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Vascular Endothelial Growth Factor Is Up-Regulatedin Vitroandin Vivoby Androgens

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Vascular Endothelial Growth Factor Is Up-Regulatedin Vitroandin Vivoby Androgens
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  Vascular Endothelial Growth Factor Is Up-Regulated in Vitro   and  in Vivo   by Androgens Sylvie Sordello, Nicolas Bertrand, 1 and Jean Ploue¨t 2  Laboratoire de Biologie Mole´culaire Eucaryote, UPR 9006 Centre National de la Recherche Scientifique,118, Route de Narbonne, 31062 Toulouse cedex, France Received August 10, 1998 Evidencefrompathophysiologicalstudiessupporttheconcept that embryonic development, tumor progres-sion, and hormonally-regulated tissue masses such asadult prostate and corpus luteum are angiogenesis-dependent. We examined if the prostatic expression of vascular endothelial growth factor (VEGF), the majorregulator of normal and pathological angiogenesis, wasregulated by testosterone. Northern blot of VEGF mes-sengerribonucleicacid(mRNA)extractedfromahumanimmortalized epithelial prostatic cell line (PNT1)showed that dihydrotestosterone (DHT) up-regulated VEGF mRNA at a level comparable to that observedupon exposure to growth factors. Polymerase chain re-action of reverse transcribed mRNA demonstrated thattheratioofthetwosplicevariantsencodingthe121and165 isoforms of VEGF were not affected by DHT. VEGFbiologicalactivity,measuredintheconditionedmediumby radio receptor assay, was increased by DHT. Injec-tion of testosterone in adult rats induced a transientincrease of the ventral lobe weight and the specific ac-tivity of prostatic VEGF, leading to a 7-fold increase inthe prostate content of VEGF.  © 1998 Academic Press  Key Words:  androgens; vascular endothelial growthfactor; angiogenesis; prostate. The progressive growth of a malignant tumor re-quires an adequate blood supply, which is provided bynewly formed vessels. Abundant evidence supports theconcept that prostate tumor growth is sustained by alocal increase of vascularization, leading to the pro-posal that vessel count is an independent prognosismarker of metastasis (1). This local hypervasculariza-tion is thought to result from the release by thesetissues of angiogenic growth factors. Several studieshave shown that vascular endothelial growth factor(VEGF) plays a major role in pathological neovascular-ization (2, 3). VEGF has been isolated from severaltumor cell lines (4, 5, 6) and cloning of the gene pro-vided evidence that at least five peptides of 121, 145,165, 189 and 206 amino acids are generated by alter-native splicing of the VEGF pre-mRNA (7, 8).In nude mice, tumor progression of prostate cancer cellline xenografts decreases after anti-angiogenic treat-ments such as angiostatin (9), neutralizing antibodiesagainstVEGF(10)orconverselyisstimulatedbyactivat-ing VEGF transduction pathways (11). There is nowsome direct evidence that organ size and tissue mass areunder the control of vascularization in adults. A recentreport has shown that castration induces a parallel de-crease of ventral lobe mass and its content in vascularendothelialcells(12).Testosteroneinjectionsrestoreden-dothelial cell content before the onset of prostate enlarg-ment thus suggesting that potent prostatic angiogenicfactors were up-regulated by androgen supply.We designed this study to demonstrate whetherprostatic expression of VEGF was controlled by andro-gens in physiological situations. We demonstrate thatandrogens up-regulate transiently VEGF expression atthe mRNA and protein level in the normal humanprostatic epithelial cell lines PNT1 without affecting the splicing of its mRNA. Furthermore testosteroneinjection in adult rats induced a moderate increase of tissue mass in the ventral lobe and a 4-fold increase in VEGF specific activity. MATERIALS AND METHODS Cell culture.  The human epithelial cell line PNT1 (Dr O. Cuss-enot, Hopital Saint-Louis, Paris) has been immortalized by transfec-tion with SV 40 large T antigen (13). These cells were cultured inDMEM medium supplemented with 100 Uml penicillin and strepto-mycin supplemented with 10% fetal calf serum and received 10  9 MDHT every other day. Prior to the onset of experiments the cellcultures were transfered to the DMEM medium supplemented with-out DHT for three days. 10  11 to 10  7 M DHT or 100 ng/ml humanrecombinant FGF1, PDGFAA, PDGFBB, EGF or 10 ng/ml FGF2were added for different time intervals. The cells were rinsed with 1 Present address: Service d’Urologie, Hopital Purpan. 2 To whom all correspondence should be addressed. Fax: (33) 561335886. E-mail: plouet@ibcg.biotoul.fr. Abbreviations: DHT, dihydrotestosterone; PNT1, human immor-talized epithelial prostatic cell line; VEGF, vascular endothelialgrowth factor. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS  251,  287–290 (1998)  ARTICLE NO . RC989328287 0006-291X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.  cold PBS and processed for RNA extraction. In parallel experimentsconfluent cell monolayers were rinsed 3 times and incubated withserum free medium supplemented with in the presence or absence of modulators (5). The conditioned media were collected 48 hours later.  Animals and treatments.  Adult male Sprague-dawley rats(weight 280–300 g) received every three days an injection of 6 mg testosterone diluted in castor oil in order to provide a slow release of active hormone (14). At different time intervals (1, 3, 7 and 10 days)the prostates were dissected into ventral or laterodorsal lobes andimmediately frozen in liquid nitrogen. Three animals were sacrificedfor each point. VEGF mRNA extraction and analysis.  Confluent cells were lysedin guanidium thiocyanate and total RNA was purified by the phenol/ chloroform method (15). Total RNA (20   g) was run on a 1.2%formaldehyde agarose gel, blotted on to a Hybond-N nylon mem-brane (Amersham) by electrotransfer and UV-crosslinked. Hybrid-ization was performed with a 605 bp BamH1 fragment of human VEGF cDNA (7) kindly provided by Dr J. Abraham (Scios Nova,Mountain View, CA). This probe was  32 P-labelled using a MegaprimeDNA labelling system (Amersham). The control for equal lane load-ing and transfer was assessed by ethidium bromide staining. Thegels were dried, analyzed by a phosphoimager and the relativeamount of each band was assessed by image analysis.Total RNA (2   g) was used for the reverse transcriptase (RT)reaction using the Superscript preamplification system (GibcoBRL)and random hexamers as primers. Polymerase chain reaction (PCR)was performed on 1/10th of the cDNAs in a 50   l volume using 2.5units of Taq polymerase and 50 pmoles of each oligonucleotide flank-ing the 5   and 3   ends of the VEGF coding sequence (CAAGTAC-CAAAGCCTCC and ACTGTTCGGCTCCGCCACT). Amplificationwas performed for 30 cycles (94°C for 1 min; 57°C for 1 min; 72°C for1.5 min). The amplified PCR products (6   l) were separated byelectrophoresis on a 1.5% agarose gel. Negative controls were per-formed in the absence of cDNA. The specificity of the amplificationwas authenticated by NcoI enzymatic restriction at the unique sitelocated at position 151 (16). The cleavage products of the VEGFcDNAs were thereafter shorter by 86 bp. VEGF measurement.  Rat tissues were powdered under liquidnitrogen immersion and extracted in RIPA buffer supplemented with10   g/ml of antiproteases (aprotinin, leupeptin, pepstatin and ben-zamidin). After 30 minutes on ice, the extracts were centifuged andthe supernatant frozen until use. VEGF content of the tissues and of the conditioned media was measured by radio receptor assay asalready described (17). The results are expressed by comparison to astandard curve of human recombinant VEGF 165 amino acids di-luted in the same vehicule (RIPA or culture medium respectively). Values are presented as means    SEM of VEGF concentration perml of conditioned medium or mg of wet weight prostate. The statis-tical analysis was performed by the test U of Mann and Whitney. RESULTS  VEGF gene expression was measured by northernblot. VEGF mRNA increased rapidly after exposure of PNT1 cells to DHT. The maximal expression (3 fold)was observed after 4 hours, persisted up to 24 hoursand decreased after 48 hours (Figure 1A). VEGFmRNA increased as a function of the dose between10  11 (x1.3) and 10  9 (x2.1) and remained constant atconcentrations of 10  8 (x1.9) and 10  7 (x1.8) M (datanot shown). A similar increase in VEGF mRNA accu-mulation was observed upon exposure of PNT1 cells toFGF1, FGF2, PDGFAA or PDGFBB (1.8, 1.9, 1.5 and1.9 fold greater than the unstimulated culture). Con-versely EGF did not increase VEGF mRNA accumula-tion (Fig 1B).To investigate the expression of the different VEGFisoforms, randomly primed first-strand cDNAs wereamplified with human VEGF primers located in exons1 and 8 to simultaneously amplify the different splicevariants. Two bands (453 and 587 bp) corresponding to VEGF 121 and 165 were observed. NcoI extensive di-gestion of RT-PCR products generated 86 bp smallerfragments. Their relative intensity was unaffected byDHT treatment (Fig 1C).The bioavailability of VEGF in PNT1 culture me-dium increased as a function of the dose of DHT (Fig 2A). Maximal stimulation was observed for 10  8 MDHT (x4.1) and remained constant for 10  7 M (x4.2).This dose-response curve shows that the increase of  FIG.1.  VEGF mRNA expression in human PNT1 epithelial cells.PNT1 cells were exposed to 10  9 M DHT for various lenghts of time(A) or to various growth factors for 24 hours (B) and total RNA wasextracted. 20  g was electrophoresed and hybridized with a  32 P-labelled human VEGF probe. Autoradiography was obtained by ex-posure to X-Omat AR film for 24h at  70°C. Position of VEGF mRNA is estimated by comigration with standard RNA marker (GibcoBRL). (C) RT-PCR analysis of VEGF, 2   g of total RNA of PNT1 cellsexposed for 24 hours to various concentrations of DHT was reversetranscribed and random single strand cDNAs were submitted toamplification using the primers described in the text, and visualizedon a 1.5% agarose gel stained with ethidium bromide, size of PCRproducts was estimated by comigration with a standard molecularweight marker (1 kb ladder, BRL) deposited in the middle lane of thegel. Vol. 251, No. 1, 1998 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS288  bioavailability requires doses of DHT an order of mag-nitude higher than that providing the maximal in-crease of VEGF mRNA. FGF2 and PDGFAA induced asimilar stimulation of VEGF bioavailability (Fig 2B),whereas exposure to FGF1 or PDGFBB resulted in amoderate but significant increase. Androgen treatment in adult rats resulted in a rapidincrease of VEGF specific activity in the ventral lobe.Its concentration increased 2-fold on the first day fol-lowing the onset of the treatment, 4-fold between day 3and day 7 and then decreased (Figure 3). Similarchanges,althoughlesspronouncedandmoretransient,were observed in laterodorsal lobe. A significant in-crease of the ventral lobe weight (x1.8) occured 3 daysafter testosterone injection. Therefore VEGF prostaticcontent is increased by 7-fold between days 3 and 6post testosterone injection. Despite a transient 3-foldinduction of VEGF specific activity in the laterodorsallobe, its weight was not significantly changed. DISCUSSION The present study shows that prostatic expression of  VEGF is up-regulated  in vitro  and  in vivo  by physio-logical concentrations of androgens.Several normal cultured cells, such as smooth mus-cle cells (7), keratinocytes, epithelial (16) or fibroblasticcells (17) express VEGF at the mRNA and proteinlevel. Although PNT1 cells are immortalized prostateepithelial cells, the expression of VEGF is unlikely dueto the immortalization but rather reflects the level of  VEGF expression observed in cultured normal epithe-lial cells. Accordingly we found that the tumoral pros-tatic cell lines PC3 and DU 145 secreted 4 to 6-foldmore VEGF than PNT1 (data not shown). However ithas been previously shown that the level of VEGFexpression was only slightly increased in immortalizedepithelial cells as compared to their normal counter-parts (16). VEGF mRNA expression is known to bestimulated by several growth factors such as FGF2(19), PDGF (20), TGF    (21), TGF   (18) or TNF   (19). FIG. 3.  Effects of testosterone on prostate weight and VEGFspecific activity. Adult male Sprague-dawley rats (weight 280-300 g)received every three days an injection of 6 mg testosterone diluted incastor oil. At different time intervals the ventral and laterodorsallobes of the prostates were dissected and immediately frozen inliquid nitrogen. The tissues were powdered under liquid nitrogenimmersion and extracted in RIPA buffer supplemented with 10  g/ml of antiproteases (aprotinin, leupeptin, pepstatin and benzami-din). After 30 minutes on ice, the extracts were centifuged and VEGFcontent was measured by radio receptor assay in the supernatants. 3animals were used for each point. The results are expressed bycomparison of a standard curve of human recombinant VEGF 165amino acids. Values are presented as means  SEM. The statisticalanalysis was performed by the test U of Mann and Whitney.(*) p  0.05. FIG. 2.  VEGF secretion in PNT1 culture medium stimulatedwith DHT. Confluent monolayers of PNT1 cells cultured withoutDHT for 3 days were rinsed and exposed to various concentrations of DHT diluted in DMEM medium or 100 ng/ml FGF1, PDGFAA,PDGFBB, EGF or 10ng/ml FGF2. Two days later the conditionedmedium was collected and the concentration of VEGF was measuredby radio receptor assay. Results are expressed as the mean. of trip-licate assays by comparison to a standard curve of recombinant VEGF 165 amino acids. Data are representative of 3 different exper-iments. Vol. 251, No. 1, 1998 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS289   Accordingly VEGF expression was up regulated byFGF2, FGF1, PDGFAA, PDGFBB in PNT1. AlthoughEGF stimulates VEGF production in glioblastoma cells(22) it has no action on PNT1 cells. The mechanisms of  VEGF up regulation by growth factors appear to in-volve interactions of transcription factors with the Sp1, AP1, AP2 sequences found in the VEGF promoter (7). Although the VEGF promoter does not contain clas-sical consensus androgen response sequences, wefound that VEGF expression is up-regulated by DHTas a function of the dose at a level comparable to thatobtained upon exposure to growth factors. RT-PCRanalysis demonstrated that both the 121 and 165amino acids isoforms encoded by alternative splicevariants were expressed in PNT1 cells and that DHTdid not seem to modify their relative expression. VEGFmRNA were actively translated in prostate epithelialcells in bioactive VEGF.The physiological role of VEGF in adult vessels re-mains to be elucidated. Androgen receptors are observed in epithelial andstromal cells of the ventral prostate (23). It was thentempting to examine the effects  in vivo  of androgens on VEGF expression. In fact testosterone injections in-creased VEGF expression in rat ventral prostate, andto a lesser extent in the other lobes. This effect ap-peared the day following the treatment and reached amaximum level within three days, corresponding to a4-fold increase of the local bioavailability of VEGF inthe ventral prostate. A transient increase of ventrallobe weight was observed between the third and thetenth day following androgen injections. However, de-spite an almost comparable increase of VEGF bioavail-ability in the laterodorsal lobe, its weight remainedunchanged, suggesting that VEGF up-regulation is notsufficient to trigger an efficient increase in tissue mass.This discrepancy suggests that in addition to VEGFoverexpression, other events, such as up-regulation of  VEGF receptors or inhibition of the expression of en-dogenous VEGF receptor antagonists, are required topromote endothelial cell proliferation. Their expressionmight be differently regulated in the ventral and thelaterodorsal lobes of the rat prostate. Further studiesare required to confirm that VEGF overexpression inthe ventral lobe of the prostate induces an increase of endothelial cell proliferation.In summary, the present study demonstrates that VEGF expression in the prostate is regulated by an-drogens. A recent report has demonstrated that pros-tate endothelial cells proliferate after androgen injec-tion in castrated rats (12) and has suggested thatangiogenesis controls prostate regrowth after castra-tion (24). These results document this hypothesis andsuggest that the angiogenesis observed in prostate re-growth upon testosterone injection might be mediatedby VEGF-VEGF receptors system. ACKNOWLEDGMENTS This work was supported by the Association de Recherche pour leCancer and the Fe´de´ration Nationale des Centres de Lutte Contre leCancer.SylvieSordelloandNicolasBertrandweresupportedbyfellow-shipsfromtheAssociationdeRecherchesurlesTumeursdelaProstateand the Fondation de la Recherche Me´dicale respectively. We wouldlike to thank J. Abraham for the gift of VEGF plasmid and O. Cussenotfor the gift of PNT1 cells. The help of B. Malavaud for dissectiontraining is greatly acknowledged. We also thank D. Villa for assistancewith illustrations and H. Hutchings with manuscript corrections. REFERENCES 1. Weidner, N., Carroll, P. R., Flax, J., Blumenfeld, W., Folkman, J.(1993)  Am. J. Pathol.  143,  401–409.2. Ferrara, N., Davis-Smyth, T. (1997)  EndocrineReviews 18, 4–25.3. Dvorak, H. F., Brown, L. F., Detmar, M., Dvorak, A. M. (1995)  Am. J. Pathol.  146,  1029–1039.4. Ferrara, N., Henzel, W. J. (1989)  Biochem. Biophys. Res. Com-mun.  161,  851–8.5. 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