FIG 7.

Expression of ICN1, HES1 and DTX1 in osteoblasts and osteosarcoma cells, and model for the regulation of NOTCH signaling by NOTCH target genes HES1 and DTX1. (A) Immunoblots demonstrate expression of ICN1, HES1 and DTX1 in normal human osteoblasts [HOSB] and a panel of human osteosarcoma cell lines. (B) Model: NOTCH1 is cleaved into ICN1 by γ–secretase, and then activates downstream target genes HES1 and DTX1. Inhibitors of γ-secretase can block this step. DTX1 causes negative feedback of ICN1 by targeting ICN1 for ubiquitination and proteasome-mediated degradation. Meanwhile, HES1 causes positive feedback of ICN1 by repressing DTX1 gene expression. HES1 promotes the invasion and metastasis of osteosarcoma. In osteosarcoma cells expressing high HES1 and low DTX1, NOTCH inhibition, such as that caused by GSI, may downregulate HES1, increasing DTX1, which further downregulates ICN1 through ubiquitination, reducing HES1-mediated invasiveness. However, in osteosarcoma cells with high DTX1 and low HES1, NOTCH inhibition may not reduce invasiveness. In fact, reducing DTX1 may stabilize low level ICN1 expression, increasing ICN1 signaling and HES1 expression, causing increased invasion, as happens with COL. Thus the impact of GSI on NOTCH signaling should take into account expression of both HES1 and DTX1.