We first showed that nitric oxide (NO), a versatile inflammatory signal, increases prolidase activity by increasing its phosphorylation on serine/threonine residues. In NIH 3T3 mouse fibroblasts stimulated with NO donors (DETA/NO) or in cells transfected with iNOS to activate endogenous NO production, prolidase activity was increased more than 2-fold, without an increase in prolidase protein. Since phosphorylation was a possibility, we immunoprecipitated prolidase and found increased immunodetectible phosphorylated serine/threonine. Since cGMP-PKG pathway is the main mediator of NO effects, we treated cells with 8-bromo-cGMP, a cGMP agonist, and found that it stimulated the phosphorylation of prolidase. Furthermore, the effects of DETA/NO could be blocked by Rp-8-pCPT-cGMP, an inhibitor of cGMP kinase, but not by UO126, an inhibitor of ERK1/2 signaling. Thus, NO activates prolidase activity through phosphorylation of serine/threonine residues mediated by cGMP-PKG signaling and not through MAP kinases. These findings provide a molecular basis for a prolidase response to inflammatory signals. The wound-healing defect in patients with prolidase deficiency has been described as angiopathic in histologic examinations postmortem. This was found both in nonhealing ulcers and at distant organ sites. Since the products of prolidase (proline, hydroxy-proline) are involved in a number of regulatory mechanisms, we asked whether prolidase might play a role in angiogenesis. To test this hypothesis, we obtained a gain-of-function experimental model by transfecting RKO colorectal cancer cells with a prolidase cDNA expression plasmid and isolating stable transfectants which express 15-20 fold that of vector controls as determined by enzyme activity and western blots. The prolidase expressing cells (PL) have increased levels of vascular endothelial growth factor (VEGF) in whole cell extracts by western blots and in conditioned media as measured by ELISA. Glucose transporter-1 (Glut-1) is also increased in PL cells. Since both VEGF and Glut-1 are target genes of HIF-1, we measured HIF-1 transcriptional activity using a hypoxia response element (HRE) -luciferase construct and found that HIF-1 was much more active in PL cells. As expected, HIF-1alpha levels were higher in nuclear extracts from PL cells. Since HIF-1alpha is regulated primarily by degradation after prolyl hydroxylation of the oxygen dependent degradation domain (ODD), we used an ODD-luciferase construct to examine the effect of PL on prolyl hydroxylase-dependent proteasomal degradation. We found that ODD degradation was markedly decreased in PL cells. That these mechanisms are due to the catalytic activity of prolidase was shown by the finding that proline and hydroxyproline could augment the effects. Furthermore, inhibition of prolidase activity by N-benzyloxycarbonyl-L-proline (Cbz-Pro) markedly decreased the angiogenic signaling with increased prolidase. In conclusion, accompanying the activation of MMP and increased ECM degradation as a source of stress substrates (proline/hydroxyproline), signals are generated to activate angiogenesis to augment the nutrient supply. The defective wound healing in patients with prolidase deficiency is of considerable interest since cancer has been considered """"""""a wound that never heals."""""""" Thus, we initiated a project to develop prolidase knockout mice. Embryonic stem cells with the prolidase gene knocked out were purchased from BayGenomics. These ES cells were targeted with a gene-trap vector containing a splice acceptor sequence upstream of a reporter gene, beta-geo, a fusion of beta-galactosidase and neomycin phosphotransferase II. The construct, randomly inserted into the murine genome was then inserted into intron 11 of the prolidase gene on chromosome 7 in the mouse genome. Following karyotyping, the targeted ES cells were microinjected into C57Bl/6 blastocysts to obtain chimeric mice. We have obtained prolidase null mice and are in the process of backcrossing with C57Bl/6 and Balb C mice to obtain knockouts isogenic in both strains. We will then perform a variety of carcinogenesis and cancer metastasis studies as well as charcterize the immune cells in these animals.
| Surazynski, Arkadiusz; Liu, Yongmin; Miltyk, Wojciech et al. (2005) Nitric oxide regulates prolidase activity by serine/threonine phosphorylation. J Cell Biochem 96:1086-94 |
