- Email: [email protected]
Dexamethasone downregulates the expression of parathyroid hormone-related protein (PTHrP) in mesenchymal stem cells
Steroids 74 (2009) 277–282
Contents lists available at ScienceDirect
Steroids journal homepage: www.elsevier.com/locate/steroids
Dexamethasone downregulates the expression of parathyroid hormone-related protein (PTHrP) in mesenchymal stem cells Mikael Ahlström ∗ , Minna Pekkinen, Christel Lamberg-Allardt Calcium Research Unit, Department of Applied Chemistry and Microbiology, P.O. Box 66, 00014 University of Helsinki, Finland
a r t i c l e
i n f o
Article history: Received 23 September 2008 Received in revised form 28 October 2008 Accepted 4 December 2008 Available online 11 December 2008 Keywords: Parathyroid hormone-related protein (PTHrP) Osteoblasts Mesenchymal stem cells Glucocorticoids PTH1R
a b s t r a c t Parathyroid hormone-related protein (PTHrP) has been shown to have anabolic effects in women with postmenopausal osteoporosis. PTHrP promotes the recruitment of osteogenic cells and prevents apoptotic death of osteoblasts and osteocytes. The receptor responsible for the effects of PTHrP is the common PTH/PTHrP receptor (PTH1R). Glucocorticoids (GC) are commonly used as drugs to treat inflammatory diseases. Long-term GC treatments are often associated with bone loss which can lead to GC-induced osteoporosis. The aim of this work was to study the effects of the glucocorticoid dexamethasone (Dex) on the expression of PTHrP and PTH1R in adult human mesenchymal stem cells, the progenitor cells of osteoblasts. Adult human mesenchymal stem cells (hMSC) were cultured and differentiated by standard methods. The expression of PTHrP and PTH1R mRNA was assayed by real-time qPCR. The PTHrP release into the culture media was measured by an immunoradiometric assay. Treatment with Dex (10 nM) resulted in an 80% drop in the PTHrP release within 6 h. A 24 h Dex treatment also reduced the expression of PTHrP mRNA by up to 90%. The expression of PTH1R receptor mRNA was simultaneously increased up to 20-fold by 10 nM Dex. The effects of Dex on PTHrP and PTH1R were dose-dependent and experiments with the GC-receptor antagonist mifepristone showed an involvement of GC-receptors in these effects. In addition to the Dex-induced effects on PTHrP and PTH1R, Dex also increased mineralization and the expression of the osteoblast markers Runx2 and alkaline phosphatase. In our studies, we show that dexamethasone decreases the expression of PTHrP and increases the expression of the PTH1R receptor. This could have an impact on PTHrP-mediated anabolic actions on bone and could also affect the responsiveness of circulating PTH. The results indicate that glucocorticoids affect the signalling pathway of PTHrP by regulating both PTHrP and PTH1R expression and these mechanisms could be involved in glucocorticoid-induced osteoporosis. © 2008 Elsevier Inc. All rights reserved.
1. Introduction Inflammatory and rheumatic diseases are commonly treated by glucocorticoids because of their effective anti-inflammatory and immunosuppressive effects. Long-term treatment with glucocorticoids can, however, have severe adverse effects; one of these being the induction of bone loss [1–4]. The reduced bone mass is manifested as an increased risk of fractures. This common and clinically relevant condition, termed glucocorticoid-induced osteopenia, has been suggested to originate from several different mechanisms [3,4]. Intermittent administration of parathyroid hormone (PTH) and PTHrP has anabolic effects on bone when administered at a specific
∗ Corresponding author. Tel.: +358 9 19158276; fax: +358 9 19158269. E-mail address: mikael.ahlstrom@helsinki.fi (M. Ahlström). 0039-128X/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2008.12.002
dose and frequency and PTH is commonly used in the treatment of glucocorticoid-induced osteoporosis [5–8]. A major difference between the physiological actions of PTH and PTHrP is that, as PTH is a circulating systemic hormone, the effects of PTHrP are local and act directly on the microenvironment of bone. Although circulating PTHrP can be the cause of bone loss in some malignant conditions [9], it now seems likely that PTHrP has an important role as an anabolic peptide in normal bone [9–13]. Recent findings show that osteoprogenitors and osteoblasts secrete PTHrP and that this peptide, in an autocrine/paracrine manner, can promote bone formation by enhancing the differentiation of committed preosteoblasts and by promoting the survival of mature osteoblasts and osteocytes [12,13]. PTHrP binds to a G-protein-coupled receptor, the PTH1R, which is common for both PTH and PTHrP. The binding to the PTH1R leads to accumulation of several intracellular second messengers, such as cAMP and diacylglycerol, which in turn activate the protein kinase A and protein kinase C signalling pathways [14,15].
278
M. Ahlström et al. / Steroids 74 (2009) 277–282
In addition to the levels of PTH and PTHrP, the magnitude of activation and the duration of their intracellular signals can depend on several other factors. One of these is the number of functional PTH1Rs and the regulation of this receptor has consequently been extensively studied [15–18]. It is, however likely that the overall balance between extracellular PTHrP/PTH levels and the number of functional PTH1Rs are central in determining the effects of PTH/PTHrP on osteoblast functions. The regulation of PTHrP and PTH1R by glucocorticoids in human mesenchymal cells has not yet been studied. As both PTHrP and PTH1R participate in the differentiation process and also takes part in the homeostasis of bone metabolism, we studied the effect of the synthetic glucocorticoid dexamethasone (Dex) on the expression of PTHrP and PTH1R.
Table 1 Oligonucleotide primers for real-time RT-PCR used to detect PTHrP, PTH1R, Runx2, ALP, osteocalcin and -actin in hMSC cells. Gene
Primer sequence
PTHrP
F: GTC TCA GCC GCC GCC TCA A R: GGA AGA ATC GTC GCC GTA AA F: GGA GTA GCC CAC GGT GTA AA R: ATC ACA AAG GCC ATG CCT AC F: TGA GAG CCG CCT CTC CAA CC R: GCG GAA GCA TTC TGG AAG GA F: ACC ATT CCC ACG TCT TCA CAT R: AGA CAT TCT CTC GTT CAC CG F: ATG AGA GCC CTC ACA CTC CTC G R: GTC AGC CAA CTC GTC ACA GTC C F: AGG CCA ACC GCG AGA AGA TGA CC R: GAA GTC CAG GGC GAC GTA GCA C
PTH1R Runx2 ALP Osteocalcin -actin
Size (bp) 93 173 266 162 255 350
2. Experimental 2.1. Cell culture Human bone marrow-derived mesenchymal stem cells (hMSC) were purchased from Cambrex. The cells were seeded at 5000–6000 cells/cm2 and cultured in ␣-MEM supplemented with 10% FBS, 100 U/ml penicillin and 100 g/ml streptomycin at 37 ◦ C in a humidified atmosphere with 5% CO2 . The differentiation of hMSC into osteoblasts was performed in ␣-MEM, supplemented as above with the addition of 10−8 M dexamethasone, 50 g/ml l-ascorbic acid, and 10 mM -glycerophosphate (referred to as OBM in the text). 2.2. Assay of immunoreactive PTHrP The PTHrP release into the culture media was measured as described earlier [19] by a commercial two-site immunoradiometric assay with an assay range of 20–2000 pg/ml (Parathyroid Hormone-Related Peptide IRMA, Diagnostic Systems Laboratories). The cells were grown to confluence on 100 mm dishes with 8 ml medium. After appropriate treatments the medium was withdrawn from the culture dishes. To avoid proteolytic cleavage, one protease inhibitor tablet (Complete, EDTA-free, Roche) was dissolved in 2 ml water, and 1% of this solution was added to the samples before centrifugation at 400 × g for 5 min at 4 ◦ C. The supernatant was used for PTHrP determinations. Samples collected for less than 24 h were concentrated by spinning the sample in 5000 molecular weight cut-off centrifugal filters (Amicon Ultra4, Millipore) at 4000 × g in order to fit the PTHrP levels within the standard curve of the IRMA. The medium was added to assay tubes with 10% of the protease inhibitor solution. The PTHrP concentration was then assayed with the immunoradiometric kit according to the manufacturer’s instructions. A standard curve was constructed by dissolving PTHrP into ␣-MEM with the same supplementations as was used in the experiments. The radioactivity of the tubes was counted with a gamma counter, and the results were analyzed with GraphPad software using non-linear regression. 2.3. RNA isolation and quantitative real-time RT-PCR Total RNA of the cells was extracted using RNeasy Protect mini kit (Qiagen). The RNA concentration was measured at 260 nm with a spectrophotometer. The integrity of the RNA was confirmed by 1.2% formaldehyde agarose gel electrophoresis. Three different RNA preparations were used for the cDNA synthesis. cDNA was synthesized from 1.5 g of total RNA as described earlier [19]. Each PCR amplification was performed in a real-time quantitative PCR engine (Mx3000P, Stratagene). The PCR reactions contained cDNA templates and components of Brilliant SYBR Green QPCR Master mix kit
(Stratagene) in a 20 l reaction volume containing 200 nM of each gene-specific primer (Table 1). Fluorescence data was collected during the annealing step and analyzed with Mx3000P software. Amplification was obtained by denaturing at 95 ◦ C for 10 min, followed by 40 cycles of denaturing at 95 ◦ C for 30 s, annealing at 55 ◦ C for 1 min, and extension at 72 ◦ C for 30 s. The amplification cycles were followed by 1 min at 95 ◦ C, 30 s at 55 ◦ C, and data for dissociation plots was collected as the temperature was returned to 95 ◦ C. cDNA from human brain total RNA (BD Biosciences) was used as a calibration standard and the data was normalized with amplification data of -actin. All reactions were run in triplicate and the mean value was used to calculate the ratio of target gene/-actin expression in each sample. Using the ratio of untreated samples as a standard, the relative ratio of treated samples was presented as the relative expression level of the target gene. 2.4. Assay of biological mineralization Mineralization was assessed by Alizarin red S staining from cells grown on the 96-well plates as described earlier [20]. For the mineralization experiments, the cells were treated with 10 nM Dex for indicated times. After staining with Alizarin red S, the dye was eluted with 10% cetylpyridinium chloride and the absorbance was measured with a spectroscopic plate reader at 540 nm. 3. Results 3.1. Effects of dexamethasone on hMSC osteoblastic differentiation Glucocorticoids are known to promote the differentiation of osteoblasts. We assayed the effects of Dex on the expression of differentiation markers and effects on biological mineralization to test the responses of our cellular model to previously reported responses. The expression of Runx2 (Cbfa1), a well known and essential transcriptional regulator of osteoblast differentiation, was upregulated over 5-fold by 10 nM Dex (Fig. 1A). The Dex treatment also upregulated the expression of the osteoblast marker alkaline phosphatase (ALP), whereas osteocalcin expression, a marker of a mature osteoblast phenotype, was downregulated by Dex (Fig. 1A). The studies of the Dex effects on mineralization were performed during a 14 days differentiation treatment in OBM. To test the effect of a Dex withdrawal on mineralization, Dex was omitted from the medium on day 4 and 7 and compared to a continuous treatment with Dex. These experiments showed that a continuous presence of Dex is more effective in supporting the mineralization as compared to if Dex is withdrawn during the differentiation process (Fig. 1B).
M. Ahlström et al. / Steroids 74 (2009) 277–282
279
Fig. 1. Effects of dexamethasone on the expression of osteoblast markers and biological mineralization. Control cells and cells treated for 24 h with 10 nM dexamethasone (Dex) were assayed for mRNA expression of the osteoblast markers Runx2, ALP and osteocalcin by real-time PCR (A). The effect of discontinued dexamethasone treatment on biological mineralization was assayed by alizarin red staining method. Cells were grown in ␣-MEM or differentiated into osteoblasts with osteogenic differentiation media containing 10 nM dexamethasone. Dexamethasone was removed from the media after indicated days of treatment and assayed for mineralization 14 days after initiation of the experiment (B).
Fig. 2. Dose- and time response studies of the dexamethasone effects on PTHrP release from hMSC. The cells were treated with indicated concentrations of dexamethasone for 24 h and the immunoreactive PTHrP was assayed from the media using an immunoradiometric assay (A). For time response studies, cells were treated with or without 10 nM dexamethasone with or without 1 M mifepristone (MIF). At indicated times media was removed from the cultures and assayed for PTHrP (B).
3.2. Effects of dexamethasone on PTHrP release and PTHrP mRNA levels Treatment with 10 nM Dex suppressed the release of immunoreactive PTHrP by up to 80% when treated for 24 h. The effect of Dex was dose-dependent and the half-maximal inhibitory effect on the PTHrP release was approximately 0.2 nM (Fig. 2A). The Dex effects on PTHrP release could be seen within 6 h of Dex treatment and remained suppressed for over 24 h (Fig. 2B). The role of glucocorticoid receptors in the Dex effects was studied by treating the cells with the glucocorticoid receptor antagonist mifepristone (MIF). MIF alone reduced the PTHrP release but the effects of MIF were significantly less pronounced compared to the effects of Dex. Furthermore, Dex did not affect the PTHrP release in the presence of MIF as compared to treatment to MIF alone (Fig. 3A).
In line with the effects on PTHrP release, Dex also significantly inhibited the levels of PTHrP mRNA in the cells (Fig. 3B). PTHrP mRNA levels were reduced by over 90% by Dex treatment, whereas MIF reduced the PTHrP mRNA levels by 15%. In accordance to the effects of Dex on PTHrP release MIF also antagonized the effects of Dex on the PTHrP mRNA levels (Fig. 3B). 3.3. Dexamethasone effects on PTH1R expression Treatment with Dex for 24 h significantly and dose-dependently increased the levels of PTH1R mRNA (Fig. 4A). The Dex effects were significant within 6 h of Dex treatment (Fig. 4B). In a similar manner as seen on the effects on PTHrP release/expression, mifepristone treatment had Dex-like effects on the PTH1R expression but the stimulating effects of Dex on PTH1R mRNA levels were completely
Fig. 3. Effects of dexamethasone and the glucocorticoid receptor antagonist mifepristone on the release of immunoreactive PTHrP and PTHrP mRNA. Cells were incubated for 24 h with/without 10 nM dexamethasone (Dex) and with/without 1 M mifepristone (MIF) as indicated. Immunoreactive PTHrP was then assayed from the media (A). The corresponding PTHrP mRNA levels were then assayed by real-time PCR (B).
280
M. Ahlström et al. / Steroids 74 (2009) 277–282
Fig. 4. The effects of dexamethasone on PTH1R expression. hMSC control cells and cells treated with indicated concentrations of dexamethasone (Dex) for 24 h (A) or with 10 nM Dex for 6 and 24 h (B) and assayed for PTH1R mRNA levels. The effects of dexamethasone and the glucocorticoid receptor antagonist mifepristone on the expression of PTH1R was also studied (C). Cells were incubated for 6 or 24 h with/without 10 nM dexamethasone (Dex) and with/without 1 M mifepristone (MIF) as indicated.
Fig. 5. Effects of dexamethasone withdrawal on the release of immunoreactive PTHrP. To test the recovery of the Dex effect on PTHrP, cells were treated for 24 h with 10 nM. Dex was removed and the PTHrP release was assayed at 24 and 72 h after the removal of Dex.
abolished by mifepristone confirming the involvement of glucocorticoid receptors (Fig. 4C). 3.4. Effects of Dex removal on PTHrP release To test the recovery of the Dex effect on PTHrP, we treated cells for 24 h with 10 nM Dex and then removed Dex from the cultures. The PTHrP release was assayed at 24 and 72 h after the removal of Dex. Removal of Dex from the cultures completely restored the rate of PTHrP release within 72 h (Fig. 5). 4. Discussion In the present study we have shown that dexamethasone can modify the release of immunoreactive PTHrP and the expression of PTHrP mRNA in human mesenchymal stem cells, the progenitor cells of the osteoblasts. Simultaneously, Dex increased the expression of PTH1R, the osteoblast-specific transcription factor Runx2 and alkaline phosphatase. The ablation of the PTHrP gene or the PTH1R receptor gene leads to abnormal bone development in mice [11]. PTHrP has also been shown to reduce osteoblastic cell death and to enhance the differen-
tiation of osteoblasts [12,13,21]. The regulation of the PTHrP gene and the PTHrP release from osteoblastic cells has been shown to be regulated by several factors. In human osteoprogenitor cells we have shown that the PTHrP expression is increased by elevated calcium levels through activation of the calcium sensing receptor (CaR) [19]. Elevated calcium levels also enhance markers of osteoblastic differentiation in primary murine osteoblasts [22] and induces mineralization in human mesenchymal stem cells [19]. Primary osteoblasts and several clonal osteoblast-like cell lines express sonic hedgehog (SHH) which also promotes primary osteoblast differentiation and simultaneously increases PTHrP mRNA expression and PTHrP release [23]. The increased PTHrP expression by both calcium and SHH therefore seems to accompany the enhancement of the osteoblastic differentiation. However, the expression and release of PTHrP does not always parallel the osteoblastic differentiation. Collagen type I, a protein associated with the osteoblastic phenotype, has been shown to alter the expression of both PTHrP and PTH1R in the same direction as Dex in the present study. When UMR106-06 osteoblast-like cells were grown on type I collagen substrate the expression of PTHrP was suppressed and the expression of PTH1R was increased [24]. Bone morphogenic protein 2 (BMP-2), which induces osteoblast differentiation in several cell types, rapidly downregulates PTHrP gene expression and increases the expression of PTH1R in mesenchymal C2C12 cells [25]. Both BMP-2 and collagen type 1 thereby seems to have similar effects on PTHrP and the PTH1R as Dex. A link between Dex treatment and BMP-2 effects also might exist, as stromal cells induced to become osteoblasts by exposure of media containing Dex show enhanced BMP-2 expression [26]. It is well known that Dex is capable of supporting osteoblastic differentiation of mesenchymal stem cells in vitro. In the present study, Dex upregulated the expression of the PTH1R-receptor. Dex also increases the PTH1R expression in several osteoblast-like cell lines, such as rat ROS 17/2 and UMR-106 osteosarcoma cells [27–29]. As the PTH1R is considered to be a marker of the osteoblastic phenotype the Dex effects on PTH1R expression seen in our study can be interpreted as a mechanism associated with osteoblastic differentiation. In accordance with the role of Dex as an agent that supports osteoblast differentiation, and in line with earlier studies [30–32] Dex increased the expression of the osteoblast associated transcrip-
M. Ahlström et al. / Steroids 74 (2009) 277–282
tion factor Runx2 and alkaline phosphatase. However, osteocalcin, which is also a known marker of osteoblast activity, was downregulated. The osteocalcin gene is known to be induced in the late stages of osteoblast differentiation, accompanying the onset of mineralization of the extracellular matrix [33,34]. Therefore, the results by us showing that Dex does not have a stimulating effect on osteocalcin expression in mesenchymal cells could well be explained by less significant role of osteocalcin in the initial induction of the osteoblastic differentiation. This is supported by other studies that also reported reduced osteocalcin expression by Dex in bone marrow-derived mesenchymal cells [30] and in primary human osteoblasts [31,32] in the early phase of osteoblastic differentiation. In these studies Dex reduced the osteocalcin expression by over 50% in the beginning of the differentiation, whereas in the later phase of the differentiation, the osteocalcin expression was increased by Dex [30–32]. A previous study using bone marrow stromal cells indicates an important role of Dex at both the early and late stages of differentiation in directing the cells toward terminal maturation [35]. However, in another study using clonal rat mesenchymal progenitor cells, Dex failed to induce Osterix expression suggesting that Dex is not necessarily needed to promote terminal osteoblast differentiation [36]. Our present study is more in line with the former results. A continuous Dex treatment was more effective in supporting biological mineralization as compared to a discontinuous treatment. One explanation of this is that the mineralizing activity of the individual differentiated cells would decrease upon the withdrawal of Dex. On the other hand, the Dex withdrawal could also cause enhanced growth of undifferentiated cells relative to differentiated cells and the increased mineralization would therefore be a reflection of a reduction in differentiation of osteoblast precursors. The glucocorticoid receptor (GR) is usually considered to be involved in the effects of glucocorticoids, although non-genomic effects by unknown mechanisms have been reported [37]. In the present study, we used the GR antagonist mifepristone to test the involvement of the GR in the effects of Dex. Mifepristone was used at concentrations 100 times higher than Dex and at these concentrations mifepristone had effects on the PTHrP and PTH1R expression that were less pronounced but similar to those of Dex. Partial agonist activity of mifepristone has earlier been described in some cell types and it has been suggested that these effects could result from a reversible DNA binding of the GR transcription complex and by binding of mifepristone to transcriptional co-activators [38,39]. In any case, when used in combination with Dex, mifepristone antagonized the effects of Dex, both in downregulating PTHrP and upregulating PTH1R. A simultaneous downregulation of PTHrP and upregulation of PTH1R by Dex reported in our present study could result in several responses in the osteoblastic progenitors and osteoblasts. Taken into account the anabolic role of PTHrP in bone, decreased levels of released PTHrP could directly attenuate bone formation by reducing differentiation and osteoblast function. An increase in PTH1R could in turn possibly counteract these effects, but might also make the cells more susceptible to the effects of circulating PTH. As the regulation of PTHrP and PTH1R is likely to be a very delicately regulated process, the disruption of these mechanisms by Dex could potentially play a role in the induction of glucocorticoid-induced osteoporosis. References [1] Gennari C, Civitelli R. Glucocorticoid-induced osteoporosis. Clin Rheum Dis 1986;12:637–54. [2] Reid IR. Glucocorticoid osteoporosis—mechanisms and management. Eur J Endocrinol 1997;137:209–17. [3] Canalis E, Delany AM. Mechanisms of glucocorticoid action in bone. Ann NY Acad Sci 2002;966:73–81.
281
[4] Mazziotti G, Angeli A, Bilezikian JP, Canalis E, Giustina A. Glucocorticoidinduced osteoporosis: an update. Trends Endocrinol Metabol 2006;17:144–9. [5] Canalis E, McCarthy TL, Centrella M. Differential effects of continuous and transitient treatment with parathyroid hormone related peptide (PTHrP) on bone collagen synthesis. Endocrinology 1990;126:1806–12. [6] Dempster DW, Cosman F, Parisien M, Shen V, Lindsay R. Anabolic actions of parathyroid hormone in bone. Endocr Rev 1993;14:690–709. [7] Horwitz MJ, Tedesco MB, Gundberg C, Garcia-Ocana A, Stewart AF. Shortterm, high-dose parathyroid hormone-related protein as a skeletal anabolic agent for the treatment of postmenopausal osteoporosis. J Clin Endocrin Metab 2003;288:569–75. [8] Saag KG, Shane E, Boonen S, Marin F, Donley DW, Taylor KA, et al. Teriparatide or alendronate in glucocorticoid-induced osteoporosis. New Engl J Med 2007;357:2028–39. [9] Asa SL, Henderson J, Goltzman D, Drucker DJ. Parathyroid hormone-like peptide in normal and neoplastic human endocrine tissues. J Clin Endocrinol Metab 1990;71:1112–8. [10] Suda N, Gillespie MT, Traianedes K, Zhou H, Ho PW, Hards DK, et al. Expression of parathyroid hormone-related protein in cells of osteoblast lineage. J Cell Phys 1996;166:94–104. [11] Lanske B, Amling M, Neff L, Guiducci J, Baron R, Kronenberg HM. Ablation of the PTHrP gene or the PTH/PTHrP receptor gene leads to distinct abnormalities in bone development. J Clin Invest 1999;104:399–407. [12] Miao D, He B, Jiang Y, Kobayashi T, Sorocéanu MA, Zhao J, et al. Osteoblastderived PTHrP is a potent endogenous bone anabolic agent that modifies the therapeutic efficacy of administered PTH 1-34. J Clin Invest 2005;115:2402–11. [13] Martin TJ. Osteoblast-derived PTHrP is a physiological regulator of bone formation. J Clin Invest 2005;115:2322–4. [14] Lanske B, Divieti P, Kovacs CS, Pirro A, Landis WJ, Krane SM, et al. The parathyroid hormone (PTH)/PTH-related peptide receptor mediates actions of both ligands in murine bone. Endocrinology 1998;139:5194–204. [15] Lanske B, Kronenberg HM. Parathyroid hormone-related peptide (PTHrP) and parathyroid hormone (PTH)/PTHrP receptor. Crit Rev Eukar Gene 1998;8:297–320. [16] Iida-Klein A, Guo J, Drake MT, Kronenberg HM, Abou-Samra AB, Bringhurst FR, et al. Structural requirements of parathyroid hormone/parathyroid hormonerelated peptide receptors for phospholipase C activation and regulation of phosphate uptake. Miner Electrol Metab 1995;21:177–9. [17] Martin TJ. Actions of parathyroid hormone-related peptide and its receptors. New Engl J Med 1996;335:736–8. [18] Yamamoto I, Shigeno C, Potts Jr JT, Segre GV. Characterization and agonistinduced down-regulation of parathyroid hormone receptors in clonal rat osteosarcoma cells. Endocrinology 1988;122:1208–17. [19] Ahlstrom M, Pekkinen M, Riehle U, Lamberg-Allardt C. Extracellular calcium regulates parathyroid hormone-related peptide expression in osteoblasts and osteoblast progenitor cells. Bone 2008;42:483–90. [20] Pekkinen M, Ahlström M, Riehle U, Huttunen M, Lamberg-Allardt C. Effects of phosphodiesterase 7 inhibition by RNA interference on the gene expression and differentiation of human mesenchymal stem cell-derived osteoblasts. Bone 2008;43:84–91. [21] Chen HL, Demiralp B, Schneider A, Koh AJ, Silve C, Wang CY, et al. Parathyroid hormone and parathyroid hormone-related protein exert both pro- and antiapoptotic effects in mesenchymal cells. J Biol Chem 2002;277:19374–81. [22] Choudhary S, Wadhwa S, Raisz LG, Alander C, Pilbeam CC. Extracellular calcium is a potent inducer of cyclo-oxygenase-2 in murine osteoblasts through an ERK signalling pathway. J Bone Miner Res 2003;18:1530–8. [23] Jemtland R, Divieti P, Lee K, Segre GV. Hedgehog promotes primary osteoblast differentiation and increases PTHrP mRNA expression and iPTHrP. Bone 2003;32:611–20. [24] Celic S, Chilco PJ, Zajac JD, Martin TJ, Findlay DM. A type I collagen substrate increases PTH/PTHrP receptor mRNA expression and suppresses PTHrP mRNA expression in UMR106-06 osteoblast-like cells. J Endocrinol 1996;150:299–308. [25] Susperregui AR, Vinals F, Ho PW, Gillespie MT, Martin TJ, Ventura F. BMP-2 regulation of PTHrP and osteoclastogenic factors during osteoblast differentiation of C2C12 cells. J Cell Physiol 2008;216:144–52. [26] Bi LX, Simmons DJ, Mainous E. Expression of BMP-2 by rat bone marrow stromal cells in culture. Calcif Tissue Int 1999;64:63–8. [27] Yamamoto I, Potts Jr JT, Segre GV. Glucocorticoids increase parathyroid hormone receptors in rat osteoblastic osteosarcoma cells (ROS 17/2). J Bone Miner Res 1988;3:707–12. [28] Yaghoobian J, Drueke TB. Regulation of the transcription of parathyroidhormone/parathyroid-hormone-related peptide receptor mRNA by dexamethasone in ROS 17/2.8 osteosarcoma cells. Nephrol Dial Transpl 1998;13:580–6. [29] Haramoto N, Kawane T, Horiuchi N. Upregulation of PTH receptor mRNA expression by dexamethasone in UMR-106 osteoblast-like cells. Oral Dis 2007;13:23–31. [30] Oshina H, Shinichi S, Yoshii T, Torigoe I, Sugata Y. Effects of continuous dexamethasone treatment on differentiation capabilities of bone marrow-derived mesenchymal cells. Bone 2007;41:575–83. [31] Viereck V, Siggelkow H, Tauber S, Raddatz D, Schutze N, Hüfner M. Differential regulation of Cbfa1/Runx2 and osteocalcin gene expression by vitamin-D3, dexamethasone, and local growth factors in primary human osteoblasts. J Cell Biochem 2002;86:348–56. [32] Subramaniam M, Colvard D, Keeting PE, Rasmussen K, Riggs LB, Spelsberg TC. Glucocorticoid regulation of alkaline phosphatase, osteocalcin, and
282
M. Ahlström et al. / Steroids 74 (2009) 277–282
proto-oncogenes in normal human osteoblast-like cells. J Cell Biochem 1992;50:411–24. [33] Owen T, Aronow M, Shalhoub V, Barone L, Wilming LM, Tassinari MS, et al. Progressive development of the rat phenotype in vitro: reciprocal relationships in expression of genes associated withosteoblast proliferation and differentiation during formation of the bone extracellular matrix. J Cell Physiol 1990;143:420–30. [34] Stein GS, Lian JB, van Wijnen AJ, Stein JL. The osteocalcin gene: a model for multiple parameters of skeletal-specific transcriptional control. Mol Biol Reports 1997;24:185–96. [35] Porter R, Huckle W, Goldstein A. Effect of dexamethasone withdrawal on osteoblastic differentiation of bone marrow stromal cells. J Cell Biochem 2003;90:13–22.
[36] Ito S, Suzuki N, Kato S, Takahashi T, Takagi M. Glucocorticoids induce the differentiation of a mesenchymal progenitor cell line, ROB-C26 into adipocytes and osteoblasts, but fail to induce terminal osteoblast differentiation. Bone 2007;40:84–92. [37] Haller J, Mikics E, Makara GB. The effects of non-genomic glucocorticoid mechanisms on bodily functions and the central neural system. A critical evaluation of findings. Front Neuroendocrinol 2008;29:273–91. [38] Mao J, Regelson W, Kalimi M. Molecular mechanism of RU 486 action: a review. Mol Cell Biochem 1992;109:1–8. [39] Mercier L, Miller PA, Simons Jr SS. Antiglucocorticoid steroids have increased agonist activity in those hepatoma cell lines that are more sensitive to glucocorticoids. J Ster Biochem 1986;25:11–20.
Contents lists available at ScienceDirect
Steroids journal homepage: www.elsevier.com/locate/steroids
Dexamethasone downregulates the expression of parathyroid hormone-related protein (PTHrP) in mesenchymal stem cells Mikael Ahlström ∗ , Minna Pekkinen, Christel Lamberg-Allardt Calcium Research Unit, Department of Applied Chemistry and Microbiology, P.O. Box 66, 00014 University of Helsinki, Finland
a r t i c l e
i n f o
Article history: Received 23 September 2008 Received in revised form 28 October 2008 Accepted 4 December 2008 Available online 11 December 2008 Keywords: Parathyroid hormone-related protein (PTHrP) Osteoblasts Mesenchymal stem cells Glucocorticoids PTH1R
a b s t r a c t Parathyroid hormone-related protein (PTHrP) has been shown to have anabolic effects in women with postmenopausal osteoporosis. PTHrP promotes the recruitment of osteogenic cells and prevents apoptotic death of osteoblasts and osteocytes. The receptor responsible for the effects of PTHrP is the common PTH/PTHrP receptor (PTH1R). Glucocorticoids (GC) are commonly used as drugs to treat inflammatory diseases. Long-term GC treatments are often associated with bone loss which can lead to GC-induced osteoporosis. The aim of this work was to study the effects of the glucocorticoid dexamethasone (Dex) on the expression of PTHrP and PTH1R in adult human mesenchymal stem cells, the progenitor cells of osteoblasts. Adult human mesenchymal stem cells (hMSC) were cultured and differentiated by standard methods. The expression of PTHrP and PTH1R mRNA was assayed by real-time qPCR. The PTHrP release into the culture media was measured by an immunoradiometric assay. Treatment with Dex (10 nM) resulted in an 80% drop in the PTHrP release within 6 h. A 24 h Dex treatment also reduced the expression of PTHrP mRNA by up to 90%. The expression of PTH1R receptor mRNA was simultaneously increased up to 20-fold by 10 nM Dex. The effects of Dex on PTHrP and PTH1R were dose-dependent and experiments with the GC-receptor antagonist mifepristone showed an involvement of GC-receptors in these effects. In addition to the Dex-induced effects on PTHrP and PTH1R, Dex also increased mineralization and the expression of the osteoblast markers Runx2 and alkaline phosphatase. In our studies, we show that dexamethasone decreases the expression of PTHrP and increases the expression of the PTH1R receptor. This could have an impact on PTHrP-mediated anabolic actions on bone and could also affect the responsiveness of circulating PTH. The results indicate that glucocorticoids affect the signalling pathway of PTHrP by regulating both PTHrP and PTH1R expression and these mechanisms could be involved in glucocorticoid-induced osteoporosis. © 2008 Elsevier Inc. All rights reserved.
1. Introduction Inflammatory and rheumatic diseases are commonly treated by glucocorticoids because of their effective anti-inflammatory and immunosuppressive effects. Long-term treatment with glucocorticoids can, however, have severe adverse effects; one of these being the induction of bone loss [1–4]. The reduced bone mass is manifested as an increased risk of fractures. This common and clinically relevant condition, termed glucocorticoid-induced osteopenia, has been suggested to originate from several different mechanisms [3,4]. Intermittent administration of parathyroid hormone (PTH) and PTHrP has anabolic effects on bone when administered at a specific
∗ Corresponding author. Tel.: +358 9 19158276; fax: +358 9 19158269. E-mail address: mikael.ahlstrom@helsinki.fi (M. Ahlström). 0039-128X/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2008.12.002
dose and frequency and PTH is commonly used in the treatment of glucocorticoid-induced osteoporosis [5–8]. A major difference between the physiological actions of PTH and PTHrP is that, as PTH is a circulating systemic hormone, the effects of PTHrP are local and act directly on the microenvironment of bone. Although circulating PTHrP can be the cause of bone loss in some malignant conditions [9], it now seems likely that PTHrP has an important role as an anabolic peptide in normal bone [9–13]. Recent findings show that osteoprogenitors and osteoblasts secrete PTHrP and that this peptide, in an autocrine/paracrine manner, can promote bone formation by enhancing the differentiation of committed preosteoblasts and by promoting the survival of mature osteoblasts and osteocytes [12,13]. PTHrP binds to a G-protein-coupled receptor, the PTH1R, which is common for both PTH and PTHrP. The binding to the PTH1R leads to accumulation of several intracellular second messengers, such as cAMP and diacylglycerol, which in turn activate the protein kinase A and protein kinase C signalling pathways [14,15].
278
M. Ahlström et al. / Steroids 74 (2009) 277–282
In addition to the levels of PTH and PTHrP, the magnitude of activation and the duration of their intracellular signals can depend on several other factors. One of these is the number of functional PTH1Rs and the regulation of this receptor has consequently been extensively studied [15–18]. It is, however likely that the overall balance between extracellular PTHrP/PTH levels and the number of functional PTH1Rs are central in determining the effects of PTH/PTHrP on osteoblast functions. The regulation of PTHrP and PTH1R by glucocorticoids in human mesenchymal cells has not yet been studied. As both PTHrP and PTH1R participate in the differentiation process and also takes part in the homeostasis of bone metabolism, we studied the effect of the synthetic glucocorticoid dexamethasone (Dex) on the expression of PTHrP and PTH1R.
Table 1 Oligonucleotide primers for real-time RT-PCR used to detect PTHrP, PTH1R, Runx2, ALP, osteocalcin and -actin in hMSC cells. Gene
Primer sequence
PTHrP
F: GTC TCA GCC GCC GCC TCA A R: GGA AGA ATC GTC GCC GTA AA F: GGA GTA GCC CAC GGT GTA AA R: ATC ACA AAG GCC ATG CCT AC F: TGA GAG CCG CCT CTC CAA CC R: GCG GAA GCA TTC TGG AAG GA F: ACC ATT CCC ACG TCT TCA CAT R: AGA CAT TCT CTC GTT CAC CG F: ATG AGA GCC CTC ACA CTC CTC G R: GTC AGC CAA CTC GTC ACA GTC C F: AGG CCA ACC GCG AGA AGA TGA CC R: GAA GTC CAG GGC GAC GTA GCA C
PTH1R Runx2 ALP Osteocalcin -actin
Size (bp) 93 173 266 162 255 350
2. Experimental 2.1. Cell culture Human bone marrow-derived mesenchymal stem cells (hMSC) were purchased from Cambrex. The cells were seeded at 5000–6000 cells/cm2 and cultured in ␣-MEM supplemented with 10% FBS, 100 U/ml penicillin and 100 g/ml streptomycin at 37 ◦ C in a humidified atmosphere with 5% CO2 . The differentiation of hMSC into osteoblasts was performed in ␣-MEM, supplemented as above with the addition of 10−8 M dexamethasone, 50 g/ml l-ascorbic acid, and 10 mM -glycerophosphate (referred to as OBM in the text). 2.2. Assay of immunoreactive PTHrP The PTHrP release into the culture media was measured as described earlier [19] by a commercial two-site immunoradiometric assay with an assay range of 20–2000 pg/ml (Parathyroid Hormone-Related Peptide IRMA, Diagnostic Systems Laboratories). The cells were grown to confluence on 100 mm dishes with 8 ml medium. After appropriate treatments the medium was withdrawn from the culture dishes. To avoid proteolytic cleavage, one protease inhibitor tablet (Complete, EDTA-free, Roche) was dissolved in 2 ml water, and 1% of this solution was added to the samples before centrifugation at 400 × g for 5 min at 4 ◦ C. The supernatant was used for PTHrP determinations. Samples collected for less than 24 h were concentrated by spinning the sample in 5000 molecular weight cut-off centrifugal filters (Amicon Ultra4, Millipore) at 4000 × g in order to fit the PTHrP levels within the standard curve of the IRMA. The medium was added to assay tubes with 10% of the protease inhibitor solution. The PTHrP concentration was then assayed with the immunoradiometric kit according to the manufacturer’s instructions. A standard curve was constructed by dissolving PTHrP into ␣-MEM with the same supplementations as was used in the experiments. The radioactivity of the tubes was counted with a gamma counter, and the results were analyzed with GraphPad software using non-linear regression. 2.3. RNA isolation and quantitative real-time RT-PCR Total RNA of the cells was extracted using RNeasy Protect mini kit (Qiagen). The RNA concentration was measured at 260 nm with a spectrophotometer. The integrity of the RNA was confirmed by 1.2% formaldehyde agarose gel electrophoresis. Three different RNA preparations were used for the cDNA synthesis. cDNA was synthesized from 1.5 g of total RNA as described earlier [19]. Each PCR amplification was performed in a real-time quantitative PCR engine (Mx3000P, Stratagene). The PCR reactions contained cDNA templates and components of Brilliant SYBR Green QPCR Master mix kit
(Stratagene) in a 20 l reaction volume containing 200 nM of each gene-specific primer (Table 1). Fluorescence data was collected during the annealing step and analyzed with Mx3000P software. Amplification was obtained by denaturing at 95 ◦ C for 10 min, followed by 40 cycles of denaturing at 95 ◦ C for 30 s, annealing at 55 ◦ C for 1 min, and extension at 72 ◦ C for 30 s. The amplification cycles were followed by 1 min at 95 ◦ C, 30 s at 55 ◦ C, and data for dissociation plots was collected as the temperature was returned to 95 ◦ C. cDNA from human brain total RNA (BD Biosciences) was used as a calibration standard and the data was normalized with amplification data of -actin. All reactions were run in triplicate and the mean value was used to calculate the ratio of target gene/-actin expression in each sample. Using the ratio of untreated samples as a standard, the relative ratio of treated samples was presented as the relative expression level of the target gene. 2.4. Assay of biological mineralization Mineralization was assessed by Alizarin red S staining from cells grown on the 96-well plates as described earlier [20]. For the mineralization experiments, the cells were treated with 10 nM Dex for indicated times. After staining with Alizarin red S, the dye was eluted with 10% cetylpyridinium chloride and the absorbance was measured with a spectroscopic plate reader at 540 nm. 3. Results 3.1. Effects of dexamethasone on hMSC osteoblastic differentiation Glucocorticoids are known to promote the differentiation of osteoblasts. We assayed the effects of Dex on the expression of differentiation markers and effects on biological mineralization to test the responses of our cellular model to previously reported responses. The expression of Runx2 (Cbfa1), a well known and essential transcriptional regulator of osteoblast differentiation, was upregulated over 5-fold by 10 nM Dex (Fig. 1A). The Dex treatment also upregulated the expression of the osteoblast marker alkaline phosphatase (ALP), whereas osteocalcin expression, a marker of a mature osteoblast phenotype, was downregulated by Dex (Fig. 1A). The studies of the Dex effects on mineralization were performed during a 14 days differentiation treatment in OBM. To test the effect of a Dex withdrawal on mineralization, Dex was omitted from the medium on day 4 and 7 and compared to a continuous treatment with Dex. These experiments showed that a continuous presence of Dex is more effective in supporting the mineralization as compared to if Dex is withdrawn during the differentiation process (Fig. 1B).
M. Ahlström et al. / Steroids 74 (2009) 277–282
279
Fig. 1. Effects of dexamethasone on the expression of osteoblast markers and biological mineralization. Control cells and cells treated for 24 h with 10 nM dexamethasone (Dex) were assayed for mRNA expression of the osteoblast markers Runx2, ALP and osteocalcin by real-time PCR (A). The effect of discontinued dexamethasone treatment on biological mineralization was assayed by alizarin red staining method. Cells were grown in ␣-MEM or differentiated into osteoblasts with osteogenic differentiation media containing 10 nM dexamethasone. Dexamethasone was removed from the media after indicated days of treatment and assayed for mineralization 14 days after initiation of the experiment (B).
Fig. 2. Dose- and time response studies of the dexamethasone effects on PTHrP release from hMSC. The cells were treated with indicated concentrations of dexamethasone for 24 h and the immunoreactive PTHrP was assayed from the media using an immunoradiometric assay (A). For time response studies, cells were treated with or without 10 nM dexamethasone with or without 1 M mifepristone (MIF). At indicated times media was removed from the cultures and assayed for PTHrP (B).
3.2. Effects of dexamethasone on PTHrP release and PTHrP mRNA levels Treatment with 10 nM Dex suppressed the release of immunoreactive PTHrP by up to 80% when treated for 24 h. The effect of Dex was dose-dependent and the half-maximal inhibitory effect on the PTHrP release was approximately 0.2 nM (Fig. 2A). The Dex effects on PTHrP release could be seen within 6 h of Dex treatment and remained suppressed for over 24 h (Fig. 2B). The role of glucocorticoid receptors in the Dex effects was studied by treating the cells with the glucocorticoid receptor antagonist mifepristone (MIF). MIF alone reduced the PTHrP release but the effects of MIF were significantly less pronounced compared to the effects of Dex. Furthermore, Dex did not affect the PTHrP release in the presence of MIF as compared to treatment to MIF alone (Fig. 3A).
In line with the effects on PTHrP release, Dex also significantly inhibited the levels of PTHrP mRNA in the cells (Fig. 3B). PTHrP mRNA levels were reduced by over 90% by Dex treatment, whereas MIF reduced the PTHrP mRNA levels by 15%. In accordance to the effects of Dex on PTHrP release MIF also antagonized the effects of Dex on the PTHrP mRNA levels (Fig. 3B). 3.3. Dexamethasone effects on PTH1R expression Treatment with Dex for 24 h significantly and dose-dependently increased the levels of PTH1R mRNA (Fig. 4A). The Dex effects were significant within 6 h of Dex treatment (Fig. 4B). In a similar manner as seen on the effects on PTHrP release/expression, mifepristone treatment had Dex-like effects on the PTH1R expression but the stimulating effects of Dex on PTH1R mRNA levels were completely
Fig. 3. Effects of dexamethasone and the glucocorticoid receptor antagonist mifepristone on the release of immunoreactive PTHrP and PTHrP mRNA. Cells were incubated for 24 h with/without 10 nM dexamethasone (Dex) and with/without 1 M mifepristone (MIF) as indicated. Immunoreactive PTHrP was then assayed from the media (A). The corresponding PTHrP mRNA levels were then assayed by real-time PCR (B).
280
M. Ahlström et al. / Steroids 74 (2009) 277–282
Fig. 4. The effects of dexamethasone on PTH1R expression. hMSC control cells and cells treated with indicated concentrations of dexamethasone (Dex) for 24 h (A) or with 10 nM Dex for 6 and 24 h (B) and assayed for PTH1R mRNA levels. The effects of dexamethasone and the glucocorticoid receptor antagonist mifepristone on the expression of PTH1R was also studied (C). Cells were incubated for 6 or 24 h with/without 10 nM dexamethasone (Dex) and with/without 1 M mifepristone (MIF) as indicated.
Fig. 5. Effects of dexamethasone withdrawal on the release of immunoreactive PTHrP. To test the recovery of the Dex effect on PTHrP, cells were treated for 24 h with 10 nM. Dex was removed and the PTHrP release was assayed at 24 and 72 h after the removal of Dex.
abolished by mifepristone confirming the involvement of glucocorticoid receptors (Fig. 4C). 3.4. Effects of Dex removal on PTHrP release To test the recovery of the Dex effect on PTHrP, we treated cells for 24 h with 10 nM Dex and then removed Dex from the cultures. The PTHrP release was assayed at 24 and 72 h after the removal of Dex. Removal of Dex from the cultures completely restored the rate of PTHrP release within 72 h (Fig. 5). 4. Discussion In the present study we have shown that dexamethasone can modify the release of immunoreactive PTHrP and the expression of PTHrP mRNA in human mesenchymal stem cells, the progenitor cells of the osteoblasts. Simultaneously, Dex increased the expression of PTH1R, the osteoblast-specific transcription factor Runx2 and alkaline phosphatase. The ablation of the PTHrP gene or the PTH1R receptor gene leads to abnormal bone development in mice [11]. PTHrP has also been shown to reduce osteoblastic cell death and to enhance the differen-
tiation of osteoblasts [12,13,21]. The regulation of the PTHrP gene and the PTHrP release from osteoblastic cells has been shown to be regulated by several factors. In human osteoprogenitor cells we have shown that the PTHrP expression is increased by elevated calcium levels through activation of the calcium sensing receptor (CaR) [19]. Elevated calcium levels also enhance markers of osteoblastic differentiation in primary murine osteoblasts [22] and induces mineralization in human mesenchymal stem cells [19]. Primary osteoblasts and several clonal osteoblast-like cell lines express sonic hedgehog (SHH) which also promotes primary osteoblast differentiation and simultaneously increases PTHrP mRNA expression and PTHrP release [23]. The increased PTHrP expression by both calcium and SHH therefore seems to accompany the enhancement of the osteoblastic differentiation. However, the expression and release of PTHrP does not always parallel the osteoblastic differentiation. Collagen type I, a protein associated with the osteoblastic phenotype, has been shown to alter the expression of both PTHrP and PTH1R in the same direction as Dex in the present study. When UMR106-06 osteoblast-like cells were grown on type I collagen substrate the expression of PTHrP was suppressed and the expression of PTH1R was increased [24]. Bone morphogenic protein 2 (BMP-2), which induces osteoblast differentiation in several cell types, rapidly downregulates PTHrP gene expression and increases the expression of PTH1R in mesenchymal C2C12 cells [25]. Both BMP-2 and collagen type 1 thereby seems to have similar effects on PTHrP and the PTH1R as Dex. A link between Dex treatment and BMP-2 effects also might exist, as stromal cells induced to become osteoblasts by exposure of media containing Dex show enhanced BMP-2 expression [26]. It is well known that Dex is capable of supporting osteoblastic differentiation of mesenchymal stem cells in vitro. In the present study, Dex upregulated the expression of the PTH1R-receptor. Dex also increases the PTH1R expression in several osteoblast-like cell lines, such as rat ROS 17/2 and UMR-106 osteosarcoma cells [27–29]. As the PTH1R is considered to be a marker of the osteoblastic phenotype the Dex effects on PTH1R expression seen in our study can be interpreted as a mechanism associated with osteoblastic differentiation. In accordance with the role of Dex as an agent that supports osteoblast differentiation, and in line with earlier studies [30–32] Dex increased the expression of the osteoblast associated transcrip-
M. Ahlström et al. / Steroids 74 (2009) 277–282
tion factor Runx2 and alkaline phosphatase. However, osteocalcin, which is also a known marker of osteoblast activity, was downregulated. The osteocalcin gene is known to be induced in the late stages of osteoblast differentiation, accompanying the onset of mineralization of the extracellular matrix [33,34]. Therefore, the results by us showing that Dex does not have a stimulating effect on osteocalcin expression in mesenchymal cells could well be explained by less significant role of osteocalcin in the initial induction of the osteoblastic differentiation. This is supported by other studies that also reported reduced osteocalcin expression by Dex in bone marrow-derived mesenchymal cells [30] and in primary human osteoblasts [31,32] in the early phase of osteoblastic differentiation. In these studies Dex reduced the osteocalcin expression by over 50% in the beginning of the differentiation, whereas in the later phase of the differentiation, the osteocalcin expression was increased by Dex [30–32]. A previous study using bone marrow stromal cells indicates an important role of Dex at both the early and late stages of differentiation in directing the cells toward terminal maturation [35]. However, in another study using clonal rat mesenchymal progenitor cells, Dex failed to induce Osterix expression suggesting that Dex is not necessarily needed to promote terminal osteoblast differentiation [36]. Our present study is more in line with the former results. A continuous Dex treatment was more effective in supporting biological mineralization as compared to a discontinuous treatment. One explanation of this is that the mineralizing activity of the individual differentiated cells would decrease upon the withdrawal of Dex. On the other hand, the Dex withdrawal could also cause enhanced growth of undifferentiated cells relative to differentiated cells and the increased mineralization would therefore be a reflection of a reduction in differentiation of osteoblast precursors. The glucocorticoid receptor (GR) is usually considered to be involved in the effects of glucocorticoids, although non-genomic effects by unknown mechanisms have been reported [37]. In the present study, we used the GR antagonist mifepristone to test the involvement of the GR in the effects of Dex. Mifepristone was used at concentrations 100 times higher than Dex and at these concentrations mifepristone had effects on the PTHrP and PTH1R expression that were less pronounced but similar to those of Dex. Partial agonist activity of mifepristone has earlier been described in some cell types and it has been suggested that these effects could result from a reversible DNA binding of the GR transcription complex and by binding of mifepristone to transcriptional co-activators [38,39]. In any case, when used in combination with Dex, mifepristone antagonized the effects of Dex, both in downregulating PTHrP and upregulating PTH1R. A simultaneous downregulation of PTHrP and upregulation of PTH1R by Dex reported in our present study could result in several responses in the osteoblastic progenitors and osteoblasts. Taken into account the anabolic role of PTHrP in bone, decreased levels of released PTHrP could directly attenuate bone formation by reducing differentiation and osteoblast function. An increase in PTH1R could in turn possibly counteract these effects, but might also make the cells more susceptible to the effects of circulating PTH. As the regulation of PTHrP and PTH1R is likely to be a very delicately regulated process, the disruption of these mechanisms by Dex could potentially play a role in the induction of glucocorticoid-induced osteoporosis. References [1] Gennari C, Civitelli R. Glucocorticoid-induced osteoporosis. Clin Rheum Dis 1986;12:637–54. [2] Reid IR. Glucocorticoid osteoporosis—mechanisms and management. Eur J Endocrinol 1997;137:209–17. [3] Canalis E, Delany AM. Mechanisms of glucocorticoid action in bone. Ann NY Acad Sci 2002;966:73–81.
281
[4] Mazziotti G, Angeli A, Bilezikian JP, Canalis E, Giustina A. Glucocorticoidinduced osteoporosis: an update. Trends Endocrinol Metabol 2006;17:144–9. [5] Canalis E, McCarthy TL, Centrella M. Differential effects of continuous and transitient treatment with parathyroid hormone related peptide (PTHrP) on bone collagen synthesis. Endocrinology 1990;126:1806–12. [6] Dempster DW, Cosman F, Parisien M, Shen V, Lindsay R. Anabolic actions of parathyroid hormone in bone. Endocr Rev 1993;14:690–709. [7] Horwitz MJ, Tedesco MB, Gundberg C, Garcia-Ocana A, Stewart AF. Shortterm, high-dose parathyroid hormone-related protein as a skeletal anabolic agent for the treatment of postmenopausal osteoporosis. J Clin Endocrin Metab 2003;288:569–75. [8] Saag KG, Shane E, Boonen S, Marin F, Donley DW, Taylor KA, et al. Teriparatide or alendronate in glucocorticoid-induced osteoporosis. New Engl J Med 2007;357:2028–39. [9] Asa SL, Henderson J, Goltzman D, Drucker DJ. Parathyroid hormone-like peptide in normal and neoplastic human endocrine tissues. J Clin Endocrinol Metab 1990;71:1112–8. [10] Suda N, Gillespie MT, Traianedes K, Zhou H, Ho PW, Hards DK, et al. Expression of parathyroid hormone-related protein in cells of osteoblast lineage. J Cell Phys 1996;166:94–104. [11] Lanske B, Amling M, Neff L, Guiducci J, Baron R, Kronenberg HM. Ablation of the PTHrP gene or the PTH/PTHrP receptor gene leads to distinct abnormalities in bone development. J Clin Invest 1999;104:399–407. [12] Miao D, He B, Jiang Y, Kobayashi T, Sorocéanu MA, Zhao J, et al. Osteoblastderived PTHrP is a potent endogenous bone anabolic agent that modifies the therapeutic efficacy of administered PTH 1-34. J Clin Invest 2005;115:2402–11. [13] Martin TJ. Osteoblast-derived PTHrP is a physiological regulator of bone formation. J Clin Invest 2005;115:2322–4. [14] Lanske B, Divieti P, Kovacs CS, Pirro A, Landis WJ, Krane SM, et al. The parathyroid hormone (PTH)/PTH-related peptide receptor mediates actions of both ligands in murine bone. Endocrinology 1998;139:5194–204. [15] Lanske B, Kronenberg HM. Parathyroid hormone-related peptide (PTHrP) and parathyroid hormone (PTH)/PTHrP receptor. Crit Rev Eukar Gene 1998;8:297–320. [16] Iida-Klein A, Guo J, Drake MT, Kronenberg HM, Abou-Samra AB, Bringhurst FR, et al. Structural requirements of parathyroid hormone/parathyroid hormonerelated peptide receptors for phospholipase C activation and regulation of phosphate uptake. Miner Electrol Metab 1995;21:177–9. [17] Martin TJ. Actions of parathyroid hormone-related peptide and its receptors. New Engl J Med 1996;335:736–8. [18] Yamamoto I, Shigeno C, Potts Jr JT, Segre GV. Characterization and agonistinduced down-regulation of parathyroid hormone receptors in clonal rat osteosarcoma cells. Endocrinology 1988;122:1208–17. [19] Ahlstrom M, Pekkinen M, Riehle U, Lamberg-Allardt C. Extracellular calcium regulates parathyroid hormone-related peptide expression in osteoblasts and osteoblast progenitor cells. Bone 2008;42:483–90. [20] Pekkinen M, Ahlström M, Riehle U, Huttunen M, Lamberg-Allardt C. Effects of phosphodiesterase 7 inhibition by RNA interference on the gene expression and differentiation of human mesenchymal stem cell-derived osteoblasts. Bone 2008;43:84–91. [21] Chen HL, Demiralp B, Schneider A, Koh AJ, Silve C, Wang CY, et al. Parathyroid hormone and parathyroid hormone-related protein exert both pro- and antiapoptotic effects in mesenchymal cells. J Biol Chem 2002;277:19374–81. [22] Choudhary S, Wadhwa S, Raisz LG, Alander C, Pilbeam CC. Extracellular calcium is a potent inducer of cyclo-oxygenase-2 in murine osteoblasts through an ERK signalling pathway. J Bone Miner Res 2003;18:1530–8. [23] Jemtland R, Divieti P, Lee K, Segre GV. Hedgehog promotes primary osteoblast differentiation and increases PTHrP mRNA expression and iPTHrP. Bone 2003;32:611–20. [24] Celic S, Chilco PJ, Zajac JD, Martin TJ, Findlay DM. A type I collagen substrate increases PTH/PTHrP receptor mRNA expression and suppresses PTHrP mRNA expression in UMR106-06 osteoblast-like cells. J Endocrinol 1996;150:299–308. [25] Susperregui AR, Vinals F, Ho PW, Gillespie MT, Martin TJ, Ventura F. BMP-2 regulation of PTHrP and osteoclastogenic factors during osteoblast differentiation of C2C12 cells. J Cell Physiol 2008;216:144–52. [26] Bi LX, Simmons DJ, Mainous E. Expression of BMP-2 by rat bone marrow stromal cells in culture. Calcif Tissue Int 1999;64:63–8. [27] Yamamoto I, Potts Jr JT, Segre GV. Glucocorticoids increase parathyroid hormone receptors in rat osteoblastic osteosarcoma cells (ROS 17/2). J Bone Miner Res 1988;3:707–12. [28] Yaghoobian J, Drueke TB. Regulation of the transcription of parathyroidhormone/parathyroid-hormone-related peptide receptor mRNA by dexamethasone in ROS 17/2.8 osteosarcoma cells. Nephrol Dial Transpl 1998;13:580–6. [29] Haramoto N, Kawane T, Horiuchi N. Upregulation of PTH receptor mRNA expression by dexamethasone in UMR-106 osteoblast-like cells. Oral Dis 2007;13:23–31. [30] Oshina H, Shinichi S, Yoshii T, Torigoe I, Sugata Y. Effects of continuous dexamethasone treatment on differentiation capabilities of bone marrow-derived mesenchymal cells. Bone 2007;41:575–83. [31] Viereck V, Siggelkow H, Tauber S, Raddatz D, Schutze N, Hüfner M. Differential regulation of Cbfa1/Runx2 and osteocalcin gene expression by vitamin-D3, dexamethasone, and local growth factors in primary human osteoblasts. J Cell Biochem 2002;86:348–56. [32] Subramaniam M, Colvard D, Keeting PE, Rasmussen K, Riggs LB, Spelsberg TC. Glucocorticoid regulation of alkaline phosphatase, osteocalcin, and
282
M. Ahlström et al. / Steroids 74 (2009) 277–282
proto-oncogenes in normal human osteoblast-like cells. J Cell Biochem 1992;50:411–24. [33] Owen T, Aronow M, Shalhoub V, Barone L, Wilming LM, Tassinari MS, et al. Progressive development of the rat phenotype in vitro: reciprocal relationships in expression of genes associated withosteoblast proliferation and differentiation during formation of the bone extracellular matrix. J Cell Physiol 1990;143:420–30. [34] Stein GS, Lian JB, van Wijnen AJ, Stein JL. The osteocalcin gene: a model for multiple parameters of skeletal-specific transcriptional control. Mol Biol Reports 1997;24:185–96. [35] Porter R, Huckle W, Goldstein A. Effect of dexamethasone withdrawal on osteoblastic differentiation of bone marrow stromal cells. J Cell Biochem 2003;90:13–22.
[36] Ito S, Suzuki N, Kato S, Takahashi T, Takagi M. Glucocorticoids induce the differentiation of a mesenchymal progenitor cell line, ROB-C26 into adipocytes and osteoblasts, but fail to induce terminal osteoblast differentiation. Bone 2007;40:84–92. [37] Haller J, Mikics E, Makara GB. The effects of non-genomic glucocorticoid mechanisms on bodily functions and the central neural system. A critical evaluation of findings. Front Neuroendocrinol 2008;29:273–91. [38] Mao J, Regelson W, Kalimi M. Molecular mechanism of RU 486 action: a review. Mol Cell Biochem 1992;109:1–8. [39] Mercier L, Miller PA, Simons Jr SS. Antiglucocorticoid steroids have increased agonist activity in those hepatoma cell lines that are more sensitive to glucocorticoids. J Ster Biochem 1986;25:11–20.