Heme oxygenase (HO) activity is vital to a host of biological functions including cellular defense mechanisms, neuronal activity, vascular tone and male reproduction. The enzyme system oxidatively cleaves the heme molecule to biliverdin and carbon monoxide, a signal molecule. Biliverdin is reduced to bilirubin, an antioxidant, by biliverdin reductase (BVR). We have identified 3 HO isozymes: HO-1 (HSP-32), HO-2 and HO-3. HO-1 is exquisitely sensitive to environmental agents that cause oxidative stress and activate MAP kinase signal transduction pathways (e.g. 02 free radicals) as well as carcinogenesis and viral infections (e.g. Herpes, HIV). HO-2 and HO-3 are constitutively expressed. We have previously shown that HO-1 levels rapidly increase in response to oxidative stress both at the transcript and protein levels. The levels, however, rapidly decline with removal of the stimulus. Recently we identified HO-1 as a phosphoprotein. This type of protein modification can be of significance to protein stability and activity. Also, we discovered BVR to be a serine/threonine kinase and an activator of protein kinase C (PKC). PKC is upstream of the MAP kinase signaling pathway. On the other hand, we found that heme degradation product, biliverdin, is an in vivo inhibitor of HO activity and, a most effective inhibitor of PKC. Collectively, these findings lead us to hypothesize that a mechanism for the regulation of HO-1 expression by environmental oxidative stress involves both the kinase and the reductase activities of BVR: As a kinase, it activates the PKC/MAP kinase signaling pathway; and, as a reductase, it circumvents inhibition of the signaling pathway by biliverdin. We further hypothesize that phosphorylation is required for HO-1 activity and/or stability; hence, control of biliverdin production. These hypotheses will be tested by: 1) investigating the significance of HO-1 phosphorylation to its activity; and, examining whether rapid turnover of the induced HO-1 reflects a difference in its phosphorylation state under normal and induced conditions; 2) determining whether HO-1 is a phosphorylation substrate for BVR and/or PKC; 3) analyzing the regulatory role of BVR on induction of HO-1 gene expression through activation of the MAP kinase pathway; and; 4) assessing the effect of biliverdin on MAP kinase pathway activation. H202 will be utilized as the generator of 02 radicals. COS cells transfected with adenoviral vectors of effectors or their mutated counterparts will be used as the experimental model.
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