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Published as doi: 10.1096/fj.07-9046com.
 (The FASEB Journal. 2008;22:355-364.)
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(The FASEB Journal. 2008;22:355-364.)
© 2008 FASEB

Rapid xenograft tumor progression in beta-arrestin1 transgenic mice due to enhanced tumor angiogenesis

Lin Zou*,{ddagger}, Rongxi Yang*,{dagger}, Jingjing Chai* and Gang Pei*,1

* Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences and

{dagger} Graduate School, Chinese Academy of Sciences, Shanghai, China; and

{ddagger} Clinical Molecular Medicine Center, Children Hospital, Chongqing University of Medical Sciences, Chongqing, China

1Correspondence: No. 320 Yueyang Rd., Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China. E-mail: gpei{at}sibs.ac.cn


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
β-arrestins (β-Arrs) are known to be associated with tumor signaling pathways such as transforming growth factor-β1 (TGF-β1), P53/Murine double minute (MDM2) and NF-{kappa}B. To investigate the role of β-Arr in tumor progression in vivo, we generated β-Arr transgenic mice by subcutaneously inoculating tumor cells in them. We found that the xenograft tumor initiated earlier and grew more rapidly in β-Arr1 transgenic mice than in both the β-Arr2 transgenic and wild-type mice after inoculating murine liver cancer Hepa1–6 cells or lymphoma EL4 cells. Moreover, matrix metalloproteinase 9 (MMP9) activity, vascular endothelial growth factor (VEGF) concentration in plasma and new small blood vessel formation in tumor tissues were enhanced in β-Arr1 transgenic mice compared with those in control mice. In addition, injection of MMP9 inhibitors in β-Arr1 transgenic mice abrogated all these effects and suppressed rapid tumor progression. Similar results were observed in human microvascular endothelial cells, where overexpressed β-Arr1 did increase MMP9 activity and small blood vessel formation. Furthermore, phosphatidylinositol 3-kinase (PI3K) inhibitors could suppress β-Arr1-enhanced MMP9 activity and the C-terminal 181–418 amino acids (aa) of β-Arr1 was largely responsible for this effect. Our data reveal a functional role for β-arrestin1 in tumor progression in vivo, in which overexpression of β-Arr1 promotes MMP9 activity and tumor angiogenesis by providing a suitable microenvironment for tumor progression.—Zou, L., Yang, R., Chai, J., Gang Pei. Rapid xenograft tumor progression in beta-arrestin1 transgenic mice due to enhanced tumor angiogenesis.


Key Words: matrix metalloproteinase 9 • MMP9 • vascular endothelial growth factor • VEGF • phosphatidylinositol 3-kinase • PI3K


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE ARRESTIN FAMILY HAS four members: β-arrestin1 (β-Arr1), β-arrestin2 (β-Arr2), x-arrestin, and s-arrestin (1) . Extensive studies have been performed on ubiquitously expressed β-Arr1 and β-Arr2, whereas x-arrestin and s-arrestin are found exclusively in the visual system. The classical functions of β-arrestins are to mediate desensitization, sequestration, and recycling of G protein-coupled receptors (GPCRs) (2) . Mounting evidence suggests that, in addition to regulation of GPCR signals, β-arrestins also serve as modulators in a number of intracellular signaling pathways, including extracellular regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK3), apoptosis-signal-regulating kinase 1 (ASK1), insulin-like growth factor I (IGF-1), phosphoinositide 3-kinase (PI3K-Akt), and wingless-type MMTV integration site family members 5A and 3A (Wnt5a and Wnt3A), which play important roles in the regulation of various cellular functions in both normal and malignant cells (3 4 5) . β-Arrs participate in some tumor-related signaling pathways such as p53/Murine double minute (MDM2), transforming growth factor β1 (TGF-β1), and insulin-like growth factor-1R (IGF1R) pathways, which function vitally as cell antiapoptosis, cell growth, and proliferation (6 7 8) . Recent reports have illustrated that the interaction of β-Arr1 and proto-oncogene tyrosine-protein kinase Src (c-Src) is critical for colorectal carcinoma cell migration (9) . Our recent research also showed that β-arrestins are involved in the NF-{kappa}B pathways and affect the secretion of cytokines, thus implying a potential role in providing a suitable microenvironment for tumor progression (10 , 11) .

Tumor growth and progression have been demonstrated to be caused by intracellular biochemical reactions combined with the influence of external factors (12) . On one hand, oncogenes and tumor suppressor genes play causal roles in the incidence, maintenance, and progression of diverse tumors (13 14 15) . On the other hand, the external factors for tumors mainly refer to the tumor microenvironment (equivalent to the "soil" in the Paget’s "seed and soil" malignant theory; 16 ). The influence of the tumor microenvironment has been the recent study of focus.

The tumor microenvironment consists of a plethora of soluble and cellular components that deliver signals to tumor cells, modulate their phenotype, and promote the progression of tumor cells (17) . Among the microenvironment components of malignant cells, the extracellular stroma and blood supply are considered to be crucial factors in tumor progression. The matrix metalloproteinase (MMP) family, which includes more than 20 Zn2+-dependent enzymes, degrades the extracellular matrix (ECM) and thereby contributes to tumor cell progression by effecting cell migration and angiogenesis (18 , 19) . MMP9, also known as gelatinase B or 92 kDa-type IV collagenase, not only participates in extracellular matrix degradation but also plays a role in angiogenesis (20) . Expression of this enzyme triggers a critical angiogenic switch during tumorigenesis by promoting the release of sequestered vascular endothelial growth factor (VEGF; 21 , 22 ). It has been reported that activation of MMP9 is correlated with PI3K signaling pathways in human epithelial cells (23 , 24) . Because these pathways could be regulated by β-Arr1, the potential relationship between β-Arr1 and MMP9 warrants investigation.

Although β-arrestins can transduce multiple tumor-related signals in cells, little is known about their participation in the modulation of the tumor microenvironment in mammals. Here we showed that rapid xenograft tumor progression in β-Arr1 transgenic mice by enhancing tumor angiogenesis and PI3K inhibitors suppressed the β-Arr1-elevated MMP9 activity and VEGF. Enhanced MMP9 activity and VEGF secretion might provide an adequate microenvironment for rapid tumor growth, suggesting the important role of β-Arr1 in tumor progression in vivo.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Polyclone antibody of β-Arr was a gift from Dr. Robert J. Lefkowitz (Duke University, Durham, NC, USA), and β-arrestin monoclonal antibody was purchased from BD Biosciences (San Jose, CA, USA). Antibodies against MMP9, MMP2, and VEGF were obtained from Chemicon (Temecula, CA, USA). Antibodies against HA (rabbit), actin (rabbit), DAPI, and HA-conjugated FITC antibody, LY294002, wortmannin, SB203580, and PD98059 were purchased from Sigma (St. Louis, MO, USA), and LH39 antibody from Lab Vision (Fremont, CA, USA). IRDyeTM800CW conjugated affinity-purified anti-mouse IgG and anti-rabbit IgG were purchased from Rockland Inc. (Gilbertsville, PA, USA).

Plasmids
3'HA β-Arr1, 3'HA β-Arr2, 3'HA β-Arr1 1–180, 181–418, 181–418, and 318–418 were constructed as described (10) . 3'HA β-Arr1 1–240, 1–320, and 241–418 were constructed per a similar strategy.

Cell culture, transfection, and treatment
Murine hepatocellular carcinoma (Hepa1–6), mouse lymphoma (EL4), and human microvascular cell line (HMEC-1) cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Hepa1–6 and EL4 cells were maintained in DMEM medium (Gibco BRL, Gaithersburg, MD, USA); HMEC-1 cells, in MCDB-131 medium (Sigma) with 10% FBS. The cells were cultured in 37°C, 5% CO2 incubator. HMEC-1 cells were transfected by Lipofectamine (Invitrogen, Carlsbad, CA, USA). After transfection for 48 h, HMEC-1 cells were treated with different inhibitors for the indicated time.

Generation of β-Arr transgenic mice
C57BL/6J mice carrying β-Arr1/2 cDNA were constructed as previously reported (23) and identified (unpublished data). Human β-Arr1/2 cDNA containing whole mRNA coding region, which was screened from the brain cDNA/{lambda} phage library, was cloned into pcDNA3 (BamHI-EcoRI) with HA tag (HindIII-BamHI) under the control of human cytomegalovirus (CMV) promoter. DNA constructions were microinjected into the fertilized eggs of C57BL/6J mice. PCR amplification and Southern blot hybridization of DNA extracted from tail biopsies verified the integration of variable copy numbers of transgene into the genomes of founder mice and their offspring. Real-time PCR analysis of mRNA samples from tissues verified the expression of transgene. Age- and sex-matched littermates were served as controls. The genomic DNA primers to identify transgenic mice were β-Arr1, sense 5'-CCTGGATGTCTTGGGTCTG-3', antisense 5'-TGATGGGTTCTCCGTGGTA-3', β-Arr2, sense 5'-CAGCCAGGACCAGAGGACA-3', antisense 5'-TGATAAGCCGCACAGAGTT-3'. All mice were housed in groups (<4 per cage) in temperature- and humidity-controlled environments with a 12-h light/12-h dark rhythm and were maintained in clean and comfortable animal rooms at the animal facility of the Shanghai Laboratory Animal Center (Shanghai, China). Mice had free access to water and a standard laboratory diet (provided by Shanghai SLAC Laboratory Animal Company, Shanghai, China).

Xenograft tumor model establishment
β-Arr transgenic mice and their littermates, 6- to 8-wk-old mice, were inoculated with 1–10 x 106 Hepa1–6 or EL4 cells (both are murine origin) at the left flank s.c. For MMP inhibitors, different groups of mice were inoculated with 2 x 106 Hepa1–6 cells at the left flank and injected with 10 µM GM6001 or 0.5 µM MMP9 specific inhibitor I (Calbiochem, San Diego, CA, USA) every 48 h at another flank, with DMSO as the control. Tumor volume was measured dynamically. At 28 days after inoculation, the mice were sacrificed and their blood, tumor, and normal tissues were collected.

Leukocyte extraction
The peripheral blood was extracted from tail veins or eyes and then centrifuged at 2000 rpm for 5 min. Cells were colleted and resuspended twice in 1 ml RBC lysis buffer (10 mM KHCO3, 0.1 M NH4Cl, 37 mg EDTA-Na2) at 4°C for 5 min, then centrifuged at 2000 rpm for 5 min. The main components in the final pellets were leukocytes.

Western blotting
Leukocytes or tissue proteins were lysed by RIPA buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1% Nonidet P-40; 0.1% SDS; and 0.5% sodium deoxycholate and boiled for 10 min in sample buffer (62.5 mM Tris-HCl, pH 6.8; 10% glycerol; 2% SDS; and 50 mM dithiothreitol). Equivalent amounts of protein were subjected to SDS-PAGE electrophoresis and then electroblotted onto nitrocellulose membrane. The membrane was incubated with primary antibody and then IRDye 800CW-conjugated secondary antibody, and the infrared fluorescence image was obtained using Odyssey infrared imaging system (Li-Cor Bioscience, Lincoln, NE, USA).

Immunoprecipitation
Tissue samples were lysed in buffer containing 1% Triton X-100, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 150 mM NaCl, 0.2 mM sodium orthovandate, 0.2 mM PMSF, 1 mM EGTA (pH 8.0), and a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA). After 15,000 g x 10 min centrifugation, the supernatant was precleared by protein G Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and then incubated at 4°C with the indicated antibodies for 12 h. The immunocomplexes were captured by rotating for 1 h with protein G Sepharose, washed four times. The immunocomplexes Sepharose G were incubated in the sample buffer at 50°C for 20 min, and the supernatants were collected.

Real-time PCR
Total RNAs were extracted from tissues with TRIzol (Invitrogen) according to the manufacturer’s instructions. Reverse transcription of purified RNA was performed using oligo (dT) primer and superscript II reverse transcriptase (Invitrogen). Then quantification of the genes was performed by real-time PCR, using Brilliant SYBR Green QPCR Master Mix (Stratagene, Garden Grove, CA, USA). Expression values were normalized to those obtained with control Actb (encoding β-actin). The primer pairs are as follows: VEGF, sense 5'-CTGCCGTCCGATTGAGACC-3', antisense 5'-CCCCTCCTTGTACCACTGTC-3'; β-actin, sense 5'-AAGTCCCTCACCCTCCCAAAAG-3', antisense 5'-AAGCAATGCTGTCACCTTCCC-3'.

SDS-PAGE substrate zymography
Plasma or accumulated culture medium was collected and sample buffer was added (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS). The samples were incubated at 37°C for 45 min. Equivalent amounts of protein were analyzed by gelatin zymography on 10% SDS-PAGE gels copolymerized with substrate (1 mg/ml gelatin). After electrophoresis, gels were washed twice for 45 min in washing buffer containing 2.5% Triton X-100 (50 mM Tris-HCl, 5 mM CaCl2, 1 µM ZnCl2, pH 7.6), then washed twice by washing buffer without Triton X-100 twice for 20 min each, incubated at 37°C for 42 h in developing buffer (50 mM Tris-HCl, 5 mM CaCl2, 1 µM ZnCl2, 0.02% Brij35, pH 7.6), and then stained with 0.5% Coomassie blue R-250 and destained with 30% methanol, 10% acetic acid. The locations of active proteinase bands were determined by the presence of negative staining.

Immunofluorescence microscopy
Tumor or liver tissues were fixed with 4% paraformaldehyde (PFA) -PBS (pH 7.4) at 4°C for 3 h, then cryoprotected in 30% sucrose at 4°C overnight and rapidly frozen in liquid nitrogen. Frozen sections (12 µm) were cut on a cryostat with OCT embedding medium. Sections were incubated in a blocking buffer (0.5% Triton X-100, 1% BSA-PBS) for 1 h at room temperature followed by incubation with 1:100 mouse HA-conjugated FITC antibody or 1:100 mouse anti-LH39 (marker for new small blood vessel) at 4°C overnight. They were then incubated with antibody conjugated with fluorophores at room temperature for 1 h, next to 5 mg/ml DAPI for 10 min for nuclear staining. The fluorescence signals were observed under a TCS NT laser confocal microscope (Leica Microsystems, Bensheim, Germany).

ELISA for VEGF concentration
The secreted concentration of VEGF in mouse plasma was determined by a "sandwich" ELISA kit (R&D Systems, San Diego, CA, USA) according to the manufacturer’s instructions. Each assay was tested in triplicate, and the results shown were from three independent experiments.

Gelatinase activity assay
Gelatinase activity in plasma and cell culture media was determined by an MMP Gelatinase Activity Assay Kit (ECM700, Chemicon), according to the protocol. Each assay condition was tested in triplicate and the results shown were from three independent experiments.

Blood tube formation assay
In vitro angiogenesis assay was followed by an Endothelia Tube Formation Assay (CBA200, Cell Biolabs Inc., San Diego, CA, USA). The ECM gel was thawed at 4°C and mixed to homogeneity using cooled pipette tips. Cell culture plates (96-well) were bottom-coated with a thin layer of ECM gel (50 µl/well), which was left to polymerize at 37°C for 60 min. HMEC-1 (5x104) in 150 µl medium was added to each well on solidified ECM gel. The plate was incubated at 37°C for 6 h, and the endothelial tubes were observed using a light microscope. Three microscopic fields were selected at random and photographed (25) . Tube formation ability was quantified by counting the total number of cell clusters and branching under three x4 fields per well. The results were expressed as mean folds of branching compared with the control groups. Each experiment was performed at least three times.

Statistical analysis
Data are presented as means ± SE. Student’s t test was used to compare two independent groups. For all tests, a value of P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of β-Arr transgenic mice
A linearized DNA construct carrying human β-Arr1 and β-Arr2 with a hemagglutinin (HA) tag under the control of CMV promoter was introduced into the C57BL/6J mouse germ line by microinjection (Fig. 1 A), according to the published procedures (26) . The insertion and integrity of the transgenic fragments were characterized by genomic DNA polymerase chain reaction (PCR) and Southern blot (data not shown). The mRNA levels of β-Arr1 and β-Arr2 were 6.38- and 6.74-fold higher than their respective littermates, tested by real-time PCR (Fig. 1B ). The protein expression of transgenes was detected by immunoprecipitation from liver tissues and showed high expression of β-Arr1 and β-Arr2 in their respective transgenic groups (Fig. 1C ). The expression and distribution of β-Arr were also observed by immunofluorescence (Fig. 1D ) in the liver. Both β-Arr1 and β-Arr2 were distributed mainly in the cytoplasma, and a small amount of nuclear β-Arr1 was detected also (data not shown). These results demonstrated a successful insertion and indicated the accurate expression of human β-Arr1 and β-Arr2 in the transgenic mice.


Figure 1
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  		Figure 1. Identification of β-arrestin transgenic mice. A) Schematic illustration of the β-arrestin transgenic vector construction. B) Real-time PCR identification of β-arrestin expression in transgenic mice. Data are represented as the fold of β-Arr/β-actin mRNA level in liver tissue (n=6). Each experiment was performed in triplicate, and means ± SE were obtained. C) Immunoprecipitation identification of β-Arr expression. Mouse liver was lysed and immunoprecipitated with anti-HA, then immunoblotted with anti-β-Arr. Two representative samples from each group are shown (n=6). D) Representative images from different mouse groups of frozen liver sections stained with HA conjugated FITC antibody. Scale bar = 40 µm.

Rapid xenograft tumor progression in β-Arr1 transgenic mice
To characterize the influence of β-Arr on xenograft tumor growth, murine Hepa1–6 hepatoma cells (27) were inoculated into the 6- to 8-wk-old male transgenic mice and their wild-type (WT) littermates. To obtain the optimal injection cell amount, we inoculated Hepa1–6 cells subcutaneously (s.c.) into C57BL/6J mice from 1 x 106 to 10 x 106 for each mouse. We found that 2 x 106 could generate a macroscopic tumor nodule after an injection period of 7–10 days, as previously reported (28) . After injection of the same amount of cells, the time of emergence of the first macroscopic tumor nodule (tumor initiation time) was observed. The results showed that the macroscopic tumor nodule in β-Arr1 transgenic mice (4.28±0.29 days) appeared much earlier than in β-Arr2 transgenic and WT mice (11.75±1.65 and 8.80±1.43 days, respectively; Fig. 2 A). To exclude the possibility that the shorter tumor initiation time was caused by gene insertion in a definite chromosome, we inoculated the same amount of Hepa1–6 cells into another founder of β-Arr transgenic mice and obtained similar results (Fig. 2B ). Further observation showed that the xenograft tumors in β-Arr1 transgenic mice grew more rapidly; the tumor volume was much larger in β-Arr1 transgenic mice than in β-Arr2 transgenic and WT mice in both founders after a 28-day injection period (Fig. 2C, D ). To confirm the result, we also injected another tumor cell line, mouse lymphoma EL4 cells, into these mice and found a similar shorter tumor initiation time and faster tumor growth (Supplemental Fig. 1A, B) in β-Arr1 transgenic mice than in β-Arr2 transgenic and WT mice. Thus, our results suggested that overexpression of β-Arr1 provides an appropriate microenvironment for tumor progression.


Figure 2
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  		Figure 2. Rapid tumor growth in β-Arr1 transgenic mice. A, B) The time of first emergence of macroscopic tumor nodules (tumor initiation time) after mice were inoculated with 2 x 106 Hepa1–6 cells. {circ} = individual tumor initiation time of each mouse; {blacktriangleup} or • = average tumor initiation time of each group. Data are presented as means ± SE. A) Founder 148 (148F) of β-Arr1 transgenic mice and founder 67 (67F) of β-Arr2 transgenic mice. B) Founder 152 (152F) of β-Arr1 transgenic mice and founder 86 (86F) of β-Arr2 transgenic mice. β-Arr1-Tg, β-Arr1 transgenic mice; β-Arr2-Tg, β-Arr2 transgenic mice; WT, wild-type mice. n = 10. C, D) Tumor dynamic growth curves in the same mice as A and B, respectively. *P < 0.05 vs. corresponding WT.

Increased tumor small blood vessels and secreted VEGF concentration in β-Arr1 transgenic mice
Considering that the optimal environment for tumor growth is dependent on the blood supply and ample nutrition, we examined new small blood vessels in tumor tissues and found many more small blood vessels in tumors from β-Arr1 transgenic mice than β-Arr2 transgenic and WT mice (Fig. 3 A), supporting our hypothesis that an adequate blood supply promotes tumor progression in β-Arr1 transgenic mice. Because the secreted VEGF, the activated form of VEGF in plasma, is believed to be the key factor in tumor angiogenesis, we further detected VEGF mRNA expression and secreted VEGF concentration in plasma by real-time PCR and ELISA, respectively (29 , 30) . The results showed that the VEGF mRNA level was not obviously changed before and after tumor cell inoculation (Fig. 3C ) in both transgenic and WT mice. In contrast to its expression, the secreted VEGF concentration in plasma was evidently increased in β-Arr1 transgenic mice after tumor cell injection (Fig. 3B ), indicating that tumor angiogenesis is more abundant in β-Arr1 transgenic mice. It was recently reported that VEGF could induce the distribution alteration of β-Arr2 (31) . We then observed the subcellular distribution of beta-arrestin in the healthy and tumor-inoculated transgenic mice. The confocal microscopy analysis showed that both β-Arr1 and β-Arr2 were found mainly in the cytoplasma and that a small amount of nuclear β-Arr1 was found in the transgenic mice with or without inoculation of tumor cells (data not shown).


Figure 3
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  		Figure 3. Increased tumor new small blood vessel formation and VEGF concentration in β-Arr1 transgenic mice. A) Representative images of frozen sections from liver (normal) and tumor tissues double-stained with 1:100 anti-LH39 (green) for new small blood vessels and 5 µg/ml DAPI (red) for nuclear staining. Scale bar = 40 µm. B) Secreted VEGF concentration in plasma was tested with ELISA. Data were represented as fold of β-Arr-Tg/normal WT mice. The VEGF level of WT in normal mice group was considered to be 1.0. C) Assessment of VEGF mRNA levels in liver (normal) or tumor tissue was performed by real-time PCR. The data (VEGF/β-actin mRNA) are represented as the fold of normal WT mice. The level of WT in normal (control) mice group was set as 1.0. Each experiment was performed in triplicate, and means ± SE were obtained; n = 6; *P < 0.05 vs. WT tumor mice.

Enhanced MMP9 activity in β-Arr1 transgenic mice
It has been reported that MMP9 releases matrix-sequestered VEGF without affecting VEGF expression (21 , 22) and that β-Arr regulating PI3K signaling pathways are involved in the activation of MMP9 (32) . We therefore examined MMP9 expression and activity in these transgenic mice. In contrast to β-Arr2 transgenic and WT mice, the expression of activated MMP9 in the leukocytes and the MMP9 mRNA level in the liver were increased in β-Arr1 transgenic mice after inoculation of tumor cells (Fig. 4 A, D). Consistently, the classical SDS-PAGE substrate zymography and gelatinase activity all showed higher levels of MMP9 activity in the plasma from β-Arr1 transgenic mice than from β-Arr2 transgenic or WT mice after inoculation of tumor cells (Fig. 4B ), whereas no obvious difference in MMP2 activity was detected (Fig. 4A, B ). Collectively, all the data from distinct assays demonstrated the enhanced MMP9 activity in β-Arr1 transgenic tumor-inoculated mice. To block the rapid tumor progression in β-Arr1 transgenic mice, we injected MMP9 inhibitors into transgenic and WT mice s.c. while inoculating tumor cells. The results illustrated that not only the broad-spectrum MMP family inhibitor GM6001 but also MMP9-specific inhibitor I suppressed the rapid xenograft tumor progression, especially in β-Arr1 transgenic mice (Fig. 5 A). Decreases in small blood vessel formation (Fig. 5B ), VEGF concentration (Fig. 5C ), and MMP9 activity (Fig. 5D ) in β-Arr1 transgenic mice were also observed after the injection of MMP9 inhibitors, suggesting the pivotal roles of MMP9 in β-Arr1-related tumor progression.


Figure 4
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  		Figure 4. Enhanced MMP9 activity in β-Arr1 transgenic mice. A) Expression of activated MMP9 in leukocytes. The samples were immunoblotted with 1:500 anti-MMP9 and 1:500 anti-MMP2, and β-actin as the loading control. Four representative samples were taken from each group (n=6). B) Representative images of MMP9 activity in plasma by SDS-PAGE substrate zymography and four representative samples from each group (n=6). C) Gelatinase activity in plasma was tested by gelatinase activity assay. Data are represented as fold of β-Arr-Tg/normal WT mice. The level of WT in normal mice was considered to be 1.0. D) Tissues were collected from liver (normal mice) or tumor (tumor-inoculated mice), RNA was purified, and then MMP9 mRNA expression was determined by real-time PCR. The mRNA level of WT in normal WT mice was set as 1.0. Each experiment was performed in triplicate, and means ± SE were obtained; n = 6; *P < 0.05 vs. WT tumor mice.


Figure 5
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  		Figure 5. Blockage of rapid tumor growth by MMP9 inhibitors in β-Arr1 transgenic mice. A) Xenograft tumor inoculation as in Materials and Methods; 10 µM GM6001 (left panel) or 0.5 µM MMP9 specific inhibitor I (right panel) was injected s.c. in another flank every 48 h. β-Arr1 transgenic mice injected with DMSO were used as the control. B) Representative images of frozen sections of tumor tissue from mice in A after injection at 28 days, immunostained with 1:100 anti-LH39 (green) and 5 µg/ml DAPI (red). Scale bar = 40 µm. C) Plasma was collected from mice in A, and VEGF concentration was detected with ELISA. The data are presented as the fold of different treatment groups/β-Arr1-Tg-DMSO mice. D) Top panel; MMP9 activity in plasma (same mice as in A) by SDS-PAGE substrate zymography. Three representative samples from each group (n=6) are shown. Bottom panel: gelatinase activity in plasma (same mice as in C). Data are presented as the fold of different treatment groups/β-Arr1-Tg-DMSO mice. Each experiment was performed in triplicate, and means ± SE were obtained.

PI3K signaling pathways involved in β-Arr1-enhanced MMP9 activity in HMEC-1 cells
To further explore the potential molecular mechanism of enhanced MMP9 activity in β-Arr1 transgenic tumor-inoculated mice, the relationship between β-Arr and MMP9 in HMEC-1 cells (33) was investigated. The results showed that only the overexpression of HA-β-Arr1 could cause the enhancement of MMP9 activity as measured by zymography (Fig. 6 A, top panel) and gelatinase activity (Fig. 6A , bottom panel), with subsequent increased small tube formation (Fig. 6B, C ). To explain the signaling pathways involved in the β-Arr1-enhanced MMP9 activity and tube formation, selective small molecule inhibitors were applied. The results showed that both wortmannin and LY294002, the inhibitors of PI3K, but not SB203580 (inhibitor of p38 MAPK) and PD98059 (MEK inhibitor, data not shown), could suppress the increase of MMP9 activity by β-Arr1 in both zymography and gelatinase activity (Fig. 6C ). Wortmannin and LY294002 could also slow down β-Arr1-accelerated small tube formation in HMEC-1 cells (Fig. 6D, E ). Together with the previous report that β-Arr1 could regulate and activate PI3K signals, we may conclude that PI3K signaling pathways are involved in the β-Arr1-enhanced MMP9 activity and angiogenesis.


Figure 6
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  		Figure 6. PI3K involved in β-Arr1-enhancing MMP9 activity and new small blood vessel formation in HMEC-1 cells. A) Top panel: representative images of MMP9 activity in culture medium of different transfected HMEC-1 cells by SDS-PAGE substrate zymography. Bottom panel: gelatinase activity of the same sample; the data are represented as the fold of different transfected cells/β-gal transfected cells. *P < 0.05 vs. cells transfected with β-gal. B) Top panel: representative images of tube formation of transfected HMEC-1 cells; 5 x 104 cells were seeded in each ECM gel-covered well and photographed 6 h later. Scale bar = 100 µm. Bottom panel: the quantified analysis of tube formation of captured images was performed by counting the total number of cell clusters and branching under three x4 fields per well. The results were expressed as mean folds of branching compared with the control groups. C) HMEC-1 cells were transfected with HA-β-Arr1, 48 h later, wortmannin (10 µM), LY294002 (100 µM), or SB203580 (10 µM) treated cells for 30 min. Then the culture media was accumulated for zymography (top panel) and gelatinase activity (bottom panel), and the cells were harvested for detection of β-Arr1 expression by Western blot with anti-HA (middle panel). DMSO was used as the control. *P < 0.05 vs. cells treated with DMSO. D) Representative images (top panel) and quantified analysis (bottom panel) of tube formation in the HMEC-1 cells treated as described in C. Scale bar = 100 µm.

The pivotal role of the C-terminal 181–418 amino acids (aa) of β-Arr1 in enhancing MMP9 activity and angiogenesis
To map the functional region of β-Arr1 in enhancing MMP9 activity and small blood tube formation, we examined the effects of a series of β-Arr1 truncations. With successful transfection in HMEC-1 cells, we found that only the C-terminal truncation (181–418 aa) of β-Arr1 completely maintained the function that HA-β-Arr1 has in enhancing MMP9 activity and gelatinase activity (Supplemental Fig. 2A ), while the N-terminal truncations of β-Arr1 (consisting of 1–180, 1–240, and 1–320 aa) and partial C-terminal truncations (consisting of 241–418, 318–418 aa) had no such effect (Supplemental Fig. 2B, C ). The results of small blood tube formation in vitro of different truncations also demonstrated that C-terminal 181–418 aa of β-Arr1 was the only one with the full function, implying that C-terminal truncation (181–418 aa) of β-Arr1 is critical for enhancing MMP9 activity and angiogenesis.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Tumor progression is the result of both the malignant cells themselves and the effect of the surrounding microenvironment (34) . Here we found that the rapid xenograft tumor progression in β-Arr1 transgenic mice is due to enhanced MMP9 activity, VEGF secretion, and subsequent increase in tumor angiogenesis. To further verify these results of the xenograft tumor growth in an overexpression microenvironment, we overexpressed β-arrestins in the whole body or in local sites by injection of adenovirus expression vectors carrying β-Arr into the local sites or tail veins of C57BL/6J mice. We observed rapid tumor progression in mice both in local site injection (Supplemental Fig. 1C ) and in whole-body vein injection (Supplemental Fig. 1D ), which was similar to the β-Arr1 transgenic mice but with weaker effect. This finding suggested that overexpression of β-Arr1 provided an appropriate microenvironment for xenograft tumor progression. In combination with previous reports that β-arrestins participate in cell antiapoptosis and chemotaxis (35 , 36) , we hypothesize that overexpression of β-Arr1 provides a suitable microenvironment for tumor progression.

In agreement with our own and other published results, β-Arr1 likely affects tumor progression by modulating multiple factors. First, after inoculation of tumor cells in mice, MMP9 activity can be enhanced in tumor cells as well as the surrounding stromal cells, such as fibroblasts, infiltrating macrophages, and endothelial cells (37) . Communication might exist between tumor cells and host-derived cells to activate MMP9, which has been shown to play a critical role in angiogenesis and progressive growth of human tumors (38) . The enhanced MMP9 activity may accelerate not only extracellular matrix degradation (18) but also tumor angiogenesis (19) to provide an appropriate microenvironment for tumor progression. Second, the increased VEGF secretion and new small blood vessel formation in β-Arr1 transgenic mice may provide more blood supply and nutrition for tumor progression (39) . Third, it has been found that β-Arr1 could induce cell growth through activation of PI3K/Akt, MDM2, or ERK signaling pathways (8 , 40 , 41) . In addition, stimulation of various 7TMRs in the absence of β-Arr could cause cell apoptosis in mouse embryonic fibroblasts, indicating the role of β-Arr role in antiapoptotic signaling (35) . β-Arr1 was also found to accelerate tumor metastasis in colorectal cancer cells by interacting with c-src (9) . Moreover, our unpublished results hinted that overexpression of β-Arr1 in HMEC-1 cells could promote cell invasion. These above-mentioned factors probably work together to provide a suitable microenvironment for rapid tumor progression in β-Arr1 transgenic mice.

Although the molecular mechanism of β-Arr1 regulation of tumor progression needs to be studied further, our data provide convincing evidence that overexpression of β-Arr1 could promote this progression. The functional difference between β-Arr1 and β-Arr2 may contribute to this divergence. β-Arr1, but not β-Arr2, has been shown to transduce PI3K and PAR2 tumor-related signals (41 42 43) to affect tumor growth. Here we also suggest that PI3K signaling pathways were involved in β-Arr1-induced MMP9 activity and subsequent blood vessel formation. Furthermore, β-arrestin1 is distributed in the nucleus on stimulation (44) and processes nuclear function to modulate the transcription and expression of some specific genes (45) . These differences between β-arrestin family members indicate their diverse effects on the regulation of microenvironment and tumor progression. In conclusion, β-arrestin1 could speed up tumor progression in mammals by enhancing tumor angiogenesis, and PI3K signaling pathways are involved in this enhancement. This finding suggests that overexpressing β-Arr1 mice might be a potential model for future studies on tumor microenvironment.


   ACKNOWLEDGMENTS
 
We thank K. J. Andersen, R. J. Lee, J. H. Kang, J. Kim, Y. Daaka, and N. Frilot for manuscript revision, and L. Teng, L. Shen, Y. Shi, S. M. Xin, and Y. L. Wu for technical assistance. This research was supported by grants from the Ministry of Science and Technology (2003CB515405, 2005CB522406), the National Natural Science Foundation of China (30621091, 30625014, 30400230, 30400166 and 30400539), the Shanghai Municipal Commission for Science and Technology (06ZR14098), the Chinese Academy of Sciences (KSCX2-YW-R-56), and the China and Shanghai Postdoctoral Science Foundation.

Received for publication June 1, 2007. Accepted for publication August 23, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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