Activating mutations in BRAF disrupt the hypothalamo-pituitary axis leading to hypopituitarism in mice and humans

Abstract

Germline mutations in BRAF and other components of the MAPK pathway are associated with the congenital syndromes collectively known as RASopathies. Here, we report the association of Septo-Optic Dysplasia (SOD) including hypopituitarism and Cardio-Facio-Cutaneous (CFC) syndrome in patients harbouring mutations in BRAF. Phosphoproteomic analyses demonstrate that these genetic variants are gain-of-function mutations leading to activation of the MAPK pathway. Activation of the MAPK pathway by conditional expression of the Braf^V600E/+ allele, or the knock-in Braf^Q241R/+ allele (corresponding to the most frequent human CFC-causing mutation, BRAF p.Q257R), leads to abnormal cell lineage determination and terminal differentiation of hormone-producing cells, causing hypopituitarism. Expression of the Braf^V600E/+ allele in embryonic pituitary progenitors leads to an increased expression of cell cycle inhibitors, cell growth arrest and apoptosis, but not tumour formation. Our findings show a critical role of BRAF in hypothalamo-pituitary-axis development both in mouse and human and implicate mutations found in RASopathies as a cause of endocrine deficiencies in humans.

Fig. 1. Mutations identified in hBRAF in patients with CFC and SOD.

a Schematic diagram of the hBRAF protein and the location of the mutations identified. The numbers indicate the location where each protein domain begins and ends. The mutations identified in the patients are labelled indicating the position of the substitution. b Electropherograms illustrating the mutations identified, indicated by an arrow and an ‘N’ in the sequence of each patient, with the corresponding wild-type (Wt) sequence below. (i) A heterozygous missense variant (c.721A>C) was identified in exon 6 of BRAF in patient 3, (ii) a heterozygous missense variant (c.770A>G) was identified in exon 6 of BRAF in patients 1 and 4, (iii) a heterozygous missense variant (c.1403T>C) was identified in exon 11 of BRAF in patient 2, (iv) a heterozygous missense variant (c.1406G>A) was identified in exon 11 of BRAF in patient 5. c Amino acid conservation of the BRAF substitutions identified in our study. (i) The threonine residue (represented by the green ‘T’) at position p.T241, (ii) the glutamine (represented by the green ‘Q’) at position p.Q257, (iii) the phenylalanine (represented by the green ‘F’) at position p.F468 and (iv) the glycine (represented by the green ‘G’) at position p.G469, and their adjacent protein sequences either side, respectively, are located at conserved regions across multiple species.

Fig. 2. The BRAF genetic variants are pathogenic and result in activation of the ERK/MAPK pathway.

a Heat map of the phosphopeptide enrichment analyses by mass spectrometry of the BRAF variants: p.T241P, p.Q257R, p.F468S and p.G469E. These mutations result in activation of the ERK/MAPK pathway, as indicated by the increase in the ERK/MAPK phosphorylated peptides BRAF and ERK1/2. Note that the p.G469E is a mild activator with most of the peptides in blue, indicating low kinase activity. b KSEA for the BRAF variants p.T241P, p.Q257R, p.F468S and p.G469E compared to Wt BRAF shows an increased activity for the kinases MEK1/2 and ERK1/2 involved in the ERK/MAPK pathway, as well as an increase for JAK2 and Ret (colours represent fold change over BRAF wild-type protein expressed as Log2). c Western blot of cell lysates from transfected HEK293T cells with BRAF p.V600E (control) and BRAF p.T241P, p.Q257R, p.F468S and p.G469E plasmids to detect levels of total ERK and phosphorylated-ERK (p-ERK), normalised to β-actin and GAPDH. d, e Graphs of the western blot quantification showing increase in the p-ERK/GAPDH (d) and p-ERK/total ERK (e) ratios associated with BRAF p.T241P, p.Q257R, p.F468S and p.G469E compared to  Wt  BRAF (****p < 0.0001, ***<0.001 and *<0.05 one-way ANOVA, data represented as mean ± SD). Twenty micrograms of each BRAF variant plasmid including Wt and empty vector were used in the experiment. NT line, non- transfected control. Images are representative of nine independent experiments.

Fig. 3. Expression of Braf^V600E in the developing anterior pituitary gland (Prop1:Cre;Braf^V600E/+) leads to severe hypopituitarism.

a Surviving mutant pups Prop1:Cre;Braf^V600E/+ exhibit dwarfism and failure to thrive compared to Prop1^+/+;Braf^V600E/+ (Wt) littermates. b, b′ Whole mount pictures of Prop1^+/+;Braf^V600E/+ Wt (b) and Prop1:Cre;Braf^V600E/+ mutant (b′) pituitaries at postnatal day P22 reveal a hypoplastic anterior lobe (b′, AL arrowheads) composed of a rudimentary layer of cells in the mutant mice compared to  Wt (b). c Growth chart illustrating growth failure of Prop1:Cre;Braf^V600E/+ mutants (n = 9) which die prematurely soon after weaning compared to  Wt littermates (n = 7). ***p < 0.001; **p < 0.01; *p < 0.05 unpaired two-tailed Student’s T-test. Data represented as mean ± SEM of n = 3–6 pups per genotype. AL anterior lobe, PL posterior lobe, P postnatal day.

Fig. 4. Activation of the ERK/MAPK pathway in the anterior pituitary gland (Prop1:Cre;Braf^V600E/+) results in defective terminal differentiation of endocrine cells.

a–j Immunohistochemistry against GH, TSH, POMC, PRL and LH in coronal sections through the pituitary gland of Prop1:Cre;Braf^V600E/+ (b, d, f, h, j) and Wt (a, c, e, g, i) embryos at E17.5 of gestation. Absence of immunoreactivity for GH, TSH, LH in Prop1:Cre;Braf^V600E/+ (b, d, j) mutant pituitaries compared to Prop1^+/+;Braf^V600E/+ (a, c, i) reveals deficient terminal differentiation. Note that the anterior pituitary in Prop1:Cre;Braf^V600E/+ is enlarged compared to Wt littermates. Prop1:Cre;Braf^V600E/+ pituitaries exhibit an increase in POMC (f) and PRL (h) expression compared to Wt littermates (e, g, respectively). d′, h′ Higher magnification views of the squared area in d and h, respectively, revealing an expanded intermediate lobe (IL arrowheads in d′ and h′) with multiple bifurcations (arrows in d′ and h′). Images are representative of three embryos per genotype. IL intermediate lobe, PL posterior lobe, GH growth hormone, TSH thyroid-stimulating hormone, POMC proopiomelanocortin, PRL prolactin, LH, luteinising hormone. Scale bar: f, 200 µm; h′ 500 µm.

Fig. 5. Activation of the ERK/MAPK results in increased expression of POMC and PRL with a portion of Sox2+ve progenitor/stem cells co-expressing POMC and PRL.

Double immunofluorescence (IF) against POMC (green, a–f), PRL (green, g–l) and Sox2 (red, a–l) on coronal sections of E18.5 Wt pituitaries (a–c, g–i) and Prop1:Cre;Braf^V600E/+ (d–f, j–l). The Prop1:Cre;Braf^V600E/+ mutant pituitaries (d) exhibit a higher number of POMC+ve cells compared to Wt (a). Enlarged merged images of the marginal zone revealed co-expression of Sox2 and POMC within a portion of POMC+ve cells (white arrowheads in f′). Increase in number of PRL+ve cells was observed in the Prop1:Cre;Braf^V600E/+ pituitaries (j) compared to Wt (g). i′ and l′ represent enlarged images of squared areas in i and l, respectively, showing the marginal zone. Cells expressing both Sox2 and PRL were observed in the MZ of the Prop1:Cre;Braf^V600E/+ mutant pituitaries (white arrowheads in l′), while no co-expression of Sox2 and PRL was detected in the cells of Wt pituitaries (I′). Images are representative of four embryos per genotype. AL anterior lobe, MZ marginal zone, PL posterior lobe. Scale bars: a, d, g, j 150 µm; c′, f′, i′, and l′ 40 µm.

Fig. 6. Abnormal terminal differentiation of hormone-producing cells in the Braf^Q241R/+ knock-in allele (CAG:Cre;Braf^Q241R/+).

IHC against GH, TSH, POMC, PRL and LH hormones on coronal sections of Wt (a–e) and mutant CAG:Cre;Braf^Q241R/+ (f–j) embryos at E18.5. GH (f), TSH (g) and LH (j) were severely reduced in CAG:Cre;Braf^Q241R/+ mutant pituitaries compared to Wt. Increase in POMC (h, h′) and PRL (i, i′) were found in mutants compared to Wt (c, d, respectively). f′–j′ represent higher magnification of the boxed areas in f–j, respectively. Note that CAG:Cre;Braf^Q241R/+ mutant pituitaries exhibit overgrowth of marginal zone (MZ) with extended growths into the pituitary lumen (arrowheads in f′–j′). Images are representative of three embryos per genotype. AL anterior lobe, IL intermediate lobe, PL posterior lobe, GH growth hormone, TSH thyroid-stimulating hormone, POMC proopiomelanocortin, PRL prolactin, LH luteinising hormone. Scale bar: j 200 µm; j′ 500 µm.

Fig. 7. Expression of Braf^V600E and Braf^Q241R leads to abnormal cell lineage specification with increase in TPit (corticotrophs and melanotrophs) and decrease in Pit1 (somatotrops, thyrotrophs and lactotrophs).

IHC against TPit (a, d, g), Pit1 (b, e, h) and α-GSU (c, f, i) on sagittal section of E15.5 embryos of Wt (a–c), Prop1:Cre;Braf^V600E/+ (d–f) and CAG:Cre;Braf^Q241R/+ (g–i). Expression of TPit was increased in the Prop1:Cre;Braf^V600E/+ and CAG:Cre;Braf^Q241R/+ pituitaries compared to Wt (arrows in d, g). Quantification of TPit-positive cells shows statistically significant increase in the % of TPit+ve cells in both Prop1:Cre;Braf^V600E/+ and CAG:Cre;Braf^Q241R/+ pituitaries compared to Wt (j). Severe reduction of Pit1 immunoreactivity was observed in Prop1:Cre;Braf^V600E/+ with only few positive foci (arrows in e) compared to Wt (b). Quantification of the Pit1-positive cells revealed a decrease in Pit1 cells in Prop1:Cre;Braf^V600E/+ and CAG:Cre;Braf^Q241R/+ mutant pituitaries (k). Mild reduction of α-GSU was observed in Prop1:Cre;Braf^V600E/+ pituitaries (arrows in f) (l). Note that Prop1:Cre;Braf^V600E/+ and CAG:Cre;Braf^Q241R/+ pituitary glands exhibited morphological abnormalities with expanded overgrowth and bifurcations of IL (arrowheads d–f and g–i) and overall enlarged size. Quantification of percentage of TPit (j), Pit1 (k) and α-GSU-positive cells (l) (***p < 0.001; **p < 0.01; *p < 0.05 one-way ANOVA, data represented as mean ± SEM from n = 4–5 pituitaries per genotype). AL anterior lobe, IL intermediate lobe, PL posterior lobe, IHC immunohistochemistry. Images are representative of four or five embryos per genotype. Scale bar: i 200 µm.

Fig. 8. Expression of Braf^V600E results in upregulation of cell cycle inhibitors p57^Kip2, p27^Kip1 and the senescence markers p16^INK4a and p21.

a–h In situ hybridisation of sagittal sections through embryonic pituitary gland of Wt (a–d) and Prop1:Cre;Braf^V600E/+ mutant pituitaries (e–h) at E16.5 reveals significantly increased p57^Kip2, p16^INK4a, p21 and p27^Kip1 mRNA transcripts in mutant pituitaries. p57^Kip2 transcripts were upregulated and its expression domain was expanded ventrally (arrows in e). p16^INK4a mRNA transcripts were upregulated in the ventral portion of the AL (arrows in f). p21 transcripts were located in the AL in mutant pituitaries (arrows in g) and absent in Wt (c), although p21 was expressed in the basisphenoid bone (bb, arrowheads in c, g) in Wt. Expression of p27^Kip1 was significantly upregulated in the ventral side of the AL (arrows in h) compared to Wt (arrows in d). The IL was negative for p27^Kip1 (arrowheads in d). i–p Representative coronal sections at P1 of Wt i–l and Prop1:Cre;Braf^V600E/+ mutant pituitaries m–p. p57^Kip2 mRNA transcripts were localised mainly in the IL and the MZ (arrowheads in i) while in the mutants expression was found ectopically throughout the AL (arrows in m). Expression of p16^INK4a (arrows in n), p21 (arrows in o) and p27^Kip1 (arrowheads in p) was upregulated compared to the corresponding Wt pituitaries (j–l). Images are representative of five embryos per genotype. Asterisks indicate tissue cavities within the AL. q Quantitative RT-qPCR from P1 pituitary glands revealed increased mRNA expression of p57^Kip2 (4.6-fold increase), p16^INK4a (17.81-fold increase), p21 and p27^Kip1 compared to Wt (****p < 0.0001; **p < 0.01; *p < 0.05 unpaired two-tailed Student’s T-test. Data represented as mean ± SEM from n = 4 pituitaries or 5 pituitaries for p16^INK4a per genotype). AL anterior lobe, IL intermediate lobe, MZ marginal zone, PL posterior lobe. Scale bars in h, n & p represents 200 µm.

Fig. 9. Activation of ERK/MAPK pathway by expression of Braf^V600E results in increased expression of the cell cycle inhibitors p57^Kip2 and of p27^Kip1 in the Sox2+ve stem cells at E18.5.

a–l Coronal sections through the pituitary gland at E18.5 of Wt (a–c, g–i) and Prop1:Cre;Braf^V600E/+ (d–f, j–l). Double immunofluorescence against cell cycle inhibitor p57^Kip2 (green, a–f) and p27^Kip1 (green, g–l) with the pituitary stem cell marker Sox2 (red, a–l). The cell cycle inhibitor p57^Kip2 was found to be upregulated in the Prop1:Cre;Braf^V600E/+ pituitaries (arrowheads in d) compared to the Wt (a). c′, f′ Merged enlarged images of squared areas in c and f reveal increased p57^Kip2 immunoreactivity co-localising with Sox2 (arrowheads in f′) in the Prop1:Cre;Braf^V600E mutant pituitaries compared to Wt (arrowheads in c′). Expression of p27^Kip1 (arrowheads in j) is observed in the marginal zone (MZ) of the mutant pituitaries compared to Wt (g). Confocal merged images of the marginal zone revealed co-localisation of Sox2 with p27^Kip1 in the mutant Prop1:Cre;Braf^V600E/+ pituitaries (yellow nuclei, arrowheads in l′), while no co-localisation of p27^Kip1 and Sox2 was seen in Wt pituitaries (arrows in i′). i′, l′ are enlarged images of the squared areas in i and l respectively. Images are representative of three embryos per genotype. AL anterior lobe, MZ marginal zone, PL posterior lobe. Scale bars in a and g represent 200 µm. Scale bars in c′ and  i' represent 25 µm.

Fig. 10. Expression of Braf p.V600E in postnatal pituitary stem cells leads to decreased proliferation and increased apoptosis in vitro.

PSC cultures from Wt and Prop1:Cre;Braf^V600E/+ at postnatal stage P4 (a) and P14 (b) reveal a significant decreased capacity in colony formation (c) and number of cells per colony (d) in mutant PSCs compared to Wt. The ability of the mutant PSCs to form colonies diminishes over time from P4 to P14 (c). Immunostaining with the PSCs marker Sox2 revealed that all the cells in culture are Sox2+ve (e, f). TUNEL immunofluorescence revealed a significant increase in apoptotic cells in the mutant PSC colonies (arrowheads in h and quantification n) while almost no apoptotic cells were seen in the Wt colonies (g, n). Immunofluorescence against pHH3 revealed a substantial decrease in pHH3+ve cells in the mutant PSCs (arrowhead in j) compared to Wt (arrowheads in i). o Quantification of the number of pHH3+ve cells per colony shows a significant decrease in the mitotic index in the mutant PSC colonies compared to Wt. The Prop1:Cre;Braf^V600E/+ mutant colonies express the senescence SA-β-galactosidase (arrowheads l) while only a few positive cells were detected in Wt (arrowheads in k). Western blotting of PSC lysate revealed expression of Braf p.V600E resulting in increased pERK in the Prop1:Cre;Braf^V600E/+ mutant PSCs compared to Wt. *** statistically significant p < 0.001, unpaired two-tailed Student’s T-test, data represented as mean ± SEM (number of colonies and cells per colony of three mutants and three Wt from three independent experiments performed in triplicates c, d); n number of TUNEL+ve cells per colony of 12 colonies from three mutants and three Wt; o number of pHH3+ve cells of 21 colonies from three mutants and three Wt. Images are representative of three independent experiments. Scale bar in e′ and e represent 50 and 10 µm respectively.