MRAP deficiency impairs adrenal progenitor cell differentiation and gland zonation

Abstract

Melanocortin 2 receptor accessory protein (MRAP) is a single transmembrane domain accessory protein and a critical component of the hypothamo-pituitary-adrenal axis. MRAP is highly expressed in the adrenal gland and is essential for adrenocorticotropin hormone (ACTH) receptor expression and function. Human loss-of-function mutations in MRAP cause familial glucocorticoid (GC) deficiency (FGD) type 2 (FGD2), whereby the adrenal gland fails to respond to ACTH and to produce cortisol. In this study, we generated Mrap-null mice to study the function of MRAP in vivo. We found that the vast majority of Mrap^-/- mice died at birth but could be rescued by administration of corticosterone to pregnant dams. Surviving Mrap^-/- mice developed isolated GC deficiency with normal mineralocorticoid and catecholamine production, recapitulating FGD2. The adrenal glands of adult Mrap^-/- mice were small, with grossly impaired adrenal capsular morphology and cortex zonation. Progenitor cell differentiation was significantly impaired, with dysregulation of WNT4/β-catenin and sonic hedgehog pathways. These data demonstrate the roles of MRAP in both steroidogenesis and the regulation of adrenal cortex zonation. This is the first mouse model of isolated GC deficiency and reveals the role of MRAP in adrenal progenitor cell regulation and cortex zonation.-Novoselova, T. V., Hussain, M., King, P. J., Guasti, L., Metherell, L. A., Charalambous, M., Clark, A. J. L., Chan, L. F. MRAP deficiency impairs adrenal progenitor cell differentiation and gland zonation.

Keywords: ACTH; accessory protein; cell fate; familial glucocorticoid deficiency; melanocortin.

Figure 1

Generation of Mrap^−/− mice. A) Schematic representation of targeting of the exon 1 of Mrap mouse for the line production. B) qRT-PCR analysis of Mrap expression in the adrenal gland of Mrap^+/+ (n = 7), Mrap^+/− (n = 7), and Mrap^−/− (n = 4) mice showing that Mrap^+/− mice express approximately half of the Mrap transcript found in the wild-type adrenals, whereas no transcript was detected in Mrap^−/−adrenals. ***P < 0.0005, Student’s t-test. C) Immunoblotting of adrenal gland lysates from Mrap^+/+ and Mrap^−/− mice with anti-MRAP and anti–actin b (ACTB) antibodies. The arrows on the left indicate the approximate MW position. The position of the main band in Mrap^+/+ corresponds to the anticipated size of a mature MRAP dimer, with the lower band representing the nonglycosylated form. D) Immunostaining of MRAP (red) in the adrenal gland of Mrap^+/+ (top panel) and Mrap^−/− mice (bottom panel). The nuclei are stained with DAPI (blue). The adrenal zonation is marked in the wild-type gland as follows: C, capsule; M, medulla; WAT, white adipose tissue; XZ, X-zone. Scale bars, 100 μm. E) Representative images of the adrenals from the Mrap^+/+ and Mrap^−/− mice sectioned though the middle of the organ, demonstrating the reduction in the gland’s size. The black boxes indicate the where the higher-magnification images were obtained. Scale bars, 200 μm. F) Adrenal gland zonation is impaired in Mrap^−/− mice (right panel) compared with Mrap^+/+ mice (left panel) as shown by the H&E staining. Panel images are from female mice, although similar adrenal gland zonation impairment is seen in male mice (data not shown). Adrenal gland zonation in male mice: C, capsule; M, medulla; XZ, X zone. The zonation in the Mrap^−/− adrenal gland is not defined. Scale bars, 50 µm.

Figure 2

Impaired zonation in the adrenal cortex of the Mrap^−/− mice. A) Severely reduced immunostaining of CYP11B1 (green) in Mrap^−/− adrenal glands. B) CYP11B2 (red) localization in Mrap^+/+ and Mrap^−/− adrenal glands. C) PNMT (red) in the adrenal medulla of Mrap^+/+ and Mrap^−/− mice. The cell nuclei are stained with DAPI (blue). CX, cortex; M, medulla. Scale bars, 200 µm.

Figure 3

ACTH resistance and isolated GC deficiency of Mrap^−/− mice. A) qRT-PCR of the adrenal gland of male Mrap^+/+ and Mrap^−/− mice (n = 4 for each group) shows down-regulation of the steroidogenic pathway. B) Plasma corticosterone levels (ng/ml) at the basal level and in response to ACTH stimulation in male Mrap^+/+ (n = 7) and Mrap^−/− (n = 7) mice. C) ACTH levels (pg/ml) in male Mrap^+/+ (n = 6) and Mrap^−/− (n = 5) mice. D) Comparison of plasma aldosterone levels (pg/ml) between the genotypes in male mice (Mrap^+/+, n = 7; Mrap^−/−, n = 7). E, F) Epinephrine (E) and norepinephrine (F) levels in the 24-h urine of male Mrap^+/+ (n = 6) and Mrap^−/− (n = 5) mice assessed as the ratio to creatinine to adjust for the kidney function. Student’s t test used for comparison of 2 groups. *P < 0.05, **P < 0.005, ***P < 0.0005. 1-way ANOVA for more than 2 groups. *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant.

Figure 4

Capsular layer is thickened in Mrap^−/− mice. A, B) Representative images of Mrap^−/− adrenal glands and of wild-type littermates showing increased capsule thickness (A; highlighted with black bars) and in response to lifetime GC treatment (B). The bottom panels show digital magnification of the adrenal gland at the capsular layer. C) Capsular layer measurements (micrometers) of the adrenals of the Mrap^−/− mice compared with the wild-type mice and in response to lifetime corticosterone replacement. D) Shh expression in the adrenal gland of male Mrap^+/+ (n = 6) and Mrap^−/− (n = 6) mice and of the animals after lifetime corticosterone replacement (GC) (Mrap^+/+, n = 4; Mrap^−/−, n = 5) shows an increase in Shh expression in Mrap^−/− mice. E, F) In situ hybridization demonstrated expression of Shh outside ZG in the adrenals of Mrap^−/− mice (E) and in the animals after lifetime corticosterone replacement (GC) (F). CX, cortex; M, medulla. Scale bars, 50 µm. One-way ANOVA with Bonferroni multiple comparison testing. *P < 0.05, **P < 0.01, ***P < 0.001. Ns, not significant.

Figure 5

Dysregulation of WNT4/β-catenin signaling in Mrap^−/− mice. A) Immunohistochemistry showing localization of β-catenin (β-CAT), WNT4, CYP11B2, and LEF1 in the adrenal glands of Mrap^+/+ and Mrap^−/− mice. B) Immunohistochemistry showing β-CAT, WNT4, CYP11B2, and LEF1 in response to lifetime corticosterone replacement. Cell nuclei are stained with DAPI as blue, with the exception if LEF1 images where the nuclei are stained green to highlight colocalization of LEF1 with nuclei. CX, cortex; M, medulla. Scale bar, 50 µm. C) Immunohistochemistry with anti-StAR antibody shows impairment of steroidogenic potential of the cells in the adrenal cortex of Mrap^−/− mice. D) Immunostaining of the adrenal glands with anti-20αHSD antibody shows staining surrounding the medulla in both wild-type and mutant mice. CX, cortex; M, medulla; XZ, X zone. Dashed line indicates medullary boundary. Scale bars, 50 µm.