Nitric oxide stimulates migration of human endothelial and prostate cancer cells through up-regulation of pleiotrophin expression and its receptor protein tyrosine phosphatase b/f Christos Polytarchou, Maria Hatziapostolou, Evangelia Poimenidi, Constantinos Mikelis, Ariadni Papadopoulou, Anastasia Parthymou and Evangelia Papadimitriou * Laboratory of Molecular Pharmacology, Department of Pharmacy, University of Patras, Patras, Greece Pleiotrophin (PTN) is a secreted growth factor involved in angio- genesis and tumor growth. We have recently shown that low con- centrations of hydrogen peroxide (HP) stimulate PTN expression, through activation of the transcription factor AP-1. In the present work, we studied the possible involvement of endothelial nitric ox- ide synthase (eNOS) and the role of nitric oxide (NO) in the regu- lation of PTN expression, as well as involvement of the latter in the NO-induced human endothelial and prostate cancer cell migration. Inhibition of eNOS or the downstream effector soluble guanylate cyclase (sGC) completely suppressed HP-induced AP-1 activities that lead to PTN expression and cell migration. The NO donor sodium nitroprusside (SNP) through activation of sGC sig- nificantly and concentration-dependently increased expression of PTN, through transcriptional activation of the corresponding gene. Moreover, SNP had no effect on the migration of stably transfected prostate cancer cells that do not express PTN and knockdown of PTN receptor protein tyrosine phosphatase b/f (RPTPb/f) completely abolished SNP-induced cell migration. NO added exogenously or produced endogenously by low concentra- tions of HP through stimulation of sGC activates extracellular sig- nal-regulated kinase[1/2] (ERK[1/2]) and leads to PTN expression and cell migration. On the other hand, p38, which also intervenes in the up-regulation of PTN expression by low concentrations of HP, seems to act upstream of eNOS and does not intervene in the SNP-induced PTN expression and cell migration. The above data suggest that PTN through its receptor RPTPb/f is a mediator of the stimulatory effects of eNOS/NO on human endothelial and prostate cancer cell migration. ' 2008 Wiley-Liss, Inc. Key words: prostate cancer cells; endothelial cells; nitric oxide; pleiotrophin; migration Nitric oxide (NO) is a highly diffusible signaling molecule that mediates a number of functions, such as blood flow, vasodilation, vascular permeability, angiogenesis, immune responses, neuro- transmission, and development of the nervous system. It is synthe- sized by the enzyme nitric oxide synthase (NOS), which catalyzes the conversion of L-arginine to L-citrulline. NOS exists as 4 iso- forms: endothelial NOS (eNOS), neuronal, inducible, and more recently mitochondrial NOS. Although 2 apparently conflicting views exist, overall there seems to be a positive correlation between NO and tumor progression and angiogenesis. 1 Among the NOS isoforms, eNOS has been demonstrated to play a predomi- nant role in tumor growth and metastasis, as well as in angiogene- sis, and to mediate vascular endothelial growth factor (VEGF)- induced endothelial cell activation. 2 In the same line, tumors implanted into eNOS2/2 mice grow slower and exhibit reduced angiogenesis 3 and eNOS-deficient mice exhibit significant impair- ment of angiogenesis in the ischemic limb, indicating that NO modulates angiogenesis in ischemic tissue. 4 We have recently shown that NO mediates the stimulatory effects of signaling con- centrations of hydrogen peroxide (HP) on angiogenesis in vivo 5 and human umbilical vein endothelial cell (HUVEC) migration in vitro. 6 The effect of NO on angiogenesis and tumor growth has been described to depend, at least partly, on activation of soluble guanylate cyclase (sGC) and production of cGMP, 1,6,7 and cGMP by itself has been also shown to have proangiogenic properties. 8,9 Pleiotrophin (PTN), also called heparin affin regulatory peptide or heparin-binding growth-associated molecule, is an 18-kDa secreted growth factor that displays high affinity for heparin. PTN is highly conserved among species, such as human, mouse, rat, bo- vine, fish, chicken, frog, and insects, and its gene is expressed in a highly restricted temporal and spatial pattern during development, suggesting that PTN may be an important protein that potentially contributes to a number of different regulating systems. A growing body of evidence indicates that PTN plays a significant role in sev- eral cellular processes and is involved in cell proliferation, migra- tion, and differentiation. PTN is detected in various carcinomas exhibiting a proto-oncogene function and seems to play a major role in physiological, as well as tumor angiogenesis. Although several data declare that PTN gene is up-regulated in several path- ological situations, very little is known on the regulation of its expression (reviewed in refs. 10, 11). We have recently shown that HP and basic fibroblast growth factor (bFGF) up-regulate the human PTN gene through activation of the transcription factor AP-1. 12,13 Interestingly, PTN seems to mediate the stimulatory effects of HP and bFGF in human prostate cancer LNCaP cell pro- liferation and migration. 12,13 The aim of the present work was to study the possible involve- ment of eNOS and the role of NO in the regulation of PTN expres- sion, as well as involvement of the latter in the NO-induced HUVEC and human prostate cancer LNCaP cell migration. The rationale for using these 2 types of cells is based on our previous findings that PTN is expressed and secreted from both and posi- tively regulates their migration. 14,15 Moreover, both types of cells are involved in tumor growth, with migrating endothelial cells supporting angiogenesis to supply the nutrients and oxygen required for tumor growth and facilitate metastasis of migrating prostate cancer cells. Our results show that NO regulates PTN expression through activation of extracellular signal-regulated ki- nase[1/2] (ERK[1/2]) and AP-1 and PTN mediates NO-induced human endothelial and prostate cancer cell migration through its receptor protein tyrosine phosphatase b/f (RPTPb/f). These data suggest that PTN may be a potential therapeutic target for NO- associated vascular or other disorders. Material and Methods Materials The NO donor sodium nitroprusside (SNP), the potent and selective inhibitor of NO sensitive sGC 1H-[1,2,4]oxadiazole[4,3- Christos Polyarchou and Maria Hatziapostolou’s current address is: Mo- lecular Oncology Research Institute, Tufts Medical Center, 800 Washing- ton Street, Boston, MA 02111. The first two authors contributed equally to this work. *Correspondence to: E. Papadimitriou, Ph.D., Laboratory of Molecular Pharmacology, Department of Pharmacy, University of Patras, GR 26504 Greece. Fax: 0030-2610-969336. E-mail: epapad@upatras.gr Received 11 April 2008; Accepted after revision 8 October 2008 DOI 10.1002/ijc.24084 Published online 20 October 2008 in Wiley InterScience (www.interscience. wiley.com). Additional Supporting Information may be found in the online version of this article. Grant sponsors: The European Social Fund (ESF), Operational Program for Educational and Vocational Training II (EPEAEK II), and particularly the Program PYTHAGORAS. Int. J. Cancer: 124, 1785–1793 (2009) ' 2008 Wiley-Liss, Inc. Publication of the International Union Against Cancer