducted. However, to date, no cognate nuclear or membrane receptor for DHEA has been identified, and its mechanisms of action remain a subject of active investigation. DHEA has been shown to exert many of its effects via the AR and/or ER after its enzymatic conversion to androgen or estrogen (21), although there are reports suggesting that DHEA could act on ERs without conversion to estrogens. Nephew et al. (22) have shown that DHEA can directly bind to ERs in vivo (even inhibiting estrogen binding to its own receptor) and cause dimerization of ERs, which are functionally active in yeast. However, these actions required approximately 1000 times higher concentrations of DHEA than estradiol. Another study has shown direct binding by DHEA to ERβ in transfected human embryonic kidney 293 cells, which do not have the required converting enzymes for its bioconversion to estrogen (23). Some other studies also have shown direct binding of DHEA to ERα (24) and DHEA-mediating ER-estrogen response element (ERE)-dependent transcriptional activity, independent of its conversion to estradiol (25).
Based on the evidence mentioned above, the aim of the present study was to investigate whether DHEA can directly affect longitudinal bone growth at the growth plate and to clarify whether this effect is mediated by AR and/or ER. We first cultured whole-rat metatarsal bones in the presence of DHEA with Casodex (AR inhibitor) or ICI 182,780 (ICI; an ER inhibitor), and studied their effects on metatarsal longitudinal growth. Then we analyzed the effects of DHEA in cultured growth plate chondrocyte proliferation, differentiation, and apoptosis. Lastly, we evaluated the mechanism under which DHEA affected growth plate chondrocytes.
Discussion
Addition of DHEA to the culture medium of rat metatarsal bones caused a dose-dependent suppression of their linear growth, suggesting a direct inhibitory effect of DHEA on mammalian longitudinal bone growth. A number of studies have demonstrated the presence of the AR and both ERs, ERα and ERβ, in growth plate tissue at the mRNA and protein level in several species, including rat, rabbit, and human (10–14). To clarify whether the effect of growth suppression induced by DHEA is mediated through ER and/or AR, we cultured the metatarsal bones in the presence of DHEA with Casodex or ICI. Unlike Casodex, which had no effect on DHEA-induced suppression of bone growth, ICI counteracted the growth suppression induce by DHEA, indicating that the suppression of DHEA on longitudinal bone growth was mediated through ER but not AR. The AR has been detected in all layers of the human growth plate at different ages (28–32), whereas in the rat it was expressed in proliferative and early hypertrophic chondrocytes at sexual maturation and only in prehypertrophic chondrocytes in older rats (14). Similar to our findings, it was demonstrated previously that modulation of AR activity in the fetal rat growth plate does not affect linear bone growth (33). In contrast, another of our published observations showed that DHEA stimulated proliferation of prostatic epithelial cell through AR (34), suggesting that effect of DHEA on cell growth and survival may depend on cell type and level of sex steroid receptor.
It was known that DHEA can exert a direct, physiologically relevant, agonistic effect on ERβ, a lesser antagonistic effect on the AR, and a modest effect on ERα in addition to its role as a precursor for androgens and estrogens. Some other studies also have shown direct binding of DHEA to ERα (28) and DHEA-mediating ER-ERE-dependent transcriptional activity, independent of its conversion to estradiol (25). Experiments with ER knockout mice have demonstrated the importance of both ERα and ERβ in the regulation of longitudinal bone growth (35, 36). In addition, both ERα and ERβ are expressed in growth plate cartilage from different species, including rat, rabbit, and human (10, 12, 32), suggesting that estrogens can act directly on growth plate chondrocytes. In fact, plasma DHEA-S levels in adult men and women are 1,000–10,000 times higher than those of estradiol, thus providing a large reservoir for conversion into estrogens in peripheral tissues. In our study, DHEA suppressed metatarsal bone growth directly at the growth plate by inhibiting chondrocyte proliferation and differentiation. To rule out the potential role played by local produced estrogen or converted from DHEA within the growth plate, we blocked estrogen synthesis by an aromatase inhibitor, letrozole, and ERs by the pure nonselective estrogen antagonist ICI. Our results clearly showed that local estrogen had no effect on chondrocyte proliferation and also longitudinal growth of fetal rat metatarsal bones. These data are in line with the observation that exogenous estradiol had no effect on HCS-2/8 chondrocytes or fetal rat metatarsal bones (37), although it was also demonstrated in the same observation that endogenous estradiol promotes metatarsal bone growth. To further confirm which subunit of ER is mediated by the effect of DHEA on chondrogenesis, we transfected the chondrocytes with ERα siRNA or ERβ siRNA. We found that addition of ERβ siRNA abolished the inhibition of chondrocyte proliferation and differentiation caused by DHEA, indicating that ERβ, other than ERα, is more important in mediating the effect of DHEA on chondrocyte. Previous studies demonstrate that ERα mediates the growth-promoting effects of estradiol in pubertal bone development in both male and female but is not involved in maintenance of trabecular bone (38, 39). The major role of ERβ during pubertal growth is to terminate the growth spurt in females, limiting longitudinal and radial bone growth (40). A normally functioning ERβ may therefore be responsible for the shorter bones and lower peak bone mass normally seen in female rodents (40). In another paper, it has confirmed that ERβ is a physiological inhibitor of appendicular- and axial-skeletal growth in young adult female mice (36).
We also found that DHEA has the capacity to induce chondrocyte apoptosis. This finding is important as apoptosis is known to play an important role in growth plate homeostasis (41). DHEA protects human keratinocytes against apoptosis through transmitting its signal via specific G protein-coupled, membrane binding sites, and prevention of mitochondrial disruption and altered balance of Bcl-2 proteins (42). DHEA was reported to act as a survival factor for endothelial cells by triggering the Gαi-PI3K/Akt-Bcl-2 pathway to protect cells against serum deprivation-induced apoptosis (43). On the other hand, DHEA increases the sensitivity of cells to γ-ray irradiation by inducing apoptosis and cell cycle arrest through glutathione-dependent regulation of the reduced form of protein phosphatase 2A to down-regulate the Akt signaling pathway (44). GT1–7 hypothalamic neurons deprived of trophic support undergo apoptosis after exposure to DHEA, as demonstrated both by morphological and biochemical criteria. This proapoptotic effect appeared to be specific to DHEA itself and not through conversion of DHEA to other steroids such as androgen or estrogen (45). The different results regarding the effect of DHEA on apoptosis might be due to the different concentrations of DHEA used [DHEA acts as a survival factor at a lower concentration (maximum up to 10 nM)], whereas DHEA acts as a proapoptotic factor at micromolar level (10–100 μM). Here we demonstrate that the addition of DHEA induces apoptosis of growth plate chondrocyte in a caspase-dependent manner. This observation suggests involvement of specific proapoptotic pathways of DHEA.
It is known that NF-κB can act as either a pro- or an antiapoptotic effector, depending on cell type and internal milieu. To shed light on the mechanism of DHEA on chondrocytes, we performed NF-κB DNA binding activity. We found that DHEA inhibited NF-κB activity via ER, indicating that the suppression of DHEA on chondrocyte proliferation and differentiation is mediated through NF-κB inactivation. Consistent with our finding, recent studies have shown that the ER and NF-κB signaling pathways are tightly linked: activated ER can inhibit both NF-κB activation and the ability of NF-κB to control gene expression, and ER-negative cancers show increased constitutive levels of nuclear NF-κB as well as increased expression of NF-κB-related genes (46). In diabetic rats, DHEA protects hippocampal neurons by preventing nitric oxide-induced activation of NF-κB (47), and in mice with experimental allergic encephalitis, DHEAS decreases demyelinization mediated by a decrease in the activation and translocation of NF-κB (48). DHEAS also protects against glutamate-induced neurotoxicity via induction of NF-κB activity (49).
Taken together, our findings indicate DHEA suppresses longitudinal bone growth by inhibiting chondrocyte proliferation and differentiation and inducing chondrocyte apoptosis. This suppression is mediated through the ER, especially ERβ, via the NF-κB signaling pathway.
Acknowledgments
Abstract
Dehydroepiandrosterone (DHEA) is produced by the adrenal cortex and is the most abundant steroid in humans. Although in some physiological and pathological conditions the increased secretion of DHEA and its sulfated form is associated with accelerated growth rate and skeletal maturation, it is unclear whether DHEA can affect longitudinal bone growth and skeletal maturation by acting directly at the growth plate. In our study, DHEA suppressed metatarsal growth, growth plate chondrocyte proliferation, and hypertrophy/differentiation. In addition, DHEA increased the number of apoptotic chondrocytes in the growth plate. In cultured chondrocytes, DHEA reduced chondrocyte proliferation and induced apoptosis. The DHEA-induced inhibition of metatarsal growth and growth plate chondrocyte proliferation and hypertrophy/differentiation was nullified by culturing metatarsals with DHEA in the presence of ICI 182,780, an inhibitor of estrogen receptor, but not in the presence of Casodex, an inhibitor of androgen receptor. Lastly, nuclear factor-κB DNA binding activity was inhibited by the addition of DHEA in the medium of cultured chondrocyte. Our findings indicate that DHEA suppressed bone growth by acting directly at growth plate through estrogen receptor. Such growth inhibition is mediated by decreased chondrocyte proliferation and hypertrophy/differentiation and by increased chondrocyte apoptosis.
The growth plate is the final target organ for longitudinal growth and results from chondrocyte proliferation and differentiation. In mammals, linear growth occurs at the long bone growth plates by endochondral ossification, a process by which cartilage is formed and then remodeled into bone (1, 2). Growth plate chondrocyte proliferation and hypertrophy/differentiation and extracellular matrix secretion all lead to formation of new cartilage, chondrogenesis (3). Because new cartilage is continuously formed, calcified cartilage at the metaphyseal border of the growth plate is replaced by bone tissue (4). The rate of longitudinal bone growth, which is determined primarily by the rate of chondrogenesis, is regulated by many hormones and growth factors. Among these, sex steroids are of crucial importance, especially during puberty (5).
Previous studies indicated that prenatal growth restrain may be followed by exaggerated adrenarche and higher dehydroepiandrosterone (DHEA)/DHEA sulfate (DHEA-S) levels (6, 7). It has been suggested that the programming of the endocrine axes occurs during critical phases of fetal development and will be affected by intrauterine growth retardation (8). DHEA is produced by the adrenal cortex and is the most abundant steroid in humans. The secretion of DHEA and DHEA-S by the adrenals increases during the adrenarche in children at the age of 6–8 yr, and elevated values of circulating DHEA-S and DHEA are maintained throughout adult life, providing high levels of substrates for conversion into potent androgens and estrogens in peripheral tissues (9). A number of studies have unequivocally demonstrated the presence of the androgen receptor (AR) and both estrogen receptors (ERs), ERα and ERβ, in growth plate tissue at the mRNA and protein level in several species, including rat, rabbit, and human (10–14), suggesting that androgens and estrogens can directly regulate processes in the growth plate. Although both androgens and estrogens are key regulators of skeletal physiology, little is known on the role played by DHEA in the regulation of bone growth. In addition, it is unclear whether DHEA can affect longitudinal bone growth and skeletal maturation by acting directly at the growth plate.
Numerous studies of DHEA in various disease conditions, such as atherosclerosis (15), cancer (16), diabetes (17), obesity (18), aging (19), and inflammatory arthritis (such as rheumatoid arthritis) (20), have been conducted. However, to date, no cognate nuclear or membrane receptor for DHEA has been identified, and its mechanisms of action remain a subject of active investigation. DHEA has been shown to exert many of its effects via the AR and/or ER after its enzymatic conversion to androgen or estrogen (21), although there are reports suggesting that DHEA could act on ERs without conversion to estrogens. Nephew et al. (22) have shown that DHEA can directly bind to ERs in vivo (even inhibiting estrogen binding to its own receptor) and cause dimerization of ERs, which are functionally active in yeast. However, these actions required approximately 1000 times higher concentrations of DHEA than estradiol. Another study has shown direct binding by DHEA to ERβ in transfected human embryonic kidney 293 cells, which do not have the required converting enzymes for its bioconversion to estrogen (23). Some other studies also have shown direct binding of DHEA to ERα (24) and DHEA-mediating ER-estrogen response element (ERE)-dependent transcriptional activity, independent of its conversion to estradiol (25).
Based on the evidence mentioned above, the aim of the present study was to investigate whether DHEA can directly affect longitudinal bone growth at the growth plate and to clarify whether this effect is mediated by AR and/or ER. We first cultured whole-rat metatarsal bones in the presence of DHEA with Casodex (AR inhibitor) or ICI 182,780 (ICI; an ER inhibitor), and studied their effects on metatarsal longitudinal growth. Then we analyzed the effects of DHEA in cultured growth plate chondrocyte proliferation, differentiation, and apoptosis. Lastly, we evaluated the mechanism under which DHEA affected growth plate chondrocytes.
Discussion
Addition of DHEA to the culture medium of rat metatarsal bones caused a dose-dependent suppression of their linear growth, suggesting a direct inhibitory effect of DHEA on mammalian longitudinal bone growth. A number of studies have demonstrated the presence of the AR and both ERs, ERα and ERβ, in growth plate tissue at the mRNA and protein level in several species, including rat, rabbit, and human (10–14). To clarify whether the effect of growth suppression induced by DHEA is mediated through ER and/or AR, we cultured the metatarsal bones in the presence of DHEA with Casodex or ICI. Unlike Casodex, which had no effect on DHEA-induced suppression of bone growth, ICI counteracted the growth suppression induce by DHEA, indicating that the suppression of DHEA on longitudinal bone growth was mediated through ER but not AR. The AR has been detected in all layers of the human growth plate at different ages (28–32), whereas in the rat it was expressed in proliferative and early hypertrophic chondrocytes at sexual maturation and only in prehypertrophic chondrocytes in older rats (14). Similar to our findings, it was demonstrated previously that modulation of AR activity in the fetal rat growth plate does not affect linear bone growth (33). In contrast, another of our published observations showed that DHEA stimulated proliferation of prostatic epithelial cell through AR (34), suggesting that effect of DHEA on cell growth and survival may depend on cell type and level of sex steroid receptor.
It was known that DHEA can exert a direct, physiologically relevant, agonistic effect on ERβ, a lesser antagonistic effect on the AR, and a modest effect on ERα in addition to its role as a precursor for androgens and estrogens. Some other studies also have shown direct binding of DHEA to ERα (28) and DHEA-mediating ER-ERE-dependent transcriptional activity, independent of its conversion to estradiol (25). Experiments with ER knockout mice have demonstrated the importance of both ERα and ERβ in the regulation of longitudinal bone growth (35, 36). In addition, both ERα and ERβ are expressed in growth plate cartilage from different species, including rat, rabbit, and human (10, 12, 32), suggesting that estrogens can act directly on growth plate chondrocytes. In fact, plasma DHEA-S levels in adult men and women are 1,000–10,000 times higher than those of estradiol, thus providing a large reservoir for conversion into estrogens in peripheral tissues. In our study, DHEA suppressed metatarsal bone growth directly at the growth plate by inhibiting chondrocyte proliferation and differentiation. To rule out the potential role played by local produced estrogen or converted from DHEA within the growth plate, we blocked estrogen synthesis by an aromatase inhibitor, letrozole, and ERs by the pure nonselective estrogen antagonist ICI. Our results clearly showed that local estrogen had no effect on chondrocyte proliferation and also longitudinal growth of fetal rat metatarsal bones. These data are in line with the observation that exogenous estradiol had no effect on HCS-2/8 chondrocytes or fetal rat metatarsal bones (37), although it was also demonstrated in the same observation that endogenous estradiol promotes metatarsal bone growth. To further confirm which subunit of ER is mediated by the effect of DHEA on chondrogenesis, we transfected the chondrocytes with ERα siRNA or ERβ siRNA. We found that addition of ERβ siRNA abolished the inhibition of chondrocyte proliferation and differentiation caused by DHEA, indicating that ERβ, other than ERα, is more important in mediating the effect of DHEA on chondrocyte. Previous studies demonstrate that ERα mediates the growth-promoting effects of estradiol in pubertal bone development in both male and female but is not involved in maintenance of trabecular bone (38, 39). The major role of ERβ during pubertal growth is to terminate the growth spurt in females, limiting longitudinal and radial bone growth (40). A normally functioning ERβ may therefore be responsible for the shorter bones and lower peak bone mass normally seen in female rodents (40). In another paper, it has confirmed that ERβ is a physiological inhibitor of appendicular- and axial-skeletal growth in young adult female mice (36).
We also found that DHEA has the capacity to induce chondrocyte apoptosis. This finding is important as apoptosis is known to play an important role in growth plate homeostasis (41). DHEA protects human keratinocytes against apoptosis through transmitting its signal via specific G protein-coupled, membrane binding sites, and prevention of mitochondrial disruption and altered balance of Bcl-2 proteins (42). DHEA was reported to act as a survival factor for endothelial cells by triggering the Gαi-PI3K/Akt-Bcl-2 pathway to protect cells against serum deprivation-induced apoptosis (43). On the other hand, DHEA increases the sensitivity of cells to γ-ray irradiation by inducing apoptosis and cell cycle arrest through glutathione-dependent regulation of the reduced form of protein phosphatase 2A to down-regulate the Akt signaling pathway (44). GT1–7 hypothalamic neurons deprived of trophic support undergo apoptosis after exposure to DHEA, as demonstrated both by morphological and biochemical criteria. This proapoptotic effect appeared to be specific to DHEA itself and not through conversion of DHEA to other steroids such as androgen or estrogen (45). The different results regarding the effect of DHEA on apoptosis might be due to the different concentrations of DHEA used [DHEA acts as a survival factor at a lower concentration (maximum up to 10 nM)], whereas DHEA acts as a proapoptotic factor at micromolar level (10–100 μM). Here we demonstrate that the addition of DHEA induces apoptosis of growth plate chondrocyte in a caspase-dependent manner. This observation suggests involvement of specific proapoptotic pathways of DHEA.
It is known that NF-κB can act as either a pro- or an antiapoptotic effector, depending on cell type and internal milieu. To shed light on the mechanism of DHEA on chondrocytes, we performed NF-κB DNA binding activity. We found that DHEA inhibited NF-κB activity via ER, indicating that the suppression of DHEA on chondrocyte proliferation and differentiation is mediated through NF-κB inactivation. Consistent with our finding, recent studies have shown that the ER and NF-κB signaling pathways are tightly linked: activated ER can inhibit both NF-κB activation and the ability of NF-κB to control gene expression, and ER-negative cancers show increased constitutive levels of nuclear NF-κB as well as increased expression of NF-κB-related genes (46). In diabetic rats, DHEA protects hippocampal neurons by preventing nitric oxide-induced activation of NF-κB (47), and in mice with experimental allergic encephalitis, DHEAS decreases demyelinization mediated by a decrease in the activation and translocation of NF-κB (48). DHEAS also protects against glutamate-induced neurotoxicity via induction of NF-κB activity (49).
Taken together, our findings indicate DHEA suppresses longitudinal bone growth by inhibiting chondrocyte proliferation and differentiation and inducing chondrocyte apoptosis. This suppression is mediated through the ER, especially ERβ, via the NF-κB signaling pathway.
Acknowledgments
