tgx Estrogen receptor related receptor ERR like other member
Estrogen receptor-related receptor γ (ERRγ), like other members of the ERR subfamily, is a constitutively active orphan nuclear receptor, though unlike ERRα and β, it is more selectively expressed in metabolically active and highly vascularized tissues such as heart, kidney, brain, and skeletal muscles (Giguère, 2008, Heard et al., 2000, Hong et al., 1999). In vitro studies suggest that ERRγ activates genes such as Pdk4 and Acadm that play a regulatory role in oxidative fat metabolism (Huss et al., 2002, Zhang et al., 2006). Furthermore, a comprehensive gene expression analysis identified ERRγ as a key regulator of multiple genes linked to both fatty tgx oxidation and mitochondrial biogenesis in cardiac muscles (Alaynick et al., 2007, Dufour et al., 2007). Expression of ERRγ is also induced in variety of tumors with hypermetabolic demands and abundant vasculature (Ariazi et al., 2002, Cheung et al., 2005, Gao et al., 2006). Therefore, we explored the potential of ERRγ in controlling the intrinsic angiogenic pathway in oxidative slow-twitch muscles. We found ERRγ to be exclusively and abundantly expressed in oxidative (type I) slow-twitch muscles. Transgenic expression of ERRγ in fast-twitch type II muscle triggers aerobic transformation, mitochondrial biogenesis, VEGF induction, and robust myofibrillar vascularization, all in the absence of exercise. These intrinsic effects of ERRγ do not depend on PGC-1α induction, but rather are linked to activation of the metabolic sensor AMPK. These findings reveal an exercise-independent ERRγ pathway that promotes and coordinates vascular supply and metabolic demand in oxidative slow-twitch muscles.
Discussion Although skeletal muscle adapts to exercise by increasing oxidative metabolism and vascular supply via induction of transcriptional regulators such as PGC-1α (Arany et al., 2008, Baar et al., 2002, Huss et al., 2002, Pilegaard et al., 2003, Russell et al., 2003, Russell et al., 2005), how type I fibers achieve intrinsic vascularization even in the absence of exercise is poorly understood. We show here that one such molecular pathway involves nuclear receptor ERRγ—highly expressed in oxidative slow-twitch muscles. Targeted expression of ERRγ to quadriceps and white gastrocnemius, where the receptor is typically not expressed, morphologically endows these muscles with dense vascular supply and numerous slow-twitch characteristics. Recently, it was reported that muscle-specific overexpression of a constitutively active ERRγ (VP16-ERRγ) imparts an oxidative metabolic phenotype to the skeletal muscle (Rangwala et al., 2010). However, the effect of VP16-ERRγ on muscle vascularization was not evaluated in these mice. Genome-wide expression analysis revealed that ERRγ acts by coordinately inducing gene networks promoting mitochondrial biogenesis, oxidative transformation, and angiogenesis. The ERRγ program includes mobilization and oxidation of fat (e.g., Acadl, Acadm, Cpt1b, Cpt2, Lpl), electron transport (e.g., Atp5h, Cox6a2, Ndufab1, Ndufb2m, Ndufv1, Sdhb), mitochondrial biogenesis (e.g., Mfn1), and formation of energy-efficient, slow-contractile muscle (e.g., Tnnc1, Tnni1, Tnnt1). The observed changes constituting transformation of the contractile apparatus to a slow phenotype and increase in oxidative metabolic genes reflected in profound increase in mitochondrial (SDH) staining represents a fiber type switch. Notably, ERRγ also induces key transcriptional inducers of oxidative metabolism including Esrrb, Ppara, Ppard, and Ppargc1b (Table S4) (Lin et al., 2002, Minnich et al., 2001, Muoio et al., 2002, Wang et al., 2004). Therefore, it is likely that ERRγ is a critical upstream genetic switch that may determine metabolic fate by presiding over the expression of multiple aerobic regulators. We hypothesize that the vascular program triggered by myocellular ERRγ activates a transcriptional program that directs secretion of paracrine signals into skeletal muscle microenvironment to induce angiogenesis. This model is strongly supported by our observation that conditioned media from ERRγ overexpressing C2C12 myotubules is able to induce endothelial cell tube formation in culture. Indeed, ERRγ transcriptionally induced all isoforms of angiokine Vegfa in C2C12 myotubes, resulting in increased Vegfa secretion into the media. Vegfa is a key regulator of angiogenesis critical for guiding endothelial cells to their targets (Grunewald et al., 2006, Springer et al., 1998). Furthermore, ERRγ stimulates the Vegfa promoter containing putative ERR binding sites that is known to transcribe all Vegfa isoforms (Arany et al., 2008). Vegfa mRNA and protein expression are also induced in ERRGO muscle. These findings collectively raise the possibility of direct transcriptional activation of angiogenic genes by ERRγ. However, it is important to note that the angiogenic effects of ERRγ cannot be solely attributed to Vegfa induction and secretion. For example, ERRγ additionally activates the expression of Fgf1 and Cxcl12, known to regulate endothelial cell proliferation and migration (Forough et al., 2006, Gupta et al., 1998, Partridge et al., 2000, Shao et al., 2008, Zheng et al., 2007), along with Efnb2, proposed to recruit mural cells that are required for vessel maturation (Foo et al., 2006). Additionally, upregulated factors such as Notch4 as well as Sox17 are transcriptional regulators of vasculogenesis (Hainaud et al., 2006, Leong et al., 2002, Matsui et al., 2006). In this aspect, ERRγ seems to serve a function similar to HIF1α, a known master regulator of angiogenesis during hypoxia (Pajusola et al., 2005). Interestingly, it was recently demonstrated that ERRs might physically interact with HIF1α in regulating its transcriptional activity (Ao et al., 2008). Whether such a mechanism is relevant to our model remains to be determined. Along these lines, HIF1α mRNA levels—a marker for chronic hypoxia—did not change in ERRGO compared to WT muscles (data not shown), indicating an absence of hypoxia or its involvement in the vascular effects of ERRγ (Hoppeler and Vogt, 2001a, Hoppeler and Vogt, 2001b). Furthermore, HIF1α is known to negatively regulate oxidative metabolism (Mason et al., 2004, Mason et al., 2007) and is therefore unlikely to contribute to ERRγ-mediated remodeling of skeletal muscles.