Rapid Insulin-like Growth Factor-1-induced Changes in Granulosa Cell Thrombospondin-1 Expression In Vitro

Thrombospondin-1 (TSP-1) is a large extracellular matrix-associated protein that is important for normal follicular development, is rapidly modulated during follicular growth and plays important roles in cellular proliferation and angiogenesis. TSP-1 mRNA is post-transcriptionally regulated, although the underlying mechanisms are largely unknown. Insulin-like growth factor-1 is a potent signalling molecule that participates in folliculogenesis. We hypothesized that IGF-1 modulates TSP-1 expression in granulosa cells, and that such modulation requires rapid turnover of the TSP-1 mRNA and protein. Spontaneously-immortalized rat granulosa cells (SIGC) were cultured in the presence or absence of IGF-1, after which the expression and turnover of TSP-1 mRNA and protein was evaluated by western blot and quantitative PCR. RNA stability reporter constructs were prepared in which wild-type and mutated AU-rich elements from the TSP-1 3'UTR were cloned downstream of the luciferase gene in a mammalian expression vector. These were transfected into SIGC cells in order to characterize mRNA elements that regulate the stability of the TSP-1 mRNA. TSP-1 expression decreased rapidly at the mRNA and protein levels in IGF-1 treated cultures. Following 12 h of IGF-I treatment, TSP-1 protein decreased by 25% and was 73% lower than in untreated cultures. The half-life of endogenous TSP-1 mRNA in SIGC was 2.0 h. This was not changed in the presence of IGF-1, however, transcription of new TSP-1 mRNA was inhibited. Reporter mRNAs with mutated AU-rich elements demonstrated a longer half-life than mRNAs in which the wild type AU-rich elements were present. These studies reveal that IGF-1 rapidly inhibits TSP-1 expression at the protein and mRNA levels in cultured granulosa cells through apparent inhibition of TSP-1 transcription. The decrease depends on an intrinsically short half-life of TSP-1 mRNA and protein. The short mRNA half life is due, at least in part, to AU-rich elements in the 3'UTR of the TSP-1 mRNA.