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. Author manuscript; available in PMC: 2012 Aug 1.
Published in final edited form as: Cell Signal. 2011 Mar 21;23(8):1299–1310. doi: 10.1016/j.cellsig.2011.03.011

Suppression of uPA and uPAR Blocks Radiation-induced MCP-1 mediated Recruitment of Endothelial Cells and Monocytes in meningioma

Arun Kumar Nalla 1, Venkateswara Rao Gogineni 1, Reshu Gupta 1, Dzung H Dinh 2, Jasti S Rao 1,2,*
PMCID: PMC3095686  NIHMSID: NIHMS286967  PMID: 21426933

Abstract

Chemokines play a vital role in recruiting various cell types in the process of tissue repair. Radiation, a major therapeutic modality in cancer treatment, has been described to induce inflammatory response that might lead to the expression of several chemokines. In the present study, we investigated the mechanism of monocyte chemoattractant protein-1 (MCP-1) induction by radiation in meningioma cell lines and the paracrine effect on human microvascular endothelial cells (HMEC). After radiation, meningioma cell lines (IOMM Lee and SF-3061) showed an increased expression of MCP-1. In addition, irradiated meningioma cancer cell conditioned medium (CM) showed increased ability to attract HMEC and stimulate MCP-1-induced protein (MCPIP), VEGF and angiogenin expression in HMEC. This chemotactic activity and angiogenic stimulator effect on HMEC was almost abrogated by depleting MCP-1 from the irradiated cancer cell CM. Further, inhibition of either ERK activation/expression or NF-κB nuclear translocation hindered radiation-induced MCP-1 expression in both meningioma cell lines. Further, supplementing cancer cells with exogenous ATF-uPA (with and without radiation) activated ERK phosphorylation, nuclear translocation of the NF-κB p65 sub-unit (Rel-A), and MCP-1 expression. Downregulation of uPA and uPAR, simultaneously by transfecting the cancer cells with bi-cistronic siRNA-expressing plasmid (pU) inhibited radiation-induced ERK activation, nuclear translocation of Rel-A, NF-κB DNA binding activity, and MCP-1 expression. In addition, pU- transfected cancer cells (with or without radiation) reduced radiation-induced MCP-1 and blocked the recruitment of other cell types during the inflammatory process induced by radiation both in in vitro and in vivo conditions.

Keywords: urokinase plasminogen activator (uPAR), uPA receptor (uPAR), Radiation, monocyte chemoattractant protein-1 (MCP-1), ERK phosphorylation, NF-κB

1. Introduction

Meningioma is the second most common central nervous system tumor with an incidence of 17-20% of all intracranial tumors. Mostly benign, meningioma rarely presents with aggressive characteristics. Typically, meningiomas are surgically removed and subsequently subjected to radiation. This treatment regimen has been reported to extend the survival rate of cancer patients. However, some patients do experience tumor recurrence after treatment with surgical resection and radiation [1].

Ionizing radiation has been utilized as a major cancer therapeutic agent for nearly a century. Apart from the cytotoxicity induced by radiation to the cancer cells, numerous negative effects stemming from radiation exposure have been documented during radiotherapy. For example, inflammatory responses are activated directly by radiation and the damage it inflicts to the cells or from the process of healing the damaged tissue [2]. Lorimore et al. [3] have reported that ionizing radiation induces inflammation. Moreover, others have reported that certain non-lethal radiation dosages might lead to neo-vascularisation via stimulation of endothelial cell signaling [4,5].

The inflammatory response is thought to primarily induce cytokines and chemokines [6], which have been reported to influence angiogenesis by stimulating the expression of various proangiogenic molecules [7-9]. Recently, monocyte chemoattractant protein-1 (MCP-1) has been included in the growing list of chemokines that have the potential to stimulate angiogenesis [10,11]. Before its recent categorization as an angiogenic stimulator, MCP-1 was well characterized for its role in modulating inflammatory response by inducing recruitment of monocytes and endothelial cells to the site of inflammation [12,13]. Recent studies have shown that ionizing radiation affect chemokine expression in human fibroblasts [14]. Further, it was also reported that lower dosage of radiation mobilizes bone marrow-derived endothelial progenitor cells (EPCs) into circulation to enhance revascularization [15,16]. Today, it is well understood that MCP-1 functions in a dual manner by recruiting monocytes and endothelial cells as well as stimulating the expression of pro-angiogenic molecules.

Although there is a considerable body of research on the role of MCP-1 as a chemokine and angiogenic stimulator, the molecular mechanism by which cells alter the expression of MCP-1 upon external stimuli, such as radiation, is still poorly understood. The importance of identifying the molecular mechanisms in response to radiation is increasing. This information will be useful in developing methods to sensitize tumors to radiation and to protect normal tissue from the effects of radiation. Recently, Malik et al. [17] suggested that the transient accumulation of granulocytes within the portal area upon radiation of the liver and the chemokines secreted by the fibroblasts present in the portal vessels are involved in neutrophil recruitment. Moreover, that particular study postulates that the inhibition of more than one chemokine might be needed to reduce leukocyte recruitment.

An increasing number of studies have highlighted the role of urokinase plasminogen activator (uPA) and its receptor, uPAR, in promoting tumor cell adhesion, migration, proliferation and angiogenesis either directly or indirectly by initiating intracellular signaling pathways [18-21]. Studies have also shown that radiation elevates uPA and uPAR levels in neuroblastomas [22] and meningiomas [23]. These studies indicate a possible correlation between radiation-induced uPA levels and the increased invasive and angiogenic properties of irradiated cells. Apart from its role in tumorigenesis, uPA also appears to be a key regulator of tissue inflammation and repair following injury. For example, uPA-null mice were reported to exhibit impaired liver, lung and skin healing, which was associated with reduced accumulation of macrophages [24,25]. Similarly, macrophage accumulation at the injured skeletal muscle was reported to be absent in uPA-null mice and was associated with severely impaired muscle regeneration [26,27]. However, the mechanism by which uPA regulates macrophage accumulation during inflammation is still poorly understood. In the present study, we sought to identify the intracellular signaling pathways that might be involved in MCP-1 regulation in meningioma cells after radiation treatment. Based on our initial results, we also downregulated uPA and uPAR using a bi-cistronic siRNA-expressing plasmid to further assess the possibility of using RNAi technology as a cancer therapeutic agent in conjunction with radiation.

2. Materials and Methods

2.1 Cell culture conditions

We used human meningioma IOMM Lee and SF-3061 cell lines, which were kindly provided by Dr. Ian E. McCutcheon (University of Texas M.D. Anderson Cancer Center, Houston, TX) and Dr. Antia Lal (UCSF), respectively. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM/High Glucose) (Thermo Scientific, South Logan, UT) supplemented with 10% fetal bovine serum (FBS), 100 U/mL streptomycin, and 100 U/mL penicillin (Invitrogen, Carlsbad, CA). Cells were treated either with MEK/ERK inhibitor, U0126 (EMD Biosciences, San Diego, CA), JNK II inhibitor, SP600125 (Calbiochem, San Diego, CA) or NF-κB activation inhibitor II, JSH-23 (Santa Cruz Biotechnology, Santa Cruz, CA) and incubated in serum free medium for the indicated time periods. siRNAs against ERK1 and JNK were purchased from Santa Cruz Biotechnology (CA). A bi-cistronic plasmid expressing siRNAs against uPA and uPAR (pU) was used as described previously[23]. We obtained antibodies for angiogenin, phospho ERK, ERK1, JNK, phospho JNK, lamin, MCP-1, MCPIP, NF-κB p65, uPA, uPAR, VEGF-A and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Santa Cruz Biotechnology (Santa Cruz, CA). ATF-uPA was purchased from American Diagnostica (Stamford, CT). Cells were serum starved before incubating overnight in serum free media supplemented with ATF-uPA.

2.2 Radiation treatment and transfection conditions

We used the RS 2000 Biological Irradiator (Rad Source Technologies, Inc., Boca Raton, FL), which was operated at 150 kV/50 mA, for the radiation treatments. All cells were given a single (10 Gy) dose of radiation that was administered either after 30 min / 2 hrs of inhibitor treatment or 48 hrs after cell transfection. Transfection experiments were performed with FuGene HD transfection reagent following the manufacturer's protocol (Roche Applied Science, Madison, WI).

2.3 Chemotaxis assay

The culture supernatants of IOMM Lee and SF-3061 cells from various treatment groups were used as chemoattractants. Human microvascular endothelial cells (HMEC) (1×105) were seeded on 8-μm Matrigel-coated transwell inserts (Greiner Bio-One, Monroe, NC) and placed in a 12-well plate with the respective cancer cell culture supernatant. After 24hrs, invaded cells were fixed, stained with Hema-3, and counted under a light microscope.

2.4 Immuno-depletion of MCP-1 from culture medium

Cancer cells culture supernatant collected from IOMM Lee and SF-3061 cells lines (with or without radiation) were either incubated with MCP-1 neutralizing antibody (R&D Systems, Minneapolis, MN) or non-specific IgG's for 1 hr at room temperature. After incubating with protein A/G agarose beads (Miltenyi Biotec, Auburn, CA) for 30 min on ice, the culture media was passed through magnetic μM columns (Miltenyi Biotec) and flow through was used for further experiments.

2.5 ELISA-based detection of MCP-1

Cancer cell culture supernatants were collected from IOMM Lee and SF-3061 cells, centrifuged and used for the detection of MCP-1 using human anti-MCP-1 ELISA kit (R&D Systems, Minneapolis, MN) following the manufacturer's instructions.

2.6 Reverse transcription polymerase chain reaction (RT-PCR)

Total RNA was extracted from cells (either irradiated or non-irradiated and with or without transfection) using TRIZOL reagent (Invitrogen, CA) as per standard protocol. Total RNA from brain tumor tissues was isolated using the RNeasy Mini Kit (Qiagen, Valencia, CA). RNA (~1 μg) was used as a template for reverse transcription reaction (Roche Applied Science, Indianapolis, IN), followed by PCR analysis using specific primers. The amplified products were analyzed on an agarose gel. Primers used in the present study and the sequences are as follows: MCP-1 (forward 5’-ctcatagcagccaccttcat-3’ and reverse 5’-gattcttgcaaagaccctca-3’), GAPDH (forward 5’-acagtcagccgcatcttctt-3’ and reverse 5’-tgacaaagtggtcgttgagg-3’), mCD31 (forward 5’-gcctggagaggttgtcagag-3’ and reverse 5’-ggtgctgagacctgcttttc-3’), mCD14 (forward 5’-ctgatctcagccctctgtcc-3’ and reverse 5’-agtgacaggttccccacttg-3’), uPA (forward 5’-cacactgcttcattgattaccc-3’ and reverse 5’-gtcggtagaattctcttttccaa-3’) and uPAR (forward 5’-cctggagcttgaaaatctgc-3’ and reverse 5’-ctgggtggttacagccactt-3’).

2.7 Western blot analysis and electro-mobility shift assay (EMSA)

Western blotting was performed as described previously [23]. Briefly, total cell lysates were collected from IOMM Lee and SF-3061 cells, which were subjected to the various treatment conditions mentioned above, using Tris-buffered lysis buffer. Equal amounts of protein were subjected to SDS-PAGE and followed by transfer of proteins to a nitrocellulose membrane (Bio-Rad, Hercules, CA). The membranes were probed with specific antibodies. Nuclear extracts were prepared using a nuclear extraction kit (Panomics, Inc., Fremont, CA) as per the manufacturer's instructions. DNA binding activity of the nuclear extracts to NFκB DNA probe III (specific for NFκB p65) was determined by electro-mobility shift assay (EMSA) using Pamomics kit (Affymetrix Inc, Fremont, CA) according to manufacturer's instructions). Briefly, nuclear extracts were incubated with biotin-labeled NFκB probe III set (with or with cold probe) as per the manufacturers’ instruction. The complexes separated on a polyacrylamide gel, transferred to a positively charged nylon membrane, detected using streptavidin-HRP IgG conjugate and developed using chemiluminescent substrate provided along with the kit.

2.8 In vivo studies

The Institutional Animal Care and Use Committee at the University Of Illinois College of Medicine at Peoria approved all experimental procedures involving the use of animals. Nude mice (4-6 weeks of age) were anesthetized, placed in a stereotactic frame (David Kopf Instruments, Tujunga, CA) and implanted with 1×105 IOMM Lee cells in 10 μL of PBS through a 27-gauge needle at 2 mm lateral and posterior to the bregma and 3 mm below the dura. After 10 days, the animals were separated into four treatment groups of 5 animals each for each cell line. Animals were treated with either pU or pSV plasmid (10 animal each) by implanting Alzet osmotic pumps (model 2001, Alzet Osmotic Pumps, Cupertino, CA) into the animals for plasmid delivery (6-8 mg/kg body weight). Among ten animals, five animal were given two doses of 10 Gy on alternate days (radiation was administered by masking the whole body with lead sheets and leaving only the skull region exposed), while the rest five animals were given any radiation treatment. All the animals were observed for 3-4 weeks, euthanized, and their brains fixed in buffered formaldehyde.

2.9 Statistical analysis

All data are presented as means ± standard error (SE) of at least three independent experiments (each performed at least in triplicate). One-way analysis of variance (ANOVA) combined with the Tukey post-hoc test of means were used for multiple comparisons in cell culture experiments. Statistical differences are presented at probability levels of p<0.05, p<0.01, and p<0.001.

3. Results

3.1 Radiation enhanced expression of MCP-1 in meningioma cells

Culture supernatant medium (CM) collected from cancer cells irradiated with 10 Gy was used to determine the expression/secretion levels of various pro-angiogenic chemokines using a human angiogenesis array. CM of irradiated IOMM Lee cells showed elevated levels of chemokines such as GRO, interleukin (IL)-8, Leptin and MCP-1 compared to CM from non-irradiated cells (Fig. 1A). Similarly radiation induced the expression of IL-6, IL-8, Leptin and MCP-1 in SF-3061 cells (Fig. 1A). By measuring the signal intensity of each spot using ImageJ software, we confirmed that the secretory levels of MCP-1 by irradiated IOMM Lee cells was ~3 folds higher than levels observed in culture medium of non-irradiated cells (Fig. 1B). While the levels of MCP-1 in irradiated SF3061 cell culture media was high by ~1.5 folds than non-irradiated SF3061 cell media (Fig. 1B). RT-PCR of the total RNA and western blot analysis of CM showed a radiation dose-dependent increase of MCP-1 at the mRNA and protein levels, respectively in both meningioma cell lines (Fig. 1C & 1D). MCP-1 transcripts in irradiated IOMM Lee and SF3061 cells were noticed to be ~3.7 and 2.5 folds, respectively when compared to their respective non-irradiated cells.

Figure 1. Radiation induces MCP-1 expression in meningioma cells.

Figure 1

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A. Meningioma cells were irradiated with 10 Gy and incubated for nearly 24 hrs before the culture supernatants were collected. Supernatants were used to determine the expression pattern of chemokines stimulated by irradiating cancer cells using a human angiogenesis antibody array. Signal band corresponding to MCP-1 was boxed B. Using ImageJ software, the intensity of signals was quantified and the percent increase of MCP-1 in irradiated cells as compared to non-irradiated meningioma cells is represented. C. RT-PCR was carried out to determine the transcripts levels of MCP-1 in meningioma cell lines (with and without radiation). Western blot analysis of culture supernatants was carried out to determine the secretory levels of MCP-1 upon radiation. D. The intensity of RT-PCR amplicons was quantified and is represented graphically. Bar represented the mean S.D values from three independent set of experiments.

3.2 Radiation-induced MCP-1 attracted endothelial cells and induced expression of MCPIP, VEGF and angiogenin

To understand the role of MCP-1 as a pro-angiogenic stimulus, we initially exposed HMEC to exogenous recombinant MCP-1 (100 ng/mL) and determined the expression of various proangiogenic molecules. Western blot analysis confirmed that MCP-1 induced the expression of MCP-1-induced protein (MCPIP; a transcription factor induced by MCP-1), VEGF and angiogenin in HMEC (Fig. 2A). Apart from induction of the pro-angiogenic molecules, MCP-1 also attracted HMEC to migrate towards the medium supplemented with recombinant MCP-1 (Fig. 2B). Further, MCP-1-mediated chemoattraction of HMEC was inhibited by 36% when MCP-1-specific IgG was added to the culture medium but not in the presence of mouse IgG (Fig. 2B).

Figure 2. Radiation induced MCP-1 stimulated endothelial cell migration and pro-angiogenic molecules.

Figure 2

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A. Induction of MCP-1-induced protein (MCPIP), VEGF and angiogenin in human microvasculature endothelial cells (HMEC) were determined by incubating HMEC with human recombinant MCP-1. Western blot analysis of total cell lysates was used to determine the levels of the pro-angiogenic molecules induced by MCP-1 in HMEC. B. Chemotaxis assay using 8 μm transwell inserts was carried out to determine the potential of MCP-1 as a chemoattractant of HMEC. The specificity of MCP-1-induced chemoattraction was determined by supplementing the medium in the lower chamber with MCP-1 (100 ng/mL) with or with anti-MCP-1 IgG. The number of cells in each representative field was counted and is represented. C. Chemoattract ability of meningioma cell culture media (either irradiated or nonirradiated) was carried out. Role of radiation-induced MCP-1 as a chemoattractant was determined by immunodepleting MCP-1 using anti-MCP-1 neutralizing antibody from the cancer cells culture media. D. The number of cells that migrated towards the cancer cell culture supernatant was counted in each representative field and is graphically presented. The experiment was repeated thrice (bars represents the mean S.D value, p<0.05) E. Western blot analysis of the total cell lysates from the HMEC incubated with either control or MCP-1 depleted (d MCP-1) culture supernatants (with and without radiation) was carried out to determine the levels of pro-angiogenic molecules such as MCPIP, VEGF and angiogenin. F. Band intensities were measured and quantified using ImageJ software, and are represented graphically. Results from three independent experiments were evaluated and presented (mean S.D value; p<0.05).

To get further insight into the role of MCP-1 released by cancer cells (with or with radiation) in attracting and stimulating pro-angiogenic molecules in HMEC, we initially carried out a chemotaxis assay using cancer cell culture supernatant as a chemoattractant for HMEC. We observed that this culture supernatant/conditioned medium from cancer cells did attract HMEC. When MCP-1 was immuno-depleted from the culture medium, this chemo-attraction was inhibited by ~35% and 43% in IOMM Lee and SF-3061, respectively. Furthermore, we observed that HMEC tend to migrate more effectively towards the irradiated cancer cell CM (by 45% and 70% in IOMM Lee and SF-3061 cells, respectively) as compared to the non-irradiated cancer cell CM (Fig. 2C & 2D). The chemotactic effect of irradiated cell culture medium was abrogated by nearly 56% (IOMM Lee CM) and 68% (SF-3061 CM) when MCP-1 was immuno-depleted from the culture medium using anti-mouse MCP-1 neutralizing antibody (Fig. 2C & 2D).

We next analyzed the paracrine role of radiation-induced MCP-1 by exposing the HMEC to CM collected from irradiated and non-irradiated cancer cells and then determining the expression levels of MCPIP, VEGF and angiogenin in HMEC. We noticed that HMEC grown in the irradiated cancer cell CM expressed elevated levels of MCP-IP, VEGF and angiogenin as compared to the HMEC grown in non-irradiated cancer cell CM (Fig. 2E). Western blot analysis confirmed that VEGF expression increased by ~59% and 62% in HMEC incubated in CM collected from irradiated IOMM Lee and SF-3061 cells, respectively as compared to the corresponding non-irradiated cancer cell CM (Fig. 2E & 2F). Similarly, HMEC exposed to irradiated cancer cell CM showed an increase in angiogenin levels by ~68% in IOMM Lee cells and 59% in SF-3061 cells when compared to the HMEC exposed to the non-irradiated cancer cell CM. Moreover the levels of MCPIP in HMEC were also observed to be elevated when incubated in irradiated cancer cell culture media by ~30% and 80% in IOMM Lee and SF3061 cells compared to non-irradiated cancer CM. However, when MCP-1 was depleted from the respective cancer cell CM (irradiated and non-irradiated), the stimulation of MCPIP, VEGF, and angiogenin expression in HMEC by irradiated IOMM Lee CM was reduced by nearly 65%, 43% and 52%, respectively. Whereas depletion of MCP-1 from the CM of irradiated SF-3061 showed a reduction in stimulation of MCPIP, VEGF and angiogenin by ~73%, 38% and 49%, respectively compared to the control (IgG) irradiated SF-3061 CM (Fig. 2E & 2F).

3.3 Radiation induced MCP-1 expression via ERK and NF-κB activation in meningioma cells

A previous study from our laboratory has shown that radiation increased phosphorylation of ERK1/2, and JNK [23]. In the present study, we attempted to determine the signaling molecule responsible for radiation-induced MCP-1 expression using specific phosphorylation (activation) inhibitors. When cancer cells were incubated with specific inhibitors for either ERK (U0126, 10 μM) or JNK activation (SP600125, 10 μM), we noticed that the MCP-1 levels were reduced significantly by inhibiting ERK phosphorylation rather than JNK phosphorylation. Furthermore, we noticed that inhibition of either ERK phosphorylation (using activation inhibitor) or expression of ERK (siRNA mediated knockdown of ERK1) reduced both basal and radiation-induced MCP-1 expression in both IOMM Lee and SF 3061 cells (Fig. 3A and supplementary Fig. S1A), suggesting that ERK activation regulates MCP-1 expression. ELISA based detection of MCP-1 in culture medium showed that inhibition of ERK activation resulted in reduced secretion of MCP-1 by nearly 30% in both IOMM Lee and SF 3061 cells. Moreover, a significant reduction in radiation-induced MCP-1 levels by 56% (IOMM Lee) and 41% (SF-3061) was noticed in the cells which were treated with ERK inhibitor prior to radiation. Apart from this, role of ERK in MCP-1 regulation was confirmed when we noticed that siRNA mediated knockdown of ERK1 reduced both the basal and radiation induced expression levels of MCP-1 (Supplementary Fig. S1A). In contrast neither inhibition of JNK phosphorylation nor siRNA mediated knockdown of JNK showed any significant effect on the radiation induced MCP-1 expression in meningioma cells (Supplementary Fig. S1B & S1C).

Figure 3. Radiation induces MCP-1 expression by ERK phosphorylation and nuclear translocation of NF-κB (p65).

Figure 3

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A. Meningioma cells were incubated with ERK phosphorylation inhibitor (U0126) for 30 minutes prior to radiation and incubated for 16 hrs. Total cell lysates from the above treated cancer cells were used to determine the basal and phosphorylated forms of ERK while the nuclear extracts were used to determine NF-κB p65 levels translocated into the nucleus. MCP-1 expression was determined in the culture supernatants. B. To confirm the role of NF-κB in regulation of MCP-1, meningioma cells were incubated with NF-κB activation inhibitor II for 2 hrs prior to radiation and incubated for 16 hrs. Levels of NF-κB p65 and phosphorylated form of ERK in the nuclear extracts, and secretory MCP-1 levels in the culture supernatants were confirmed by western blot analysis using specific antibodies. C. ELISA-based assay was also used to confirm the secretory levels of MCP-1 in the cancer cell culture supernatants treated with or without radiation and with or without a prior incubation of inhibitors of ERK/NF-κB nuclear translocation. Experiments were repeated for three times and the bar column represented mean S.D value, p<0.001.

Many of the growth factors and chemokines that are transcriptionally regulated by NF-κB activation during the inflammatory response are stimulated by sources like radiation [28]. Therefore, we next examined the role of NF-κB activation in radiation-induced MCP-1 expression. Western blotting of nuclear extracts isolated from irradiated meningioma cells showed increased nuclear levels of NF-κB (p65) as compared to nuclear extracts of nonirradiated cancer cells (Fig. 3B). However, incubating the cells with a NF-κB activation inhibitor-II (JSH-23, 25 μM) prior to radiation significantly reduced the nuclear translocation of NF-κB as well as radiation-induced MCP-1 expression in both the cancer cell lines. ELISA based detection of MCP-1 levels in CM of NF-κB inhibitor treated cells showed a reduction in secretory MCP-1 levels by 68% and 76% in IOMM Lee and SF-3061 cells, respectively (Fig. 3C). Taken together, these data suggest that radiation regulates MCP-1 expression by activating ERK and nuclear translocation of NF-κB p65 in both IOMM Lee and SF-3061 cells.

3.4 Radiation-induced uPA and uPAR activated ERK and NF-κB, which in turn regulated expression of MCP-1

Considering the potential role of uPA/uPAR interaction in ERK activation [29] and the induction of uPA within 6 hrs of radiation [23], we next attempted to characterize the possible role of radiation-induced uPA in activating ERK as well as NF-κB nuclear translocation—both of which were identified to regulate MCP-1 expression in meningioma cells. Initially we confirmed a radiation dosage dependent increase in the ERK phosphorylation and uPA expression in both the meningioma cells (Supplementary Fig. S2A). Further to establish the role of radiation induced uPA in activation of ERK is via the interaction with its receptor, we attempted to hinder the uPA-uPAR interaction by blocking the uPA receptor by incubating the cells with uPAR blocking antibody (10 ug/ml) for 2 hrs at 4° C, before irradiated the cells. We noticed that blocking uPA receptor prior to radiation significantly reduced ERK phosphorylation (Supplementary Fig. S2B), suggesting the importance of uPA/uPAR interaction in activating ERK in irradiated meningioma cells. To study and confirm the downstream effect of uPA/uPAR binding, we used the amino terminal fragment of uPA (ATF), a motif known to interact with uPAR and induce the downstream signaling cascade [30,31]. We noticed that supplementing ATF-uPA in culture medium resulted in a dose-dependent activation of ERK phosphorylation in IOMM Lee and SF 3061 cells (Supplementary Fig. S2C). Further, we noticed that exposure of meningioma cells to exogenous ATF-uPA not only increased phosphorylation levels of ERK in IOMM Lee and SF-3061 cells, respectively (Fig. 4A) but also showed increased nuclear NF-κB p65 levels in meningioma cells supplemented with ATF-uPA in culture medium. When the levels of MCP-1 in CM were examined by ELISA, we noticed that ATF-uPA induced MCP-1 expression by 67% and 54% in IOMM Lee and SF-3061 cells, respectively. Moreover, addition of ATF-uPA followed by irradiation to cancer cells resulted in a significant cumulative increase in the secretory levels of MCP-1 (Fig. 4B) in both cell lines. Further, inhibiting ERK phosphorylation by incubating the cancer cells with ERK inhibitor prior to ATF-uPA stimulation reduced ATF-uPA-induced ERK phosphorylation, nuclear translocation of NF-κB p65 and MCP-1 expression (Fig. 4C & 4D). Overall, these results suggest that the activation of ERK and NF-κB p65 nuclear translocation, either by supplementation with ATF-uPA or radiation-induced uPA, plays a key role in regulating MCP-1 expression. To confirm the specificity of ATF-uPA in inducing ERK and NF-κB activation, we next examined the effect of ATF-uPA in activating ERK and NF-κB in the presence of soluble uPAR (suPAR), which acts as a competitor for uPA in interacting with surface bound uPAR[32]. The addition of suPAR to the medium significantly inhibited MCP-1 expression, which was induced by either ATF-uPA or radiation in both cancer cell lines (Fig. 4E).

Figure 4. Radiation-induced uPA levels regulate MCP-1 expression by activating ERK phosphorylation and NF-κB p65 nuclear translocation.

Figure 4

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A. Meningioma cells were incubated in media supplemented with 250 ng of amino terminal fragment (ATF) of uPA and irradiated as mentioned in Materials and methods. Western blot analysis was carried out to determine the levels of ERK phosphorylation, NF-κB p65, and MCP-1. B. An ELISA-based assay was carried out on the culture supernatants collected from meningioma cells either incubated with or without ATF-uPA and with or without radiation. Expression levels in pg/mL are represented graphically (bar column represented mean S.D value from three independent experiments). C. To confirm that ATF-uPA-induced ERK activation is essential for MCP-1 expression, meningioma cells were incubated with ERK inhibitor prior to ATF-uPA stimulation. Total cell lysates were used to confirm the inhibition of ERK phosphorylation. Secretory levels of MCP-1 were measured by western blot analysis of the culture supernatants. D. ELISA was carried out using the culture supernatant of ATF-uPA-treated cancer cells with or without prior incubation of ERK inhibitor. E. The secretory levels of MCP-1 in culture supernatants from meningioma cells incubated with or without suPAR along with or without ATF-uPA and with or without radiation treatment were determined. Bar represents the mean S.D values from three independent experiments.

3.5 siRNA-mediated downregulation of uPA and uPAR inhibited radiation-induced MCP-1 by suppressing the DNA binding activity of NF-κB

Since uPA/uPAR interaction is the major upstream event that activated ERK and NF-κB after radiation treatment in meningioma cells, we attempted to block the expression of uPA and uPAR and studied its effect on radiation-induced MCP-1 expression. To block uPA and uPAR expression, we transfected meningioma cells with a plasmid expressing siRNA against both uPA and uPAR (pU) prior to radiation. siRNA-mediated downregulation of uPA and uPAR not only inhibited the basal expression levels of uPA and uPAR in meningioma cells, but also inhibited radiation-induced uPA and uPAR levels by ~70% and 75% in IOMM Lee cells, and 59% and 68% in SF-3061 cells, respectively when compared to the respective irradiated control cancer cells (Fig. 5A & 5B). Apart from the target molecules, we noticed that MCP-1 levels in both cell lines were also significantly reduced in pU-transfected cells as compared to control cells. Western blot analysis of CM collected from uPA and uPAR-downregulated cancer cells reduced the secretion of MCP-1 by ~38% and 58% in IOMM Lee and SF-3061 cells, respectively when compared to pSV-transfected cells (Fig. 5A). In addition, pU transfection followed by radiation reduced the radiation-induced MCP-1 levels by ~64% and 75% in IOMM Lee and SF-3061 cells, respectively (Fig. 5A & 5B). Downregulation of uPA and uPAR also reduced the phosphorylation of ERK in both non-irradiated and irradiated cancer cells (Fig. 5A & 5B). ELISA of the CM collected from the transfected cells showed that pU transfected cells reduced the secretory MCP-1 levels by nearly 64% and 57% in IOMM Lee and SF3061 cells, respectively compared to the levels in the control CM (Fig. 5C).

Figure 5. siRNA-mediated downregulation of uPA and uPAR inhibits the phosphorylation of ERK, DNA binding activity of NF-κB, and MCP-1 expression.

Figure 5

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Meningioma cells were transfected with either a scrambled vector (pSV) or a vector expressing siRNA against uPA and uPAR (pU) and followed with radiation treatment. A. Western blot analysis of total cell lysates extracted from the irradiated and non-irradiated transfected cells was carried out to determine the expression levels of uPA, uPAR, phosphorylated ERK and total ERK using specific antibodies. Levels of MCP-1 in the irradiated and non-irradiated transfected cancer cell culture supernatants were determined by western blot analysis using MCP-1 specific IgG. B. The relative expression levels were quantified by measuring the band intensities and represented graphically. Results from three independent experiments were evaluated with a mean S.D value (p<0.05). C. ELISA-based assay was carried out to determine MCP-1 levels in the culture supernatants of cancer cells transfected with either pSV or pU (with or without radiation). D. DNA binding activity of nuclear extracts to NF-κB probe was carried out using nuclear extracts from the meningioma cells transfected with either pSV or pU (with and without radiation). Lane 1 is probe only. Lanes 2 and 3 are nuclear extracts from pSV-transfected cells with NF-κB probe alone or in combination with the cold probe, respectively. Lanes 4 and 5 are nuclear extracts from pU-transfected cells with NF-κB probe alone or in combination with the cold probe, respectively. Lanes 6 and 7 are nuclear extracts from irradiated pSV-transfected cells with NF-κB probe alone or in combination with the cold probe, respectively. Lanes 8 and 9 are nuclear extracts from irradiated pU-transfected cells with NF-κB probe alone or in combination with the cold probe, respectively.

Our previous experiments revealed that the inhibition of nuclear translocation of NF-κB led to a decrease in the expression of MCP-1 induced either by ATF-uPA supplementation or radiation. So, we next attempted to determine the DNA binding activity of NF-κB in the nuclear extracts from irradiated and non-irradiated pU-transfected cells using EMSA. As shown in Figure 5D, the NF-κB probe/DNA binding activity of nuclear extracts from uPA/uPAR-downregulated cells was significantly reduced when compared to cells transfected with pSV. Moreover, the nuclear extracts from irradiated cells enhanced the ability to bind to the NF-κB DNA probe as compared to nuclear extracts from non-irradiated cells. Furthermore, radiation-induced NF-κB activity was reduced in cells treated with pU and radiation by nearly 50% and 87% in IOMM Lee and SF-3061 cells, respectively.

3.6 Conditioned medium from uPA/uPAR-downregulated meningioma cells reduced the potential to attract and stimulate endothelial cells to express pro-angiogenic molecules

Using the CM of cancer cells transfected with the respective plasmid as a chemoattractant, we carried out a chemotaxis assay to examine the migration of HMEC towards the cancer cell CM. We noticed the CM from pU-transfected cancer cells significantly reduced the ability to attract HMEC towards them as compared to CM collected from pSV-transfected cancer cells. Moreover, CM from pU-transfected and irradiated cancer cells (combination treatment) reduced the ability to attract HMEC by nearly 51% and 43% when compared to CM from irradiated IOMM Lee and SF-3061 cells, respectively (Fig. 6A). Taken together, the present data suggest that downregulation of uPAR and uPA reduced the potential of cancer cells to attract endothelial cells, especially during radiation treatment conditions.

Figure 6. Chemoattractant and angiogenic stimulatory ability of culture supernatants from cancer cells transfected with pSV and pU (with or without radiation).

Figure 6

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A. Culture supernatants from cancer cells transfected with pSV or pU (with and without radiation) were used as chemoattractants in the chemotaxis assay. HMEC were seeded on the upper chamber of Matrigel-coated transwell inserts and the lower chamber was filled with culture supernatants. Invaded cells were fixed, stained and counted in each of the representative wells. Percent of HMEC invaded are represented graphically. B. Angiogenic stimulation capacity of transfected cancer cell culture supernatants (with or without radiation) was determined by incubating HMEC with the culture supernatants. Western blot analysis of the total cell lysates isolated from HMEC grown in the transfected cancer cell supernatants was carried out to determine the levels of MCPIP, VEGF and angiogenin using specific antibodies. Experiment was repeated at least three times.

With the reduced chemoattractant potential and MCP-1 levels in CM of pU-transfected cancer cells, we next examined the effect of HMEC grown in the CM collected from cancer cells transfected with either pSV or pU (both irradiated and non-irradiated). Western blot analysis confirmed that HMEC grown in CM from pU-transfected cancer cells showed a significant reduction in expression of MCPIP, VEGF and angiogenin as compared to HMEC grown in CM from pSV-transfected cells (Fig. 6B), indicating the reduction in angiogenic potential of pU transfected cancer cells. Moreover, the elevated levels of MCPIP, VEGF and angiogenin observed in HMEC grown in CM of irradiated cancer cells were noticed to be reduced in HMEC grown in CM from pU-transfected irradiated cancer cells.

3.7 Downregulation of uPA/uPAR reduced the radiation-induced recruitment of endothelial cells and monocytes towards intracranial tumors

We intracranially implanted IOMM Lee cells in nude mice to determine the effect of radiation and pU treatment on intracranial tumor development and cell type recruitment in an in vivo tumor model. Hematoxylin and Eosin (H&E) staining confirmed the occurrence of tumor by injecting IOMM Lee cells intracranially. Treating the mice with pU showed relatively lower tumor area compared to pSV treated mice (Fig. 7A). Moreover, the tumors in the mice treated with radiation alone showed scattered tumor appearance, whereas the combination of pU and radiation has reduced the scattering of the tumor mass. Further we carried out RT-PCR analysis of the total RNA isolated from the brains collected from mice implanted with IOMM Lee cells treated with either pSV or pU (with and without radiation). Based on RT-PCR analysis, we observed that CD31/PECAM-1 (an endothelial cell marker) mRNA levels were high (by ~80%) in brain tumors developed from irradiated cancer cells as compared to tumors developed from non-irradiated cancer cells (Fig. 7B). This result indicated that irradiated cancer cells increased the recruitment of endothelial cells by ~80% as compared to non-irradiated cancer cells, which was consistent with our in vitro results. Morevoer, mRNA levels of CD31 in the pU-treated tumors (with or with radiation) were reduced by nearly 77% compared to pSV-treated tumors. We made a similar observation with mRNA levels of CD14 (a monocyte/macrophage marker) in brain tumor samples. CD14 transcripts levels were more noticeable (~57%) in tumors grown by implanting irradiated cancer cells as compared to tumors grown by implanting non-irradiated cells (Fig. 7B). However, pU treatment of intracranial tumors resulted in ~80% reduction of CD14 mRNA transcript levels, suggesting that knockdown of uPA and uPAR significantly reduced the recruitment of monocytes under in vivo conditions (Fig.7C). Imunnohistochemical analysis of the paraffin embedded brain section using mouse specific CD31 and CD14 confirmed the RT-PCR results (Fig. 7D). Brain sections from tumors treated with radiation alone showed increased accumulation of CD31 and CD14 cells around the tumor region when compared to untreated tumor. Further more, brain sections from pU treated tumors (with and with radiation treatment) showed a decreased number of CD31 and CD14 cells compared to tumor treated with pSV.

Figure 7. Effect of radiation and pU treatment on pre-established intracranial tumor.

Figure 7

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Tumors were established in nude mice by injecting IOMM Lee cells followed treating with either pSV or pU (with and without radiation). After the time period mentioned, the brain were either fixed in buffered formalin or snap frozen. A. Formalin fixed brain were paraffin embedded and used for hematoxylin and eosin staining, and visualized under light microscope. Representative figures from each treatment were shown. B. Total RNA extracted from the pSV- and pU-transfected snap frozen brain tumor samples (with and without radiation) was used to detect transcript levels of CD31 (an endothelial cell marker) and CD14 (a monocyte marker) C. The relative expression levels of mRNA transcripts were quantified by densitometry analysis and represented (n=3). D. Immunohistochemical analysis of the paraffin embedded brain section using mouse specific CD31 and CD14 to observe the endothelial cell and monocyte recruitment towards the tumors.

4. Discussion

Recent studies have demonstrated that ionizing radiation modulates chemokine expression [14,15,33,34]. The results of the present study confirm that radiation alters chemokine expression in human meningioma cells. In addition, our study provides insight into the mechanisms responsible for MCP-1 induction in irradiated meningioma cells as well as the role of MCP-1 as a chemoattractant and angiogenic stimulator of endothelial cells. Angiogenic antibody array analysis highlighted the elevated expression of several chemokines, such as IL-8, MCP-1 and Leptin upon radiation in both meningioma cell lines. Aside from the antibody array, further experiments demonstrated that MCP-1 increased in a radiation dose-dependent manner at both the mRNA and secretory levels. Among these three chemokines, we chose to limit the current study to radiation-induced MCP-1 since this molecule plays a major role in facilitating angiogenesis via its recruitment of various cell types to the site of inflammation or peri-tumor regions [10,35,36]. Recently, Chang et al. [33] reported that conditioned medium from irradiated endothelial cells significantly increased the chemotaxis of bone marrow-derived monocytes. Likewise, the present study also provides evidence that CM of irradiated meningioma cancer cells increased the tendency to attract HMEC when compared to CM from non-irradiated cancer cells. We further observed that immuno-depletion of MCP-1 using anti-human neutralizing antibody abolished the chemotactic effect of the irradiated cancer cell supernatant. This strengthens the possibility that the radiation-induced increase of MCP-1 into the culture medium might induce endothelial migration towards cancer cells.

In addition to MCP-1 being a major chemoattractant, several studies have highlighted the role of MCP-1 in directly inducing angiogenesis[10,37,38]. Hong et al. [39] reported that MCP-1-induced angiogenesis is mediated through pathways involving the vascular endothelial growth factor (VEGF). Similarly, in the present study, we observed that immunodepletion of MCP-1 from the conditioned medium of irradiated cancer cells reduced the potential to stimulate proangiogenic molecules, such as VEGF-A and angiogenin. Taken together, the present data suggest that radiation-induced MCP-1 by meningioma cells not only attracts HMEC towards the irradiated cancer cells, but also plays a paracrine role in promoting the angiogenic process of HMEC.

Knowing the role of radiation-induced MCP-1 in regulating angiogenesis, efforts where made to understand the mechanism by which meningioma cells induces MCP-1 expression upon receiving radiation dosage. Radiation generates reactive oxygen species, which activates RAS proteins and enhances protein tyrosine kinases to activate signaling pathways like ERK, Akt and JNK, which regulate several transcriptional factors [40]. Similarly, previous work from our own laboratory has shown that radiation activates the ERK and JNK signaling pathway in IOMM Lee cells [23]. In the process of identifying the role of these molecules during radiation in regulating MCP-1 expression, we incubated meningioma cells with a specific inhibitor prior to radiation and then checked the expression of MCP-1 upon radiation. The results indicated a positive correlation between MCP-1 expression and radiation-induced ERK phosphorylation, but not radiation-induced JNK phosphorylation. These results were further confirmed by knocking down ERK1 using specific siRNA.

Recent studies have demonstrated the role of NF-κB as a molecular link between inflammation and cancer [41,42]. NF-κB plays an important role by inducing the expression of genes coding for antigen receptors on immune cells, adhesion molecules, pro-inflammatory cytokines or chemoattractants (such as MCP-1) for inflammatory cells [43]. In addition, an interesting body of evidence suggests that generation of reactive oxygen species during the inflammatory process (e.g., ionizing radiation) activates NF-κB, which transcriptionally regulates several growth factors and chemokines [44]. It was reported that inflammatory stimuli activates NF-κB leading to translocation of NF-κB into the nucleus for binding to DNA motifs in gene promoters [45]. Similarly, in the present study, we observed that the nuclear translocation of NF-κB p65 molecules was enhanced with radiation, indicating the possibility of an increased regulatory/transcriptional role for NF-κB. Inhibition of radiation-induced MCP-1 expression by inhibiting nuclear translocation of NF-κB using a specific inhibitor suggested that radiation-induced NF-κB activity might play a key role in regulating MCP-1 expression. The NF-κB transcriptional pathway in protein-induced chemokine production was supported by the finding that inhibitors of NF-κB activation reduced MCP-1 upregulation[46]. Furthermore, the lower levels of NF-κB p65 in nuclear extracts of irradiated cancer cells treated with ERK phosphorylation inhibitor indicated that radiation-induced ERK phosphorylation might activate NF-κB nuclear translocation. Overall, the results of the present study suggest that radiation activates the ERK signaling pathway and subsequently enhances nuclear translocation of NF-κB, which in turn, transcriptionally regulates MCP-1 expression.

Previous work from our laboratory has shown that radiation-induced uPA expression in IOMM Lee cells was mediated through the ERK-dependent signaling pathway [23]. Similarly, in the current study, radiation induced uPA expression and activated ERK in IOMM Lee and SF-3061 cell lines. In continuation of our earlier discussion regarding ERK phosphorylation, there is an increasing body of evidence indicating that uPA—by interacting with its receptor uPAR—can activate ERK phosphorylation in various cancer cells [47-49]. Therefore, we next aimed to determine the autocrine effect of radiation-induced uPA on meningioma cells in activating ERK. To gain insight on this front, we used ATF-uPA, a non-enzymatic amino-terminal fragment of uPA, which interacts with uPAR and activates downstream signaling cascades. Our results demonstrated increased MCP-1 expression with the addition of ATF-uPA, which clearly suggests that uPA/uPAR interaction can indeed regulate MCP-1 expression. The cumulative increase in MCP-1 expression observed with the exogenous supplementation of ATF-uPA to irradiated cancer cells, when compared to cancer cells supplemented with either ATF-uPA or radiation alone, suggests that radiation-induced uPA might also play an important role in regulating MCP-1 expression. Further supporting evidence that the uPA/uPAR interaction regulates MCP-1 expression via ERK and NF-κB activation was provided by inhibiting the uPA/uPAR interaction using a natural competitor, suPAR. Adding suPAR to the medium along with ATF-uPA inhibited the phosphorylation of ERK, nuclear translocation of NF-κB and MCP-1 expression, which were induced by either ATF-uPA or radiation. Taken together, these results suggest that interaction of uPA with uPAR regulates MCP-1 expression.

The results of the present study demonstrate that radiation induces uPA expression, which elevates the availability of uPA to interact with uPAR. Further, radiation-induced uPA/uPAR interaction, as any other ligand/receptor interaction at the cell surface, leads to activation of the intracellular signaling pathway, which in the present case, activates ERK and nuclear translocation of NF-κB to regulate MCP-1 expression. Given the importance of uPA/uPAR in the induction of MCP-1 in meningioma cells, we next tried to knockdown uPA/uPAR by transfecting the cancer cells with plasmids expressing siRNA specific to uPA and uPAR. Various researchers have previously demonstrated that downregulation or inhibition of uPA/uPAR inhibited the expression of pro-angiogenic molecules [23,50,51]. The inhibition of MCP-1 (with and without radiation) in pU-transfected cancer cells demonstrates that targeting uPA and uPAR using siRNA-expressing plasmids is indeed effective. Further, the reduction in NF-κB DNA binding activity in pU-transfected cells suggests that downregulation of uPA/uPAR inhibited the transcriptional activity of nuclear translocated NF-κB (p65). The role of the uPA/uPAR system in angiogenesis is well established, and previous studies have shown that the tumor conditioned medium from uPA and uPAR-downregulated cancer cells reduced the potential to stimulate HMEC to form capillary network-like structures [23,51,52]. In the present study, we have shown that HMEC grown in tumor CM collected from pU-transfected meningioma cells reduced the potential to stimulate pro-angiogenic molecules like VEGF and angiogenin. Our in vivo results showed that radiation augmented the recruitment of endothelial cells and monocytes towards the tumor site. This cell recruitment was noticeably reduced when the tumors were treated with pU.

5. Conclusion

Overall, the results of our study demonstrate that downregulation of uPA/uPAR inhibited the secretion of angiogenic stimulators, like MCP-1, which in turn, inhibited the migration and secretion of pro-angiogenic molecules in HMEC. Previously, siRNA plasmids against uPA and uPAR have been shown to suppress tumor growth in experimental mice models. In the present study, we demonstrated that targeting uPA and uPAR not only inhibited established intracranial tumors but also radiation-induced recruitment of endothelial cells and monocytes towards the tumor region.

Supplementary Material

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Supplementary Figure legends

Supplementary Figure 1. Radiation induces MCP-1 expression in an ERK dependent and JNK independent pathway in meningioma cells. A. Meningioma cells were transfected with siRNA against ERK1 and incubated for 24 hrs. The transfected cells were irradiated and incubated for another 16 hrs in serum free media. Total cell lysates were analyzed by western blotting analysis to determine the basal and phosphorylated forms of ERK1/2. MCP-1 expression levels were determined in the culture supernatants by western blotting. B. Total cell lysates from meningioma cells transfected with JNK siRNA (with and without radiation treatment) were used to determine basal and phosphorylated JNK levels using specify antibodies. Secretory levels of MCP-1 in the culture supernatants were confirmed by western blot analysis. C. Further confirmation on independency of JNK pathways in MCP-1 expression in irradiated cells was provided by incubating meningioma cells with JNK inhibitor for 30 minutes prior to radiation. Total cell lysates from the above treated cancer cells were used to determine the basal and phosphorylated forms of JNK. MCP-1 expression was determined in the culture supernatants by western blot analysis. All the experiments were repeated for three times and the bar column represented mean S.D value, p<0.001.

Supplementary Figure 2. Radiation induces uPA expression and the interaction with its receptor (uPAR) activates ERK phosphorylation. A. IOMM Lee and SF 3061 cells were irradiated at two different dosages (5 and 10 Gy) and incubated overnight in serum free media. The enzymatic activity of uPA in the conditioned medium was determined by fibrinogen/plasminogen zymography. Total cell lysate was used to determine the levels of uPA, ERK and phosphorylated forms of ERK. B. IOMM Lee and SF3061 cells were pre-incubated with either uPAR blocking antibody or non-specific (NS) IgGs (negative control) prior to radiation treatment. After 2 hrs of incubation at 4° C, the cells were irradiated and incubated overnight. Basal and phosphorylated ERK levels were determine by western analysis of the total cell lysates. C. To demonstrate that ATF-uPA activates ERK phosphorylation, meningioma cells were incubated overnight in serum free medium supplemented with increasing concentrations of recombinant ATF-uPA. Total cell lysate was extracted and analyzed by western blotting analysis to determine the ERK and phosphorylated ERK levels.

Acknowledgements

We thank Noorjehan Ali for heir technical assistance in preparing the paraffin embedded brain section. We also thank Shellee Abraham for manuscript preparation, Diana Meister and Sushma Jasti for manuscript review.

This research was supported by a grant from N.I.N.D.S., NS061835 (to J.S.R.) The contents are solely the responsibility of the authors and do not necessarily represent the official views of NIH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Footnotes

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Associated Data

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Supplementary Materials

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NIHMS286967-supplement-01.tif (860KB, tif)
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NIHMS286967-supplement-02.tif (538.9KB, tif)
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Supplementary Figure legends

Supplementary Figure 1. Radiation induces MCP-1 expression in an ERK dependent and JNK independent pathway in meningioma cells. A. Meningioma cells were transfected with siRNA against ERK1 and incubated for 24 hrs. The transfected cells were irradiated and incubated for another 16 hrs in serum free media. Total cell lysates were analyzed by western blotting analysis to determine the basal and phosphorylated forms of ERK1/2. MCP-1 expression levels were determined in the culture supernatants by western blotting. B. Total cell lysates from meningioma cells transfected with JNK siRNA (with and without radiation treatment) were used to determine basal and phosphorylated JNK levels using specify antibodies. Secretory levels of MCP-1 in the culture supernatants were confirmed by western blot analysis. C. Further confirmation on independency of JNK pathways in MCP-1 expression in irradiated cells was provided by incubating meningioma cells with JNK inhibitor for 30 minutes prior to radiation. Total cell lysates from the above treated cancer cells were used to determine the basal and phosphorylated forms of JNK. MCP-1 expression was determined in the culture supernatants by western blot analysis. All the experiments were repeated for three times and the bar column represented mean S.D value, p<0.001.

Supplementary Figure 2. Radiation induces uPA expression and the interaction with its receptor (uPAR) activates ERK phosphorylation. A. IOMM Lee and SF 3061 cells were irradiated at two different dosages (5 and 10 Gy) and incubated overnight in serum free media. The enzymatic activity of uPA in the conditioned medium was determined by fibrinogen/plasminogen zymography. Total cell lysate was used to determine the levels of uPA, ERK and phosphorylated forms of ERK. B. IOMM Lee and SF3061 cells were pre-incubated with either uPAR blocking antibody or non-specific (NS) IgGs (negative control) prior to radiation treatment. After 2 hrs of incubation at 4° C, the cells were irradiated and incubated overnight. Basal and phosphorylated ERK levels were determine by western analysis of the total cell lysates. C. To demonstrate that ATF-uPA activates ERK phosphorylation, meningioma cells were incubated overnight in serum free medium supplemented with increasing concentrations of recombinant ATF-uPA. Total cell lysate was extracted and analyzed by western blotting analysis to determine the ERK and phosphorylated ERK levels.

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