Skeletal muscle growth and fiber composition in mice are regulated through the transcription factors STAT5a/b: linking growth hormone to the androgen receptor

Abstract

In skeletal muscle, STAT5a/b transcription factors are critical for normal postnatal growth, whole-animal glucose homeostasis, and local IGF-1 production. These observations have led us to hypothesize that STAT5a/b are critical for maintenance of normal muscle mass and function. To investigate this, mice with a skeletal muscle-specific deletion of the Stat5a/b genes (Stat5MKO) were used. Stat5MKO mice displayed reduced muscle mass, altered fiber-type distribution and reduced activity. On a molecular level, gene expression in skeletal muscle of Stat5MKO and control mice was analyzed by microarrays and real-time PCR, both in the presence and absence of growth hormone (GH) stimulation. Expression of several genes involved in muscle growth and fiber type were significantly changed. Specifically, in the quadriceps, a muscle almost exclusively composed of type II fibers, the absence of STAT5a/b led to increased expression of several genes associated with type I fibers and the de novo appearance of type I fibers. In addition, it is shown here that expression of the androgen receptor gene (Ar) is controlled by GH through STAT5a/b. The link between STAT5a/b and Ar gene is likely through direct transcriptional regulation, as chromatin immunoprecipitaion of the Ar promoter region in C2C12 myoblasts was accomplished by antibodies against STAT5a. These experiments demonstrate an important role for STAT5a/b in skeletal muscle physiology, and they provide a direct link to androgen signaling.

Figure 1.

Muscle mass and fiber cross-sectional area in quadriceps of Stat5MKO and control mice. A) Quadriceps from 3-mo-old male Stat5MKO mice were reduced ∼35% in weight compared to controls (n=12), while quadriceps from females were reduced only 8% (n=12) B) Body weight of male Stat5MKO mice in panel A was reduced ∼15%, while body weight of females was reduced by 10%. C) Cross-sectional area of individual gastrocnemius and quadriceps muscle fibers (average of 100 fibers).

Figure 2.

Motor activity and energy expenditure in Stat5MKO mice. A) Activity of Stat5MKO and control mice, measured by breaks of a laser beam over 24 h (n=8). B) Oxygen consumption in Stat5MKO and control mice before adjusting for body weight. C) Energy expenditure in Stat5MKO and control mice before adjusting for body weight. D) Oxygen consumption did not differ between Stat5MKO mice adjusted for body weight. *P < 0.05; n = 8.

Figure 3.

Confirmation of the GH and STAT5a/b dependence of Igf1, Socs2, and Ar genes. RNA was extracted from quadriceps of Stat5MKO and control animals injected i.p. in the morning with GH for 3 h. RNA was converted to cDNA and used for TaqMan real-time PCR. Graphs show relative gene expression of Stat5MKO compared to controls. A, B) GH-induced expression of expected STAT5a/b target genes Igf1 (A) and Socs2 (B). C) GH-induced expression of Ar. *P < 0.05, n = 5–8.

Figure 4.

Type I fiber gene expression in quadriceps muscle fibers from Stat5MKO and control mice. A) Real-time PCR gene expression analysis of selected slow-isoform genes (n=3). B) Western blot analysis of 4 control and 4 Stat5MKO animals probed for slow-twitch myosin heavy chain (Myh7, top panel), STAT5a/b (middle panel) and b-actin (bottom panel) to show loading consistency. C, D) Control and Stat5MKO quadriceps muscles from 14-wk-old animals from each group were analyzed. Transverse sections were stained for myosin heavy chain slow isoform. C) Type I myosin staining in quadriceps muscle of control mice. D) Type I myosin staining in quadriceps muscle of Stat5MKO shows mostly fast-twitch; however, tissue contains areas of abundant type I fibers not seen in controls. View shows area of abundant type I fibers.

Figure 5.

Ar expression is reduced in Stat5MKO soleus muscle, while slow-twitch fibers are unchanged. A, B) Transverse soleus muscle frozen sections stained for slow-twitch myosin heavy chain slow isoform. A) Control mice have abundant type I slow-twitch fibers. B) Stat5MKO soleus has similar slow-twitch fibers compared to control. C, D) Real-time PCR analysis of Myh7 and Ar expression in soleus muscle from untreated 3-mo-old male Stat5MKO and control mice. C) Myh7 expression is similar in control and Stat5MKO soleus, confirming that a fiber shift is not present in this muscle (n=6). D) Ar expression in soleus of Stat5MKO mice (n=6).

Figure 6.

Existence and binding of STAT5a to a conserved GAS site in the Ar gene promoter. A) Schematic of the Ar gene. Vertical boxes indicate translated exon. Location of the conserved GAS sequence is indicated. Gray region containing GAS sequence represents an ∼340-bp region conserved between species. B) Conserved GAS sequence in the AR promoter from multiple mammalian species. C) Results from chromatin immunoprecipitation assays in C2C12 cells. Cells were infected with a constitutively active STAT5a (STAT5a R20)- or GFP-expressing retrovirus. Cross-linked protein/DNA complexes were sonicated and immunoprecipitated with anti-STATa or control antibodies. DNA was amplified from STAT5a-precipitated complexes using specific primers near known (Socs2) and suspected (Ar) GAS regions, respectively, and for a region outside of a known STAT5a/b binding site (outside). Data represent averages of 2 separate experiments with the range indicated.

Figure 7.

Diagram of GH-STAT5a/b-AR regulation in muscle. We propose a model in which GH signals through STAT5a/b to induce AR expression in skeletal muscle tissues. GH can induce autocrine/paracrine IGF-1 production in skeletal muscle through STAT5a/b signaling that may act on skeletal muscle IGF-1 receptors (IGF-1R). In addition, GH/STAT5a/b activation induces expression of the AR. Androgens testosterone and dihydroxytestosterone in males then send an additional growth signal through the AR, controlling male-specific skeletal muscle growth.