C-cell hyperplasia, pheochromocytoma and sympathoadrenal malformation in a mouse model of multiple endocrine neoplasia type 2B

Abstract

Dominantly inherited multiple endocrine neoplasia type 2B (MEN2B) is characterized by tumors of the thyroid C-cells and adrenal chromaffin cells, together with ganglioneuromas of the gastrointestinal tract and other developmental abnormalities. Most cases are caused by substitution of threonine for Met918 in the RET receptor tyrosine kinase, which is believed to convert the RET gene to an oncogene by altering the enzyme's substrate specificity. We report the production of a mouse model of MEN2B by introduction of the corresponding mutation into the ret gene. Mutant mice displayed C-cell hyperplasia and chromaffin cell hyperplasia progressing to pheochromocytoma. Homozygotes did not develop gastrointestinal ganglioneuromas, but displayed ganglioneuromas of the adrenal medulla, enlargement of the associated sympathetic ganglia and a male reproductive defect. Surprisingly, homozygotes did not display any developmental defects attributable to a loss-of-function mutation. Thus, while our results support the conclusion that the Met918Thr substitution is responsible for MEN2B, they suggest that the substrate specificity of the RET kinase does not interfere with its normal role in the development of the kidneys and enteric nervous system.

Fig. 1. Introducing a MEN2B mutation into the ret locus. (A) Structure of the normal protein encoded by ret. The location of Met919 (equivalent to human Met918) is indicated. (B) Site-directed mutagenesis to introduce the Met919Thr mutation. In addition, two silent base changes were introduced to destroy a MunI restriction enzyme site, so that the mutant allele could be detected easily in ES cells and mice. (C) Schematic representation of the targeting strategy. (i) A segment of the normal ret gene including exons 15–17 (black boxes). (ii) The targeting vector included two segments of genomic ret DNA, one of which includes exon 16 (with the Thr919 mutation), separated by the loxP-neo-loxP gene. LoxP sites are indicated by triangles. (iii) Homologous recombination between the ret genomic locus and the targeting construct introduced the mutant exon 16 and the loxP-neo-loxP gene in the adjacent intron, to produce the RET^MEN2B-neo allele. (iv) The ret^MEN2B allele produced by Cre-mediated excision of loxP-neo-loxP. The restriction enzyme sites shown are M, MunI; X, XbaI; B, BamHI; K, KpnI; S, ScaI; H, HindIII; and P, PstI. (D) Southern blot analysis of targeted ES cell clone #10 and the parental ES cell line W9.5. Left: DNA digested with MunI and hybridized with probe A. A 15 kb mutant band and a 6.5 kb wild-type band were observed. Right: DNA digested with XbaI and hybridized with probe B to check the integrity of the 3′ end. A 12 kb wild-type band and a 7.4 kb mutant band resulted. (E) Southern blot (left) and PCR (right) analysis of mouse tail DNAs after germline transmission and Cre-mediated excision of loxP-neo-loxP. Left: lanes 1 and 2, two progeny from a mouse chimeric for ret^MEN2B-neo/+ ES cells. Lane 1, wild-type; lane 2, ret^MEN2B-neo/+. Lane 3, a ret^MEN2B/+ mouse derived from ret^MEN2B-neo/+ crossed with a β-actin/Cre transgenic. Lane 4, a ret^MEN2B/ret ^MEN2B homozygote. The DNAs were digested with XbaI and hybridized with probe A. The 12 kb band was derived from the wild-type allele, the 6 kb band from the ret^MEN2B-neo allele and the 4 kb band from the ret^MEN2B allele. Right: PCR analysis on three newborn pups from a ret^MEN2B intercross, using primers p5 and p6. The 280 bp amplification product was generated from the wild-type allele and the 350 bp product from the ret^MEN2B allele (the difference, 70 bp, is due to a 34 bp loxP site and 36 bp of polylinker sequence). Lane 5, heterozygote; lane 6, ret^MEN2B homozygote; lane 7, wild-type. M, molecular weight markers. (F) RT–PCR analysis of the expression of the ret^MEN2B allele. Left: schematic representation of the mutated locus, indicating exons 16 and 17. Primers p9 and p10 were used to amplify a 632 bp cDNA fragment spanning the two exons. The product of the wild-type ret allele is cleaved by MunI to yield two fragments of 300 and 332 bp, while the product of the mutant allele is not cleaved by MunI. Right: total brain cDNA from adult wild-type, ret^MEN2B/+ and ret^MEN2B/ret^MEN2B was amplified and the product analyzed before and after MunI digestion. In ret^MEN2B heterozygotes, half of the PCR product is cleaved, indicating that the wild-type and mutant alleles produce similar amounts of mRNA.

Fig. 2. Nodular and diffuse C-cell hyperplasia in the ret^MEN2B/+ mutant thyroid. Calcitonin-stained sections through the thyroid of an 8-month-old wild-type mouse (a and b) showing the normal follicular pattern; intrafollicular C-cells are indicated by an arrow and f represents the follicular space. (c) Diffuse C-cell hyperplasia (DCCH) and (d and e) nodular C-cell hyperplasia (NCCH) in 8-month-old heterozygous mice. Magnification bars = 120 μm.

Fig. 3. Histological sections of ret^MEN2B mutant adrenals: (a) wild-type adrenal; c, cortex; m, medulla; (b) heterozygous mutant adrenal with nodular chromaffin cell hyperplasia (nh); (c) heterozygous mutant with pheochromocytoma (p) replacing the medulla. (d) A homozygous mutant with a ganglioneuroma-like area (g) extending into and abutting the pheochromocytoma (p). (e) A higher magnification view of a pheochromocytoma in a homozygous mutant, in which the normal medulla tends to be replaced by sheets and wide cords of cells. (f) Wild-type medulla, showing the nested or ‘alveolar’ pattern that predominates in the normal mouse adrenal medulla. Magnification bars = 120 μm.

Fig. 4. Nodular hyperplastic adrenal cells and pheochromocytoma are noradrenergic. (a, c, e and g) Sections of heterozygous ret^MEN2B adrenal medulla with a region of nodular chromaffin cell hyperplasia (nh) occupying part of the medulla (m); (b, d, f, and h) a pheochromocytoma. Distinction of nodular hyperplasia from pheochromocytoma in the veterinary pathology literature is arbitrary and is based on small size (<50% of medullary volume) and absence of significant compression or invasion of the cortex (Longeart, 1996). (a and b) H&E staining; (c and d) tyrosine hydroxylase; (e and f) chromogranin A; (g and h) PNMT. PNMT is not expressed in cells of the hyperplastic nodule or the pheochromocytoma. Small islands of PNMT-negative cells in (g) are normal norepinephrine cells. c, cortex.

Fig. 5. Enlarged sympathetic ganglia and ganglioneuroma-like areas in ret^MEN2B/ret^MEN2B mice. (a and b) H&E-stained sections through the adrenal medulla of newborn wild-type (a) and homozygous mutant (b) newborn mice. (c) Tyrosine hydroxylase-stained section through the adrenal medulla of a 4-month-old homozygous mutant, showing the continuity between cells of the medulla (m), the enlarged sympathetic ganglia (sg) and the ganglioneuroma-like areas (g). (d) A representative ganglioneuroma-like area (g) is shown at a higher magnification (arrows point to nerve fibers while the arrowhead points to a ganglion cell). c, adrenal cortex; m, adrenal medulla; sg, enlarged sympathetic ganglia; g, ganglioneuroma. Magnification bars = 80 μm.