Extracellular signal-regulated kinase 1 (ERK1) and ERK2 play essential roles in osteoblast differentiation and in supporting osteoclastogenesis

Abstract

Osteoblasts and chondrocytes arise from common osteo-chondroprogenitor cells. We show here that inactivation of ERK1 and ERK2 in osteo-chondroprogenitor cells causes a block in osteoblast differentiation and leads to ectopic chondrogenic differentiation in the bone-forming region in the perichondrium. Furthermore, increased mitogen-activated protein kinase signaling in mesenchymal cells enhances osteoblast differentiation and inhibits chondrocyte differentiation. These observations indicate that extracellular signal-regulated kinase 1 (ERK1) and ERK2 play essential roles in the lineage specification of mesenchymal cells. The inactivation of ERK1 and ERK2 resulted in reduced beta-catenin expression, suggesting a role for canonical Wnt signaling in ERK1 and ERK2 regulation of skeletal lineage specification. Furthermore, inactivation of ERK1 and ERK2 significantly reduced RANKL expression, accounting for a delay in osteoclast formation. Thus, our results indicate that ERK1 and ERK2 not only play essential roles in the lineage specification of osteo-chondroprogenitor cells but also support osteoclast formation in vivo.

FIG. 1.

ERK1^−/−; ERK2^flox/flox; Prx1-Cre embryos and mice. (A) Skeletal preparation after alizarin red and alcian blue staining at P1. Limbs were severely deformed in ERK1^−/−; ERK2^flox/flox; Prx1-Cre mice. (B) Skeletal preparation of the cranium after alizarin red staining. ERK1^−/−; ERK2^flox/flox; Prx1-Cre mice showed bone defects in the calvaria at P12. (C) Hematoxylin, eosin, and alcian blue staining of the humerus showing an absence of the primary ossification center and cortical bone formation in ERK1^−/−; ERK2^flox/flox; Prx1-Cre mice at P5. (D) Real-time PCR showed ERK2 inactivation in the tibiae and humeri of ERK1^−/−; ERK2^flox/flox; Prx1-Cre embryos at E16.5. (E) Immunohistochemistry using anti-ERK1 and ERK2 antibody showed reduced immunoreactivity in chondrocytes and cells in the bone-forming region of tibiae of ERK1^−/−; ERK2^flox/flox; Prx1-Cre mice at P5. (a) ERK1^−/+; ERK2^flox/flox mice. The boxed area in the upper panel is magnified in the lower panel. (b) ERK1^−/−; ERK2^flox/flox; Prx1-Cre mice. The upper left panel shows immunostaining for ERK1 and ERK2. Boxed areas 1 and 2 are magnified in the corresponding right panels. While chondrocytes showed reduced immunoreactivity (boxed area 1), endothelial cells show positive staining (boxed area 2). The lower left panel shows alcian blue, hematoxylin, and eosin staining of a neighboring section. Bars indicate 100 μm.

FIG. 2.

In situ hybridization analyses of the femur (A, B) and calvaria (C), showing normal levels of Col1a1, Runx2, and Osterix (Osx) expression and markedly decreased Osteocalcin (OCN) expression in ERK1^−/−; ERK2^flox/flox; Prx1-Cre embryos and mice. (A) E15.5; (B) E16.5 and P5; (C) P1.

FIG. 3.

(A) Semiquantitative reverse transcription-PCR showing reduced Osteocalcin (OCN) and Bone sialoprotein (BSP) expression in primary ERK1^−/−; ERK2^flox/flox calvarium mesenchymal cells that were infected with Ad expressing Cre recombinase (Ad-Cre). Primary calvarium mesenchymal cells were isolated from E15.5 ERK1^−/−; ERK2^flox/flox embryos and infected with Ad-Cre or Ad expressing GFP (Ad-GFP). RNA was extracted 20 days after infection. (B) Western blot analysis showing Osterix (OSX), ATF4, and RSK2 expression in primary ERK1^−/−; ERK2^flox/flox calvarium cells that were infected with Ad-Cre or Ad-GFP. ERK2 expression was inhibited 80% by Ad-Cre infection, while Osterix, ATF4, and RSK2 expression remained largely unaffected. Total cell lysates were prepared 10 days after infection. (C) von Kossa staining of ERK1^−/−; ERK2^flox/flox calvarium cell cultures 20 days after infection with Ad-Cre or Ad-GFP. Ad-Cre infection inhibited mineralization. (D) Real-time PCR analysis showed reduced Krox20, c-Fos, Fra1, Fra2, and Cbfb expression in the humeri of ERK1^−/−; ERK2^flox/flox; Prx1-Cre embryos at E16.5, while JunB was not affected. Real-time PCR analysis of the tibia and femur showed similar results. (E) Real time PCR analysis showed reduced Erk2, Krox20, and c-Fos expression in ERK1^−/−; ERK2^flox/flox calvarium cells infected with Ad-Cre. ERK1^−/−; ERK2^flox/flox calvarium cells were infected with Ad-Cre or Ad-GFP, and RNA was extracted 9 days after infection.

FIG. 4.

Ectopic cartilage formation in the perichondria of ERK1^−/−; ERK2^flox/flox; Prx1-Cre embryos. (A) Alcian blue staining and in situ hybridization of the femur at E15.5. The ectopic cartilage (arrowheads) in the perichondrium expresses Sox9, Col2a1, and Indian hedgehog (Ihh). (B) Alcian blue staining (top panel) and immunohistochemical staining of the radius for β-catenin (middle panel). ERK1^−/−; ERK2^flox/flox; Prx1-Cre embryos showed reduced β-catenin protein levels in the perichondrium at E16.5 (arrows). The boxed area is magnified in the bottom panel. (C) Tcf1 and Dkk1 expression quantitated by real-time PCR. ERK1^−/−; ERK2^flox/flox; Prx1-Cre embryos showed reduced Tcf1 and Dkk1 expression in the tibia and humerus at E16.5.

FIG. 5.

Delayed formation of primary ossification centers in ERK1^−/−; ERK2^flox/flox; Prx1-Cre mice. (A) Immunohistochemistry for type X collagen showed a widening of the zone of hypertrophic chondrocytes in the tibiae of ERK1^−/−; ERK2^flox/flox; Prx1-Cre mice at P0. (B) In situ hybridization for Col10a1, Vegf, Mmp-13, and Osteopontin showed an expansion of terminally differentiated chondrocytes in the hypertrophic zone of the tibia at P0. (C) TRAP staining showed an absence of TRAP-positive cells in the tibiae of ERK1^−/−; ERK2^flox/flox; Prx1-Cre embryos at E16.5. Arrows indicate TRAP-positive cells. The upper images in panels A, B, and C show ERK1^−/−, and the lower images show ERK1^−/−; ERK2^flox/flox; Prx1-Cre mice. (D) Real-time PCR showed reduced RANKL expression in the tibiae and humeri of ERK1^−/−; ERK2^flox/flox; Prx1-Cre embryos at E16.5. (E, F) ERK2, RANKL, and Osteoprotegerin (OPG) expression examined by real-time PCR. Inactivation of ERK2 strongly inhibited RANKL expression in ERK1^−/−; ERK2^flox/flox rib chondrocytes (E) and calvarium osteoblasts (F) in vitro. RNA was extracted from ERK1^−/−; ERK2^flox/flox chondrocytes and osteoblasts 5 days and 8 days after infection with Ad expressing Cre recombinase or GFP. (G) ELF97-based fluorescent TRAP staining (green fluorescence) in combination with immunofluorescence for ERK protein (red fluorescence), showing the presence of ERK protein in TRAP-positive osteoclasts (arrows) in the femoral metaphysis of an ERK1^−/−; ERK2^flox/flox; Prx1-Cre mouse at P0. The immunofluorescent signal for ERK protein was indistinguishable from that of ERK1^−/−; ERK2^flox/flox mice (data not shown). The lower panels show a magnification of an osteoclast indicated by arrowheads in the upper panels. Nuclei were visualized by DAPI (4′,6-diamidino-2-phenylindole). (H) X-Gal staining followed by TRAP staining showing no β-galactosidase activity in TRAP-positive osteoclast-like cells derived from spleen cells of Prx1-Cre mice harboring the ROSA-LacZ reporter allele (right panel). The staining results were indistinguishable from those observed for TRAP-positive osteoclast-like cells derived from control ROSA-LacZ reporter mice (left panel). (I) Spleen cells from ERK1^−/−; ERK2^flox/flox; Prx1-Cre mice formed TRAP-positive multinucleated osteoclast-like cells in the presence of M-CSF and RANKL (left panel). Nuclei were visualized by DAPI. These osteoclast-like cells express ERK protein (right panel), and the staining intensity was indistinguishable from that of osteoclast-like cells generated from spleen cells of ERK1^−/−; ERK2^flox/flox mice (data not shown).

FIG. 6.

ERK1^−/−; ERK2^flox/flox; Col2a1-Cre embryos. (A) Immunofluorescence using anti-ERK1 and ERK2 antibody showed reduced immunoreactivity in chondrocytes in the tibiae of ERK1^−/−; ERK2^flox/flox; Col2a1-Cre embryos at E18.5. (B) ELF97-based fluorescent TRAP staining in combination with immunofluorescence for ERK protein, showing the presence of ERK protein in TRAP-positive osteoclasts (arrows) of ERK1^−/−; ERK2^flox/flox; Col2a1-Cre embryos. (C) ELF97-based fluorescent alkaline phosphatase staining in combination with immunofluorescence for ERK protein, showing the presence of ERK protein in alkaline phosphatase-positive osteoblasts (arrows) of ERK1^−/−; ERK2^flox/flox; Col2a1-Cre embryos. (D) Skeletal preparation after alizarin red and alcian blue staining. ERK1^−/−; ERK2^flox/flox; Col2a1-Cre embryos showed severe kyphotic deformity in the thoracic spine at E18.5. (E, F) Hematoxylin, eosin, and alcian blue staining of the spine (E) and cranial base (F) showing an absence of ossification centers in ERK1^−/−; ERK2^flox/flox; Col2a1-Cre embryos at E18.5. * indicates the ossification center in the vertebral body, and arrowheads indicate the corresponding areas in ERK1^−/−; ERK2^flox/flox; Col2a1-Cre embryos. VB, vertebral body; D, intervertebral disc; SC, spinal cord; Pi, pituitary gland; wt, wild type.

FIG. 7.

(A) Hematoxylin, eosin, and alcian blue staining of the femur showing a dosage-dependent widening of the zone of hypertrophic chondrocytes at E16.5. Bars indicate the width of the zone of hypertrophic chondrocytes. (B) In situ hybridization of the tibia showing an expansion of Col10a1-expressing domains in ERK1^−/−; ERK2^flox/flox; Col2a1-Cre embryos at E18.5. (C) Primary ERK1^−/−; ERK2^flox/flox chondrocytes were infected with Ad expressing Cre recombinase or GFP at a multiplicity of infection of 200. Col10a1 expression was examined by real-time PCR at 5 days after Ad infection. ERK2 inactivation in ERK1^−/−; ERK2^flox/flox chondrocytes increased Col10a1 expression. (D) Disorganization of columnar structures in the tibial growth plates of ERK1^−/−; ERK2^flox/flox; Col2a1-Cre embryos at E18.5. (E) BrdU incorporation of the distal femoral growth plate. ERK1^−/−; ERK2^flox/flox; Col2a1-Cre embryos showed reduced chondrocyte proliferation at E18.5. Data represent means ± standard deviations. N.S., not significant; wt, wild type. Analysis of variance was used to detect significant difference. *, P < 0.01.

FIG. 8.

(A) TRAP staining of the femur showing decreased TRAP-positive cells in ERK1^−/−; ERK2^flox/flox; Col2a1-Cre embryos at E16.5. (B) ELF97-based fluorescent TRAP staining in combination with immunofluorescence for ERK protein. Spleen cells from ERK1^−/−; ERK2^flox/flox (a) and ERK1^−/−; ERK2^flox/flox; Col2a1-Cre (b,c) embryos formed TRAP-positive, multinucleated osteoclast-like cells in the presence of M-CSF and RANKL. Nuclei were visualized by DAPI (4′,6-diamidino-2-phenylindole). Lower panels show immunofluorescence using anti-ERK antibody (a′ and b′) or nonimmune immunoglobulin G (IgG) (c′) in corresponding cells.

FIG. 9.

(A) Schematic representation of the construct that drives the expression of a constitutively active mutant of MEK1 and LacZ under the control of a 2.4-kb prx1 promoter. (B) X-Gal staining of an E15.5 embryo showing transgene expression in the limb and cranium. (C) X-Gal staining of the distal ulna of an E15.5 embryo showing transgene expression in the periarticular chondrocytes, periosteum, and perichondrium. (D) Immunostaining of the FLAG-tagged MEK1(S218/222E, Δ32-51) using anti-M5 FLAG antibody showing transgene expression in the periarticular chondrocytes (arrows), periosteum (arrowheads), and perichondrium of the distal radius of a Prx1-MEK1 transgenic embryo at E15.5 (middle panel). No immunoreactivity was observed in a wild-type (Wt) littermate embryo (left panel). The immunostained section was further stained with alcian blue and eosin (right panel). The cartilaginous matrix of transgene-expressing periarticular chondrocytes (arrows) shows reduced alcian blue staining. (E) Skeletal preparation of the forelimbs after alizarin red and alcian blue staining. Transgenic mice showed a thickening and shorting of long bones at P8. Wt, wild type; Tg, transgenic. (F) Skeletal preparation of the cranium after X-Gal and alizarin red staining showing transgene expression in the mesenchyme of the lambdoid suture. Transgenic mice showed an accelerated closure of the lambdoid suture at E17.5.

FIG. 10.

(A) Cross section of the forelimb stained with hematoxylin and eosin. Prx1-MEK1 transgenic mice showed increased bone formation and fusion of long bones (arrowhead) at P10. Wt, wild type. (B) Hematoxylin, eosin, and alcian blue staining and in situ hybridization of the tibia at E15.5. Prx1-MEK1 transgenic embryos showed a thickening of the perichondrium, which expresses Runx2, Osterix (Osx), and Bone sialoprotein (BSP) (arrowheads). (C) Hematoxylin, eosin, and alcian blue staining of the foot. Prx1-MEK1 transgenic embryos showed a delay in the formation of cartilage anlage at E15.5. Ti, tibia; Ta, talus; Ca, calcaneus. (D) In situ hybridization of the carpal bones. Prx1-MEK1 transgenic embryos showed reduced Col2a1 expression at E15.5. (E) Primary wild-type chondrocytes were infected with Ad expressing a constitutively active mutant of MEK1 (Ad-MEK1) or empty virus (Ad-Null). Col2a1 expression was examined by real-time PCR at 48 h after Ad infection. Expression of a constitutively active mutant of MEK1 strongly inhibited Col2a1 expression. (F) FGF18 treatment (20 ng/ml) downregulated Col2a1 expression in wild-type primary chondrocytes at 24 h. The downregulation was inhibited by U0126. C, control; F, FGF18; U, U0126; F+U, FGF18 plus U0126.

FIG. 11.

Model for the roles of MAPK in chondrocyte and osteoblast differentiation. While MAPK inhibits chondrogenic differentiation of osteo-chondroprogenitor cells, MAPK enhances osteoblast differentiation. MAPK is essential for osteoblast differentiation into Osteocalcin-expressing mature osteoblasts. MAPK also inhibits hypertrophic chondrocyte differentiation.