Placenta Growth Factor Gene Expression Is Induced by Hypoxia in Fibroblasts: a Central Role for Metal Transcription Factor-11

[CANCER RESEARCH 61, 2696–2703, March 15, 2001] Placenta Growth Factor Gene Expression Is Induced by Hypoxia in Fibroblasts: A Central Role for Metal Transcription Factor-11 Christopher J. Green,2 Peter Lichtlen,2 Nhung T. Huynh, Marianna Yanovsky, Keith R. Laderoute, Walter Schaffner, and Brian J. Murphy3 The Pharmaceutical Discovery Division, SRI International, Menlo Park, California 94025 [C. J. G., N. T. H., M. Y., K. R. L., B. J. M.]; the Institut fur Molekularbiologie der Universitat Zurich, CH-8057 Zurich, Switzerland [W. S.]; and ESBATech AG, CH-8057 Zurich, Switzerland [P. L.] ABSTRACT hPlGF isoforms in that it contains an additional 21 basic amino acid region at the COOH terminus that confers a heparin-binding ability Placenta growth factor (PlGF) is a mitogen for endothelial cells that can similar to that of VEGF (3, 5). However, whereas all VEGF potentiate the growth and permeabilizing effects on endothelium of vas- 165 isoforms bind to the tyrosine kinase receptors flt-1 and KDR/flk-1, cular endothelial growth factor. Here we report that hypoxia induces the PlGF homodimers are believed to bind only to flt-1 (see Ref. 6), expression of both PlGF mRNA and protein in immortalized/transformed mouse embryonic fibroblasts (mEFs) and in NIH 3T3 cells. Importantly, suggesting a role for the homodimers in endothelial cell-cell or the magnitude of the induction of PlGF expression by hypoxia is enhanced cell-matrix interactions (7). PlGF may also potentiate the angiogenic by the presence of oncogenic Ras. To investigate the transcriptional effects of VEGF (8) because the two proteins have the ability to form component of hypoxia-inducible PlGF expression, we cloned and se- heterodimers with each other (2, 9–11) that can activate the KDR/ quenced a 1350-bp fragment of the 5؅-flanking region of the mouse gene. flk-1 receptor (9). Like VEGF, PlGF-2 has also been shown to bind to Analysis of the promoter region indicated the presence of putative con- and activate neuropilin-1 (6, 12), a receptor found on many cell types sensus sequences for known hypoxia-responsive regulatory sites, including in addition to endothelial cells. This receptor can bind at least five metal response elements and Sp1-like sites. In the present study, we show different ligands including VEGF165 and has established roles in a that the induction of PlGF expression by hypoxia is dependent on the wide variety of cellular processes including axonal guidance (13). presence of the metal response element-binding transcription factor 1 These findings suggest that PlGF is a component of extensive com- (MTF-1). Thus, in mEFs with targeted deletions of both MTF-1 alleles, binatorial interactions involving the VEGF superfamily. hypoxia-induced increases of PlGF mRNA and protein levels were greatly In contrast to the widespread distribution of VEGF, PlGF expres- attenuated compared with those in wild-type mEFs. Moreover, transient transfection of a PlGF promoter reporter gene into NIH 3T3 cells resulted sion was originally considered to be restricted to cell types of placen- in hypoxia-responsive transcriptional activation of the reporter. Finally, tal origin such as primary cytotrophoblasts and in vitro differentiated ectopic expression of MTF-1 resulted in increased basal transcriptional syncytiotrophoblasts (e.g., see Ref. 14). However, accumulating evi- activity of a PlGF promoter reporter. Together, these findings demon- dence indicates that PlGF is synthesized in many nonplacental cells strate that the PlGF gene is responsive to hypoxia and that this response and other tissues including human thyroid, brain, lung, and skeletal is mediated by MTF-1. It remains to be determined whether this activa- muscle (reviewed in Ref. 15). PlGF is also highly expressed in dermal tion is the result of direct and/or indirect transcriptional activation by microvascular endothelial cells and retinal pericytes (16), whereas MTF-1. The stimulatory effect of oncogenic Ras on the induction of PlGF conditioned medium from cultured human keratinocytes was found to expression in hypoxic cells suggests that PlGF could be an important contain both PlGF homodimers and PlGF/VEGF heterodimers (11). It proangiogenic factor in the tumor microenvironment. has also been reported that migrating keratinocytes from healing wounds contain significant levels of PlGF from the third to the seventh day after injury (11). Other studies indicate that PlGF may be INTRODUCTION involved in the inflammatory process because PlGF homodimers and PlGF4 is a member of the VEGF family of proangiogenic factors PlGF/VEGF heterodimers were detected in the synovial fluid of (1). Both PlGF and VEGF form glycosylated homodimers that share patients with inflammatory arthopathies (17). PlGF is also expressed significant homology at the amino acid level within their platelet- in several types of solid tumors (18–21). Furthermore, PlGF is be- derived growth factor-like regions, including the conservation of eight lieved to be involved in the pathogenesis of PDR and other ischemic cysteine residues involved in intra- and interchain disulfide bond retinal diseases (22–24). formation (1, 2). hPlGF is also similar to VEGF (and platelet-derived Oxygen deprivation (hypoxia) appears to be a common feature of growth factor) in that alternative splicing of the mRNA from a single many of the pathophysiological conditions in which PLGF is ex- copy gene produces different isoforms of the protein [PlGF-1, PlGF-2, pressed. For example, hypoxia is a characteristic common to many solid tumors and is believed to contribute to increased expression of and PlGF-3 (3, 4)]. PlGF-2, or PlGF170, differs from the other two proangiogenic proteins (e.g., VEGF) and subsequent activation of angiogenesis (25, 26). However, the role of hypoxia in the regulation Received 9/7/00; accepted 1/12/01. The costs of publication of this article were defrayed in part by the payment of page of PlGF expression is currently unclear because the majority of cell charges. This article must therefore be hereby marked advertisement in accordance with lines used to examine its expression appear to be unresponsive to 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by NCI Grants CA57692 (to B. J. M.) and CA73807 and CA67166 (to oxygen deprivation (10, 16, 27, 28). The findings presented here K. R. L.). demonstrate that immortalized/transformed fibroblasts express PlGF 2 C. J. G. and P. L. contributed equally to these studies. and that exposure of these cells to physiologically relevant levels of 3 To whom requests for reprints should be addressed, at The Pharmaceutical Discovery Division, SRI International, 333 Ravenswood Avenue, Menlo Park, CA 94025. Phone: hypoxia results in significant inductions of the steady-state levels of (650) 859-4213; Fax: (650) 859-5816; E-mail: [email protected]. both PlGF mRNA and protein. We also show that this activation is 4 The abbreviations used are: PlGF, placental growth factor; hPlGF, human PlGF; mPlGF, mouse PlGF; HRE, hypoxia response element; MRE, metal response element; dependent on the presence of the redox-sensitive transcription factor MT, metallothionein; MTF-1, metal response element-binding transcription factor 1; MTF-1 (29, 30) and that the hypoxia-inducible increase in PlGF PDR, proliferative diabetic retinopathy; RT-PCR, reverse transcription-PCR; VEGF, vascular endothelial growth factor; mEF, mouse embryonic fibroblast; Tag, T antigen; expression is due at least in part to increased transcription of the PlGF CMV, cytomegalovirus. gene, particularly in the presence of oncogenic ras. 2696 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2001 American Association for Cancer Research. PlGF INDUCTION BY HYPOXIA IS MTF-1 DEPENDENT MATERIALS AND METHODS been cleaved with a variety of restriction enzymes and ligated with linkers containing primer sites for PCR amplification. Two nested PCR primers Cell Culture and Hypoxia. All cell cultures, including mEFs and NIH complementary to the 5Ј-end of the longest known mouse cDNA for PlGF 3T3, JAR (a human choriocarcinoma cell line), and HepG2 (a human hepa- (GenBank accession number X96793) were used. The two primers were toma cell line) cells, were maintained in DMEM containing 10% dialyzed fetal complementary to bases 268–291 and 295–318 of this cDNA sequence. Only bovine serum. Both SV40 large TAg and TAg/Ras-transformed wild-type one 500-bp fragment was obtained using this kit. Because only slightly more ϩ/ϩ Ϫ/Ϫ (MTF-1 ) and MTF-1 null (MTF-1 ) mEFs were used in these studies. To than 200 bp of this sequence were from the upstream region of the known obtain the mEFs, primary embryonic fibroblasts were isolated from 12.5-day- mPlGF cDNA, it was considered unlikely that this fragment would contain all old mouse embryos using standard techniques (31). PCR genotyping for of the necessary regulatory elements to function as a promoter in reporter gene MTF-1 null cells was performed using genomic DNA prepared from yolk sacs studies. Therefore, inverse PCR was used to clone a larger sequence of the and early passages of the primary cells. Wild-type and MTF-1 null primary mPlGF gene. Mouse genomic DNA (1 ␮g) was cleaved with BamHI, EcoRI, cells were then transfected with either 10 ␮g of plasmid CMV-TAg or 10 ␮g HindIII, KpnI, SstI, or XbaI. Each of the cleaved DNA preparations was of plasmid CMV-TAg and 10 ␮g of plasmid c-H-ras(A) (32) per 100-mm- dissolved in 0.5 ml of 50 mM Tris-HCl (pH 7.8), 10 mM MgCl2,20mM DTT, diameter cell culture plate. The plasmid CMV-TAg directs expression of TAg, and1mM ATP, and then 2 units of T4 DNA ligase (Life Technologies, Inc., and c-H-ras(A) directs expression of oncogenic human H-ras. Cell foci were Rockville, MD) were added. The ligation reactions were then incubated at 5°C isolated, and immortalized cell lines were derived.

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