Achaete-scute-like-3 (ASCL3) Regulates Vascular Muscle Cell Proliferation

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DESCRIPTION (provided by applicant): Numerous cascades involved in cell fate determination during development and governing cell proliferation, differentiation, apoptosis and migration are regulated by transcription factors (TF) in the basic-helix-loop-helix class (bHLH), including those in the achaete-scute family of bHLH, which has been highly conserved from invertebrates to humans. Although the mammalian ASCL1 has been the object of extensive studies in neurobiology, yet very little information is available on the role of the other members of the family, ASCL2 to 5, particularly the ubiquitously expressed ASCL3. Our preliminary data show that ASCL3, ASCL2 and ASCL1 are expressed in vascular smooth muscle cells (VSMC). Focusing on ASCL3, we found that its expression is increased upon stimulation of proliferation with angiotensin II, and to a lesser extent PDGF-BB, while being reduced in the context of growth inhibition by Notch1 activation or BMP2 treatment. Furthermore, stable over-expression of ASCL3 leads to increased proliferation of VSMC in vitro and ASCL3 is clearly up-regulated in neointima upon vascular lesion in vivo. These data suggest that ASCL3 contributes actively to proliferation in VSMC with a potential role in development of pathological vascular remodeling, defining it as a "vasculopatic" factor. These observations led to our central hypothesis that ASCL3 is a growth-promoting bHLH transcription factor regulating VSMC proliferation. To address the implications and predictions of this hypothesis we propose to undertake: Specific aim 1: Characterization of ASCL3 expression profiles in relation to VSMC proliferation through (1.1) analysis of ASCL3 expression in response to different stimuli in vitro, focusing on factors of relevance in vascular pathologies to correlate it to the (1.2) analysis of ASCLS expression in vascular remodeling in vivo, and Specific aim 2: Analysis of the effects of ASCL3 on proliferation of VSMC in vitro through characterization of the (2.1) effects of gain- and loss-of-function of ASCLS on VSMC proliferation and, naturally derived, the (2.2) identification of ASCL3 target genes regulating VSMC proliferation. These specific aims will be addressed using immunochemistry and quantitative analysis of expression in vivo and in vitro in combination with growth factors of relevance in vascular remodeling as well as in the context of gain- and loss-of function through genetic and biochemical approaches. Development of this proposal will define ASCL3 as a novel bHLH TF with an active role in vascular proliferation and remodeling. This work is bound to transcend the field of vascular biology by shedding light on the general roles of this ubiquitous TF in cellular proliferation at large, with a potential to impact other fields where deregulated cell growth is at the roots of the involved biological processes as it is the case for cancer or developmental biology.