Time course of the osteoprotegerin gene expression in human primary osteoblasts as well as MG-63 cell lines and the effect of 17β-estradiol
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《中华医药杂志》英文版
[Abstract] Objective Osteoprotegerin (OPG) is a key factor regulating bone resorption and play an important role in the episode of osteoporosis.This study was designed to observe the time course of the expression of osteoprotegerin (OPG) in adult human osteoblasts (HOB) and MG-63 osteosarcoma cells cultured in vitro, analyse the effects of 17-beta-estradiol (E2) on it and to explore the mechanisms of E2 acts on osteoblasts. Methods The activity of alkaline phosphatase (ALP) and osteocalcin (OC) content in cultured cells were measured at various stages of culture. Stages of cell proliferation, bone matrix maturity and mineralization were determined by Van Gieson Staining. Mature cells were treated with E2, the expression of OPG mRNA and protein quantified by semi-quantitative RT-PCR and Western blot analysis respectively.Results For both types of cells, expression of OPG peaked on the 12th day of culture without treatment, and this was the stage of bone matrix maturity. E2 of 1×10-8 mol/L increased OPG mRNA expression to a maximum of about 4 folds (P<0.001) in MG-63 cells, while the expression of OPG mRNA was increased by a maximum of 3 folds (P<0.01) and 2 folds in HOB cells. E2 of 1×10-8 mol/L increased the OPG protein expression obviously.Conclusions OPG can be differentiatedly expressed by both human osteoblasts and MG-63 cells at different culture stage, and reachs its maximal expression at the stage of bone matrix maturity, E2 exhibits positive effects on osteoblasts to upregulate OPG gene expression in vitro.
[Key words] osteoprotegerin;time course; estradiol; osteoblast.
INTRODUCTION Osteoprotegerin (OPG), also known as osteoclastoge nesis inhibitory factor (OCIF), plays an important role in regulating bone resorption. OPG functions as a soluble decoy receptor for the receptor activator of nuclear kappa-B ligand (RANKL or OPG ligand) and shares homologies with other members of the tumor necrosis factor receptor superfamily [1, 2].OPG is expressed in osteoblasts and regulates the activities of osteoclasts through OPG ligand (osteoclast differentiating factor, ODF). Therefore, OPG is one of the coupling factors for the inter-regulation between osteoblasts and osteoclasts. Osteoporosis in postmenopausal women is closely associated with the lack of E2, but the mechanism for this relationship has not been clarified and suppo sedly results from an imbalance between osteoblastic and osteoclastic activity. Estrogen inhibits the activity of osteoclasts so that bone resorption is limited. The expression of OPG mRNA and protein in the mouse stromal ST-2 cell line was reported to be upregulated by E2 in research by Saika et al[3]. Hofbauer et al[4] reported that 17-beta-estradiol increased OPG mRNA and protein levels to maximal levels of 370% and 320%, respectively. Co-treatment with “pure” anti-estrogen ICI 182 780 abrogated these effects completely. 17-beta-estradiol also dose-dependently increased OPG mRNA and protein levels in normal human osteoblasts with approximately 400 ER/nucleus by 60% and 73%, respectively. However, the gene expression of different stages maybe variant and so that the drug effect on it. This study was designed to observe the time course of the expression of osteoprotegerin (OPG) in adult human osteoblasts (HOB) and MG-63 osteosarcoma cells cultured in vitro, analyse the effects of 17-beta-estradiol (E2) on the osteoblasts at bone matrix maturity stage and to explore the mechanisms of E2 acts on osteoblasts.
Institute of Endocrinology & Metabolism Research, The Second Xiangya Hospital of Central South University,Changsha 410011,Hunan Province, China
Correspondence to Dr.ZHOU Hou-De,Institute of Endocrinology & Metabolism,The Second Xiangya Hospital of Central South University,139# Middle Renmin Road,Changsha 410011, Hunan Province, China
Tel: +86-731-5592151, Fax:+86-731-5361472, E-mail: houdezhou@hotmail.com.
METHODS
Cell Culture
The MG-63 cell lines were obtained from American Type Culture Collection (ATCC,No. CRL-1427) and cultured in modified Eagle's medium (Sigma) cont aining no phenolsulfonphthalein and supplemented with 10% fetal bovine serum (FBS), 50 u/ml penicillin and 50 g/ml cidomycin. The medium was changed every 3 days and sub-cultured after confluence. Human osteoblasts (HOB) were cultured in the following described manner. Spongy bone at the anterior superior iliac spine of 4 patients, aged 35~55 years, with iliac fracture and no other diseases was obtained by surgical procedure with consent from patients. The bone was cut into pieces of about 2 mm3 and washed 3 times in PBS, followed by the addition of 1% type IV collagenase (Sigma) and incubating at 37℃ with gentle shaking until the cancellous bone whitened and assumed a honeycomb appearance. The addition of Modified Eagle's medium (MEM) with 20% FBS terminated the enzymatic digestion and bone fragments were washed 3 times with MEM containing 10% FBS until no cells remained in the washing solution. The bone pieces adhered to the wall of 25 cm2 culture flasks for 1 hour at 37℃. The addition of MEM with 15% FBS to the flasks was then did. The medium was first changed 2 days later and subsequently changed every other day. Cell growth was monitored and confluence was reached about 20 days later. Cells were then harvested with 0.25% trypsin-EDTA and transferred into 25 cm2 flasks at the density of 5×105 cells/flask.
Characterization of Cultured Cells
Seed the cells to 25 cm2 flasks and 3 days later, the cells reach its confluents, after culturing for 3, 6, 12,18 days, cells were washed twice in PBS and disrupted by ultrasonic in Tris-Triton X-100 solution (20 mM Tris, 2mM MgCl2, 150 mM NaCl and 1% Triton X-100). Supernatant was collected after centrifugation and the activity of ALP measured by HITACHI 7000 automatic biochemistry analyzer with α-nitrophenyl phosphate used as substrate. Osteocalcin concentrations in the supernatant were determined by ELISA assay. Intracellular total protein content was measured by the Bradford method and used to adjust ALP and osteocalcin levels. Type I collagen in cultured cells at different stages was stained by using the Van Gieson staining method.
Management of the 17β-estradiol
For 17β-estradiol intervene, the medium of cells grown in flasks was changed every 2 days and transferred into MEM containing only 0.1% bovine serum albumin 3 days before the addition of E2. According to approximate values of physiological E2 concentration in the human body, 4 concentrations of E2 (Sigma) including 0, 1×10-6, 1×10-8 and 1×10-10 mol/L were employed to treat cultured HOB and MG-63 cells at the stage of bone matrix maturity.
Detecting the mRNA Expression by RT-PCR
RNA was extracted according to the manufacture instructions for TRIZOL (GIBCO/BRL, USA), then was treated by RNase-free DNase I，after electrophoresis on 1.2% agarose gel, the optical density ratio of 28S:18S is 1.6∶1, then optical density (OD) ratio (A260/A280) determined by ultraviolet spectrophotometer, RNA concentrations were then calculated. A 219 bp fragment (496 to 714 base pair) of full-length cDNA of human OPG was amplified with primers as follows: (+) 5'AACCCCAGAGCGAAATAC3'; (-) 5'AAGAATGCCTCCTCACAC3'. β-actin gene (620 bp) was simultaneously amplified as an internal control. One-step RT-PCR was carried out on the PE2400 Thermal Cycler (Perkin-Elmer, Inc., U.S.A.) with an annealing temperature of 56℃ and a total cycle number of 26. RT-PCR products were electrophoresed on a 1.5% agarose gel and visualized by ethidium bromide staining. Optical density of electrophoretic bands was measured by the Bio-Rad Gel Doc 2000 Image Analyzing System and the OD percentages of OPG to β-actin were then calculated.
Western-blot and Gel Imaging Analysis
The cultured cells were washed 2 times by PBS, then lysed the cell by lysate buffer. Centrifuge the lysate at 4℃ for 20 min, extracted protein samples were electrophoresed on a 9.5% SDS-polyacrylamide gel (60 μg in each lane) and transferred to PVDF membrane. The blot was blocked by non-fat dried milk for non-specific binding and incubated with rabbit anti-human OPG antibody for 1 h. The membrane was washed and subsequently incubated with a secondary goat anti-rabbit horseradish peroxidase (HRP)-labeled antibody for 1h, followed by incubation with ECL substrate for 1 min. The blots were then analyzed by Image Master VDS-CL using the ECL tray.
Statistical Analysis
All experiments above were replicated 3 to 4 times. Data in this study were analyzed by SPSS 9.0 for Windows statistical software. The results were expressed in x±s and differences between groups were determined by ANOVA.
RESULTS
Alkaline Phosphatase Activities
Figure 1A illustrates the changes of ALP activity over time. The expression of ALP was evident in both HOB and MG-63 cells; secreted amounts of ALP in HOB were 13~20 times those in MG-63 cells (19.6~74.3 mu/mg protein vs 1.23~3.58 mu/mg protein,P<0.01). The activity of ALP in both cell types reached the highest levels by the 12th day of culture and then diminished gradually. The maximal expression levels of ALP in HOB and MG-63 cells were 74.3 mu/mg protein and 3.58 mu/mg protein, respectively, being 3~4 and 2~3 times greater than those levels at initiation of culture, i.e. (74.3 ± 4.7)mu/mg vs(19.6 ±1.6)mu/mg protein and (3.58 ± 0.28)protein vs (1.24 ± 0.19)mu/mg protein, respectively.
Osteocalcin
The expression of osteocalcin in MG-63 cells was too weak to be detected by radioimmunoassay in this study while its expression was detected in HOB, which had a slight increase at initiation of culture [(3.4 ± 0.30)ng/mg protein vs (3.84 ± 0.39)ng/mg protein)], but began a decrease after 6 days of culture (Figure 1B).
Figure 1 Time course of alkaline phosphatase (ALP) activity (A) and osteoclacin (B) changes in MG-63 cells and human primary osteoblasts (HOB)
Ossein Staining
No obvious bone nodules were observed by microscopy after 6 days of culture and Van Gieson staining for cultured cells revealed pale red collagen suggesting low collagen production. By 12 days of culture, red-stained collagen and bone nodules were visible. By 18 days, cells had become very dense without clear outline; numerous nodules of various size and scarlet-stained collagen with more intensity at nodules than other areas were observed (Figure 2).
OPG Expression
The expression of OPG mRNA in MG-63 cells was weak after 6 days of culture and increased 3~4 times by the 12th day of culture (P<0.01), and decreased slightly after 18 days of culture (P<0.01)(Figure 3). E2 increased OPG gene expression in MG-63 cells with the concentration of 1×10-8 mol/L obviously being the most effective by increasing OPG mRNA expression about 4 times (P<0.001) in MG-63 cells (Figure 4). OPG was expressed in HOB in a time-dependent way, which reached the highest level by the 12th day of culture about 2 times that on the 6th day of culture (42% vs 84%)(Figure 3),but the production of OPG in HOB was less than that in MG-63 cells. The expression of OPG gene in HOB was elevated by E2, especially at the concentration of 1×10-8 mol/L, which increased OPG mRNA expression 3 times (483% vs 66%,P<0.01) compared to control (Figure 5). Western blot analysis showed that E2 increased OPG protein expression obviously both in MG-63 and HOB cells.
Figure 2 Time course of collagen content changes in cultured MG-63 cells by Van Gieson staining. Cells cultured for 6 days (A), 12 days (B), 18 days (C)
Figure 3 Time course of OPG mRNA expression in cultured MG-63 cells and human primary osteoblasts. The cells were cultured for 3, 6,12 and 18 days, the OPG mRNA expression was determined by RT-PCR
Figure 4 Effects of 17β-estradiol on the expression of OPG mRNA in the osteoblast-like MG-63 cell and human primary osteoblasts
Figure 5 Effects of 17β-estradiol on the expression of OPG protein in the osteoblast-like MG-63 cell and human primary osteoblasts (HOB) analyzed by Western blot.
DISCUSSION
OPG is a member of TNF receptor superfamily. It has been demonstrated that OPG / receptor activator of nuclear factor kappa-B (RANK)/RANK-ligand (RANKL) system is effective in regulating the differentiation, growth, fusion and activation of osteoclasts. OPG and RANKL are regulated by many hormones including glucocorticosteroid, vitamin D and estrogen and so on,and cytokines such as TNF-α and interleukin-1, 4, 6, 11 and 17, etc. and transcription factors such as cbfa-1 and PPAR-γ and so on [1, 2,5, 6]. Estrogen has been confirmed to greatly influence the activities of osteoblasts and osteoclasts in experiments both in vitro and in vivo, but mechanisms for this effect have not been clarified. There have been no reports on the effects of E2 on cultured cells at different stages of growth. In our previous studies, we have found that E2 increased bone mineral density in female rats undergoing ovariectomy and suppressed expression of matrix metalloproteinase-1 (MMP-1) in osteoblasts with different effects on MMP expression at different stages of culture [7, 8]. By determining different stages of cell differentiation based on amounts of ALP and osteocalcin release, collagen staining and numbers of bone nodules in cultured cells, osteoblastic cells were found to mature by the 12th to 15th day of culture and to begin mineralization after about 18 d of culture. Therefore, the 6th, 12th and 18th day were selected as 3 key time points for determination of osteoblast maturation [9] for the further study of the effects of E2 on the expression of OPG mRNA in osteoblasts. OPG was expressed in osteoblasts and its N-terminal domains (D1-D4) effectively inhi bited osteoclastogenesis and reduced the activity and survival rate of adult multi-nuclear osteoclasts resulting in decreased bone resorptive activity of osteoclasts [10]. A variety of hormones such as 1,25(OH)2D3, parathyroid hormone (PTH), PG-E2, dexamethasone and immune factors including IL-1 and TNF and so on have the ability to regulate OPG gene expression in osteoblasts [11, 12]. Disruption of the OPG gene in the OPG knockout mouse produced severe osteoporosis leading to susceptibility to bone fracture indicating the importance of this molecule in normal bone physiology [13]. Treatment of rats undergoing ovariectomy with purified OPG protein was able to prevent bone loss [14]. Transgenic overexpression of OPG in mice not only reversed osteoporosis but also produced an osteopetrotic phenotype [15]. Postmenopausal osteoporotic patients have normal blood levels of ALP, osteocalcin and calcium, but have higher OPG levels than healthy individuals [16] suggesting that OPG plays a more important role in bone metabolism than other mediators.
In this study, we chose cultured cells at different stages to observe OPG gene expression and observed OPG gene expression to decrease over time in the absence of E2 interference. The non-mineralized osteosarcoma MG-63 cell line expressed high levels of OPG which reached the highest levels 12 days after confluence being 3~4 times levels on the 6th day and maintained this level thereafter. Normal human osteoblasts expressed lower levels of OPG than MG-63 cells (about 40% of that in MG-63 cells) and the time-dependent changes of OPG expression were less apparent than those in MG-63 cells. After 12 days of culture, there was only a slight increase in OPG expression in HOB at about 2 times that on the 6th day. OPG inhibits differentiation of osteoclastic precursors into mature osteoclasts and suppresses the activity of adult osteoclasts. The weak expression of OPG gene in normal HOB may be due to their greater capability to support the survival of mature osteoclasts than MG-63 cells or other immortal cell lines. Time courses of OPG expression in both HOB and MG-63 cells were similar to those of ALP and osteocalcin indicating that OPG production in mature osteoblasts is greater than that in proliferating ones and so likewise the ability to inhibit osteoclasts is greater in adult osteoblasts.
In the present study, E2 was confirmed to increase the expression of OPG gene in both types of cells with the concentration of 1×10-8 mmol/L, which is close to the physiological concentration in microenvironment of bone tissue, having the strongest effect and exerting stronger effects on MG-63 than HOB cells. Hofbaner et al[4] reported that E2 increased the expression of OPG mRNA and protein only 3.7 and 3.2 times, respectively, in hFOB/ER-3 and hFOB/ER-9 cells.The reason for the differences between the increased amounts of OPG mRNA and protein expression caused by E2 is not clear. The MG-63 cell line was derived from a 16-year-old girl and belonged to early differentiated osteoblasts while HOB were obtained from iliac bone of adult women which had far greater levels of ALP expression than MG-63 cells, indicating that HOB had a higher degree of differentiation than the MG-63 cells. E2 had greater effects on the expression of OPG mRNA in MG-63 than in HOB cells. This suggests that the effects of E2 on osteoblasts are associated with their differentiating phases, identical to the relationship between OPG expression in normal osteoblasts and their differentiating phases. Whether the time-dependent effects of E2 on osteoblasts were related to osteogenesis, growth and senescence of bone skeleton remains to be elucidated? In this study, E2 was shown to have stronger effects on mRNA expression of OPG than on protein expression suggesting that expressions of OPG at transcriptional and translational levels in osteoblasts are not consistent. Therefore, effects of medicines for the prevention and treatment of osteoporosis on OPG gene expression in osteoblasts should not be evaluated solely at the transcriptional level. Estrogen increased the OPG gene expression in experiments in vitro, which might account for the decrease in OPG expression with aging [3, 17, 18]. However, some studies have found that circulating OPG levels in postmenopausal women were elevated and even higher than those in osteoporotic patients and older people with low bone mineral content. Further studies are needed to elucidate the differences between these in vivo and in vitro results.
Funding:This work was supported by National Natural Science,China (No.30400218)
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(Editor Jaque)(ZHOU Hou-de, BU Yan-hong,)
[Key words] osteoprotegerin;time course; estradiol; osteoblast.
INTRODUCTION Osteoprotegerin (OPG), also known as osteoclastoge nesis inhibitory factor (OCIF), plays an important role in regulating bone resorption. OPG functions as a soluble decoy receptor for the receptor activator of nuclear kappa-B ligand (RANKL or OPG ligand) and shares homologies with other members of the tumor necrosis factor receptor superfamily [1, 2].OPG is expressed in osteoblasts and regulates the activities of osteoclasts through OPG ligand (osteoclast differentiating factor, ODF). Therefore, OPG is one of the coupling factors for the inter-regulation between osteoblasts and osteoclasts. Osteoporosis in postmenopausal women is closely associated with the lack of E2, but the mechanism for this relationship has not been clarified and suppo sedly results from an imbalance between osteoblastic and osteoclastic activity. Estrogen inhibits the activity of osteoclasts so that bone resorption is limited. The expression of OPG mRNA and protein in the mouse stromal ST-2 cell line was reported to be upregulated by E2 in research by Saika et al[3]. Hofbauer et al[4] reported that 17-beta-estradiol increased OPG mRNA and protein levels to maximal levels of 370% and 320%, respectively. Co-treatment with “pure” anti-estrogen ICI 182 780 abrogated these effects completely. 17-beta-estradiol also dose-dependently increased OPG mRNA and protein levels in normal human osteoblasts with approximately 400 ER/nucleus by 60% and 73%, respectively. However, the gene expression of different stages maybe variant and so that the drug effect on it. This study was designed to observe the time course of the expression of osteoprotegerin (OPG) in adult human osteoblasts (HOB) and MG-63 osteosarcoma cells cultured in vitro, analyse the effects of 17-beta-estradiol (E2) on the osteoblasts at bone matrix maturity stage and to explore the mechanisms of E2 acts on osteoblasts.
Institute of Endocrinology & Metabolism Research, The Second Xiangya Hospital of Central South University,Changsha 410011,Hunan Province, China
Correspondence to Dr.ZHOU Hou-De,Institute of Endocrinology & Metabolism,The Second Xiangya Hospital of Central South University,139# Middle Renmin Road,Changsha 410011, Hunan Province, China
Tel: +86-731-5592151, Fax:+86-731-5361472, E-mail: houdezhou@hotmail.com.
METHODS
Cell Culture
The MG-63 cell lines were obtained from American Type Culture Collection (ATCC,No. CRL-1427) and cultured in modified Eagle's medium (Sigma) cont aining no phenolsulfonphthalein and supplemented with 10% fetal bovine serum (FBS), 50 u/ml penicillin and 50 g/ml cidomycin. The medium was changed every 3 days and sub-cultured after confluence. Human osteoblasts (HOB) were cultured in the following described manner. Spongy bone at the anterior superior iliac spine of 4 patients, aged 35~55 years, with iliac fracture and no other diseases was obtained by surgical procedure with consent from patients. The bone was cut into pieces of about 2 mm3 and washed 3 times in PBS, followed by the addition of 1% type IV collagenase (Sigma) and incubating at 37℃ with gentle shaking until the cancellous bone whitened and assumed a honeycomb appearance. The addition of Modified Eagle's medium (MEM) with 20% FBS terminated the enzymatic digestion and bone fragments were washed 3 times with MEM containing 10% FBS until no cells remained in the washing solution. The bone pieces adhered to the wall of 25 cm2 culture flasks for 1 hour at 37℃. The addition of MEM with 15% FBS to the flasks was then did. The medium was first changed 2 days later and subsequently changed every other day. Cell growth was monitored and confluence was reached about 20 days later. Cells were then harvested with 0.25% trypsin-EDTA and transferred into 25 cm2 flasks at the density of 5×105 cells/flask.
Characterization of Cultured Cells
Seed the cells to 25 cm2 flasks and 3 days later, the cells reach its confluents, after culturing for 3, 6, 12,18 days, cells were washed twice in PBS and disrupted by ultrasonic in Tris-Triton X-100 solution (20 mM Tris, 2mM MgCl2, 150 mM NaCl and 1% Triton X-100). Supernatant was collected after centrifugation and the activity of ALP measured by HITACHI 7000 automatic biochemistry analyzer with α-nitrophenyl phosphate used as substrate. Osteocalcin concentrations in the supernatant were determined by ELISA assay. Intracellular total protein content was measured by the Bradford method and used to adjust ALP and osteocalcin levels. Type I collagen in cultured cells at different stages was stained by using the Van Gieson staining method.
Management of the 17β-estradiol
For 17β-estradiol intervene, the medium of cells grown in flasks was changed every 2 days and transferred into MEM containing only 0.1% bovine serum albumin 3 days before the addition of E2. According to approximate values of physiological E2 concentration in the human body, 4 concentrations of E2 (Sigma) including 0, 1×10-6, 1×10-8 and 1×10-10 mol/L were employed to treat cultured HOB and MG-63 cells at the stage of bone matrix maturity.
Detecting the mRNA Expression by RT-PCR
RNA was extracted according to the manufacture instructions for TRIZOL (GIBCO/BRL, USA), then was treated by RNase-free DNase I，after electrophoresis on 1.2% agarose gel, the optical density ratio of 28S:18S is 1.6∶1, then optical density (OD) ratio (A260/A280) determined by ultraviolet spectrophotometer, RNA concentrations were then calculated. A 219 bp fragment (496 to 714 base pair) of full-length cDNA of human OPG was amplified with primers as follows: (+) 5'AACCCCAGAGCGAAATAC3'; (-) 5'AAGAATGCCTCCTCACAC3'. β-actin gene (620 bp) was simultaneously amplified as an internal control. One-step RT-PCR was carried out on the PE2400 Thermal Cycler (Perkin-Elmer, Inc., U.S.A.) with an annealing temperature of 56℃ and a total cycle number of 26. RT-PCR products were electrophoresed on a 1.5% agarose gel and visualized by ethidium bromide staining. Optical density of electrophoretic bands was measured by the Bio-Rad Gel Doc 2000 Image Analyzing System and the OD percentages of OPG to β-actin were then calculated.
Western-blot and Gel Imaging Analysis
The cultured cells were washed 2 times by PBS, then lysed the cell by lysate buffer. Centrifuge the lysate at 4℃ for 20 min, extracted protein samples were electrophoresed on a 9.5% SDS-polyacrylamide gel (60 μg in each lane) and transferred to PVDF membrane. The blot was blocked by non-fat dried milk for non-specific binding and incubated with rabbit anti-human OPG antibody for 1 h. The membrane was washed and subsequently incubated with a secondary goat anti-rabbit horseradish peroxidase (HRP)-labeled antibody for 1h, followed by incubation with ECL substrate for 1 min. The blots were then analyzed by Image Master VDS-CL using the ECL tray.
Statistical Analysis
All experiments above were replicated 3 to 4 times. Data in this study were analyzed by SPSS 9.0 for Windows statistical software. The results were expressed in x±s and differences between groups were determined by ANOVA.
RESULTS
Alkaline Phosphatase Activities
Figure 1A illustrates the changes of ALP activity over time. The expression of ALP was evident in both HOB and MG-63 cells; secreted amounts of ALP in HOB were 13~20 times those in MG-63 cells (19.6~74.3 mu/mg protein vs 1.23~3.58 mu/mg protein,P<0.01). The activity of ALP in both cell types reached the highest levels by the 12th day of culture and then diminished gradually. The maximal expression levels of ALP in HOB and MG-63 cells were 74.3 mu/mg protein and 3.58 mu/mg protein, respectively, being 3~4 and 2~3 times greater than those levels at initiation of culture, i.e. (74.3 ± 4.7)mu/mg vs(19.6 ±1.6)mu/mg protein and (3.58 ± 0.28)protein vs (1.24 ± 0.19)mu/mg protein, respectively.
Osteocalcin
The expression of osteocalcin in MG-63 cells was too weak to be detected by radioimmunoassay in this study while its expression was detected in HOB, which had a slight increase at initiation of culture [(3.4 ± 0.30)ng/mg protein vs (3.84 ± 0.39)ng/mg protein)], but began a decrease after 6 days of culture (Figure 1B).
Figure 1 Time course of alkaline phosphatase (ALP) activity (A) and osteoclacin (B) changes in MG-63 cells and human primary osteoblasts (HOB)
Ossein Staining
No obvious bone nodules were observed by microscopy after 6 days of culture and Van Gieson staining for cultured cells revealed pale red collagen suggesting low collagen production. By 12 days of culture, red-stained collagen and bone nodules were visible. By 18 days, cells had become very dense without clear outline; numerous nodules of various size and scarlet-stained collagen with more intensity at nodules than other areas were observed (Figure 2).
OPG Expression
The expression of OPG mRNA in MG-63 cells was weak after 6 days of culture and increased 3~4 times by the 12th day of culture (P<0.01), and decreased slightly after 18 days of culture (P<0.01)(Figure 3). E2 increased OPG gene expression in MG-63 cells with the concentration of 1×10-8 mol/L obviously being the most effective by increasing OPG mRNA expression about 4 times (P<0.001) in MG-63 cells (Figure 4). OPG was expressed in HOB in a time-dependent way, which reached the highest level by the 12th day of culture about 2 times that on the 6th day of culture (42% vs 84%)(Figure 3),but the production of OPG in HOB was less than that in MG-63 cells. The expression of OPG gene in HOB was elevated by E2, especially at the concentration of 1×10-8 mol/L, which increased OPG mRNA expression 3 times (483% vs 66%,P<0.01) compared to control (Figure 5). Western blot analysis showed that E2 increased OPG protein expression obviously both in MG-63 and HOB cells.
Figure 2 Time course of collagen content changes in cultured MG-63 cells by Van Gieson staining. Cells cultured for 6 days (A), 12 days (B), 18 days (C)
Figure 3 Time course of OPG mRNA expression in cultured MG-63 cells and human primary osteoblasts. The cells were cultured for 3, 6,12 and 18 days, the OPG mRNA expression was determined by RT-PCR
Figure 4 Effects of 17β-estradiol on the expression of OPG mRNA in the osteoblast-like MG-63 cell and human primary osteoblasts
Figure 5 Effects of 17β-estradiol on the expression of OPG protein in the osteoblast-like MG-63 cell and human primary osteoblasts (HOB) analyzed by Western blot.
DISCUSSION
OPG is a member of TNF receptor superfamily. It has been demonstrated that OPG / receptor activator of nuclear factor kappa-B (RANK)/RANK-ligand (RANKL) system is effective in regulating the differentiation, growth, fusion and activation of osteoclasts. OPG and RANKL are regulated by many hormones including glucocorticosteroid, vitamin D and estrogen and so on,and cytokines such as TNF-α and interleukin-1, 4, 6, 11 and 17, etc. and transcription factors such as cbfa-1 and PPAR-γ and so on [1, 2,5, 6]. Estrogen has been confirmed to greatly influence the activities of osteoblasts and osteoclasts in experiments both in vitro and in vivo, but mechanisms for this effect have not been clarified. There have been no reports on the effects of E2 on cultured cells at different stages of growth. In our previous studies, we have found that E2 increased bone mineral density in female rats undergoing ovariectomy and suppressed expression of matrix metalloproteinase-1 (MMP-1) in osteoblasts with different effects on MMP expression at different stages of culture [7, 8]. By determining different stages of cell differentiation based on amounts of ALP and osteocalcin release, collagen staining and numbers of bone nodules in cultured cells, osteoblastic cells were found to mature by the 12th to 15th day of culture and to begin mineralization after about 18 d of culture. Therefore, the 6th, 12th and 18th day were selected as 3 key time points for determination of osteoblast maturation [9] for the further study of the effects of E2 on the expression of OPG mRNA in osteoblasts. OPG was expressed in osteoblasts and its N-terminal domains (D1-D4) effectively inhi bited osteoclastogenesis and reduced the activity and survival rate of adult multi-nuclear osteoclasts resulting in decreased bone resorptive activity of osteoclasts [10]. A variety of hormones such as 1,25(OH)2D3, parathyroid hormone (PTH), PG-E2, dexamethasone and immune factors including IL-1 and TNF and so on have the ability to regulate OPG gene expression in osteoblasts [11, 12]. Disruption of the OPG gene in the OPG knockout mouse produced severe osteoporosis leading to susceptibility to bone fracture indicating the importance of this molecule in normal bone physiology [13]. Treatment of rats undergoing ovariectomy with purified OPG protein was able to prevent bone loss [14]. Transgenic overexpression of OPG in mice not only reversed osteoporosis but also produced an osteopetrotic phenotype [15]. Postmenopausal osteoporotic patients have normal blood levels of ALP, osteocalcin and calcium, but have higher OPG levels than healthy individuals [16] suggesting that OPG plays a more important role in bone metabolism than other mediators.
In this study, we chose cultured cells at different stages to observe OPG gene expression and observed OPG gene expression to decrease over time in the absence of E2 interference. The non-mineralized osteosarcoma MG-63 cell line expressed high levels of OPG which reached the highest levels 12 days after confluence being 3~4 times levels on the 6th day and maintained this level thereafter. Normal human osteoblasts expressed lower levels of OPG than MG-63 cells (about 40% of that in MG-63 cells) and the time-dependent changes of OPG expression were less apparent than those in MG-63 cells. After 12 days of culture, there was only a slight increase in OPG expression in HOB at about 2 times that on the 6th day. OPG inhibits differentiation of osteoclastic precursors into mature osteoclasts and suppresses the activity of adult osteoclasts. The weak expression of OPG gene in normal HOB may be due to their greater capability to support the survival of mature osteoclasts than MG-63 cells or other immortal cell lines. Time courses of OPG expression in both HOB and MG-63 cells were similar to those of ALP and osteocalcin indicating that OPG production in mature osteoblasts is greater than that in proliferating ones and so likewise the ability to inhibit osteoclasts is greater in adult osteoblasts.
In the present study, E2 was confirmed to increase the expression of OPG gene in both types of cells with the concentration of 1×10-8 mmol/L, which is close to the physiological concentration in microenvironment of bone tissue, having the strongest effect and exerting stronger effects on MG-63 than HOB cells. Hofbaner et al[4] reported that E2 increased the expression of OPG mRNA and protein only 3.7 and 3.2 times, respectively, in hFOB/ER-3 and hFOB/ER-9 cells.The reason for the differences between the increased amounts of OPG mRNA and protein expression caused by E2 is not clear. The MG-63 cell line was derived from a 16-year-old girl and belonged to early differentiated osteoblasts while HOB were obtained from iliac bone of adult women which had far greater levels of ALP expression than MG-63 cells, indicating that HOB had a higher degree of differentiation than the MG-63 cells. E2 had greater effects on the expression of OPG mRNA in MG-63 than in HOB cells. This suggests that the effects of E2 on osteoblasts are associated with their differentiating phases, identical to the relationship between OPG expression in normal osteoblasts and their differentiating phases. Whether the time-dependent effects of E2 on osteoblasts were related to osteogenesis, growth and senescence of bone skeleton remains to be elucidated? In this study, E2 was shown to have stronger effects on mRNA expression of OPG than on protein expression suggesting that expressions of OPG at transcriptional and translational levels in osteoblasts are not consistent. Therefore, effects of medicines for the prevention and treatment of osteoporosis on OPG gene expression in osteoblasts should not be evaluated solely at the transcriptional level. Estrogen increased the OPG gene expression in experiments in vitro, which might account for the decrease in OPG expression with aging [3, 17, 18]. However, some studies have found that circulating OPG levels in postmenopausal women were elevated and even higher than those in osteoporotic patients and older people with low bone mineral content. Further studies are needed to elucidate the differences between these in vivo and in vitro results.
Funding:This work was supported by National Natural Science,China (No.30400218)
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(Editor Jaque)(ZHOU Hou-de, BU Yan-hong,)