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Oncogene (2002) 21, 844 ± 848 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc

Inhibition of human endothelial cell proliferation by ShIF, a vacuolar H+-ATPase-like protein Edgardo E Tulin1,2, Nobuhisa Onoda1, Masakazu Hasegawa1, Hitoshi Nomura1 and Toshio Kitamura3 1

Chugai Research Institute for Molecular Medicine Incorporated, 153-2 Nagai, Niihari, Ibaraki, 300-4101, Japan; 2Department of Hematopoietic Factors, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, 108, Japan; 3Division of Cellular Therapy, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, 108, Japan

ShIF is a bone marrow stroma cell-derived factor originally identi®ed to support proliferation of bone marrow cells in vitro. This protein shares high sequence homology to the yeast vacuolar H+-ATPase subunit, Vph1p, and the 116 kDa proton pump of the rat and bovine synaptic vesicle, Vpp1. We examined the function of ShIF in the proliferation of human umbilical vein endothelial cells (HUVEC). ShIF inhibited HUVEC proliferation in a dose-dependent manner. Recombinant ShIF added at 10 and 20 ng/ml inhibited HUVEC proliferation by 21.6 and 44.3%, respectively and increasing the concentration of ShIF to 100 ng/ml inhibited proliferation by as much as 55.5%. When HUVEC cells were cultured at various concentrations of ShIF in the presence of anti-ShIF antibody, the inhibitory e€ects of ShIF to HUVEC proliferation were abrogated by 89 ± 91% indicating that the activity of ShIF to HUVEC was speci®c. HUVEC cultured in the presence of ShIF and ba®lomycin, a speci®c inhibitor of ATPase, resulted to a 90% growth inhibition. Thus, ShIF may act as an antagonist to the ATPase complex by disrupting the production of cellular ATP thereby decreasing the ability of HUVEC to proliferate. Oncogene (2002) 21, 844 ± 848. DOI: 10.1038/sj/onc/ 1205114 Keywords: ShIF; V-ATPase; endothelial cell proliferation; HUVEC

ShIF is a 27 kDa secreted and membrane-bound protein isolated from mouse bone marrow stroma (Tulin et al., 2001). This protein was originally identi®ed based on its ability to support proliferation of a mutant clone S21, which was established from interleukin-3-dependent Ba/F3 cells but became dependent on a stroma cell line ST2 after chemical mutagenesis. ShIF corresponds to a naturally occurring

*Correspondence: E Tulin, Department of Hematopoietic Factors, Institute of Medical Science, University of Tokyo, Minato-Ku, Tokyo, 108-8639, Japan; E-mail: [email protected] Received 8 June 2001; revised 16 October 2001; accepted 29 October 2001

short form of ISF protein (Lee et al., 1990) produced by proteolytic processing and supports proliferation of bone marrow cells in vitro suggesting that this protein is a growth stimulatory peptide involved in the regulation of cell growth. Interestingly, ShIF has a high sequence homology to a C-terminal part of a 95 kDa yeast vacuolar H+ATPase subunit, Vph1p (39%) (Manolson et al., 1992) and a 116 kDa proton pump (54%) of the rat and bovine synaptic vesicle (Perin et al., 1991). Vacuolar H+-ATPases are a distinct class of proton translocating enzymes found on a variety of organelles including the plasma membrane of specialized cell types (renal epithelia, macrophages, and neutrophils) where they pump protons out of the cell (Forgac, 1989). On the basis of co-puri®cation and immunoprecipitations, the V-ATPAses complex is composed of at least eight subunits (Kane et al., 1989); ®ve of the subunits are part of the peripherally bound ATP-binding V1 sector, and the remaining three subunits are part of the poreforming hydrophobic Vo sector (Kane, 1992). How VATPases are targeted and assembled onto di€erent membranes and how they are regulated according to the speci®c needs of the cell are not fully understood. V-ATPases have important consequences at multiple physiological levels and have positive and negative regulatory functions in a variety of cell types. In Tcells, V-ATPases play a positive role by activating NFAT transcriptional factors leading to production of IL-2 to stimulate cell growth (Rao et al., 1997). In contrast, in macrophages, V-ATPases suppress NF-kB activation and negatively regulate macrophage cell proliferation (Conboy et al., 1999). It has also been demonstrated that addition of ATP synthase to cultures of tumor cell lines induced membrane depolarization and eventual lysis of transformed cells (Di Virgilio et al., 1989; Rozengurt et al., 1977). Recently, angiostatin, a potent antagonist of angiogenesis, was reported to bind to ATP synthase on the surface of human endothelial cells and this binding inhibited endothelial cell migration and proliferation (Moser et al., 1999). This implies a potential regulatory role of ATP synthase on endothelial cell proliferation. In the light of the high sequence similarity of ShIF to V-ATPase, we investigated the

Antiproliferative function of ShIF EE Tulin et al

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Figure 1 Endogenous expression of ShIF. (a) Analysis of ShIF expression in HUVEC and ST2 cells by RT ± PCR. Total RNA was prepared from HUVEC and ST2 cells and ®rst strand cDNA synthesis was performed using OligodT primer. The PCR was performed using primers, 5'-AACCACTTGCACTTTAGGAAG-3' (forward) and 5'-GGTGTATCTTACAGAACCATT-3' (reverse), for a total of 30 cycles (20 s at 948C and 1 min at 708C for ®ve cycles and 20 s at 948C and 1 min at 688C for 25 cycles) using Advantage polymerase (Clontech). G3PDH control primers were used as loading controls. (b) Subcellular localization of ShIF in HUVEC and ST2 cells. HUVEC and ST2 cells were grown in tissue culture cover slips for 48 h. Cells were washed with PBS, incubated with 100-fold diluted anti-ShIF polyclonal antibody for 2 h then with Cy3-conjugated secondary antibody for 1 h under dark conditions. Anti-ShIF antibody was a rabbit polyclonal antiserum raised against ShIF, residues 81 ± 95 (LHNGRNCFGMSRSGY) of the ShIF protein. Upper panel, left, HUVEC under epi-illumination showing no surface staining of ShIF and right, the same ®eld of HUVEC under visible light. Lower panel, left, ST2 cells under epi-illumination showing immuno¯uorescent surface staining of ShIF and right, same ®eld of ST2 cells under visible light. Control experiments using antiFLAG alone and secondary antibody alone showed no staining of ShIF (photographs not shown). (c) Detection of ShIF protein in culture supernatants of HUVEC and ST2 cells. HUVEC and ST2 cells were grown for 5 days and culture supernatants were harvested and immunoprecipitated with 100-fold diluted anti-ShIF antibody for 2 h at 48C. The immunoprecipitate was collected by centrifugation at 5000 r.p.m. for 5 min and washed with the immunoprecipitation bu€er (1% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA, 0.2 mM NaVO4, 0.2 mM PMSF and 0.5% NP-40) three times. The bound proteins were eluted by the sample bu€er, applied to an SDS ± PAGE and electroblotted onto a PVDF membrane (Millipore). The blots were probed with a 1000-fold diluted anti-ShIF antibody, incubated with HRP-conjugated complex and subsequently developed with ECL Western blotting detection reagents (Amersham Japan) and exposed to ECL Hyper®lm. Molecular weight markers shown on the left are in kDa Oncogene

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function of ShIF on the proliferation of human umbilical vein endothelial cells. First, we examined the endogenous expression of ShIF in HUVEC using speci®c primers to ShIF and by immuno¯uorescence staining using polyclonal antibodies generated from ShIF protein (Figure 1). HUVE cells did not express the endogenous form of ShIF (Figure 1a,b) and the ShIF protein was not detected in the supernatant (Figure 1c) suggesting that both secreted and membranebound forms of the protein were not expressed by HUVEC. The bone marrow stroma cell line ST2 expressed both the membrane-bound protein (Figure 1b) as well as the 27 kDa protein in the supernatant (Figure 1c). To test the e€ect of ShIF on the proliferation of HUVEC, we cultured HUVEC in the presence or absence of ShIF. The ShIF protein was derived either from supernatant of COS7 cells transfected with a ShIF cDNA construct or from puri®ed recombinant ShIF. As shown in Table 1, when the culture medium was added with 20% (by vol) of ShIF-containing supernatant, HUVEC proliferation was inhibited by about 30%. When the amount of ShIF-containing supernatant was doubled to 40% of the total culture volume, this inhibitory e€ect increased to nearly 50%. Moreover, recombinant ShIF added at 1/10 and 1/5 of the total assay volume equivalent to 50 and 100 ng/ml protein, inhibited HUVEC proliferation by as much as 46.5 and 55.5%, respectively. The growth of cells in endothelial cell media (Figure 2a) was not a€ected by the addition of COS7 cell control supernatant (Figure 2b) or in PBS bu€er (Figure 2c). However, a marked reduction in cell proliferation phenotype was observed when the cells were cultured in the presence of ShIF (Figure 2d to g). The dose-dependent e€ect of ShIF on HUVEC proliferation is shown in Table 2. In the absence of Table 1

ShIF inhibits the proliferation of HUVEC cells

Growth medium

Volume (ml) of ShIF added Absorbance Per cent (per100 ml total) 595 nm inhibition

EBM only

0

0.708

EBM + ShIF supernatant from COS7 cells

20 40

0.498 0.365

29.7 48.4

EBM + COS7 cells control supernatant

20 40

0.688 0.683

2.8 3.5

EBM + purified ShIF

10 20

0.379 0.315

46.5 55.5

EBM + PBS

10 20

0.696 0.682

1.7 3.7

HUVEC were plated at a density of 1000 cells/well in 96-well ¯at bottom plates using endothelial cell basal media (EBM) with or without ShIF. The ShIF protein was either derived from culture supernatant of COS7 cells transfected with ShIF cDNA construct or from puri®ed recombinant ShIF. The amount of recombinant protein in the 10 and 20 microliter puri®ed fraction was 50 and 100 ng/ml, respectively. Cells were grown for 72 h at 5% CO2 and cell proliferation was determined using a Cell Titre proliferation assay kit by Promega. Results present three separate experiments performed in duplicate. Per cent inhibition indicates the ability of ShIF to inhibit proliferation of HUVEC Oncogene

anti-ShIF antibody, recombinant ShIF added at 10 ng/ ml inhibited HUVEC proliferation by 21.6%. When this concentration was increased to 20 and 50 ng/ml, inhibition of cell proliferation was correspondingly increased to 44.3 and 51.9%, respectively. Moreover, a higher dose of 100 ng/ml inhibited proliferation by as much as 55%. To con®rm the speci®city of the inhibitory e€ect of ShIF to HUVEC proliferation, we cultured HUVEC at various concentrations of ShIF in the presence of a rabbit polyclonal antiserum raised against ShIF. The inhibitory e€ects of ShIF to HUVEC proliferation were abrogated by 89 ± 91% (Table 2) indicating that the inhibitory e€ect of ShIF to HUVEC proliferation was speci®c. How does ShIF inhibit proliferation of HUVEC? Endothelial cells play a strategic role within the vasculature, serving as a barrier between the intravascular compartment and the underlying tissues. Relative to other cell types, endothelial cells are more resistant to hypoxic challenge by their ability to maintain a high level of intracellular ATP (Bodin and Burnstock, 1995). However, when the production of ATP is disrupted, endothelial cells become vulnerable to hypoxia and irreversible cell damage. We performed experiments to see if ShIF acts through ATP synthase by using a speci®c inhibitor to ATPase, ba®lomycin A. We cultured HUVEC in the presence of ShIF with or without the addition of ba®lomycin and found that in cells cultured with ba®lomycin alone, HUVEC proliferation was inhibited by 65% and in cultures with both ba®lomycin and ShIF present, a 90% growth inhibition was observed (data not shown). Thus, we propose a following mechanism of ShIF inhibitory action to HUVEC proliferation. ShIF may disrupt the production of ATP by acting as an antagonist through binding to the a/bsubunits of plasma membrane-localized ATP synthase thus inhibiting the production of cellular ATP. ShIF is highly identical to vacuolar-ATPase and it is most likely that binding of this protein to the a/b-subunits of the ATPase complex through a feedback mechanism is responsible for the inhibitory e€ect of ShIF to HUVEC. Several recent reports appear relevant to our ®ndings and support our proposed mechanism. It was previously demonstrated that the a- and b-subunits of ATP synthase constitute the endothelial cell binding site of angiostatin, an inhibitor of endothelial cell migration and proliferation (Moser et al., 1999). An extension of this work also showed that the ATP synthase is catalytically active and angiostatin-mediated inhibition of this activity correlates with inhibition of proliferation (Moser et al., 2001). In yeast, the H subunit (Vma13p) of the yeast vacuolar-ATPase has been found to inhibit the ATPase activity of the cytosolic V1 complexes thus, decelerating the production of ATP (Parra et al., 2000). An ATP analogue, adenosine 5'-[beta, gamma-imino]triphosphate (AMP-PNP), was also found to bind to the A subunit of the plant V-ATPase and this binding induced conformational changes in the E subunit of the ATPase complex. Consequently, this conformational change reduced the catalytic activity of the A subunit (Kawamura et al., 2001).

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847

Figure 2 Inhibition of HUVEC proliferation by ShIF. HUVEC were plated at a density of 1000 cells/well in 96-well ¯at bottom plates using endothelial cell basal media (EBM) with or without ShIF. Cells were grown for 72 h at 378C and 5% CO2. (a) medium only, (b) with control supernatant obtained from COS7 cells (2/5 by volume), (c) with control PBS bu€er (1/10 by volume), (d) with ShIF-containing supernatant of COS7 cells (1/5 by volume), (e) with ShIF-containing supernatant of COS7 cells (2/5 by volume), (f) with recombinant ShIF (1/10 by volume equivalent to 50 ng/ml), (g) with recombinant ShIF (1/5 by volume equivalent to 100 ng/ ml). Results represent three separate experiments performed in duplicate. Photographs were taken after 72 h of culture

Table 2 The inhibitory e€ect of ShIF to HUVEC proliferation was restored by anti-ShIF antibody Concentration of ShIF added (ng/ml)

Per cent proliferation inhibited, +s.e.m. Without With Per cent antibody antibody Recovery

0 10 20 50 100

0 21.6+1.2 44.3+0.6 51.9+1.1 55.0+0.2

0 1.9+1.9 3.8+1.1 5.5+2.7 6.0+1.1

0 91.2 91.4 89.4 89.0

HUVEC were plated at a density of 1000 cells/well in endothelial cell basal media containing ShIF at a ®nal concentration of 10, 20, 50 and 100 ng/ml. Anti-ShIF antibody was added concomitantly at a dilution of 1 : 100. Cells were grown for 72 h at 5% CO2 and cell proliferation was determined using a Cell Titre proliferation assay kit by Promega. Results represent three separate experiments performed in duplicate. Per cent recovery indicates the ability of the anti-ShIF antibody to block the antiproliferative e€ects of ShIF and thereby restore proliferation

In addition to angiostatin and certain antibodies directed against the a- and b-subunits of ATP synthase that function as angiostatin mimetics, there are other known inhibitors of ATP synthase that exhibit antitumor e€ects such as piceatannol (Zheng and Ramirez, 1999) and resveratrol (Zheng and Ramirez, 2000). Thus, because of the ability of ShIF to inhibit proliferation of cultured human endothelial cells, it could be considered a potential molecule with antiproliferative function. In the microenvironment of a growing tumor, tissue hypoxia provides a powerful stimulus for the production of angiogenic growth factors such as vascular endothelial growth factor, basic ®broblast growth factor and angiopoietin. The ability of host endothelial cells to respond to these growth factors by increased proliferation likely depends on their ability to survive hypoxic challenge. In summary, the ®ndings of the present paper suggest Oncogene

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848

that ShIF decreased the ability of the cells to survive hypoxic challenge by inhibiting the production of cellular ATP thereby reducing the capacity of the cells to proliferate.

Acknowledgments We thank Drs Y Hirata for HUVEC and C SchoÈenbach for valuable discussion and encouragement and M Yoshida for preparation of ®gures.

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