Anti-TIMP-1 antibody (clone 63515) and recombinant human TIMP-1 were purchased from R&D Systems. Anti-TIMP-1 ab-2 from Sigma. Anti-CD63 antibody is from Abcam, integrin β1 from Millipore. HRP-conjugated mouse IgG and anti-rabbit IgG from Zymed. Transfection reagent was Lipofectamine from Invitrogen.
Hygromycin B from Invitrogen. Fluorescein-labeled affinity purified antibody to mouse IgG (KPL 01-18-06).
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.
Ⅲ RESULTS
Expression pattern of TIMP-1 in human brain tumors
As noted above, Tissue inhibitor of metalloproteinase-1(TIMP-1) can function as an anti-apoptotic and survival factor, and its levels increase in response to inflammation.
However recently report show that such processes may contribute to exacerbation of the malignant process. Higher levels of TIMP-1 correlate with poor clinical outcome for patients (Kossakowskaet al., 2000). And our previous data show that TIMP-1 is over expression in human brain tumors than normal brain tissue of microarray and proteomics.
So we essentiality need that to definite expression pattern of timp-1 in human brain tumor.
Relationship between TIMP-1 expression and clinic pathological features Tissue inhibitor of metalloproteinase-1 expression in human brain tumors was investigated by immunohistochemical analysis. Positive staining for TIMP-1 was restricted to tumor cells.
When comparing the expression patterns of the brain tumors (Fig. 1E-H) and normal brain specimens (Fig 1.A-D). TIMP-1 showed a correlation with brain tumor over the range of Brain Tumors tissue. For TIMP-1, expression was never detectable by IHC in normal brain tissue but expressed dramatically for brain tumor tissues. Intensity of staining varied among different patients and in different areas of the tumor.
Immunoreactive products had a fine granular appearance and localized in the cytoplasm and displayed a heterogeneous of tumor cells. We detected excessive expression area (Indication arrows in Fig1). Previous reported that TIMP-1 has effects on ECM breakdown, angiogenesis, cell morphology, and cell survival/death, it is the net outcome
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of the balance of these functions that would determine the pathophysiology of glioma cells (Gardner & Ghorpade, 2003). So we supposed that this area is looks like blood vessel and we suggest that TIMP-1 is over expression in blood vessel. Therefore, TIMP-1 secreted when brain tumor metastasis. We hypothesized that Timp-1 induced the migration of neural stem cell. We arrived at conclusion, TIMP-1 was the only expression protein in the human Brain Tumors. Furthermore our data are consistent with a potential chemoattract molecule function for TIMP-1.
TIMP-1 induced Human Neural stem migration.
previous reported that neural stem cell migration toward glioma in vitro(Heese et al, 2005). These findings suggest that soluble factors secreted by Brain tumor are potent inducers of NSC migration, but that the responsible factors are produced to a varying extent by the different cell lines. However, the factors responsible for NSC migration in the adult CNS are unknown. Our previous data show that Brain tumor secreted TIMP-1.
TIMP-1 is up regulated in regions of Brain tumor injury and inflammation and attracts migratory cells. So we investigated the neural stem cell migration after stimulation with the TIMP-1. Boyden chamber assays was performed for measuring the Human Neural stem cell movement. Boyden chamber assays were used to assay TIMP-1 in concentrations ranging from 50 ng/ml to 500 ng/ml. TIMP-1 induced the typical bell- shape curve for the human neural stem cell chemoattraction. The effects were concentration dependent, and strongest motogenic responses were consistently observed at the highest protein concentration. (100 ng/ml). Stimulatory effects ranged from
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fold stimulation (50ng/ml) to 1.6 fold stimulation (100ng/ml). (Fig. 2) TIMP- 1 specific factor responsible for NSC chemoattraction migration.
Migration capacity of F3 and NIH3T3 Cells in Vitro
We observed that TIMP-1 induced human neural stem cells. Recent studies have shown that showing the remarkable migratory capacity of NSCs to the infiltrating tumor cell in the normal brain. In contrast, the NIH 3T3 fibroblast cells did not migrate and remained localized at the area of the injection site (Kim et al, 2006). We next compared the in vitro migratory capacities of the two cell lines Human neural stem cell (F3) and Mouse fibroblast cell (NIH3T3) in TIMP-1 by using the boyden chamber assay as described in materials and methods. Our previous data showed the maximum activity at a concentration of 100 ng/ml (Fig. 2), accordingly we treated TIMP-1 100ng/ml. F3 Stimulatory effects 1.3-fold stimulation but NIH3T3 effects -8.7 fold stimulation (100ng/ml). Human neural stem cell (F3) migrated with the typical bell- shape curve for the TIMP-1 chemoattraction. But mouse fibroblast cell (NIH3T3) does not migrate. (Fig.
3) Importantly, the migration capacity of human neural stem cells was significantly higher than that of the mouse fibroblast cells (NIH3T3) on TIMP-1. (Fig. 3).
Expression of TIMP-1 receptors in various cells
Pevious migration data show that only HNSC migrated by TIMP-1. We guess reason of this situation. So we hypothesize that they have different receptor expression pattern.
Recent studies from our laboratory found that TIMP-1 expression correlated with the
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levels of active integrin β1 on the cell surface in a CD63-dependent manner. And TIMP-1 affects CD63’s regulation of the integrin signaling pathway in a cell-type specific manner (Chirco et al, 2006). Our lab had microarray data in HNSC. So we compare expression patterns of tetraspanins (CD9, CD81, CD63) and integrins (β1, α3, α6), in microarray data. Almost cell line is expression tetraspanins (CD9, CD81, and CD63) and integrins (β1, α3, α6). Especially F3 is most expression of CD63 and β1. Next we compared expression tetraspanins (CD9, CD81, and CD63) and integrins (β1, α3, α6) by PT-PCR analysis in HNSCs, HEK293 and NIH3T3. We performed RT-PCR analysis using primers specific for tetraspanins (CD9, CD81, and CD63) and integrins (β1, α3, α6) cDNA to compare the expression in HNSCs, HEK293 and NIH3T3. The bands of these integrins and TM4 were clearly detected both in HNSCs, HEK293 and NIH3T3 by RT-PCR. As a control, the presence of mRNA for GAPDH and β–actin was examined. All integrin and TM4 mRNA revel expression is NIH3T3 better than HNSCs and HEK293 expect CD81.
Especially CD63 is extremely expression in NIH3T3 rather than HNSC and HEK293. We investigated that of expression pattern of CD63 and integrin β1 in protein level. We performed western blot analysis using mouse monoclonal Antibody CD63 (abcam) and rabbit polyclonal anti-body integrin β1 (Millipore). HNSC (F3), NIH3T3 and Human embryonic kidney cell (HEK293) were examined for surface expression of CD63 and integrin β1 by western-blot (Fig.4 C, D). The CD63 protein with 30–70% of its size (30–
60 kDa) being heavily glycosylated (Stippet al, 2003) exhibited a diffuse distribution on SDS–PAGE under a nonreducing condition Surprising F3 and HEK293 expressed CD63 and integrin β1 but NIH3T3 was not detected. And CD63 and integrin β1 was extremely
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expression in F3 rather than HEK293. This results show that HNSC (F3) has most powerful migration ability. Due to F3 have a lot of receptors.
Interaction of with TIMP-1 and integrin β1 and CD63 in HNSC Cells
Previous reports show that CD63 interacts with the C-terminal, but not with the N-terminal domain of TIMP-1, suggesting TIMP-1 interaction with CD63. Here, we asked whether TIMP-1 interacts with CD63/integrin complex, and if so, whether these interactions are critical for TIMP-1 regulation of cell migration. Since integrin β1 is thought to be one of the main tetraspanin-interacting integrins (Berditchevski et al, 1995, 1996, 1997), we first examined CD63 interactions with integrin. As shown in Fig. 4E and F, anti-CD63 antibodies immunoprecipitated integrin β1 and TIMP-1 from lysates of F3 cells, which suggests that these proteins are forming a complex. We observed that CD63 is interacted with integrin β1. In addition, TIMP-1 is interacted with CD63. These interactions may be very importance for NSC migration.
Efficient silencing of CD63 expression by shRNA system
Our data show that TIMP-1 and CD63 and integrin complex are important for HNSC migration. Specifically, CD63 is mediator in TIMP-1/CD63/integrin β1 complex. To evaluate the significance of CD63 for TIMP-1 binding on the cell surface, we established CD63-knockdown F3 cells using a vector-based small hairpin RNA (shRNA) strategy. To examine the role of CD63 in mediating the surface binding of TIMP-1 and integrin β1.
We had knocked down CD63 in HNSC cells using a shRNA approach. CD63 shRNA
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sequence is showed in Method. The shCD63-F3 cells were stably transfected with the Prep4 vector containing CD63 target sequences (Fig6.A).Western-blot analyses confirmed significant down regulation of CD63 expression in cells receiving specific CD63 shRNA target sequences (Fig6.B). Compared with wild-type control cells, cell lines bearing two of the CD63 shRNAs (shCD63-1, and shCD63-2) shRNA displayed almost loss of cell surface CD63. Conceivably, cell surface expression of CD63 could be more sensitive to suppression by RNAi than total CD63 expression. So we evaluated CD63 expression in with wild-type control cell and two of the shCD63-F3 cells populations by flow cytometry (Fig6.C). FACS Results show that cell surface expression, total levels of CD63 in wild-type, shCD63-1, and shCD63-2 cells. shRNA displayed a almost loss of cell surface CD63. As a result our shCD63-F3 cell lines are low expression CD63.
CD63 is a key mediator for NSC migration by TIMP-1
We hypothesis that Cd63 is key mediator in Human Neural stem cell migration. we next compared the in vitro migratory capacities of the 4 kinds of cell lines Human neural stem cell (F3), shCD63-F3-1, and shCD63-F3-2 cells and pREP4 cell line in TIMP-1 by using the boyden chamber assay as described under Experimental Methods. our previous data showed the maximum activity at a concentration of 100 ng/ml (Fig. 2), accordingly we treated TIMP-1 100ng/ml. F3 and pREP4 cell Stimulatory effects 2-fold stimulation but shCD63 cells effects -0.2 fold stimulation (100ng/ml). These results show that CD63 is key mediator in NSC migration by TIMP-1(Fig 4)
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ERK and CDK5 are independent pathways for stimulating TIMP-1-mediated NSC migration
Previous paper reported TIMP-1 inhibits both the intrinsic and extrinsic cell death pathways through activation of the FAK-PI3K pathway. These effects are mediated by activation of the extracellular regulated kinase (ERK). (Stetler-Stevenson, 2008) and Erk has been implicated in the migration of numerous cell types. The in response to cell matrix proteins, such as fibronectin, vitronectin (Liu et al, 2007) and collagen (Anand-Apte et al., 1997; Klemke et al., 1997; Webb et al., 2000). We hypothesis that Erk has been implicated in the migration of Human neural stem cell. Then we using Erk pathway inhibitors PD98059inhibite the migration of Human neural stem cells. When cells were pre-treated TIMP-1 with the MEK inhibitor, PD098059 does-dependent (10 ~40μM/ml), the response to human neural stem cell migration was entirely blocked. When TIMP-1 stimulated cell migration was maximally inhibited by PD098059 40 μM. and simultaneous addition of PD098059 40 μM had a 37.5% effect. Only pre-treated PD98059 20 μM/ml had a 50% inhibition effect. But it doesn’t have dramatically effect, so we find another signal pathway. previous paper reported CDK5 is a member of the cyclin-dependent kinasefamily of proline-directed protein kinases CDK5 is an essential role in regulating the complex migration Several other neuronal functions of CDK5 have also been identified, including cell-cell adhesion (Liu et al, 2007), cell-matrix adhesion (Tang et al, 1998), neurite extension(Cruz & Tsai, 2004), and cytoskeletal regulation (Miyamoto et al, 2007). CDK5 deficiency on cell migration is consistent with a role for CDK5 in regulating cell-to matrix adhesion. We hypothesis that CDK5 has been
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implicated in the migration of Human neural stem cell. Then we using CDK5 pathway inhibitors Roscovitin inhibit the migration of Human neural stem cells. When cells were pre-treated TIMP-1 with the CDK5 inhibitor, Roscovitin does-dependent (10 ~40 μM/ml), the response to human Neural stem cell migration was entirely blocked. When TIMP-1 stimulated cell migration was maximally inhibited by Roscovitin 40 μM. And`
simultaneous addition of Roscovitin 40μM had a 97% decrease effect. Only pre-treated Roscovitin 20 μM/ml had a 80% inhibitaion effect. Thus Erk signaling is an important factor in the regulation of cell migration. But CDK5 signaling is more powerful factor in the regulation of cell migration.
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Fig. 2. TIMP-1 expression in human glioblastoma tissues. Immunohistochemical staining was used to detect the expression of TIMP-1 in normal human brain tissues (A-D) and human glioblastoma tissues (E-H). After DAB staining, sections were counter stained with haematoxylin.
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Fig. 3. TIMP-1 elicited a concentration-dependent migration of HNSCs. Chemotactic migration of HNSC was induced by TIMP-1 in concentrations ranging from 50 ng to 500 ng in Boyden chamber chemotaxis assays. The Boyden chamber was incubated at 37 ℃ to allow for cell migration. The cells that migrated through the membrane were stained and counted. Included on membrain are representative images of migrated HNSC from greater than three independently performed experiments done in triplicate (n > 3). Error bars indicate SEM. Magnification, X100.
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Fig. 4. TIMP-1 induces migration in NSC-type specific manner. The migratory ability of hNSC (F3), mouse fibroblast cells (NIH 3T3), or embryonic kidney cell (HEK293) by TIMP-1 was determined using a Boyden chamber assay. TIMP-1 significantly stimulates the directional migration of F3 cells compared with NIH3T3 and HEK293 cells. The migrating cells were stained and counted. In the lower panel are representative images of migrated cells in each cell line of three independent experiments done in triplicate (n > 3).
Error bars indicate SEM. Magnification, X100.
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Fig. 5. CD63 mediates TIMP-1 binding to the cell surface and TIMP-1 co-localization with integrin β1. (A) Expression profiles of tetraspanin superfamily and integrin genes from microarray data of human NSC lines (F3,A4,F5). (B) RT-PCR analysis of tetraspanin superfamily genes and integrin genes in hNSC lines, NIH 3T3, HEK293 cells. GAPDH; internal control. (C) Expression of CD63 and integrin β1 proteins. Each cells was analyzed by western blot. β-actin and tubulin were used as internal control. D. Interaction of CD63 with TIMP1 or integrinβ. Lysates of F3 cells were immunoprecipitated with anti-CD63 or anti-TIMP-1antibody, followed by immunoblotting with anti-integrin β1 or anti-CD63 antibody, respectively.
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Fig. 6. Efficient silencing of CD63 expression in HNSCs. (A) Structural of shCD63 knockdown vector. (B) Expression of CD63 by western blot. NSCs were stably transfected with shCD63 plasmids. P1 and P2; F3 cells transfected with Prep4 vector, 63-1 and 63-2; transfected with shCD63 knockdown plasmids. (C) FACS analysis of CD63 expression in F3 and CD63 knockdown cells. CD63 expression was evaluated by flow cytometry. Isotype background staining was donw with nonimmune mouse IgG1.
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Fig. 7. CD63 is a mediator of migration of NSC. In all cases, migration was measured after 12h and is expressed relative to the migration of unstimulated cells. Included on membrane are representative images of migrated F3 from greater than two independently performed experiments done in triplicate (n > 3).Error bars indicate SD. Magnification, X100.
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Fig. 8. The migration of NSC is mediated by ERK and Cdk5 siganling pathways.
HNSC were preincubated with 10% FBS for 12h and then treated does-dependent PD98059 or Roscovitin. Other cell groups were treated PD98059 or Roscovitin with TIMP-1. In all cases, migration was measured after 12h and is expressed relative to the migration of unstimulated cells. Included on membrane are representative images of migrated F3 from greater than three independently performed experiments done in triplicate (n > 3).Error bars indicate SD. Magnification, X100.
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. D
Ⅳ ISCUSSION
our previously report reported one common secreted proteins that are up-regulated in human brain tumor from microarray data and proteomics data actually expressed highly in brain tumor samples compared to normal tissues with real-time PCR. We hypothesized that these secreted genes from human glioma may be putative chemioattracts and provide clues to the molecular pathway involved in neural stem cell migration to glioma. First we definite expression of TIMP-1 in human brain tumor tissue. Expression of TIMP-1 is increased in human glioma compared with normal tissues using and immunohistochemistry. TIMP-1 is expression in only human brain tissue, but not normal brain tissue did not expression. We investigated for one candidate protein tissue inhibitor of metalloproteinase-1(TIMP-1) to induce NSCs migration and the molecular mechanisms involved in such migration of human neural stem cells. TIMP-1 induced dramatic increase the migration of F3 cells, but not of mouse fibroblasts NIH3T3 cells in a dose-dependent manner, and showed the maximum activity at a concentration of 100 ng/ml. So we investigated as to how TIMP-1 regulates migration activity. We hypothesize that receptor expression is different in cell type specific manner. Therefore we investigated expression of receptors. In previous paper show that TIMP-1 regulates the integrin signaling complex via its interaction with CD63, a member of the tetraspanins.
TIMP-1 affects CD63’s regulation of integrin signaling in a cell-type specific manner.
TIMP-1 interaction with the tetraspanin member CD63 implies potentially diverse effects of TIMP-1 signaling on many cellular processes (Jung et al, 2006).We experiment
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expression of tetraspanins and integrins by RT-PCR and microarray data mining. Most abundance expression of CD63 and integrin is F3. And NIH3T3 in mRNA revel. We experiment expression of tetraspanins and integrins by western-blot. Surprising expression of CD63 and integrin was detected only from F3 but not NIH3T3.We identification that migration ability is decided by different expression of receptors.
Forward receptor’s interaction. Next confirmed interaction of CD63, integrin and TIMP-1 in F3. CD63 is mediator of migration by TIMP-1 in NSCs. So we performed knock-out CD63 in F3. We established shCD63-F3 cell lines using short hairpin-activated gene silencing system (Lee, Y. J et al., 2004). We confirmed that CD63 protein did not detected in shCD63-F3 cell lines by western blot and FACS. We performed boyden chamber assay using shCD63-F3, F3, prep4-F3. These result show that shCd63-F3 cell lines are dramatic decease at migration than F3, pREP4-1. So we identification CD63 is very important for NSC migration.CD63 interacts with signaling molecules. Increasing evidence suggests that CD63 regulates FAK, Src, Gab2, PI3K, Akt, and PI4K signaling pathways as shown by modulation of these pathways upon anti-CD63 antibody binding to CD63 in breast carcinoma cells and immune cells (Berditchevski and Odintsova, 1999; Sugiura and Berditchevski, 1999; Pfistershammer et al, 2004; Kraft et al,)
2005). These results demonstrated that TIMP-1 is powerful chemoattract molecule for NSC, and may be promising drug for brain tumor therapy. In addition, TIMP-1 may provide critical cues in understanding the migration of neural stem cells in physiological and pathopysiological conditions.
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Ⅴ. CONCLUSION
This study showed that TIMP-1 is induced NSCs. And we find mechanism of migration effect, cell signaling and different expression of receptor. These results demonstrated that TIMP-1 is powerful chemoattract molecule for NSC, and may be promising drug for brain tumor therapy. In addition, TIMP-1 may provide critical cues in understanding the migration of neural stem cells in physiological and pathopysiological conditions.
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