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Occurrence of cGMP/nitric oxide-sensitive store-operated calcium entry in fibroblasts and its effect on matrix metalloproteinase secretion.

AIM
To examine the existence of Nitric oxide/cGMP sensitive store-operated Ca(2+) entry in mouse fibroblast NIH/3T3 cells and its influence on matrix metalloproteinase (MMP) production and adhesion ability of fibroblasts.


METHODS
NIH/3T3 cells were cultured. Confocal laser scanning microscopy was used to examine the existence of thapsigargin-induced store-operated Ca(2+) entry in fibroblasts. Gelatin zymography and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) were employed to detect the involvement of [Ca(2+)]i and NO/cGMP in MMP secretion. The involvement of NO/cGMP-sensitive Ca(2+) entry in adhesion was determined using matrigel-coated culture plates.


RESULTS
8-bromo-cGMP inhibited the thapsigargin-induced Ca(2+) entry in 3T3 cells. The cGMP-induced inhibition was abolished by an inhibitor of protein kinase G, KT5823 (1 micromol/L). A similar effect on the Ca(2+) entry was observed in 3T3 cells in response to a NO donor, (+/-)-S-nitroso-N-acetylpenicillamine (SNAP). The inhibitory effect of SNAP on the thapsigargin-induced Ca(2+) entry was also observed, indicating NO/cGMP-regulated Ca(2+) entry in 3T3 cells. Results of gelatin zymography assay showed that addition of extracellular Ca(2+) concentration induced MMP release and activation in a dose-dependent manner. RT-PCR also showed that cGMP and SNAP reduced the production of MMP mRNA in 3T3 cells. Experiments investigating adhesion potentials demonstrated that cGMP and SNAP could upgrade 3T3 cell attachment rate to the matrigel-coated culture plates.


CONCLUSION
NO/cGMP sensitive store-operated Ca(2+) entry occurs in fibroblasts, and attenuates their adhesion potentials through its influence on MMP secretion.


INTRODUCTION
Members of matrix metalloproteinase (MMP) family have been broadly implicated in both physiological and pathophysiological tissue remodeling [1] , and play a key role in tumor invasion and metastasis. Since degradation of extracellular matrix (ECM) components by MMP is critical for tumor cell invasion and metastasis [2,3] , MMP-2 degrades type Ⅳ collagen, a major component of the basement membrane because of its activity. Recent studies have revealed that tumor cells utilize MMP-2 produced by neighboring stromal cells including fibroblasts rather than by tumor cells themselves [4,5] for tumor progression, invasion, and metastasis, and that tumor cells can stimulate MMP production by stromal cells via soluble factors such as cytokines [6][7][8][9] or through cell-cell interaction mediated by cell adhesion molecules such as CD147 [10,11] .
Studies have proved that stromal cells mainly release MMP around tumor cells, and that calcium is a second intracellular messenger which mediates a wide range of cellular responses. These studies aroused our interest in whether calcium ions are involved in MMP secretion. In non-excitable cells, the influx of Ca 2+ is a biphasic process, which consists of an initial transient phase followed by a large and sustained phase. Under Ca 2+ -free conditions, the response is only a small and transient rise in [Ca 2+ ]i, which should reflect the release of intracellular Ca 2+ stores. When extracellular Ca 2+ is reintroduced in the absence of the agonist, there is a large rise in [Ca 2+ ]i. Since the depletion of intracellular Ca 2+ stores is apparently the single Occurrence of cGMP/nitric oxide-sensitive store-operated c a l c i u m e n t r y i n f i b r o b l a s t s a n d i t s e f f e c t o n m a t r i x metalloproteinase secretion METHODS: NIH/3T3 cells were cultured. Confocal laser scanning microscopy was used to examine the existence of thapsigargin-induced store-operated Ca 2+ entry in fibroblasts. Gelatin zymography and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) were employed to detect the involvement of [Ca 2+ ]i and NO/cGMP in MMP secretion. The involvement of NO/ cGMP-sensitive Ca 2+ entry in adhesion was determined using matrigel-coated culture plates.
RESULTS: 8-bromo-cGMP inhibited the thapsigargininduced Ca 2+ entry in 3T3 cells. The cGMP-induced inhibition was abolished by an inhibitor of protein kinase G, KT5823 (1µmol/L). A similar effect on the Ca 2+ entry was observed in 3T3 cells in response to a NO donor, (±)-S -nitroso-N -acetylpenicillamine (SNAP). The inhibitory effect of SNAP on the thapsigargin-induced Ca 2+ entry was also observed, indicating NO/cGMPregulated Ca 2+ entry in 3T3 cells. Results of gelatin zymography assay showed that addition of extracellular Ca 2+ concentration induced MMP release and activation in a dose-dependent manner. RT-PCR also showed that cGMP and SNAP reduced the production of MMP mRNA in 3T3 cells. Experiments investigating adhesion potentials demonstrated that cGMP and SNAP could upgrade 3T3 cell attachment rate to the matrigel-coated culture plates. mechanism at work under such a circumstance, it has been hypothesized that the Ca 2+ entry is activated by the depletion of Ca 2+ stores and this Ca 2+ entry is thus called store-operated Ca 2+ entry (SOC) [12] .
Nitric oxide (NO) is also known to inhibit Ca 2+ entry through L-type Ca 2+ channels (LTCC) in SMCs via cGMPdependent mechanisms or via membrane hyperpolarization due to cGMP-dependent activation of Ca 2+ -dependent K + channels. Besides the direct effects of NO on Ca 2+ entry, it can also activate heme-containing soluble guanylyl cyclase (sGC) and catalyze the production of cGMP from GTP, thus playing a role in NO/cGMP/protein kinase G (PKG) pathway. Although many reports have described the regulating mechanisms on the calcium channels, they all focused on the end result--increase in [Ca 2+ ]i. Thus further investigation is certainly required to clarify the role of NO/cGMP in SOC and MMP secretion.
The aim of the present study was to demonstrate the existence of NO/cGMP-regulated SOC in NIH/3T3 cells and to further explore the influence of store-operated Ca 2+ entry on MMP secretion and adhesion potential in NIH/3T3 cells.

Measurement of [Ca2+]i in single cells by confocal laser scanning microscopy
After an overnight attachment, the cells were rinsed in 0.01 mol/L PBS and loaded with 5 µmol/L Fluo3/AM for 45 min in dark at 37℃ in normal PBS (NPBS) containing 2 mmol/L CaCl2, pH 7.4. The cells were then washed and resuspended in NPBS. To start the experiment, the cells were pretreated with 4 µmol/L thapsigargin for 2 min. Then the cells were washed with and maintained briefly in PBS containing no Ca 2+ and 2 mmol/L EGTA. Unless stated otherwise, the cells were pretreated with or without chemicals (i.e. 8-bromo-cGMP, KT5823, SNAP, L-NAME or NiCl2) for 1-2 min. The fluorescent signal of cytoplasmic calcium ion concentration in 3T3 cells was determined by fluorescence using Bio-Rad MRC 1024 visible light CLSM (Bio-Rad, Hercules, CA) and argon (488/526 nm) laser light.

Gelatin zymography
The conditioned media were collected by centrifugation, concentrated and dialyzed. T he dialyzed samples containing an equal amount (20 µg) of total protein were mixed with the sample buffer, incubated in water bath (about 55℃) for 3-5 min and loaded onto the zymographic 100 g/L SDS-polyacrylamide gel containing gelatin (1g/L) and run under standard conditions (2 h at constant voltage of 100V). Afterwards, the gels were washed once with 50 mmol/L Tris (pH 7.4), containing 25 mL/L Triton X-100 for 30 min, and twice with 50 mmol/L Tris (pH 7.4). Gels were then incubated for 16-18 h in 50 mmol/L Tris (pH 7.5), 0.15 NaCl, 10 mmol/L CaCl2, 1 mL/L Triton X-100 and 0.2 mg/L sodium azide. Finally, the gels were stained with Coomassie blue and washed with 75 mL/L acetic acid containing 200 mL/L methanol. The gels were subjected to densitometric analysis to quantitate the gelatinase activity by obtaining volumograms on a photo documentation system from UVItec (Cambridge, UK) using the UVIchrom acquisition software.

Semiquantitative reverse transcriptase-polymerase chain reaction
Total RNA was extracted from cGMP-pretreated 3T3 cells using Trizol agents. First-strand cDNA synthesis was performed using 1 µg of total RNA, 40 MU/L of RNase inhibitor, 20 MU/L sense and anti-sense primers of MMP-2, MMP-9 and β-actin, 10 mmol/L dNTP mixture, 25 mmol/L MgCl2, 5 MU/L AMV Rtase XL, 5 MU/L AMV-optimized Taq. MMP-2, MMP-9 and β-actin primers were reported in Material section. According to One Step RNA PCR kit (AMV) instructions, the following conditions were used for MMP-2: 28 cycles of PCR amplification, denaturation at 94℃ for 1 min, annealing at 60℃ for 1 min, extension at 72℃ for 1 min; MMP-9: 30 cycles of PCR amplification, denaturation at 94℃ for 50 s, annealing at 60℃ for 1 min, and extension at 72℃ for 2 min; β-actin: 23 cycles of PCR amplification, denaturation at 94℃ for 30 s, annealing at 53℃ for 30 s, and extension at 72℃ for 1 min. Amplification was carried out in a GeneAmp 2400 PCR system (Perkins-Elmer, Foster City, CA). PCR products were resolved on 25 g/L agarose gel in the presence of 0.6 mg/L ethidium bromide. The intensities of the cDNA bands for each protein were normalized to β-actin band intensities. Images of the ethidium bromide (EB)-stained agarose gels were acquired with a digital Kodak camera (Eastman Kodak Company, Rochester, NY, USA) and quantification of the bands was performed by imaging analysis software from Kodak.

Cell adhesion assay
The wells of 96-well culture plates were coated with matrigel at a concentration of 5 mg/L and incubated at 4℃ overnight. The coated wells were blocked with PBS containing 20 g/L bovine serum albumin for 30 min and then washed with PBS. Cell suspension in serumfree medium containing 1 g/L bovine serum albumin was added to the wells (2 × 10 4 /well) and incubated at 37℃ in 50 mL/L CO2 for 30-60 min with or without test agents (8-bromo-cGMP or SNAP). After the medium and nonattached cells were removed, 2 g/L crystal violet was added for 10 min. The plate was gently washed with tap water and dried in air for 24 h. Then 0.1mL of 50 g/L SDS with 500 mL/L ethanol was added for 20 min, the plate was read at 540 nm on an ELISA reader (Microplate Co., EL311SX).

Statistical analysis
Data were expressed as mean ± SD. Intracellular Ca 2+ fluorescence ratio and percentages of attached cells were estimated with Student′s t-test or analysis of variance followed by Student-Newman-Keuls test. P < 0.05 was considered statistically significant.

Thapsigargin-induced store-operated Ca 2+ entry in fibroblast NIH/3T3 cells
Thapsigargin was used to deplete intracellular Ca 2+ stores and induce Ca 2+ entry from extracellular space. After treatment with 4 µmol/L thapsigargin in Ca 2+ -free and 2 mmol/L EGTA-containing medium for 2 min, the addition of 2 mmol/L CaCl2 induced a rise in [Ca 2+ ]i about 120 later (Figure 1). The thapsigargin-induced elevation of [Ca 2+ ]i was completely blocked by Ni 2+ (3 mmol/L), a potent blocker of Ca 2+ entry that competes for Ca 2+binding sites in 3T3 cells (Figure 2), confirming the presence of capacitive Ca 2+ entry in this cell type.

Effect of SOC on MMP mRNA expression
The expression and release of MMPs (such as MMP-2 and MMP-9) work through kinase signaling pathways [17][18][19] , like NO-cGMP-Ca 2+ signaling pathway [20][21][22] . The present study tested the involvement of SOC in regulating MMP release in vitro. RT-PCR showed that synthesis of MMP-2 and MMP-9 mRNA in cGMP-pretreated 3T3 cells was less than that in 3T3 cells not pretreated. Gelatin zymography showed that production of MMP-2 and MMP-9 in 3T3 medium culture was more than that in cGMP-pretreated 3T3 medium culture ( Figure 5).

Involvement of [Ca 2+ ]i and NO/cGMP in MMP secretion
Z y m o g r a p hy r e s u l t s s h owe d t h a t t h e i n c r e a s i n g extracellular calcium concentrations (0, 0.09, 0.4, 0.8, 1.2 mmo/L) enhanced MMP-2 and MMP-9 secretion in a dose-dependent manner ( Figure 6). In the present study, 4 µmol/L thapsigargin was used to deplete the intracellular Ca 2+ store in order to induce Ca 2+ entry. Zymography results showed pretreatment with 8-bromo-cGMP (2 mmol/L) or SNAP (200 µmol/L) significantly reduced the production of MMP-2 and MMP-9 in 3T3 cells, and 1 µmol/L KT5823 or 100 µmol/L L-NAME enhanced the production of MMP-2 and MMP-9 in 3T3 cells. These results indicated that NO/cGMP sensitive SOC was  involved in MMP secretion of fibroblasts ( Figure 7).

Involvement of NO/cGMP-sensitive Ca 2+ entry in adhesion
We used matrigel-coated plates as models for investigation of possible signaling pathways involved in extracellular matrix-induced metastasis. Matrigel is a soluble basement membrane preparation containing almost all extracellular matrix components [23] . The present study was carried out with matrigel as an adhesion substratum. A significant difference in the amount of cells attached to the matrigelcoated plates was observed in 3T3 cells pre-treated and not pre-treated with cGMP (2 mmol/L) for 30 min (71.4% ± 0.5% vs 44.7% ± 2.5%) (P < 0.05). Pre-treatment with SNAP (200 µmol/L) showed that the percent of attached and non-attached 3T3 cells was 64.7% ± 1.2% and 46.3% ± 2.0%, respectively (P < 0.05, Figure 8).

DISCUSSION
Collagenolytic degradation of endothelial and parenchymal basement membranes is a necessary step in the process of tumor invasion and angiogenesis. Neutral MMP-2 and MMP-9 mainly secreted locally by stromal cells, degrade the basement membrane and extracellular matrix collagens.
However, little is known about the signaling pathways that regulate the production of these enzymes. Previous studies have demonstrated that transforming growth factor-β1 (TGF-β1) plays a role in transmembrane signal transduction of MMP-2 regulation [24] . The present study described a role of calcium mobilization in the regulation of MMP expression. The present study demonstrated that the existence of NO/cGMP-sensitive store-operated Ca 2+ entry in mouse fibroblast NIH/3T3 cells, which is allergic to NO/cGMP. The thapsigargin-induced SOC appears to be evidenced by the magnitude of the Ca 2+ entry and its blockage by Ni 2+ , cGMP or SNAP inhibits the thapsigargin-induced Ca 2+ entry in 3T3 cells and can be reversed by KT5823 (highly specific PKG inhibitor), indicating a negative regulatory mechanism involving NO/cGMP via a PKG-dependent pathway.
Characterization of the regulation of interstitial collagenase (MMP-1) and stromelysidtransin (MMP-3) indicates that there are at least two known regulatory arms for the secretion of these MMPs. Treatment with TGF-β1 inhibits the production of these MMP genes through TIE, a novel CIS-acting transcriptional element [25,26] , whereas phorbol ester-mediated stimulation of transcription is

A B C
through phorbol ester-responsive elements (TREs) and AP-1 sites [27,28] . Regulation of matrix metalloproteinases has also been linked to the second cAMP messenger signal transduction pathway [29,30] . Several investigators have demonstrated the induction of MMP-1 production in response to cAMP either directly or after cell stimulation with cytokines that signal their effects through cAMP [29,31] . However, in contrast to MMP-1 and MMP-3, neither MMP-2 nor MMP-9 has TIE or cAMP-regulated elements defined in their 5' sequences [27,32] . The TRE mechanism has been implicated in the regulation of MMP-9 but not MMP-2 [27] . AP-1 sites have not been documented for MMP-2, although AP-1 and AP-2 sites are in the upstream of MMP-9 [27,32,33] . It was reported that there is a NF-κB binding site in the upstream sequence of MMP-9 [34] . Besides the factors influencing MMP secretion, calciumactivated signal transduction steps are also a common thread underlying important signal transduction pathways, including protein kinase C, phosphoinositide metabolism, and generation of arachidonic acid. However, few calciummediated transcriptional pathways have been identified for MMP secretion. The present study was to identify NO/cGMP-sensitive production and activation of MMP through SOC. In our study, adding extracellular Ca 2+ concentration induced release and activation of MMP (MMP-2, MMP-9) in a dose-dependent manner, cGMP and SNAP reduced MMP secretion in 3T3 cells. RT-PCR displayed that MMP-2 and MMP-9 mRNA synthesis in cGMP-pretreated 3T3 cells was significantly less than that in non cGMP-pretreated 3T3 cells. Tumor invasion is greatly dependent on the permissive action of the microenvironment. One critical factor is the production of proteolytic enzymes involved in the degradation and remodeling of ECM. Among these enzymes, MMPs represent a large family playing a key role in cell proliferation, angiogenesis, tumor invasion and metastasis [6] . These enzymes principally degrade the ECM components and attenuate cell-cell adhesion ability so as to promote tumor invasion and metastasis.
Our experiments investigating adhesion potentials demonstrated that the attachment rate of 3T3 cells living in high extracellular Ca 2+ concentration of the matrigel-coated culture plates was lower than that in low extracellular Ca 2+ concentration of the matrigelcoated culture plates, suggesting that increased cellular Ca 2+ concentration attenuates the adhesion potentials in fibroblasts by enhancing MMP secretion. The attachment rate could be upgraded by cGMP and SNAP in 3T3 cells, indicating that interference with NO/cGMP/PKG pathway sensitivity of fibroblasts reduces SOC and inhibits MMP secretion at a considerable level, thus strengthening the adhesion potential of fibroblasts and decreasing the chance of HCC metastasis.
In conclusion, store-operated Ca 2+ channels exist in mouse fibroblast cells and are involved in MMP secretion. NO/cGMP/PKG pathway negatively regulates this Ca 2+ entry. Increased extracellular Ca 2+ concentration may promote degradation of ECM components by increasing MMP secretion and attenuating fibroblast adhesion potential.