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. Author manuscript; available in PMC: 2010 Mar 15.
Published in final edited form as: Mol Endocrinol. 2007 Aug 30;21(12):2929–2940. doi: 10.1210/me.2007-0153

Mitogen-Activated Protein Kinase Phosphatase 1/Dual Specificity Phosphatase 1 Mediates Glucocorticoid Inhibition of Osteoblast Proliferation

Kay Horsch 1, Heidi de Wet 1, Macé M Schuurmans 1, Fatima Allie-Reid 1, Andrew C B Cato 1, John Cunningham 1, Jacky M Burrin 1, F Stephen Hough 1, Philippa A Hulley 1
PMCID: PMC2838148  EMSID: UKMS28492  PMID: 17761948

Abstract

Steroid-induced osteoporosis is a common side effect of long-term treatment with glucocorticoid (GC) drugs. GCs have multiple systemic effects that may influence bone metabolism but also directly affect osteoblasts by decreasing proliferation. This may be beneficial at low concentrations, enhancing differentiation. However, high-dose treatment produces a severe deficit in the proliferative osteoblastic compartment. We provide causal evidence that this effect of GC is mediated by induction of the dual-specificity MAPK phosphatase, MKP-1/DUSP1. Excessive MKP-1 production is both necessary and sufficient to account for the impaired osteoblastic response to mitogens. Overexpression of MKP-1 after either GC treatment or transfection ablates the mitogenic response in osteoblasts. Knockdown of MKP-1 using either immunodepletion of MKP-1 before in vitro dephosphorylation assay or short interference RNA transfection prevents inactivation of ERK by GCs. Neither c-jun N-terminal kinase nor p38 MAPK is activated by the mitogenic cocktail in 20% fetal calf serum, but their activation by a DNA-damaging agent (UV irradiation) was inhibited by either GC treatment or overexpression of MKP-1, indicating regulation of all three MAPKs by MKP-1 in osteoblasts. However, an inhibitor of the MAPK/ERK kinase-ERK pathway inhibited osteoblast proliferation whereas inhibitors of c-jun N-terminal kinase or p38 MAPK had no effect, suggesting that ERK is the MAPK that controls osteoblast proliferation. Regulation of ERK by MKP-1 provides a novel mechanism for control of osteoblast proliferation by GCs.

Chronic glucocorticoid (GC) therapy is frequently required for the management of inflammatory conditions but has multiple adverse side effects. Increased incidence of bone fractures is one of the main concerns with prolonged use of even relatively low doses of GC, because fracture risk increases with use of more than 5 mg/d (1). Understanding the molecular basis of both beneficial and negative effects of GCs is essential if improvements are to be made to the safety profile of this class of drugs (2). Although several pivotal molecular regulators of the immune modulatory effects of GCs have been described, progress in identifying equivalent mediators in bone has been slow. Bone cells and cells of the immune system have in common a tight endogenous regulation by GC and exceptional sensitivity to GC drugs. In bone, normal osteoblast differentiation is dependent on GC and is induced in vitro by a wide range of GC concentrations (3, 4). However, sustained high-dose treatment inhibits osteoblast proliferation (3, 5), enhances adipocyte differentiation from mesenchymal stem cell precursors at the expense of osteoblasts (4, 6), increases apoptosis of both osteoblasts and osteocytes (7, 8), and impairs matrix synthesis (4, 9), all of which contribute to decreased bone formation. There is also an initial increase in osteoclastic bone resorption, due in part to systemic effects on calcium metabolism and also to a possible GC-induced decrease in osteoblastic osteoprotegerin production and an increase in receptor activator of NFκB ligand secretion, with a net transient increase in osteoclast activity (10, 11).

MKP-1 (DUSP1) has recently emerged as a major regulatory target of GCs. This classical dual-specificity phosphatase is one of an 11-member family that inhibits activity of the MAPKs, ERK, c-jun N-terminal kinase (JNK), and p38 (12). MKP-1-deficient mice are viable and healthy but display aberrant immune responses such as enhanced lethality due to lipopolysaccharide-induced endotoxic shock (13, 14), overactive signaling downstream of Toll-like receptors (14, 15), and a higher incidence and severity of autoimmune arthritis (15). Most of these effects appear to be mediated by dysregulation of the inhibitory feedback of MKP-1 on p38 and JNK (13-15). MKP-1 has also now been identified as a required effector for GC-mediated repression of TNF, cyclooxygenase-2, and IL-1α and -1β in macrophages, with MKP-1-deficient mice displaying reduced response to the antiinflammatory effects of dexamethasone (Dex) on zymosan-induced inflammation (16). This provides the first causal link between MKP-1 induction, JNK, and p38 MAPK inhibition and the antiinflammatory effects of GC.

Skeletal development is normal in MKP-1-deficient mice (17) but immune system phenotypes have only emerged after challenges, and equivalent skeletal studies have not yet been performed. Up-regulation of MKP-1 in osteoblasts in association with decreased proliferation has been described after treatment with GC (18, 19) or with PTH (20). Both GC and PTH enhance osteoblast differentiation at low doses or with intermittent treatment, and both are also markedly antianabolic at high doses and/or with sustained duration of treatment (7, 20, 21). Further work is needed to delineate the role of MKP-1 and ERK activity in normal and pathological response of osteoblasts to GC.

Of potential relevance to the skeletal role of MKP-1 is the recent observation of decreased adiposity with age in MKP-1-deficient mice, accompanied by enhanced resistance to diet-induced obesity (22). Osteoblasts and adipocytes develop from common precursors in the bone marrow, and enhanced adiposity of bone marrow is a feature of steroid osteoporotic bones (23). Bone marrow studies have not yet been performed in MKP-1-deficient mice but MKP-1 is strongly up-regulated in differentiating adipocytes and causes decreased ERK activity and consequent cell-cycle exit (24). In addition, more pluripotential marrow cells differentiate into marrow adipocytes when transplanted into GC-treated mice than when transplanted into untreated mice (25).

Because GC-induced bone damage is a major disadvantage in the clinical use of GCs, we set out to examine the functional consequences of MKP-1 up-regulation in osteoblasts.

RESULTS

Dex Treatment Leads to Up-Regulation of MKP-1 Protein in a Dose-Dependent Manner, Starting at Subnanomolar Concentrations

We previously reported up-regulation of MKP-1 expression at both mRNA and protein levels in mouse and human osteoblasts after treatment with high doses of the long-acting GC, Dex (1 μm) (9). To investigate whether the Dex-induced up-regulation of MKP-1 is limited to the pharmacological range of GC doses, we treated MG-63 human osteoblasts, growing rapidly in 10% fetal calf serum (FCS), for 5 h with Dex at doses between 0.01 nM and 10 μm and monitored MKP-1 expression by Western blot (Fig. 1). The 5-h time point and the presence of 10% FCS were chosen because maximal MKP-1 expression occurs under these conditions in MG-63 osteoblasts (18). We consistently detect the regulated MKP-1 band (the lower band indicated in all figures) and a higher molecular weight band (possibly MKP-2), which is not regulated and has been retained in the images because it serves as a convenient loading reference. Induction of MKP-1 expression was first detected at 0.01 nm, the lowest concentration used, and increased in a dose-dependent manner up to 1 μm Dex where the expression was found to be maximal. Therefore, although pharmacological doses of Dex have the most potent effects on MKP-1 up-regulation, subnanomolar doses also cause up-regulation.

Fig. 1. Dex Leads to Dose-Dependent Up-Regulation of MKP-1 Protein, Starting at Subnanomolar Concentrations.

Fig. 1

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Human MG-63 osteoblastic cells, growing rapidly in 10% FCS, were subjected to 5 h treatment with 10−11 to 10−5 m Dex. Equal amounts of total protein were loaded on the gel, and MKP-1 protein was detected by Western blot (lower band indicated by arrow). Membranes were stripped and reprobed for total ERK as equal loading control. This is stable in these cells and under these conditions. Levels of both proteins were quantified by densitometry, and MKP-1 was normalized to ERK. Result shown is representative of two experimental repeats

Mouse and Human Osteoblasts Show Strong, Comparable Turnover of MKP-1

To test whether the low MKP-1 expression levels, particularly in mouse osteoblasts, were due to low stability and high turnover of the protein, we used the 26S proteasome inhibitor MG132 to block protein degradation in mouse (MBA-15.4) and human (MG-63) osteoblasts. Cotreatment of MG-63 and MBA-15.4 cells with Dex and MG132 strongly enhanced the Dex-induced accumulation of MKP1 (Fig. 2A). The murine MBA-15.4 cell line responds more slowly to Dex treatment (18); hence the selected treatment time of 8 h. This mouse cell line frequently displays a regulated doublet instead of the single MKP-1 band in MG-63 cells (see Fig. 2B). This may represent a splice variant or phosphorylated form of MKP-1, but validated mouse MKP-1 siRNA was not available to confirm this. Both cell lines showed no basal MKP-1, but after 2 h of proteasome inhibition MKP-1 protein began to accumulate detectably even in the absence of Dex (Fig. 2B). With increasing incubation time the protein levels rapidly accumulated in both cell lines, demonstrating strong synthesis of MKP-1, although this is not normally detectable due to equally rapid degradation.

Fig. 2. Proteasomal Degradation of MKP-1 in Human MG-63 and Mouse MBA-15.4 Osteoblasts Is Strong and Comparable.

Fig. 2

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A, Human and mouse osteoblasts, rapidly growing in 10% FCS, were subjected to 1 h pretreatment with the 26S proteasome inhibitor, MG132 (30 μm), before addition of Dex (1 μm) for 5 h (MG-63) or 8 h (MBA-15.4). Cells were lysed in NP-40 buffer and MKP-1 protein (arrow) was detected by Western blot of equal protein samples. Membranes were stripped and reprobed for total ERK, levels of both proteins were quantified by densitometry, and MKP-1 was normalized to ERK. B, MG132 was added at 30 μm for different time periods, and accumulation of MKP-1 was monitored by Western blot of equal protein samples. Results shown are representative of two experimental repeats.

Dex-Induced Up-Regulation of MKP-1 Is Regulated by GC-Glucocorticoid Receptor (GR) Interaction and Depends Primarily on Protein Synthesis

Because we have previously shown (18) that Dex induction of MKP-1 expression occurs within 30 min (mRNA) and 1 h (protein), it is unclear whether the up-regulation is controlled by a classical transcriptional mechanism requiring GC-GR interaction or whether a rapid, nongenomic mechanism is involved. We therefore pretreated MG-63 cells with the GR antagonist RU486. Cells were then stimulated with Dex, and MKP-1 protein levels were monitored by Western blot. The results (Fig. 3, left panel) show a strong inhibition of Dex-induced MKP-1 by the GR antagonist, demonstrating that up-regulation requires GC-GR interaction. Kassel et al. (26) reported that in mast cells GCs induce up-regulation of MKP-1 by a dual mechanism consisting of both increased expression and decreased degradation. To determine whether the same mechanism is active in osteoblasts, we treated MG-63 cells with Dex in combination with the protein translation inhibitor cycloheximide. As shown in the Western blot (Fig. 3, right panel) cycloheximide completely blocks MKP-1 up-regulation in osteoblasts, in contrast to mast cells. Although MKP-1 is also clearly subjected to proteasomal degradation, this may indicate that in osteoblasts the GC-induced rise in MKP-1 levels is primarily dependent on de novo protein synthesis. If decreased degradation accounted for the major part of the GC-induced MKP-1 increase, we should see residual MKP-1 levels in the presence of cycloheximide alone.

Fig. 3. Dex Induced Up-Regulation of MKP-1 Is Dependent on GC-GR Interaction and de Novo Protein Synthesis.

Fig. 3

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Human MG-63 osteoblasts were grown with 10% FCS and subjected to 1 h pretreatment with the GR antagonist RU486 (1 μm) or the protein translation inhibitor cycloheximide (CHX) (40 μg/ml), before addition of Dex (1 μm) for an additional 4 h. Cell lysates were prepared in NP-40 buffer, and MKP-1 protein (arrow) was detected by Western blot. Membranes were stripped and reprobed for total ERK, levels of both proteins were quantified by densitometry, and MKP-1 was normalized to ERK as loading control. Result shown is representative of two experimental repeats.

MKP-1 Is the Only Dex-Induced Phosphatase Responsible for Dephosphorylation of ERK

Synthesis of a phosphatase does not necessarily mean that it is responsible for the observed concurrent dephosphorylation of a substrate. To provide evidence that Dex-induced up-regulation of MKP-1 does account for the Dex-induced dephosphorylation of ERK, we developed an in vitro dephosphorylation assay using phosphorylated ERK as substrate. An MKP-1-containing [protein tyrosine phosphatase (PTP)] lysate was added to an active ERK-enriched substrate lysate, both derived from MG-63 osteoblasts. The substrate lysate, containing phosphorylated ERK, was obtained by O-tetradecanoylphorbol 13-acetate (TPA) stimulation of serum-starved cells (Fig. 4A, right panel). Two different PTP lysates were generated, either basal (Fig. 4A, left lane) or enriched in MKP-1 (Fig. 4A, right lane). Basal lysate was obtained from untreated cells, i.e. with low MKP-1 levels (Fig. 4A, left panel), whereas PTP lysate enriched in MKP-1 was generated by treatment of nonstarved MG-63 cells with Dex to up-regulate MKP-1 expression. The Dex-treated lysate inevitably has slightly lower levels of ERK phosphorylation. However, ERK in both basal and PTP lysates is phosphorylated at much lower levels than the TPA-stimulated substrate lysate because cells were growing steadily and unsynchronized in 10% serum. These are conditions that minimize the ERK difference and maximize MKP-1 expression (data not shown). Equal quantities of protein from PTP and substrate lysates were mixed and incubated to permit any active phosphatases to dephosphorylate the substrate P-ERK (Fig. 4B). Lane 2 shows dephosphorylation of ERK by the MKP-1-containing Dex-treated lysate, compared with no dephosphorylation induced by the basal lysate in lane 1. Addition of vanadate (lane 3) to the reaction restores the ERK phosphorylation, suggesting that the loss of P-ERK in lane 2 is due to PTP activity. However vanadate inhibits all PTPs and DUSPs and therefore this does not prove the exclusive action of MKP-1. To rule out other phosphatases, we repeated the experiment and compared the MKP-1 containing lysate (lane 5) with the same lysate from which MKP-1 had been immunodepleted (lane 6). The complete restoration of the signal by immunodepletion suggests that MKP-1 may be the only phosphatase up-regulated by Dex that is responsible for dephosphorylation of ERK in osteoblasts.

Fig. 4. MKP-1 Is the Primary Phosphatase Responsible for Dex-Induced Dephosphorylation of the MAPK ERK.

Fig. 4

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As a source of substrate and phosphatase lysates, two sets of cells were grown as follows: to obtain MKP-1-enriched PTP lysate, MG-63 osteoblasts were grown in 10% serum, stimulated with Dex (1 μm) for 5 h, and lysed in phosphatase lysis buffer (A). As a substrate lysate for the in vitro dephosphorylation assay, active ERK was generated by growing MG-63 cells in 10% FCS, synchronizing cell cycle over night in 1% FCS, stimulating with TPA (100 ng/ml) for 30 min, and lysing in phosphatase lysis buffer (A). Quality of PTP and substrate lysate was shown by Western blot using specific antibodies for MKP-1 and P-ERK. B, For in vitro dephosphorylation, 50 μg of PTP lysate was added to 50 μg substrate lysate and incubated at 30 C for 45 min. Where indicated, 2 mm vanadate was added (lane 3) or MKP-1 was immunodepleted (lane 6) from the PTP lysate before mixing (the 50-kDa heavy chain of the immunodepleting antibody is indicated with an arrowhead). Phosphatase reactions were stopped by addition of an appropriate amount of sample buffer and boiling. Aliquots of each reaction mix were subjected to Western blotting with P-ERK-specific antibody, and membranes were stripped and reprobed for ERK to show relative dephosphorylation levels. Results shown are representative of three independent experiments.

MKP-1 Dephosphorylates ERK, p38, and JNK in Osteoblasts with Distinct Sensitivity

Because MKP-1 has such a strong impact on ERK phosphorylation, we were interested to determine whether it also dephosphorylates other known substrates, p38 and JNK, in osteoblasts. We extended the in vitro dephosphorylation assay above to compare the dephosphorylation of p38, JNK, and ERK by Dex-induced MKP-1. The substrate lysates had to be prepared differently, because p38 and JNK could not be activated by TPA or serum (data not shown). We therefore chose to activate both stress kinases by UV irradiation, which leads to strong phosphorylation between 45 and 60 min of treatment. All assays were performed under equivalent conditions and for constant times, and dephosphorylation of all three MAPKs was detected with phospo-specific antibodies for each of the substrates. Specificity of the antibodies was checked using the selective inhibitors U0126, SB203580, and SP600125 to block ERK, p38, and JNK phosphorylation, respectively. Because kinetics and specificity of enzymes can vary between species and cell types, we compared mouse and human osteoblasts and for statistical relevance we repeated each dephosphorylation at least three times. Figure 5 shows representative Western blots for MG-63 human (Fig. 5A) and MBA-15.4 mouse osteoblasts (Fig. 5C), in addition to the summaries of all the experiments performed with MG-63 (Fig. 5B) and MBA-15.4 (Fig. 5D) cell lines. As previously demonstrated, both cell lines show strong dephosphorylation of ERK (hatched bars) by the Dex-treated lysate, which reaches at least 70% over the period of incubation. This dephosphorylation was prevented by vanadate. In MG-63 the dephosphorylation of p38 (white bars) was almost as strong as that of ERK, whereas in MBA-15.4 it was equivalent. However, in contrast to ERK, the phosphorylation of p38 was only partially protected by vanadate. This might be due to dephosphorylation by a vanadate-insensitive Ser/Thr phosphatase, in addition to MKP-1. JNK (black bars) was not strongly dephosphorylated by MKP-1-enriched lysate, indicating possible regulation by a different phosphatase in osteoblasts or inaccessibilty due to a scaffolding complex. This depletion was more apparent, however, in MG-63 cells and was prevented by vanadate.

Fig. 5. ERK, p38, and JNK Are All Sensitive to in Vitro Dephosphorylation by Dex-Induced MKP-1.

Fig. 5

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To obtain active MAPK substrate for dephosphorylation, human MG-63 or mouse MBA-15.4 osteoblast cells were starved in 1% serum overnight, activated by stimulation with TPA (100 ng/ml) for 30 min (ERK) or UV irradiation for 45 min, and lysed in PTP buffer. A second, nonstarved set of cells was treated with Dex [1 mm for 5 h (MG-63) or 8 h (MBA-15.4)] and lysed in the same way to generate PTP lysates. A 21-mm concentration of vanadate was added to the PTP lysates where indicated. For in vitro dephosphorylation, 50 μg protein of phosphatase lysate was added to 50 μg protein of each substrate lysate (ERK, JNK, p38) and incubated at 30 C for 45 min. Phosphatase reactions were stopped by addition of sample buffer and boiling. Aliquots of each reaction mix were subjected to Western blotting and detection with antibodies specific for the phosphorylated forms of ERK, JNK, or p38. Membranes were then stripped and reprobed for total ERK, and levels of all proteins were quantified by densitometry and normalized to ERK. Representative blots for four independent experiments are shown for MG-63 (A) and MBA-15.4 (C) cell lines. All four repeats were quantified with densitometry, and means ± SD for MG-63 (B) and MBA-15.4 (D) are shown.

Overexpression of MKP-1 Is Sufficient for the Dephosphorylation of ERK, p38, and JNK in Osteoblasts

In vitro dephosphorylation of the MAPK substrates, although useful, may not be specific. We therefore transiently transfected a myc-tagged MKP-1 expression vector into MG-63 cells and examined the effects on phosphorylation of ERK in comparison with Dex treatment (Fig. 6A). As already demonstrated, Dex caused expression of MKP-1 and strong inhibition of TPA-induced ERK phosphorylation (lane 3). Overexpression of exogenous MKP-1 had an even stronger effect and completely abolished ERK phosphorylation (lane 4), whereas the vector control had no effect (lane 5). Dephosphorylation by exogenous MKP-1 might be stronger than that caused by Dex treatment due to higher expression levels of MKP-1 or due to a higher activity of the construct. The protein levels could not be directly compared due to a slight truncation [first 314 amino acids (aa) only of 367 aa; with 10-aa myctag added] of the myc-tagged MKP-1 (27). This truncation includes the C-terminal epitope recognized by the reliable and well-characterized anti-MKP-1 antibody. We therefore used anti-myc antibody to detect exogenous MKP-1 (lane 4). Reprobing for total ERK showed equal loading. These data demonstrate that expression of MKP-1 is sufficient for the dephosphorylation of ERK seen in Dex-treated osteoblasts, although this does not necessarily exclude secondary mechanisms.

Fig. 6. Overexpression of myc-MKP-1 Is Sufficient to Mimic the Effect of Dex on Phosphorylation of ERK, p38, and JNK in MG-63 Osteoblasts.

Fig. 6

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MG-63 osteoblasts were grown to 50% confluence and transiently transfected with either myc-MKP-1 expression vector or control vector. Forty-eight hours after transfection, cells were treated with Dex (1 μm) for 5 h, and TPA (100 ng/ml) was added for the last 30 min of incubation, before lysis in NP-40 buffer. Phosphorylation of ERK was detected by Western blot using phospho-specific ERK antibody, and total ERK levels were visualized after stripping of the membranes. Dex-induced expression of MKP-1 was examined by Western blot analysis with anti-MKP-1 antibody (arrowhead), and the overexpressed MKP-1 was detected with myc-specific monoclonal antibody (open arrow). A, Comparison between the effect of Dex and overexpression of MKP-1. B, Transfected cells were stimulated with either TPA (100 ng/ml) for 30 min or UV-irradiated for 45 or 60 min to activate the different MAPKs. The phosphorylation status of ERK, p38, and JNK was investigated by Western blot using phospho-specific antibodies for each of the kinases, and overexpression of MKP-1 (open arrow) was controlled using the myc antibody.

We also tested the ability of exogenous MKP-1 to dephosphorylate JNK and p38 (Fig. 6B). JNK and p38 were activated by UV irradiation for 45 or 60 min for optimal phosphorylation. Similar to its effect on ERK, overexpression of myc-MKP-1 completely dephosphorylated p38 (lane 9), whereas the control vector had no effect (lane 8). In contrast to the in vitro dephosphorylation experiments, JNK was clearly dephosphorylated (lane 6). However, dephosphorylation was still reduced in comparison with ERK or p38.

Overexpression of MKP-1 Blocks Osteoblast Proliferation

To see whether MKP-1 overexpression also blocks the downstream effects of ERK phosphorylation in osteoblasts, we transiently transfected MG-63 cells with myc-MKP-1 expression vector and control vector and examined the effect on TPA-induced proliferation (Fig. 7). In the control cells, proliferation was increased by 80% after TPA stimulation (black bars). In contrast, cells overexpressing MKP-1 did not respond to TPA at all. These data demonstrate that overexpression of the immediate early gene phosphatase MKP-1 alone can abolish osteoblast-proliferative response to a mitogen.

Fig. 7. Overexpression of MKP-1 Leads to Full Inhibition of TPA-Induced Osteoblast Proliferation.

Fig. 7

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MG-63 osteoblastic cells were grown in 10% FCS, transiently transfected with MKP-1 expression vector or control vector, and grown for 24 h, after which they were stimulated with TPA (100 ng/ml) for an additional 24 h. Two hours before the reaction was terminated, 2 μCi/ml [3H]thymidine was added to the medium and DNA synthesis was quantified. A representative experiment of three identical repeats is shown (mean ± SD; *, P < 0.05).

ERK, but Not p38 and JNK, Is Important for Osteoblast Proliferation

Despite the importance of all three MAPKs and their multiple and varying functions in all cell types, not much is known about their functions in osteoblasts. Although ERK is generally accepted to be the primary mitogenic effector in most cell types, both p38 and JNK may also be involved. Because MKP-1 also dephosphorylates ERK, p38, and JNK and inhibits the proliferation of osteoblasts, we examined the contribution made by each of the three kinases to osteoblast proliferation. Mouse MBA-15.4 (Fig. 8A) and human MG-63 osteoblasts (Fig. 8B) were grown in the presence of pharmacological inhibitors for each of the kinases and compared with normal cells undergoing rapid growth. For comparison, cells were also starved in low serum or grown in the presence of 1 μm Dex to inhibit proliferation of both cell lines.

Fig. 8. ERK Is the Primary MAPK Responsible for Proliferation of Mouse and Human Osteoblasts.

Fig. 8

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Cells were grown in 10% FCS for 24 h before treatment with MEK inhibitor U0126 (0.1 μm for MG-63 or 10 μm for MBA-15.4) (designated U), p38 inhibitor SB203580 (1 μm) (designated SB), or JNK inhibitor SP600125 (10 μm) (designated SP) for an additional 24 h, as indicated. Two hours before the incubation was terminated, 2 μCi/ml [3H]thymidine were added to the medium and DNA synthesis was quantified. Dex (1 μm) or medium containing only 0.5% (MG-63) or 1% (MBA-15.4) FCS was also included as control. A representative of three identical repeats is shown (mean ± SD). A, MBA-15.4 mouse; B, human MG-63 osteoblasts. C, Selective inhibition of kinase activity in human MG-63 osteoblasts with inhibitor concentrations as above. The phosphorylation status of ERK, p38, and JNK was investigated by Western blot using phospho-specific antibodies for each of the kinases, and membranes were stripped and reprobed for total ERK as loading control.

Growth factor withdrawal by starvation in 1% FCS led to a strong inhibition of proliferation of both cell lines with proliferation being reduced by 50% in MBA-15.4 (Fig. 8A) and more than 70% in MG-63 (Fig. 8B). As previously reported (18), Dex treatment also led to a dramatic decrease in proliferation, with MBA-15.4 being more sensitive (75% inhibition) than MG-63 (30% inhibition). Inhibition of JNK (SB203580) or p38 (SP600125) signaling with specific inhibitors at doses that block kinase activity (illustrated in Fig. 8C for MG-63) had no antiproliferative effect in either cell line, demonstrating that these kinases do not make a major contribution. However, JNK and p38 are likely to be important for other functions in osteoblasts, which may well be regulated by MKP-1 and therefore affected by Dex. In contrast, blocking of MAPK/ERK kinase (MEK) and the downstream ERK pathway in both cell lines with U0126 had strong inhibitory effects on proliferation. Interestingly, this decrease in proliferation was comparable to that produced by Dex treatment, illustrating the link between Dex effect and ERK signaling.

Knockdown of MKP-1 with siRNA Prevents Dex-Induced ERK Dephosphorylation

To further demonstrate the relevance of Dex-induced MKP-1 expression to the inhibition of the ERK signaling pathway, we used siRNA technology to knock down MKP-1 expression and to analyze the effect of Dex on ERK phosphorylation. We transiently transfected human MG-63 cells with a double-stranded RNA sequence previously shown to be effective and specific for silencing human MKP-1 (28), induced MKP-1 expression with a 5-h Dex treatment, and then activated ERK for 30 min with TPA (Fig. 9). Western blot for MKP-1 demonstrates induction of MKP-1 by Dex alone (lane 3) and in the presence of scrambled control siRNA (lane 6). MKP-1 siRNA decreased the amount of MKP-1 protein induced by Dex treatment (lane 4). When the blot was stripped and reprobed with antiactive ERK antibody, there was an inverse correlation between MKP-1 levels and ERK phosphorylation. The complete recovery of ERK activity after MKP-1 silencing illustrates that MKP-1 is the primary dephosphorylating agent induced by Dex.

Fig. 9. Knockdown of MKP-1 with siRNA Is Able to Significantly Reduce the Negative Effect of Dex on ERK Phosphorylation.

Fig. 9

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MG-63 cells were transiently transfected with either MKP-1-specific siRNA or a nonspecific control and incubated for 48 h. Dex (1 μm) was added for the last 5 h and TPA (100 ng/ml) was added for the last 30 min before lysis in NP-40 buffer. Expression of MKP-1 was examined by Western blot analysis with anti-MKP-1 antibody, phosphorylation of ERK was detected by Western blot using phospho-specific ERK antibody, and ERK levels were visualized after stripping of the membranes. Representative Western blots of three independent experiments are shown. SiC, Scrambled control short interference RNA; siMKP-1, MKP-1-specific short interference RNA.

DISCUSSION

MKP-1 is currently emerging as a fundamental regulator of the immune system and particularly of GC response in vivo and in vitro. We provide the first causal evidence that MKP-1 mediates a direct GC effect on osteoblasts, which are the principal cell type affected in GC-induced osteoporosis. Like the immune system, bone is highly responsive to endogenous GC and therefore very susceptible to bystander damage after systemic immunosuppressive GC therapy. GCs up-regulate MKP-1 with immediate-early gene kinetics in osteoblastic cells in vitro (18), and we find that this up-regulation is both necessary and sufficient to account for decreased ERK activation and attenuated mitogenic response. Vector-driven overexpression of MKP-1 markedly attenuates mitogenic response by the centrally regulating kinase, ERK, and abrogates cell proliferation. We also find that knockdown of GC-induced MKP1 by RNA interference completely restores normal ERK response to mitogens in osteoblasts, providing evidence that MKP-1 is nonredundant for this function in osteoblastic cells. In contrast to studies performed in mouse embryonic fibro-blasts from the MKP-1-deficient mouse (17, 29), bone marrow-derived macrophages (13-15), HeLa cells (30), U937 human leukemic cells (31), alveolar macrophages (32), and rheumatoid arthritis synoviocytes (33), we observed that in osteoblasts the effects of MKP-1 overexpression are mediated primarily by dephosphorylation of ERK. Although JNK and p38 MAPKs can also be dephosphorylated by GC-induced MKP-1, we find no evidence of their involvement in mitogenic response and osteoblast proliferation.

The causes of GC-induced osteoporosis are unclear, although multiple contributing factors have been identified. Although there is an initial rise in bone resorption, the dominant feature is a sustained impairment of new bone formation, which is thought to be caused by decreased osteoblast progenitor recruitment, decreased proliferation, impaired function, and enhanced apoptosis of osteoblasts and osteocytes (34, 35). ERK is involved in each of these processes but the effects of the sustained up-regulation of MKP-1 induced by GC treatment still need to be studied. ERK activity is essential for proliferation and also, to some extent, for differentiation because it promotes the expression and function of both AP1 and cbfa1/Runx2, which in turn activate expression of all the major osteoblastic markers including osteocalcin, osteopontin, bone sialoprotein, and collagen I (36-38). ERK activity is a critical repressor of adipogenesis, because it phosphorylates and represses activity of peroxisome proliferator-activated receptor-γ and prevents adipocyte differentiation, while also promoting osteoblast differentiation in bone marrow precursors (6, 24, 39). MKP-1 is an essential regulator of both adipocyte size and increase of body fat with age and fatty diet, because adult MKP-1-deficient mice are significantly leaner than wild-type littermates and are also protected from diet-induced obesity (22). The mechanism of protection involves enhanced responsiveness of insulin-sensitive tissues via hyperactivation of the nuclear pool of ERK, p38, and JNK, but whether this extends to bone marrow adipocytes remains to be seen. It is also clearly apparent that high dose GC trigger premature apoptosis of both osteo-blasts and osteocytes in vivo (34, 40) and that activation of ERK by bisphosphonates, estrogen, mechanical loading, and cell-substrate adhesion are protective, at least in vitro (41-43).

Separating the normal vs. pathological effects of GC on osteoblasts has proved difficult. It is clear that lower doses (nanomolar range) are essential for normal differentiation, and it is likely that GC-induced MKP-1 and consequent cell cycle exit contribute positively to this process. One of the recent bone anabolic mechanisms proposed for PTH is induction of MKP-1 and subsequent repression of ERK, down-regulation of cyclin D1, and up-regulation of the cell cycle inhibitor, p21cip (20). The resultant decrease in proliferation is thought to enhance osteoblastic differentiation. Intriguingly, whereas all stages of osteoblasts express GC receptor (GR), stromal cells barely express PTH receptor, and expression levels increase with differentiation into mature osteoblasts and osteocytes (44, 45). Therefore MKP-1 is unlikely to be strongly up-regulated by PTH in osteoblast precursors but should increase in more mature osteoblast stages, and these are also stages that respond positively to GC treatment by up-regulating differentiation markers and secretion of a mineralized matrix. Because GR is present not only in mature cells but also in mesenchymal stem cells and immature, proliferating osteoblasts, GCs have the potential for wide-ranging effects across the differentiation spectrum. We provide evidence that MKP-1 is up-regulated in a dose-dependent manner by picomolar through to micromolar concentrations of Dex, with the highest level seen at 1–10 μm. A recent study reports differentiation of primary dermal fibro-blasts into bone-forming osteoblasts after transfection with cbfa1/Runx2 type II and treatment with the standard in vitro osteogenic dose of 10 nm Dex (46). This study correlates expression of bone markers with increased MKP-1 expression and decreased cbfa1/Runx2 phosphorylation, although the intermediate MAPK was not identified.

To date no in vivo studies have been published in either mouse or human demonstrating that up-regulation of MKP-1 occurs after direct systemic GC administration, although there are data after ex vivo culture of clinical material (33). Both wild-type and MKP-1-deficient mice were injected with GCs in the recent study by Abraham et al. (16), but MKP-1/DUSP1 expression was not studied and dosage was based on an estimated EC50. We demonstrate that MKP-1 is significantly up-regulated in vivo in the circulating white blood cells of stable chronic obstructive pulmonary disease patients after standard clinical treatment with GCs, indicating a chronic shift in transcriptional pattern that is likely to affect multiple GC-sensitive cell compartments (see the figure in the supplemental data published on The Endocrine Society's Journals Online web site at http://mend.endojournals.org). The fact that MKP-1 levels are elevated on d 28 of a daily high-dose regimen means that either MKP-1 is up-regulated in a sustained manner or that it is capable of daily, transient up-regulation. This is a response in circulating white blood cells and not bone, but bone biopsies are invasive and not routinely used to diagnose GC-induced osteoporosis. However, the demonstration that MKP-1 mRNA can be accurately measured in patient specimens should facilitate a closer investigation of bone effects. Resistance to GC therapy is a major clinical problem in a subset of patients with asthma and inflammatory bowel disease, and a role for MKP-1 has been proposed (47-49). Approximately 50% of our patients responded with a strong MKP-1 up-regulation, although this did not correlate with airway response to GCs in this study (50).

Given the response of MKP-1 to an in vitro dose of 10−11 m Dex, it seems likely that MKP-1 may be regulated normally in many GC-sensitive body cells in response to circadian GC rhythm and also to both acute and chronic stress-induced GC elevations. Different cells respond at different rates and with varying intensities. The MG-63 human osteoblast cell line responded more quickly than mouse MBA-15.4 cells (5 vs. 8 h to peak protein induction) and expressed a higher level of Dex-induced MKP-1. However, the addition of proteasome inhibitor profoundly boosted MBA-15.4 expression levels, possibly indicating more active proteasomal degradation in this mouse cell line. Although transfection of MKP-1 into MG-63 cells abolished mitogenic response, MBA-15.4 cells were more responsive to Dex in terms of inhibition of proliferation than MG-63 cells. This could be due to the induction of second GC-sensitive phosphatase in MBA-15.4 cells but does suggest that the higher proteasomal turnover of MKP-1 in these cells has little effect on its activity and that the rather short half-life of MKP-1 does not impair its ability to dephosphorylate ERK and trigger growth arrest.

In conclusion, GC-induced osteoporosis is caused by both direct and indirect effects of GCs on bone cells. The initial sharp increase in osteoclast activity can be partially prevented by treatment with bisphosphonates and calcitriol, with debatable benefit from calcium and vitamin D supplementation (51, 52). The profound impairment of osteoblastic bone formation reverses provided GC treatment is rapidly stopped and not resumed (1, 51). Because this is rarely clinically feasible, the inevitable sustained treatment and repeated exposures produce a severe low formation osteopenia, associated with early fractures, that is currently untreatable. The up-regulation of MKP-1 by GCs in osteoblastic cells has clear functional consequences in terms of depleted mitogenic response and modulation of MAPK family signaling. Identification of such novel GC-regulated gene targets provides mechanistic insight into tissue-specific effects of this class of drugs and provides the essential basis for the development of less damaging compounds. This study provides the first causal link between MKP-1 induction, ERK inhibition, and the antiproliferative effects of GC on osteoblasts.

MATERIALS AND METHODS

Enhanced chemiluminescence detection reagents and molecular weight markers were obtained from Amersham Biosciences (Amersham Pharmacia Biotech, Uppsala, Sweden) and [3H]thymidine from Amersham Pharmacia Biotech (Amersham, UK). Ready Gel aqueous scintillation fluid was from Beckman Coulter, Inc. (Fullerton, CA). FCS was from Delta Bioproducts (Johannesburg, South Africa). RU486 was a kind gift from Professor Janet Hapgood (Department of Biochemistry, University of Stellenbosch, Stellenbosch, South Africa). Protein A-Sepharose was from Pierce Biotechnology (Rock-ford, IL). Polyclonal rabbit MKP-1 (sc-1102) and ERK antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Polyclonal phospho-specific antibodies for ERK, JNK, and p38 and monoclonal anti-Myc-tag antibody were from Cell Signaling Technology, Inc. (Beverly, MA). Polyclonal anti-JNK and anti-p38 antibodies were from Sigma (Munich, Germany). Secondary peroxidase-coupled antirabbit and antimouse antibodies were from Amersham Biosciences (Uppsala, Sweden). Immobilon-P membrane was from Millipore Corp. (Bedford, MA). U0126 was from Promega (Southampton, UK), and SP600125 and SB-203580 were from Biomol, Inc. (Plymouth Meeting, PA). GeneJuice transfection reagent was obtained from Novagen (San Diego, CA). The pcDNA MKP-1 expression vector and pcDNA control vector were a kind gift from Professor Jacky Burrin (St. Barts Hospital, London, UK), and RNA oligos for RNA interference were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA). mRNA isolation kit for blood/bone marrow was from Roche (Mannheim, Germany), and magnetic poly-dT Dyna-beads were from Dynal Biotech (Hamburg Germany). Fast-Start DNA Master SYBR Green I came from Roche. All other chemicals, including tissue culture media, TPA, cycloheximide, MG132, and vanadate were the highest grade, obtained from Sigma.

Cell Culture

MBA-15.4 mouse bone marrow stromal cells were a kind gift from Professor S. Wientraub (Tel Aviv University, Tel Aviv, Israel). They express osteoblastic markers such as alkaline phosphatase and collagen type I but very low levels of PTH receptors in vitro and can be induced to differentiate in response to GCs and produce bone in vivo (53). MBA-15.4 cells were grown in bicarbonate-buffered DMEM with 10% heat-inactivated FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin. MG-63 cells were grown in MEM containing the same supplements. For experiments, rapidly growing, subconfluent cells were seeded into 24-well plates (proliferation studies), 30-mm culture dishes (overexpression and siRNA), or 100-mm culture dishes (all other experiments). For stimulation of MAPKs, cells were grown to 70% confluence and either treated with TPA (100 ng/ml) for 30 min to activate ERK or exposed to UV light with open lid in a laminar flow cabinet for 45 or 60 min to activate JNK and p38. Dex (1 μm) was added for 4 h (MG-63) or 8 h (MBA-15.4), and cycloheximide (40 μg/ml) or RU486 (1 μm) was added for 5 h before lysis of the cells. For experiments on proteasomal degradation, the inhibitor MG132 was used (30 μm).

Western Blotting

Whole-cell lysates were obtained by solubilizing the cells in Nonidet P-40 (NP-40) buffer [1% NP-40, 50 mm Tris (pH 7.5), 120 mm NaCl, 5 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 0.5 mm phenylmethylsulfonylfluoride, 5 mm sodium fluoride, 20 μm β-glycerophosphate, and 2 mm vanadate] for 5 min on ice. The lysates were clarified by centrifugation and for immunoprecipitations, equal volume lysates with equal protein content were incubated with specific antibody overnight on ice. Immune complexes were collected with protein A-Sepharose and immunoprecipitated proteins were analyzed by SDS-PAGE. Proteins were electroblotted onto polyvinylidine difluoride membranes and detected with specific antisera after blocking with 5% nonfat dry milk. Proteins were visualized with peroxidase-coupled secondary antibody using the enhanced chemiluminescence detection system. For reprobing, membranes were stripped in 62.5 mm Tris (pH 6.8), 2% sodium dodecyl sulfate, and 100 mm β-mercaptoethanol for 30 min at 60 C.

Cell Proliferation

Cell proliferation was measured by [3H]thymidine incorporation into acid-insoluble material, as previously described (5). Briefly, MBA-15.4 or MG-63 cells were grown in 24-well plates in DMEM with 10% FCS. To investigate the role of different MAPKs in control of proliferation, the medium was removed from 50% confluent cultures and replaced with fresh medium containing 10% FCS to which 1 μm Dex, 1 μm SB203580 (p38 inhibitor), 10 μm SP600125 (JNK inhibitor), 0.4 μm (MG-63), or 10 μm (MBA-15.4) U0126 (MEK inhibitor) was added for 24 h. As an additional control, cells were serum starved in medium containing only 1% (MBA-15.4) or 0.5% (MG-63) FCS, concentrations that drastically reduce proliferation of each cell line without causing immediate cell death. [3H]thymidine (2 mCi/ml) was added to the medium for the last 2 h of incubation. Cells were lysed by snap freezing at −70 C, followed by incubation with 0.1 m NaOH-0.1% sodium dodecyl sulfate at room temperature. Protein and DNA were precipitated overnight at 4 C with 20% cold trichloroacetic acid, and the pellet was dissolved in 0.1 m NaOH before scintillation counting for incorporated thymidine.

In Vitro Dephosphorylation Assay

MBA-15.4 or MG-63 cells were grown under standard conditions and divided into two groups at 70% confluency. The substrate group was starved in medium containing 1% serum overnight (to synchronize cells and subsequently achieve maximum kinase phosphorylation). To generate the substrate lysate, MAPKs were activated by TPA (100 ng/ml) treatment for 30 min (ERK) or stimulation with UV light for 45 min (JNK and p38) (activation shorter than 2 h to avoid MKP-1 expression). As a control, plates were left untreated. The PTP-enriched lysate group (cultured unsynchronized for >24 h in 10% serum to minimize activity of ERK and enhance MKP-1 expression) was treated with 1 μm Dex for 5 h (MG-63) or 8 h (MBA-15.4) to achieve maximal expression of MKP-1.

Substrate and phosphatase lysates were obtained by detaching the cells with a cell scraper in PTP lysis buffer [50 mm HEPES (pH 7.6), 10 mm EDTA, 10 mm EGTA, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mm phenylmethylsulfonylfluoride, 5 mm sodium fluoride] and sonicating for 30 sec. The lysates were clarified by centrifugation, and protein concentrations were determined. For in vitro dephosphorylation, 50 μg protein or phosphatase lysate were added to 50 μg of substrate lysate, in a total volume of 200 μl PTP lysis buffer, and incubated at 30 C for 45 min. For control experiments 2 mm vanadate was added to the lysate mix or MKP-1 was immunodepleted from the PTP lysate. For immunodepletion the lysate was incubated with specific antiserum, protein A sepharose beads were added, and immune complexes were removed by centrifugation. For complete depletion the procedure was repeated three times. Dephosphorylation reaction was stopped by addition of sample buffer and heating at 95 C for 10 min. Samples were subjected to SDS-PAGE, Western blotting, and detection with phospho-specific polyclonal antibodies for ERK, JNK, and p38. Membranes were stripped as described above and reprobed with pan ERK, JNK, or p38 antibodies as equal loading controls.

MKP-1 Overexpression

MG-63 cells were grown to 60% confluence and transiently transfected with MKP-1 expression vector or empty pcDNA vector using GeneJuice transfection reagent according to the manufacturer's protocol. Medium was changed 24 h after transfection, and 48 h after transfection cells were harvested. Dex (1 μm) was added for the last 5 h and TPA (100 ng/ml) was added for the last 30 min before lysis in Nonidet P-40 buffer.

RNA Interference

Double-stranded siRNA oligonucleotides targeting MKP-1 (5′-GGACAUGCUGGAUGCCUUTT-3′; 5′-AAGGCAUCCAGCAUGUCCTT-3′) were synthesized based on the sequences kindly provided by Dr. Ellis R. Levin, Division of Endocrinology, Long Beach Veterans Affairs Medical Center, Long Beach, CA (28). Scrambled RNA of the inverse sequence served as negative control. siRNA was introduced into cells by the scrape loading technique (54). Briefly, cells were grown to 30% confluence in 30-mm dishes and then provided with fresh serum-containing medium. siRNA was added to the dish to a final concentration of 100 nm and equally distributed by rocking the plate. Cells were induced to internalize siRNA by being scraped off the plate with a cell scraper, using a rotating movement, and then allowed to reattach to the same plate under standard culture conditions. After 24 h medium was replaced with fresh DMEM supplemented with 10% FCS. Cells were harvested 48 h after transfection. Dex (1 μm) was added for the last 5 h of incubation, and TPA (100 ng/ml) was added 30 min before lysis in NP-40 buffer. MKP-1 expression and ERK phosphorylation were monitored by Western blot.

Statistical Analysis

All results are from multiple experimental replicates as indicated in the figure legends and are presented as the mean ± sd. Data were analyzed using GraphPad Prism version 2.01 (GraphPad Software, Inc., San Diego, CA; 50 μg protein of phosphatase lysate) by one-way ANOVA with Tukey's post hoc test for multigroup comparisons or paired t test as appropriate. Differences were considered statistically significant at P < 0.05.

Supplementary Material

S1

Acknowledgments

Sisters Dorothy Steyn and Zelda Williams, Lung Unit, Tygerberg Hospital, are gratefully acknowledged for their coordination and collection of patient blood samples.

This work was supported by grants from the South African Medical Research Council (to P.A.H. and F.S.H.), South African National Research Foundation GUN 2047295 (to P.A.H.), and the Wellcome Trust Collaborative Research Initiative Grant 064335 (to F.S.H., J.M.B., J.C., and P.A.H.).

A.C.B.C. consults for Bayer Schering Pharma and received grant support from Schering AG (1999–2001), Boehringer Ingelheim Pharma AG (2000–2001), and ByK Gulden (1998–1999). J.C. has received lecture fees from Amgen and Shire and advised Amgen, Genzyme, Shire, Abbott, and Ineos. F.S.H. has received consulting/advisory and lecture fees.

Abbreviations

aa

Amino acid

Dex

dexamethasone

DUSP

dual-specificity phosphatase

FCS

fetal calf serum

GC

glucocorticoid

GR

glucocorticoid receptor

JNK

c-jun N-terminal kinase

MKP

MAPK phosphatase

MEK

MAPK/ERK kinase

NP-40

Nonidet P-40

PTP

protein tyrosine phosphatase

siRNA

short interference RNA

TPA

O-tetradecanoylphorbol 13-acetate

Footnotes

Disclosure Statement: K.H., H.d.W., M.M.S., F.A.R., J.M.B., and P.A.H. have nothing to disclose.

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