Abstract
Limited information is available on the role of MAPK phosphatase 1 (MKP1) signaling in osteoblasts. We have recently reported distinct roles for MKP1 during osteoblast proliferation, differentiation, and skeletal responsiveness to parathyroid hormone (PTH). As MKP1 regulates the phosphorylation status of MAPKs, we investigated the involvement of P-ERK and P-p38 MAPKs in MKP1 knockout (KO) early and mature osteoblasts with respect to mineralization and PTH response. Calvarial osteoblasts from 9–14-week-old WT and MKP1 KO male and female mice were examined. Western blot analysis revealed downregulation and sustained expressions of P-ERK and P-p38 with PTH treatment in differentiated osteoblasts derived from KO males and females respectively. Exposure of early osteoblasts to p38 inhibitor, SB203580 (S), markedly inhibited mineralization in WT and KO osteoblasts from both genders as determined by von Kossa assay. In osteoblasts from males, ERK inhibitor U0126 (U), not p38 inhibitor (S), prevented the inhibitory effects of PTH on mineralization in early or mature osteoblasts. In osteoblasts from KO females, PTH sustained mineralization in early osteoblasts and decreased mineralization in mature cells. This effect of PTH was attenuated by S in early osteoblasts and by U in mature KO cells. Changes in matrix Gla protein expression with PTH in KO osteoblasts did not correlate with mineralization, indicative of MKP1-dependent additional mechanisms essential for PTH action on osteoblast mineralization. We conclude that PTH regulation of osteoblast mineralization in female mice is maturation stage specific and involves MKP1 modulation of P-ERK and P-p38 MAPKs.
Introduction
Bone homeostasis is maintained by a balance between bone-building osteoblasts and bone-resorbing osteoclasts, a process known as bone remodeling (Feng & McDonald 2011, Kular et al. 2012). Age-related bone loss is associated with changes in bone remodeling characterized by decreased bone formation relative to bone resorption, resulting in bone fragility, increased risk of fractures, and osteoporosis (Khosla & Riggs 2005, Raisz 2005, Feng & McDonald 2011). Bisphosphonates are widely prescribed anti-resorptive agents that inhibit osteoclast attachment to bone matrix and enhance osteoclast apoptosis (Dominguez et al. 2011). However, there are risks associated with bisphosphonate therapy (Khosla et al. 2012). A new therapeutic approach for treating osteoporosis includes anabolic agents that promote osteoblastic differentiation leading to increased bone formation. Daily injection of parathyroid hormone (PTH), PTH (1–34), has been shown to be anabolic (Greenfield 2012) and increases bone mass through the stimulation of trabecular and cortical bone formation and decreases fracture incidences in severe cases of osteoporosis (for review, see Datta (2011)). However, the therapeutic use of PTH is limited by the principal side effects of hypercalcemia (Miller et al. 2007) and possible bone cancer concerns (Cappuzzo & Delafuente 2004).
As osteoporosis is a disorder of remodeling in which bone resorption outstrips formation, any insights into pathways that control bone formation and PTH molecular action have potential therapeutic implications. PTH plays important roles at multiple stages during bone formation having both anabolic and catabolic effects in bone (Qin et al. 2004, Silva et al. 2011). The anabolic actions of PTH involve direct effects on osteoblasts such as cell survival (Manolagas 2000), osteoblast proliferation, differentiation (Nishida et al. 1994, Datta et al. 2005, 2007, 2010a), and mature osteoblast function (Qin et al. 2004). The indirect effects include activation of skeletal growth factors (Lombardi et al. 2010, Bikle & Wang 2012, Fei & Hurley 2012) and inhibition of growth factor antagonists, such as sclerostin (Yu et al. 2011). The mediators of PTH intracellular signaling pathways are not well defined relative to specific actions. PTH regulates bone remodeling by activating distinct signaling pathways including MAPK pathways in osteoblasts via PTH receptor-1 (PTH1R; for review, see Datta & Abou-Samra (2009)). MAPK cascades are conserved signal transduction pathways that are regulated in response to diverse stimuli and mediate physiological responses including cell proliferation, differentiation, and transformation (Dong et al. 2002, Wada & Penninger 2004). MAPK signaling is both diverse and complex and dependent on a balance between activation by kinases and inactivation by MAPK phosphatases (MKP; Owens & Keyse 2007). It has been suggested that signaling through multiple MAPK pathways may be integrated at the level of regulation by MKPs (Owens & Keyse 2007). We have previously demonstrated that PTH and PTH-related peptide regulate osteoblast cell cycle by regulating the expressions of cyclin D1, CDK1, and JunB and the activity of ERK–MAPK (Datta et al. 2005, 2007, 2010b, 2012). Recent observations by us and others implicating MKP1 in maintaining bone homeostasis and in PTH regulation brings the field one step closer to finding another possible treatment (Carlson et al. 2009, Datta et al. 2010a, Mahalingam et al. 2011).
Bone mineralization is an essential process that improves hardness and strength of the skeleton of the vertebrates. Osteoblasts pass through a series of stages before reaching maturity and bone matrix mineralization. Differentiation of osteoblasts in vitro and in vivo can be characterized in three stages: i) cell proliferation, ii) matrix maturation, and iii) matrix mineralization (Stein & Lian 1993). The mineralization process of osteoblasts in in vitro culture has been used as a model for testing PTH regulation on bone cell differentiation and bone formation (Kostenuik et al. 1999). In this paper, we explored the involvement of MKP1 in ERK- and p38 MAPK-mediated osteoblast mineralization and PTH action by studying primary osteoblasts derived from MKP1 knockout (KO) mice. The role of the MKP1–MAPK pathway in osteoblast proliferation and differentiation may depend on the maturation stage in which PTH signaling is studied (Datta et al. 2005, 2007). It is therefore important to consider the precise stage of osteoblast maturation in which MKP1 signaling is presented. Here, we show that osteoblast mineralization requires MKP1 and P-p38-MAPK depending on the maturation stages of the osteoblasts. Our data support the notion that MKP1 in osteoblasts from females plays a key role in mineral homeostasis predominantly mediated by p38 MAPK. PTH signal transduction differs from early to late osteoblasts, targeting both P-ERK and P-p38-MAPK pathways. Furthermore, these data suggest a disparate PTH regulation of osteoblast mineralization in male and female MKP1 KO mice.
Materials and methods
Animals
MKP1 KO mice were obtained through a Material Transfer Agreement from Bristol-Myers Squibb as previously described (Dorfman et al. 1996, Zhao et al. 2005, Mahalingam et al. 2011). These mice, containing a disruption within exon 2 of MKP1, were bred in-house either intercrossing heterozygous or homozygous KO breeders to yield both WT and KO mice. The experiments in this study were performed with 9–14-week-old WT and KO male and female mice fed with rodent chow (Lab diet, Brentwood, MD, USA). For genotyping, real-time PCR analysis was performed by Transnetyx (Cordova, TN, USA) (Mahalingam et al. 2011). All animals were maintained in facilities operated by Wayne State University, and all animal experimental procedures were approved by the Institutional Animal Care and Use Committee for the Use and Care of Animals (IACUC).
Antibodies and reagents
Antibodies to phospho-ERK, total ERK, and phospho-p38 were obtained from Cell Signaling (Beverly, MA, USA). Cyclin D1 was from Santa Cruz Biotechnology and glyceraldehyde 3-phospho-dehydrogenase (GAPDH) was from Sigma. Secondary antibody HRP conjugates to rabbit or mouse immunoglobulins were obtained from GE Healthcare Life Sciences (Pittsburg, PA, USA). Tissue culture medium and fetal bovine serum (FBS) were from Invitrogen. Human PTH (PTH1-34) was purchased from Bachem (Torrance, CA, USA). U0126, the MEK inhibitor, was from Promega and SB203580, the phospho-p38 inhibitor, was obtained from EMD Biosciences (Darmstadt, Germany). 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was from Sigma–Aldrich.
Primary osteoblast cell culture
Primary osteoblasts were isolated from calvaria by serial digestion (Datta et al. 2005). Briefly, calvaria were dissected, isolated, and subjected to sequential digestions in collagenase A (2 mg/ml) and trypsin (0.25%) for 20, 40, and 90 min. Cells from the third digest were rinsed, counted, and plated in primary culture medium containing αMEM, 10% FBS, 100 U/ml penicillin, and 1 μg/ml streptomycin.
Cell proliferation
Cell proliferation was determined by plating primary osteoblasts at first passage at a density of 10 000 cells/well in 24-well plates. Media were changed every other day until cell enumeration. Cell-doubling was calculated either from hemocytometer counts using the trypan blue exclusion method (not shown) or by the MTT reduction to formazan in living cells as a measure of mitochondrial activity. PBS solution of MTT (0.5 mg/ml) was incubated with cells in 24-well plates for 4 h. The resulting formazan crystals were dissolved by addition of 500 μl isopropyl alcohol to each well and optical density read at a wavelength of 595 nm.
Osteoblast differentiation, SDS–PAGE, and western blot analysis
Calvarial osteoblasts were differentiated for 7 days with ascorbic acid (50 μg/ml) and β-glycerophosphate (10 mM) and induced with 100 nM PTH for 10 min to 5 h for subsequent experiments. SDS–PAGE was performed in 10–12% polyacrylamide and western blot analysis was performed as described previously (Datta et al. 2005). Each lane was loaded with 40–80 μg protein of cell lysates. Prestained molecular weight standards were run in parallel lanes. After electrophoresis, the proteins were transferred to a PVDF membrane (Bio-Rad Laboratories) in 25 mM Tris–HCl, 192 mM glycine, 20% v/v methanol, and 0.01% SDS (pH 8.5) using a semi-dry transfer system (Hoeffer, Holliston, MA, USA). Residual protein binding sites on the membrane were blocked by TBST (20 mM Tris–HCl, pH 7.6, 137 mM NaCl, and 0.5% Tween 20) containing 5% nonfat dry milk for 1 h at room temperature. The membranes were then incubated with the primary antiserum for either 1–2 h at room temperature or overnight at 4 °C. After washing with TBST, a HRP-conjugated secondary antibody was added for 20–60 min. The protein bands were visualized by autoradiography using an ECL detection system (Pierce, Rockford, IL, USA). The protein band intensities on the autoradiograms (all with exposures within the linear range of the film) were quantified using Scion software (Frederick, MD, USA).
Osteoblast differentiation and PTH, U0126, and SB203580 administrations
Freshly isolated calvarial osteoblasts were plated at a density of 200 000 cells/well in six-well plates and allowed cell attachment onto culture dishes for 3 days. After removal of nonadherent cells, primary osteoblasts were subjected to differentiation, as earlier, in osteogenic medium containing ascorbic acid (50 μg/ml) and β-glycerophosphate (10 mM). Two treatment plans were initiated as follows.
Plan 1
Adherent early osteoblasts were exposed to U0126 (20 μM), or SB203580 (20 μM), either alone or in the presence of PTH (1–34) (100 nM) from day 3 until days 18–20 in differentiation medium. Media with the inhibitors and/or PTH were replaced every other day.
Plan 2
Adherent osteoblasts were exposed to differentiation medium without inhibitors or PTH for 7 days from days 3 to day 10. At day 10, differentiated osteoblasts were exposed to U0126 (20 μM) or SB203580 (20 μM), either alone or in the presence of PTH (1–34) (100 nM) until days 20–23 in differentiation medium as earlier. Media with the inhibitors and/or PTH were replaced every other day.
Osteoblast mineralization
The mineralization assays were performed using the von Kossa method as described previously (Mahalingam et al. 2011). At the end of the culture period, the cells were fixed with phosphate buffer formalin, 95% EtOH, and stained with AgNO3 to detect phosphate deposits in bone nodules as described (Marsh et al. 1995). The calcium phosphate deposits were observed by the formation of opaque mineralized nodules. The numbers of nodules were counted and plotted as percent relative expression compared to untreated WT cells. Undifferentiated osteoblasts were used as a negative control.
Analysis of mRNA by real-time PCR
mRNA isolation of cells were performed with TRIZOL reagent (Invitrogen), and cDNAs were prepared using the TaqMan Reverse Transcription assay system (Applied Biosystems) as described (Mahalingam et al. 2011). Real-time PCR was performed using StepOne Plus real-time PCR system (Applied Biosystems) with FAM-labeled primer assay systems (matrix Gla protein, MGP #Mm00485009, GAPDH # 99999915) from Applied Biosystems. GAPDH was used as an internal control.
Statistical analysis
All values were expressed as mean±s.e.m. Comparisons between groups were made by either unpaired two-tailed Student's t-test or ANOVA with the Instat Biostatistics program (GraphPad Software, San Diego, CA, USA) to determine significance between groups. P values <0.05 were considered statistically significant.
Results
Increased proliferation of calvarial osteoblasts derived from MKP1 KO mice
To determine the role of MKP1 on the growth of primary calvarial osteoblasts, cells were derived from 9–10-week-old WT and MKP1 KO male and female mice. Cell proliferation was examined by the MTT assay. The MTT reduction activity was increased (∼1.5- to 2-fold) in KO cells from both sexes compared with respective WT controls, suggesting increased proliferation in KO osteoblasts (Fig. 1A and B). Proliferation of osteoblasts derived from male mice linearly increased during the first 3 days and then plateaued between days 4 and 10. The KO osteoblasts reached a significantly higher plateau (∼1.2- to 1.3-fold) at days 4 and 5 compared with WT cells (Fig. 1A). By contrast, proliferation of cells derived from KO female mice increased in a linear fashion up to day 10 (Fig. 1B). The KO osteoblasts had a significantly higher (∼1.2- to 1.8-fold) MTT reduction activity between days 2 and 10 compared with WT controls (Fig. 1B). Similar to osteoblasts from WT and KO males, changes in the number of osteoblasts from WT females beyond day 4 were not statistically significant. These results are consistent with our recent finding that loss of MKP1 delays or attenuates osteoblast differentiation in terms of activity and expressions of alkaline phosphatase (ALKP), OCN, ON, and Runx2 (Mahalingam et al. 2011) when compared with osteoblasts from WT females. We have also reported that PTH induces P-ERK and cyclin D1 in proliferating osteoblastic cells (Datta et al. 2007). We hypothesized that increased P-ERK upregulates cyclin D1 and stimulates osteoblast proliferation. As expected, in this study, the degree of proliferation in the MKP1 KO osteoblasts (where ERK remains phosphorylated and continued to be active) was not diminished following PTH administration (data not shown).
Analysis of MKP1 KO osteoblast proliferation in vitro. Calvarial osteoblasts were derived from 9–10-week-old (A) male and (B) female WT and MKP1 KO mice. Cell proliferation was assayed by MTT reduction activity as described in Materials and methods section. Data from three to four independent experiments are plotted. Values are mean±s.e.m. (n=2–3 for male and female WT and KO mice in each independent experiment), bP<0.02; cP<0.05.
Citation: Journal of Endocrinology 216, 3; 10.1530/JOE-12-0372
Effect of MKP1 deletion on cyclin D1, MAPK expression, and PTH regulation
Our previous studies reported that PTH1R activation downregulates P-ERK1/2 and cyclin D1 in differentiated MC3T3-E1 osteoblastic cells (Chen et al. 2004, Datta et al. 2005), and this effect was attenuated in the absence of MKP1 in primary osteoblasts derived from female mice (Mahalingam et al. 2011). In this study, we examined the effects of PTH on P-p38, P-ERK, and cyclin D1 in 7-day differentiated MKP1 KO primary osteoblasts derived from both male and female mice. In osteoblasts derived from male mice, PTH decreased (∼40–60%) P-ERK and P-p38 expression similarly in both WT and KO osteoblasts (Fig. 2A). On the other hand, in osteoblasts derived from female mice, the effects of PTH on P-ERK and P-p38 expression were attenuated in MKP1 KO osteoblasts when compared with WT cells (Fig. 2B), similar to our previous report (Mahalingam et al. 2011). PTH decreased (70–80%) cyclin D1 protein expression in osteoblasts derived from both WT and MKP1 KO male mice (Fig. 2A). By contrast, the effect of PTH on cyclin D1 protein expression was attenuated in cells derived from female KO animals when compared with WT controls (Fig. 2B), similar to our previous observation (Mahalingam et al. 2011). These studies reveal a distinct molecular role for MKP1 in PTH signaling in differentiated osteoblasts derived from male and female mice.
Effect of PTH on P-ERK, P-p38 MAPK, and cyclin D1 in MKP1 KO osteoblasts. Seven-day differentiated calvarial osteoblasts from 12–14-week-old WT and KO (A) male and (B) female mice were treated with 100 nM PTH (P) or vehicle (V) for either 10 min (P-ERK), 1–2 h (P-p38), or 5 h (cyclin D1). Total cellular protein was harvested. Western blot analyses were performed for P-ERK1/2, total ERK, P-p38, cyclin D1, and GAPDH. Total ERK and GAPDH were used as loading controls. Densitometric values were normalized and plotted as fold changes with respect to WT-V-treated cells as described in Materials and methods section. Representative data from at least three to four independent experiments are shown. Data are expressed as mean±s.e.m. from three to four independent experiments (n=2–3 for male and female WT and KO mice in each experiment). aP<0.001 vs V; bP<0.02 vs V; cP<0.05 vs V; eP<0.05 vs WT-V; fP<0.05 vs WT-P.
Citation: Journal of Endocrinology 216, 3; 10.1530/JOE-12-0372
Disparate roles for MKP1 in PTH-regulated bone matrix mineralization of osteoblast cultures derived from male and female mice
Continuous PTH administration inhibits osteoblast mineralization in vitro (Gopalakrishnan et al. 2005) and regulates matrix Gla protein (MGP), an inhibitor of mineralization (Gopalakrishnan et al. 2001). To evaluate the effects of MKP1 deletion on in vitro osteoblastogenesis and cell maturation, calvarial osteoblasts from 12–14-week-old male and female WT and KO mice following isolation and cell attachment were differentiated in the presence or absence of PTH and mineralized nodule formation was assessed by von Kossa staining. In osteoblasts derived from male mice, PTH dramatically decreased mineralization and increased Mgp mRNA to similar degrees (90–95%) in WT and KO cells (Fig. 3A). By contrast, osteoblasts derived from female mice PTH inhibited mineralization by only 15–20% in MKP1 KO cells and had no effect on Mgp mRNA expression (Fig. 3B; Mahalingam et al. 2011). The effects of PTH on WT osteoblasts from females were similar to those derived from males (90–95% inhibition of mineralization and several fold stimulation of Mgp mRNA expression, Fig. 3A and B).
Effect of PTH on MKP1 KO osteoblast mineralization and MGP expression: Osteoblasts were isolated from calvaria of 12–14-week-old WT and MKP1 KO (A) male and (B) female mice. After cell attachment, differentiation of the cells was initiated with ascorbic acid and β-glycerophosphate with or without 100 nM PTH for 15–21 days. Mineralized nodule formation was examined by von Kossa staining as described in Materials and methods section. Representative wells from six to eight individual experiments with similar results for nodule formation are shown. The number of nodules was counted and plotted as % expression of nodules with respect to PTH-untreated WT cells. Total RNA was isolated from triplicate independent cultures at day 15 with and without PTH treatment, and Mgp mRNA expression was quantified in (A) male and (B) female WT and KO osteoblasts by real-time PCR analysis as described in Materials and methods section. Results are graphically represented after normalization with GAPDH as fold change with respect to PTH-untreated differentiated cells from WT cultures. Values are expressed as mean±s.e.m. from six to eight independent experiments (n=2–3 for male and female WT and KO mice in each experiment). aP<0.001 vs untreated cells; bP<0.05 vs untreated cells; cP<0.05 vs untreated WT control cells; dP<0.001 vs PTH treated WT control cells. NC, negative control, undifferentiated osteoblasts.
Citation: Journal of Endocrinology 216, 3; 10.1530/JOE-12-0372
Roles of MKP1, P-p38, and P-ERK signaling in PTH effect on mineralization in early vs mature osteoblasts
Our previous studies suggested that MKP1 regulates osteoblast proliferation/differentiation and mineralization (Datta et al. 2010a, Mahalingam et al. 2011). As MKP1 regulates P-ERK and P-p38, we examined the involvement of ERK and p38 MAPKs in PTH–MKP1 signaling. WT and KO osteoblasts were treated with PTH in the presence or absence of pharmacological inhibitors during differentiation. In this study, the specific inhibitors were titrated in WT osteoblasts for maximal suppression of P-ERK and P-p38 (not shown), and for least nonspecific toxic effects (Xiao et al. 2002). We designed our experiments with two different treatment plans (Fig. 4). In treatment plan 1, following primary calvarial osteoblast isolation and cell attachment onto culture dishes, differentiation was initiated at day 3 and specific inhibitors for P-ERK and P-p38 with or without PTH were added until the end of experiment (Fig. 4A). In treatment plan 2, the cells were first allowed to differentiate from days 3 to 10 and then the respective inhibitors were added, with or without PTH, from day 10 until the end of experiment (Fig. 4B). Thus, treatment plan 1 examined the involvement of P-ERK and P-p38 in PTH–MKP1 signaling in early osteoblasts whereas plan 2 examined their effects on mature osteoblasts.
Treatment plans for primary osteoblast differentiation. Freshly isolated calvarial osteoblasts from male and female WT and KO mice were plated for 3 days for cell attachment. After removing the nonadherent cells, these were differentiated in osteogenic media and subjected to two different treatment plans as described in Materials and methods section. (A) In treatment plan 1, adherent early osteoblasts were exposed to 20 μM U0126, ERK–MAPK inhibitor or 20 μM SB203580, p38 MAPK inhibitor in the presence or absence of 100 nM PTH from day 3 until days 18–20 in differentiation media, and processed. Media with the inhibitors and/or PTH were replaced every other day. (B) In treatment plan 2, adherent cells were first allowed to differentiate for 7 days without inhibitors or PTH from day 3 to day 10. At day 10, the cells were exposed to inhibitors in the presence or absence of PTH, as in treatment plan 1, until days 18–20 in differentiation media and processed.
Citation: Journal of Endocrinology 216, 3; 10.1530/JOE-12-0372
Figure 5 shows the results in osteoblasts isolated from male WT (Fig. 5A) and KO (Fig. 5B) mice. The MEK/ERK inhibitor, U0126 (U), showed an increased tendency of mineralization compared with untreated differentiated cells (AB vs ABU), either in treatment plan 1 or in treatment plan 2 similarly in WT (Fig. 5A) and KO (Fig. 5B) osteoblasts. Also, the inhibitory effect of PTH was blocked in the presence of U equally in WT and KO cells (ABP vs ABUP, Fig. 5A and B, plans 1 and 2). By contrast, SB203580 (S), a p38/MAPK inhibitor, dramatically blocked basal mineralization by ∼70% and did not influence the inhibitory effect of PTH in early osteoblasts (Fig. 5A and B, plan 1). On the other hand, in the more mature cells, S had no effect on basal mineralization and attenuated the inhibitory effect of PTH in both WT and KO osteoblasts (ABP vs ABSP, Fig. 5A and B, plan 2). These results suggest MKP1-independent regulation of ERK/MAPK and p38/MAPK in osteoblasts from male mice.
Involvement of MKP1, ERK, and p38 MAPK in PTH action on matrix mineralization in early and mature osteoblasts derived from male mice. Mineralized nodule formation was examined in osteoblasts derived from 11–14-week-old male (A) WT and (B) KO mice. von Kossa assay of mineralization was performed in early (plan 1) and mature (plan 2) osteoblasts as described in Fig. 4. Representative wells from three to four individual experiments with similar results for nodule formation are shown. The number of nodules were counted and plotted as % expression of nodules with respect to PTH-untreated cells. AB, differentiated cells without PTH or inhibitors; ABP, differentiated cells with PTH (P); ABU, differentiated cells with U0126 (U), an ERK inhibitor; ABUP, differentiated cells with U and P; ABS, differentiated cells with SB203580 (S), a p38 inhibitor; ABSP, differentiated cells with S and P. Values are expressed as mean±s.e.m. from three to four independent experiments (n=2–3 for male WT and KO mice in each experiment). aP<0.001 vs untreated with or without inhibitor; bP<0.05 vs untreated with or without inhibitor; eP<0.05 vs untreated cells (AB); fP<0.05 vs PTH-treated cells (ABP).
Citation: Journal of Endocrinology 216, 3; 10.1530/JOE-12-0372
Figure 6 describes the results in osteoblasts derived from female WT (Fig. 6A) and KO (Fig. 6B) mice. In plan 1, osteoblasts from WT females showed similar results as observed in WT males. An increased tendency in basal mineralization in the presence of U and significant downregulation (∼50%) in the presence of S was noted (AB vs ABU or ABS, Fig. 6A, plan 1). Also, the inhibitory effect of PTH was blocked in the presence of U while presence of S did not influence PTH action (Fig. 6A, plan 1). By contrast, in MKP1 KO osteoblasts from females, PTH, as shown previously (Mahalingam et al. 2011), did not inhibit mineralization in KO osteoblasts in plan 1 (Fig. 6B). In these cells, U alone did not influence basal mineralization while S alone drastically diminished mineralization. Furthermore, the effect of PTH was blocked in the presence of S but not in the presence of U (Fig. 6B, plan 1). In more mature osteoblasts from WT females, effects of individual inhibitors or PTH alone showed only 15–20% inhibition. The inhibitory effect was not altered in the presence of inhibitors (Fig. 6A, plan 2). Conversely, in KO osteoblasts from females, PTH inhibited (80%) mineralization and this effect of PTH was blocked by U and not altered by S (Fig. 6B, plan 2). Taken together, these results suggested that p38 contribute to in vitro mineralization of osteoblasts. The disparate effect of PTH on osteoblast mineralization from female mice is MKP1 dependent involving maturation stage-specific P-ERK and P-p38 MAPKs.
Involvement of MKP1, ERK, and p38 MAPK in PTH action on matrix mineralization in early and mature osteoblasts derived from female mice. Mineralized nodule formation was examined in osteoblasts derived from 11–14-week-old female (A) WT and (B) KO mice. von Kossa assay of mineralization was performed in early (plan 1) and mature (plan 2) osteoblasts as described above in Fig. 4. Representative wells from three to four individual experiments with similar results for nodule formation are shown. The number of nodules were counted and plotted as % expression of nodules with respect to PTH-untreated cells. AB, differentiated cells without PTH (P) or inhibitors; ABP, differentiated cells with PTH (P); ABU, differentiated cells with U0126 (U), an ERK inhibitor; ABUP, differentiated cells with U and P; ABS, differentiated cells with SB203580 (S), a p38 inhibitor; ABSP, differentiated cells with S and P. Values are expressed as mean±s.e.m. from three to four independent experiments (n=2–3 for female WT and KO mice in each experiment). aP<0.001 vs untreated with or without inhibitor; bP<0.05 vs untreated with or without inhibitor; eP<0.05 vs untreated cells (AB); fP<0.001 vs PTH-treated cells (ABP).
Citation: Journal of Endocrinology 216, 3; 10.1530/JOE-12-0372
Roles of MKP1, P-p38, and P-ERK signaling on PTH regulation of Mgp expression in early vs mature osteoblasts
Studies shown in Fig. 7 examined the effect of PTH on MGP expression in the presence or absence of MAPK inhibitors in early and mature osteoblasts from WT and MKP1 KO male (Fig. 7A) and female (Fig. 7B) mice. PTH treatment of osteoblasts derived from WT and KO male mice upregulated (approximately threefold) Mgp mRNA similarly in plans 1 and 2 (Fig. 7A). MGP expression was increased 4.5-fold in the presence of ERK inhibitor, U, in early WT osteoblasts and the effect of PTH was further increased in the presence of U (Fig. 7A, plan 1). By contrast, the effect of PTH was blocked in the presence of U in KO osteoblasts (Fig. 7A, plan 1). In the presence of the p38 inhibitor, S, basal expression of MGP was increased 1.5- to 3-fold in WT and KO cells. Treatment with S blocked the effects of PTH on MGP expression in WT osteoblasts (Fig. 7A, plan 1). By contrast, S augmented (approximately twofold) the effects of PTH on MGP expression in KO cells (Fig. 7A, plan 1). In plan 2, in mature WT cells from males, a similar effect of PTH was observed in the presence or absence of U and the effect of PTH was augmented (∼1.5- to 2-fold) in the presence of S (Fig. 7A, plan 2). By contrast, in KO osteoblasts, the stimulatory effect of PTH was attenuated in the presence of U and remained unchanged in the presence of S (Fig. 7A, plan 2).
Involvement of MKP1, ERK, and p38 MAPK in PTH action on MGP expression in early and mature osteoblasts derived from male and female mice. Osteoblasts derived from 11–14-week-old (A) male and (B) female WT and KO mice were subjected to two treatment plans as described in Fig. 4. Total RNA was isolated from cultures at day 15 with and without inhibitors and/or PTH treatment, and Mgp mRNA expression was quantified by real-time PCR analysis as described in Materials and methods section. Results are graphically represented after normalization with GAPDH as fold change with respect to PTH-untreated differentiated cells. AB, differentiated cells without PTH (P) or inhibitors; ABP, differentiated cells with PTH (P); ABU, differentiated cells with U0126 (U), an ERK inhibitor; ABUP, differentiated cells with U and P; ABS, differentiated cells with SB203580 (S), a p38 inhibitor; ABSP, differentiated cells with S and P. Values are expressed as mean±s.e.m. from three independent experiments (n=2–3 for female WT and KO mice in each experiment). aP<0.001 vs untreated with or without inhibitor; bP<0.05 vs untreated with or without inhibitor; eP<0.01 vs untreated cells (AB); fP<0.05 vs PTH treated cells (ABP).
Citation: Journal of Endocrinology 216, 3; 10.1530/JOE-12-0372
In osteoblasts derived from female WT mice, PTH upregulated Mgp mRNA in early osteoblasts (Fig. 7B, plan 1) but had no effect in more mature cells (Fig. 7B, plan 2). On the other hand, consistent with our previous report (Mahalingam et al. 2011), PTH had no effect on MGP expression in MKP1 KO osteoblasts (Fig. 7B, plan 1 and plan 2). No significant effects of ERK inhibitor U were noted except that MGP expression was stimulated in the presence of U alone and PTH in the presence of U in mature KO osteoblasts (Fig. 7B, plan 2). Presence of p38 inhibitor, S, increased (twofold) the basal expression of MGP in treatment plan 2 for WT cells (Fig. 7B). PTH in the presence of S augmented (twofold) MGP expression in plan 1 (Fig. 7B), and no effect was observed in mature WT osteoblasts (Fig. 7B, plan 2). In the KO osteoblasts from females, S alone did not influence basal expression in early osteoblasts and the effect of PTH remained unchanged in the presence of S (plan 1). However, MGP expression was increased approximately four- to fivefold in the presence of S in mature KO cells. Although this effect was attenuated when cells were treated with PTH in the presence of S, MGP expression remained high compared with PTH alone (plan 2).
Discussion
In this study, we observed increased proliferation of calvarial osteoblasts obtained from either male or female MKP1 KO mice. This is consistent with a preliminary report using bone marrow stromal cells from male KO mice (Ma et al. 2009). Continued proliferation of KO osteoblasts from female mice beyond 10 days in culture when proliferation plateaued in osteoblasts from KO males suggested differences in MKP1 regulation and MAPK activation between cells from different genders. Sex-related differences in MAPK activation and cell proliferation/survival of rat astrocytes, rat cardiomyocytes, or mouse liver have been previously reported (Nuedling et al. 1999, Zhang et al. 2002, Vilatoba et al. 2005). Selective modulation of MAPKs was suggested to be involved in gender-based differences in cardiac diseases (Nuedling et al. 1999) and in cardioprotection (Vilatoba et al. 2005). It was also suggested that regulation of MAPKs may result in differential regulation of cell proliferation and death in astrocytes and possibly contributes to sexual dimorphisms in brain development (Zhang et al. 2002).
MAPKs regulate cyclin D1 (Terada et al. 1999), an unique regulator of cell cycle progression and cell proliferation (Sherr & Roberts 2004, Gladden & Diehl 2005, Musgrove 2006). The disparate regulation of P-ERK, P-p38 MAPKs, and cyclin D1 in male and female MKP1 KO osteoblasts in this study implies that cyclin D1 and other canonical regulators of the G1/S transition coordinate key players of cell cycle progression to achieve the sexually dimorphic cell division (Tilmann & Kimble 2005) and PTH response in osteoblasts. In spite of the very well-established role of cyclin D1-CDK4/6 in the cell cycle, cyclin D1 may perform CDK-independent functions by binding, activating, or repressing several transcription factors including DMP1 (Inoue & Sherr 1998), STAT3 (Bienvenu et al. 2001), and hormone receptors such as estrogen receptor (McMahon et al. 1999, Lamb et al. 2000), androgen receptor (Reutens et al. 2001, Petre et al. 2002), and thyroid hormone receptor (Lin et al. 2002) in a ligand-dependent or -independent manner. Among other transcription factors, Jun B, a member of the AP-1 family, regulates cell proliferation and differentiation (Piechaczyk & Farras 2008). Previous work by us demonstrated that posttranslational stabilization of Jun B by decreased phosphorylation leads to differential expression of cyclin D1 protein and growth arrest in MC3T3-E1 osteoblastic cells (Datta et al. 2005). Furthermore, novel function of JunB as a regulator of osteoclast and osteoblast activities in osteopenia has been reported (Kenner et al. 2004). Future studies involving receptors of estrogen, androgen, and/or PTH with AP-I transcription factors, MKP1, MAPKs, and cell cycle machinery in bone cells are necessary to substantiate our hypothesis on regulation by PTH.
Both magnitude and duration of MAPK activity are responsible for a number of physiological responses (Marshall 1995, Ebisuya et al. 2005). Using primary osteoblasts and pharmacological inhibitors, our studies demonstrate that p38 MAPK is required for in vitro matrix mineralization in both early and mature osteoblasts. This is in agreement with previous studies by Hu et al. (2003) using osteoblastic cells in vitro and with a recent report showing that cells derived from osteoblast-specific p38 KO mice have defective mineralization in vitro, reduced endocortical and trabecular bone formation, and decreased expression of osteoblast differentiation marker genes (Thouverey & Caverzasio 2012). We also observed a tendency toward increased osteoblast mineralization when P-ERK activation was blocked, irrespective of osteoblast differentiation stage. Interestingly, recent evidence suggests that chronic inhibition of ERK signaling decreases unmineralized osteoid volume and increased mineralization of trabecular and cortical bone in hypophosphatemic mice (Zhang et al. 2012). Collectively, these findings and our observation provide evidence that p38 and ERK MAPKs have opposite roles in osteoblast mineralization. Activation of p38 MAPK is critical for calcification of bone matrix from early to late stages of differentiation and inhibition of ERK–MAPK improves mineralization of osteoblasts.
Our novel findings include the dependency of MKP1 signaling on PTH regulation of primary osteoblast mineralization. Consistent with the report that continuous PTH administration inhibits mineralization of osteoblasts in vitro (Gopalakrishnan et al. 2005), we observed inhibition of mineralization with PTH in early and mature osteoblasts derived from WT or MKP1 KO males and this effect of PTH was dependent on ERK, not on p38. The inhibitory effect of PTH on osteoblast mineralization in WT females in early osteoblasts was not replicated when PTH treatment was initiated in more mature cells. Furthermore, we observe an opposite effect of PTH in osteoblasts derived from KO females showing continued mineralization in early osteoblast and decreased mineralization in more mature cells when compared with respective WT females. Taken together, these studies show that the effect of PTH on mineralization of osteoblasts from female mice is MKP1 dependent and involves p38 MAPK in early osteoblasts and ERK–MAPK in more mature cells. Thus, MKP1-dependent disparate regulation of PTH in female osteoblasts is maturation stage specific. The involvement of p38 MAPK in PTH regulation of mineralization (Rey et al. 2007) was only obvious in early osteoblasts derived from MKP1 KO females. We do not know the biological relevance of this disparate response at the present time. Perhaps PTH regulation of mineralization requires continuous presence of MKP1; thus, it could regulate MAPK pathway activities at various stages of differentiation. This is supported by the fact that PTH regulation of osteoblast differentiation and molecular action is maturation stage specific (Datta et al. 2005, 2007, 2010a).
Commitment to the mature osteoblast stage is confirmed by the upregulation of mineralization genes (Hartmann 2009). MGP is regulated by PTH, FGF, and ERK MAPK in osteoblasts (Gopalakrishnan et al. 2005, Khoshniat et al. 2011, Kyono et al. 2012). We hypothesized that modulation of MGP expression with PTH and/or MAPK inhibitors would correlate mineralization in WT and MKP1 KO osteoblasts at different stages of maturation. However, our data using the present experimental conditions did not show such a correlation. Differentiation-dependent hormonal regulation of MGP has been suggested during chondrogenesis and osteogenesis in rat (Barone et al. 1991). Two conclusions may be drawn from this study: first, critical changes at the molecular level occur at the earlier stages of the differentiation process, particularly in the presence or absence of MAPKs and MKP1, and these changes influence MGP expression without any association with mineralization. Secondly, although MGP is a major regulator of PTH inhibition of mineralization (Gopalakrishnan et al. 2005), MKP1, P-ERK, and p38 MAPKs govern this process via additional mechanisms unrelated to MGP. Collectively, our observations suggest that MKP1 signaling coordinately with cell maturation stage regulates mineralization in the osteoblast lineage and that MKP1, ERK, and p38 signaling is essential for PTH action on mineralization in addition to regulation of MGP expression.
At the present time, the physiological and pathological significance of the MKP1-dependent maturation stage-specific effect of PTH are not known. Timely activation/inactivation of MAPKs is essential for the generation of appropriate cellular outcomes and for restoration of responsiveness. Studies suggest that c-Raf regulates ERK activity at an earlier time point and MKP1 may contribute to the regulation over a prolonged period of time (Lai & Mitchell 2009). Thus, time course is critical in this regulation. The current study provides important evidence of sexual dimorphism even in vitro, indicating that MKP1 plays a relevant role in the maintenance of skeletal homeostasis in female mice. The mechanism by which gender influences cortical bone size may be overly complicated and is still a matter of investigation. Evidence shows that skeletal dimorphism is dependent on time-specific effects of sex steroids and GH–insulin-like growth factor 1 (GH–IGF1; Callewaert et al. 2010). Interaction of IGF1 and ERK (Perrini et al. 2008) may alter osteoblast proliferation and mineralization response. Sexually dimorphic behavior may therefore arise from finely tuned modifications at every level of different signaling cascades.
It should be pointed out that change in MKP1 expression (Datta et al. 2010a) is only one aspect of the highly complex osteoblast response to PTH. This response also involves the induction of several modulators of osteoblast signaling in addition to MKP1. However, these studies clearly suggest that MKP1 plays an important role during the differentiation process and matrix mineralization. We propose that PTH control of osteoblast mineralization involves more than one pathway, which may be MKP1-dependent ERK/p38 signaling, or MKP1-independent ERK/pp38 signaling (Fig. 8). At present, it is not known to what extent other phosphatases contribute to the osteoblast differentiation pathway and at what levels MKP1-mediated regulation of MAPKs influences osteoblast differentiation and mineralization gene expression.
Hypothetical responses to PTH signal and P-ERK-mediated and P-p38-mediated matrix mineralization of osteoblasts derived from female mice. Both MKP1-dependent and MKP1-independent responses of ERK/MAPK and p38/MAPK to PTH are proposed in early and late osteoblasts. Major responses are depicted by large arrows.
Citation: Journal of Endocrinology 216, 3; 10.1530/JOE-12-0372
MKPs differentially recognize, bind, and dephosphorylate MAPKs to integrate MAPK signaling (Owens & Keyse 2007). Some details of the molecular mechanism involved in MKPs and MAPKs in regulating specific functions, e.g. regulation of immune function is partially understood (Zhang et al. 2004, 2005, Chi et al. 2006). However, there remains considerable complexity despite the apparent specificity for ERK and p38 (Jeffrey et al. 2006). Considering the perplexity of this network, comprehensive understanding of MKPs function, in bone biology will require extensive future investigations. Nevertheless, in conclusion, our studies provide evidence that the MKP1-P-p38 and MKP1-P-ERK circuits play important roles in controlling the balance between osteoblast maturation, function/mineralization, and PTH signal transduction. Understanding the role of MKP1 in the anabolic activities of bone in female mice may lead to the identification of potentially novel therapeutic targets to reduce the risk of postmenopausal osteoporosis.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by funding from WSU OVPR and NIH DK087848 to N S D.
Acknowledgements
MKP1 KO mice were obtained through Material Transfer Agreement between Bristol-Myers Squibb and NSD (Wayne State University [WSU]). Acknowledgement is due to animal care facilities, Division of Laboratory and Animal Research, WSU, for taking care of our mice, and Alemu Fite for assistance with mice handling.
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