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Gene expression changes accompanying p53 activity during estrogen treatment of osteoblasts
Life Sciences 75 (2004) 2045 – 2055 www.elsevier.com/locate/lifescie
Gene expression changes accompanying p53 activity during estrogen treatment of osteoblasts Nalini Chandar*, Dan Logan, Ann Szajkovics, Wesley Harmston Department of Biochemistry, Chicago College of Osteopathic Medicine, Midwestern University, 555, 31st street, Downers Grove, IL 60515, United States Received 2 December 2003; accepted 24 March 2004
Abstract Estrogen is known to be anabolic for bone and we have used estrogen treatment as a paradigm to understand how p53 may affect osteoblast differentiation. In previous studies we have shown estrogen treatment to increase p53 functional activity in osteoblasts. Estrogen has been suggested to inhibit apoptosis in osteoblasts. Since the significance of a p53 increase during estrogen treatment is not apparent, we investigated the environment within osteoblasts after treatment with estrogen. We observed two peaks of p53 activity during continuous treatment of 17-[beta]-estradiol (E2) for 72h. The gene expression profile of different cell cycle regulators and apoptosis related genes at different times during treatment with 17-[beta]-estradiol were tested using gene arrays. There was an early increase in expression of several genes involved in apoptosis. This was followed by changes in expression of several genes involved in cell survival and stress response. The second peak of activity was associated with increase in expression of cell cycle regulators. Our results suggest that p53 activity may be a result of activation of several signaling pathways involving apoptosis, cell survival and cell cycle arrest. P53 may have a role in integrating these responses, which eventually results in cell cycle arrest and expression of differentiation markers. D 2004 Elsevier Inc. All rights reserved. Keywords: Osteoblast differentiation; p53; Estrogen; Gene expression
* Corresponding author. Fax: +1 630 971 6414. E-mail address: [email protected] (N. Chandar). 0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.03.028
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Introduction Postmenopausal osteoporosis due to estrogen deficiency is largely believed to be a result of increased osteoclastic activity (Frost, 1999; Romas and Martin, 1997), due to removal of estrogenmediated inhibition on osteoclast activity. However a number of studies have suggested an anabolic role for estrogen on osteoblast activity (Ernst et al., 1989; Ernst and Rodan, 1991; Scheven et al., 1992). In the case of osteoclasts and osteoblasts, mechanism of estrogen action probably involves regulation of apoptosis in these cells (Frost, 1999; Romas and Martin, 1997). Estrogen mediates its action through intracellular receptors and recent identification of estrogen receptors a and h has allowed several of the actions to be attributed to either of the two receptors. While both these receptors have been identified in human and rat osteoblast cells, their exact function must await further studies. The p53 tumor suppressor gene is inactivated in a number of tumors and the major role attributed to p53 is in maintaining the integrity of the genome by its ability to regulate various aspects of cell growth and apoptosis (Bargonetti and Manfredi, 2002). We have previously demonstrated that osteosarcomas lose p53 function (Chandar et al., 1992) and replacement of wild type p53 into these cells can inhibit tumor growth (Chandar et al., 1993). In other studies we have demonstrated a role for wild type p53 tumor suppressor gene in bone differentiation (Chandar et al., 1993; Schwartz et al., 1999; Chandar et al., 2000). These studies have shown that functional loss of p53 in osteosarcomas affects the expression of a bone specific differentiation related protein, osteocalcin (Chandar et al., 1993). Osteocalcin expression is not seen in the absence of p53 and replacement of wild type p53 into osteosarcoma cells, not only produced inhibition of tumor formation in mice, but also further differentiation and mineralization of the tumor (Chandar et al., 1993 and unpublished observations). We have demonstrated that this ability of p53 to allow expression of osteocalcin may be mediated at the level of gene transcription (Schwartz et al., 1999). Using primary osteoblasts from p53 knockout mice, we have also been able to demonstrate that a deficiency of p53 causes a deficiency in the differentiation capacity of osteoblasts (Chandar et al., 2000). In more recent studies we have been able to show that functional p53 activity is increased in osteoblasts on treatment with E2 (Bovenkerk et al., 2003), and this increase may mediate bone differentiation, rather than a cell cycle arrest or apoptosis, roles that are commonly attributed to p53 (Scheven et al., 1992). On further analysis of p53 levels after E2 treatment, we found a second peak of activity to occur 48 to 72 hours after continuous E2 treatment. Recently it has been demonstrated that E2 inhibits apoptosis in osteoblasts using non-classical mechanisms that does not involve the steroid binding to its cognate DNA sequence, rather involving perturbation of plasma membrane signaling mechanisms (Kousteni et al., 2001). In light of this information, we have attempted to look at gene expression related to apoptosis and other signaling pathways to determine if changes in apoptosis related genes occur during treatment. We have focused on the two time points when p53 levels are elevated to determine the association of p53 expression to changes gene expression related to apoptosis, cell cycle arrest with emphasis on p53 regulated genes. Through utilization of gene expression profiling, we have a preliminary understanding of how pathways of signal transduction and apoptosis may interact to eventually produce the responses seen during E2 treatment. This we hope will throw some light on how E2 and p53 influence bone differentiation, and also the possible interrelationships between estrogen and p53.
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Materials and methods ROS17/2.8 cells (provided by Dr. G.Rodan, Merck, Research Laboratory, West Point, PA) was grown in minimal essential medium with a-F12 with 10% fetal bovine serum in a modified atmosphere of 95% air and 5% CO2 at 37C. This line contains a wild type endogenous p53 (Schwartz et al., 1999) and can be induced to mineralize in culture and express genes associated with advanced stages of differentiation. The ROS17/2.8 cells were stably transfected with the plasmid PG-13-CAT or MG15-CAT (a kind gift of Dr. B. Vogelstein, Johns Hopkins University, Baltimore, MD) as described earlier (Schwartz et al., 1999) and used for these studies. Cell Culture conditions and Treatment with 17b-Estradiol Cells for E2 treatment were exposed to phenol red free media before and during treatment with E2. The water-soluble form 17h-Estradiol (Sigma, St. Louis, MO) was used at the concentrations indicated in the results section. Cells used for E2 treatment were exposed to 2% charcoal treated serum containing media (phenol red free) for 24–48 hours before treatment with E2. For experiments requiring E2 for longer than 24hours, fresh media with E2 was maintained on cells. Unless otherwise mentioned all experiments were done using E2 to a final concentration of 10 11M. This concentration is based on results obtained with our previous studies where we saw maximal induction of p53 at 10 11M–10 12M (Bovenkerk et al., 2003). Measurement of CAT activity Harvested cells were suspended in buffered saline and then in a 0.25M Tris buffer pH 7.8, disrupted by 3 freeze-thaw cycles, and supernatants were collected after centrifugation and heated at 658C for 10 minutes to inactivate cellular acetylase activity. Protein concentrations were measured with the Bradford method and equal amounts of protein were used in the assays. CAT activity was determined by means of thin layer chromatography and liquid scintillation counting, and was measured over a linear range of chloramphenicol acetylation such that the fraction acetylated was proportional to actual activity. cDNA gene expression microarray analysis RNA isolated from cells treated with E2 at 0 h, 6h, 16h and 48h were utilized to compare the changes in gene expression related to apoptosis and other signaling pathways. Three different arrays were used in these experiments: GEArray mouse Apoptosis, a GEArray mouse signal transduction pathway finder and p53 signaling pathway were used for the analyses. (Superarray, Inc.). Each array contained about 23–96 marker genes. Many of the genes were repeated in these different arrays and served as internal controls. For the generation of cDNA probes, 10 ug of total RNA was mixed with 2ul of GEA-primer mix and incubated at 70C for 2 minutes. Then 20ul of labeling mix containing dNTP mic, 1M DTT, MMLV reverse transcriptase (50U/ul; 5ul of [a-32P-dCTP (370MBq/ml, 10Mci/ml; Amersham Pharmacia Biotech,) and RNAse inhibitor (40U/ul) was added and incubated at 42C for 25 minutes according to the manufacturer’s instructions. Denatured cDNA probes were then hybridized at 68C and washed according to instructions. Quantitation of radioactivity on blots was done on a Molecular Dynamics Phosphorimager using ImageQuant software. The intensity of each spot was calculated using
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local median background subtraction. The relative expression of each gene was determined by normalizing the signal intensity of each gene in the membrane to the signal of the internal control gene GAPDH. The change in expression for each spot was calculated as fold change over control. The cDNA microarray was done with 3 independent total RNA samples. Western blot analyses Cells were lysed in RIPA buffer containing a protease inhibitor cocktail (Boehringer Mannheim, Germany) and protein aliquots were analyzed by western blotting with a monoclonal antibody specific to mouse Bax, RB and tubulin (Oncogene Research Products Inc, USA). Detection was carried out using enhanced chemiluminescence. (Amersham Corporation, Arlington Heights, IL).
Results 17B-estradiol treatment results in a biphasic change in p53 transcription activating activity We have previously shown p53 functional activity to be increased during 16h after E2 treatment (Bovenkerk et al., 2003). Recently we found a second peak of activity to be present around 48 h after treatment (Fig. 1). This was studied with the use of ROS17/2.8 cell line stably carrying a construct containing several copies of the p53 response element (ROS-PG13) as described earlier (Schwartz et al., 1999). We have previously confirmed that p53 activity results in changes to genes downstream to it as evidenced by increase in activity of p21 and mdm-2 gene expression (Bovenkerk et al., 2003). This however, did not give us any insight into why p53 activity is induced by 17-h-estradiol. In order to understand the significance of p53 increase, we carried out gene expression analyses using macroarrays of genes involved in apoptosis, cell cycle arrest, cell survival and p53 regulation.
Fig. 1. Changes in p53 transcription activating activity during treatment with E2. ROS17/2.8 osteoblastic cells stably carrying PG13-CAT was used to determine p53 transcription activating activity during treatment with 10–12M E2. CAT activity was measured are different time intervals and expressed as fold elevation compared to control cells. Figure is representative of an experiment repeated thrice.
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The environment in E2 treated osteoblasts 17-h-Estradiol is not known to be associated with apoptosis. In fact studies have shown an increase in osteoblast proliferation when treated with h-estradiol. Recent studies have shown that E2 might inhibit apoptosis in osteoblasts by signaling through pathways not involving classical steroid mediated DNA binding and gene transcription (Kousteni et al., 2001). In order to define the reason for p53 expression in osteoblasts treated with E2, we first compared the gene expression profiles within cells at time different time points to determine the environment during E2 treatment within osteoblasts. We chose to look for differences in genes involved in apoptosis, specific signal transduction pathways and genes that are known to be involved in p53 mediated signaling pathways. The gene arrays were hybridized using RNA from 0, 6, 16 and 48h E2 treated samples. In order to establish the sequence of events we compared different treatments to untreated samples, and compared the different time intervals to each other (0 to 16. 0 to 48h, 6h to 16h and 16h to 48h). This gave us a better idea of the genes that showed sustained expression during the treatment intervals. Apoptosis related genes are increased early during treatment with E2 Several genes known to be associated with apoptosis were increased at 16h when compared to 0 h control (Fig. 2). However, when the same expression was compared between 6h and 16h there was little difference in the level of gene expression, showing that changes in expression of apoptosis genes occurred early during E2 treatment (Table 1).
Fig. 2. Changes in apoptosis and related genes during E2 treatment of ROS17/2.8 cells: The gene array was performed with RNA from 0 and 16h time interval and reported as fold change at 16h when compared to untreated controls. Other details are as described under material and methods.
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Table 1 Analyses of timing of apoptosis gene expression during E2 treatment Gene
6h
16h
Bad Bax Bcl2 Caspase Caspase Caspase Caspase Caspase DR5
2.4 2.7 1.9 1 2 1 1 1 1
1 1 1 4 1 1 1 1 2.5
1 2 3 7 8
The gene array experiments were carried out with 0, 6 h and 16 h samples and fold change obtained. The 6h interval gene expression was then compared to 16h time interval to determine the timing of changes in gene expression. Details are described under methods.
Expression of genes involved in the survival pathway is increased during 16h after E2 treatment Increase in expression of NFKB, c-myc and iNOS genes was observed at 16h after E2 treatment (Table 2A) showing that signals for cell survival is activated and follows an increase in apoptosis related gene expression. Expression of genes related to cell cycle arrest is increased during E2 treatment Several of the genes involved in cell cycle regulation especially CDK inhibitors (p21, p57Kip2) were increased (Table 2B). Several of them belonged to the TGF-beta pathway (p21, p27Kip, p57Kip2). E2 is known to increase TGF-beta expression (Heino et al., 2002) and it is possible that up regulation of some of these genes is mediated by TGF-beta. P53 regulated genes are increased in activity 16h after E2 treatment We compared a set of 6 genes that are regulated by p53 during the time interval of 6 and 16 h to determine if the environment within cells changed as a result of p53 expression at 16h (Fig. 3). Except for Bax and E124 all the other genes ( mdm2, p21, caspase1 and GADD45) were increased in activity at 16h when compared to 6h. Table 2 A and B: Analyses of changes in cell cycle arrest and cell survival genes during E2 treatment: Some of the genes that showed alteration during E2 treatment were classified according to their established functional roles A. Genes involved in survival pathway (16h)
Fold Change
B. Genes involved in cell cycle arrest (16h)
Fold Change
NfKB iNOS C-myc
3.3 3.9 2.0
P21 GADD45 P57/Kip2 RB
3.0 2.0 2.9 2.9
Fold change was calculated based on comparison to 0 time untreated controls.
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Fig. 3. P53 responsive genes expressed during E2 treatment compared at 6 and 16h. Changes in expression of a set of p53 responsive genes were compared at the two different time intervals to demonstrate a p53-mediated effect.
Increase in activity of several of the genes are sustained In order to determine changes associated with cell cycle arrest and p53 regulated gene expression was sustained during the 48h time interval when p53 activity was high, we chose a few genes belonging to these categories to demonstrate an association with E2 treatment and p53 function (Kip1, Kip2, mdm2, BMP-4) (Fig. 4). The increase in activity that occurred at 16h was sustained at 48 h showing that the environment after E2 treatment results in an arrest to the cell cycle. BMP-4 was recently shown to cause growth arrest and osteoblast differentiation (Kinyamu and Archer, 2003) and an sustained increase in expression of BMP-4 also suggests that the
Fig. 4. Changes in gene expression of cell cycle arrest genes is sustained over 48h.
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Table 3 A and B: Gene array containing 96-p53 related genes were analyzed for changes in expression at 16h when compared to untreated controls A. Downstream effectors
Fold Change compare to 16h
Mdm2 TSP-1 Cyclin G Tnfaip 1 Sfn Gtse-1 Hsp-70 EF1A
2.7 2.45 1.9 3.23 2.25 2.6 3.43 2.0
B. P53 modifiers
Fold Change compared to 16h
Cyclin H Casein Kinase 1a1 Casein Kinase 2a2 Cathepsin Wrn
2.7 2.6 2.5 2.1 2.5
Genes that showed a change in expression in osteoblasts were classified according to whether they were downstream effectors or p53 function modifiers.
environment within the cells may be conducive for differentiation, a process we have shown to result on E2 treatment (Bovenkerk et al., 2003). P53 modifiers as well as downstream effectors are increased during E2 treatment: Using gene arrays of p53 regulated genes we found that several of the p53 regulated genes were increased during E2 treatment. These genes belong to groups that are known to modify p53 gene expression and groups that are downstream effectors. Both types of genes were increased in expression (Table 3A and B) when compared to 16h time interval.
Fig. 5. Western blot of proteins that showed alteration in gene expression during E2 treatment: Protein lysates from E2 treated cells were separated on SDS-PAGE and probed with antibodies to Bax and RB. Antibodies to tubulin were used to demonstrate equal loading of lanes.
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Several p53- regulated genes undergo changes in expression during E2 treatment In order to independently confirm changes seen in some of the genes, we used western blot analyses to establish the expression of Bax (marker for apoptosis function) and RB (cell cycle arrest) during E2 treatment. As shown in Fig. 5, Bax expression was higher early during treatment (about 2-fold higher during the 0–6h time interval when compared to 16–24h) and RB expression increased during 16h treatment period. RB expression is high during the 0 time and probably is a reflection of cell cycle arrest seen with switching cells to 2% serum containing media at the beginning of the experiment. The levels then drop after E2 treatment is initiated. While Bax is known to be p53 regulated, the change in Bax expression corresponded better to an increase in other apoptosis markers rather than to p53 and corresponds well to the activation of apoptosis that is seen early during treatment. At a later point (16h) RB expression is increased and this corresponded well with the gene array data on RB as well as with other genes that are important in mediating cell cycle arrest that were altered around 16h. The protein expression data therefore independently corroborated with the data obtained from gene arrays.
Discussion We have investigated the environment within osteoblasts during treatment with E2 to define the reason for increase in wild type p53 function. We focused on two time intervals during E2 treatment when p53 activity was maximal. The first increase occurred at 16h after treatment with 10 11M E2 and the second peak was seen at 48h after E2 treatment. When changes in gene expression of genes involved in apoptosis, signal transduction pathways and p53 related genes (total of about 200) were compared, we found that specific changes in genes related to p53 correlated to the presence of increased p53 expression. Apoptosis related gene expression was increased early during treatment but was not accompanied by cell death. Clearly, the increase in apoptosis related genes did not result in apoptosis even though expression of several of these genes was high 16h after treatment with E2. Two possibilities exist to account for this observation. On one hand, osteoblasts may be programmed to die without adequate survival signals and treatment with E2 perhaps lifts the block to apoptosis. It is also possible that expression of caspases and several of the genes involved in DNA damage is part of the osteoblast differentiation process unrelated to apoptosis (Mogi and Togari, 2003). This increase is followed by expression of several genes involved in the cell cycle arrest and survival pathway during the 16h period. This may also relate to the ability of E2 to extend the lifespan of osteoblasts (Kousteni et al., 2001; Manolagas et al., 2002). Some of these genes are regulated by p53 and an increase in expression of these genes may be a result of activation by p53. The second peak of increase in p53 is probably important to sustain the cell cycle arrest during this time. While several proteins important for modification of p53 function are increased (mdm-2, Casein Kinase etc), it does not appear to affect p53 function and might reflect the fact that the role of p53 and mdm2 in the control of E2 mediated cellular physiology might be different from events that occur during apoptosis due to DNA damage ( Kinyamu and Archer, 2003; Kannan et al., 2000). However it is also possible that the biphasic response seen in p53 represents a down regulation of p53 after the initial increase by these p53 modifiers. Based on these observations, two possibilities exist for the role of p53 during E2 treatment: p53 increase may be a part of an apoptosis trigger, as several of the genes regulated by p53 are increased at 16h (Bax, DR5, caspase 1 etc). On the other hand, p53 may be necessary to integrate signals and produce
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cell cycle arrest. This possibility is in agreement with changes seen in p53- regulated genes involved in cell cycle arrest p21, Gadd45 etc. It is known that E2 modulates the production of TGF-beta in osteoblasts and osteocytes (Kannan et al., 2000). The increase in RB expression may also be related to the cell cycle arrest of these cells. We have used estrogen treatment as a paradigm to demonstrate a role for p53 in osteoblast differentiation. We believe that the changes seen in p53 during E2 treatment are a reflection of its role in facilitating osteoblast differentiation. We have shown in previous studies that full differentiation of osteoblasts requires the presence of p53. This inference is supported by recent studies where interaction of E2 with p53 has been observed (Kinyamu and Archer, 2003; Liu et al., 2000; Qin et al., 2002). These studies have indicated a role for p53’s downstream effector mdm-2 in E2’s responses. P53 is shown to remain active in these studies (Kinyamu and Archer, 2003; Liu et al., 2000) which indicates that mdm-2 does not degrade p53 under physiological conditions. The larger increase seen in the second peak may indicate that the amount of p53 dictates the outcome of the cell. While these types of studies have been important to gain an initial understanding of the environment within osteoblasts during E2 treatment, further studies are necessary to gain a better understanding and dissect out pathways in which E2 and p53 may cooperate to regulate outcome in osteoblasts.
Acknowledgements We thank Drs. Vogelstein and Rodan for reagents. Funding from Midwestern University to NC and American Osteopathic Association fellowship to DL and WH is gratefully acknowledged.
References Bargonetti, J., Manfredi, J.J., 2002. Multiple roles of the tumor suppressor p53. Current Opinion Oncology 14, 86 – 91. Bovenkerk, S., Lanciloti, N., Chandar, N., 2003. Induction of p53 expression and function by estrogen in osteoblasts. Calcified Tissue International 73 (33) 274 – 280. Chandar, N., Billig, B., McMaster, J., Novak, J., 1992. Inactivation of p53 gene in human and murine osteosarcoma cells. British Journal of Cancer 65, 208 – 214. Chandar, N., Campbell, P., Novak, J., Smith, M., 1993. Dependence of induction of osteocalcin gene expression on the presence of wild-type p53 in a murine osteosarcoma cell line. Molecular Carcinogenesis 8, 299 – 305. Chandar, N., Donehower, L., Lanciloti, N., 2000. Reduction in p53 dosage diminishes differentiation capacity of osteoblasts. Anticancer Research 20, 2555 – 2562. Ernst, M, Heath, J.K., Schmid, C., Froesch, R.E., Rodan, G.A., 1989. Evidence for a direct effect of estrogen on bone cells in vitro. Journal of Steroid Biochemistry 34, 279 – 284. Ernst, M., Rodan, G.A., 1991. Estradiol regulation of insulin-like growth factor-I expression in osteoblastic cells: evidence for transcriptional control. Molecular Endocrinology 8, 1081 – 1089. Frost, H.M, 1999. On the estrogen-bone relationship and postmenopausal bone loss: A new model. Journal of Bone and Mineral Research 14, 1473 – 1477. Heino, T.J, Hentunen, T.A., Vaananen, H.K., 2002. Osteocytes inhibit osteoclastic bone resorption through transforming growth factor-beta: enhancement by estrogen. J. Cellular Biochemistry 85 (1) 185 – 197. Kannan, K., Amariglio, N., Rechavi, G., Givol, D, 2000. Profile of gene expression regulated by induced p53: connection to the TGF-beta family. FEBS Lett. 470 (1) 77 – 82. Kinyamu, H.K., Archer, T.K., 2003. Estrogen receptor-dependent proteosomal degradation of the glucocorticoid receptor is coupled to an increase in mdm2 protein expression. Molecular and Cellular Biology 16, 5667 – 5681.
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Kousteni, S., Bellido, T., Plotkin, L.I., O’Brien, C.A., Bodenner, D.L., Han, L., Han, K., DiGregorio, G.B., Katzenellenbogen, J.A., Katzenellenbogen, B.S., Roberson, P.K., Weinstein, R.S., Jilka, R.L., Manolagas, S.C., 2001. Nongenotropic, sexnonspecific signaling through the estrogen or androgen receptors: dissociation from transcriptional activity. Cell 104 (5) 719 – 730. Liu, G., Schwartz, J.A., Brooks, S.C., 2000. Estrogen receptor protects p53 from deactivation by human double minute-2. Cancer Research 60, 1810 – 1814. Manolagas, S.C., Kousteni, S., Jilka, R.L., 2002. Sex steroids and bone. Recent Progress in Hormone Research 57, 385 – 409. Mogi, M., Togari, A., 2003. Activation of caspases is required for osteoblastic differentiation. Journal of Biological Chemistry 278 (48) 47477 – 47482. Qin, C., Nguyen, T., Stewart, J., Samudio, I., Burghardt, R., Safe, S., 2002. Estrogen up-regulation of p53 gene expression in MCF-7 breast cancer cells is mediated by calmodulin kinase IV-dependent activation of a nuclear factor kappaB/CCAATbinding transcription factor-1 complex. Molecular Endocrinology 16, 1793 – 1809. Romas, E., Martin, T.J., 1997. Cytokines in the pathogenesis of osteoporosis. Osteoporos International 7, (Suppl 3) S47–53. Scheven, B.A., Damen, C.A., Hamilton, N.J., Verhaar, H.J., Duursma, S.A., 1992. Stimulatory effects of estrogen and progesterone on proliferation and differentiation of normal human osteoblast-like cells in vitro. Biochemical Biophysical Research Communications 186, 54 – 60. Schwartz, K.A., Campione, A.L., Moore, M.K., Chandar, N., 1999. P53 transactivity during osteoblast differentiation. Molecular Carcinogenesis 25, 132 – 138.
Gene expression changes accompanying p53 activity during estrogen treatment of osteoblasts Nalini Chandar*, Dan Logan, Ann Szajkovics, Wesley Harmston Department of Biochemistry, Chicago College of Osteopathic Medicine, Midwestern University, 555, 31st street, Downers Grove, IL 60515, United States Received 2 December 2003; accepted 24 March 2004
Abstract Estrogen is known to be anabolic for bone and we have used estrogen treatment as a paradigm to understand how p53 may affect osteoblast differentiation. In previous studies we have shown estrogen treatment to increase p53 functional activity in osteoblasts. Estrogen has been suggested to inhibit apoptosis in osteoblasts. Since the significance of a p53 increase during estrogen treatment is not apparent, we investigated the environment within osteoblasts after treatment with estrogen. We observed two peaks of p53 activity during continuous treatment of 17-[beta]-estradiol (E2) for 72h. The gene expression profile of different cell cycle regulators and apoptosis related genes at different times during treatment with 17-[beta]-estradiol were tested using gene arrays. There was an early increase in expression of several genes involved in apoptosis. This was followed by changes in expression of several genes involved in cell survival and stress response. The second peak of activity was associated with increase in expression of cell cycle regulators. Our results suggest that p53 activity may be a result of activation of several signaling pathways involving apoptosis, cell survival and cell cycle arrest. P53 may have a role in integrating these responses, which eventually results in cell cycle arrest and expression of differentiation markers. D 2004 Elsevier Inc. All rights reserved. Keywords: Osteoblast differentiation; p53; Estrogen; Gene expression
* Corresponding author. Fax: +1 630 971 6414. E-mail address: [email protected] (N. Chandar). 0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.03.028
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Introduction Postmenopausal osteoporosis due to estrogen deficiency is largely believed to be a result of increased osteoclastic activity (Frost, 1999; Romas and Martin, 1997), due to removal of estrogenmediated inhibition on osteoclast activity. However a number of studies have suggested an anabolic role for estrogen on osteoblast activity (Ernst et al., 1989; Ernst and Rodan, 1991; Scheven et al., 1992). In the case of osteoclasts and osteoblasts, mechanism of estrogen action probably involves regulation of apoptosis in these cells (Frost, 1999; Romas and Martin, 1997). Estrogen mediates its action through intracellular receptors and recent identification of estrogen receptors a and h has allowed several of the actions to be attributed to either of the two receptors. While both these receptors have been identified in human and rat osteoblast cells, their exact function must await further studies. The p53 tumor suppressor gene is inactivated in a number of tumors and the major role attributed to p53 is in maintaining the integrity of the genome by its ability to regulate various aspects of cell growth and apoptosis (Bargonetti and Manfredi, 2002). We have previously demonstrated that osteosarcomas lose p53 function (Chandar et al., 1992) and replacement of wild type p53 into these cells can inhibit tumor growth (Chandar et al., 1993). In other studies we have demonstrated a role for wild type p53 tumor suppressor gene in bone differentiation (Chandar et al., 1993; Schwartz et al., 1999; Chandar et al., 2000). These studies have shown that functional loss of p53 in osteosarcomas affects the expression of a bone specific differentiation related protein, osteocalcin (Chandar et al., 1993). Osteocalcin expression is not seen in the absence of p53 and replacement of wild type p53 into osteosarcoma cells, not only produced inhibition of tumor formation in mice, but also further differentiation and mineralization of the tumor (Chandar et al., 1993 and unpublished observations). We have demonstrated that this ability of p53 to allow expression of osteocalcin may be mediated at the level of gene transcription (Schwartz et al., 1999). Using primary osteoblasts from p53 knockout mice, we have also been able to demonstrate that a deficiency of p53 causes a deficiency in the differentiation capacity of osteoblasts (Chandar et al., 2000). In more recent studies we have been able to show that functional p53 activity is increased in osteoblasts on treatment with E2 (Bovenkerk et al., 2003), and this increase may mediate bone differentiation, rather than a cell cycle arrest or apoptosis, roles that are commonly attributed to p53 (Scheven et al., 1992). On further analysis of p53 levels after E2 treatment, we found a second peak of activity to occur 48 to 72 hours after continuous E2 treatment. Recently it has been demonstrated that E2 inhibits apoptosis in osteoblasts using non-classical mechanisms that does not involve the steroid binding to its cognate DNA sequence, rather involving perturbation of plasma membrane signaling mechanisms (Kousteni et al., 2001). In light of this information, we have attempted to look at gene expression related to apoptosis and other signaling pathways to determine if changes in apoptosis related genes occur during treatment. We have focused on the two time points when p53 levels are elevated to determine the association of p53 expression to changes gene expression related to apoptosis, cell cycle arrest with emphasis on p53 regulated genes. Through utilization of gene expression profiling, we have a preliminary understanding of how pathways of signal transduction and apoptosis may interact to eventually produce the responses seen during E2 treatment. This we hope will throw some light on how E2 and p53 influence bone differentiation, and also the possible interrelationships between estrogen and p53.
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Materials and methods ROS17/2.8 cells (provided by Dr. G.Rodan, Merck, Research Laboratory, West Point, PA) was grown in minimal essential medium with a-F12 with 10% fetal bovine serum in a modified atmosphere of 95% air and 5% CO2 at 37C. This line contains a wild type endogenous p53 (Schwartz et al., 1999) and can be induced to mineralize in culture and express genes associated with advanced stages of differentiation. The ROS17/2.8 cells were stably transfected with the plasmid PG-13-CAT or MG15-CAT (a kind gift of Dr. B. Vogelstein, Johns Hopkins University, Baltimore, MD) as described earlier (Schwartz et al., 1999) and used for these studies. Cell Culture conditions and Treatment with 17b-Estradiol Cells for E2 treatment were exposed to phenol red free media before and during treatment with E2. The water-soluble form 17h-Estradiol (Sigma, St. Louis, MO) was used at the concentrations indicated in the results section. Cells used for E2 treatment were exposed to 2% charcoal treated serum containing media (phenol red free) for 24–48 hours before treatment with E2. For experiments requiring E2 for longer than 24hours, fresh media with E2 was maintained on cells. Unless otherwise mentioned all experiments were done using E2 to a final concentration of 10 11M. This concentration is based on results obtained with our previous studies where we saw maximal induction of p53 at 10 11M–10 12M (Bovenkerk et al., 2003). Measurement of CAT activity Harvested cells were suspended in buffered saline and then in a 0.25M Tris buffer pH 7.8, disrupted by 3 freeze-thaw cycles, and supernatants were collected after centrifugation and heated at 658C for 10 minutes to inactivate cellular acetylase activity. Protein concentrations were measured with the Bradford method and equal amounts of protein were used in the assays. CAT activity was determined by means of thin layer chromatography and liquid scintillation counting, and was measured over a linear range of chloramphenicol acetylation such that the fraction acetylated was proportional to actual activity. cDNA gene expression microarray analysis RNA isolated from cells treated with E2 at 0 h, 6h, 16h and 48h were utilized to compare the changes in gene expression related to apoptosis and other signaling pathways. Three different arrays were used in these experiments: GEArray mouse Apoptosis, a GEArray mouse signal transduction pathway finder and p53 signaling pathway were used for the analyses. (Superarray, Inc.). Each array contained about 23–96 marker genes. Many of the genes were repeated in these different arrays and served as internal controls. For the generation of cDNA probes, 10 ug of total RNA was mixed with 2ul of GEA-primer mix and incubated at 70C for 2 minutes. Then 20ul of labeling mix containing dNTP mic, 1M DTT, MMLV reverse transcriptase (50U/ul; 5ul of [a-32P-dCTP (370MBq/ml, 10Mci/ml; Amersham Pharmacia Biotech,) and RNAse inhibitor (40U/ul) was added and incubated at 42C for 25 minutes according to the manufacturer’s instructions. Denatured cDNA probes were then hybridized at 68C and washed according to instructions. Quantitation of radioactivity on blots was done on a Molecular Dynamics Phosphorimager using ImageQuant software. The intensity of each spot was calculated using
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local median background subtraction. The relative expression of each gene was determined by normalizing the signal intensity of each gene in the membrane to the signal of the internal control gene GAPDH. The change in expression for each spot was calculated as fold change over control. The cDNA microarray was done with 3 independent total RNA samples. Western blot analyses Cells were lysed in RIPA buffer containing a protease inhibitor cocktail (Boehringer Mannheim, Germany) and protein aliquots were analyzed by western blotting with a monoclonal antibody specific to mouse Bax, RB and tubulin (Oncogene Research Products Inc, USA). Detection was carried out using enhanced chemiluminescence. (Amersham Corporation, Arlington Heights, IL).
Results 17B-estradiol treatment results in a biphasic change in p53 transcription activating activity We have previously shown p53 functional activity to be increased during 16h after E2 treatment (Bovenkerk et al., 2003). Recently we found a second peak of activity to be present around 48 h after treatment (Fig. 1). This was studied with the use of ROS17/2.8 cell line stably carrying a construct containing several copies of the p53 response element (ROS-PG13) as described earlier (Schwartz et al., 1999). We have previously confirmed that p53 activity results in changes to genes downstream to it as evidenced by increase in activity of p21 and mdm-2 gene expression (Bovenkerk et al., 2003). This however, did not give us any insight into why p53 activity is induced by 17-h-estradiol. In order to understand the significance of p53 increase, we carried out gene expression analyses using macroarrays of genes involved in apoptosis, cell cycle arrest, cell survival and p53 regulation.
Fig. 1. Changes in p53 transcription activating activity during treatment with E2. ROS17/2.8 osteoblastic cells stably carrying PG13-CAT was used to determine p53 transcription activating activity during treatment with 10–12M E2. CAT activity was measured are different time intervals and expressed as fold elevation compared to control cells. Figure is representative of an experiment repeated thrice.
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The environment in E2 treated osteoblasts 17-h-Estradiol is not known to be associated with apoptosis. In fact studies have shown an increase in osteoblast proliferation when treated with h-estradiol. Recent studies have shown that E2 might inhibit apoptosis in osteoblasts by signaling through pathways not involving classical steroid mediated DNA binding and gene transcription (Kousteni et al., 2001). In order to define the reason for p53 expression in osteoblasts treated with E2, we first compared the gene expression profiles within cells at time different time points to determine the environment during E2 treatment within osteoblasts. We chose to look for differences in genes involved in apoptosis, specific signal transduction pathways and genes that are known to be involved in p53 mediated signaling pathways. The gene arrays were hybridized using RNA from 0, 6, 16 and 48h E2 treated samples. In order to establish the sequence of events we compared different treatments to untreated samples, and compared the different time intervals to each other (0 to 16. 0 to 48h, 6h to 16h and 16h to 48h). This gave us a better idea of the genes that showed sustained expression during the treatment intervals. Apoptosis related genes are increased early during treatment with E2 Several genes known to be associated with apoptosis were increased at 16h when compared to 0 h control (Fig. 2). However, when the same expression was compared between 6h and 16h there was little difference in the level of gene expression, showing that changes in expression of apoptosis genes occurred early during E2 treatment (Table 1).
Fig. 2. Changes in apoptosis and related genes during E2 treatment of ROS17/2.8 cells: The gene array was performed with RNA from 0 and 16h time interval and reported as fold change at 16h when compared to untreated controls. Other details are as described under material and methods.
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Table 1 Analyses of timing of apoptosis gene expression during E2 treatment Gene
6h
16h
Bad Bax Bcl2 Caspase Caspase Caspase Caspase Caspase DR5
2.4 2.7 1.9 1 2 1 1 1 1
1 1 1 4 1 1 1 1 2.5
1 2 3 7 8
The gene array experiments were carried out with 0, 6 h and 16 h samples and fold change obtained. The 6h interval gene expression was then compared to 16h time interval to determine the timing of changes in gene expression. Details are described under methods.
Expression of genes involved in the survival pathway is increased during 16h after E2 treatment Increase in expression of NFKB, c-myc and iNOS genes was observed at 16h after E2 treatment (Table 2A) showing that signals for cell survival is activated and follows an increase in apoptosis related gene expression. Expression of genes related to cell cycle arrest is increased during E2 treatment Several of the genes involved in cell cycle regulation especially CDK inhibitors (p21, p57Kip2) were increased (Table 2B). Several of them belonged to the TGF-beta pathway (p21, p27Kip, p57Kip2). E2 is known to increase TGF-beta expression (Heino et al., 2002) and it is possible that up regulation of some of these genes is mediated by TGF-beta. P53 regulated genes are increased in activity 16h after E2 treatment We compared a set of 6 genes that are regulated by p53 during the time interval of 6 and 16 h to determine if the environment within cells changed as a result of p53 expression at 16h (Fig. 3). Except for Bax and E124 all the other genes ( mdm2, p21, caspase1 and GADD45) were increased in activity at 16h when compared to 6h. Table 2 A and B: Analyses of changes in cell cycle arrest and cell survival genes during E2 treatment: Some of the genes that showed alteration during E2 treatment were classified according to their established functional roles A. Genes involved in survival pathway (16h)
Fold Change
B. Genes involved in cell cycle arrest (16h)
Fold Change
NfKB iNOS C-myc
3.3 3.9 2.0
P21 GADD45 P57/Kip2 RB
3.0 2.0 2.9 2.9
Fold change was calculated based on comparison to 0 time untreated controls.
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Fig. 3. P53 responsive genes expressed during E2 treatment compared at 6 and 16h. Changes in expression of a set of p53 responsive genes were compared at the two different time intervals to demonstrate a p53-mediated effect.
Increase in activity of several of the genes are sustained In order to determine changes associated with cell cycle arrest and p53 regulated gene expression was sustained during the 48h time interval when p53 activity was high, we chose a few genes belonging to these categories to demonstrate an association with E2 treatment and p53 function (Kip1, Kip2, mdm2, BMP-4) (Fig. 4). The increase in activity that occurred at 16h was sustained at 48 h showing that the environment after E2 treatment results in an arrest to the cell cycle. BMP-4 was recently shown to cause growth arrest and osteoblast differentiation (Kinyamu and Archer, 2003) and an sustained increase in expression of BMP-4 also suggests that the
Fig. 4. Changes in gene expression of cell cycle arrest genes is sustained over 48h.
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Table 3 A and B: Gene array containing 96-p53 related genes were analyzed for changes in expression at 16h when compared to untreated controls A. Downstream effectors
Fold Change compare to 16h
Mdm2 TSP-1 Cyclin G Tnfaip 1 Sfn Gtse-1 Hsp-70 EF1A
2.7 2.45 1.9 3.23 2.25 2.6 3.43 2.0
B. P53 modifiers
Fold Change compared to 16h
Cyclin H Casein Kinase 1a1 Casein Kinase 2a2 Cathepsin Wrn
2.7 2.6 2.5 2.1 2.5
Genes that showed a change in expression in osteoblasts were classified according to whether they were downstream effectors or p53 function modifiers.
environment within the cells may be conducive for differentiation, a process we have shown to result on E2 treatment (Bovenkerk et al., 2003). P53 modifiers as well as downstream effectors are increased during E2 treatment: Using gene arrays of p53 regulated genes we found that several of the p53 regulated genes were increased during E2 treatment. These genes belong to groups that are known to modify p53 gene expression and groups that are downstream effectors. Both types of genes were increased in expression (Table 3A and B) when compared to 16h time interval.
Fig. 5. Western blot of proteins that showed alteration in gene expression during E2 treatment: Protein lysates from E2 treated cells were separated on SDS-PAGE and probed with antibodies to Bax and RB. Antibodies to tubulin were used to demonstrate equal loading of lanes.
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Several p53- regulated genes undergo changes in expression during E2 treatment In order to independently confirm changes seen in some of the genes, we used western blot analyses to establish the expression of Bax (marker for apoptosis function) and RB (cell cycle arrest) during E2 treatment. As shown in Fig. 5, Bax expression was higher early during treatment (about 2-fold higher during the 0–6h time interval when compared to 16–24h) and RB expression increased during 16h treatment period. RB expression is high during the 0 time and probably is a reflection of cell cycle arrest seen with switching cells to 2% serum containing media at the beginning of the experiment. The levels then drop after E2 treatment is initiated. While Bax is known to be p53 regulated, the change in Bax expression corresponded better to an increase in other apoptosis markers rather than to p53 and corresponds well to the activation of apoptosis that is seen early during treatment. At a later point (16h) RB expression is increased and this corresponded well with the gene array data on RB as well as with other genes that are important in mediating cell cycle arrest that were altered around 16h. The protein expression data therefore independently corroborated with the data obtained from gene arrays.
Discussion We have investigated the environment within osteoblasts during treatment with E2 to define the reason for increase in wild type p53 function. We focused on two time intervals during E2 treatment when p53 activity was maximal. The first increase occurred at 16h after treatment with 10 11M E2 and the second peak was seen at 48h after E2 treatment. When changes in gene expression of genes involved in apoptosis, signal transduction pathways and p53 related genes (total of about 200) were compared, we found that specific changes in genes related to p53 correlated to the presence of increased p53 expression. Apoptosis related gene expression was increased early during treatment but was not accompanied by cell death. Clearly, the increase in apoptosis related genes did not result in apoptosis even though expression of several of these genes was high 16h after treatment with E2. Two possibilities exist to account for this observation. On one hand, osteoblasts may be programmed to die without adequate survival signals and treatment with E2 perhaps lifts the block to apoptosis. It is also possible that expression of caspases and several of the genes involved in DNA damage is part of the osteoblast differentiation process unrelated to apoptosis (Mogi and Togari, 2003). This increase is followed by expression of several genes involved in the cell cycle arrest and survival pathway during the 16h period. This may also relate to the ability of E2 to extend the lifespan of osteoblasts (Kousteni et al., 2001; Manolagas et al., 2002). Some of these genes are regulated by p53 and an increase in expression of these genes may be a result of activation by p53. The second peak of increase in p53 is probably important to sustain the cell cycle arrest during this time. While several proteins important for modification of p53 function are increased (mdm-2, Casein Kinase etc), it does not appear to affect p53 function and might reflect the fact that the role of p53 and mdm2 in the control of E2 mediated cellular physiology might be different from events that occur during apoptosis due to DNA damage ( Kinyamu and Archer, 2003; Kannan et al., 2000). However it is also possible that the biphasic response seen in p53 represents a down regulation of p53 after the initial increase by these p53 modifiers. Based on these observations, two possibilities exist for the role of p53 during E2 treatment: p53 increase may be a part of an apoptosis trigger, as several of the genes regulated by p53 are increased at 16h (Bax, DR5, caspase 1 etc). On the other hand, p53 may be necessary to integrate signals and produce
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cell cycle arrest. This possibility is in agreement with changes seen in p53- regulated genes involved in cell cycle arrest p21, Gadd45 etc. It is known that E2 modulates the production of TGF-beta in osteoblasts and osteocytes (Kannan et al., 2000). The increase in RB expression may also be related to the cell cycle arrest of these cells. We have used estrogen treatment as a paradigm to demonstrate a role for p53 in osteoblast differentiation. We believe that the changes seen in p53 during E2 treatment are a reflection of its role in facilitating osteoblast differentiation. We have shown in previous studies that full differentiation of osteoblasts requires the presence of p53. This inference is supported by recent studies where interaction of E2 with p53 has been observed (Kinyamu and Archer, 2003; Liu et al., 2000; Qin et al., 2002). These studies have indicated a role for p53’s downstream effector mdm-2 in E2’s responses. P53 is shown to remain active in these studies (Kinyamu and Archer, 2003; Liu et al., 2000) which indicates that mdm-2 does not degrade p53 under physiological conditions. The larger increase seen in the second peak may indicate that the amount of p53 dictates the outcome of the cell. While these types of studies have been important to gain an initial understanding of the environment within osteoblasts during E2 treatment, further studies are necessary to gain a better understanding and dissect out pathways in which E2 and p53 may cooperate to regulate outcome in osteoblasts.
Acknowledgements We thank Drs. Vogelstein and Rodan for reagents. Funding from Midwestern University to NC and American Osteopathic Association fellowship to DL and WH is gratefully acknowledged.
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