We have observed that the cytokines, tumor necrosis factor alpha (TNF, cachectin) and interleukin-1 alpha (IL-1), induce the synthesis of the H subunit of ferritin, a major intracellular iron binding protein. The overall goals of our experiments are to elucidate the mechanism of the response of ferritin to these cytokines, and to assess the implications of altered ferritin composition on intracellular iron balance and selected iron-dependent pathways. Specifically, our first aim is to determine whether the increase in synthesis of H subunit-rich ferritins which occurs as a consequence of TNF or IL- 1 treatment modulates intracellular iron levels. This will be assessed by measuring the effects of TNF and IL-1 treatment on the iron content of ferritin and on chelatable iron pools. Ferritin H expression vectors will be used to distinguish effects on iron metabolism directly attributable to the increase in ferritin H from other effects of cytokine treatment. Our second aim is to determine the consequences of altered ferritin subunit composition on specific cellular functions. For these experiments we will use cells transfected with ferritin H expression vectors to measure the effect of altered ferritin subunit composition on the response to oxidant stress and the cytotoxic response to TNF. Our third aim is to explore the molecular mechanisms underlying the response of the ferritin H gene to TNF . Using chimeric genes, gel retardation assays and methylation interference, we will assess the role of specific transcription factors in mediating the response of the ferritin H gene to TNF and IL-1. Our fourth aim is to explore our very recent observation that treatment of myoblasts with iron induces the synthesis of a 23 kDa protein with properties thus far consistent with those predicted for a secreted ferritin: induction by iron, precipitation with anti-ferritin antibodies, glycosylation, and correct molecular mass. We propose to definitively assess whether this protein is a component of secreted ferritin, in what cell types it is synthesized, and what factors (including cytokines) influence its synthesis. This 23 kDa protein will be isolated and purified and its relationship to known ferritin subunits assessed. Taken together, these experiments will be used to clarify the effects exerted on intracellular iron metabolism by the cytokines TNF and IL-1.