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Cross-talk between vascular endothelial growth factor and semaphorin-3A pathway in the regulation of normal and malignant mesothelial cell proliferation¹

ALFONSO CATALANO^*,2, PAOLA CAPRARI^*, SABRINA RODILOSSI^*, PIERGIACOMO BETTA, MARIO CASTELLUCCI^¶, ANDREA CASAZZA, LUCA TAMAGNONE and ANTONIO PROCOPIO^*

^* Department of Molecular Pathology and Innovative Therapies, Laboratory of Experimental Pathology, and the
^¶ Institute of Normal Human Morphology, Polytechnic University of Marche, Ancona, Italy; the
Pathology Unit, Department of Oncology, City Hospital, Alessandria, Italy; and the
Institute for Cancer Research (IRCC), University of Torino, Candiolo, Italy

² Correspondence: Dipartimento di Patologia Molecolare e Terapie Innovative, Laboratorio di Patologia Sperimentale, Università Politecnica delle Marche, Via Ranieri 31, 60131, Ancona, Italy. E-mail: catgfp{at}yahoo.it

SPECIFIC AIMS

We investigated the relationship between vascular endothelial growth factor (VEGF)/VEGF receptor system and the secreted semaphorin-3A (Sema-3A) pathways in human mesothelial cells with the intent to identify molecular mechanisms differentially activated in malignant cells.

PRINCIPAL FINDINGS

1. VEGF induces cell growth in malignant but not in normal mesothelial cells
The Met-5A mesothelial cell line, three different primary normal mesothelial cells (NM1, NM2, NM3), and three mesothelioma (MM) cell lines derived from untreated MM patients (MM1, MM2, MM3) constitutively express VEGF-R1, -R2, Np-1, and Np-2 at analogous levels by Western blot analysis. However, VEGF dose- and time-dependently reduced [³H]-thymidine uptake and cell growth in NM cells but increased DNA synthesis and cell growth in MM cells. Similar time-dependent VEGF-R2 phosphorylation by VEGF was observed in NM and MM cells. Thus, we found an opposite regulation of DNA synthesis rate by VEGF in NM cells and MM cells that could not be explained by a compromised expression/activation of its receptor.

2. VEGF up-regulates Sema-3A expression in NM cells
We tested whether the lack of mitogenic activity of VEGF on NM cells could be explained by the activation of antagonistic signaling mechanisms, e.g., those mediated by inhibitory semaphorins. VEGF increased Sema-3A mRNA and protein levels in NM1 cultures, with maximum stimulation after 48 h. In contrast, expression of Sema-3A was lower in a base form and did not significantly increase in VEGF-treated MM1 cells. The gene transcription inhibitor, actinomycin D (25 µg/mL), suppressed VEGF-induced Sema-3A mRNA in NM1 cells, whereas the protein synthesis inhibitor cycloheximide (10 µM) had no effect. Accordingly, an increase in levels of Sema-3A secreted protein was detected in the additional NM cells and in Met-5A upon VEGF treatment but was not found in MM cells, further indicating that Sema-3A is selectively up-regulated by VEGF in NM cells.

3. p38 MAPK signal transduction pathways mediates VEGF-induced Sema-3A
In NM cells, the increase in Sema-3A protein levels induced by VEGF was blocked by 10 µM SU-1498, an inhibitor of VEGF-R2. p38 MAPK inhibitors SB202190 and SB203580 repressed VEGF-induced Sema-3A up-regulation, whereas SB202474, an inactive structural analog of SB202190, the PI3-kinase inhibitor LY294002, the nitric oxide synthase inhibitor L-NAME, and the ERK1/2 kinase (MEK) inhibitor PD98059 were ineffective. The effect of p38 MAPK inhibitors was dose-dependent between 0.5 and 10 µM. VEGF induced only a transient activation of p38 MAPK in MM cells, whereas it sustained a prolonged activation in NM cells. VEGF sustained the phosphorylation of one of p38 MAPK endogenous substrates (namely, ATF-2) in NM1 but not in MM1 cells. MM1 cells transiently cotransfected with the specific p38 MAPK kinases MKK3 and MKK6 increased p38 MAPK activity and Sema-3A production in a VEGF-dependent manner that was abrogated by SB202190. Thus, VEGF strongly induces p38 MAPK signaling pathway in NM cells, but not in MM cells, and this leads to enhanced Sema-3A production.

4. p38 MAPK activation negatively controls VEGF-induced mesothelial cell proliferation via Sema-3A-dependent pathways
When NM cells were preincubated with SB202190 or SB203580, VEGF could stimulate [³H]thymidine incorporation up to 300–320%, whereas the SB202474 was ineffective. Conversely, MKK3/6 cotransfection reduced by 35–40% VEGF-induced [³H]thymidine uptake in MM cells, which was rescued by SB202190. Thus, activated p38 MAPK exerts a negative role on both normal and malignant mesothelial cell proliferation in response to VEGF.

We then postulated that Sema-3A mediates some of these effects. Transient transfection with c-myc-tagged Sema-3A decreased by 45–50% VEGF-induced DNA synthesis in MM cells, demonstrating that either p38 MAPK- and Sema-3A-dependent pathway can reduce mesothelial cell growth upon VEGF. To support the dependence on Sema-3A autocrine/paracrine loop for p38 MAPK-mediated cell proliferation, we established clones that express wild-type plexin-A1 (WT-plexin-A1) or its dominant-negative form (DN-plexin-A1) in Met-5A cell line. When DN-plexin-A1 cells were treated with VEGF, the proliferative effects were increased by 220–230% compared with WT-plexin-A1 cells or Met-5A parental cells. The expression of WT- or DN-plexin-A1 left unchanged VEGF-induced p38 MAPK activation and Sema-3A up-regulation compared with Met-5A parental cells. Thus, activation of Sema-3A-dependent pathway by VEGF-induced p38 MAPK signal is a final effector for p38 MAPK-controlled mesothelial cell growth.

5. Cyclin D1 is a target for VEGF/ Sema-3A cross-talk
We then investigated what protein involved in the cell cycle progression is the target for VEGF/Sema-3A cross-talk. Increased cyclin D1 transcription is stimulated by VEGF-induced mitotic signal, suggesting that Sema-3A could play a role at this level. Unlike NM1 cells, cyclin D1 protein synthesis was stimulated by VEGF in MM cells, and transient expression of Sema-3A-myc markedly inhibited this activation (Fig. 1 a). VEGF did not increase cyclin D1 synthesis in Met-5A parental cells and WT-plexin-A1 cells but this was stimulated in DN-plexin-A1 cells (Fig. 1b ). Thus, events important for VEGF-induced cell cycle progression, such as cyclin D1 up-regulation, are reduced through Sema-3A action. A diagrammatic representation of our findings is reported.

View larger version (33K): [in this window] [in a new window]   Figure 1. VEGF-stimulated cyclin D1 synthesis is inhibited by Sema-3A-dependent signals. a) NM1 and MM1 cells were transiently transfected with 2 µg of either control vector (pcDNA3) or c-myc-tagged Sema-3A construct. After 24 h, cells were then incubated for an additional 24 h with or without 10 ng/mL of VEGF. Cell lysate protein was immunoblotted with anti-c-Myc (top) or anti-cyclin D1 (middle) antibody. ß-Actin served as loading controls. b) Met-5A cell line, WT-plexin-A1 cells, and DN-plexin-A1 cells were treated for 24 h with VEGF (10 ng/mL). Cellular proteins were then extracted, and immunoblotted with anti-cyclin D1 antibody and normalized with respect to ß-actin protein. Representative experiments (n=3) are shown (a, b). Fold are indicated as reported in Figure 2 . The relative ratio measured in the control without VEGF is arbitrarily presented as 1. Numbers represent the mean ± SD (n=3). *P < 0.05 vs. control (b).

CONCLUSIONS

The studies aimed at elucidating the role of VEGF in tumor biology, have focused primarily on the ability of VEGF to induce angiogenesis. Recently, we and others have shown that VEGF directly influences, by an autocrine mechanism, cell growth of several cancer cells, such as mesothelioma, melanoma, and Kaposi sarcoma. This implies that VEGF signaling regulates a variety of processes involved in tumor progression, including cell proliferation, migration, degradation of extracellular matrix, as well as angiogenesis. However, the function of VEGF/VEGF receptor system is unlikely to be restricted to malignant cells. Expression of VEGF receptors and the absence of VEGF autocrine growth factor activity in several nonendothelial cell lines have been described. These observations suggest that only tumor cells are configured to carry a mitogenic VEGF signal to the nucleus.

We have hypothesized that during neoplastic transformation, mesothelial cells may acquire aberrant expression/activation of the downstream signaling to VEGF, which contributes to increasing metastatic potential and poor outcome. Here, we show that VEGF increases the expression of a secreted class III semaphorin, namely, Sema-3A, in normal, but not in malignant mesothelial cells. We further demonstrated that activation of VEGF-R2 receptor tyrosine kinase and, subsequently, of p38 MAPK were required in Sema-3A up-regulation. Consistently, coexpression of a constitutively active form of MKK6 and MKK3 increased p38 MAPK activity and, in turn, Sema-3A production in mesothelioma cells. Thus, p38 MAPK-dependent signaling appears the dominant pathway involved in VEGF-induced Sema-3A up-regulation, thought additional work will be required to definitively conclude which p38 MAPK isoform(s) are implicated.

The general importance of p38 MAPK in cell cycle control has been established recently in endothelial cells. Our unexpected results on different p38 MAPK activation between normal and malignant mesothelial cells, still await a mechanistic explanation. However, the role of p38 MAPK-dependent pathway in mesothelial cells is mainly to increase Sema-3A expression when activated by a potential mitogenic growth factor, such as VEGF. A regulation of Sema-3A levels following treatment with angiogenic factors, such as VEGF, had not been reported before. Our finding that Sema-3A is an important mediator for VEGF-induced p38 MAPK to control cell proliferation, was unanticipated. Our identified role of Sema-3A and the importance of VEGF/Sema-3A cross-activation is specifically seen for the stimulation of important G₁ cell cycle events that lead to progression to S phase (i.e., DNA synthesis), such as up-regulation of cyclin D1.

Widely distributed in many tissues and organs, Sema-3A was first identified as a repelling cue in growth cone guidance during neural development. Sema-3A is believed to act as a morphogenic protein, but little is known about its regulation and functional relevance in tumor formation and progression. Sema-3A binds Np-1/plexin-A1 receptor complexes and promotes phosphorylation of specific downstream targets. Whether any are required for Sema-3A-dependent growth inhibition induced by VEGF remains to be seen. Recent evidence suggests that two other secreted class III semaphorins, Sema-3B and -3F, may function as tumor suppressor genes, implicated in small-cell lung cancer. Sema-3A and -3F have been shown to antagonize several biological effects of VEGF in endothelial and neuronal cells, likely via its competition with a binding site on Np-1. Our results indicate a possible link between p38 MAPK-dependent cell cycle control and the regulatory effects of different semaphorins. Sema-3A, being transcriptionally induced by VEGF-activated p38 MAPK-dependent pathway, may create a negative feedback loop to modulate growth promoting VEGF signaling in mesothelial cells. In contrast, the absence of this negative regulatory pathway in tumor cells could allow their uncontrolled proliferation in response to VEGF, sustaining tumor growth.

View larger version (21K): [in this window] [in a new window]   Figure 2. Schematic diagram.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0513fje

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