Dusp6 is a genetic modifier of growth through enhanced ERK activity - PubMed (original) (raw)

. 2019 Jan 15;28(2):279-289.

doi: 10.1093/hmg/ddy349.

Kayleigh A Swaggart 2, Anna Woo 3, Quan Q Gao 3, Alexis R Demonbreun 3, Katherine S Fallon 3, Mattia Quattrocelli 3, Michele Hadhazy 3, Patrick G T Page 3, Zugen Chen 4, Ascia Eskin 4, Kevin Squire 4, Stanley F Nelson 4, Elizabeth M McNally 3

Affiliations

Dusp6 is a genetic modifier of growth through enhanced ERK activity

Andy H Vo et al. Hum Mol Genet. 2019.

Abstract

Like other single-gene disorders, muscular dystrophy displays a range of phenotypic heterogeneity even with the same primary mutation. Identifying genetic modifiers capable of altering the course of muscular dystrophy is one approach to deciphering gene-gene interactions that can be exploited for therapy development. To this end, we used an intercross strategy in mice to map modifiers of muscular dystrophy. We interrogated genes of interest in an interval on mouse chromosome 10 associated with body mass in muscular dystrophy as skeletal muscle contributes significantly to total body mass. Using whole-genome sequencing of the two parental mouse strains combined with deep RNA sequencing, we identified the Met62Ile substitution in the dual-specificity phosphatase 6 (Dusp6) gene from the DBA/2 J (D2) mouse strain. DUSP6 is a broadly expressed dual-specificity phosphatase protein, which binds and dephosphorylates extracellular-signal-regulated kinase (ERK), leading to decreased ERK activity. We found that the Met62Ile substitution reduced the interaction between DUSP6 and ERK resulting in increased ERK phosphorylation and ERK activity. In dystrophic muscle, DUSP6 Met62Ile is strongly upregulated to counteract its reduced activity. We found that myoblasts from the D2 background were insensitive to a specific small molecule inhibitor of DUSP6, while myoblasts expressing the canonical DUSP6 displayed enhanced proliferation after exposure to DUSP6 inhibition. These data identify DUSP6 as an important regulator of ERK activity in the setting of muscle growth and muscular dystrophy.

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Figures

Figure 1

Figure 1

Chromosomal intervals contributing to body mass in muscular dystrophy identified by genome-wide mapping. QTL mapping was conducted using the Sgcg mouse muscular dystrophy model from the D2 and 129 strains. (A) The QTL plot for total body mass identified two loci that were significantly associated with total body mass on chromosome 1 (P < 0.01) and chromosome 10 (P < 0.05). (B) Genetic map of markers used for the QTL analysis on chromosome 10. The region associated with total body mass is from 40.67 to 54.96 cM on chromosome 10. (C) Single-QTL plot for chromosome 10 and markers associated with total body mass.

Figure 2

Figure 2

Genetic background influences body and muscle mass. WT (left) and _Sgcg_-null (right) mice were evaluated at 14–15 weeks of age. (A) Body mass was measured in D2 and 129 mice from WT and Sgcg animals. D2 mice have significantly increased body mass compared with 129 mice in the WT background but not in the _Sgcg_-null background (males, P < 0.0001; females, P < 0.0002). In WT mice, the quadriceps (B) and gastrocnemius (C) muscles were larger for female mice but not for males in the D2 compared with the 129 background strain (P = 0.011 female quadriceps and P = 0.006 female gastrocnemius). Conversely, muscle mass was significantly decreased in _Sgcg_-null D2 mice compared with _Sgcg_-null 129 mice for both quadriceps (males and females; P < 0.0001) and gastrocnemius (males P = 0.0004; females P = 0.0002). Comparisons by two-tailed student’s _t_-test.

Figure 3

Figure 3

DUSP6 contains a Met62Ile substitution in the D2 strain that reduces ERK2 interaction. (A) The Dusp6 Met62Ile variant (rs13480726) was identified in the D2 strain but not in other common mouse strains. Met62Ile is also conserved in human DUSP6 and overall this domain, which mediates ERK binding, is highly conserved. (B) Genotypes of SNPs flanking the Dusp6 region from a cohort of more than 175 intercross Sgcg F3 mice from a SgcgD2 X _Sgcg_129 cross (14). SNPs flanking DUSP6, JAX00187391 and UNC101149923, from the F3 Sgcg intercross were analyzed for their association with body mass. Their relative position to Dusp6 is shown on the top bar. Mice with the D2 genotype at both markers showed significant association with increased body mass (P = 0.044, P = 0.042). The most proximal marker to Dusp6, JAX00187391, showed a significant association between the D2 genotype and increased quadriceps muscle mass (P = 0.003). Comparisons by one-way ANOVA. (C) A GST-pulldown assay was performed to measure the interaction between ERK and DUSP6 using the ERK2 binding domain. DUSP6 Met62Ile displayed a significantly reduced interaction with ERK2 compared with DUSP6 Met62 (P = 0.0009, n = 3, two-tailed student’s _t_-test.).

Figure 4

Figure 4

The DUSP6 Met62Ile substitution exhibits a partial loss of function as a regulator of ERK phosphorylation. (A) Immunoblotting was performed on C2C12 cell lysates to measure p-ERK levels following transfection of plasmids expressing DUSP6 Met62 or DUSP6 Met62Ile. Cells expressing DUSP6 Met62Ile exhibited significantly higher p-ERK levels compared with cells transfected with DUSP6 Met62 (P = 0.04, n = 3). (B) Immunoblotting was performed on protein lysates extracted from quadriceps muscle harvested from WT and Sgcg mice from the D2 and 129 backgrounds. Sgcg129 mice showed significantly higher p-ERK levels compared with _Sgcg_D2 mice (P = 0.03, n = 3). (C) RNA-seq analysis from WT and Sgcg quadriceps muscles revealed that expression of the Dusp6 was significantly higher in the D2 strain than the 129 strains in both WT and Sgcg muscles (P < 0.05, n = 3). (D) Immunoblotting of quadriceps muscles showed significantly higher DUSP6 protein expression in the D2 strain from Sgcg muscle (P = 0.03, n = 3) but not WT muscle. Comparisons by two-tailed student’s _t_-test.

Figure 5

Figure 5

Myoblasts from the D2 background were insensitive to the DUSP6 inhibitor BCI. Myoblasts from D2 and 129 muscles were isolated, cultured and treated with the DUSP6-specific small molecule inhibitor BCI (also known as NSC 150117) (31). Myoblasts were treated with BCI at multiple concentrations, and proliferation was measured. (A) Myoblast growth curves from D2 and 129 WT and _Sgcg-_null mice treated with 0 μm BCI or 2.0 μm BCI. BCI treatment increased the proliferation of 129 myoblasts but had little to no effect on D2 myoblast proliferation. This increase in proliferation was seen for both myoblasts isolated from WT and _Sgcg-_null 129 mice. (B) Graphical representation of myoblast growth is shown measuring 4 days post-treatment and comparing D2 and 129 myoblasts treated with 0, 0.5 or 2.0 μm BCI. WT and _Sgcg-_null 129 myoblasts showed significantly increased growth from BCI treatment compared with WT and Sgcg D2 myoblasts. (*P < 0.05, by two-way ANOVA with Bonferonni correction).

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