(620q) Ectopic Expression of NANOG Restores the Actin Filamentous Organization and Contractile Capacity of Senescent Cells

Actin filamentous organization is critical for mediating cellular contraction. In the present work we hypothesized that cellular senescence decreases actin polymerization whereas ectopic expression of Nanog completely restores actin reorganization. To address this hypothesis, human hair follicle Mesenchymal Stem Cells and human dermal fibroblasts were isolated and transduced with a tetracycline regulatable vector that carries the Nanog gene. In this system the transgene is expressed only in the presence of the tetracycline analogue, Doxycycline. To induce cellular senescence, Early Passage (EP) carrying the Nanog gene were serially passaged in the absence of Doxycycline until they reached Late Passage (LP) or senescence. Subsequently Doxycycline was added to the culture medium (LP NANOG). Genome wide transcription analysis in EP, LP and LP NANOG revealed that upon senescence several components of the actin cytoskeleton pathway were affected. In agreement with this, we report that senescent cells failed to polymerize actin in respond to myogenic stimuli as evidenced by the complete absence of ACTA2 fibers as well as the weak F-Actin staining. The reduced filamentous actin was also associated with reduced contractile potential.  Interestingly, Nanog expression in senescent cells restored the actin polymerization and the force generation capacity. To further elucidate the mechanism through which NANOG elicits its function, we studied the ROCK/ MRTF-A and the TGF-beta pathways, which are known to regulate the contractile phenotype. Interestingly, NANOG increased key components of the ROCK pathway such as ROCK1 and LIMK2, and activated the TGF-beta pathway by upregulating SMAD2. We also show that upon ROCK activation MRTF-A translocated into the nucleus and activated SRF-dependent myogenic gene transcription. Last but not least, inhibition of the ROCK and/or the TGF-beta pathway with either chemical inhibitors or shRNA abolished actin polymerization and myogenic differentiation.  Taken together, our data provide novel insight into the mechanism of cellular senescence and propose a novel strategy to regain the lost properties, thereby increasing the potential of adult cells for tissue regeneration.