Cytokines and polypeptide hormones bind to cell surface receptors and generate second messenger molecules, via signal transduction, that alter the metabolic direction of the cell. In addition, the receptor-ligand complexes are internalized by receptor-mediated endocytosis and localized within endosome and lysosomes in the cytoplasm of the cell. We postulated that the internalized ligand may have an intracellular role and tested this idea by directly microinjecting cytokines into the cytoplasmic compartment. Microinjection of IFNgamma induced Ia expression across the species barrier and injection of TNFalpha induced c-Jun/AP-1 transcriptional activity, rapid cytotoxicity and DNA fragmentation (apoptosis). The bacterial invasion, IpaB, was shown to induce apoptosis after injection into macrophages through direct association with the interleukin-1beta converting enzyme (ICE). Activation of the ICE protease was blocked with ICE specific inhibitors and this prevented Shigella-induced apoptosis. A role for protein kinase C (PKC) ras, and phospholipase C (PLC) has been identified in the induction of macrophage surface Ia. These studies demonstrate a direct role for PKC, ras, and PLC in the biochemical pathways that lead to Ia expression. Injection of protein kinase A (PKA) and phospholipase A2 (PLA2) had no effect. Thus, the PKC pathway can control expression of macrophage surface Ia, possibly by regulating the genes of the major histocompatibility complex. Recently, we have focused on determining the mechanism of action of tumor suppressor genes during the regulation of cell cycle progression. We have shown that TIMP-3 expression in DLD human colon carcinoma reverts this tumorigenic cell to a non-tumorigenic variant. TIMP-3 maybe inhibiting the activity of a metalloproteinase in the extracellular matrix that cleaves TNFalpha receptors. The accumulation of TNFalpha receptors on the cell surface reconnects the TNFalpha signal transduction and apoptosis pathways in the DLD cells.
