β-Arrestin scaffolds and signaling elements essential for the obestatin/GPR39 system that determine the myogenic program in human myoblast cells

Abstract

Obestatin/GPR39 signaling stimulates skeletal muscle repair by inducing the expansion of satellite stem cells as well as myofiber hypertrophy. Here, we describe that the obestatin/GPR39 system acts as autocrine/paracrine factor on human myogenesis. Obestatin regulated multiple steps of myogenesis: myoblast proliferation, cell cycle exit, differentiation and recruitment to fuse and form multinucleated hypertrophic myotubes. Obestatin-induced mitogenic action was mediated by ERK1/2 and JunD activity, being orchestrated by a G-dependent mechanism. At a later stage of myogenesis, scaffolding proteins β-arrestin 1 and 2 were essential for the activation of cell cycle exit and differentiation through the transactivation of the epidermal growth factor receptor (EGFR). Upon obestatin stimulus, β-arrestins are recruited to the membrane, where they functionally interact with GPR39 leading to Src activation and signalplex formation to EGFR transactivation by matrix metalloproteinases. This signalplex regulated the mitotic arrest by p21 and p57 expression and the mid- to late stages of differentiation through JNK/c-Jun, CAMKII, Akt and p38 pathways. This finding not only provides the first functional activity for β-arrestins in myogenesis but also identify potential targets for therapeutic approaches by triggering specific signaling arms of the GPR39 signaling involved in myogenesis.

Obestatin controls skeletal muscle fiber-type determination

“…KM155C25 myoblast cells were switched to DM supplemented with obestatin (10 nM) for 7 days 11 . Myoblasts in GM was used as control.…”

“…Additionally, Zn +2 treatment was described to diminish GPR39 basal phosphorylation 40 . Having all this information in mind, these data could imply Zn +2 in GPR39 signaling, probably due to the activation of the MMPs in EGFR pathways, since obestatin needs EGFR transactivation and MMPs activity 11,27 . Indeed, Zn +2 induces EGFR phosphorylation through the extracellular release of EGF-like ligands that are mediated by MMPs 39,41 .…”

“…Furthermore, to the best of our knowledge, there is no data supporting the role of GLP-1R, i.e. siRNA technology, on obestatin signaling unlike GPR39 9–11 . First, knockdown of GPR39 by siRNA significantly inhibited obestatin signaling in murine and human myoblasts 9,11 .…”

“…This action is associated to the regulation on the different stages of myogenesis: proliferation, migration, fusion and myofiber growth, and more generally enhancing the regeneration process with the formation of fibers of larger caliber in mouse skeletal muscle 10 . In a human context in vitro , the obestatin/GPR39 system exerts an autocrine effect on the skeletal myogenic process promoting myogenic differentiation and fusion of human myoblasts 11 . At the molecular level, the action of obestatin involves both G-protein-dependent and -independent mechanisms linking the activated GPR39 receptor with distinct sets of accessory and effector proteins, thereby controlling the specificity and efficiency of the signals.…”

“…siRNA technology, on obestatin signaling unlike GPR39 9–11 . First, knockdown of GPR39 by siRNA significantly inhibited obestatin signaling in murine and human myoblasts 9,11 . This is further supported by siRNA assays developed in murine adipocytes 25 , human gastric adenocarcinoma 43 and retinal-pigmented epithelium cells 23 .…”