Nedd4 controls animal growth by regulating IGF-1 signaling

Abstract

The ubiquitin ligase Nedd4 has been proposed to regulate a number of signaling pathways, but its physiological role in mammals has not been characterized. Here we present an analysis of Nedd4-null mice to show that loss of Nedd4 results in reduced insulin-like growth factor 1 (IGF-1) and insulin signaling, delayed embryonic development, reduced growth and body weight, and neonatal lethality. In mouse embryonic fibroblasts, mitogenic activity was reduced, the abundance of the adaptor protein Grb10 was increased, and the IGF-1 receptor, which is normally present on the plasma membrane, was mislocalized. However, surface expression of IGF-1 receptor was restored in homozygous mutant mouse embryonic fibroblasts after knockdown of Grb10, and Nedd4(-/-) lethality was rescued by maternal inheritance of a disrupted Grb10 allele. Thus, in vivo, Nedd4 appears to positively control IGF-1 and insulin signaling partly through the regulation of Grb10 function.

Fig. 1

Generation of gene-trapped insertional alleles for the mouse Nedd4 gene. (A) A gene-trapping vector was used to disrupt this gene into two independent cell lines: in the XA209 allele, the gene trap vector is inserted in the middle of the HECT domain (after exon 25), whereas in the XB398 allele, the gene trap vector is inserted after the first WW domain (after exon 12). (B) PCR genotyping of mice potentially harboring one of the alleles. (C) RT-PCR analysis of mice carrying the XA209 allele. Transcript containing exon 25 and the gene trap vector (exon 25-βGeo) was not detected in wild-type mice, whereas the endogenous transcript (exons 25/27) was not detected in mice homozygous for the XA209 allele. GAPDH was used as an internal control. (D) Immunoblot confirming the absence of Nedd4 protein in cells derived from XA209 homozygous mutants. Total cell lysate (50 µg protein) obtained from passage number–matched cultures was loaded in each lane. Primary antibody against the WW2 domain of Nedd4 (Upstate) and goat–anti-rabbit secondary antibody (Super Signal Femto, Pierce) were used.

Fig. 2

Nedd4^−/− mice die immediately after birth, and Nedd4^+/− and Nedd4^−/− mice exhibit intrauterine growth retardation. No mice homozygous for disruption of the Nedd4 gene were found 2 or 3 weeks after birth. Ratios of heterozygotes and homozygous mutants were thus assessed at earlier time points: (A) 12.5 dpc, (B and C) 18.5 dpc, and (D) immediately after birth. Both heterozygotes and homozygous mutants showed signs of intrauterine growth retardation as early as 12.5 dpc (A) and at late gestation [18.5 dpc (B) and (C)]. At the time of birth [post-natal day 1 (D)], the body weights among three genotypes differed significantly:Nedd4^−/− body weight averaged 64 to 68% lower relative to that of wild-type littermates; heterozygote body weight averaged about 15 to 20% reduction in body weight relative to that of wild-type littermates. In (C) and (D), the numbers of animals used for the analyses are shown in parentheses; the body weight was significantly different between groups of mice, with P values indicated.

Fig. 3

Mice heterozygous for Nedd4 disruption show significant growth retardation postnatally. Postweaning mice were weighed weekly until they were 13 weeks old. (A and B) Heterozygotes at 3 weeks of age [both male (M) and female (F)] were significantly smaller than control mice, and the body weight difference persisted until at least 3 months of age. The numbers of animals are identical in (A) and (B). The femur length, which was determined by x-ray radiography of 2-month-old mice, showed significant differences between the wild-type and heterozygous mice [(C), P = 0.01]. However, femur length as a function of body weight did not differ significantly between these groups (D). There were 12 mice in each group in (C) and (D).

Fig. 4

Nedd4^−/− mice are developmentally delayed. Tissue samples were taken from mice on postnatal day 1 and processed for histology. Wild-type littermates were used as controls. More than six mice were used for each genotype, and typical histology is shown. (A and B) Skin: In the mutant mouse, the hair follicles are immature and the superficial skeletal muscle (platysma) is underdeveloped. The width of the platysma muscle layer is indicated by a red bar in each panel. (C and D) Spinal cord: In the mutant mouse, the spinal cord tends to be hypocellular, with fewer mature ganglion cells in the anterior horns than in the control (see arrowhead). (E and F) Skeletalmuscle: In the mutant mouse, fiber size varies significantly and internal nuclei are common [arrows in (F)]. Also, the interstitium tends to be more immature. Scale bar, 100 µm.

Fig. 5

MEFs isolated from Nedd4^−/− embryos show reduced mitogenic activity. MEFs were isolated from mid-gestation embryos (13.5 dpc) and passage number–matched MEFs were used for each experiment. (A) MEFs from Nedd4^−/− embryos exhibit passage number–dependent decreases in growth rate, whereas Nedd4^+/+ MEFs do not. (B) Passage 4 Nedd4^−/− MEFs grew more slowly than did Nedd4^+/+ MEFs. (C) Nedd4^−/− MEFs exhibit reduced colony-forming activity after low-density seeding at passage 2.Data shown are representative of three independent experiments. (D) Wild-type MEFs require high concentrations of exogenous growth factors from serum for Nedd4-dependent stimulation of cell growth to take place.

Fig. 6

Reduced IGF-1–induced and insulin-induced signaling in Nedd4-deficient MEFs. (A) IGF-1 signaling: MEFs of the indicated genotypes were serum-starved for 2 hours and then left untreated or stimulated with either 10 or 100 ng IGF-1/ml for 5 min. Cell lysates were then immunoblotted as indicated. (B) Insulin signaling: As in (A), except that cells were stimulated with either 10 or 100 ng insulin/ml. (C) Restoration of IGF-1 signaling in Nedd4^−/− MEFs by reintroduction of Nedd4. A Nedd4 expression vector was transiently transfected in MEFs as indicated, cells were treated with IGF-1, and lysates were blotted as in (A). The results from two separate transfection experiments are shown. “p,” phospho-.

Fig. 7

Cell surface IGF-1R concentrations change when Nedd4 and Grb10 protein expression are altered. (A) Decreased cell surface IGF-1R and IR in Nedd4-deficient MEFs. Cell surface proteins were isolated by biotin labeling and affinity purification and detected by immunoblotting. (B to I) IGF-1R concentrations at the plasma membranes of Nedd4^+/+ and Nedd4^−/− MEFs were determined in the absence or presence of an siRNA against Grb10 (rabbit anti–IGF-1Rα antibody was used at 1:100, Santa Cruz Biotechnology). (B and C) No IGF-1 stimulation. (D and E) Cells stimulated with recombinant IGF-1 (at 200 ng/ml, Sigma) for 4 hours. (F and G) Cells stably transfected with siRNA targeting Grb10. (H and I) Cells stably transfected with control siRNA. IGF-1R was not detectable on Nedd4^−/− cells, but Grb10 knockdown restored IGF-1R to the plasma membrane. (J and K) Grb10 and IGF-1R are partially colocalized in Nedd4^−/− MEF cells. MEFs grown in DMEM + 10% fetal bovine serum were fixed with 2% paraformaldehyde for 30 min at room temperature. Grb10 (green) and IGF-1R (red) were detected with monoclonal antibody against Grb10 and rabbit polyclonal antibody against IGF-1Rβ (Santa Cruz Biotechnology). Scale bar, 10 µm in the main figures and 2 µm in the insets.

Fig. 8

The amount of cellular Grb10 changes in response to amount of Nedd4. (A) The abundance of Grb10 in Nedd4^−/− MEFs is much higher than in Nedd4^+/+ MEFs. (B) Pretreatment of Nedd4^+/+ MEFs with the proteasome inhibitor MG132 increases the abundance of Grb10. Cells were treated with chloroquine (Chlq) (40 µM) or MG132 (20 µM) for 4 hours before harvesting of cells and preparation of cell lysates. The β-actin immunoblot signals serve as protein loading controls.