The induction of proline oxidase accompanying p53-dependent induction of apoptosis suggests that the proline metabolic pathway plays a role in programmed cell death and carcinogenesis. An important source of proline is from degradation of extracellular matrix (ECM) by metalloproteinases. We are studying these metabolic pathways at the level of : a) proline oxidase - its induction and its role in apoptosis; b) pyrroline 5-carboxylate (P5C) and P5C reductase in cell signaling; and c) prolidase: a mechanism linking extracellular matrix degradation, inflammation and apoptosis ? ? a) Proline oxidase - its induction and its role in apoptosis. In cells undergoing apoptosis, proline oxidase is induced and added proline stimulates the formation of reactive oxygen species (ROS). Importantly, in p53 negative cells transfected to overexpress proline oxidase, the addition of proline is sufficient to induce apoptosis. To define the specific reactive oxygen species, we co-expressed Ad-MnSOD, Ad-CuZnSOD or Ad-catalase in cells expressing POX. These experiments showed that POX generates superoxide radicals which is the mediator of apoptosis. Hydrogen peroxide, on the other hand, appears to be antiapoptotic perhaps due to its involvement in proliferative signaling. POX expression not only activates the intrinsic mitochondrial apoptosis pathway, but also the extrinsic, death receptor pathway. Recently, it has been shown that the expression of proline oxidase is silenced in certain tumors in the face of normal expression of p53, p21 and BAX suggesting that proline oxidase may act as a cancer suppressor. We are characterizing the proline oxidase promoter to determine its regulation on a molecular level. PPARgamma and its ligands are very active in upregulating POX expression. These ligands not only activate the POX promoter in luciferase assays, but also increase endogenous POX expression, findings which link POX with bioenergetics and obesity. In addition, we are monitoring the expression of genes participating in this metabolic paradigm using real-time PCR and panels of cancer cells and tumors. There is differential expression of the two oxidase enzymes, PRODH which codes for proline oxidase and PRODH2 which codes for hydroxyproline oxidase. PRODH and PRODH2 are expressed at high levels in renal tissue. In contrast, several tumor cell lines have low expression of both oxidase genes. ? ? b) Pyrroline 5-carboxylate (P5C) as a signaling and regulatory molecule. P5C, the product of proline oxidase, ornithine aminotransferase and glutamate synthase, has been shown to have regulatory activities. Recent work suggests that P5C reductase (P5CR) may play a role in receptor-mediated regulation. The two known isozymes of P5CR may serve distinct functions. P5CR1 is a NADH-dependent enzyme regulated to produce proline, whereas P5CR2 is a NADPH-dependent enzyme coupled to redox regulation. We are defining the interaction of P5CR2 with regulatory proteins using the yeast two-hybrid system (Myriad). Using P5CR2 as bait, several interesting proteins have been identified and are currently being characterized. ? ? c) Prolidase: a mechanism linking extracellular matrix degradation, inflammation and apoptosis The degradation of extracellular matrix (ECM) by matrix metalloproteinases (MMPs) is associated with inflammation, carcinogenesis and tumor invasion. Matrix proteins, especially collagen, can be a source of proline and hydroxyproline as substrate for bioenergetics under conditions of nutritional stress. The metabolic consequences of the release of the constituents of matrix proteins, especially proline and hydroxyproline, have received little attention. Prolidase catalyzes the final step in matrix degradation because it uniquely hydrolyzes imidodipeptides containing proline or hydroxyproline at the carboxyl terminus.
