Mex3c regulates insulin-like growth factor 1 (IGF1) expression and promotes postnatal growth

Abstract

Insulin-like growth factor 1 (IGF1) mediates the growth-promoting activities of growth hormone. How Igf1 expression is regulated posttranscriptionally is unclear. Caenorhabditis elegans muscle excess 3 (MEX-3) is involved in cell fate specification during early embryonic development through regulating mRNAs involved in specifying cell fate. The function of its mammalian homologue, MEX3C, is unknown. Here we show that MEX3C deficiency in Mex3c homozygous mutant mice causes postnatal growth retardation and background-dependent perinatal lethality. Hypertrophy of chondrocytes in growth plates is significantly impaired. Circulating and bone local production of IGF1 are both decreased in mutant mice. Mex3c mRNA is strongly expressed in the testis and the brain, and highly expressed in resting and proliferating chondrocytes of the growth plates. MEX3C is able to enrich multiple mRNA species from tissue lysates, including Igf1. Igf1 expression in bone is decreased at the protein level but not at the mRNA level, indicating translational/posttranslational regulation. We propose that MEX3C protein plays an important role in enhancing the translation of Igf1 mRNA, which explains the perinatal lethality and growth retardation observed in MEX3C-deficient mice.

FIGURE 1:

Mutation of Mex3c in mouse by gene trapping. (A) Structure of normal Mex3c gene and its gene products. The black line indicates the introns of mouse Mex3c gene, and the solid boxes indicate the three Mex3c exons. Exons 2 and 3 contain coding regions. The N-terminal KH RNA-binding domains (KH) and the C-terminal ZNF domain (zinc F) are indicated. The gene structure is based on NCBI EST sequence CJ072395.1 and mRNA sequence BC125427, and is submitted to DDBJ (DNA Data Bank of Japan) with accession number BR000945. Positions of primers used to amplify authentic Mex3c mRNA (Mex3cF and Mex3cR) are indicated by two unfilled arrows. (B) Structure of the trapped Mex3c allele and its gene products. The empty box indicates the gene trap vector containing lacZ cDNA. The transcript from the trapped allele translates into a peptide containing the N-terminal 56 AA residues from MEX3C and the full-length β-gal. Positions of primers used to amplify the gene trap vector DNA (TrapF and TrapR) are indicated by two solid arrows. (C) qRT-PCR comparison of authentic Mex3c mRNA levels in +/+, +/tr, and tr/tr mice (n = 3 mice for each genotype). Means ± SEM are presented. *** p < .001 by Tukey post hoc tests following analysis of variance. Total RNA was isolated from hypothalami. (D) Mutant mice have poorly expanded alveoli after birth. Shown are lung sections of 1-d-old pups stained with hematoxylin and eosin. Scale bar: 50 μm.

FIGURE 2:

Examination of Mex3c expression in newborn mice by tracing β-gal activity. (A) High β-gal activity was detected in skeletal muscle. Bodies of 1-d-old pups were eviscerated followed by whole-mount X-Gal staining. The muscle tissues of +/tr and tr/tr pups were strongly positive for β-gal activity. The residual skin around the mouth and paws was negative, demonstrating the specificity of the staining. (B) Detecting β-gal activity in internal organs of newborn mice. β-Gal activity was high in most internal organs except for the liver. (C) All cells in the lung of newborn pups were β-gal positive. Scale bar: 50 μm. (D) Detecting β-gal activity in tibia of newborn mice. Cryosections of the proximal ends of tibia from +/tr mice were stained by X-Gal, followed by eosin counterstaining. Scale bar: 100 μm.

FIGURE 3:

Analysis of growth plates from control and Mex3c mutant mice. (A) Proximal tibia growth plates of 20-d-old control and mutant mice. The hypertrophic zone (HZ), but not the resting zone (RZ) or the proliferating zone (PZ), was attenuated in the mutants. (B) High magnification showing the hypertrophic zone from (A). For (A) and (B), Masson's trichrome staining was performed on tibia sections from 20-d-old mice. (C) Growth plates of 8-wk-old control and mutant mice. Shown were hematoxylin and eosin–stained sections of proximal tibia. Scale bars in A and C equal 100 μm; in B, 50 μm.

FIGURE 4:

Reduced IGF1 expression in bone cells of Mex3c mutant mice. (A) IGF1 immunohistochemical analysis of proximal tibia growth plates (GPs) of 20-d-old mice. The three boxes indicate the areas shown in B, C, and D. (B) High magnification showing the proliferating zone (PZ) of the growth plate. (C) High magnification showing the hypertrophic zone (HZ) of the GP. (D) High magnification of the spongiosa region. SO, secondary ossification center; PO, primary ossification center.

FIGURE 5:

Analysis of Mex3c expression in mouse tissues. (A) Expression of Mex3c in tissues of developing mice. Tissues from 4-wk-old heterozygous and normal mice were stained for β-gal; positive tissues were stained blue. The epididymis were positive even in normal mice (+/+) due to endogenous β-gal activity, which indicates the reliability of the staining condition. Lu: lung; Spl: spleen; Kid: kidney; Liv: liver; Musc: skeletal muscle; Fat/epi: gonadal fat/epididymis; Ov/Ut: ovary/uterus; Tes: testis; He: heart; Bla/Ure: bladder/ureter. (B) qRT-PCR analysis of Mex3c mRNA in various tissues from normal mice. Means ± SEM of data from four mice are presented. (C) Mex3c expression in chondrocytes of proximal tibia growth plates of 20-d-old mice indicated by tracing β-gal activity. β-Gal–positive cells were stained blue. No β-gal–positive cells were observed in control (+/+) mice. The sections were counterstained with eosin to show the matrix in red. RZ, resting zone; PZ, proliferating zone; HZ, hypertrophic zone. (D) Expression of Mex3c in stromal cells of the spongy bone. (E) Expression of Mex3c in stromal cells of the compact bone. Scale bars: 50 μm.

FIGURE 6:

qRT-PCR analysis of Igf1 mRNA in mutant mice and in GST pull-down assays. (A) Comparison of Igf1 mRNA levels in liver, kidney, spleen, testis, and muscle tissues from +/+ and tr/tr mice. Four 4∼5-wk-old male mice for each genotype were analyzed. Expression in tissues of control mice was set as 1. Means ± SEM are presented. (B) Igf1 mRNA expression in +/+ and tr/tr tibia. (C) Diagram showing domains in MEX3C, MEX3C/N, and MEX3C/C. KH: KH RNA-binding domain. (D) GST pull-down analysis of Igf1 mRNA enrichment by partial and full-length MEX3C proteins. Means ± SEM of three independent assays are presented. mRNA levels in GST-only assays were set as 1. Numbers above the columns are means ± SEM of Ct numbers.