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Keywords:

  • prostate cancer cells;
  • endothelial cells;
  • nitric oxide;
  • pleiotrophin;
  • migration

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Pleiotrophin (PTN) is a secreted growth factor involved in angiogenesis and tumor growth. We have recently shown that low concentrations 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 oxide synthase (eNOS) and the role of nitric oxide (NO) in the regulation 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 significantly 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 β/ζ (RPTPβ/ζ) completely abolished SNP-induced cell migration. NO added exogenously or produced endogenously by low concentrations of HP through stimulation of sGC activates extracellular signal-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 RPTPβ/ζ is a mediator of the stimulatory effects of eNOS/NO on human endothelial and prostate cancer cell migration. © 2008 Wiley-Liss, Inc.

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, neurotransmission, and development of the nervous system. It is synthesized by the enzyme nitric oxide synthase (NOS), which catalyzes the conversion of L-arginine to L-citrulline. NOS exists as 4 isoforms: 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 predominant role in tumor growth and metastasis, as well as in angiogenesis, and to mediate vascular endothelial growth factor (VEGF)-induced endothelial cell activation.2 In the same line, tumors implanted into eNOS−/− mice grow slower and exhibit reduced angiogenesis3 and eNOS-deficient mice exhibit significant impairment 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 concentrations of hydrogen peroxide (HP) on angiogenesis in vivo5 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, bovine, 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 several cellular processes and is involved in cell proliferation, migration, 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 pathological 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 proliferation and migration.12, 13

The aim of the present work was to study the possible involvement of eNOS and the role of NO in the regulation of PTN expression, 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 positively 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 kinase[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 β/ζ (RPTPβ/ζ). These data suggest that PTN may be a potential therapeutic target for NO-associated vascular or other disorders.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Materials

The NO donor sodium nitroprusside (SNP), the potent and selective inhibitor of NO sensitive sGC 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), and the nonselective inhibitor of NOS activity Nω-nitro-L-arginine methyl ester (L-NAME) were purchased from Sigma. The mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor U0126 and the potent inhibitor of eNOS activity N5-(1-iminoethyl)-L-ornithine dihydrochloride (L-NIO) were purchased from Tocris Cookson Ltd. The selective p38 inhibitor 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole (SB202190) and the selective c-Jun NH2-terminal kinase (JNK) inhibitor anthra[1,9cd]pyrazol-6(2H)-one (SP600125), were purchased from Biosource. All inhibitors at the concentrations used were not toxic to the cells. All cell culture media and reagents were purchased from Biochrom AG and Sigma.

Cell culture

HUVEC were isolated and cultured as previously described6 and used only at passage 1. HUVEC were grown in M199 medium supplemented with 15% fetal bovine serum (FBS), 150 μg/mL endothelial cell growth supplement, 4 units/mL heparin sodium, 100 units/mL penicillin, 100 μg/mL streptomycin, 50 μg/mL gentamycin, and 2.5 μg/mL amphotericin B. The human prostate cancer epithelial cell line LNCaP (ATCC) was grown routinely in RPMI-1640 medium supplemented with 10% FBS, 100 units/mL penicillin, 100 μg/mL streptomycin, 50 μg/mL gentamycin, and 2.5 μg/mL amphotericin B. Cultures were maintained at 37°C, 5% CO2, and 100% humidity. For treatment with HP or SNP, cells were grown in their corresponding medium supplemented with 2% FBS. Other treatments were initiated 30 min before HP or SNP treatment. Development of stably transfected LNCaP cell lines, PC-LNCaP bearing the appropriate control vector (pCDNA3.1) and AS-LNCaP expressing antisense PTN has been described previously.14

Transient transfection and luciferase assay

HUVEC or LNCaP cells were transfected with wild-type hPTNpro2.3-Luc reporter plasmid containing 2.3 kb of the human PTN 5′-flanking region, as previously described12 with small modifications. Briefly, at 80% confluency, cells were incubated with 3 μg of DNA and 9.6 μL of jetPEI™ (Polyplus Transfection) in 2 mL M199 supplemented with 5% FBS/well in 6-well plates for 2 hrs at 37°C. After addition of FBS to a final concentration of 10%, the cells were incubated for 2 hrs at 37°C. The transfection medium was removed and cells were grown in M199 supplemented with 15% FBS at 37°C. After 16 hrs, the culture medium was replaced with M199 containing 5% FBS and substances tested. Cells were harvested and luciferase activity was determined using the Luciferase Reporter Gene Assay (Roche Applied Science). Cell lysates were analyzed for protein content using the Bradford method and luminescence units were normalized for total protein content.

Down-regulation of eNOS expression using antisense oligonucleotides

Antisense oligonucleotides for eNOS were obtained from VBC Biotech. The following sequences were used as previously described16: eNOS antisense: 5′-CTT CAA GTT GCC CAT-3′ and eNOS sense: 5′-ATG GGC AAC TTG AAG-3′. A total of 5 × 105 HUVEC were plated onto 60-mm tissue culture dishes and left for 16 hr. Oligonucleotides were introduced into HUVEC using JetPEI™-HUVEC (Polyplus Tranfection), according to the manufacturer's protocol. After 4 hrs, HUVEC were incubated for 18 hrs in fresh serum-free medium containing 0.25% bovine serum albumin (BSA), before stimulation with HP.

Migration assays

Migration assays were performed as previously described,6, 12 in a 24-well microchemotaxis chamber (Costar), using untreated polycarbonate membranes with 8 μm pores. The bottom chamber was filled with 0.6 mL of medium containing 0.25% BSA and the tested agents at the concentrations indicated. The upper chamber was loaded with 105 cells/0.1 mL and incubated for 4 hrs at 37°C. Filters were fixed with saline-buffered formalin and stained using 0.03% toluidine blue. The cells that migrated through the filter were quantified by counting the whole area of each filter, using a grid and an Optech microscope at a 20× magnification.

Evaluation of DNA binding activity of AP-1 by enzyme-linked immunosorbent assay

The DNA binding activity of AP-1 was quantified by enzyme-linked immunosorbent assay (ELISA) using the Trans-AM AP-1 Transcription Factor Family Assay kit (Active Motif Europe), as previously described.12 Briefly, nuclear extracts were prepared using Nuclear Extract Kit (Active Motif Europe) and incubated in 96-well plates coated with immobilized oligonucleotide containing a consensus binding site for AP-1 (wild-type TPA Response Element, TRE). AP-1 binding to the target oligonucleotide was detected by incubation with primary antibodies specific for c-Fos, Fos-B, Fra-1, Fra-2, JunB, JunD, or the activated form of c-Jun, visualized with anti-IgG horseradish peroxidase conjugate and developing solution, and quantified at 450 nm with a reference wavelength of 655 nm. The specificity of the assay was verified using soluble oligonucleotides containing TRE.

Western blot analysis

The presence of PTN secreted in the cell culture medium was investigated as described previously.12 Briefly, the conditioned medium of the cells was incubated overnight with 100 μL of heparin-Sepharose (GE Healthcare, UK) at 4°C with continuous agitation. Bound proteins were eluted with 50 μL of Laemmli sample buffer under reducing conditions, fractionated on 17.5% SDS-PAGE, and transferred to Immobilon-P membranes. Blocking was performed by incubating the membranes with 3% BSA in Tris-buffered saline (TBS) containing 0.05% Tween 20 (TBS-T). The membranes were then incubated with 45 ng/mL affinity purified anti-PTN antibody in TBS-T for 1 hr at room temperature under continuous agitation and then with horseradish peroxidase-conjugated rabbit anti-goat IgG (Sigma) at a dilution of 1:7,500 in TBS-T, for 1 hr at room temperature under continuous agitation.

To study activation of ERKs [1/2] and p38, cell lysates were analyzed by SDS-PAGE and transferred to Immobilon-P membranes, as previously described.6, 13 Blocking was performed by incubating the membranes with 5% nonfat dry milk in TBS, pH 7.4, containing 0.1% Tween 20 (TBS-T), for 2 hrs at room temperature. Membranes were further incubated in primary antibodies for 18 hrs at 4 °C under continuous agitation, as follows: anti-phosho-ERK[1/2] antibody (phospho-44/42 mitogen-activated protein kinase MAPK, on Thr202/Tyr204, Cell Signaling), or anti-phospho-p38 antibody (phosphop38 MAP kinase on Thr180/Tyr182, Cell Signaling), at 1:1,000 dilution in TBS-T containing 5% BSA. Blots for phospho-ERK[1/2] and phospho-p38 were stripped and subjected to subsequent Western blotting for the corresponding total proteins as follows: anti-ERK[1/2] antibody (Upstate Biotechnology) at 1:5,000 dilution in TBS-T containing 3% nonfat dry milk and anti-p38 antibody (Cell Signaling) at 1:1,000 dilution in TBS-T containing 5% BSA, for 1 hr at room temperature under continuous agitation. Membranes were further incubated with horseradish peroxidase-conjugated anti-rabbit IgG (Cell Signaling) at a dilution of 1:2,000 in TBS-T containing 5% nonfat dry milk for 1 hr at room temperature under continuous agitation. Detection of immunoreactive bands was performed by the Chemilucent detection system kit (Chemicon International), according to the manufacturer's instructions. The protein levels that corresponded to each band were quantified using the ImagePC image analysis software (Scion Corp., Frederick, MD).

RNA interference

RNA oligonucleotide primers for RPTPβ/ζ were obtained from VBC Biotech. The following sequences were used, as previously described15: RPTPβ/ζ sense, 5′-AAAUGCGAAUCCUAAAGC GUU-3′; RPTPβ/ζ antisense, 5′-AACGCUUUAGGAUUCG CAUUU-3′. Cells were passaged and grown to a confluence of 40% in medium without antibiotics. Transfection of cells was performed in serum-free medium for 4 hrs using annealed RNA at the concentration of 50 nM and jetSI-ENDO (Polyplus-Transfection) as transfection reagent. Cells were incubated for another 24 hrs in serum-containing medium and serum-starved before further experiments. Transfection efficiency was evaluated by RT-PCR and Western blot analysis, as previously described.15 Double-stranded negative control siRNA from Ambion was also used in all experiments.

Statistical analysis

The significance of variability between the results from various groups was determined by one-way analysis of variance (ANOVA). Each experiment included triplicate measurements for each condition tested, unless otherwise indicated. All results are expressed as mean ± S.E.M. from at least 3 independent experiments.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Inhibition of eNOS inhibits HP-induced PTN expression and secretion through inhibition of AP-1 activation

Since we have previously shown that eNOS6 and PTN12 intervene in the HP-induced migration of HUVEC and LNCaP cells respectively, in the present work we initially studied whether eNOS affects HP-induced PTN secretion by HUVEC and LNCaP cells. Both types of cells are known to express eNOS5, 17 and this was verified in the present study (data not shown). As shown in Figure 1, preincubation of cells with the NOS inhibitors L-NAME (1 mM) and L-NIO (100 μM) abolished HP-induced PTN protein secretion, as well as transcriptional activation of the ptn gene, as evidenced by the reporter activity of a plasmid construct containing the full-length promoter of the human ptn gene fused to a luciferase reporter gene. Interestingly, the sGC inhibitor ODQ (5μM), which has been shown to inhibit HP-induced HUVEC migration,6 also abolished HP-induced PTN protein secretion and ptn gene transcriptional activation.

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Figure 1. Effect of eNOS and sGC inhibition on HP-induced expression and secretion of PTN by HUVEC (a and b) and LNCaP cells (c and d). (a) and (c) Show representative pictures of Western blot analysis of cell culture medium for PTN of 4 independent experiments. PTN protein amounts were quantified by densitometric analysis of the corresponding band in each lane. Results are expressed as mean ± S.E.M. of the percent change of the amounts of secreted PTN protein in treated when compared with the untreated cells (set as default = 100). (b) and (d) show the reporter activities of hPTNpro2.3-Luc, which are expressed as mean ± S.E.M. of relative luciferase units (RLU) per mg of total protein (d) or as mean ± S.E.M. of the percent change of relative luciferase activity per mg of total protein in treated compared with the untreated cells (set as default = 100) (b). Asterisks in all cases denote a statistically significant difference from cells treated with HP. **p < 0.01.

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We have recently shown that AP-1 activation is responsible for HP-induced PTN expression and secretion in LNCaP cells and the AP-1 complexes that bind to the corresponding sites of the PTN promoter consist of Fra-1, JunD, and c-Jun.12, 13 As shown in Figure 2, inhibition of eNOS or sGC abolished HP-induced activation of Fra-1, JunD, and c-Jun in both types of cells, suggesting that the NO/cGMP pathway acts up-stream of AP-1 activation that leads to PTN expression and secretion.

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Figure 2. Effect of eNOS and sGC inhibition on the binding activities of Jun and Fos family members in HP (10 μM) stimulated HUVEC (a) and LNCaP cells (b). Results are expressed as mean ± S.E.M. of the percentage change of the binding of each AP-1 subunit when compared with the untreated cells (set as default = 100). Asterisks denote a statistically significant difference from cells treated with HP. *p < 0.05, **p < 0.01, ***p < 0.001. L-NAME, cells treated with L-NAME 1 mM; ODQ, cells treated with ODQ 5 μM.

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Exogenous NO increases the expression and secretion of PTN

To verify that NO affects PTN expression, we incubated HUVEC with different concentrations of the NO donor SNP and measured protein amounts of PTN secreted into the cell culture medium, as well as transcriptional activation of the ptn gene. PTN protein levels secreted into the culture medium of HUVEC were significantly increased in a concentration-dependent manner 24 hrs after addition of SNP (Fig. 3a). Similarly, SNP significantly induced transcriptional activation of the ptn gene. Reporter activity was increased since the first 3 hrs after SNP addition into the cell culture medium and was maximal at 6 hrs (Fig. 3b).

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Figure 3. Effect of SNP on PTN secretion (a) and transcriptional activation of the ptn gene (b). (a) Shows a representative picture of Western blot analysis of HUVEC culture medium for PTN of 4 independent experiments. PTN protein amounts were quantified by densitometric analysis of the corresponding band in each lane. Results are expressed as mean ± S.E.M. of the percent change of the amounts of secreted PTN protein in treated when compared with the untreated cells (set as default = 100). (b) Shows the reporter activities of the human hPTNpro2.3-Luc, which are expressed as mean ± S.E.M. of the percent change of relative luciferase activity per mg of total protein in treated when compared with the untreated cells (set as default = 100). Asterisks in all cases denote a statistically significant difference from the corresponding untreated cells. *p < 0.05, ***p < 0.001.

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PTN expression is required for NO-induced cell migration

We have recently shown that endogenous PTN expression plays a key role in LNCaP cell migration.14 To evaluate the role of PTN in NO-induced LNCaP cell migration, we used LNCaP cells expressing either antisense PTN (AS-LNCaP) or the appropriate control vector (PC-LNCaP), as previously described.14 SNP significantly increased PC-LNCaP cell migration, similar to its effect on nontransfected LNCaP cells. In contrast, it had no effect on AS-LNCaP cell migration (Fig. 4a).

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Figure 4. PTN through its receptor RPTPβ/ζ is required for NO-induced cell migration. (a) Effect of SNP on LNCaP cell migration. (b) Effect of RPTPβ/ζ down-regulation on HP- or SNP-induced HUVEC migration. (c) Effect of RPTPβ/ζ down-regulation on SNP-induced LNCaP cell migration. Results are expressed as mean ± S.E.M. of the percent change in the number of migrating cells when compared with the nontransfected, untreated cells (set as default = 100). LNCaP, non-transfected LNCaP cells; PC-LNCaP, LNCaP cells transfected with the plasmid containing only the neomycin resistance gene; AS-LNCaP, LNCaP cells transfected with the plasmid containing antisense PTN. Asterisks denote a statistically significant difference from the nontransfected, untreated cells. **p < 0.01, ***p < 0.001.

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RPTPβ/ζ knockdown by RNA interference interrupts HP-induced cell migration

We have recently shown that RPTPβ/ζ is a receptor for PTN in HUVEC and mediates PTN-induced migration.15 To evaluate whether RPTPβ/ζ is responsible for the stimulatory effects of HP and NO on cell migration, we performed migration assays after down-regulation of RPTPβ/ζ by RNA interference and stimulation with low concentrations of HP or SNP. RPTPβ/ζ down-regulation totally inhibits HUVEC migration induced by HP or SNP (Fig. 4b) and LNCaP cell migration induced by SNP (Fig. 4c).

MAPKs act both upstream and downstream of eNOS and mediate HP-induced PTN secretion and cell migration

We studied whether MAPKs are involved in the signaling pathway that is induced by low concentrations of HP and eNOS, and leads to PTN secretion and cell migration. The inhibitors of ERK[1/2] activation U0126 (100 nM) and p38 activity SB202190 (10 μM), but not the inhibitor of JNKs activity SP600125 (10μM), completely reversed HP-induced AP-1 activation (data not shown), PTN secretion (Fig. 5a), and HUVEC migration (Fig. 5b). Blockade of eNOS or sGC by L-NAME or ODQ, respectively, significantly attenuated HP-induced activation of ERK[1/2] (Fig. 6a) but not that of p38 (Fig. 6b) and inhibition of p38 activity by SB202190 (10 μM) completely prevented HP-induced ERK[1/2] activation (Fig. 6c), suggesting that eNOS lies up-stream of ERK[1/2] but down-stream of p38 in this pathway. The latter was confirmed by down-regulation of eNOS expression with the use of antisense oligonucleotides. In this case, HP induced activation of ERK[1/2] but not of p38 was attenuated (Fig. 6d), similarly to what has been observed with the pharmacological inhibitors.

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Figure 5. Implication of MAPKs in HP-induced PTN secretion (a) and cell migration (b). (a) Shows a representative picture of Western blot analysis of HUVEC culture medium for PTN of 4 independent experiments. PTN protein amounts were quantified by densitometric analysis of the corresponding band in each lane. Results are expressed as mean ± S.E.M. of the percent change of the amounts of secreted PTN protein in treated when compared with the untreated cells (set as default = 100). (b) Shows the effect of MAPKs inhibition on HP-induced HUVEC migration. Results are expressed as mean ± S.E.M. of the percent change in the number of migrating cells compared with the untreated cells (set as default = 100). U, cells treated with U0126; SB, cells treated with SB202190; SP, cells treated with SP600125. Asterisks in all cases denote a statistically significant difference from the cells treated with HP. *p < 0.05, **p < 0.01.

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Figure 6. Role of eNOS in HP-induced signaling in HUVEC. (a) and (b) Effect of eNOS and sGC inhibition on HP-induced ERK[1/2] and p38 activation, respectively. Representative pictures of Western blot analysis of HUVEC lysates for phospho and total ERK[1/2] and p38 of 4 independent experiments are shown. Protein amounts were quantified by densitometric analysis of the corresponding bands and the ratio pERK[1/2]/ERK[1/2] or pp38/p38 was calculated in each lane. (c) Effect of p38 inhibition on HP-induced ERK[1/2] activation. Representative pictures of Western blot analysis of HUVEC lysates for phospho and total ERK[1/2] are shown. Protein amounts were quantified by densitometric analysis of the corresponding bands and the ratio pERK[1/2]/ERK[1/2] was calculated in each lane. Results in a, b, and c are expressed as mean ± S.E.M. of the percent change of the ratio of phosphorylated to the corresponding total protein in treated when compared with the untreated cells (set as default = 100). Asterisks denote a statistically significant difference from the cells treated with HP. **p < 0.01. (d) Effect of down-regulation of eNOS by antisense oligonucleotides on HP-induced ERK[1/2] and p38 activation. Representative pictures of Western blot analysis of HUVEC lysates for eNOS and actin (up) or phospho and total ERK[1/2] and p38 (down) are shown. SB, cells treated with SB202190; wt, untrasfected cells; S, cells transfected with sense oligonucleotides for eNOS; AS, cells transfected with antisense oligonucleotides for eNOS.

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ERK[1/2] but not p38 mediate NO-induced PTN secretion and cell migration

We further studied whether and how the inhibitors of ERK[1/2] activation U0126 (100 nM) or p38 activity SB202190 (10 μM) affect SNP-induced PTN secretion and cell migration. As shown in Figure 7, inhibition of p38 activity had no effect, whereas inhibition of ERK[1/2] completely abolished SNP-induced PTN secretion and HUVEC migration. The stimulatory effect of SNP on PTN secretion and HUVEC migration seems to be mediated by sGC, since it was completely inhibited by ODQ (5 μM).

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Figure 7. Effect of ERK[1/2] and p38 inhibition on NO-induced PTN secretion (a) and cell migration (b). (a) Shows a representative picture of Western blot analysis of HUVEC culture medium for PTN of 3 independent experiments. PTN protein amounts were quantified by densitometric analysis of the corresponding band in each lane. Results are expressed as mean ± S.E.M. of the percent change of the amounts of secreted PTN protein in treated when compared with the untreated cells (set as default = 100). (b) Shows the effect of MAPKs inhibition on SNP-induced HUVEC migration. Results are expressed as mean ± S.E.M. of the percent change in the number of migrating cells when compared with the untreated cells (set as default = 100). SB, cells treated with SB202190; U, cells treated with U0126. Asterisks in all cases denote a statistically significant difference from the cells treated with SNP. *p < 0.05, **p < 0.01.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

In the present study we showed that (i) eNOS and NO regulate transcriptional activation of the ptn gene and PTN secretion through sequential activation of sGC, ERK[1/2] and AP-1, and (ii) PTN mediates NO-induced human endothelial and prostate cancer cell migration through its receptor RPTPβ/ζ. To our knowledge, this is the first study that shows the involvement of eNOS/NO in PTN transcription and secretion, as well as involvement of the latter in NO-induced cell migration.

Although the human ptn gene has been considered a potent proto-oncogene, very little is known on the regulation of PTN expression.10, 11 We have recently shown that the PTN promoter contains 2 AP-1 binding sites responsible for induction of PTN expression by HP and bFGF in human prostate cancer LNCaP cells.12, 13 The present study verifies induction of PTN expression by HP in HUVEC, which is inhibited by pharmacological inhibition of AP-1 by curcumin (supplementary file 1), similarly to LNCaP cells.12, 13 Interestingly, among the MAPKs known to be involved in AP-1 activation,18 only p38 and ERK[1/2] seem to participate in the pathway activated by low concentrations of HP and leading to AP-1 activation, PTN transcription/secretion and cell migration. JNKs are not involved (present study and 13), in line with the notion that JNKs are activated only by high concentrations of HP and participate in the signaling pathway induced by oxidative stress,19, 20 but are not affected by signaling levels of HP13 and are not involved in cell migration.21 The involvement of ERK[1/2] and p38 in the effects of signaling concentrations of HP has been previously shown13, 22; however, in the present study we further show that in the pathway leading to PTN expression, p38 lies up-stream of eNOS and ERK[1/2], in line with studies suggesting a p38-dependent eNOS23 activation, which then activates ERK[1/2].24, 25 HP activates eNOS through activation of Akt,26, 27 which in the studied pathway lies down-stream of p38 (supplementary file 2) and has been also implicated in the regulation of PTN expression.28

We have recently shown that NO mediates HP-induced angiogenesis in vivo5 and endothelial cell migration in vitro.6 The results of the present study suggest that this may be due to eNOS/NO mediated PTN expression, which is also angiogenic in the same systems.15, 29 Besides endothelial cells, NO seems to also affect PTN expression in human prostate cancer cells (present study) as well as C6 rat glioma cells (supplementary file 3), suggesting that this may be a universal pathway playing significant role(s) in cell functions, considering the multiple actions that have been attributed to both NO and PTN. Since eNOS/NO are activated by several growth factors and participate in their actions,2 it would be tempting to speculate that PTN may mediate the effects of several growth factors on different types of cells.

The effect of NO on PTN transcription and secretion seems to be mediated by sGC and activation of ERK[1/2], but not p38, in line with data showing that SNP-induced p38 activation is not mediated by cGMP and is not involved in NO-induced inhibition of cardiomyocyte apoptosis,25 although cGMP mediates NO-induced ERK[1/2] activation.24, 25 The present study, together with our observation that exogenous bromo-cGMP induces PTN secretion in glioma cells (supplementary file 3), suggest that PTN may also mediate the stimulatory effects of cGMP on angiogenesis8, 9 or/and tumor growth.24 It is not known, however, which cellular targets of cGMP affect PTN secretion.

As already mentioned, both PTN10, 11 and NO1 are considered proangiogenic and tumorigenic molecules, which fits with the notion that PTN mediates NO-induced endothelial and tumor cell migration, a key step in angiogenesis and tumor growth and metastasis. On the other hand, both PTN and NO have been mentioned to be inhibitory on angiogenesis and tumor growth under certain circumstances.1, 10, 11, 30, 31 Although the reasons for these discrepancies remain unclear, it is interesting to note that at least in some cases, the existing data suggest common effects of both molecules. For example, PTN acts as an angiostatic factor in an in vivo neuroblastoma model resistant to the DNA-topoisomerase I inhibitor irinotecan,32 and NO has been also reported as an anti-angiogenic molecule in neuroblastomas.33 The mRNA levels of both PTN and RPTPβ/ζ are decreased in colorectal cancers as compared with those in adjacent normal mucosa34 and eNOS expression in colorectal carcinoma is significantly lower than in non-neoplasm colorectal mucosa,35 while NO induces apoptosis in colon cancer cells36, 37 via ERK[1/2] activation.37 PTN is increased in non–tumor-bearing skin adjacent to excised squamous cell carcinomas38 and eNOS-transfected oral carcinoma SCC-25 cells have decreased growth rate in vitro and in vivo compared with the wild-type parental or vector control-transfected cells.39 Over-expression of PTN in NIH 3T3 cells has been implicated in cellular quiescence rather than an oncogenic phenotype40 and PTN has been identified as a confluence specific protein in several types of cells,10, 40, 41 among which endothelial cells.10 Similarly, activity of eNOS is significantly increased in quiescent compared with growing endothelial cells.42–44 Based on the above, it would be worthwhile to further elucidate this point and examine whether PTN/PTN receptors system is the determinant of the pro- or anti-angiogenic or/and tumorigenic effects of NO in vitro and in vivo.

On the opposite direction, the role of NO in PTN-induced cell migration has not been clarified yet. Heiss et al.45 suggest that PTN-induced migration of HUVEC or circulating endothelial progenitor cells is NO-dependent, in contrast to Souttou et al.46 who observe no dependence on NO in HUVEC. Results on the effect of PTN on NO production are also contradictory, with one study suggesting increased45 and another decreased47 NO production by PTN. We have observed a PTN-induced concentration-dependent increase in NO production in GH3 rat pituitary tumor cells, but no effect in rat glioma C6 cells (unpublished observation). What is(are) the reason(s) for these discrepancies is not known and further, more detailed studies are required to elucidate if NO is implicated in PTN-induced migration as well as which NOS isoforms may be involved.

In summary, in the present study we describe the signaling pathway involved in the regulation of PTN expression by eNOS/NO and suggest that PTN mediates NO-induced human endothelial and prostate cancer cell migration through its receptor RPTPβ/ζ (Fig. 8). These results strengthen the importance of PTN for angiogenesis and prostate cancer cell functions and the notion that PTN or/and its receptor RPTPβ/ζ may be new targets for anti-angiogenic or/and anti-tumor therapies.

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Figure 8. Schematic diagram of the suggested pathway for the eNOS/NO-dependent up-regulation of PTN, which leads to human endothelial and prostate cancer cell migration through its receptor RPTPβ/ζ.

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Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The authors thank the Private Maternity Clinic of Patras for providing us with umbilical cords.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
IJC_24084_sm_suppinfofig1.tif887KSupplementary Figure 1
IJC_24084_sm_suppinfofig2.tif736KSupplementary Figure 2
IJC_24084_sm_suppinfofig3.tif299KSupplementary Figure 3

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