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. 2004 Feb 1;18(3):290-305.
doi: 10.1101/gad.1179104.

Constitutive activation of MEK1 in chondrocytes causes Stat1-independent achondroplasia-like dwarfism and rescues the Fgfr3-deficient mouse phenotype

Shunichi Murakami  1 , Gener Balmes, Sandra McKinney, Zhaoping Zhang, David Givol, Benoit de Crombrugghe
Affiliations

Constitutive activation of MEK1 in chondrocytes causes Stat1-independent achondroplasia-like dwarfism and rescues the Fgfr3-deficient mouse phenotype

Shunichi Murakami et al. Genes Dev. .

Abstract

We generated transgenic mice that express a constitutively active mutant of MEK1 in chondrocytes. These mice showed a dwarf phenotype similar to achondroplasia, the most common human dwarfism, caused by activating mutations in FGFR3. These mice displayed incomplete hypertrophy of chondrocytes in the growth plates and a general delay in endochondral ossification, whereas chondrocyte proliferation was unaffected. Immunohistochemical analysis of the cranial base in transgenic embryos showed reduced staining for collagen type X and persistent expression of Sox9 in chondrocytes. These observations indicate that the MAPK pathway inhibits hypertrophic differentiation of chondrocytes and negatively regulates bone growth without inhibiting chondrocyte proliferation. Expression of a constitutively active mutant of MEK1 in chondrocytes of Fgfr3-deficient mice inhibited skeletal overgrowth, strongly suggesting that regulation of bone growth by FGFR3 is mediated at least in part by the MAPK pathway. Although loss of Stat1 restored the reduced chondrocyte proliferation in mice expressing an achondroplasia mutant of Fgfr3, it did not rescue the reduced hypertrophic zone, the delay in formation of secondary ossification centers, and the achondroplasia-like phenotype. These observations suggest a model in which Fgfr3 signaling inhibits bone growth by inhibiting chondrocyte differentiation through the MAPK pathway and by inhibiting chondrocyte proliferation through Stat1.

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Figures

Figure 1.
Figure 1.
(A,B) Prolonged phosphorylation of ERK1, ERK2, and MEK1 after FGF18 treatment in primary chondrocytes. Chondrocytes were prepared from the ribs of newborn mice. Confluent culture was serum-starved for 24 h and treated with 20 ng/mL FGF18, 20 ng/mL EGF, 100 ng/mL PDGF-BB, 20 ng/mL TGF-β, and 100 ng/mL IGF-I. Cells were harvested at indicated periods of time after treatment. Total and phosphorylated ERK1, ERK2, and MEK1 proteins, Sox9, and c-Fos were detected by Western blot analysis. (C) Immunostaining of MEK1 (left) and its phosphorylated form (middle) and Sox9 (right) in the proximal tibia of a wild-type mouse at P7. Sox9 staining in hypertrophic chondrocytes is magnified for subcellular localization. (D) Immunostaining of phosphorylated MEK1 in the proximal tibial growth plates in Fgfr3 mutant littermates at P21. Increased staining for phosphorylated MEK1 was observed in the growth plate chondrocytes of heterozygous and homozygous mice expressing an achondroplasia mutant of Fgfr3 (G374R) compared with heterozygous and homozygous mice carrying a hypomorphic allele of Fgfr3 (G374R neo+). Similar results were observed in the femur and radius. Fgfr3 mutant mice homozygous for the hypomorphic G374R neo+ allele show skeletal overgrowth similar to Fgfr3-null mice, whereas heterozygous mice show normal bone growth. The Prx1-Cre transgenic mouse line was used to delete the neomycin cassette that interfered with normal splicing of Fgfr3, causing an achondroplasia mutant Fgfr3 G374R to be expressed. Bars indicate the position of the zone of hypertrophic chondrocytes. (E) Schematic representation of the construct that drives expression of a constitutively active mutant of MEK1 in chondrocytes. The original initiation codon of Col2a1 was mutated to CTG to facilitate translation from downstream cDNA. (F) X-gal staining of an E14.5 embryo showing cartilage-specific expression of the transgene (left). X-gal staining of the distal femoral growth plate of a transgenic mouse at P1 (middle) and in situ hybridization of Fgfr3 of the corresponding area in a wild-type littermate (right). (G) Northern blot analysis using a probe for MEK1. A transcript of ∼7 kb was detected in the limb and rib cartilages of transgenic mice. The MEK1 transgene expression was 5% of the endogenous MEK1 expression. The lower panels show ethidium bromide staining of RNA as a loading control. (H) Western blot analysis using an anti-Flag M5 antibody. The Flag-tagged mutant MEK1 protein is expressed in chondrocytes isolated from the ribs. A cell lysate of C3H10T1/2 cells stably transfected with the Flag-tagged MEK1 was run as a control. (Wt) Wild type; (Tg) transgenic.
Figure 2.
Figure 2.
(A) Transgenic and wild-type littermates in line A mice at 3 wk of age. Transgenic mice expressing a constitutively active MEK1(S218/222E, Δ32-51) in chondrocytes showed an achondroplasia-like dwarf phenotype. (Tg) Transgenic; (Wt) wild-type. (B) Skeletal preparation of newborn line C mice after alizarin red and alcian blue staining. Transgenic mice expressing a constitutively active mutant of MEK1 in chondrocytes showed an achondroplasia-like phenotype characterized by shortened axial and appendicular skeletons, midfacial hypoplasia, and a rounded cranium. (C) Caudal view of the head showing remarkable hypoplasia of the sphenoid (sp), basisphenoid (bs), and basioccipital (bo) bones, along with an anteriorly displaced foramen magnum. Anterior (D) and lateral (E) views of the cervical spine, showing a delay in ossification of vertebral bodies and fusions of lamina (arrow) with neighboring vertebrae in transgenic mice expressing MEK1 (S218/222E, Δ32-51) in chondrocytes. (F) Long bones and limb girdles of transgenic and wild-type littermates in line A mice at 6 wk of age. Transgenic mice expressing MEK1(S218/222E, Δ32-51) in chondrocytes showed proportional shortening of long bones and limb girdles.
Figure 3.
Figure 3.
Alcian blue staining of proximal tibial growth plates of wild-type (A) and transgenic (B) littermates in line A at P4. Transgenic mice expressing MEK1(S218/222E, Δ32-51) in chondrocytes showed smaller than normal hypertrophic chondrocytes in the growth plate. (C-E) Alcian blue staining of proximal tibial growth plates of wild-type (C), heterozygous (D), and homozygous (E) Fgfr3 mutant mice that express Fgfr3 G374R at P4. Mice expressing Fgfr3 G374R show dosage-dependent reduction in the size of hypertrophic chondrocytes and width of the hypertrophic zone. (F,G) Type X collagen immunostaining of proximal tibial growth plates of wild-type (F) and transgenic (G) male littermates in line B at P7, showing a reduced zone of hypertrophic chondrocytes in transgenic mice. (H-K) Hematoxylin and eosin staining of the proximal tibia at the level of cruciate ligament insertion. Transgenic mice expressing MEK1(S218/222E, Δ32-51) in chondrocytes (I) showed a delay in the formation of secondary ossification centers compared with their wild-type littermates (H) at P7. Similar delay is observed in heterozygous Fgfr3 mutant mice that express Fgfr3 G374R at P8; (J) wild-type; (K) Fgfr3 G374R. (L,M) Carpal bones of wild-type (L) and transgenic (M) female littermates in line A at P9. Ossification of carpal bones was delayed in transgenic mice. (N,O) Indian hedgehog (Ihh) expression in fibulae of E15.5 wild-type (N) and double-transgenic (O) embryos that harbor the transgenes of both lines A and B. Ihh expression was unaltered in transgenic mice expressing MEK1(S218/222E, Δ32-51) in chondrocytes. (P,Q) There was no difference in BrdU incorporation in chondrocytes of the proximal tibial growth plates between wild-type (P) and transgenic (Q) littermates in line A at P8. (R,S) Localization of BrdU-labeled cells 26 h after administration of BrdU at P13. Because cells in the hypertrophic zone do not proliferate and do not incorporate BrdU, the presence of BrdU-labeled cells in the hypertrophic zone would indicate that these cells have undergone hypertrophic differentiation after incorporating BrdU in the proliferating zone. Within 26 h after administration of BrdU, BrdU-labeled cells advanced to the upper 1/3 of the hypertrophic zone in wild-type mice (R), while BrdU-positive cells stayed in the prehypertrophic region in transgenic mice that express MEK1(S218/222E, Δ32-51) in chondrocytes (S). (T) X-gal staining of the proximal femoral growth plate of a heterozygous female E15.5 embryo in line B, showing a mosaic pattern of transgene expression consistent with inactivation of one of the X-chromosomes in females. Chondrocytes that stained positive for X-gal were smaller than neighboring chondrocytes that did not stain with X-gal, indicating that the MAPK pathway inhibited chondrocyte hypertrophy in a cell-autonomous manner.
Figure 4.
Figure 4.
Delayed endochondral ossification of the cranial base in mice expressing MEK1(S218/222E, Δ32-51) in chondrocytes. Sagittal sections of the cranial base were stained with alcian blue (A,B,E,F,K,L) or stained immunohistochemically for type X collagen (C,D), Sox9 (I,J,M,N), osteopontin (G,H), and BrdU (O,P). (A,C) The basioccipital region of E15.5 wild-type mice; (B,D) the basioccipital region of E15.5 transgenic mice. (Pi) Pituitary glands. Alcian blue staining of the basioccipital region in E15.5 wild-type (A) and transgenic (B) embryos showed a delay in hypertrophic differentiation of chondrocytes in transgenic mice. Immunohistochemical staining of type X collagen in E15.5 wild-type (C) and transgenic (D) embryos showed reduced staining only in transgenic mice. Delayed ossification of the cranial base was noted at E16.5 for (E,G) wild-type and (F,H) transgenic embryos and at E17.5 for (K) wild-type and (L) transgenic embryos. Transgenic embryos showed persistent expression of Sox9 in chondrocytes at E16.5 for (I) wild-type and (J) transgenic embryos and at E17.5 for (M) wild-type and (N) transgenic embryos. (G and I), (H and J), (K and M), and (L and N) are neighboring sections. There was no obvious difference in BrdU incorporation in chondrocytes of the cranial base at E16.5 for (O) wild-type and (P) transgenic embryos.
Figure 5.
Figure 5.
(A) Expression of a constitutively active mutant of MEK1 in the chondrocytes of Fgfr3-deficient mice inhibited skeletal overgrowth. The distance between the proximal and distal growth plates in the tibia was measured in males at 3 wk of age. The tibiae of Fgfr3-deficient mice expressing the MEK1 transgene were shorter than those of Fgfr3-deficient mice without the MEK1 transgene (p < 0.05). No statistically significant difference was detected between transgenic mice in the Fgfr3-deficient and wild-type backgrounds. The number of samples in each group is indicated in parentheses. Values are the mean ± SD. (N.S.) Not significant. (B) Gross appearance of 4-wk-old female littermates with or without the transgene in the Fgfr3 homozygous mutant background. (C) The tibia, fibula, and talus of 4-wk-old male littermates. Overgrowth of bones in Fgfr3-deficient mice was inhibited by the expression of a constitutively active mutant of MEK1 in chondrocytes. (D) Alcian blue staining of the proximal tibia of Fgfr3 homozygous mutant male mice with or without the transgene at P5. Expression of a constitutively active mutant of MEK1 in the chondrocytes of Fgfr3-deficient mice reduced the hypertrophic zone in the growth plates.
Figure 6.
Figure 6.
Expression of a constitutively active mutant of MEK1 in the chondrocytes of Stat1-null mice and the resulting achondroplasia-like phenotype. (A) Gross appearance of 8-d-old male littermates with or without the transgene in the Stat1-null background. Transgenic mice in the Stat1-null background showed a rounded cranium and a dwarf phenotype. (B) Alcian blue staining of distal ulnar growth plates of mice shown in A. Stat1-null transgenic mice show reduced size of hypertrophic of chondrocytes and a narrower hypertrophic zone compared with Stat1-null mice without the transgene. (C) The distance between the proximal and distal growth plates in the tibia was measured in males at 3 wk of age. Expression of a constitutively active mutant of MEK1 in chondrocytes caused shortening of tibiae in Stat1-null mice (p < 0.01). No statistically significant difference was detected between transgenic mice in the Stat1-null and wild-type backgrounds. The number of samples in each group is indicated in parentheses. (N.S.) Not significant. (D,E) Primary chondrocytes were isolated from the rib cages of wild-type and Stat1-null mice. Cells were treated with 10 ng/mL FGF2 or 20 ng/mL FGF18. (D) Sox9 expression was strongly up-regulated at 1 h after FGF2 treatment both in wild-type and Stat1-null chondrocytes. This up-regulation was strongly inhibited by the MAPK pathway inhibitor, U0126. (E) c-fos and p21 expression were up-regulated at 3 h after FGF18 treatment both in wild-type and Stat1-null chondrocytes. This up-regulation was strongly inhibited by U0126.
Figure 7.
Figure 7.
The G374R mutation in Fgfr3 causes an achondroplasia-like phenotype in Stat1-null mice. (A) Mating scheme to express Fgfr3 G374R in Stat1-null mice. Zp3-Cre transgenic mouse line was used to delete the neomycin cassette that interfered with normal splicing of Fgfr3. Zp3-Cre transgenic mice express Cre recombinase in the oocyte. (B) Gross appearance of 14-d-old female littermates with or without the G374R mutation in Fgfr3 in the Stat1-null and wild-type backgrounds. The G374R mutation in Fgfr3 caused an achondroplasia-like phenotype in the Stat1-null background. (C) Alizarin red staining of tibia and fibula of 7-d-old male littermates. The G374R mutation in Fgfr3 caused shortening of long bones and a delay in the formation of secondary ossification centers in the Stat1-null background. (D) Alcian blue staining of proximal tibia of 12-d-old male littermates. Mice carrying the G374R mutation in Fgfr3 in the Stat1-null background showed a reduced hypertrophic zone in the growth plates and a delayed formation of secondary ossification centers. (E,F) BrdU incorporation of proximal tibia of 12-d-old male littermates. BrdU-positive cells were counted and the percentage of positive cells in the proliferating zone was calculated (F). The G374R mutation in Fgfr3 caused a significant reduction in BrdU incorporation in mice carrying wild-type Stat1 (p < 0.01). There was no statistically significant difference in the number of BrdU-incorporating cells between mice expressing Fgfr3 G374R and mice that were wild-type for Fgfr3 in the Stat1-null background. (N.S.) Not significant. (G) The distance between the proximal and distal growth plates in the tibia was measured in males (□) and females (•) at 2 wk of age. Expression of Fgfr3 G374R caused reduction in the tibial bone length both in mice that were wild type for Stat1 and Stat1-null mice. Loss of Stat1 corrected the bone length to a minor extent.
Figure 8.
Figure 8.
Model for downstream signaling pathways that mediate the inhibitory effects of FGFR3 signaling on bone growth. The MAPK pathway mediates inhibition of hypertrophic chondrocyte differentiation, whereas Stat1 mediates inhibition of chondrocyte proliferation.
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