Ras1^CA-Upregulated bcpi Inhibits Cathepsin Activity to Prevent Tissue Destruction of the Bombyx Posterior Silk Gland

Using the GAL4/UAS transgenic system established in the silkworm, Bombyx mori, we have previously reported that overexpression of the Ras1^CA oncogene specifically in the posterior silk gland (PSG) resulted in improved fibroin synthesis, silk yield, and other phenotypic effects. However, the detailed molecular mechanism remains to be fully elucidated. Using 2D-DIGE-MS/MS analyses, we compared the proteomic profiles of PSGs from the wild type (WT) and Ras1^CA-overexpressed silkworms. Among the 24 Ras1^CA-enhanced proteins, the Bombyx cysteine protease inhibitor (BCPI) was increased 2.4-fold at the protein level and 3.4-fold at the mRNA level. Consistent with the developmental profiles, injection of recombinant BCPI into the WT silkworms at the early wandering stage inhibited cathepsin activity, prevented tissue destruction of the PSG, and delayed pupation. Moreover, injection of small-molecule inhibitors of cathepsin into the WT silkworms prevented PSG destruction and delayed pupation, confirming the role of BCPI in inhibiting cathepsin activity. Furthermore, injection of chemical inhibitors of the Ras downstream effectors into the Ras1^CA-overexpressed and WT silkworms revealed that both Raf-MAPK and PI3K-TORC1 pathways were required for Ras1 to induce bcpi expression. Taken together, we conclude that via the downstream Raf-MAPK and PI3K-TORC1 pathways, Ras1^CA upregulates bcpi, which inhibits cathepsin activity thus preventing PSG destruction in Bombyx.