The ERK MAPK Pathway Is Essential for Skeletal Development and Homeostasis

Mitogen-activated protein kinases (MAPKs) are a family of protein kinases that function as key signal transducers of a wide spectrum of extracellular stimuli, including growth factors and pro-inflammatory cytokines. Dysregulation of the extracellular signal-regulated kinase (ERK) MAPK pathway is associated with human skeletal abnormalities including Noonan syndrome, neurofibromatosis type 1, and cardiofaciocutaneous syndrome. Here, we demonstrate that ERK activation in osteoprogenitors is required for bone formation during skeletal development and homeostasis. Deletion of Mek1 and Mek2, kinases upstream of ERK MAPK, in osteoprogenitors (Mek1OsxMek2−/−), resulted in severe osteopenia and cleidocranial dysplasia (CCD), similar to that seen in humans and mice with impaired RUNX2 function. Additionally, tamoxifen-induced deletion of Mek1 and Mek2 in osteoprogenitors in adult mice (Mek1Osx-ERTMek2−/−) significantly reduced bone mass. Mechanistically, this corresponded to decreased activation of osteoblast master regulators, including RUNX2, ATF4, and β-catenin. Finally, we identified potential regulators of osteoblast differentiation in the ERK MAPK pathway using unbiased phospho-mass spectrometry. These observations demonstrate essential roles of ERK activation in osteogenesis and bone formation.


Introduction
Homeostasis in the skeletal system is maintained by coupling between bone-forming osteoblasts and bone-resorbing osteoclasts through a process of continual remodeling. Regulation of the anabolic component of this homeostasis occurs through the controlled hierarchical differentiation of skeletal stem cells, osteoprogenitors, and mature osteoblasts [1]. A number of critical signaling pathways, such as transforming growth factor-beta (TGFβ)/bone morphogenic protein (BMP) signaling, wingless-type MMTV integration site (WNT) signaling, and fibroblast growth factors (FGFs), regulate the lineage commitment of skeletal stem cells to osteoprogenitors and subsequent maturation of osteoprogenitors to osteoblasts [2].
As important mediators of cellular signaling, mitogen-activated protein kinases (MAPKs) are evolutionally-conserved serine/threonine kinases that regulate diverse cellular functions including cell proliferation, differentiation, and apoptosis [3]. MAPKs are part of a phospho-relay system in

The ERK MAPK Pathway is Highly Activated in Osteoblasts In Vitro and In Vivo
To examine ERK activation in osteoblasts in vivo, immunohistochemistry was performed for phosphorylation of ERK1/2 in the femur of eight-week-old mice ( Figure 1A). Phosphorylated ERKs were mainly detected in the trabecular bone area under the growth plate that has a high bone remodeling activity. Specifically, ERK1/2 were highly phosphorylated in osteoblasts on the bone surface, while osteocytes within the bone matrix showed modest phosphorylation of ERK1/2, suggesting dynamic ERK activation in osteoblast-lineage cells at different differentiation stages in vivo ( Figure 1A). To test this hypothesis, we examined the kinetics of ERK phosphorylation during osteoblast differentiation in vitro ( Figure 1B). Primary calvarial osteoblasts (COBs, osteoprogenitor) were isolated from mouse calvaria at Postnatal Day 5 (P5), cultured under osteogenic conditions for 21 days, and phosphorylation of ERK1/2 at different osteoblast differentiation stages was assessed by immunoblotting. Phosphorylation levels of ERK peaked at Day 12 of osteogenic culture and gradually decreased during later differentiation stages, demonstrating that ERK is highly activated in mature osteoblasts. Thus, these results suggest that ERK activation plays a role in osteoblast differentiation and function in vivo and in vitro. Immunohistochemistry for phospho-ERK1/2 was performed in the femur of eight-week-old male mice. TB, trabecular bone; CB, cortical bone; BM; bone marrow. Scale bar, 500 μm (left) and 100 μm (right). (B) Primary calvarial osteoblasts (COBs) were isolated from mouse calvaria at Postnatal Day 5 and cultured under osteogenic conditions. Phosphorylation of ERK1/2 was determined by immunoblotting with anti-P-ERK1/2 antibody. ERK1/2 was used as a loading control.

Inactivation of ERK in Osteoprogenitors Causes Severe Osteopenia and Cleidocranial Dysplasia
To investigate the role of the ERK MAPK pathway in osteoblasts in vivo, mice lacking Mek1 and Mek2, mitogen-activated protein kinase kinases (MAP2Ks) upstream of ERK, in osteoprogenitors were generated by crossing mice harboring a floxed allele of Mek1 (Mek1 fl/fl ) with Osx-Cre [17,18] in the Mek2-germline null background (Mek2 −/− ) [19,20]. Immunoblotting analysis validated deletion of Mek1 and Mek2 in the calvarium of P5 Mek1 Osx Mek2 −/− neonates ( Figure. 2A). While no obvious skeletal phenotypes were observed in mice with a deletion of Mek1 (Mek1 Osx ) or Mek2 (Mek2 −/− ) alone, Mek1 Osx Mek2 −/− mice displayed severe growth retardation and skeletal defects and died at approximately four weeks old ( Figure 2B). Mek1 Osx Mek2 −/− mice were born at the expected Mendelian ratio. Skeletal preparations using Alizarin red/Alcian blue staining and microCT analysis of three-week-old Mek1 Osx Mek2 −/− mice showed multiple rib fractures ( Figure 2C), hypoplasia of the hyoid bone and clavicle ( Figure 2D,E), and hypomineralization of the calvarium ( Figure 2F). These phenotypes resemble those seen in human and murine cleidocranial dysplasia (CCD), characterized by open fontanelles, hypoplastic clavicles, and short stature [15,16]. Additionally, microCT analysis was performed in the femurs of WT, Mek1 Osx , Mek2 −/− , and Mek1 Osx Mek2 −/− mice to assess bone mass ( Figure 3A,B). While bone mass in Mek2 −/− femurs was comparable to that in wildtype femurs, Mek1 Osx femurs displayed a modest decrease in trabecular bone mass and midshaft cortical thickness. When Mek1 and Mek2 were both deleted in osteoprogenitors, femoral trabecular bone mass, number, and thickness in addition to cortical thickness were all significantly reduced, demonstrating severe osteopenia ( Figure 3A,B). Accordingly, expression of osteoblast differentiation genes, including alkaline phosphatase (Alpl), osteocalcin (Bglap), osterix (Sp7), bone sialoprotein (Ibsp), and type 1 collagen α1 (Col1a1), was markedly decreased relative to WT femurs ( Figure 3C). Histological analysis demonstrated that trabecular bone mass and cortical thickness were both reduced in Mek1 Osx Mek2 −/− femurs, while bone marrow was filled with hypertrophic chondrocytes, suggesting delayed endochondral ossification and impaired osteoclast-mediated remodeling of the growth plate due to impaired expression of the Rank ligand ( Figure 3D). Finally, serum levels of the bone formation marker, procollagen type I N-terminal propeptide (P1NP), and the bone resorption marker, C-terminal telopeptide of type I collagen (CTx-I), were measured in Mek1 Osx Mek2 −/− mice, demonstrating a decrease in bone formation activity without any alteration in bone resorption activity ( Figure 3E and F). Notably, these skeletal defects seen in Mek1 Osx Mek2 −/− mice are more severe than those in mice with Runx2 haploinsufficiency [15], suggesting that the ERK MAPK pathway has additional functions beyond RUNX2 regulation in osteoblasts. Taken together, ERK activation in osteoprogenitors is critical for skeletal development during the early postnatal stage.

Inducible Inactivation of the ERK Pathway in Osteoprogenitors Results in Osteopenia in Adult Mice
To investigate the role of ERK activation in bone formation during later postnatal life, we generated an inducible, osteoblast-specific Mek1/2-knockout by crossing Mek1 fl/fl mice with osterix-CreERT mice expressing a tamoxifen-inducible Cre recombinase in osteoprogenitors in the Mek2-germline null background (Mek1 Osx-ERT Mek2 −/− ) [21]. Eight-week-old Mek1 fl/fl Mek2 −/− and Mek1 Osx-ERT Mek2 −/− mice were treated with tamoxifen for five consecutive days to induce Cre-mediated deletion of Mek1 in osteoprogenitors, and nine weeks later, skeletal phenotypes were assessed by microCT ( Figure 4A). Similar to Mek1 Osx Mek2 −/− mice, trabecular bone mass, number, and thickness and midshaft cortical thickness were markedly reduced in Mek1 Osx-ERT Mek2 −/− mice relative to those in Mek1 fl/fl Mek2 -/mice ( Figure 4B,C), demonstrating that ERK activation in osteoprogenitors is also important for bone formation during skeletal homeostasis.

Mek1
Osx-ERT Mek2 −/− mice were treated with tamoxifen for five consecutive days to induce Cre-mediated deletion of Mek1 in osteoprogenitors, and nine weeks later, skeletal phenotypes were assessed by microCT ( Figure 4A). Similar to Mek1 Osx Mek2 −/− mice, trabecular bone mass, number, and thickness and midshaft cortical thickness were markedly reduced in Mek1 Osx-ERT Mek2 −/− mice relative to those in Mek1 fl/fl Mek2 -/-mice ( Figure 4B,C), demonstrating that ERK activation in osteoprogenitors is also important for bone formation during skeletal homeostasis.    To identify novel regulators of osteoblast differentiation in the ERK MAPK pathway, unbiased phospho-mass spectrometry using the post translational modification (PTM) scan direct technology was performed in WT and ∆Mek1/2 COBs ( Figure 5D). Three days after osteogenic culture, cell lysates were immunoprecipitated using a mixture of antibodies specific to phosphorylated proteins regulated by MAPK, CDK, PKA, AKT, and AMPK, and the immunoprecipitates were subjected to mass spectrometry. Gene-set enrichment analysis (GSEA) using the intensity of identified proteins showed an enrichment of phosphorylated proteins in the pathways of SHP2 (encoded by Ptpn11), FGF (fibroblast growth factor), and SCF-KIT (stem cell factor-kit) in WT COBs, and that phosphorylation of the proteins in these pathways was markedly decreased in ∆Mek1/2 COBs ( Figure 5E and Supplementary Figure S1). The protein-tyrosine phosphatase SHP2 has been known to function upstream of MEK1/2 MAP2Ks and to be important for osteoblast maturation [28]. Additionally, FGF signaling activates the ERK MAPK pathway and functions as a key regulator of osteoblast differentiation and skeletal development [12,29]. Finally, the SCF-KIT pathway is found to protect osteoblasts from oxidative stress through activating c-Kit-AKT signaling [30]. However, further study is needed to understand the contribution of these pathways to the overall functions of the ERK pathway in osteoblasts.

Discussion
In this study, we demonstrated that the ERK MAPK pathway is required for bone formation during skeletal development and homeostasis. Inactivation of ERK in osteoprogenitors (Mek1 Osx Mek2 −/− ) at an early postnatal stage of skeletal development results in severe osteopenia and CCD phenotypes. Similarly, inducible, postnatal inactivation of ERK in osteoprogenitors (Mek1 Osx-ERT Mek2 −/− ) significantly decreased bone mass. These results suggest that ERK activation plays a critical role in both skeletal development and homeostasis. Mechanistically, this corresponded to decreased activation of RUNX2, WNT/β-catenin, and RSK-ATF4 signaling pathways. Furthermore, unbiased phospho-mass spectrometry was performed to identify potential new regulators of osteoblast differentiation in the ERK MAPK pathway.
The ERK MAPK pathway has been implicated as a positive regulator for osteoblast differentiation and bone formation [11,13,14]. A previous study has demonstrated that dominant-negative Mek1 expression in osteoblasts results in CCD in mice, while constitutively-active Mek1 expression in osteoblasts partially rescues CCD phenotypes caused by Runx2 haploinsufficiency [5], suggesting that the MEK-ERK MAPK pathway regulates RUNX2 in osteoblasts. Consistent with this study, CCD phenotypes were also observed in mice lacking Mek1 and Mek2 in osteoprogenitors (Mek1 Osx Mek2 −/− ) ( Figure 2). Additionally, Mek1 Osx Mek2 −/− mice showed delayed ossification of long bones along with increased number of hypertrophic chondrocytes ( Figure 3D), a phenotype similar to that seen in Erk1 −/− Erk2 Osx mice [31].
ERK has several other known substrates besides RUNX2 that contribute to osteogenesis including, RSK2 [23]. RSK2 phosphorylates and activates the transcription factor ATF4, which is required for procollagen gene transcription in the later stages of osteoblast differentiation [24]. Additionally, ERK interacts with a wide range of parallel signal-transduction pathways in osteoblasts, such as the WNT/β-catenin pathway. ERK-mediated phosphorylation of GSK3β at Ser9 decreases its kinase activity, resulting in β-catenin accumulation through a reduction in ubiquitin-mediated proteasomal degradation [27]. Our data demonstrate that inactivation of ERK in osteoblasts leads to a significant decrease in the phosphorylation of RUNX2 and RSK2 and the corresponding transcriptional activity of RUNX2 and ATF4 ( Figure 5A-C). Intriguingly, while GSK3β phosphorylation was not altered in the absence of Mek1 and Mek2, ∆Mek1/2 COBs displayed a substantial decrease in protein levels and transcription activity of β-catenin, implicating additional ERK-mediated mechanisms that regulate β-catenin stability in osteoblasts.
Our unbiased phospho-mass spectrometry revealed a high enrichment of phosphorylated proteins in the pathways of SHP2, FGF, and SCF-KIT in WT COBs, and that phosphorylation of the proteins in these pathways was markedly decreased in ∆Mek1/2 COBs. Among these proteins, phosphorylation of a member of the FOS family, FRA2 (FOSL2) [32], was substantially reduced in the absence of Mek1 and Mek2. FRA2 is important for skeletal mineralization, as overexpression of Fra2 (Fosl2) in transgenic mice increased bone formation, though understanding the mechanism regulating FRA2 in osteoblasts requires further study [33]. Our data identified potential regulators of osteogenic differentiation and bone formation and will thereby improve understanding of ERK-mediated molecular mechanisms in osteogenesis and bone-related disease.

Mice
Mek1 fl/fl mice and Mek2 −/− mice were generated as previously reported, respectively [19,35], and maintained in a 129/SvEv background. To generate osteoprogenitor-specific double knockout (Mek1 Osx Mek2 −/− ), mice were crossed with Osx-cre [17,18], and littermate control was used for all skeletal analyses. To generate mice harboring inducible deletion of Mek1 in osteoprogenitors, Mek1 fl/fl Mek2 −/− mice were crossed with Osterix-CreERT [21]. For postnatal activation of CreERT, 75 mg/kg of tamoxifen (Sigma, T5648) in corn oil (Sigma) were intraperitoneally injected into 8-week-old mice once a day for five consecutive days. All animals including Mek1-floxed mice and Mek2 knockout mice were used in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were handled according to the animal protocol approved by the University of Massachusetts Medical School on animal care (IACUC).

MicroCT and Skeletal Preparation
MicroCT was used for qualitative and quantitative assessment of trabecular and cortical bone microarchitecture and performed by an investigator blinded to the genotypes of the animals under analysis. Femurs excised from the indicated mice were scanned using a microCT 35 (Scanco Medical, Brüttisellen, Switzerland) with a spatial resolution of 7 µm. Briefly, trabecular bone mass of the distal femur was measured in an upper 2.1-mm region beginning 280 µm proximal to the growth plate, and cortical bone thickness was measured in a midshaft region of 0.6 mm in length. MicroCT analysis of 3-week-old skulls was performed at isotropic voxel sizes of 12 µm. 3D reconstruction images were generated from contoured 2D images using microCT software (Brüttisellen, Switzerland). Alternatively, the Inveon multimodality 3D visualization program was used to generate fused 3D views of multiple static or dynamic volumes of microCT modalities (Siemens Medical Solutions USA, Inc., Norwood, MA, USA). All images presented are representative of the respective genotypes (n > 4).
For skeletal preparation, mice were skinned, eviscerated, and fixed in 95% EtOH for a day, and the skeletons were transferred in acetone for 2 days. Then, skeletons were stained with 0.1% of Alizarin red s and 0.3% of Alcian blue (Sigma, A3157) solution for 3 days as previously described [36]. After staining, samples were washed with 95% EtOH, and soft tissue was cleared by a 1.5% KOH solution. Soft tissue was subsequently further cleared in 1% KOH. All images presented are representative of the respective genotypes (n > 5).

Histology and Immunohistochemistry
For histological analysis, 3-or 8-week-old femurs were fixed in 10% neutral formalin at 4 • C for 2 days and then decalcified in 15% tetrasodium EDTA (pH 8.0) at 4 • C for 2 weeks. Tissues were dehydrated in different concentrations of EtOH, incubated in xylene, and embedded in paraffin. Paraffin sections were performed at a 7-µm thickness along the coronal plate and stained with hematoxylin and eosin (H&E).

RT-PCR and Immunoblotting
Total RNAs were purified using QIAzol (Qiagen, Germantown, MA, USA), and cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Beverly, MA, USA). Quantitative RT-PCR was performed using SYBR ® Green PCR Master Mix (Bio-Rad, Hercules, CA, USA) with the Bio-Rad CFX Connect Real-Time PCR detection system. The primers used for PCR are described in the Supplementary Table S1.

Phospho-Mass Spectrometry-Based Antibody Enrichment
Post translational modification (PTM) scan direct technology from Cell Signaling Technology (CST, Danvers, MA, USA) was performed in WT and ∆Mek1/2 COBs to identify phosphorylated proteins regulated by ERK. Briefly, two sets of WT and ∆Mek1/2 COBs were cultured under osteogenic conditions for 3 days. Cell extracts were incubated with an antibody mixture (Phospho-Akt/AMPK/MAPK/CDK/PKA Substrate Motif Antibodies Mix, Cell Signaling, 9614/10001/ 759/2325/9624, respectively) and antibodies were immobilized to protein A (or G) agarose. After immunoprecipitation, eluted proteins were analyzed by LC-MS/MS using LTQ-Orbitrap-Velos, ESI-CID. PTM scan results in WT and ∆Mek1/2 COBs are shown in Supplementary Table S2.

Gene Set Enrichment Analysis
Proteins with more than two unique peptides derived from the parent ion intensity were defined as qualified proteins for GSEA analysis. Gene sets from the Broad Institute Molecular Signatures Database were used, and multiple lists of enriched gene sets were generated using the GSEA algorithm as previously described [37]. Enrichment for up-/down-regulated proteins in ∆Mek1/2 COBs was assessed against a rank list of all the available expression values from ∆Mek1/2 COBs to expression from WT COBs. The permutation type was set to phenotype, and other settings were set as default. A nominal p-value <0.05 and FDR (false discovery rate) q-value <0.25 were considered as a significantly enriched pathway.

Statistical Analysis
All data are shown as the mean ± standard deviation (SD). We first performed the Shapiro-Wilk normality test for checking normal distributions of the groups. If normality tests passed, two-tailed, unpaired Student's t-tests, and if normality tests failed, Mann-Whitney tests were used for the comparisons between two groups. To compare four groups, one-way ANOVA was used if normality tests passed, and then Tukey's multiple comparison test was used for all pairs of groups. Statistical analysis was performed using the GraphPad PRISM software (v7.0a, La Jolla, CA, USA). p < 0.05 was considered statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Conclusion
In this study, we demonstrated ERK MAPK pathway is required for skeletal development and bone homeostasis through analysis of genetic deletion of ERK upstream, Mek1 and Mek2 in osteoprogenitors in developmental stage (Mek1 Osx Mek2 −/− ) and postnatal stage (Mek1 Osx-ERT Mek2 −/− ). This study strongly supports previous studies on ERK MAPK function in osteoblast differentiation and bone formation, phenotypically and mechanistically. Moreover, unbiased phospho-mass spectrometry provides putative downstream of ERK MAPK pathway in osteoblast differentiation and further study will improve knowledge regarding the role of ERK MAPK in bone biology.