Abstract

Cardio-facio-cutaneous (CFC) syndrome is one of the ‘RASopathies’, a group of phenotypically overlapping syndromes caused by germline mutations that encode components of the RAS–MAPK pathway. Germline mutations in BRAF cause CFC syndrome, which is characterized by heart defects, distinctive facial features and ectodermal abnormalities. To define the pathogenesis and to develop a potential therapeutic approach in CFC syndrome, we here generated new knockin mice (here BrafQ241R/+) expressing the Braf Q241R mutation, which corresponds to the most frequent mutation in CFC syndrome, Q257R. BrafQ241R/+ mice manifested embryonic/neonatal lethality, showing liver necrosis, edema and craniofacial abnormalities. Histological analysis revealed multiple heart defects, including cardiomegaly, enlarged cardiac valves, ventricular noncompaction and ventricular septal defects. BrafQ241R/+ embryos also showed massively distended jugular lymphatic sacs and subcutaneous lymphatic vessels, demonstrating lymphatic defects in RASopathy knockin mice for the first time. Prenatal treatment with a MEK inhibitor, PD0325901, rescued the embryonic lethality with amelioration of craniofacial abnormalities and edema in BrafQ241R/+ embryos. Unexpectedly, one surviving pup was obtained after treatment with a histone 3 demethylase inhibitor, GSK-J4, or NCDM-32b. Combination treatment with PD0325901 and GSK-J4 further increased the rescue from embryonic lethality, ameliorating enlarged cardiac valves. These results suggest that our new Braf knockin mice recapitulate major features of RASopathies and that epigenetic modulation as well as the inhibition of the ERK pathway will be a potential therapeutic strategy for the treatment of CFC syndrome.

INTRODUCTION

Cardio-facio-cutaneous (CFC) syndrome is an autosomal dominant congenital anomaly syndrome, characterized by a distinctive facial appearance, short stature, congenital heart defects, intellectual disability and ectodermal abnormalities such as sparse, fragile hair, hyperkeratotic skin lesions and a severe generalized ichthyosis-like condition (1). The cardiac defects observed in CFC syndrome include pulmonary valve stenosis, hypertrophic cardiomyopathy and atrial septal defects. Increased nuchal translucency/fatal cystic hygroma colli due to lymphatic defects is also occasionally observed in affected individuals (2). Our group as well as another group has identified germline BRAF mutations in 50–75% of patients with CFC syndrome (3–6). Other known CFC-causative genes include KRAS as well as MAP2K1 and MAP2K2 (MEK1 and MEK2, respectively) (3–6), all located in the same RAS–MAPK pathway that regulates cell differentiation, proliferation, survival and apoptosis (7). Germline mutations associated with RAS–MAPK pathway components cause partially overlapping disorders, including Noonan syndrome, Costello syndrome, LEOPARD syndrome, neurofibromatosis type 1 and Legius syndrome (neurofibromatosis type 1-like syndrome). These syndromes are now collectively termed RASopathies or RAS–MAPK syndromes (8–10).

BRAF is a serine threonine kinase which regulates the RAS–MAPK signaling pathway. Somatic BRAF mutations have been identified in 7% of human tumors, including melanoma, papillary thyroid carcinoma, colon cancer and ovarian cancer (11). The BRAF V600E mutation, located in the catalytic kinase domain (conserved region (CR) 3 domain), accounts for 90% of all somatic BRAF mutations. In contrast, BRAF V600E mutation has not been identified in CFC syndrome. Germline BRAF mutations in CR3 kinase domain, including G464R, G469E and L597V, were overlapping those in somatic mutations (4,5,12,13). In contrast, germline mutations in the CR1 domain have been rarely identified in somatic cancers. The most frequent mutations identified in CFC syndrome patients are substitutions of the residue Gln257 (p.Q257R and p.Q257K) in the CR1 domain, which account for ∼40% (13). Previous studies have shown that the activation of downstream signaling, including ELK transactivation, is weaker in cells expressing the Q257R mutation than in those expressing V600E (3).

Braf is ubiquitously expressed in murine organs at mid-gestation, and high levels of its expression are found in the brain and testes at adult stage (14,15). Braf knockout mice have been found to die at mid-gestation from vascular defects due to enlarged blood vessels and apoptotic death of dif ferentiated endothelial cells (16). Heterozygous knockin mice constitutively expressing V600E mutation have been found to exhibit embryonic lethality (17). Knockin mice expressing a hypomorphic BRAF V600E allele have been reported to show phenotypes partially overlapping those of CFC syndrome patients, including small size, craniofacial abnormalities and epileptic seizures (18). However, no mouse model for CFC syndrome expressing a Braf mutation in the CR1 domain has been generated and no therapeutic approach has been developed. In the present study, we generated knockin mice expressing CFC syndrome-associated Braf Q241R mutation, corresponding to BRAF Q257R mutation, in order to investigate the molecular pathogenesis and potential therapeutic possibilities for CFC syndrome.

RESULTS

Generation of a CFC syndrome mouse model

We have previously reported that the transcriptional activity of ELK, downstream of ERK, was enhanced by the transient overexpression of human BRAF Q257R in NIH3T3 cells (3). To verify whether the expression of mouse Braf Q241R enhances ELK transcription as BRAF Q257R, reporter assays were performed in NIH3T3 cells. The expression of Braf Q241R and that of V637E, which corresponds to BRAF V600E, were ∼2.7- and 8.4-fold higher than that of Braf WT, respectively (Fig. 1A). These results suggest that the Braf Q241R mutation is a gain-of-function mutation, although the activation is weaker than that observed in Braf V637E.

Figure 1.

Generation of Braf Q241R knockin mice. (A) NIH 3T3 cells were transfected with the ELK1-GAL4 vector, the GAL4-luciferase trans-reporter vector, phRLnull-luc control vector and each mouse Braf expression plasmid, and reporter activities were determined as described in Materials and Methods. Luciferase activities were normalized with phRLnull-luc activities, containing distinguishable R. reniformis luciferase. Data are the means ± SD (n = 4). p.V637E in mouse Braf corresponds to oncogenic p.V600E in human BRAF. *, P < 0.05, **, P < 0.01 versus WT. WT, wild type. (B) Exons (solid boxes), PGK-Neomycin (PGK-Neo) cassette (gray box), STOP transcriptional sequences (open box), loxp sites (arrowheads) and Flp recombination target sites (ellipses) are indicated. Cleavage sites for diagnostic enzymes (SacI, NcoI and AflII) and the probes (5′-, 3′- and Neo probe) used to identify the homologous recombination are indicated. The PGK-Neo cassette was removed by crossing with CAG-Cre transgenic mice (Braf+/+; Cre). The arrow indicates the positions of PCR primers used for genotyping of positive ES cells and mice. p. Q241R in mouse Braf corresponds to p.Q257R in human BRAF. DTA, diphtheria toxin A. (C) Southern blotting of ES cell clones. Genomic DNA from Braf+/+ and BrafQ241R Neo/+ ES cells was digested with SacI (5′ probe), NcoI (3′ probe) or AflII (Neo probe) and subjected to Southern blotting with a 5′, 3′ or Neo probe. The 5′, 3′ or Neo probe detects the 9.1-kb (Braf WT) and 6.1-kb (BrafQ241R Neo/+) SacI fragments, the 15.9 kb (Braf WT) and 9.2 kb (BrafQ241R Neo/+) NcoI fragments or the 8.6 kb (BrafQ241R Neo/+) AflII fragment, respectively. (D) RNA was isolated from the brain of Braf+/+; Cre and BrafQ241R/+; Cre embryos at E18.5, and reverse transcribed into cDNA. Sanger sequencing was carried out using the cDNA. The arrow indicates the Q241R mutation in Braf exon 7. (E and F) Protein extracts from whole-mouse embryos (E12.5 and E14.5) and brain (E16.5) (n = 4–5 of each genotype) were subjected to western blotting with the indicated antibodies. β-Actin is shown as a loading control. The arrowheads indicate the bands corresponding to each protein.

Figure 1.

Generation of Braf Q241R knockin mice. (A) NIH 3T3 cells were transfected with the ELK1-GAL4 vector, the GAL4-luciferase trans-reporter vector, phRLnull-luc control vector and each mouse Braf expression plasmid, and reporter activities were determined as described in Materials and Methods. Luciferase activities were normalized with phRLnull-luc activities, containing distinguishable R. reniformis luciferase. Data are the means ± SD (n = 4). p.V637E in mouse Braf corresponds to oncogenic p.V600E in human BRAF. *, P < 0.05, **, P < 0.01 versus WT. WT, wild type. (B) Exons (solid boxes), PGK-Neomycin (PGK-Neo) cassette (gray box), STOP transcriptional sequences (open box), loxp sites (arrowheads) and Flp recombination target sites (ellipses) are indicated. Cleavage sites for diagnostic enzymes (SacI, NcoI and AflII) and the probes (5′-, 3′- and Neo probe) used to identify the homologous recombination are indicated. The PGK-Neo cassette was removed by crossing with CAG-Cre transgenic mice (Braf+/+; Cre). The arrow indicates the positions of PCR primers used for genotyping of positive ES cells and mice. p. Q241R in mouse Braf corresponds to p.Q257R in human BRAF. DTA, diphtheria toxin A. (C) Southern blotting of ES cell clones. Genomic DNA from Braf+/+ and BrafQ241R Neo/+ ES cells was digested with SacI (5′ probe), NcoI (3′ probe) or AflII (Neo probe) and subjected to Southern blotting with a 5′, 3′ or Neo probe. The 5′, 3′ or Neo probe detects the 9.1-kb (Braf WT) and 6.1-kb (BrafQ241R Neo/+) SacI fragments, the 15.9 kb (Braf WT) and 9.2 kb (BrafQ241R Neo/+) NcoI fragments or the 8.6 kb (BrafQ241R Neo/+) AflII fragment, respectively. (D) RNA was isolated from the brain of Braf+/+; Cre and BrafQ241R/+; Cre embryos at E18.5, and reverse transcribed into cDNA. Sanger sequencing was carried out using the cDNA. The arrow indicates the Q241R mutation in Braf exon 7. (E and F) Protein extracts from whole-mouse embryos (E12.5 and E14.5) and brain (E16.5) (n = 4–5 of each genotype) were subjected to western blotting with the indicated antibodies. β-Actin is shown as a loading control. The arrowheads indicate the bands corresponding to each protein.

To investigate the gain-of-function effect of the Braf Q241R mutation on development, Braf Q241R knockin mice were generated (Fig. 1B). The targeting vector (Fig. 1B) was electroporated into ES cells and targeted clones were identified by Southern blotting (Fig. 1C). Appropriate ES cells were injected into BALB/c blastocysts and chimeras were obtained from six independent ES cell clones (hereafter referred to as BrafQ241R Neo/+). To induce ubiquitous expression of Braf Q241R in germ cells, the BrafQ241R Neo/+ mice were crossed with CAG-Cre transgenic mouse (Braf+/+; Cre) and genotyping was confirmed by PCR (Supplementary Material, Fig. S1). Furthermore, sequencing was performed to confirm that Cre recombination resulted in Braf Q241R expression (Fig. 1D).

To examine if cell signaling pathways, including ERK, JNK, p38 and PI3K–AKT pathways, were altered in BrafQ241R/+; Cre embryos, western blotting analysis was performed using cell extracts derived from whole-mouse embryos and brain. Protein levels of BRAF, CRAF, phosphorylated MEK and ERK in BrafQ241R/+; Cre whole embryos were similar to those of Braf+/+; Cre (Fig. 1E; Supplementary Material, Table S1), whereas phosphorylated MEK protein levels were higher in the brain of BrafQ241R/+; Cre embryos (Fig. 1F; Supplementary Material, Table S2). Unexpectedly, phosphorylated p38 and AKT (Thr308) protein levels were somewhat lower in BrafQ241R/+; Cre whole embryos at embryonic day (E) 14.5 (Fig. 1E; Supplementary Material, Table S1). These results suggest that BrafQ241R/+; Cre embryos at E14.5 show a decrease of phosphorylated p38 and AKT (Thr308) protein levels.

Germline expression of Braf Q241R results in embryonic/neonatal lethality

Genotype analysis of embryos from an intercross between Braf+/+; Cre and BrafQ241R Neo/+ mice showed no surviving BrafQ241R/+; Cre littermates at weaning, whereas Braf+/+, Braf+/+; Cre and BrafQ241R Neo/+ littermates survived (Table 1). A normal Mendelian ratio was observed by E14.5. However, the survival rate of BrafQ241R/+; Cre embryos dropped after E16.5. At E16.5, ∼9.8% of embryos (4 of 41) were grossly hemorrhagic and edematous such as nuchal translucency (Fig. 2A, Table 1). Other BrafQ241R/+; Cre embryos appeared normal (Fig. 2B) with no difference in body weight (data not shown). BrafQ241R/+; Cre embryos, which were delivered by cesarean section at E18.5 and E19.5, remained pale and without movement or gasped for breath with cyanotic appearance, resulting in death within a few hours. A few embryos showed mandibular hypoplasia (2 of 39, 5.1%) and kyphosis (Fig. 2C and D).

Table 1.

Genotyping of pups resulting from intercross between Braf+/+; Cre and BrafQ241R Neo/+ mice

Age Braf+/+ Braf+/+; Cre BrafQ241R Neo/+ BrafQ241R/+; Cre n P 
E12.5 24 29 23 23 99 0.80 
E13.5 14 6 (2 [1]) 31 0.08 
E14.5 19 22 (1) 23 11 (1 [1]) 75 0.19 
E16.5 57 60 55 34 (7 [4]) 206 0.04 
E18.5 16 23 20 0 (17 [4]) 59 <0.0001 
E19.5 11 16 11 0 (11 [1]) 38 <0.01 
Weaning (P21) 56 54 56 166 <0.0001 
Expected 25% 25% 25% 25%   
Age Braf+/+ Braf+/+; Cre BrafQ241R Neo/+ BrafQ241R/+; Cre n P 
E12.5 24 29 23 23 99 0.80 
E13.5 14 6 (2 [1]) 31 0.08 
E14.5 19 22 (1) 23 11 (1 [1]) 75 0.19 
E16.5 57 60 55 34 (7 [4]) 206 0.04 
E18.5 16 23 20 0 (17 [4]) 59 <0.0001 
E19.5 11 16 11 0 (11 [1]) 38 <0.01 
Weaning (P21) 56 54 56 166 <0.0001 
Expected 25% 25% 25% 25%   

Deviation from the expected Mendelian ratios was assessed by the χ2 test. The number of dead embryos is shown in parentheses. The number of edematous embryos is shown in brackets. P: postnatal day.

Figure 2.

Lethal phenotypes of BrafQ241R/+; Cre embryos. (A and B) Gross appearance of Braf+/+; Cre and BrafQ241R/+; Cre embryos at E16.5. (A) Arrowheads and arrows indicate hemorrhage and edema, respectively. The right panel shows BrafQ241R/+; Cre embryos with transmitted illumination. (C) Craniofacial structure of BrafQ241R Neo/+ and BrafQ241R/+; Cre embryos at E19.5. The arrow indicates mandibular hypoplasia. (D) Alcian Blue/Alizarin Red staining of BrafQ241R Neo/+ and BrafQ241R/+; Cre embryos at E19.5. The arrow, solid arrowhead and open arrowhead indicate mandibular hypoplasia, kyphosis and ossification in the interparietal bone, respectively. (E) H&E staining of liver sections of Braf+/+; Cre and BrafQ241R/+; Cre embryos at E18.5. The arrow indicates hepatic necrosis. The lower panel shows higher magnification views of hepatic necrosis. Scale bars in upper panels = 200 μm and in lower panels = 50 μm.

Figure 2.

Lethal phenotypes of BrafQ241R/+; Cre embryos. (A and B) Gross appearance of Braf+/+; Cre and BrafQ241R/+; Cre embryos at E16.5. (A) Arrowheads and arrows indicate hemorrhage and edema, respectively. The right panel shows BrafQ241R/+; Cre embryos with transmitted illumination. (C) Craniofacial structure of BrafQ241R Neo/+ and BrafQ241R/+; Cre embryos at E19.5. The arrow indicates mandibular hypoplasia. (D) Alcian Blue/Alizarin Red staining of BrafQ241R Neo/+ and BrafQ241R/+; Cre embryos at E19.5. The arrow, solid arrowhead and open arrowhead indicate mandibular hypoplasia, kyphosis and ossification in the interparietal bone, respectively. (E) H&E staining of liver sections of Braf+/+; Cre and BrafQ241R/+; Cre embryos at E18.5. The arrow indicates hepatic necrosis. The lower panel shows higher magnification views of hepatic necrosis. Scale bars in upper panels = 200 μm and in lower panels = 50 μm.

Gross observation showed increased heart size in BrafQ241R/+; Cre embryos at E16.5. At E18.5, BrafQ241R/+; Cre embryos revealed severe peripheral liver necrosis (15 of 17, 88%) with decreased liver size and liver weight (Fig. 2E; Supplementary Material, Fig. S2). At E16.5, decreased liver weight was already observed (data not shown), although the gross appearance of the liver appeared normal. To examine if delayed lung maturation causes neonatal lethality, the histology of lung in BrafQ241R/+; Cre embryos was examined at E18.5 and E19.5. Lungs of the mutant embryos appeared normal and were able to inflate, but ∼11.1% of embryos (1 of 9) showed alveolar hemorrhage (Supplementary Material, Fig. S3). Thyroid transcription factor-1 (TTF-1; lung epithelial cells marker), pro-surfactant protein C and PAS staining showed similar levels in BrafQ241R Neo/+ and BrafQ241R/+; Cre embryos (Supplementary Material, Fig. S4), suggesting that lung development and maturation are normal. Gross observation suggests that BrafQ241R/+; Cre embryos show embryonic/neonatal lethality, cardiomegaly, liver necrosis, edema and craniofacial abnormalities.

BrafQ241R/+; Cre embryos display various heart defects

Because BrafQ241R/+; Cre embryos showed cardiomegaly and liver necrosis, possibly due to heart failure (Fig. 2E), detailed histological analysis of the heart at different embryonic stages was conducted. At E12.5, the hearts of BrafQ241R/+; Cre embryos appeared normal (Supplementary Material, Fig. S5A), but showed an enlarged pulmonary valve and a dramatic increase in the density of trabeculae (hypertrabeculation) at E14.5 (Supplementary Material, Fig. S5B). At E16.5, 13 of 14 (93%) BrafQ241R/+; Cre embryos (excluding edematous embryos) had various heart defects (Supplementary Material, Tables S3 and S4). Hypertrophy of pulmonary, tricuspid and mitral valves was present in 7, 8 and 9 of 14 embryos, respectively (Fig. 3A; Supplementary Material, Tables S3 and S4). In particular, hypertrophy in pulmonary valve leaflets was prominent, plugging the entire space of the pulmonary valve ring (Fig. 3B). Other heart defects observed in BrafQ241R/+; Cre embryos included ventricular septal defect (VSD) in 2 of 14 embryos (Fig. 3A), abnormal endocardial cushion in 2 (Fig. 3A), hypertrabeculation in 3 (Fig. 3A), epicardial blisters in 2 (Fig. 3A and C), a thickened trabecular layer and thinned compact layer in the left, right or combined myocardium (noncompaction: one case of cardiomyopathy accompanied by cardiac hypertrophy) in 4 (Fig. 3D) and hypoplasia of the coronary arteries in 3. The ventricular radius and the thickness of the pulmonary and tricuspid valves were significantly higher in BrafQ241R/+; Cre embryos, suggesting cardiac enlargement and thickened pulmonary and tricuspid valves (Fig. 3E). These results suggest that BrafQ241R/+; Cre embryos develop various congenital heart defects, which almost certainly contributes to embryonic lethality.

Figure 3.

Cardiac phenotype of BrafQ241R/+; Cre embryos. (AD) H&E staining of sequential anterior to posterior sections of embryonic hearts from Braf+/+; Cre and BrafQ241R/+; Cre at E16.5. A dramatic increase in density of trabeculae (arrows), enlarged valves (solid arrowheads), VSD (open arrowhead) and abnormal endocardial cushion (asterisk) are observed. Scale bars 500 μm (A) and 100 μm (B–D). (B) Higher magnification of the pulmonary valves in Braf+/+; Cre and BrafQ241R/+; Cre embryos. (C) Higher magnification of the boxed region in Figure 3A showing the epicardial blisters (asterisks) in BrafQ241R/+; Cre embryos at E16.5. (D) Representative image of noncompaction in hearts from BrafQ241R/+; Cre embryos at E16.5. (E) The ventricular radius and the thicknesses of the cardiac valve leaflets were measured at their largest diameter in serial sections of Braf+/+; Cre and BrafQ241R/+; Cre embryos at E16.5. Data are the means ± SD (Braf+/+; Cre (n = 9) and BrafQ241R/+; Cre (n = 14)). *P < 0.05, **P < 0.01 versus Braf+/+; Cre. LV, left ventricle; RV, right ventricle; LA, left atrium; RA, right atrium; PV, pulmonary valve; AV, aortic valve; TV, tricuspid valve; MV, mitral valve; CL, compact layer; TL, trabecular layer.

Figure 3.

Cardiac phenotype of BrafQ241R/+; Cre embryos. (AD) H&E staining of sequential anterior to posterior sections of embryonic hearts from Braf+/+; Cre and BrafQ241R/+; Cre at E16.5. A dramatic increase in density of trabeculae (arrows), enlarged valves (solid arrowheads), VSD (open arrowhead) and abnormal endocardial cushion (asterisk) are observed. Scale bars 500 μm (A) and 100 μm (B–D). (B) Higher magnification of the pulmonary valves in Braf+/+; Cre and BrafQ241R/+; Cre embryos. (C) Higher magnification of the boxed region in Figure 3A showing the epicardial blisters (asterisks) in BrafQ241R/+; Cre embryos at E16.5. (D) Representative image of noncompaction in hearts from BrafQ241R/+; Cre embryos at E16.5. (E) The ventricular radius and the thicknesses of the cardiac valve leaflets were measured at their largest diameter in serial sections of Braf+/+; Cre and BrafQ241R/+; Cre embryos at E16.5. Data are the means ± SD (Braf+/+; Cre (n = 9) and BrafQ241R/+; Cre (n = 14)). *P < 0.05, **P < 0.01 versus Braf+/+; Cre. LV, left ventricle; RV, right ventricle; LA, left atrium; RA, right atrium; PV, pulmonary valve; AV, aortic valve; TV, tricuspid valve; MV, mitral valve; CL, compact layer; TL, trabecular layer.

BrafQ241R/+; Cre embryo hearts exhibit enhancement of cell proliferation, ERK signaling activation and decrease of phosphorylated p38 and AKT

To examine if heart defects observed in BrafQ241R/+; Cre embryos are caused by increased cell proliferation and/or reduced cell death, cell proliferation was analyzed by phospho-histone H3 (pHH3) immunostaining and cell death by TUNEL assay. At E13.5, regarding heart abnormalities in each embryos, the number of pHH3-positive-stained cells varied. pHH3-positive-stained cells in the interventricular septum and myocardium increased in BrafQ241R/+; Cre embryos (Fig. 4A and B). At E16.5, the nucleus of pHH3-positive cells increased in the interventricular septum in embryos with VSD (Fig. 4C). BrafQ241R/+; Cre embryos had more pHH3-positive cells in pulmonary valves (Fig. 4D). In contrast to cell proliferation, hardly any cells undergoing apoptosis were observed in either Braf+/+; Cre or BrafQ241R/+; Cre at E13.5 and E16.5 (data not shown). These results suggest that the cell proliferation state depends on heart abnormalities in each embryo at E16.5 and that the increased staining for pHH3 in the interventricular septum was associated with VSD.

Figure 4.

Increased cell proliferation and altered multiple signaling pathways in BrafQ241R/+; Cre embryo hearts. (AD) Immunostaining for pHH3 in the myocardium (A), interventricular septum (B and C) and pulmonary valves (D) of Braf+/+; Cre and BrafQ241R/+; Cre embryos at E13.5 (A and B) and E16.5 (C and D). The arrows indicate representative positive cells. BrafQ241R/+; Cre embryos with or without VSD are shown in closed cycles or open circles, respectively. Scale bars 50 μm (A–C). Data are means ± SD (A and B) Braf+/+; Cre (n = 5) and BrafQ241R/+; Cre (n = 5). (C and D) Braf+/+; Cre (n = 3) and BrafQ241R/+; Cre (n = 6). *P < 0.05 versus Braf+/+; Cre. (E) Protein extracts (400 μg) of the hearts from Braf+/+; Cre and BrafQ241R/+; Cre embryos at E16.5 were subjected to Phospho-Kinase Antibody Array. Results are representative of gene spots that showed significant changes in 45 phosphorylated proteins. (F) Western blotting of the hearts from Braf+/+; Cre and BrafQ241R/+; Cre embryos at E16.5 (pooled samples; Braf+/+; Cre (n = 5), BrafQ241R/+; Cre (n = 5)). β-Actin is shown as a loading control. The arrowheads indicate the bands corresponding to each protein. (G) Cardiac mRNA levels were determined by quantitative reverse transcription–PCR. mRNA levels were normalized by those of Gapdh, and those in Braf+/+; Cre at E18.5 are set at 1. Data are the means ± SD (Braf+/+; Cre (n = 10) and BrafQ241R/+; Cre (n = 6)). ***P < 0.001, ****P < 0.0001 versus Braf+/+; Cre. Etv1, Etv4, Etv5, Myh6 and Myh7 encode ER81, Pea3, ERM, α-MHC and β-MHC, respectively.

Figure 4.

Increased cell proliferation and altered multiple signaling pathways in BrafQ241R/+; Cre embryo hearts. (AD) Immunostaining for pHH3 in the myocardium (A), interventricular septum (B and C) and pulmonary valves (D) of Braf+/+; Cre and BrafQ241R/+; Cre embryos at E13.5 (A and B) and E16.5 (C and D). The arrows indicate representative positive cells. BrafQ241R/+; Cre embryos with or without VSD are shown in closed cycles or open circles, respectively. Scale bars 50 μm (A–C). Data are means ± SD (A and B) Braf+/+; Cre (n = 5) and BrafQ241R/+; Cre (n = 5). (C and D) Braf+/+; Cre (n = 3) and BrafQ241R/+; Cre (n = 6). *P < 0.05 versus Braf+/+; Cre. (E) Protein extracts (400 μg) of the hearts from Braf+/+; Cre and BrafQ241R/+; Cre embryos at E16.5 were subjected to Phospho-Kinase Antibody Array. Results are representative of gene spots that showed significant changes in 45 phosphorylated proteins. (F) Western blotting of the hearts from Braf+/+; Cre and BrafQ241R/+; Cre embryos at E16.5 (pooled samples; Braf+/+; Cre (n = 5), BrafQ241R/+; Cre (n = 5)). β-Actin is shown as a loading control. The arrowheads indicate the bands corresponding to each protein. (G) Cardiac mRNA levels were determined by quantitative reverse transcription–PCR. mRNA levels were normalized by those of Gapdh, and those in Braf+/+; Cre at E18.5 are set at 1. Data are the means ± SD (Braf+/+; Cre (n = 10) and BrafQ241R/+; Cre (n = 6)). ***P < 0.001, ****P < 0.0001 versus Braf+/+; Cre. Etv1, Etv4, Etv5, Myh6 and Myh7 encode ER81, Pea3, ERM, α-MHC and β-MHC, respectively.

To examine if the cardiac signaling pathways were altered in BrafQ241R/+; Cre embryos, the activation of kinases was screened in various signaling pathways using a phospho-kinase array followed by western blotting of the lysates from hearts of BrafQ241R/+; Cre embryos at E16.5 (Fig. 4E and F; Supplementary Material, Fig. S6). No changes in phosphorylated ERK protein levels in both the phospho-kinase array and western blotting were observed. In contrast, phosphorylated p38, AKT (Ser473) and AKT (Thr308) protein levels, which are not direct targets of BRAF, were relatively lower in BrafQ241R/+; Cre embryos than in Braf+/+; Cre, which was confirmed by western blotting. To verify the activation of transcription factors downstream of ERK, the expression of ELK1 and the PEA3 (polyoma enhancer activator 3) subfamily Ets transcription factors were examined by quantitative real-time PCR, these expressions being known as transcriptional targets of FGF signaling-mediated activation of ERK in heart and oncogenic BRAF signaling in melanoma (19,20). At E13.5, E16.5 and E18.5, cardiac mRNA levels of Etv1, Etv4 and Etv5, but not Elk1, were significantly higher in BrafQ241R/+; Cre embryos than those in Braf+/+; Cre (Fig. 4G; Supplementary Material, Fig. S7). Next, we investigated the influence of genes responsible for hypertrophic cardiomyopathy and cardiac development in BrafQ241R/+; Cre embryos at E18.5, which exhibited a cardiomyopathy phenotype, such as cardiac enlargement and noncompaction (Fig. 3D and E) and structural abnormalities, including VSD. No differences in mRNA levels of cardiomyopathy-specific genes (Myh6 and Myh7) and genes related to the heart formation and development (Gata4 and Nkx2.5) were observed (Fig. 4G). These results suggest that ERK activation, including increased mRNA levels of Ets transcription factors, and decreased levels of p38 and AKT exist in heart tissues of BrafQ241R/+; Cre embryos.

BrafQ241R/+; Cre embryos develop lymphangiectasia

Patients with RASopathies, including CFC syndrome and Noonan syndrome, exhibit nuchal translucency, which is subcutaneous fluid collection in the fetal neck visualized by ultrasonography. Nuchal translucency is caused by distended jugular lymphatic sacs (JLSs), which result from a disturbance in differentiation of lymphatic endothelial cells (21,22). We hypothesized that the hemorrhage and edema in BrafQ241R/+; Cre embryos may be caused by defective lymphatic development. Histological examination revealed distended and blood-filled JLSs in BrafQ241R/+; Cre embryos but not in Braf+/+; Cre embryos at E12.5 and E16.5 (Fig. 5A and B; Supplementary Material, Fig. S8A). The primary lymphatic sacs are remodeled to produce a hierarchically organized network of lymphatic capillaries and collecting lymph vessels at stages between E14.5 and postnatal stages (23). The JLSs are hardly observed in mouse embryos at E16.5. In BrafQ241R/+; Cre embryos at E16.5, cavities such as the JLSs of mouse embryos from E12.5 to E14.5 were observed (Fig. 5B), suggesting defective lymphatic development from the cardinal vein in BrafQ241R/+; Cre embryos. To examine the network formation of blood and lymphatic vessels, we performed immunostaining using antibodies against lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1; lymphatic endothelial cell-specific marker), α-SMA for staining of vessels with smooth muscle and CD31 (platelet-endothelial cell adhesion molecule-1, PECAM-1) for staining of vascular endothelial cells. At E12.5, the cells lining JLSs in both Braf+/+; Cre and BrafQ241R/+; Cre embryos were positive for LYVE-1 (Fig. 5C), whereas slightly CD31-positive cells were detected in JLSs and the jugular vein (Fig. 5D). No α-SMA expression was observed (Supplementary Material, Fig. S8B). At E16.5, the cavities such as the JLSs in BrafQ241R/+; Cre embryos were negative for LYVE-1, α-SMA and CD31 (Fig. 5E; Supplementary Material, Fig. S8C and D), but the subcutaneous lymphatic vessels were markedly positive for LYVE-1 (Fig. 5F; Supplementary Material, Fig. S8E). These results indicate that BrafQ241R/+; Cre embryos show defective lymphatic development from the cardinal vein, leading to distention of the JLSs, dilated lymphatic vessels and edema.

Figure 5.

Abnormal lymphatic development in BrafQ241R/+; Cre embryos. (A and B) Transverse sections of Braf+/+; Cre and BrafQ241R/+; Cre embryos at E12.5 (A) and E16.5 (B) stained with H&E. Lower panels show high-magnification views of jugular lymph sac (A). The arrows (A) and arrowheads (B) indicate blood cells in jugular lymph sacs and the regions which are similar to the jugular lymph sacs or jugular veins of embryos at E12.5, respectively. Scale bars 1 mm (in upper panels, A), 100 μm (in lower panels, A) and 2 mm (B). (C–F) Sections of Braf+/+; Cre and BrafQ241R/+; Cre embryos at E12.5 (C and D) and E16.5 (E and F) stained with antibodies against lymphatic endothelial markers, LYVE-1 (C, E and F) or CD31 (D). (F) Subcutaneous lymphatic vessels. jls, jugular lymph sac; jv, jugular vein.

Figure 5.

Abnormal lymphatic development in BrafQ241R/+; Cre embryos. (A and B) Transverse sections of Braf+/+; Cre and BrafQ241R/+; Cre embryos at E12.5 (A) and E16.5 (B) stained with H&E. Lower panels show high-magnification views of jugular lymph sac (A). The arrows (A) and arrowheads (B) indicate blood cells in jugular lymph sacs and the regions which are similar to the jugular lymph sacs or jugular veins of embryos at E12.5, respectively. Scale bars 1 mm (in upper panels, A), 100 μm (in lower panels, A) and 2 mm (B). (C–F) Sections of Braf+/+; Cre and BrafQ241R/+; Cre embryos at E12.5 (C and D) and E16.5 (E and F) stained with antibodies against lymphatic endothelial markers, LYVE-1 (C, E and F) or CD31 (D). (F) Subcutaneous lymphatic vessels. jls, jugular lymph sac; jv, jugular vein.

Treatment with a MEK inhibitor and/or histone demethylase inhibitors prevents embryonic lethality in BrafQ241R/+; Cre embryos

MEK inhibitor, PD0325901, treatment is known to rescue the embryonic lethality of Noonan syndrome model mice (24). Pregnant Braf+/+; Cre mice were treated with various compounds to see whether this would result in recovery from embryonic lethality (Table 2). Male BrafQ241R Neo/+ mice were crossed with female Braf+/+; Cre mice, and pregnant mice were intraperitoneally injected with dimethylsulfoxide (vehicle), PD0325901 [0.5 or 1.0 mg of body weight (mg/kg)], MAZ51 (VEGFR3 inhibitor; 1.0, 2.0 or 5.0 mg/kg), sorafenib (BRAF, VEGFR, PDGFR multikinase inhibitor; 5.0 mg/kg), lovasatin (HMG-CoA reductase and farnesyl transferase inhibitor; 5.0 mg/kg) or everolimus (mTOR inhibitor; 0.1 mg/kg), daily from E10.5 to E18.5. PD0325901 treatment (0.5 mg/kg) modestly rescued the embryonic lethality of BrafQ241R/+; Cre mice (2 of 30). Seven embryos also survived for 3 weeks with prenatal treatment of PD0325901 (1.0 mg/kg) (7 of 37, P = 0.32, χ2 test for deviation from the Mendelian ratios). PD0325901-treated BrafQ241R/+; Cre embryos appeared normal without edema and mandibular hypoplasia (0 of 31 at E16.5 to P0), whereas other genotype mice, excluding BrafQ241R/+; Cre treated with PD0325901, showed teratogenic effects, including open eyes (Supplementary Material, Fig. S9), edema, enlarged semilunar valves and atrioventricular valves (data not shown). Other compounds had no effect on the recovery of embryonic lethality in BrafQ241R/+; Cre embryos. Thus, PD0325901 treatment prevented embryonic lethality in BrafQ241R/+; Cre embryos and could ameliorate edema and mandibular hypoplasia.

Table 2.

Rescue of embryonic lethality in BrafQ241R/+; Cre embryos by MEK inhibitor, histone demethylase inhibitor or these combined treatment

Compound Dose (mg/kg body weight) Genotype (3 weeks)
 
na nb nc P 
Braf+/+ Braf+/+; Cre BrafQ241R Neo/+ BrafQ241R/+; Cre 
DMSO (vehicle) – 14 30 5.0 <0.01 
PD0325901 0.5 14 30 13 2.3 0.02 
 1.0 11 13 37 14 2.6 0.32 
MAZ51 1.0 11 28 4.7 0.02 
 2.0 10 14 31 5.2 <0.01 
 5.0 10 11 28 11 2.5 0.01 
Sorafenib 5.0 12 15 35 13 2.7 <0.01 
Lovastatin 5.0 19 17 44 10 4.4 <0.01 
Everolimus 0.1 21 2.3 0.04 
NCDM-32b 2.0 12 25 2.8 <0.01 
 5.0 10 10 14 35 11 3.2 0.02 
 10.0 11 10 19 40 4.4 <0.01 
GSK-J4 5.0 18 14 41 11 3.7 <0.01 
 10.0 16 26 20 62 23 2.7 <0.01 
PD0325901 + GSK-J4 0.5 + 5.0 13 31 10 3.1 0.14 
Compound Dose (mg/kg body weight) Genotype (3 weeks)
 
na nb nc P 
Braf+/+ Braf+/+; Cre BrafQ241R Neo/+ BrafQ241R/+; Cre 
DMSO (vehicle) – 14 30 5.0 <0.01 
PD0325901 0.5 14 30 13 2.3 0.02 
 1.0 11 13 37 14 2.6 0.32 
MAZ51 1.0 11 28 4.7 0.02 
 2.0 10 14 31 5.2 <0.01 
 5.0 10 11 28 11 2.5 0.01 
Sorafenib 5.0 12 15 35 13 2.7 <0.01 
Lovastatin 5.0 19 17 44 10 4.4 <0.01 
Everolimus 0.1 21 2.3 0.04 
NCDM-32b 2.0 12 25 2.8 <0.01 
 5.0 10 10 14 35 11 3.2 0.02 
 10.0 11 10 19 40 4.4 <0.01 
GSK-J4 5.0 18 14 41 11 3.7 <0.01 
 10.0 16 26 20 62 23 2.7 <0.01 
PD0325901 + GSK-J4 0.5 + 5.0 13 31 10 3.1 0.14 

Male BrafQ241R Neo/+ mice were crossed with female Braf+/+; Cre mice, and pregnant mice were intraperitoneally injected with vehicle or various compounds shown daily from E10.5 to E18.5. Deviation from the expected Mendelian ratios was assessed by χ2 test. na, the total number of acquired pups. nb, the total number of treated female Braf+/+; Cre mice. nc, the average number of survived pups at weaning (na/nb).

Epigenetic regulation of gene expression, such as histone acetylation and histone methylation, plays a crucial role in the transcriptional regulation of cell differentiation, development, the inflammatory response and cancer (25). Recently, a histone deacetylase inhibitor, SAHA [vorinostat (Zolinza)], has been used in the treatment of lymphomas and solid tumors. Recent studies have suggested the association of UTX and JMJD3, a histone H3 lysine 27 (H3K27) demethylase, with heart development (26–28). We therefore tested whether treatment using these compounds leads to the rescue of embryonic lethality (Table 2). SAHA treatment had no effect (data not shown); however, one embryo survived for 3 weeks with prenatal treatment of GSK-J4 (inhibitors of histone H3K27 demethylase UTX and JMJD3; 5.0 mg/kg) (25) or NCDM-32b (inhibitor of histone H3K9 demethylase JMJD2C; 5.0 mg/kg) (29). Moreover, co-treatment with GSK-J4 (5.0 mg/kg) and PD0325901 (0.5 mg/kg) further increased the number of BrafQ241R/+; Cre mice alive at weaning (5 of 31, P = 0.14). The teratogenic effects, which were frequently observed in PD0325901 treatment, were not observed in the co-treatment with GSK-J4 and PD0325901.

We further investigated whether co-treatment with PD0325901 and GSK-J4 prevented heart defects in BrafQ241R/+; Cre embryos. Co-treatment with PD0325901 and GSK-J4, but not PD0325901 treatment (1.0 mg/kg) alone, ameliorated enlarged pulmonary, tricuspid and mitral valves in BrafQ241R/+; Cre embryos (Fig. 6A and B). However, no difference in the frequency of heart defects, including VSD, hypertrabeculation, epicardial blisters and noncompaction, was observed. It is noteworthy that treatment with PD0325901 or GSK-J4 alone or the co-treatment reversed the decrease of phosphorylated p38 protein levels (Fig. 6C; Supplementary Material, Fig. S10). These results suggest that combination treatment with PD0325901 and GSK-J4 prevents embryonic lethality, enlarged cardiac valves and decreased phosphorylated p38 in BrafQ241R/+; Cre embryos.

Figure 6.

Influence of co-treatment with PD0325901 and GSK-J4 on the cardiac phenotype and signaling of BrafQ241R/+; Cre embryos. (A and B) Sequential sections of embryonic hearts from Braf+/+; Cre and BrafQ241R/+; Cre at E16.5 stained H&E. (A) Histological sections of pulmonary valves. (B) The ventricular radius and the thicknesses of the cardiac valve leaflets were measured at their largest diameter in serial sections. Data are means ± SD (vehicle; Braf+/+; Cre (n = 5), BrafQ241R/+; Cre (n = 5). PD0325901 + GSK-J4; Braf+/+; Cre (n = 7), BrafQ241R/+; Cre (n = 11)). *P < 0.05, ***P < 0.001 (Tukey–Kramer test). NS, not significant. (C) Western blotting of the hearts from Braf+/+; Cre and BrafQ241R/+; Cre embryos at E16.5 (vehicle-treated pooled samples; Braf+/+; Cre (n = 8), BrafQ241R/+; Cre (n = 8). PD0325901 + GSK-J4-treated pooled samples; Braf+/+; Cre (n = 8), BrafQ241R/+; Cre (n = 7)). β-Actin is shown as a loading control. The arrowheads indicate the bands corresponding to each protein.

Figure 6.

Influence of co-treatment with PD0325901 and GSK-J4 on the cardiac phenotype and signaling of BrafQ241R/+; Cre embryos. (A and B) Sequential sections of embryonic hearts from Braf+/+; Cre and BrafQ241R/+; Cre at E16.5 stained H&E. (A) Histological sections of pulmonary valves. (B) The ventricular radius and the thicknesses of the cardiac valve leaflets were measured at their largest diameter in serial sections. Data are means ± SD (vehicle; Braf+/+; Cre (n = 5), BrafQ241R/+; Cre (n = 5). PD0325901 + GSK-J4; Braf+/+; Cre (n = 7), BrafQ241R/+; Cre (n = 11)). *P < 0.05, ***P < 0.001 (Tukey–Kramer test). NS, not significant. (C) Western blotting of the hearts from Braf+/+; Cre and BrafQ241R/+; Cre embryos at E16.5 (vehicle-treated pooled samples; Braf+/+; Cre (n = 8), BrafQ241R/+; Cre (n = 8). PD0325901 + GSK-J4-treated pooled samples; Braf+/+; Cre (n = 8), BrafQ241R/+; Cre (n = 7)). β-Actin is shown as a loading control. The arrowheads indicate the bands corresponding to each protein.

DISCUSSION

In this study, we generated heterozygous Braf Q241R-expressing mice, which exhibited embryonic and postnatal lethality due to liver necrosis, skeletal abnormalities, lymphatic defects and various cardiac defects, including cardiomegaly, noncompaction, enlarged cardiac valves and hypertrabeculation. Increased expression of Ets transcription factors and decreased expression of cardiac phosphorylated p38 in embryonic heart tissues were observed. PD0325901 treatment, in part, rescued embryonic and postnatal lethality in BrafQ241R/+; Cre mice. One pup in BrafQ241R/+; Cre also survived until P21 with treatment of GSK-J4 or NCDM-32b. PD0325901 treatment, but not GSK-J4 and NCDM-32b treatment, ameliorated edema and mandibular hypoplasia. Moreover, PD0325901 co-treatment with GSK-J4 further rescued embryonic lethality with recovered hypertrophy of pulmonary, tricuspid and mitral valves and the decreased expression of phosphorylated p38. Taken together, mice expressing a development-specific Braf Q241R mutation will be useful to further clarify the pathogenesis of CFC syndrome and to develop therapeutic approaches.

Patients with RASopathies are characterized by generalized abnormalities of lymphatic development. Fetuses with RASopathies have been shown to be characterized by hydrops, pleural effusions, increased nuchal translucency due to distended JLS and cystic hygroma in utero (30–32). Children and adults with RASopathies show generalized lymphedema, peripheral lymphoedema or pulmonary lymphangiectasia (33). Our new model, BrafQ241R/+; Cre mice, showed embryonic and postnatal lethality and exhibited multiple developmental defects in the lymphatic system, including hydrops, distended JLS and subcutaneous lymphatic vessels. In contrast, mice of other knockin mouse models for RASopathies survived to adulthood and have not shown the defects in lymphatic system (34–36). Thus, for the first time our new model BrafQ241R/+; Cre mice demonstrated the developmental lymphatic defects, which are the common features observed in RASopathies, in knockin mouse models for RASopathies.

Dysregulation of the RAS–MAPK pathway is a common underlying mechanism of RASopathies. However, a variety of compounds, including the RAS–MAPK pathway and other signaling pathways, has been effective for ameliorating the defects in previous knockin mouse models of RASopathies. MEK inhibitors have been found to ameliorate the cardiac defects and skeletal features in mice expressing SOS1 and RAF1 mutations (24,35). Angiotensin II inhibitor ameliorates the phenotypes of hypertension, vascular remodeling and fibrosis of the kidney and heart in mice expressing HRAS G12V mutation (36), and mTOR inhibitor ameliorates hypertrophic cardiomyopathy in a mouse model of LEOPARD syndrome, expressing a catalytically inactive mutation in SHP2 (34). We examined a variety of compounds, including anti-cancer agents, MEK inhibitor, mTOR inhibitor, VEGFR3 inhibitor, BRAF inhibitor and farnesyl transferase inhibitor using our BrafQ241R/+; Cre mice. Treatment with MEK inhibitor, but not mTOR inhibitor, in BrafQ241R/+; Cre mice ameliorated embryonic lethality and skeletal abnormalities, suggesting that the pathogenesis of the disease is similar to those in SOS1 and RAF1 mutations. Thus, our new BrafQ241R/+; Cre mice will be useful to screen various compounds for therapeutic approaches to RASopathies.

The exact mechanisms by which the single treatment of histone demethylase inhibitor or co-treatment of MEK inhibitor and histone demethylase inhibitor were effective for BrafQ241R/+; Cre mice have not yet been characterized. Lysine modification of histone 3, acetylation and methylation, is associated with gene activation or silencing (37). In gene expression, inactive genes show methylation at lysine 27, and permanently silenced genes frequently are characterized by methylation at lysine 9 (37). Histone H3K27 methylase, Ezh2, conditional knockout mice in cardiomyocytes have been reported to show abnormal heart development, such as noncompaction and excessive trabeculation (38). Meanwhile, deletion of histone H3K27 demethylase, UTX, has been identified in individuals with Kabuki syndrome, who showed distinctive facial appearance and congenital heart disease (39). H3K9 methyltransferases, G9a and GLP, have been shown to be essential for cardiac morphogenesis (40). It is of note that the balance between methylation and demethylation of H3 is required for normal cardiac differentiation. De novo mutations in SMAD2, a transcription factor which regulates H3K27 methylation in embryonic left–right organizer, have been identified in children with congenital heart disease (28). SMAD2, which is regulated by ERK (41), has been found to bind to H3K27 demethylase JMJD3, and regulate H3K27 methylation (28), suggesting that the histone demethylase JMJD3 is associated with heart development in humans by indirect regulation of ERK. In addition, constitutively activated BRAF and RAS mutants, through ERK activation, have been shown to induce JMJD3 and EZH2 expression (42,43). These observations suggest that activation of BRAF or ERK is associated with histone H3K27 modification, regulating cardiac development. In this study, the total content of the H3K27me3 in heart tissues of BrafQ241R/+; Cre or BrafQ241R/+; Cre mice after GSK-J4 co-treatment with PD0325901 was comparable with that of Braf+/+; Cre mice (data not shown). Furthermore, the histone H3K27 demethylase activity of lysates from BrafQ241R/+; Cre embryos at E14.5 was comparable with that of Braf+/+; Cre (data not shown). Further analysis of H3K9 and H3K27 modification status on individual genes will clarify the mechanism by which histone demethylase inhibitor is effective against embryonic and postnatal lethality and developmental defects in BrafQ241R/+; Cre mice.

MEK inhibitor treatment or crossing with ERK1 knockout mice has improved the hypertrophy of cardiac valves in Noonan syndrome model mice with a SOS1 or PTPN11 mutation (24,44). In contrast, treatment of MEK inhibitor did not lead to the amelioration of enlarged cardiac valves in BrafQ241R/+; Cre embryos. Furthermore, other mice, excluding BrafQ241R/+; Cre, treated with MEK inhibitor showed enlarged cardiac valves (data not shown), suggesting that the vital nature of MEK/ERK signaling balance in cardiac valve development. Given that no MEK inhibitor activity nor the inhibition activity of other protein kinases has been reported in GSK-J1 (GSK-J4 sodium salt) (25), these results suggest that not only MEK/ERK signaling balance but also histone H3K27 modification can play a crucial role in the normal development of cardiac valve in BrafQ241R/+; Cre embryos.

The natural history and the frequency of tumors in adult CFC patients have not been fully elucidated (6). Since molecular analysis became available, three individuals with BRAF mutation have been reported to have developed acute lymphoblastic leukemia and non-Hodgkin lymphoma (6). Knockin mice expressing BRAF L597V mutation survived to adulthood and showed multiple Noonan syndrome/CFC syndrome phenotypes, including short stature, facial dysmorphia and cardiac enlargement (12). The L597V is located in the CR3 kinase domain and leads to 2-fold elevated BRAF kinase activity (45). The L597V mutation has been identified in 11 somatic cancers (COSMIC; http://cancer.sanger.ac.uk/cancergenome/projects/cosmic/) and three patients with Noonan syndrome (13,46,47), which generally shows milder phenotype than that in CFC syndrome. In contrast, Q257R mutation is located in the CR1 domain and has been identified in 40% of CFC syndrome, not in cancers. Our ELK transactivation study has shown that level of ELK transactivation in Q257R was a half of V600E (3). The previous report showed that BRAF Q257R has increased BRAF kinase activity compared with WT and the activity was as high as that of the V600E (4). It is possible that differences in kinase activity and/or the effect on downstream pathways could cause the phenotypic differences in these knockin mice. Surviving BrafQ241R/+; Cre mice in the PD0325901 treatment showed distinctive facial appearance, abnormal dental occlusion, reduced postnatal length and weight, kyphosis and skin disease, which are similar to CFC syndrome phenotype (data not shown) (1,48). BrafQ241R/+; Cre mice also survived to adulthood when these mice (C57BL/6J background) were crossed with ICR or BALB/c mice (unpublished data). Further studies will be necessary to examine if adult BrafQ241R/+; Cre mice show phenotypes similar to patients with CFC syndrome, including seizures and tumor development.

The potential mechanism of activation and downregulation of multiple signaling pathways in BrafQ241R/+; Cre embryos is unclear. In additional studies, we performed microarray analysis and quantitative real-time PCR using heart tissues from BrafQ241R/+; Cre embryos at E13.5 or E16.5. Interestingly, mRNA levels of dual specificity phosphatase (Dusp) 2, 4 and 6, that inactivate ERK, p38 or JNK, and Spry 1, which inhibits the RAS–MAPK signaling pathway, were significantly higher in BrafQ241R/+; Cre embryos than those in Braf+/+; Cre (data not shown). In the present study, constitutive activation of phosphorylated ERK was not clearly observed in whole embryos and heart tissues from BrafQ241R/+; Cre. These results suggest that increased mRNA levels of Dusp 2, 4, 6 and Spry 1 and decreased expression of phosphorylated p38 in embryonic heart could represent a negative feedback mechanism for normalizing constitutive ERK activation in BrafQ241R/+; Cre embryos.

In summary, Braf Q241R-expressing mice provided an effective tool for studying the pathogenesis of CFC syndrome. It was found for the first time that combination treatment with PD0325901 and GSK-J4 is efficacious for the treatment of mice with the activation of the RAS–MAPK pathway. At present, clinical trials of a new MEK inhibitor, MEK162, are now being conducted to investigate the efficacy and safety of its use in Noonan syndrome with hypertrophic cardiomyopathy as well as in individuals with solid tumors, while no clinical trial of histone H3K27 demethylase inhibitor has been performed. Given that BRAF mutations cause cancer, combination therapy with MEK inhibitors and histone H3K27 demethylase inhibitors can be effective not only for the treatment of patients with RASopathies but also for the treatment of BRAF mutation-associated cancer in the future.

MATERIALS AND METHODS

Generation of BrafQ241R knockin mice

To construct the targeting vector for BrafQ241R knockin mice, a short arm containing Braf exon 5 and 6 (NotI-SacII genomic DNA fragment), a long arm including exon 7, 8 (XmaI–BamHI genomic DNA fragment) and the downstream of exon 8 (BamHI–SaclI) were amplified using a Roswell Park Cancer Institute-23 BAC clone. The DNA fragments were ligated into the pBSIISK+ vector. The Braf Q241R (exon 7) mutation was introduced by site-directed mutagenesis. The Psp0MI–XhoI site was used to insert PGK-Neo-STOP cassette flanked by loxP sites. The targeting vector was linearized with SalI and electroporated into ES cells (C57BL/6J background). To confirm correctly targeted ES clones, we performed genotyping, sequencing and the test of the Cre-mediated recombination system. Furthermore, homologous recombinants were confirmed by Southern blotting using 5′, 3′ and Neo probes. For this experiment, genomic DNA was digested with SacI (5′ probe), NcoI (3′ probe) or AflII (Neo probe). The probe sequences are shown in Supplementary Material, Table S5. Screened ES clones were then microinjected into BALB/c blastocytes and the resulting chimeras were crossed with C57BL/6J mice to obtain BrafQ241R Neo/+ heterozygotes mice. Excisions of the PGK-Neo cassette and STOP codon were achieved by crossing of BrafQ241R Neo/+ heterozygotes with CAG-Cre transgenic mice (Braf+/+; Cre) on C57BL/6J background (RIKEN BioResource Center, Tsukuba, Japan; RBRC01828) (49). Animal experiments were approved by the Animal Care and Use Committee of Tohoku University.

Genotyping

Genomic DNA was prepared from tail tissue with DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) or Maxwell 16 Mouse Tail DNA Purification Kit (Promega, Madison, WI, USA). Genotyping of the Braf+/+, Braf+/+; Cre, BrafQ241R Neo/+ and BrafQ241R/+; Cre was carried out by PCR using KOD FX Neo (TOYOBO, Osaka, Japan) or TaKaRa Taq (Takara Bio, Otsu, Japan) with the primers shown in Supplementary Material, Table S6.

Sequencing

Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsland, CA, USA), and cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). The exonic region in Braf was amplified by PCR using TaKaRa Taq with the primers including M13 sequences: 5′-GTAAAACGACGGCCAGTGAAGTACTGGAGAATGTCCC-3′ and 5′-AGGAAACAGCTATGACCCCACATGTTTGACAACGGAAACCC-3′. The PCR products were purified with QIAquick Gel Extraction Kit (Qiagen, Tokyo, Japan) and sequenced on an ABI 3500xl automated DNA sequencer (Applied Biosystems).

Quantitative reverse transcription–PCR

Quantitative PCR was performed using FastStart Universal Probe Master (ROX) (Applied Biosystems) with StepOnePlus (Applied Biosystems). Amplification primers and hydrolysis probes were designed using Universal ProbeLibrary Assay Design Center (https://qpcr.probefinder.com/roche3.html).

Alcian Blue/Alizarin Red staining

After embryos were placed in water for a day, the skin and viscera were removed. The eviscerated embryos were then fixed in 95% ethanol for at least 3 days and stained with 150 mg/l Alcian Blue 8GX (Sigma-Aldrich, St Louis, MO, USA), 80% ethanol and 20% acetic acid for 16–24 h. The stained embryos were rinsed with 95% ethanol and kept in 2% KOH for 16–24 h. They were then stained with 50 mg/l Alizarin Red (Sigma-Aldrich) and 1% KOH for 3 h, kept in 2% KOH for 12–48 h, placed in 20% glycerin/1% KOH for at least 5 days and stored in 50% glycerin.

Plasmid construction

The expression construct, including mouse Braf cDNA, was purchased from Origene (Rockville, MD, USA). PCR was performed using primers designed to introduce HindIII sites and the V5 epitope (C terminus). The PCR fragment was subcloned into pCR4-TOPO Vector (Invitrogen). The entire cDNA was verified by sequencing. The mutant constructs for Braf Q241R and V637E were generated using QuikChange Lightning Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) with the primers, 5′-CCGAAAGCTGCTTTTCCGGGGTTTCCGTTGTCAAA-3′ and 5′-TTTGACAACGGAAACCCCGGAAAAGCAGCTTTCGG-3′, and 5′-CTTTGGTCTAGCCACAGAGAAATCTCGGTGGAGTG-3′ and 5′-CACTCCACCGAGATTTCTCTGTGGCTAGACCAAAG-3′, respectively. All mutant constructs were verified by sequencing. The cDNAs were digested with HindIII, blunt-ended with T4 DNA polymerase and ligated into blunt-ended EcoRI site of pCAGGS vector (50).

Reporter assay

NIH 3T3 cells (ATCC, Rockville, MD, USA) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% newborn calf serum, 50 U/ml penicillin and 50 μg/ml of streptomycin. The cells were seeded in 24-well plates at 3 × 105 cells/well 24 h before transfection. The cells were then transiently transfected using Lipofectamine and PLUS Reagent (Invitrogen) with 400 ng of pFR-luc, 25 ng of pFA2-Elk1, 5 ng of phRLnull-luc and 5 ng of WT or mutant expression constructs of Braf. Forty-eight hours after transfection, the cells were harvested in passive lysis buffer, and luciferase activity was assayed using Dual-Luciferase Reporter Assay System (Promega). Renilla luciferase expressed by phRLnull-luc was used to normalize the transfection efficiency.

Western blotting and phospho-kinase-antibody array

Whole-mouse embryos and brain were lysed in lysis buffer (10 mM Tris–HCl, pH 8.0 and 1% SDS), or genotype-confirmed hearts were pooled and lysed in the same buffer. These lysates were centrifuged at 14 000g for 15 min at 4°C and the protein concentration was determined by the Bradford method with Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA). Lysates were subjected to SDS–polyacrylamide gel electrophoresis (5–20% gradient gel; ATTO, Tokyo, Japan) and transferred to nitrocellulose membrane. Antibodies used were as follows (with catalog numbers in parentheses): B-RAF (9434), ERK1/2 (9102), phospho-ERK1/2 (9101), phospho-MEK (9121), p38 (9212), phospho-p38 (4511), phospho-SAPK/JNK (4668), AKT (9272), phospho-AKT (on Ser473; 9018) and phospho-AKT (on Thr308; 2965) from Cell Signaling (Danvers, MA, USA). C-RAF (610152), MEK (sc-219) and β-actin (A5316) were from BD Transduction Laboratories (San Jose, CA, USA), Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Sigma-Aldrich, respectively. All the membranes were visualized using Western Lightning ECL-Plus Kit (PerkinElmer, Waltham, MA, USA). The band intensities were quantified using ImageJ software (http://rsbweb.nih.gov/ij/) and normalized to β-actin. Phosphorylated protein was measured to determine the ratios of phosphorylated protein to non-phosphorylated protein and then normalized to β-actin.

For kinase-antibody arrays, protein extracts of embryonic hearts (400 μg) were incubated with the Phospho-Kinase Antibody Array Kit (Proteome Profiler Antibody Array; R&D systems, Minneapolis, MN, USA) following the manufacturer's instructions.

Histology and immunohistochemistry

Embryonic hearts were perfused with phosphate-buffered saline and 10% neutral buffered formalin from the placenta. The fixed hearts and whole-mouse embryos fixed in 10% neutral buffered formalin were embedded in paraffin. Embedded tissues were sectioned at 6 μm (hearts) or 3 μm (whole-mouse embryos and lungs). Sections were stained with hematoxylin and eosin. In hearts from embryos at E16.5, the largest diameters of the ventricular radius were measured in serial coronal sections where a four-chamber view was observed. The largest thicknesses of cardiac valve leaflets in serial sections were measured. Edematous and dead embryos were excluded from these analyses.

For immunohistochemistry, the antibodies used were as follows (with catalog numbers in parentheses): phospho-Histone H3 (9701) from Cell Signaling, LYVE-1 (103-PA50AG) from RELIA Tech GmbH (Braunschweig, Germany), α-SMA (M0851) from DAKO (Glostrup, Denmark), PECAM-1 (CD31; sc-1506) from Santa Cruz Biotechnology and TTF-1 (MS-669-P1ABX) from Thermo Fisher Scientific (Fremont, CA, USA). Signals were amplified by Histofine Simple Stain (Nichirei Bio Sciences, Tokyo, Japan) and color was developed by DAB Substrate Kit (Nichirei Bio Sciences). Sections were counterstained with hematoxylin.

PAS staining

Deparaffinized lung sections were incubated in 0.5% periodic acid for 10 min at 60°C, rinsed with distilled water and stained in Schiff's reagent (Muto Pure Chemicals, Tokyo, Japan) for 10 min. Stained slides were counterstained with hematoxylin, dehydrated and mounted.

Animal treatment

Stock solution of PD0325901 (Sigma-Aldrich) was prepared using ethanol, whereas those of MAZ-51 (Calbiochem, San Diego, CA, USA), Sorafenib (Toronto Research Chemicals, North York, ON, USA), Lovastatin (Calbiochem), Everolimus (Selleckchem, Houston, TX, USA), NCDM-32b (Wako Pure Chemicals, Osaka, Japan), GSK-J4 (Cayman Chemical) and the combination of PD0325901 and GSK-J4 were prepared using dimethylsulfoxide. PD0325901 was resuspended in saline while and all other reagents were resuspended in 0.5% hydroxypropylmethylcellulose with 0.2% Tween80, respectively. The prepared reagents or vehicles were i.p. injected into pregnant mice daily, beginning on E10.5 and continuing till E15.5 or E18.5.

Statistical analysis

All statistical analysis was performed using Prism software (ver. 6.01; GraphPad Software, Inc., San Diego, CA, USA). Data analysis were performed with Student's t-test for unpaired samples, one-way analysis of variance followed by the Tukey–Kramer test for comparison of multiple experimental groups and the χ2 test for differences between observed and expected distributions. Differences were considered significant at a P-value of < 0.05.

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG online.

FUNDING

This work was supported by the Funding Program for the Next Generation of World-Leading Researchers (NEXT Program) from the Ministry of Education, Culture, Sports, Science and Technology of Japan to Y.A. (LS004), by Grants-in-Aids from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Ministry of Health, Labor and Welfare, and the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant number 26293241 to Y.A., and by JSPS KAKENHI Grant number 25860839 to S.I.

ACKNOWLEDGEMENTS

We are grateful to Jun-ichi Miyazaki, Osaka University, for supplying the pCAGGS expression vector. We thank Riyo Takahashi, Kumi Kato, Yoko Tateda and Daisuke Akita for technical assistance and Fumiko Date for technical assistance and for discussion of the experimental data. We also acknowledge the support of the Biomedical Research Core of Tohoku University Graduate School of Medicine. We thank RIKEN BioResource Center for providing us with B6.Cg-Tg(CAG-Cre)CZ-MO2Osb mice (RBRC01828).

Conflict of Interest statement. None declared.

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Supplementary data