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TIMP-1 inhibits apoptosis in breast carcinoma cells via a pathway involving pertussis toxin-sensitive G protein and c-Src
BBRC Biochemical and Biophysical Research Communications 312 (2003) 1196–1201 www.elsevier.com/locate/ybbrc
TIMP-1 inhibits apoptosis in breast carcinoma cells via a pathway involving pertussis toxin-sensitive G protein and c-Src Seo-Jin Lee,a Ho Jung Yoo,b Yun Soo Bae,b Hwa-Jung Kim,c and Seung-Taek Leea,* a b
Department of Biochemistry, College of Science, and Protein Network Research Center, Yonsei University, Seoul, Republic of Korea Department of Life Sciences, Division of Molecular Life Sciences and Center for Cell Signaling Research, Ewha Womans University, Seoul, Republic of Korea c College of Pharmacy, Ewha Womans University, Seoul, Republic of Korea Received 3 November 2003
Abstract In addition to inhibiting matrix metalloproteinases, tissue inhibitor of metalloproteinase-1 (TIMP-1) is involved in the regulation of cell growth and survival. To determine its mechanism of action, we investigated effects of TIMP-1 on cell proliferation and survival and signaling pathways induced by TIMP-1 in the human breast carcinoma T-47D cell line. Treatment of T-47D cells with TIMP-1 strongly inhibited apoptosis induced by serum deprivation, but did not affect cell proliferation. TIMP-1 induced phosphorylation of Akt and extracellular signal-regulated protein kinases (ERKs), but pertussis toxin and specific inhibitors of Src family tyrosine kinases, protein tyrosine kinases, and phosphatidylinositol-3 kinase (PI3 kinase) blocked the ability of TIMP-1 to activate Akt and ERKs as well as the anti-apoptotic effect of TIMP-1. We found that TIMP-1 enhanced the kinase activities of c-Src and PI3 kinase and that this enhancement was inhibited by pertussis toxin. Inhibition of ERK activation, however, resulted in a slight decrease of the TIMP-1-induced anti-apoptotic effect. These findings demonstrate that the ability of TIMP-1 to inhibit apoptosis in T-47D cells is mediated by the sequential activation of pertussis toxin-sensitive G protein, c-Src, PI3 kinase, and Akt. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Apoptosis; Breast cancer; c-Src; Pertussis toxin; Signaling pathway; TIMP-1
Tissue inhibitors of metalloproteinases (TIMPs) are a family of four proteins, TIMP-1 through TIMP-4, that act as endogenous inhibitors of matrix metalloproteinases (MMPs) and, thus, have a potentially important function in regulating the composition and integrity of the extracellular matrix. TIMPs have also been shown to modulate cell growth and survival [1]. Following the demonstration that TIMP-1 has erythroid-potentiating activity [2], TIMP-1 was shown to stimulate the proliferation of many types of cells [3]. In the human osteosarcoma MG-63 cell line, TIMP-1 has been found to activate protein tyrosine kinases (PTKs) and extracellular signal-regulated protein kinase (ERK)2 [4]. Similarly, in the human breast carcinoma BC-61 cell line, TIMP-1 stimulates cell proliferation, accompanied by the activation of PTKs [5]. More recently, * Corresponding author. Fax: +82-2-362-9897. E-mail address: [email protected] (S.-T. Lee).
0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.11.050
TIMP-1 was found to activate Ras through a PTKmediated pathway, leading to the formation of a complex between activated Ras and Raf-1 [6]. In addition to its effects on cell proliferation, TIMP1 is involved in the inhibition of apoptosis [3]. In Burkitt’s lymphoma JD38 cell line, overexpression of TIMP-1 blocked apoptosis via the downregulation of expression of NF-jB and the upregulation of expression of Bcl-XL [7]. It was reported that TIMP-1 inhibition of apoptosis involves janus kinase-2 (JAK2), phosphatidylinositol-3 kinase (PI 3-kinase), and Akt in the human erythroleukemic UT-7 cell line [8] and focal adhesion kinase (FAK), PI 3-kinase, and mitogenactivated protein (MAP) kinases in the human breast epithelial MCF10A cell line [9,10]. However, the signaling mechanisms by which TIMP-1 exerts its effects on cell proliferation and survival have not been clearly elucidated. We therefore investigated effects of TIMP-1 on proliferation and apoptosis in the human breast
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carcinoma T-47D cell line. We analyzed signaling pathways important for the anti-apoptotic activity of TIMP-1 by assaying the phosphorylation and kinase activities of signaling proteins in the presence of various signaling blockers. Materials and methods Cells and materials. The human breast carcinoma T-47D cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal bovine serum. Anti-phospho-Akt antibody was purchased from New England Biolabs (Beverly, MA, USA) and antiAkt antibody was the generous gift of Dr. Bahk (Yonsei University, Korea). Anti-c-Src and anti- PI3-kinase p85 antibodies were purchased from Upstate Biotechnology (Lake Placid, NY, USA), whereas antibodies to Bcl-2, Bcl-XL , phospho-ERK1/2, and ERK2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-b-actin antibody and pertussis toxin (PTX) were obtained from Sigma– Aldrich (St. Louis, MO, USA), and PD98059, U0126, PP1, genistein, U73122, W-7, GF109203X, and LY294002 were purchased from Biomol (Plymouth Meeting, PA, USA). Purification of recombinant TIMP-1. Human TIMP-1 cDNA corresponding to nucleotides 37–735 [11] was generated by PCR from a human fibroblast cDNA library using the 50 primer, 50 -AGATCC AGCGCCCAGAGAGA-30 , and the 30 primer, 50 -GGACACTGTG CAGGCTTCAG-30 , plus Taq polymerase. The PCR product was ligated into pGEM-T vector (Promega, Madison, WI, USA), and the resultant plasmid (pGEM-T-TIMP-1) was sequenced to ascertain that there were no PCR errors. The pGEM-T-TIMP-1 was digested with Eco52I, blunt-ended with Klenow fragment, and digested with SpeI to isolate a 0.7-kb TIMP-1 cDNA fragment containing a half SpeI sequence at the 50 end and a blunt end at the 30 end. This 0.7-kb TIMP-1 cDNA fragment was ligated into the baculovirus transfer vector, pBlueBac4 (Invitrogen, Carlsbad, CA, USA), which had been digested with XhoI, blunt-ended with Klenow fragment, and digested with NheI. Procedures for transfection, plaque isolation, and multiplication of the recombinant TIMP-1 baculovirus were as described in the Bac-N-Blue Transfection manual (Invitrogen). Recombinant human TIMP-1, of at least 99% purity, was purified as described previously [12]. Cell survival assay. To examine the effect of TIMP-1 on cell survival in response to apoptosis induced by serum deprivation, subconfluent T-47D cells were incubated for 48 h with serum-free DMEM containing various concentrations of TIMP-1. Viability of the cells was determined by 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay as described previously [13]. Immunoprecipitation and immunoblot analysis. Cells were washed with ice-cold PBS and lysed by incubation in lysis buffer (50 mM Tris– HCl, pH 7.4, 150 mM NaCl, 0.25% sodium deoxycholate, and 1% Nonidet P-40), supplemented with 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 lg/ml leupeptin, 1 lg/ml aprotinin, 1 lg/ ml pepstatin, and 100 lM Na3 VO4 , for 20 min on ice. Insoluble material was removed by centrifugation at 4 °C for 20 min at 10,000g. Protein concentration in the lysate was determined with a BCA protein assay kit (Pierce, Rockford, IL, USA). For immunoprecipitation, lysate aliquots containing 500 lg protein were incubated with antibody for 2 h and then with protein A– or protein G–Sepharose for 2 h at 4 °C. Immunoprecipitates were washed three times with lysis buffer and resuspended in 2 sample buffer. For immunoblotting, lysate aliquots containing 50 lg protein were electrophoresed in 7.5% sodium dodecyl sulfate–polyacrylamide gels (SDS–PAGE) and transferred to nitrocellulose membranes. Specific proteins were detected using the appropriate antibodies and the enhanced chemiluminescence system (Amersham Bioscience, Uppsala, Sweden) as described [14].
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Kinase assay. For PI3-kinase assay, cells were washed twice with PBS containing 1 mM CaCl2 , 1 mM MgCl2 , and 100 lM Na3 VO4 and lysed in the above lysis buffer supplemented with 1 mM CaCl2 , 1 mM MgCl2 , 1 mM PMSF, 1 mM Na3 VO4 , 5 lg/ml aprotinin, and 5 lg/ml leupeptin. Lysate aliquots containing 500 lg protein were each immunoprecipitated with 2 ll of rabbit antiserum against the p85 subunit of PI3 kinase, and PI3-kinase enzymatic activity was assayed as described previously [15]. For Src tyrosine kinase assay, lysate aliquots containing 500 lg protein were each immunoprecipitated with 1 lg of anti-c-Src antibody. Src tyrosine kinase activity in 1/16 of each immunoprecipitate was assayed using a biotinylated PTK peptide substrate (AEEEIYG ELEA) as described previously [16]. Data analysis and statistics. All data are presented as means SD of three independent experiments. Statistical comparisons between groups were performed using Student’s t test.
Results Effects of TIMP-1 on apoptosis and proliferation of T-47D cells To determine the effect of TIMP-1 on apoptosis induced by serum deprivation, T-47D cells were cultured in serum-free DMEM containing various concentrations of TIMP-1 for 48 h, and cell survival was determined by an MTT assay. We found that TIMP-1 inhibited serum starvation-induced apoptosis in a dose-dependent manner (Fig. 1A). Enhanced cell survival was observed at TIMP-1 concentrations as low as 1 ng/ml (p < 0:01), with a maximal effect at TIMP-1 concentrations above 10 ng/ml (Fig. 1A). However, TIMP-1 had no effect on proliferation of T-47D cells which was determined by [3 H]thymidine incorporation (data not shown). These results suggest that TIMP-1 enhances cell survival without altering cell proliferation. It has been reported that TIMP-1 induces expression of an apoptotic protein Bcl-XL in Burkitt’s lymphoma JD38 and erythroleukemic UT-7 cell lines [7,8]. We thus tested the effects of TIMP-1 on expression of the antiapoptotic proteins, Bcl-2 and Bcl-XL . We found that treatment of serum-starved cells with TIMP-1 stimulated the expression of Bcl-2 and Bcl-XL proteins in a dose-dependent manner (Fig. 1B), suggesting that TIMP-1 inhibits apoptosis through upregulation of Bcl2 and Bcl-XL in T-47D cells. Effects of signaling blockers on TIMP-1-induced activation of Akt and ERK1/2 Since Akt and MAP kinases are important for cell survival [17–19], we assayed the ability of TIMP-1 to induce the phosphorylation of these proteins. We found that TIMP-1 stimulated phosphorylation of Akt and ERK1/2 (Fig. 2). Phosphorylation of Akt and ERK1/2 was induced in a time-dependent manner, with maximal effects 10 min after TIMP-1 treatment. Treatment of
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Fig. 1. Effects of TIMP-1 on survival (A) and induction of anti-apoptotic proteins (B) in breast carcinoma T-47D cells. T-47D cells were serum-starved in the presence of various concentrations of TIMP-1 for 48 h. (A) Cell survival was measured by an MTT assay as described in “Materials and methods.” Survival of each culture was expressed relative to the number of live cells cultured in DMEM supplemented with 5% FBS (100%). Data are means SD of three independent experiments. *p < 0:05, **p < 0:01 versus cell survival in the absence of TIMP-1. (B) Cell lysates were subjected to Western blot analysis using antibodies to Bcl-2 and Bcl-XL as well as anti-human b-actin antibody as a loading control.
these cells with TIMP-1, however, had no effect on the phosphorylation of p38 or c-Jun N-terminal kinase (data not shown). To determine whether signaling molecules upstream of Akt and ERK1/2 are activated by TIMP-1, we tested effects of signaling blockers on TIMP-1-induced phosphorylation of Akt and ERK1/2. TIMP-1-induced phosphorylation of Akt and ERK1/2 was blocked by PTX, a selective inhibitor of the Gi=o subclass of heterotrimeric G proteins; LY294002, a specific inhibitor of PI3 kinase; PP1, an inhibitor of the Src family of tyrosine kinases; or genistein, a general inhibitor of PTKs (Fig. 2). In contrast, TIMP-1-induced phosphorylation was not inhibited by U73122, an inhibitor of phospholipase C; W-7, an inhibitor of calmodulin-regulated enzymes; GF109203X, an inhibitor of protein kinase C; or H89, an inhibitor of protein kinase A. We also found that PD98059 and U0126, which are inhibitors of MEKs, abolished TIMP-1-induced ERK1/2 phosphorylation but not Akt phosphorylation. These results suggest that the TIMP-1-induced Akt and ERK1/2 activation pathways involve a PTX-sensitive G protein, PTKs (including at least one member of the Src family of tyrosine kinases), and PI3 kinase.
Fig. 2. Effects of signaling blockers on TIMP-1-induced phosphorylation of Akt and ERK1/2. T-47D cells were serum-starved for 24 h and pretreated with 380 ng/ml PTX for 3 h, or with 5 lM PP1, 50 lM genistein, 10 lM LY294002, 5 lM PD98059, 5 lM U0126, 1 lM U73122, 25 lM W-7, 1 lM GF109203X, or 30 lM H89 for 30 min. Cells were subsequently treated with serum-free medium containing 10 ng/ml TIMP-1 for 10 min. Cell lysates were subjected to immunoblot analysis using antibody to phospho-Akt (A, upper panel) or phospho-ERK1/2 (B, upper panel). The membranes were stripped and reprobed with antibody to Akt (A, lower panel) or ERK-2 (B, lower panel) as the respective loading controls.
Effects of signaling blockers on TIMP-1-mediated cell survival The previous results prompted us to determine whether the inhibitors of signaling molecules involved in TIMP-1-induced phosphorylation of Akt and ERK1/2 can block the anti-apoptotic activity of TIMP-1. The MTT assay showed that the ability of TIMP-1 to enhance cell survival was significantly inhibited by PTX, LY294002, PP1, and genistein (Fig. 3), suggesting that the anti-apoptotic effect of TIMP-1 is mediated, at least in part, by a PTX-sensitive G protein; by PTKs, including at least one member of the Src family of tyrosine kinases; and by PI3 kinase. Although TIMP-1 strongly stimulated the phosphorylation of both ERK1/2 and Akt, PD98059, and U0126 only slightly inhibited the effect of TIMP-1 on cell survival, suggesting that Akt activation plays a major role in the anti-apoptotic effect of TIMP-1 on T-47D cells. Effect of TIMP-1 on PI3-kinase activity Our finding, that a PI3-kinase-specific inhibitor, LY294002, abolished the phosphorylation of Akt and ERK1/2 and the anti-apoptotic effect of TIMP-1, indicated that PI3 kinase was involved in the TIMP-1-induced anti-apoptotic signaling pathway. We therefore assayed the effect of TIMP-1 on PI3-kinase activity, as measured by phosphorylation of PI in the presence of
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[c-32 P]ATP. Treatment of T-47D cells with TIMP-1 resulted in a significant, time-dependent increase in PI(3)P production (Fig. 4A) as reported in the erythroleukemic
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UT-7 cell line [8]. The TIMP-1-induced PI(3)P production was almost completely blocked by PTX, PP1, genistein, or LY294002 (Fig. 4B). Effect of TIMP-1 on Src kinase activity
Fig. 3. Effects of signaling blockers on TIMP-1-induced cell survival. T-47D cells were pretreated with the indicated signaling blockers, as described in the legend to Fig. 2, and cell survival was determined by the MTT assay described in the legend to Fig. 1. Survival of each culture was expressed relative to the number of live cells cultured in the presence of 10 ng/ml TIMP-1 (100%). Data are means SD of three independent experiments. *p < 0:05, **p < 0:01 versus cell survival in the presence of TIMP-1.
Fig. 4. Activation of PI3 kinase by TIMP-1. (A) T-47D cells were treated with 10 ng/ml TIMP-1 for the indicated time periods and PI3kinase activity was assayed as described in “Materials and methods.” (B) T-47D cells were pretreated with the indicated signaling blockers, as described in the legend to Fig. 2, and PI3-kinase activity was assayed.
Our finding, that PP1, an inhibitor of the Src family of tyrosine kinases, strongly inhibited the TIMP-1induced production of PI(3)P, the phosphorylation of Akt and ERK1/2, and the anti-apoptotic effect of TIMP-1, suggested that TIMP-1 may activate c-Src in T47-D cells. Since c-Src is highly expressed in breast carcinomas [20] and is activated by G proteins [21], we tested whether TIMP-1 activates c-Src in T-47D cells. We found that TIMP-1 enhanced the activity of c-Src kinase in a time-dependent manner (Fig. 5A), with maximal c-Src kinase activity observed 10 min after treatment with TIMP-1. PTX, genistein, and PP1, but not LY194002, abolished the TIMP-1-induced c-Src kinase activity (Fig. 5B), indicating that c-Src is present in the TIMP-1 signaling pathway, downstream of the PTX-sensitive G protein and upstream of PI3 kinase.
Fig. 5. Activation of c-Src tyrosine kinase by TIMP-1. (A) T-47D cells were treated with 10 ng/ml TIMP-1 for the indicated time periods, and c-Src kinase activity was assayed as described in “Materials and methods.” **p < 0:01, ***p < 0:001 versus kinase activity in the absence of TIMP-1. (B) Cells were pretreated with the indicated signaling blockers, as described in the legend to Fig. 2, and c-Src tyrosine kinase activity was assayed. Data are expressed as means SD of three independent experiments. **p < 0:01, ***p < 0:001 versus kinase activity in the presence of TIMP-1.
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Discussion In this study, we have shown that TIMP-1 inhibited apoptosis without affecting proliferation in the breast carcinoma T-47D cells. Although TIMP-1 stimulated phosphorylation of both Akt and ERK1/2, our results suggest that Akt is more important for the TIMP-1induced anti-apoptotic effect in T-47D cells. In Burkitt’s lymphoma and breast epithelial cell lines, the anti-apoptotic activity of TIMP-1 was shown to be independent of its ability to inhibit MMP activity [7,10]. In contrast, in activated hepatic stellate cells, the ability of TIMP-1 to inhibit apoptosis was found to be mediated by MMP inhibition [22]. To determine whether the anti-apoptotic activity of TIMP-1 in T-47D cells was associated with its inhibition of MMP, we assayed the TIMP-1-induced phosphorylation of Akt and ERK1/2 after pretreating the cells with actinonin, a natural hydroxamate-pseudopeptide MMP inhibitor. We found, however, that actinonin had no effect on TIMP-1-induced activation of Akt and ERK1/2 (data not shown), suggesting that, in the T47-D cell line, the anti-apoptotic effect of TIMP-1 is independent of its effects on MMPs. We did observe that PTX-sensitive G protein and c-Src are involved in the TIMP-1-induced activation of PI3 kinase. Activation of many G-protein-coupled receptors has been shown to increase the activities of Src family members by physical interaction with the Ga or Gbc subunit [21]. In addition, c-Src has been shown to activate PI3 kinase, by direct binding to the 85-kDa regulatory subunit of the PI3 kinase as well as indirectly [23–25]. We did not detect c-Src in the immunoprecipitate of the p85 subunit of PI3 kinase, or the p85 subunit in the immunoprecipitate of c-Src, in lysates of TIMP1-treated T-47D cells (data not shown), suggesting that c-Src indirectly activates PI3 kinase in the TIMP-1 anti-apoptotic signaling pathway. Cross-linking studies have shown putative TIMP-1 receptors with apparent molecular weights of 32 and 80 kDa in the human erythroleukemic K562 cell line [26] and the human breast carcinoma BC-61 cell line [5], respectively. Our results suggest the involvement of a PTX-sensitive G protein in the TIMP-1-induced antiapoptotic signaling pathway. It is thus likely that a Gprotein-coupled receptor is a receptor of TIMP-1. TIMP-1 overexpression in the renal carcinoma Caki-1 cell line was observed to increase cellular adhesive and invasive capacity on extracellular matrix components, probably due to an increase in the binding affinity of integrins, suggesting that TIMP-1 induces conformational changes of integrins [27]. In addition, the complex of integrin with thrombospondin receptor or urokinase receptor has been shown to activate G proteins [28,29]. Therefore, it is possible that an integrin may function as a component of the TIMP-1 receptor complex.
Our results have shown that the anti-apoptotic effect of TIMP-1 in T47-D cells utilizes a major signaling cascade mediated by the sequential activation of PTXsensitive G protein, c-Src, PI3 kinase, and Akt. In the MCF10A breast epithelial cell line, overexpression of TIMP-1 was shown to inhibit apoptosis after the loss of cell adhesion, as well as to induce constitutive activation of FAK through tyrosine phosphorylation [9]. More recently, TIMP-1-induced survival of MCF10A cells was observed to involve the activation of PI3 kinase and ERKs [10]. In hematopoietic cell lines, TIMP-1 has been reported to induce cell survival via the JAK2/PI3-kinase/ Akt pathway [8]. However, we did not detect any changes in FAK and JAK2 phosphotyrosine levels in the TIMP-1-treated T-47D cells (data not shown). Therefore, further investigations need to understand the discrepancy of TIMP-1 signaling pathways in different cell types. High levels of TIMP-1 mRNA in primary breast carcinomas have shown significant correlations with the presence of lymph node metastases, development of distant metastases, and death from the disease [30]. In transgenic mice, TIMP-1 rescued mammary epithelial cells from apoptosis [31], and in vitro studies have shown that breast cancer cell lines secrete larger amounts of TIMP-1 than non-neoplastic breast epithelial cell lines [32]. The results presented here provide further evidence for the involvement of TIMP-1 in the development and metastasis of breast cancers.
Acknowledgments This work was supported by grants from the NRL Program of MOST NRDP, MOST/KOSEF through PNRC at Yonsei University, and the Brain Korea 21 Project. Seo-Jin Lee is a pre-doctoral fellow supported by KRF.
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TIMP-1 inhibits apoptosis in breast carcinoma cells via a pathway involving pertussis toxin-sensitive G protein and c-Src Seo-Jin Lee,a Ho Jung Yoo,b Yun Soo Bae,b Hwa-Jung Kim,c and Seung-Taek Leea,* a b
Department of Biochemistry, College of Science, and Protein Network Research Center, Yonsei University, Seoul, Republic of Korea Department of Life Sciences, Division of Molecular Life Sciences and Center for Cell Signaling Research, Ewha Womans University, Seoul, Republic of Korea c College of Pharmacy, Ewha Womans University, Seoul, Republic of Korea Received 3 November 2003
Abstract In addition to inhibiting matrix metalloproteinases, tissue inhibitor of metalloproteinase-1 (TIMP-1) is involved in the regulation of cell growth and survival. To determine its mechanism of action, we investigated effects of TIMP-1 on cell proliferation and survival and signaling pathways induced by TIMP-1 in the human breast carcinoma T-47D cell line. Treatment of T-47D cells with TIMP-1 strongly inhibited apoptosis induced by serum deprivation, but did not affect cell proliferation. TIMP-1 induced phosphorylation of Akt and extracellular signal-regulated protein kinases (ERKs), but pertussis toxin and specific inhibitors of Src family tyrosine kinases, protein tyrosine kinases, and phosphatidylinositol-3 kinase (PI3 kinase) blocked the ability of TIMP-1 to activate Akt and ERKs as well as the anti-apoptotic effect of TIMP-1. We found that TIMP-1 enhanced the kinase activities of c-Src and PI3 kinase and that this enhancement was inhibited by pertussis toxin. Inhibition of ERK activation, however, resulted in a slight decrease of the TIMP-1-induced anti-apoptotic effect. These findings demonstrate that the ability of TIMP-1 to inhibit apoptosis in T-47D cells is mediated by the sequential activation of pertussis toxin-sensitive G protein, c-Src, PI3 kinase, and Akt. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Apoptosis; Breast cancer; c-Src; Pertussis toxin; Signaling pathway; TIMP-1
Tissue inhibitors of metalloproteinases (TIMPs) are a family of four proteins, TIMP-1 through TIMP-4, that act as endogenous inhibitors of matrix metalloproteinases (MMPs) and, thus, have a potentially important function in regulating the composition and integrity of the extracellular matrix. TIMPs have also been shown to modulate cell growth and survival [1]. Following the demonstration that TIMP-1 has erythroid-potentiating activity [2], TIMP-1 was shown to stimulate the proliferation of many types of cells [3]. In the human osteosarcoma MG-63 cell line, TIMP-1 has been found to activate protein tyrosine kinases (PTKs) and extracellular signal-regulated protein kinase (ERK)2 [4]. Similarly, in the human breast carcinoma BC-61 cell line, TIMP-1 stimulates cell proliferation, accompanied by the activation of PTKs [5]. More recently, * Corresponding author. Fax: +82-2-362-9897. E-mail address: [email protected] (S.-T. Lee).
0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.11.050
TIMP-1 was found to activate Ras through a PTKmediated pathway, leading to the formation of a complex between activated Ras and Raf-1 [6]. In addition to its effects on cell proliferation, TIMP1 is involved in the inhibition of apoptosis [3]. In Burkitt’s lymphoma JD38 cell line, overexpression of TIMP-1 blocked apoptosis via the downregulation of expression of NF-jB and the upregulation of expression of Bcl-XL [7]. It was reported that TIMP-1 inhibition of apoptosis involves janus kinase-2 (JAK2), phosphatidylinositol-3 kinase (PI 3-kinase), and Akt in the human erythroleukemic UT-7 cell line [8] and focal adhesion kinase (FAK), PI 3-kinase, and mitogenactivated protein (MAP) kinases in the human breast epithelial MCF10A cell line [9,10]. However, the signaling mechanisms by which TIMP-1 exerts its effects on cell proliferation and survival have not been clearly elucidated. We therefore investigated effects of TIMP-1 on proliferation and apoptosis in the human breast
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carcinoma T-47D cell line. We analyzed signaling pathways important for the anti-apoptotic activity of TIMP-1 by assaying the phosphorylation and kinase activities of signaling proteins in the presence of various signaling blockers. Materials and methods Cells and materials. The human breast carcinoma T-47D cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal bovine serum. Anti-phospho-Akt antibody was purchased from New England Biolabs (Beverly, MA, USA) and antiAkt antibody was the generous gift of Dr. Bahk (Yonsei University, Korea). Anti-c-Src and anti- PI3-kinase p85 antibodies were purchased from Upstate Biotechnology (Lake Placid, NY, USA), whereas antibodies to Bcl-2, Bcl-XL , phospho-ERK1/2, and ERK2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-b-actin antibody and pertussis toxin (PTX) were obtained from Sigma– Aldrich (St. Louis, MO, USA), and PD98059, U0126, PP1, genistein, U73122, W-7, GF109203X, and LY294002 were purchased from Biomol (Plymouth Meeting, PA, USA). Purification of recombinant TIMP-1. Human TIMP-1 cDNA corresponding to nucleotides 37–735 [11] was generated by PCR from a human fibroblast cDNA library using the 50 primer, 50 -AGATCC AGCGCCCAGAGAGA-30 , and the 30 primer, 50 -GGACACTGTG CAGGCTTCAG-30 , plus Taq polymerase. The PCR product was ligated into pGEM-T vector (Promega, Madison, WI, USA), and the resultant plasmid (pGEM-T-TIMP-1) was sequenced to ascertain that there were no PCR errors. The pGEM-T-TIMP-1 was digested with Eco52I, blunt-ended with Klenow fragment, and digested with SpeI to isolate a 0.7-kb TIMP-1 cDNA fragment containing a half SpeI sequence at the 50 end and a blunt end at the 30 end. This 0.7-kb TIMP-1 cDNA fragment was ligated into the baculovirus transfer vector, pBlueBac4 (Invitrogen, Carlsbad, CA, USA), which had been digested with XhoI, blunt-ended with Klenow fragment, and digested with NheI. Procedures for transfection, plaque isolation, and multiplication of the recombinant TIMP-1 baculovirus were as described in the Bac-N-Blue Transfection manual (Invitrogen). Recombinant human TIMP-1, of at least 99% purity, was purified as described previously [12]. Cell survival assay. To examine the effect of TIMP-1 on cell survival in response to apoptosis induced by serum deprivation, subconfluent T-47D cells were incubated for 48 h with serum-free DMEM containing various concentrations of TIMP-1. Viability of the cells was determined by 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay as described previously [13]. Immunoprecipitation and immunoblot analysis. Cells were washed with ice-cold PBS and lysed by incubation in lysis buffer (50 mM Tris– HCl, pH 7.4, 150 mM NaCl, 0.25% sodium deoxycholate, and 1% Nonidet P-40), supplemented with 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 lg/ml leupeptin, 1 lg/ml aprotinin, 1 lg/ ml pepstatin, and 100 lM Na3 VO4 , for 20 min on ice. Insoluble material was removed by centrifugation at 4 °C for 20 min at 10,000g. Protein concentration in the lysate was determined with a BCA protein assay kit (Pierce, Rockford, IL, USA). For immunoprecipitation, lysate aliquots containing 500 lg protein were incubated with antibody for 2 h and then with protein A– or protein G–Sepharose for 2 h at 4 °C. Immunoprecipitates were washed three times with lysis buffer and resuspended in 2 sample buffer. For immunoblotting, lysate aliquots containing 50 lg protein were electrophoresed in 7.5% sodium dodecyl sulfate–polyacrylamide gels (SDS–PAGE) and transferred to nitrocellulose membranes. Specific proteins were detected using the appropriate antibodies and the enhanced chemiluminescence system (Amersham Bioscience, Uppsala, Sweden) as described [14].
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Kinase assay. For PI3-kinase assay, cells were washed twice with PBS containing 1 mM CaCl2 , 1 mM MgCl2 , and 100 lM Na3 VO4 and lysed in the above lysis buffer supplemented with 1 mM CaCl2 , 1 mM MgCl2 , 1 mM PMSF, 1 mM Na3 VO4 , 5 lg/ml aprotinin, and 5 lg/ml leupeptin. Lysate aliquots containing 500 lg protein were each immunoprecipitated with 2 ll of rabbit antiserum against the p85 subunit of PI3 kinase, and PI3-kinase enzymatic activity was assayed as described previously [15]. For Src tyrosine kinase assay, lysate aliquots containing 500 lg protein were each immunoprecipitated with 1 lg of anti-c-Src antibody. Src tyrosine kinase activity in 1/16 of each immunoprecipitate was assayed using a biotinylated PTK peptide substrate (AEEEIYG ELEA) as described previously [16]. Data analysis and statistics. All data are presented as means SD of three independent experiments. Statistical comparisons between groups were performed using Student’s t test.
Results Effects of TIMP-1 on apoptosis and proliferation of T-47D cells To determine the effect of TIMP-1 on apoptosis induced by serum deprivation, T-47D cells were cultured in serum-free DMEM containing various concentrations of TIMP-1 for 48 h, and cell survival was determined by an MTT assay. We found that TIMP-1 inhibited serum starvation-induced apoptosis in a dose-dependent manner (Fig. 1A). Enhanced cell survival was observed at TIMP-1 concentrations as low as 1 ng/ml (p < 0:01), with a maximal effect at TIMP-1 concentrations above 10 ng/ml (Fig. 1A). However, TIMP-1 had no effect on proliferation of T-47D cells which was determined by [3 H]thymidine incorporation (data not shown). These results suggest that TIMP-1 enhances cell survival without altering cell proliferation. It has been reported that TIMP-1 induces expression of an apoptotic protein Bcl-XL in Burkitt’s lymphoma JD38 and erythroleukemic UT-7 cell lines [7,8]. We thus tested the effects of TIMP-1 on expression of the antiapoptotic proteins, Bcl-2 and Bcl-XL . We found that treatment of serum-starved cells with TIMP-1 stimulated the expression of Bcl-2 and Bcl-XL proteins in a dose-dependent manner (Fig. 1B), suggesting that TIMP-1 inhibits apoptosis through upregulation of Bcl2 and Bcl-XL in T-47D cells. Effects of signaling blockers on TIMP-1-induced activation of Akt and ERK1/2 Since Akt and MAP kinases are important for cell survival [17–19], we assayed the ability of TIMP-1 to induce the phosphorylation of these proteins. We found that TIMP-1 stimulated phosphorylation of Akt and ERK1/2 (Fig. 2). Phosphorylation of Akt and ERK1/2 was induced in a time-dependent manner, with maximal effects 10 min after TIMP-1 treatment. Treatment of
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Fig. 1. Effects of TIMP-1 on survival (A) and induction of anti-apoptotic proteins (B) in breast carcinoma T-47D cells. T-47D cells were serum-starved in the presence of various concentrations of TIMP-1 for 48 h. (A) Cell survival was measured by an MTT assay as described in “Materials and methods.” Survival of each culture was expressed relative to the number of live cells cultured in DMEM supplemented with 5% FBS (100%). Data are means SD of three independent experiments. *p < 0:05, **p < 0:01 versus cell survival in the absence of TIMP-1. (B) Cell lysates were subjected to Western blot analysis using antibodies to Bcl-2 and Bcl-XL as well as anti-human b-actin antibody as a loading control.
these cells with TIMP-1, however, had no effect on the phosphorylation of p38 or c-Jun N-terminal kinase (data not shown). To determine whether signaling molecules upstream of Akt and ERK1/2 are activated by TIMP-1, we tested effects of signaling blockers on TIMP-1-induced phosphorylation of Akt and ERK1/2. TIMP-1-induced phosphorylation of Akt and ERK1/2 was blocked by PTX, a selective inhibitor of the Gi=o subclass of heterotrimeric G proteins; LY294002, a specific inhibitor of PI3 kinase; PP1, an inhibitor of the Src family of tyrosine kinases; or genistein, a general inhibitor of PTKs (Fig. 2). In contrast, TIMP-1-induced phosphorylation was not inhibited by U73122, an inhibitor of phospholipase C; W-7, an inhibitor of calmodulin-regulated enzymes; GF109203X, an inhibitor of protein kinase C; or H89, an inhibitor of protein kinase A. We also found that PD98059 and U0126, which are inhibitors of MEKs, abolished TIMP-1-induced ERK1/2 phosphorylation but not Akt phosphorylation. These results suggest that the TIMP-1-induced Akt and ERK1/2 activation pathways involve a PTX-sensitive G protein, PTKs (including at least one member of the Src family of tyrosine kinases), and PI3 kinase.
Fig. 2. Effects of signaling blockers on TIMP-1-induced phosphorylation of Akt and ERK1/2. T-47D cells were serum-starved for 24 h and pretreated with 380 ng/ml PTX for 3 h, or with 5 lM PP1, 50 lM genistein, 10 lM LY294002, 5 lM PD98059, 5 lM U0126, 1 lM U73122, 25 lM W-7, 1 lM GF109203X, or 30 lM H89 for 30 min. Cells were subsequently treated with serum-free medium containing 10 ng/ml TIMP-1 for 10 min. Cell lysates were subjected to immunoblot analysis using antibody to phospho-Akt (A, upper panel) or phospho-ERK1/2 (B, upper panel). The membranes were stripped and reprobed with antibody to Akt (A, lower panel) or ERK-2 (B, lower panel) as the respective loading controls.
Effects of signaling blockers on TIMP-1-mediated cell survival The previous results prompted us to determine whether the inhibitors of signaling molecules involved in TIMP-1-induced phosphorylation of Akt and ERK1/2 can block the anti-apoptotic activity of TIMP-1. The MTT assay showed that the ability of TIMP-1 to enhance cell survival was significantly inhibited by PTX, LY294002, PP1, and genistein (Fig. 3), suggesting that the anti-apoptotic effect of TIMP-1 is mediated, at least in part, by a PTX-sensitive G protein; by PTKs, including at least one member of the Src family of tyrosine kinases; and by PI3 kinase. Although TIMP-1 strongly stimulated the phosphorylation of both ERK1/2 and Akt, PD98059, and U0126 only slightly inhibited the effect of TIMP-1 on cell survival, suggesting that Akt activation plays a major role in the anti-apoptotic effect of TIMP-1 on T-47D cells. Effect of TIMP-1 on PI3-kinase activity Our finding, that a PI3-kinase-specific inhibitor, LY294002, abolished the phosphorylation of Akt and ERK1/2 and the anti-apoptotic effect of TIMP-1, indicated that PI3 kinase was involved in the TIMP-1-induced anti-apoptotic signaling pathway. We therefore assayed the effect of TIMP-1 on PI3-kinase activity, as measured by phosphorylation of PI in the presence of
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[c-32 P]ATP. Treatment of T-47D cells with TIMP-1 resulted in a significant, time-dependent increase in PI(3)P production (Fig. 4A) as reported in the erythroleukemic
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UT-7 cell line [8]. The TIMP-1-induced PI(3)P production was almost completely blocked by PTX, PP1, genistein, or LY294002 (Fig. 4B). Effect of TIMP-1 on Src kinase activity
Fig. 3. Effects of signaling blockers on TIMP-1-induced cell survival. T-47D cells were pretreated with the indicated signaling blockers, as described in the legend to Fig. 2, and cell survival was determined by the MTT assay described in the legend to Fig. 1. Survival of each culture was expressed relative to the number of live cells cultured in the presence of 10 ng/ml TIMP-1 (100%). Data are means SD of three independent experiments. *p < 0:05, **p < 0:01 versus cell survival in the presence of TIMP-1.
Fig. 4. Activation of PI3 kinase by TIMP-1. (A) T-47D cells were treated with 10 ng/ml TIMP-1 for the indicated time periods and PI3kinase activity was assayed as described in “Materials and methods.” (B) T-47D cells were pretreated with the indicated signaling blockers, as described in the legend to Fig. 2, and PI3-kinase activity was assayed.
Our finding, that PP1, an inhibitor of the Src family of tyrosine kinases, strongly inhibited the TIMP-1induced production of PI(3)P, the phosphorylation of Akt and ERK1/2, and the anti-apoptotic effect of TIMP-1, suggested that TIMP-1 may activate c-Src in T47-D cells. Since c-Src is highly expressed in breast carcinomas [20] and is activated by G proteins [21], we tested whether TIMP-1 activates c-Src in T-47D cells. We found that TIMP-1 enhanced the activity of c-Src kinase in a time-dependent manner (Fig. 5A), with maximal c-Src kinase activity observed 10 min after treatment with TIMP-1. PTX, genistein, and PP1, but not LY194002, abolished the TIMP-1-induced c-Src kinase activity (Fig. 5B), indicating that c-Src is present in the TIMP-1 signaling pathway, downstream of the PTX-sensitive G protein and upstream of PI3 kinase.
Fig. 5. Activation of c-Src tyrosine kinase by TIMP-1. (A) T-47D cells were treated with 10 ng/ml TIMP-1 for the indicated time periods, and c-Src kinase activity was assayed as described in “Materials and methods.” **p < 0:01, ***p < 0:001 versus kinase activity in the absence of TIMP-1. (B) Cells were pretreated with the indicated signaling blockers, as described in the legend to Fig. 2, and c-Src tyrosine kinase activity was assayed. Data are expressed as means SD of three independent experiments. **p < 0:01, ***p < 0:001 versus kinase activity in the presence of TIMP-1.
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Discussion In this study, we have shown that TIMP-1 inhibited apoptosis without affecting proliferation in the breast carcinoma T-47D cells. Although TIMP-1 stimulated phosphorylation of both Akt and ERK1/2, our results suggest that Akt is more important for the TIMP-1induced anti-apoptotic effect in T-47D cells. In Burkitt’s lymphoma and breast epithelial cell lines, the anti-apoptotic activity of TIMP-1 was shown to be independent of its ability to inhibit MMP activity [7,10]. In contrast, in activated hepatic stellate cells, the ability of TIMP-1 to inhibit apoptosis was found to be mediated by MMP inhibition [22]. To determine whether the anti-apoptotic activity of TIMP-1 in T-47D cells was associated with its inhibition of MMP, we assayed the TIMP-1-induced phosphorylation of Akt and ERK1/2 after pretreating the cells with actinonin, a natural hydroxamate-pseudopeptide MMP inhibitor. We found, however, that actinonin had no effect on TIMP-1-induced activation of Akt and ERK1/2 (data not shown), suggesting that, in the T47-D cell line, the anti-apoptotic effect of TIMP-1 is independent of its effects on MMPs. We did observe that PTX-sensitive G protein and c-Src are involved in the TIMP-1-induced activation of PI3 kinase. Activation of many G-protein-coupled receptors has been shown to increase the activities of Src family members by physical interaction with the Ga or Gbc subunit [21]. In addition, c-Src has been shown to activate PI3 kinase, by direct binding to the 85-kDa regulatory subunit of the PI3 kinase as well as indirectly [23–25]. We did not detect c-Src in the immunoprecipitate of the p85 subunit of PI3 kinase, or the p85 subunit in the immunoprecipitate of c-Src, in lysates of TIMP1-treated T-47D cells (data not shown), suggesting that c-Src indirectly activates PI3 kinase in the TIMP-1 anti-apoptotic signaling pathway. Cross-linking studies have shown putative TIMP-1 receptors with apparent molecular weights of 32 and 80 kDa in the human erythroleukemic K562 cell line [26] and the human breast carcinoma BC-61 cell line [5], respectively. Our results suggest the involvement of a PTX-sensitive G protein in the TIMP-1-induced antiapoptotic signaling pathway. It is thus likely that a Gprotein-coupled receptor is a receptor of TIMP-1. TIMP-1 overexpression in the renal carcinoma Caki-1 cell line was observed to increase cellular adhesive and invasive capacity on extracellular matrix components, probably due to an increase in the binding affinity of integrins, suggesting that TIMP-1 induces conformational changes of integrins [27]. In addition, the complex of integrin with thrombospondin receptor or urokinase receptor has been shown to activate G proteins [28,29]. Therefore, it is possible that an integrin may function as a component of the TIMP-1 receptor complex.
Our results have shown that the anti-apoptotic effect of TIMP-1 in T47-D cells utilizes a major signaling cascade mediated by the sequential activation of PTXsensitive G protein, c-Src, PI3 kinase, and Akt. In the MCF10A breast epithelial cell line, overexpression of TIMP-1 was shown to inhibit apoptosis after the loss of cell adhesion, as well as to induce constitutive activation of FAK through tyrosine phosphorylation [9]. More recently, TIMP-1-induced survival of MCF10A cells was observed to involve the activation of PI3 kinase and ERKs [10]. In hematopoietic cell lines, TIMP-1 has been reported to induce cell survival via the JAK2/PI3-kinase/ Akt pathway [8]. However, we did not detect any changes in FAK and JAK2 phosphotyrosine levels in the TIMP-1-treated T-47D cells (data not shown). Therefore, further investigations need to understand the discrepancy of TIMP-1 signaling pathways in different cell types. High levels of TIMP-1 mRNA in primary breast carcinomas have shown significant correlations with the presence of lymph node metastases, development of distant metastases, and death from the disease [30]. In transgenic mice, TIMP-1 rescued mammary epithelial cells from apoptosis [31], and in vitro studies have shown that breast cancer cell lines secrete larger amounts of TIMP-1 than non-neoplastic breast epithelial cell lines [32]. The results presented here provide further evidence for the involvement of TIMP-1 in the development and metastasis of breast cancers.
Acknowledgments This work was supported by grants from the NRL Program of MOST NRDP, MOST/KOSEF through PNRC at Yonsei University, and the Brain Korea 21 Project. Seo-Jin Lee is a pre-doctoral fellow supported by KRF.
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