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. 2011 Nov;211(2):145-56.
doi: 10.1530/JOE-11-0144. Epub 2011 Aug 18.

Mitogen-activated protein kinase phosphatase 1 regulates bone mass, osteoblast gene expression, and responsiveness to parathyroid hormone

Chandrika D Mahalingam  1 , Tanuka Datta, Rashmi V Patil, Jaclynn Kreider, R Daniel Bonfil, Keith L Kirkwood, Steven A Goldstein, Abdul B Abou-Samra, Nabanita S Datta
Affiliations

Mitogen-activated protein kinase phosphatase 1 regulates bone mass, osteoblast gene expression, and responsiveness to parathyroid hormone

Chandrika D Mahalingam et al. J Endocrinol. 2011 Nov.

Abstract

Parathyroid hormone (PTH) signaling via PTH 1 receptor (PTH1R) involves mitogen-activated protein kinase (MAPK) pathways. MAPK phosphatase 1 (MKP1) dephosphorylates and inactivates MAPKs in osteoblasts, the bone-forming cells. We previously showed that PTH1R activation in differentiated osteoblasts upregulates MKP1 and downregulates pERK1/2-MAPK and cyclin D1. In this study, we evaluated the skeletal phenotype of Mkp1 knockout (KO) mice and the effects of PTH in vivo and in vitro. Microcomputed tomography analysis of proximal tibiae and distal femora from 12-week-old Mkp1 KO female mice revealed osteopenic phenotype with significant reduction (8-46%) in bone parameters compared with wild-type (WT) controls. Histomorphometric analysis showed decreased trabecular bone area in KO females. Levels of serum osteocalcin (OCN) were lower and serum tartrate-resistant acid phosphatase 5b (TRAP5b) was higher in KO animals. Treatment of neonatal mice with hPTH (1-34) for 3 weeks showed attenuated anabolic responses in the distal femora of KO mice compared with WT mice. Primary osteoblasts derived from KO mice displayed delayed differentiation determined by alkaline phosphatase activity, and reduced expressions of Ocn and Runx2 genes associated with osteoblast maturation and function. Cells from KO females exhibited attenuated PTH response in mineralized nodule formation in vitro. Remarkably, this observation was correlated with decreased PTH response of matrix Gla protein expression. Expressions of pERK1/2 and cyclin D1 were inhibited dramatically by PTH in differentiated osteoblasts from WT mice but much less in osteoblasts from Mkp1 KO mice. In conclusion, MKP1 is important for bone homeostasis, osteoblast differentiation and skeletal responsiveness to PTH.

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Conflict of interest statement

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.

Figures

Figure 1
Figure 1
Mkp1 deletion results in decreased bone phenotype of female mice. (A) Representative 3D microCT images from 12-week-old wild-type (WT) and Mkp1 knockout (KO) mice tibiae and femora are shown. Mkp1 KO females show osteopenia compared with WT controls. (B) Histomorphometric analysis, (C) serum osteocalcin (OCN), and (D) serum TRAP5b assays were performed and plotted as mean±S.E.M. (n = 4–5). b, P<0.01 and c, P<0.05 versus respective WT control.
Figure 2
Figure 2
Mkp1 deletion attenuates PTH anabolic response in growing female mice. (A) Representative microCT images of distal femoral trabecular bone from 3-week-old wild-type (WT) and Mkp1 knockout (KO) female mice with vehicle (0 9% saline) and PTH treatment. (B) Bone volume/total volume (BV/TV), (C) bone surface/bone volume (BS/BV), (D) trabecular thickness (Tb.Th), (E) trabecular number (Tb.N), and (F) trabecular spacing (Tb.Sp) were analyzed and plotted as fold changes with respect to saline-treated WT control group. The values for saline-treated WT control are BV/TV (%), 0 235±0 031; BS/BV (mm/mm2), 64 75±3 40; Tb.Th (μm), 0 033±0 005; Tb.N (1/mm), 7 45±0.61; Tb.Sp (mm), 0.118±0.017. All data are expressed as mean±S.D. (n=4–5). a, P<0.001; b, P<0.01; and c, P<0.05 versus respective saline control; # P<0 01 versus WT saline.
Figure 3
Figure 3
Altered osteoblast differentiation in calvarial cells from Mkp1 knockout (KO) female mice with and without PTH treatment. Cells were isolated from calvaria of 4–5-week-old WT and Mkp1 KO mice. The cells were differentiated with ascorbic acid and β-glycerophosphate. (A) Pth1R mRNA expression in differentiated primary calvarial osteoblasts derived from WT and Mkp1 KO mice at day 7 with and without 100 nM PTH treatment. Total RNA was isolated from triplicate independent cultures. Results are graphically represented after normalization with Gapdh as fold change (mean±S.D.) with respect to PTH untreated differentiated cells from WT cultures. (B) Histochemical staining of ALKP. Representative wells of 4–6 individual experiments with similar results for ALKP staining at day 5, with or without 100 nM PTH, are shown. (C) Time course of ALK activity in primary calvarial osteoblasts with or without PTH treatment. Results are expressed as fold change with respect to undifferentiated wild-type (WT) control cells at day 3. The basal ALKP activity for undifferentiated WT control cells at day 3 is 1 15±0 18 units/mg protein per min. (D) Time course of Alkp mRNA expression in primary calvarial osteoblasts with or without PTH treatment. Total RNA was isolated from triplicate independent cultures from day 5 to day 15. Results are graphically represented after normalization with Gapdh as fold change with respect to undifferentiated wild-type (WT) control cells at day 5. UD, undifferentiated cells; D, differentiated cells. Values are expressed as mean±S.D. from 4 to 6 individual experiments. a, P<0 001 versus UD; c, P<0 05 versus UD; *P<0 01 versus D without PTH, # P<0 001 versus WT-D cells at same time point.
Figure 4
Figure 4
Effects of Mkp1 deletion on osteoblast mineralization and MGP mRNA expression with or without PTH treatment. Cells were isolated from calvaria of 11-week-old wild-type (WT) and Mkp1 knockout (KO) mice. The cells were differentiated with ascorbic acid and β-glycerophosphate for 21 days with or without 100 nM PTH. Mineralized nodule formation was examined by von Kossa staining as described in the Materials and Methods section. (A) Representative wells of 5–7 individual experiments with similar results for nodule formation are shown. The number of nodules was counted and plotted as percentage (%) expression of nodules with respect to PTH untreated WT cells from 5 to 6 individual experiments. Values are expressed as mean±S.D. a, P<0 001 versus untreated cells; b, P<0 01 versus untreated cells; c, P<0 01 versus untreated WT control cells; # P<0 001 versus PTH-treated WT control cells. The number of mineralized nodules in osteoblasts derived from KO animals without PTH treatment was 30% lower compared with PTH untreated WT cultures. (B) Mgp expression in primary calvarial osteoblasts derived from WT and Mkp1 KO mice at day 15 with and without PTH treatment. Total RNA was isolated from triplicate independent cultures. Results are graphically represented after normalization with Gapdh as fold change (mean±S.D.) with respect to PTH untreated differentiated cells from WT cultures. D, differentiated cells; a, P<0 001 versus D.
Figure 5
Figure 5
Temporal expressions of osteoblast differentiation marker genes from 11-week-old female wild-type (WT) and Mkp1 knockout (KO) mice. Time course of (A) osteocalcin (Ocn), (B) osteonectin (On), and (C) Runx2 mRNA expressions in primary calvarial osteoblasts, with or without PTH treatment. Total RNA was isolated from triplicate independent cultures from day 5 to day 15. Results are graphically represented after normalization with Gapdh as fold change (mean±S.D.) with respect to undifferentiated cells at day 7 (A) or day 5 (B and C) from WT control. UD, undifferentiated cells; D, differentiated cells; a, P<0 001 versus UD; b, P<0 01 versus UD; # P<0 05 versus WT-D cells at the same time point; *P<0 05 versus WT-D without PTH; **P<0 02 versus KO-D without PTH.
Figure 6
Figure 6
Effect of Mkp1 deletion on PTH-mediated pERK1/2 and cyclin D1 expression in osteoblasts derived from wild-type (WT) and Mkp1 knockout (KO) female mice. Seven days differentiated calvarial osteoblasts from either WT or KO female mice were treated with 100 nM PTH (P) or vehicle (V) for (A) 10 min, (B) 5 h, or (C) 1 h. Total cellular protein or RNA was harvested. Western blot analyses were performed for (A) pERK1/2 and total ERK and (B) cyclin D1 and actin. Total ERK and actin were used as loading controls. Densitometric values were normalized and plotted as fold change with respect to WT-V treated cells. (C) Real-time PCR analysis was performed for cyclin D1 mRNA, normalized with Gapdh as described in the Materials and Methods section and plotted as fold changes with respect to WT-V treated cells. Representative data from at least 3 to 4 independent experiments are shown. Data are expressed as mean±S.D. a, P<0 001 versus V; b, P<0 05 versus V; # P<0 05 versus WT-V.
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References

    1. Aghaloo TL, Pirih FQ, Shi A, Bezouglaia O, Tetradis S. Parathyroid hormone induces mitogen-activated kinase phosphatase 1 in murine osteoblasts primarily through cAMP-protein kinase A signaling. Journal of Periodontology. 2006;77:21–30. doi: 10.1902/jop.2006.77.1.21. - DOI - PubMed
    1. Alves de Oliveira EC, Szejnfeld VL, Pereira da Silva N, Coelho Andrade LE, Heldan de Moura Castro C. Intermittent PTH1–34 causes DNA and chromosome breaks in osteoblastic and nonosteoblastic cells. Calcified Tissue International. 2010;87:424–436. doi: 10.1007/s00223-010-9396-6. - DOI - PubMed
    1. Andreassen TT, Fledelius C, Ejersted C, Oxlund H. Increases in callus formation and mechanical strength of healing fractures in old rats treated with parathyroid hormone. Acta Orthopaedica Scandinavica. 2001;72:304–307. doi: 10.1080/00016470152846673. - DOI - PubMed
    1. Beamer WG, Donahue LR, Rosen CJ. Genetics and bone. Using the mouse to understand man. Journal of Musculoskeletal & Neuronal Interactions. 2002;2:225–231. - PubMed
    1. Beamer WG, Shultz KL, Ackert-Bicknell CL, Horton LG, Delahunty KM, Coombs HF, III, Donahue LR, Canalis E, Rosen CJ. Genetic dissection of mouse distal chromosome 1 reveals three linked BMD QTLs with sex-dependent regulation of bone phenotypes. Journal of Bone and Mineral Research. 2007;22:1187–1196. doi: 10.1359/jbmr.070419. - DOI - PubMed

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