Studio del ruolo svolto da Pin1 nelle cellule satelliti del muscolo adulto.

Muscle differentiation is finely modulated by the synergistic action of two main families of transcription factors: Myogenic Regulatory Factors (MRFs) such as MyoD, Myogenin, Myf5 and MRF4, and the Myocyte Enhancer Factor 2 family of proteins (MEF2), MEF2A- D. The function of these factors is regulated through different mechanisms ranging from alternative splicing to post-translational modification of proteins and interaction with cofactors. Both MRFs and MEF2 proteins are phosphorylated on numerous serine and threonine residues by protein kinases activated by intracellular signaling pathways in response to external stimuli. It has been shown that the function of many phosphorylated proteins is regulated by the enzyme Pin1, a prolyl cis-trans isomerase that, with its N-terminal WW domain, is able to bind proteins phosphorylated on serine or threonine residues followed by a proline and induce reversible cis/trans isomerization with consequent conformational change of the target proteins. Previous studies in our laboratory have shown that Mef2C is a Pin1 target in C2C12 cells, a murine muscle cell line. The consequence of the Pin1/MEF2C interaction is the reduction of MEF2C stability, with inhibition of myogenic differentiation. Pin1 is expressed in skeletal muscle and the protein is particularly abundant in muscle stem cells present in adult muscle, the satellite cells (SC). Since the expression of Pin1 is highest during the first 21 days of life of the mouse, period in which satellite cells are in intense activity to ensure post-natal muscle growth in rodents, we hypothesized that PIN1 might play a role in the function of stem cells. This hypothesis finds support in the analysis of quiescent, proliferating, differentiating or self-renewing SC in fibers isolated from Extensor Digitorum Longus (EDL) muscle of mice KO for Pin1 and WT control mice of about 8 weeks. This analysis showed a lower percentage of MyoD-positive proliferating myoblasts (MyoD +) between the PAX7 + SC after isolation of the fibers from the muscle of KO mice compared to WT. Similar results were obtained by treating SC with All-Trans Retinoic Acid (ATRA), a Pin1 enzyme inhibitor. Since the progression of SC to myoblasts requires the expression of the myogenic determinant MyoD, we investigated whether Pin1 could influence the levels of MyoD protein in the cells. Pin1 was over-expressed or silenced with siRNA in C2C12 cells. From these experiments we found that the levels of MyoD protein change proportionally to the levels of Pin1 expressed in the cell, without significant changes in transcript levels. Comparable results were obtained using Pin1 inhibitors: ATRA, Juglone and Epigallocatechin gallate (EGCG). Co-immunoprecipitation experiments conducted in our laboratory indicate a physical interaction of Pin1 with MyoD, a process that is necessary for Pin1 catalytic activity. Globally these data suggest a stabilizing effect of Pin1 on the MyoD protein, a key factor for cell cycle progression. In light of the results obtained, Pin1 would seem to influence the activity of SC favoring their exit from the quiescent state, probably through a stabilizing action on MyoD. Next, with the aim to study the effects of Pin1 deficiency in vivo we performed cadiotoxin induced muscle regeneration experiments. The preliminary data obtained indicate that muscle regeneration is not inhibited in muscles depleted for Pin1. A second aspect that reflects the activity of SC in vivo is represented by post-natal growth, where instead we found that KO mice have a reduced body weight compared to controls. The lower body weight is, at least partially, due to a reduction in muscle mass: the muscles are composed of a smaller number of fibers with a mean cross sectional area (CSA) lower than that found in control muscles. In summary our data indicate that the activity of Pin1 is important for the myogenic progression of satellite cells in culture and in vivo.