Functional analysis of Hic-5/ARA55 isoforms in C2C12 myogenesis
Hic-5 is a focal adhesion protein of paxillin superfamily that was initially cloned from mouse osteoblasts as a TGF-β or H2O2 inducible cDNA. As well, Hic-5 was independently identified as an Androgen receptor activator (ARA55). Conflicting data have implicated Hic-5 in opposing processes. With two Hic-5 isoforms documented, I hypothesized that multiple Hic-5 isoforms may exist that have both overlapping and isoform-specific functions, which may explain those discrepancies. To test this hypothesis, I have utilized C2C12 myoblasts and analyzed the roles of Hic-5 isoforms in development and homeostasis.
1. I have confirmed the presence of the two previous reported Hic-5 isoforms (α and β) and uncovered 10 additional novel Hic-5 transcripts. Conceptually translated proteins from these transcripts significantly differ at the N-terminal region and likely have distinct binding properties and functions. Hic-5 isoforms have distinct tissue distribution and are developmentally regulated in the mouse mammary gland in vivo (Chapter two).
2. I found that: (a) myoblasts express multiple Hic-5 isoforms; (b) the two predominant isoforms, Hic-5α and Hic-5β, are differentially expressed during myogenesis; (c) any experimentally-induced change in Hic-5 expression results in a substantial increase in apoptosis during differentiation; (d) ectopic expression of Hic-5α is permissive to differentiation while expression of either Hic-5β or antisense Hic-5 reduces myoblast chemo-differentiation and blocks fusion; (e) Hic-5 localizes to focal adhesion in C2C12 myoblasts and perturbation of Hic-5 leads to defects in cell spreading; (f) perturbations of Hic-5 expression interfere with the normal expression dynamics of laminin; and (g) the rescue of myoblast survival and differentiation by laminin but not fibronectin suggests that Hic-5 isoforms differentially regulate myogenesis due to their different impacts on cell-ECM interaction, focal adhesion dynamics and integrin signaling (Chapter Three).
In summary, the roles Hic-5 may assume in development and homeostasis are complex and the different Hic-5 isoforms may mediate distinct physiological and/or pathological responses in cells. Therefore, a more precise analysis of Hic-5 isoforms is required to more fully understand the roles of not only Hic-5, but also integrin signaling in normal and diseased cells ( Chapter four and five).