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Paracrine effect of TGF-β1 on downregulation of gap junctional intercellular communication between human dermal fibroblasts
BBRC Biochemical and Biophysical Research Communications 319 (2004) 321–326 www.elsevier.com/locate/ybbrc
Paracrine effect of TGF-b1 on downregulation of gap junctional intercellular communication between human dermal fibroblastsq Dominik Stuhlmann,1 Holger Steinbrenner, Bernhard Wendlandt, Dragana Mitic, Helmut Sies, and Peter Brenneisen* Institute for Biochemistry and Molecular Biology I, Heinrich-Heine-University, D-40225 D€usseldorf, Germany Received 29 April 2004 Available online 14 May 2004
Abstract Disruption of gap junctional intercellular communication (GJIC) is associated with tumor progression during multistage carcinogenesis. A coordinated interaction of epithelial tumor cells with the stromal environment via growth factors is a prerequisite for tumor invasion. Here, the involvement of growth factors in downregulation of homologous GJIC of dermal fibroblasts, used as model for stromal cells, was examined. Tumor cell derived transforming growth factor-b1 (TGF-b1), having oncogenic activities at late stages of carcinogenesis, was identified as being responsible for downregulation of GJIC via an increase in the level of reactive oxygen species in stromal fibroblasts. Lowering the level of reactive oxygen species by antioxidants, such as the cell-permeable N-acetyl-L -cysteine, prevented TGF-b1-mediated downregulation of intercellular communication between confluent fibroblasts. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Gap junction; Intercellular communication; Fibroblast; Squamous tumor cell; Tumor–stroma; Transforming growth factor-b; Reactive oxygen species; Skin cancer
Disruption of homologous and heterologous gap junctional intercellular communication (GJIC) is a hallmark of carcinogenesis [1], while induction of GJIC in some types of cancer cells leads to a reversal of the cancer phenotype [2,3]. This direct pathway of cellular crosstalk is provided by gap junctions, each comprised of two hemichannels (connexons) which connect the cytoplasm of neighboring cells. The carcinogenic process involves the transition from a normal, GJIC-competent cell to one which is defective in GJIC, finally
q
Abbreviations: TGF-b1, transforming growth factor-b1; GJIC, gap junctional intercellular communication; Cx, connexin; HDF, human dermal fibroblasts; FCS, fetal calf serum; H2DCF-DA, 20 ,70 dichlorodihydrofluorescein diacetate; ROS, reactive oxygen species; NAC, N-acetyl-L -cysteine. * Corresponding author. Fax: +49-211-811-3029. E-mail address: [email protected] (P. Brenneisen). 1 This work is part of D. Stuhlmann’s Ph.D. thesis at the University of D€ usseldorf. 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.05.004
resulting in breakdown of intercellular communication between tumor cells during tumor promotion [1,2,4]. In cancer, interactions of tumor cells with their microenvironment and the influence of tumor on stroma and vice versa are essential for tumor survival and drive invasion and metastasis [5,6]. In melanoma and carcinoma, a wide variety of different cytokines and growth factors are expressed by tumor cells and stromal cells such as endothelial cells and fibroblasts which promote tumor growth as well as migration during tumor invasion [7–9]. Among the autocrine and paracrine acting growth factors involved in molecular processes of tumor–stroma interaction, transforming growth factor-b1 (TGFb1), a 25 kDa homodimeric protein, plays a pivotal role. Many studies led to the elucidation of a TGF-b1 signal transduction network indicating that this growth factor has a pleiotropic effect mediating a diversity of biological processes on different cell types [10–12]. Reactive oxygen species (ROS) generated by TGF-b1 may initiate
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signaling pathways [13,14]. Furthermore, it has been postulated that TGF-1 acts as tumor promoter at late stages of carcinogenesis promoting invasion and metastasis [15,16]. Based on our recent data on downregulation of homologous GJIC of stromal fibroblasts in tumor–stroma interaction [17] which is mediated by tumor cell derived factor(s) of 20–30 kDa, we addressed the question of whether TGF-b1 might be the factor mediating this effect on fibroblast via a paracrine mechanism. Based on our results, we add a novel aspect of TGF-b1 effects, namely, that tumor cell derived TGF-b1 downregulates homologous GJIC of dermal fibroblasts in vitro.
Materials and methods Materials. Cell culture medium (Dulbecco’s modified Eagle’s medium (DMEM)) was purchased from Invitrogen GmbH (Karlsruhe, Germany) and the defined fetal calf serum (FCS gold) was from PAA Laboratories (Linz, Austria). All chemicals and biochemicals were obtained from Sigma (Deisenhofen, Germany) unless otherwise indicated. The protein assay kit (Bio-Rad DC, detergent compatible) was from Bio-Rad Laboratories GmbH (M€ unchen, Germany). The dye 20 ,70 -dichlorodihydrofluorescein diacetate (H2 DCF-DA) was supplied from MoBiTec (G€ ottingen, Germany). TGF-b1 ELISA was delivered by R&D Systems (Wiesbaden, Germany) and N-acetyl-L -cysteine (NAC) by Merck Biosciences (Schwalbach, Germany). Culture of human dermal fibroblasts. Human dermal fibroblasts (HDF) were established from foreskin biopsies of healthy donors [18] with an age of 4–6 years. Cells were used at passages 4–9 corresponding to cumulative population doubling levels of 7–20 and passaged as described earlier [19]. Fibroblasts and the squamous carcinoma cell line SCL-1, originally derived from facial skin of a 74year-old woman [20] (generously provided by Dr. Norbert E. Fusenig, DKFZ Heidelberg, Germany), were maintained in DMEM supplemented with glutamine (2 mM), penicillin (400 U/ml), streptomycin (50 mg/ml), and 10% defined FCS in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. SCL-1 cells were passaged twice a week. Enzyme linked immunosorbent assay of TGF-b1. Supernatants of confluent HDF and SCL-1 cells were harvested at 48 h after serum starvation. The amount of secreted active and latent TGF-b1 was measured by a 96-well microplate enzyme linked immunosorbent assay (ELISA) system for active TGF-b1 according to the manufacturer’s instruction. Briefly, total TGF-b1 was determined by acid activation of latent TGF-b1. One hundred microliters per sample of acid activated or untreated (active) TGF-b1 was captured by a mouse anti-human TGF-b1 antibody. The captured growth factor was detected by a biotinylated chicken anti-human TGF-b1 antibody followed by incubation with streptavidin conjugated horseradish peroxidase, substrate incubation, and measurement of optical density at 450 nm. The percentage of active versus latent TGF-b1 in the supernatants was calculated by the amount of TGF-b1 of untreated samples compared with the total TGF-b1 amount. Gap junctional communication assay (Lucifer Yellow transfer). Serum-free supernatants of confluent fibroblasts, which are called “conditioned medium,” and of subconfluent SCL-1 cells were harvested after 24 or 48 h and put on confluent HDF monolayer cultures which were grown in complete DMEM + 10% FCS. Prior to addition of the supernatants, serum-containing medium was removed and the cells were washed with phosphate-buffered saline (PBS). At different time points thereafter, the cells were used for microinjection. GJIC was measured by microinjection of the fluorescent dye Lucifer Yellow CH (10% in 0.33 M LiCl) into selected fibroblasts by means of a micro-
manipulator and a microinjector system (Eppendorf, Hamburg). One minute after injection, the number of fluorescent cells around a single cell loaded with the dye was counted. Ten individual cells per dish were injected and medians, 25% quartile, and 75% quartile were calculated (see below). Measurement of intracellular ROS. The intracellular ROS level was measured using the fluorescent dye H2 DCF-DA which is readily diffusible into cells, where it is hydrolyzed to the non-fluorescent polar derivative H2 DCF and thereby trapped within cells [21]. In the presence of an oxidant, H2 DCF is converted into the highly fluorescent 20 ,70 -dichlorofluorescein (DCF). For assays, confluent fibroblast monolayer cultures were loaded with 20 lM H2 DCF-DA in PSG buffer (100 mM KH2 PO4 , 10 mM NaCl, and 5 mM glucose; pH 7.4) for 15 min in the dark. After washing three times with PSG buffer, the loaded cells were subjected to 10 ng TGF-b1/ml PSG or supernatants of tumor cells which were diluted in that buffer. ROS generation was detected as a result of the oxidation of H2 DCF and the fluorescence (excitation 488 nm; emission 515–540 nm), given in arbitrary units, was followed with a Zeiss axiovert fluorescent microscope with a chargecoupled device (CCD) camera (ORCA II, Hamamatsu, Herrsching, Germany) for additional 20 min. To prevent growth factor-mediated increase in intracellular ROS level, confluent fibroblasts were incubated with 5 mM N-acetyl-L -cysteine for 4 h prior to treatment with H2 DCF-DA, recombinant TGFb1, or supernatants. The used concentrations of H2 DCF-DA and NAC neither showed any cytotoxic effect nor a change in cell morphology (data not shown). Statistical analysis. In order to quantify the distribution of the number of communicating cells, box-and-whisker plots (box plot) were used which highlight and combine important parameters describing the scatter of the sample data: median (line inside the box), 25% quartile (lower boundary of the box), 75% quartile (upper boundary of the box), minimum and maximum values (vertical lines from the box, “whiskers”). In this study, box plots indicate non-Gaussian distribution. Therefore, for analysis of statistical significance, non-parametric tests such as Kruskal–Wallis test were applied [22]. Before the study, P < 0:05, P < 0:01, and P < 0:001 were selected as the level of significance.
Results and discussion Tumor cells release high amounts of active TGF-b1 responsible for downregulation of homologous GJIC of fibroblasts As tumor progression is concomitant with activation of the local host stroma via molecular crosstalk between tumor and stromal cells mediated by cytokines and growth factors, among them TGF-b1 [5], we measured the amounts of TGF-b1 in the supernatants of dermal fibroblast as well as of SCL-1 tumor cells. For fibroblasts, 19% of total TGF-b1, which was set at 100%, reflected the fraction of active TGF-b1, whilst the active fraction released from the tumor cells was about 8%. However, tumor cells released a 16-fold higher amount of total TGF-b1, and, therefore, a 6-fold higher amount of active TGF-b1 compared to stromal fibroblasts (Fig. 1). The bioactive TGF-b1 results from dissociation of the latent inactive TGF-b complex consisting of the non-covalently bound latency associated peptide (LAP) dimer to the active TGF-b1 dimer which are secreted as
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Fig. 1. Constitutive TGF-b1 levels of stromal and tumor cells. Supernatants of human dermal fibroblasts (HDF) and SCL-1 tumor cells were harvested 24 h after incubation of the cells with serum-free cell culture medium and subjected to TGF-b1 ELISA. Active and total TGF-b1 amounts were determined as described in Materials and methods. Data represent means standard deviation for three independent experiments.
LAP/TGF-b1 complex by epithelial and mesenchymal cells [23,24]. Furthermore, released latent TGF-b1 can be activated by thrombospondin, integrins, and matrix metalloproteinases [25], which are generated by stromal [26] as well as malignant cells [27]. Both epithelial cells and their fibroblast neighbors secrete TGF-b1 keeping cell proliferation in check, but, if the sensitive balance is disturbed by genetic or epigenetic events, cancer may develop [28,29]. Recently, Lewis et al. [30] demonstrated a TGF-b1-dependent transdifferentiation of fibroblasts secreting high levels of hepatocyte growth factor/scatter factor which promoted tumor invasion. This high amount of active TGF-b1 of tumor cells is responsible for downregulation of homologous GJIC of fibroblasts. To test this hypothesis, serum-free (-FCS) supernatants of subconfluent SCL-1 monolayer cultures were harvested after 2 days and added to fibroblast monolayer cultures for 24 h. As controls, serum-free conditioned medium was harvested after 2 days and added to confluent fibroblasts which were subjected to gap junctional communication assays (Fig. 2). In comparison with these controls (median ¼ 9.5), confluent fibroblasts incubated with supernatants of the tumor cells or serum-free medium containing recombinant TGF-b1 exhibited a significant lowering of GJIC, reflected by a median of 3.0 and 1.0, respectively, and interquartile ranges (indicated by the box length) of 2–4 and 0–1.5 which represent the middle 50% of the values. Treatment of the SCL-1 supernatants with anti-TGF-b1 neutralizing antibody or immunoprecipitation of the growth factor maintained homologous GJIC of fibroblasts compared with cells treated with SCL-1 supernatants or recombinant TGF-b1 (P < 0:01), which is indicated by the medians and the interquartile ranges from 7.0 to 14.5 and 9.0 to 11.5, respectively (Fig. 2).
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Fig. 2. TGF-b1-mediated modulation of homologous GJIC. Confluent fibroblasts (HDF) were incubated with serum-free (-FCS) conditioned medium (SN(2d) HDF) and supernatants of SCL-1 cells (SN(2d) SCL-1), both harvested after 2 days, or with 10 ng of recombinant TGF-b1/ml medium. In addition, TGF-b1 was neutralized or immunoprecipitated (IP, 2 h) with 1 lg anti-TGF-b1/ml medium. Twentyfour hours after treatment the number of Lucifer Yellow stained cells adjacent to the injected cell was used as a measure of GJIC. The upper and lower boundaries of the boxes are the upper and lower quartiles. The box length is the interquartile distance so the box contains the middle 50% of values (the horizontal line inside the box indicates the median). The vertical lines extend to maximum and minimum values. The values of the box plots represent the number of communicating cells of 20 microinjected cells (10 cells/dish). Three independent experiments were performed; **P < 0:01 (Kruskal–Wallis test).
Some contrary results of the effect of TGF-b1 on gap junctional intercellular communication were described which may result from different studied cell types. Robe et al. [31] demonstrated a TGF-b1-dependent increase in GJIC of rat astrocytes, although the GJIC of C6 glioblastoma cells, the tumorigenic counterpart, was inhibited by TGF-b1. While TGF-b1 triggered an increase in GJIC of bovine aortic endothelial cells [32], the opposite effect of TGF-b1 was observed for human keratinocytes [33] and rat kidney fibroblasts (NRK) [34]. In the osteoblastic cell line MC3T3-E1, TGF-b1 inhibited GJIC by decreasing the phosphorylated form of connexin 43 [35]. In our study, we demonstrate a lowering of homologous GJIC of fibroblasts which is triggered by TGF-b1 secreted by a highly invasive squamous tumor cell line. Supernatants of the cervix carcinoma cell line HeLa, the squamous cell line A431, and melanoma cells A375 showed similar results (data not shown) which indicates a crucial role of TGF-b1 in tumor progression. TGF-b1 mediates downregulation of homologous GJIC of fibroblasts via reactive oxygen species As TGF-b1 was shown to produce reactive oxygen species in human lung fibroblasts [13], we addressed the
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question of whether this factor mediates downregulation of homologous GJIC of fibroblasts via the involvement of ROS. Therefore, time course analysis of ROS generation after treatment of confluent HDF with recombinant TGF-b1 or SCL-1 supernatants was performed (Fig. 3). Incubation with both the TGF-b1 containing supernatant and recombinant TGF-b1 resulted in significant increase in DCF fluorescence which was maintained over the studied time range. This suggests that TGF-b1 increases intracellular ROS level in fibroblasts. A non-toxic concentration of 1 mM H2 O2 , which was used as technical control, further increased the intracellular ROS level. A non-toxic concentration of the antioxidant NAC (5 mM) prevented the TGF-b1and H2 O2 -mediated increase in the ROS level (Fig. 3). NAC is a thiol compound and a precursor of L -cysteine and reduced glutathione (GSH). As a source of sulfhydryl groups in cells, NAC may act directly on detoxification of reactive oxygen species such as hydrogen peroxide, hydroxyl radical, and superoxide [36,37]. NAC has beneficial effects in pathological conditions characterized by decreased GSH or oxidative stress, such as HIV infection, cancer, and heart disease [38]. However, to exclude a direct alteration of the structure of TGF-b1 by P10 mM NAC [39] resulting in a ROSindependent downregulation of homologous GJIC of fibroblasts, the water-soluble vitamin E analog Trolox
Fig. 3. Time course of TGF-b1 induced intracellular ROS production from human dermal fibroblasts. Confluent cells were incubated either with SCL-1 supernatants or 10 ng recombinant TGF-b1/ml medium, and 1 mM H2 O2 for the indicated time. Increase of DCF fluorescence was followed over 20 min. N-Acetyl-L -cysteine (NAC) at a concentration of 5 mM was added 4 h prior to measurement of DCF fluorescence. The experiment was performed in duplicate. Arrows (!) indicate incubation with SCL-1 supernatants, harvested after 2 days (SN(2d) SCL-1), or TGF-b1, or H2 O2 .
and butylated hydroxytoluene (BHT), a lipid-soluble antioxidant, were applied which protected the fibroblasts against TGF-b1-mediated inhibition of GJIC (data not shown). In accordance with this finding, Stuhlmann et al. [17] demonstrated that preincubation of the fibroblasts with selenite prevents tumor cellmediated downregulation of GJIC between fibroblasts dealing with selenium-dependent antioxidative enzymes such as phospholipid hydroperoxide glutathione peroxidase (PHGPX) or glutathione peroxidase (GPX) to be involved in this mechanism. To test the effect of ROS on intercellular communication, confluent fibroblasts were pretreated with a nontoxic concentration of NAC prior to incubation with conditioned medium or SCL-1 supernatants. The interquartile ranges of NAC pretreated cells overlapped with that of fibroblasts treated with conditioned medium (control) which suggests that ROS play a role in downregulation of GJIC between fibroblasts. By contrast, SCL-1 supernatants lowered this intercellular communication (Fig. 4A). Furthermore, we demonstrated that H2 O2 may be a potential candidate for a
Fig. 4. Effect of ROS on homologous GJIC of fibroblasts. Confluent fibroblasts (HDF) were incubated with serum-free conditioned medium (SN(1d) HDF) or serum-free supernatants of SCL-1 cells (SN(1d) SCL-1), both harvested after 1 day (A,B). In addition, the cells were preincubated with 5 mM NAC for 4 h (A) or with 0.1 and 1 mM H2 O2 for 1 h (B). The values of the box plots (see Fig. 2), measured after 24 h (A) or 1 h (B) after treatment, represent the number of communicating cells of 30 microinjected cells (10 cells/dish). Similar results were obtained in two independent experiments. ***P < 0:001, **P < 0:01 (Kruskal–Wallis test).
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dose-dependent downregulation of this homologous GJIC, because treatment of confluent fibroblasts with non-toxic concentrations of H2 O2 significantly lowered GJIC to control cells (Fig. 4B). Our data correspond to earlier results showing H2 O2 mediated inhibition of intercellular communication of WB-F344 rat liver epithelial cells [40]. Furthermore, a TGF-b1-mediated increase in intracellular H2 O2 levels in human lung fibroblasts [41] and murine embryo fibroblasts (NIH 3T3) [42] was measured. The network of TGF-b1 signaling involves receptor serine/threonine kinases at the cell surface [43] and their substrates, the so-called Smad proteins, which move into the nucleus and activate target gene transcription in association with further transcription factors [11,44]. In addition, TGF-b1 induces non-Smad signaling mechanisms [25]. In that context, TGF-b1 stimulation of human lung fibroblasts resulted in a transient burst of ROS which regulate downstream events such as Ca2þ -influx, mitogen-activated protein kinase (MAPK) activation, and phosphorylation-dependent activation of activating protein-1 (AP-1) which induced interleukin-6 gene expression [13]. Recently, it was shown that paracrine factor-mediated downregulation of GJIC between fibroblasts is independent of phosphorylation of the gap junctional protein connexin 43 [17]. In conclusion, we identified TGF-b1 to be the tumor cell derived growth factor which is responsible for the downregulation of homologous GJIC of stromal cells in vitro via the involvement of reactive oxygen species. As invasion and metastatic spread of tumor cells continue to be a barrier to cure cancer, understanding of the cellular interaction between tumor cells and its surrounding stroma could assist in the development of novel therapeutic strategies to combat metastasis more efficiently in the future.
Acknowledgments We are grateful to Claudia Wyrich for excellent technical assistance. H.S. is a Fellow of the National Foundation for Cancer Research (NFCR), Bethesda, MD, USA. This work was supported by Deutsche Krebshilfe e.V. (10-2223) and Deutsche Forschungsgemeinschaft SPP Selenoproteine (Si 255/11-3) and SFB 575/B4.
References [1] J.E. Trosko, R.J. Ruch, Cell–cell communication in carcinogenesis, Front. Biosci. 3 (1998) D208–D236. [2] M. Mesnil, Connexins and cancer, Biol. Cell 94 (2002) 493–500. [3] J.E. Trosko, R.J. Ruch, Gap junctions as targets for cancer chemoprevention and chemotherapy, Curr. Drug Targets 3 (2002) 465–482. [4] W.R. Loewenstein, Junctional intercellular communication and the control of growth, Biochim. Biophys. Acta 560 (1979) 1–65.
325
[5] L.A. Liotta, E.C. Kohn, The microenvironment of the tumour– host interface, Nature 411 (2001) 375–379. [6] M.M. Mueller, N.E. Fusenig, Tumor–stroma interactions directing phenotype and progression of epithelial skin tumor cells, Differentiation 70 (2002) 486–497. [7] O. de Wever, M. Mareel, Role of tissue stroma in cancer cell invasion, J. Pathol. 200 (2003) 429–447. [8] N.E. Fusenig, P. Boukamp, Multiple stages and genetic alterations in immortalization, malignant transformation, and tumor progression of human skin keratinocytes, Mol. Carcinog. 23 (1998) 144–158. [9] E. Lazar-Molnar, H. Hegyesi, S. Toth, A. Falus, Autocrine and paracrine regulation by cytokines and growth factors in melanoma, Cytokine 12 (2000) 547–554. [10] E.P. Bottinger, J.J. Letterio, A.B. Roberts, Biology of TGF-beta in knockout and transgenic mouse models, Kidney Int. 51 (1997) 1355–1360. [11] J. Massague, TGF-beta signal transduction, Annu. Rev. Biochem. 67 (1998) 753–791. [12] M.S. Pepper, Transforming growth factor-beta: vasculogenesis, angiogenesis, and vessel wall integrity, Cytokine Growth Factor Rev. 8 (1997) 21–43. [13] E. Junn, K.N. Lee, H.R. Ju, S.H. Han, J.Y. Im, H.S. Kang, T.H. Lee, Y.S. Bae, K.S. Ha, Z.W. Lee, S.G. Rhee, I. Choi, Requirement of hydrogen peroxide generation in TGF-beta 1 signal transduction in human lung fibroblast cells: involvement of hydrogen peroxide and Ca2þ in TGF-beta 1-induced IL-6 expression, J. Immunol. 165 (2000) 2190–2197. [14] V.J. Thannickal, R.M. Day, S.G. Klinz, M.C. Bastien, J.M. Larios, B.L. Fanburg, Ras-dependent and -independent regulation of reactive oxygen species by mitogenic growth factors and TGF-beta1, FASEB J. 14 (2000) 1741–1748. [15] C. Berking, R. Takemoto, H. Schaider, L. Showe, K. Satyamoorthy, P. Robbins, M. Herlyn, Transforming growth factor-beta1 increases survival of human melanoma through stroma remodeling, Cancer Res. 61 (2001) 8306–8316. [16] X.J. Wang, Role of TGFbeta signaling in skin carcinogenesis, Microsc. Res. Tech. 52 (2001) 420–429. [17] D. Stuhlmann, N. Ale-Agha, R. Reinehr, H. Steinbrenner, M.C. Ramos, H. Sies, P. Brenneisen, Modulation of homologous gap junctional intercellular communication of human dermal fibroblasts via a paracrine factor(s) generated by squamous tumor cells, Carcinogenesis 24 (2003) 1737–1748. [18] R. Fleischmajer, J.S. Perlish, T. Krieg, R. Timpl, Variability in collagen and fibronectin synthesis by scleroderma fibroblasts in primary culture, J. Invest. Dermatol. 76 (1981) 400–403. [19] K. Bayreuther, P.I. Francz, J. Gogol, K. Kontermann, Terminal differentiation, aging, apoptosis, and spontaneous transformation in fibroblast stem cell systems in vivo and in vitro, Ann. N. Y. Acad. Sci. 663 (1992) 167–179. [20] P. Boukamp, W. Tilgen, R.T. Dzarlieva, D. Breitkreutz, D. Haag, R.K. Riehl, A. Bohnert, N.E. Fusenig, Phenotypic and genotypic characteristics of a cell line from a squamous cell carcinoma of human skin, J. Natl. Cancer Inst. 68 (1982) 415– 427. [21] Y.S. Bae, S.W. Kang, M.S. Seo, I.C. Baines, E. Tekle, P.B. Chock, S.G. Rhee, Epidermal growth factor (EGF)-induced generation of hydrogen peroxide. Role in EGF receptor-mediated tyrosine phosphorylation, J. Biol. Chem. 272 (1997) 217–221. [22] E. Whitley, J. Ball, Statistics review 6: nonparametric methods, Crit. Care 6 (2002) 509–513. [23] R. Govinden, K.D. Bhoola, Genealogy, expression, and cellular function of transforming growth factor-beta, Pharmacol. Ther. 98 (2003) 257–265. [24] E. Piek, C.H. Heldin, P. ten Dijke, Specificity, diversity, and regulation in TGF-beta superfamily signaling, FASEB J. 13 (1999) 2105–2124.
326
D. Stuhlmann et al. / Biochemical and Biophysical Research Communications 319 (2004) 321–326
[25] R. Derynck, R.J. Akhurst, A. Balmain, TGF-beta signaling in tumor suppression and cancer progression, Nat. Genet. 29 (2001) 117–129. [26] K. Airola, N.E. Fusenig, Differential stromal regulation of MMP1 expression in benign and malignant keratinocytes, J. Invest. Dermatol. 116 (2001) 85–92. [27] Q. Yu, I. Stamenkovic, Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-beta and promotes tumor invasion and angiogenesis, Genes Dev. 14 (2000) 163–176. [28] A. Balmain, R.J. Akhurst, Dangerous liaisons, Nature 428 (2004) 271–272. [29] N.A. Bhowmick, A. Chytil, D. Plieth, A.E. Gorska, N. Dumont, S. Shappell, M.K. Washington, E.G. Neilson, H.L. Moses, TGFbeta signaling in fibroblasts modulates the oncogenic potential of adjacent epithelia, Science 303 (2004) 848–851. [30] M.P. Lewis, K.A. Lygoe, M.L. Nystrom, W.P. Anderson, P.M. Speight, J.F. Marshall, G.J. Thomas, Tumour-derived TGF-beta1 modulates myofibroblast differentiation and promotes HGF/SFdependent invasion of squamous carcinoma cells, Br. J. Cancer 90 (2004) 822–832. [31] P.A. Robe, B. Rogister, M.P. Merville, V. Bours, Growth regulation of astrocytes and C6 cells by TGFbeta1: correlation with gap junctions, Neuroreport 11 (2000) 2837–2841. [32] D.M. Larson, T.G. Christensen, G.D. Sagar, E.C. Beyer, TGFbeta1 induces an accumulation of connexin43 in a lysosomal compartment in endothelial cells, Endothelium 8 (2001) 255–260. [33] B.V. Madhukar, S.Y. Oh, C.C. Chang, M. Wade, J.E. Trosko, Altered regulation of intercellular communication by epidermal growth factor, transforming growth factor-beta and peptide hormones in normal human keratinocytes, Carcinogenesis 10 (1989) 13–20. [34] P.E. Maldonado, B. Rose, W.R. Loewenstein, Growth factors modulate junctional cell-to-cell communication, J. Membr. Biol. 106 (1988) 203–210.
[35] L.E. Wyatt, C.Y. Chung, B. Carlsen, A. Iida-Klein, G.H. Rudkin, K. Ishida, D.T. Yamaguchi, T.A. Miller, Bone morphogenetic protein-2 (BMP-2) and transforming growth factor-beta1 (TGFbeta1) alter connexin 43 phosphorylation in MC3T3-E1 cells, BMC Cell. Biol. 2 (2001) 14. [36] O.I. Aruoma, B. Halliwell, B.M. Hoey, J. Butler, The antioxidant action of N-acetylcysteine: its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid, Free Radic. Biol. Med. 6 (1989) 593–597. [37] M. Zafarullah, W.Q. Li, J. Sylvester, M. Ahmad, Molecular mechanisms of N-acetylcysteine actions, Cell. Mol. Life Sci. 60 (2003) 6–20. [38] G.S. Kelly, Clinical applications of N-acetylcysteine, Altern. Med. Rev. 3 (1998) 114–127. [39] A.C. White, E.K. Maloney, S.L. Lee, J.J. Lanzillo, B.L. Fanburg, Reduction of endothelial cell related TGFbeta activity by thiols, Endothelium 6 (1999) 231–239. [40] B.L. Upham, K.S. Kang, H.Y. Cho, J.E. Trosko, Hydrogen peroxide inhibits gap junctional intercellular communication in glutathione sufficient but not glutathione deficient cells, Carcinogenesis 18 (1997) 37–42. [41] V.J. Thannickal, K.D. Aldweib, B.L. Fanburg, Tyrosine phosphorylation regulates H2 O2 production in lung fibroblasts stimulated by transforming growth factor beta1, J. Biol. Chem. 273 (1998) 23611–23615. [42] R.M. Liu, Y. Liu, H.J. Forman, M. Olman, M.M. Tarpey, Glutathione regulates transforming growth factor-beta-stimulated collagen production in fibroblasts, Am. J. Physiol. Lung Cell. Mol. Physiol. 286 (2004) L121–L128. [43] M. de Caestecker, The transforming growth factor-beta superfamily of receptors, Cytokine Growth Factor Rev. 15 (2004) 1–11. [44] C.H. Heldin, K. Miyazono, P. Ten Dijke, TGF-beta signalling from cell membrane to nucleus through SMAD proteins, Nature 390 (1997) 465–471.
Paracrine effect of TGF-b1 on downregulation of gap junctional intercellular communication between human dermal fibroblastsq Dominik Stuhlmann,1 Holger Steinbrenner, Bernhard Wendlandt, Dragana Mitic, Helmut Sies, and Peter Brenneisen* Institute for Biochemistry and Molecular Biology I, Heinrich-Heine-University, D-40225 D€usseldorf, Germany Received 29 April 2004 Available online 14 May 2004
Abstract Disruption of gap junctional intercellular communication (GJIC) is associated with tumor progression during multistage carcinogenesis. A coordinated interaction of epithelial tumor cells with the stromal environment via growth factors is a prerequisite for tumor invasion. Here, the involvement of growth factors in downregulation of homologous GJIC of dermal fibroblasts, used as model for stromal cells, was examined. Tumor cell derived transforming growth factor-b1 (TGF-b1), having oncogenic activities at late stages of carcinogenesis, was identified as being responsible for downregulation of GJIC via an increase in the level of reactive oxygen species in stromal fibroblasts. Lowering the level of reactive oxygen species by antioxidants, such as the cell-permeable N-acetyl-L -cysteine, prevented TGF-b1-mediated downregulation of intercellular communication between confluent fibroblasts. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Gap junction; Intercellular communication; Fibroblast; Squamous tumor cell; Tumor–stroma; Transforming growth factor-b; Reactive oxygen species; Skin cancer
Disruption of homologous and heterologous gap junctional intercellular communication (GJIC) is a hallmark of carcinogenesis [1], while induction of GJIC in some types of cancer cells leads to a reversal of the cancer phenotype [2,3]. This direct pathway of cellular crosstalk is provided by gap junctions, each comprised of two hemichannels (connexons) which connect the cytoplasm of neighboring cells. The carcinogenic process involves the transition from a normal, GJIC-competent cell to one which is defective in GJIC, finally
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Abbreviations: TGF-b1, transforming growth factor-b1; GJIC, gap junctional intercellular communication; Cx, connexin; HDF, human dermal fibroblasts; FCS, fetal calf serum; H2DCF-DA, 20 ,70 dichlorodihydrofluorescein diacetate; ROS, reactive oxygen species; NAC, N-acetyl-L -cysteine. * Corresponding author. Fax: +49-211-811-3029. E-mail address: [email protected] (P. Brenneisen). 1 This work is part of D. Stuhlmann’s Ph.D. thesis at the University of D€ usseldorf. 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.05.004
resulting in breakdown of intercellular communication between tumor cells during tumor promotion [1,2,4]. In cancer, interactions of tumor cells with their microenvironment and the influence of tumor on stroma and vice versa are essential for tumor survival and drive invasion and metastasis [5,6]. In melanoma and carcinoma, a wide variety of different cytokines and growth factors are expressed by tumor cells and stromal cells such as endothelial cells and fibroblasts which promote tumor growth as well as migration during tumor invasion [7–9]. Among the autocrine and paracrine acting growth factors involved in molecular processes of tumor–stroma interaction, transforming growth factor-b1 (TGFb1), a 25 kDa homodimeric protein, plays a pivotal role. Many studies led to the elucidation of a TGF-b1 signal transduction network indicating that this growth factor has a pleiotropic effect mediating a diversity of biological processes on different cell types [10–12]. Reactive oxygen species (ROS) generated by TGF-b1 may initiate
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signaling pathways [13,14]. Furthermore, it has been postulated that TGF-1 acts as tumor promoter at late stages of carcinogenesis promoting invasion and metastasis [15,16]. Based on our recent data on downregulation of homologous GJIC of stromal fibroblasts in tumor–stroma interaction [17] which is mediated by tumor cell derived factor(s) of 20–30 kDa, we addressed the question of whether TGF-b1 might be the factor mediating this effect on fibroblast via a paracrine mechanism. Based on our results, we add a novel aspect of TGF-b1 effects, namely, that tumor cell derived TGF-b1 downregulates homologous GJIC of dermal fibroblasts in vitro.
Materials and methods Materials. Cell culture medium (Dulbecco’s modified Eagle’s medium (DMEM)) was purchased from Invitrogen GmbH (Karlsruhe, Germany) and the defined fetal calf serum (FCS gold) was from PAA Laboratories (Linz, Austria). All chemicals and biochemicals were obtained from Sigma (Deisenhofen, Germany) unless otherwise indicated. The protein assay kit (Bio-Rad DC, detergent compatible) was from Bio-Rad Laboratories GmbH (M€ unchen, Germany). The dye 20 ,70 -dichlorodihydrofluorescein diacetate (H2 DCF-DA) was supplied from MoBiTec (G€ ottingen, Germany). TGF-b1 ELISA was delivered by R&D Systems (Wiesbaden, Germany) and N-acetyl-L -cysteine (NAC) by Merck Biosciences (Schwalbach, Germany). Culture of human dermal fibroblasts. Human dermal fibroblasts (HDF) were established from foreskin biopsies of healthy donors [18] with an age of 4–6 years. Cells were used at passages 4–9 corresponding to cumulative population doubling levels of 7–20 and passaged as described earlier [19]. Fibroblasts and the squamous carcinoma cell line SCL-1, originally derived from facial skin of a 74year-old woman [20] (generously provided by Dr. Norbert E. Fusenig, DKFZ Heidelberg, Germany), were maintained in DMEM supplemented with glutamine (2 mM), penicillin (400 U/ml), streptomycin (50 mg/ml), and 10% defined FCS in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. SCL-1 cells were passaged twice a week. Enzyme linked immunosorbent assay of TGF-b1. Supernatants of confluent HDF and SCL-1 cells were harvested at 48 h after serum starvation. The amount of secreted active and latent TGF-b1 was measured by a 96-well microplate enzyme linked immunosorbent assay (ELISA) system for active TGF-b1 according to the manufacturer’s instruction. Briefly, total TGF-b1 was determined by acid activation of latent TGF-b1. One hundred microliters per sample of acid activated or untreated (active) TGF-b1 was captured by a mouse anti-human TGF-b1 antibody. The captured growth factor was detected by a biotinylated chicken anti-human TGF-b1 antibody followed by incubation with streptavidin conjugated horseradish peroxidase, substrate incubation, and measurement of optical density at 450 nm. The percentage of active versus latent TGF-b1 in the supernatants was calculated by the amount of TGF-b1 of untreated samples compared with the total TGF-b1 amount. Gap junctional communication assay (Lucifer Yellow transfer). Serum-free supernatants of confluent fibroblasts, which are called “conditioned medium,” and of subconfluent SCL-1 cells were harvested after 24 or 48 h and put on confluent HDF monolayer cultures which were grown in complete DMEM + 10% FCS. Prior to addition of the supernatants, serum-containing medium was removed and the cells were washed with phosphate-buffered saline (PBS). At different time points thereafter, the cells were used for microinjection. GJIC was measured by microinjection of the fluorescent dye Lucifer Yellow CH (10% in 0.33 M LiCl) into selected fibroblasts by means of a micro-
manipulator and a microinjector system (Eppendorf, Hamburg). One minute after injection, the number of fluorescent cells around a single cell loaded with the dye was counted. Ten individual cells per dish were injected and medians, 25% quartile, and 75% quartile were calculated (see below). Measurement of intracellular ROS. The intracellular ROS level was measured using the fluorescent dye H2 DCF-DA which is readily diffusible into cells, where it is hydrolyzed to the non-fluorescent polar derivative H2 DCF and thereby trapped within cells [21]. In the presence of an oxidant, H2 DCF is converted into the highly fluorescent 20 ,70 -dichlorofluorescein (DCF). For assays, confluent fibroblast monolayer cultures were loaded with 20 lM H2 DCF-DA in PSG buffer (100 mM KH2 PO4 , 10 mM NaCl, and 5 mM glucose; pH 7.4) for 15 min in the dark. After washing three times with PSG buffer, the loaded cells were subjected to 10 ng TGF-b1/ml PSG or supernatants of tumor cells which were diluted in that buffer. ROS generation was detected as a result of the oxidation of H2 DCF and the fluorescence (excitation 488 nm; emission 515–540 nm), given in arbitrary units, was followed with a Zeiss axiovert fluorescent microscope with a chargecoupled device (CCD) camera (ORCA II, Hamamatsu, Herrsching, Germany) for additional 20 min. To prevent growth factor-mediated increase in intracellular ROS level, confluent fibroblasts were incubated with 5 mM N-acetyl-L -cysteine for 4 h prior to treatment with H2 DCF-DA, recombinant TGFb1, or supernatants. The used concentrations of H2 DCF-DA and NAC neither showed any cytotoxic effect nor a change in cell morphology (data not shown). Statistical analysis. In order to quantify the distribution of the number of communicating cells, box-and-whisker plots (box plot) were used which highlight and combine important parameters describing the scatter of the sample data: median (line inside the box), 25% quartile (lower boundary of the box), 75% quartile (upper boundary of the box), minimum and maximum values (vertical lines from the box, “whiskers”). In this study, box plots indicate non-Gaussian distribution. Therefore, for analysis of statistical significance, non-parametric tests such as Kruskal–Wallis test were applied [22]. Before the study, P < 0:05, P < 0:01, and P < 0:001 were selected as the level of significance.
Results and discussion Tumor cells release high amounts of active TGF-b1 responsible for downregulation of homologous GJIC of fibroblasts As tumor progression is concomitant with activation of the local host stroma via molecular crosstalk between tumor and stromal cells mediated by cytokines and growth factors, among them TGF-b1 [5], we measured the amounts of TGF-b1 in the supernatants of dermal fibroblast as well as of SCL-1 tumor cells. For fibroblasts, 19% of total TGF-b1, which was set at 100%, reflected the fraction of active TGF-b1, whilst the active fraction released from the tumor cells was about 8%. However, tumor cells released a 16-fold higher amount of total TGF-b1, and, therefore, a 6-fold higher amount of active TGF-b1 compared to stromal fibroblasts (Fig. 1). The bioactive TGF-b1 results from dissociation of the latent inactive TGF-b complex consisting of the non-covalently bound latency associated peptide (LAP) dimer to the active TGF-b1 dimer which are secreted as
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Fig. 1. Constitutive TGF-b1 levels of stromal and tumor cells. Supernatants of human dermal fibroblasts (HDF) and SCL-1 tumor cells were harvested 24 h after incubation of the cells with serum-free cell culture medium and subjected to TGF-b1 ELISA. Active and total TGF-b1 amounts were determined as described in Materials and methods. Data represent means standard deviation for three independent experiments.
LAP/TGF-b1 complex by epithelial and mesenchymal cells [23,24]. Furthermore, released latent TGF-b1 can be activated by thrombospondin, integrins, and matrix metalloproteinases [25], which are generated by stromal [26] as well as malignant cells [27]. Both epithelial cells and their fibroblast neighbors secrete TGF-b1 keeping cell proliferation in check, but, if the sensitive balance is disturbed by genetic or epigenetic events, cancer may develop [28,29]. Recently, Lewis et al. [30] demonstrated a TGF-b1-dependent transdifferentiation of fibroblasts secreting high levels of hepatocyte growth factor/scatter factor which promoted tumor invasion. This high amount of active TGF-b1 of tumor cells is responsible for downregulation of homologous GJIC of fibroblasts. To test this hypothesis, serum-free (-FCS) supernatants of subconfluent SCL-1 monolayer cultures were harvested after 2 days and added to fibroblast monolayer cultures for 24 h. As controls, serum-free conditioned medium was harvested after 2 days and added to confluent fibroblasts which were subjected to gap junctional communication assays (Fig. 2). In comparison with these controls (median ¼ 9.5), confluent fibroblasts incubated with supernatants of the tumor cells or serum-free medium containing recombinant TGF-b1 exhibited a significant lowering of GJIC, reflected by a median of 3.0 and 1.0, respectively, and interquartile ranges (indicated by the box length) of 2–4 and 0–1.5 which represent the middle 50% of the values. Treatment of the SCL-1 supernatants with anti-TGF-b1 neutralizing antibody or immunoprecipitation of the growth factor maintained homologous GJIC of fibroblasts compared with cells treated with SCL-1 supernatants or recombinant TGF-b1 (P < 0:01), which is indicated by the medians and the interquartile ranges from 7.0 to 14.5 and 9.0 to 11.5, respectively (Fig. 2).
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Fig. 2. TGF-b1-mediated modulation of homologous GJIC. Confluent fibroblasts (HDF) were incubated with serum-free (-FCS) conditioned medium (SN(2d) HDF) and supernatants of SCL-1 cells (SN(2d) SCL-1), both harvested after 2 days, or with 10 ng of recombinant TGF-b1/ml medium. In addition, TGF-b1 was neutralized or immunoprecipitated (IP, 2 h) with 1 lg anti-TGF-b1/ml medium. Twentyfour hours after treatment the number of Lucifer Yellow stained cells adjacent to the injected cell was used as a measure of GJIC. The upper and lower boundaries of the boxes are the upper and lower quartiles. The box length is the interquartile distance so the box contains the middle 50% of values (the horizontal line inside the box indicates the median). The vertical lines extend to maximum and minimum values. The values of the box plots represent the number of communicating cells of 20 microinjected cells (10 cells/dish). Three independent experiments were performed; **P < 0:01 (Kruskal–Wallis test).
Some contrary results of the effect of TGF-b1 on gap junctional intercellular communication were described which may result from different studied cell types. Robe et al. [31] demonstrated a TGF-b1-dependent increase in GJIC of rat astrocytes, although the GJIC of C6 glioblastoma cells, the tumorigenic counterpart, was inhibited by TGF-b1. While TGF-b1 triggered an increase in GJIC of bovine aortic endothelial cells [32], the opposite effect of TGF-b1 was observed for human keratinocytes [33] and rat kidney fibroblasts (NRK) [34]. In the osteoblastic cell line MC3T3-E1, TGF-b1 inhibited GJIC by decreasing the phosphorylated form of connexin 43 [35]. In our study, we demonstrate a lowering of homologous GJIC of fibroblasts which is triggered by TGF-b1 secreted by a highly invasive squamous tumor cell line. Supernatants of the cervix carcinoma cell line HeLa, the squamous cell line A431, and melanoma cells A375 showed similar results (data not shown) which indicates a crucial role of TGF-b1 in tumor progression. TGF-b1 mediates downregulation of homologous GJIC of fibroblasts via reactive oxygen species As TGF-b1 was shown to produce reactive oxygen species in human lung fibroblasts [13], we addressed the
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question of whether this factor mediates downregulation of homologous GJIC of fibroblasts via the involvement of ROS. Therefore, time course analysis of ROS generation after treatment of confluent HDF with recombinant TGF-b1 or SCL-1 supernatants was performed (Fig. 3). Incubation with both the TGF-b1 containing supernatant and recombinant TGF-b1 resulted in significant increase in DCF fluorescence which was maintained over the studied time range. This suggests that TGF-b1 increases intracellular ROS level in fibroblasts. A non-toxic concentration of 1 mM H2 O2 , which was used as technical control, further increased the intracellular ROS level. A non-toxic concentration of the antioxidant NAC (5 mM) prevented the TGF-b1and H2 O2 -mediated increase in the ROS level (Fig. 3). NAC is a thiol compound and a precursor of L -cysteine and reduced glutathione (GSH). As a source of sulfhydryl groups in cells, NAC may act directly on detoxification of reactive oxygen species such as hydrogen peroxide, hydroxyl radical, and superoxide [36,37]. NAC has beneficial effects in pathological conditions characterized by decreased GSH or oxidative stress, such as HIV infection, cancer, and heart disease [38]. However, to exclude a direct alteration of the structure of TGF-b1 by P10 mM NAC [39] resulting in a ROSindependent downregulation of homologous GJIC of fibroblasts, the water-soluble vitamin E analog Trolox
Fig. 3. Time course of TGF-b1 induced intracellular ROS production from human dermal fibroblasts. Confluent cells were incubated either with SCL-1 supernatants or 10 ng recombinant TGF-b1/ml medium, and 1 mM H2 O2 for the indicated time. Increase of DCF fluorescence was followed over 20 min. N-Acetyl-L -cysteine (NAC) at a concentration of 5 mM was added 4 h prior to measurement of DCF fluorescence. The experiment was performed in duplicate. Arrows (!) indicate incubation with SCL-1 supernatants, harvested after 2 days (SN(2d) SCL-1), or TGF-b1, or H2 O2 .
and butylated hydroxytoluene (BHT), a lipid-soluble antioxidant, were applied which protected the fibroblasts against TGF-b1-mediated inhibition of GJIC (data not shown). In accordance with this finding, Stuhlmann et al. [17] demonstrated that preincubation of the fibroblasts with selenite prevents tumor cellmediated downregulation of GJIC between fibroblasts dealing with selenium-dependent antioxidative enzymes such as phospholipid hydroperoxide glutathione peroxidase (PHGPX) or glutathione peroxidase (GPX) to be involved in this mechanism. To test the effect of ROS on intercellular communication, confluent fibroblasts were pretreated with a nontoxic concentration of NAC prior to incubation with conditioned medium or SCL-1 supernatants. The interquartile ranges of NAC pretreated cells overlapped with that of fibroblasts treated with conditioned medium (control) which suggests that ROS play a role in downregulation of GJIC between fibroblasts. By contrast, SCL-1 supernatants lowered this intercellular communication (Fig. 4A). Furthermore, we demonstrated that H2 O2 may be a potential candidate for a
Fig. 4. Effect of ROS on homologous GJIC of fibroblasts. Confluent fibroblasts (HDF) were incubated with serum-free conditioned medium (SN(1d) HDF) or serum-free supernatants of SCL-1 cells (SN(1d) SCL-1), both harvested after 1 day (A,B). In addition, the cells were preincubated with 5 mM NAC for 4 h (A) or with 0.1 and 1 mM H2 O2 for 1 h (B). The values of the box plots (see Fig. 2), measured after 24 h (A) or 1 h (B) after treatment, represent the number of communicating cells of 30 microinjected cells (10 cells/dish). Similar results were obtained in two independent experiments. ***P < 0:001, **P < 0:01 (Kruskal–Wallis test).
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dose-dependent downregulation of this homologous GJIC, because treatment of confluent fibroblasts with non-toxic concentrations of H2 O2 significantly lowered GJIC to control cells (Fig. 4B). Our data correspond to earlier results showing H2 O2 mediated inhibition of intercellular communication of WB-F344 rat liver epithelial cells [40]. Furthermore, a TGF-b1-mediated increase in intracellular H2 O2 levels in human lung fibroblasts [41] and murine embryo fibroblasts (NIH 3T3) [42] was measured. The network of TGF-b1 signaling involves receptor serine/threonine kinases at the cell surface [43] and their substrates, the so-called Smad proteins, which move into the nucleus and activate target gene transcription in association with further transcription factors [11,44]. In addition, TGF-b1 induces non-Smad signaling mechanisms [25]. In that context, TGF-b1 stimulation of human lung fibroblasts resulted in a transient burst of ROS which regulate downstream events such as Ca2þ -influx, mitogen-activated protein kinase (MAPK) activation, and phosphorylation-dependent activation of activating protein-1 (AP-1) which induced interleukin-6 gene expression [13]. Recently, it was shown that paracrine factor-mediated downregulation of GJIC between fibroblasts is independent of phosphorylation of the gap junctional protein connexin 43 [17]. In conclusion, we identified TGF-b1 to be the tumor cell derived growth factor which is responsible for the downregulation of homologous GJIC of stromal cells in vitro via the involvement of reactive oxygen species. As invasion and metastatic spread of tumor cells continue to be a barrier to cure cancer, understanding of the cellular interaction between tumor cells and its surrounding stroma could assist in the development of novel therapeutic strategies to combat metastasis more efficiently in the future.
Acknowledgments We are grateful to Claudia Wyrich for excellent technical assistance. H.S. is a Fellow of the National Foundation for Cancer Research (NFCR), Bethesda, MD, USA. This work was supported by Deutsche Krebshilfe e.V. (10-2223) and Deutsche Forschungsgemeinschaft SPP Selenoproteine (Si 255/11-3) and SFB 575/B4.
References [1] J.E. Trosko, R.J. Ruch, Cell–cell communication in carcinogenesis, Front. Biosci. 3 (1998) D208–D236. [2] M. Mesnil, Connexins and cancer, Biol. Cell 94 (2002) 493–500. [3] J.E. Trosko, R.J. Ruch, Gap junctions as targets for cancer chemoprevention and chemotherapy, Curr. Drug Targets 3 (2002) 465–482. [4] W.R. Loewenstein, Junctional intercellular communication and the control of growth, Biochim. Biophys. Acta 560 (1979) 1–65.
325
[5] L.A. Liotta, E.C. Kohn, The microenvironment of the tumour– host interface, Nature 411 (2001) 375–379. [6] M.M. Mueller, N.E. Fusenig, Tumor–stroma interactions directing phenotype and progression of epithelial skin tumor cells, Differentiation 70 (2002) 486–497. [7] O. de Wever, M. Mareel, Role of tissue stroma in cancer cell invasion, J. Pathol. 200 (2003) 429–447. [8] N.E. Fusenig, P. Boukamp, Multiple stages and genetic alterations in immortalization, malignant transformation, and tumor progression of human skin keratinocytes, Mol. Carcinog. 23 (1998) 144–158. [9] E. Lazar-Molnar, H. Hegyesi, S. Toth, A. Falus, Autocrine and paracrine regulation by cytokines and growth factors in melanoma, Cytokine 12 (2000) 547–554. [10] E.P. Bottinger, J.J. Letterio, A.B. Roberts, Biology of TGF-beta in knockout and transgenic mouse models, Kidney Int. 51 (1997) 1355–1360. [11] J. Massague, TGF-beta signal transduction, Annu. Rev. Biochem. 67 (1998) 753–791. [12] M.S. Pepper, Transforming growth factor-beta: vasculogenesis, angiogenesis, and vessel wall integrity, Cytokine Growth Factor Rev. 8 (1997) 21–43. [13] E. Junn, K.N. Lee, H.R. Ju, S.H. Han, J.Y. Im, H.S. Kang, T.H. Lee, Y.S. Bae, K.S. Ha, Z.W. Lee, S.G. Rhee, I. Choi, Requirement of hydrogen peroxide generation in TGF-beta 1 signal transduction in human lung fibroblast cells: involvement of hydrogen peroxide and Ca2þ in TGF-beta 1-induced IL-6 expression, J. Immunol. 165 (2000) 2190–2197. [14] V.J. Thannickal, R.M. Day, S.G. Klinz, M.C. Bastien, J.M. Larios, B.L. Fanburg, Ras-dependent and -independent regulation of reactive oxygen species by mitogenic growth factors and TGF-beta1, FASEB J. 14 (2000) 1741–1748. [15] C. Berking, R. Takemoto, H. Schaider, L. Showe, K. Satyamoorthy, P. Robbins, M. Herlyn, Transforming growth factor-beta1 increases survival of human melanoma through stroma remodeling, Cancer Res. 61 (2001) 8306–8316. [16] X.J. Wang, Role of TGFbeta signaling in skin carcinogenesis, Microsc. Res. Tech. 52 (2001) 420–429. [17] D. Stuhlmann, N. Ale-Agha, R. Reinehr, H. Steinbrenner, M.C. Ramos, H. Sies, P. Brenneisen, Modulation of homologous gap junctional intercellular communication of human dermal fibroblasts via a paracrine factor(s) generated by squamous tumor cells, Carcinogenesis 24 (2003) 1737–1748. [18] R. Fleischmajer, J.S. Perlish, T. Krieg, R. Timpl, Variability in collagen and fibronectin synthesis by scleroderma fibroblasts in primary culture, J. Invest. Dermatol. 76 (1981) 400–403. [19] K. Bayreuther, P.I. Francz, J. Gogol, K. Kontermann, Terminal differentiation, aging, apoptosis, and spontaneous transformation in fibroblast stem cell systems in vivo and in vitro, Ann. N. Y. Acad. Sci. 663 (1992) 167–179. [20] P. Boukamp, W. Tilgen, R.T. Dzarlieva, D. Breitkreutz, D. Haag, R.K. Riehl, A. Bohnert, N.E. Fusenig, Phenotypic and genotypic characteristics of a cell line from a squamous cell carcinoma of human skin, J. Natl. Cancer Inst. 68 (1982) 415– 427. [21] Y.S. Bae, S.W. Kang, M.S. Seo, I.C. Baines, E. Tekle, P.B. Chock, S.G. Rhee, Epidermal growth factor (EGF)-induced generation of hydrogen peroxide. Role in EGF receptor-mediated tyrosine phosphorylation, J. Biol. Chem. 272 (1997) 217–221. [22] E. Whitley, J. Ball, Statistics review 6: nonparametric methods, Crit. Care 6 (2002) 509–513. [23] R. Govinden, K.D. Bhoola, Genealogy, expression, and cellular function of transforming growth factor-beta, Pharmacol. Ther. 98 (2003) 257–265. [24] E. Piek, C.H. Heldin, P. ten Dijke, Specificity, diversity, and regulation in TGF-beta superfamily signaling, FASEB J. 13 (1999) 2105–2124.
326
D. Stuhlmann et al. / Biochemical and Biophysical Research Communications 319 (2004) 321–326
[25] R. Derynck, R.J. Akhurst, A. Balmain, TGF-beta signaling in tumor suppression and cancer progression, Nat. Genet. 29 (2001) 117–129. [26] K. Airola, N.E. Fusenig, Differential stromal regulation of MMP1 expression in benign and malignant keratinocytes, J. Invest. Dermatol. 116 (2001) 85–92. [27] Q. Yu, I. Stamenkovic, Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-beta and promotes tumor invasion and angiogenesis, Genes Dev. 14 (2000) 163–176. [28] A. Balmain, R.J. Akhurst, Dangerous liaisons, Nature 428 (2004) 271–272. [29] N.A. Bhowmick, A. Chytil, D. Plieth, A.E. Gorska, N. Dumont, S. Shappell, M.K. Washington, E.G. Neilson, H.L. Moses, TGFbeta signaling in fibroblasts modulates the oncogenic potential of adjacent epithelia, Science 303 (2004) 848–851. [30] M.P. Lewis, K.A. Lygoe, M.L. Nystrom, W.P. Anderson, P.M. Speight, J.F. Marshall, G.J. Thomas, Tumour-derived TGF-beta1 modulates myofibroblast differentiation and promotes HGF/SFdependent invasion of squamous carcinoma cells, Br. J. Cancer 90 (2004) 822–832. [31] P.A. Robe, B. Rogister, M.P. Merville, V. Bours, Growth regulation of astrocytes and C6 cells by TGFbeta1: correlation with gap junctions, Neuroreport 11 (2000) 2837–2841. [32] D.M. Larson, T.G. Christensen, G.D. Sagar, E.C. Beyer, TGFbeta1 induces an accumulation of connexin43 in a lysosomal compartment in endothelial cells, Endothelium 8 (2001) 255–260. [33] B.V. Madhukar, S.Y. Oh, C.C. Chang, M. Wade, J.E. Trosko, Altered regulation of intercellular communication by epidermal growth factor, transforming growth factor-beta and peptide hormones in normal human keratinocytes, Carcinogenesis 10 (1989) 13–20. [34] P.E. Maldonado, B. Rose, W.R. Loewenstein, Growth factors modulate junctional cell-to-cell communication, J. Membr. Biol. 106 (1988) 203–210.
[35] L.E. Wyatt, C.Y. Chung, B. Carlsen, A. Iida-Klein, G.H. Rudkin, K. Ishida, D.T. Yamaguchi, T.A. Miller, Bone morphogenetic protein-2 (BMP-2) and transforming growth factor-beta1 (TGFbeta1) alter connexin 43 phosphorylation in MC3T3-E1 cells, BMC Cell. Biol. 2 (2001) 14. [36] O.I. Aruoma, B. Halliwell, B.M. Hoey, J. Butler, The antioxidant action of N-acetylcysteine: its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid, Free Radic. Biol. Med. 6 (1989) 593–597. [37] M. Zafarullah, W.Q. Li, J. Sylvester, M. Ahmad, Molecular mechanisms of N-acetylcysteine actions, Cell. Mol. Life Sci. 60 (2003) 6–20. [38] G.S. Kelly, Clinical applications of N-acetylcysteine, Altern. Med. Rev. 3 (1998) 114–127. [39] A.C. White, E.K. Maloney, S.L. Lee, J.J. Lanzillo, B.L. Fanburg, Reduction of endothelial cell related TGFbeta activity by thiols, Endothelium 6 (1999) 231–239. [40] B.L. Upham, K.S. Kang, H.Y. Cho, J.E. Trosko, Hydrogen peroxide inhibits gap junctional intercellular communication in glutathione sufficient but not glutathione deficient cells, Carcinogenesis 18 (1997) 37–42. [41] V.J. Thannickal, K.D. Aldweib, B.L. Fanburg, Tyrosine phosphorylation regulates H2 O2 production in lung fibroblasts stimulated by transforming growth factor beta1, J. Biol. Chem. 273 (1998) 23611–23615. [42] R.M. Liu, Y. Liu, H.J. Forman, M. Olman, M.M. Tarpey, Glutathione regulates transforming growth factor-beta-stimulated collagen production in fibroblasts, Am. J. Physiol. Lung Cell. Mol. Physiol. 286 (2004) L121–L128. [43] M. de Caestecker, The transforming growth factor-beta superfamily of receptors, Cytokine Growth Factor Rev. 15 (2004) 1–11. [44] C.H. Heldin, K. Miyazono, P. Ten Dijke, TGF-beta signalling from cell membrane to nucleus through SMAD proteins, Nature 390 (1997) 465–471.