Removal of cell surface heparan sulfate increases TACE activity and cleavage of ErbB4 receptor
© Määttä et al; licensee BioMed Central Ltd. 2009
Received: 06 June 2008
Accepted: 26 January 2009
Published: 26 January 2009
Nuclear localization of proteolytically formed intracellular fragment of ErbB4 receptor tyrosine kinase has been shown to promote cell survival, and nuclear localization of ErbB4 receptor has been described in human breast cancer. Tumor necrosis factor alpha converting enzyme (TACE) initiates the proteolytic cascade leading to ErbB4 intracellular domain formation. Interactions between matrix metalloproteases and heparan sulfate have been described, but the effect of cell surface heparan sulfate on TACE activity has not been previously described.
As indicated by immunodetection of increased ErbB4 intracellular domain formation and direct enzyme activity analysis, TACE activity was substantially amplified by enzymatic removal of cell surface heparan sulfate but not chondroitin sulfate.
In this communication, we suggest a novel role for cell surface heparan sulfate. Removal of cell surface heparan sulfate led to increased formation of ErbB4 intracellular domain. As ErbB4 intracellular domain has previously been shown to promote cell survival this finding may indicate a novel mechanism how HS degradation active in tumor tissue may favor cell survival.
Heparan sulfate (HS) is a sulfated polysaccharide which consists of glucosamine and glucuronic or iduronic acid disaccharide units. Several HS chains are attached to a syndecan or glypican protein core. HS has been found to bind and regulate the activity of various extracellular matrix metalloproteases (MMP) such as MMP-1, MMP-2, MMP-7, MMP-9 and MMP-13 . In Alzheimer's disease the activity of BACE1, an enzyme responsible for the production of the amyloidogenic peptide, has been shown to be directly regulated by interactions with HS .
Cell surface proteases take part in cell signaling by i) producing soluble extracellular mediators such as growth factors, chemokines and cytokines from membrane bound precursors  and ii) generating intracellular signaling molecules from transmembrane protein receptors [4, 5]. One such cell surface protease is tumor necrosis factor alpha (TNF-α) converting enzyme, TACE .
TACE has also been shown to cleave various cell surface receptors including ErbB4 . ErbB4 receptor belongs to the EGF receptor family of receptor tyrosine kinases (RTKs), which share homology with the avian erythroblastosis virus oncogenic factor v-erbB. It has been shown to stimulate cell survival, proliferation and differentiation [7, 8].
Four different ErbB4 isoforms can be generated by alternative splicing [9, 10]. The juxtamembrane isoform JM-a is recognized and cleaved by TACE, whereas JM-b is not cleavable . The cytoplasmic isoforms either bind (JM-a CYT-1, JM-b CYT-1) or can not bind (JM-a CYT-2, JM-b CYT-2 phosphoinositide 3-kinase (PI 3-K) . Proteolytically produced ErbB4 CYT-2 80 kDa fragments has been shown to favor cell survival .
The expression of heparan sulfate cleaving β-endoglucuronidase, heparanase, is tightly controlled in normal tissues [11, 12]. However, in inflammed or cancer tissue and in several cancer cell lines the expression of heparanase has been shown to be elevated [13–15] and the high expression of heparanase has been linked to highly invasive cancers [15–17]. In breast cancer nuclear localization of ErbB4 receptor has been demonstrated and nuclear ErbB4 expression has been shown to be associated with unfavorable disease prognosis when compared to membraneous ErbB4 expression .
In this communication we describe for the first time that removal of cell surface HS increases membrane bound ErbB4 80 kDa fragment formation by TACE-like activity. Further, removal of cell surface HS enhanced the capability of living cells to cleave synthetic TACE substrate peptide suggesting that cell surface HS regulates TACE activity.
Preparation of expression constructs
Hemagglutinin (HA) -tagged human TACE encoding vector  was a generous gift from Professor A. Ullrich, Max Planck Institute, Germany. HA-tagged ErbB4 JM-a and JM-b CYT-2 receptor constructs were generated as follows: Sequence encoding the aminoterminal part of the full length receptor  was joined in front of a sequence encoding HA-tag at the C-terminus of ErbB4 CYT2 80 kDa fragment . Flag-tagged syndecan-4 expression construct in pcDNA3.1 Neo vector (Promega, USA) was generated by trimming the flag-peptide encoding sequence to the 3'-terminus of syndecan-4 cDNA derived from MCF-7 cells.
Generation and maintenance of the MCF-7 human breast cancer cells expressing human ErbB4 JM-a CYT-2 has been described previously . T47D cells were maintained in RPMI-1640 medium supplemented with 10 FCS and glutamine. For immunoblot analysis cells were grown to 40–50% confluency on 6-well tissue culture plates. For confocal microscopy, cells were grown on coverslips in flat-bottomed 24-well tissue culture plates. Transfections were done by using Fugene-6 transfection reagent (Roche, Switzerland).
Preparation and analysis of cell lysates
Cells grown to 70% confluency on 6-well tissue culture plates were washed twice with PBS prior to treatments. Incubations with enzymes and control treatments were performed at +37°C for 30 min in PBS supplemented with 10 μM calcium acetate and 10 mM glucose. Heparitinase and chondroitinase (Seikagaku, Japan) were used at 0.01 U/ml. After incubation cells were lysed as described previously  in the presence of TACE inhibitor 2 mM 1,10 orto-phenanthroline (Sigma-Aldrich, USA) . For immunoprecipitation the NaCl concentration of the lysates were adjusted to 150 mM. The precipitations and immunoblots were performed as previously described .
Measurement of TACE activity
Recombinant human TACE and TACE substrate peptide (Fluorescent substrate peptide III) were purchased from R&D Systems (USA). The amount of generated fluorescence was measured according to manufacturer's instructions after 60 min incubation at +37°C in the presence or absence of increasing concentrations of bovine lung heparin (Sigma-Aldrich, USA) or bovine kidney HS (Sigma-Aldrich, USA). TACE activity on living MCF-7 cells was measured after cells were grown to 80% confluency on flat-bottomed 96-well plates. Cells were incubated for 30 min in PBS containing 10 mM glucose in the presence or absence of 0.001 U/ml heparitinase or chondroitinase (Seikagaku, Japan). After incubation, cells were washed and the generation of fluorescent end product was followed at 5 min intervals. The enzyme assay buffer was supplemented with 0.2 mM phenylmethyl sulphonium fluoride and 5 mM EDTA.
Cells grown on coverslips were fixed and permeabilized with ice-cold methanol. Primary antibodies were used as following dilutions: HFR-1 monoclonal mouse anti-ErbB4 intracellular fragment (Neomarkers, USA) at 1:50; monoclonal rat anti-hemagglutinin 12CA5 epitope and monoclonal mouse anti-myc 9E10 epitope at 1:100 (Zymed, USA). Secondary antibodies were diluted in 10% FBS-PBS as follows: Alexa-568 goat anti-rat at 1:400; Alexa-488 goat anti-mouse at 1:400 (Molecular Probes, USA). Coverslips were embedded on Vecta Shield Hard Set mounting solution containing DAPI. Samples were analyzed with Zeiss LSM-510 Meta confocal microscope.
Results and Discussion
Two mechanisms for HS-mediated regulation of TACE-activity can be postulated. HS may hinder TACE-mediated ErbB4 cleavage by forming steric barriers between the enzyme and its substrate cleavage site or directly inhibiting TACE activity with interactions between the enzyme and HS side chains. As soluble heparin and HS inhibited cleavage of fluorescent substrate peptide by recombinant TACE enzyme, the presence of the latter mechanism is suggested. This is further supported by the fact that exogenous heparin and HS hindered the HS degradation induced ErbB4 cleavage.
In this communication removal of cell surface HS was shown to increase TACE activity and TACE-dependent formation of ErbB4 80 kDa intracellular domain. The high HS degrading activity reportedly present in tumor tissues [13–17] thus probably favors TACE-activity which may lead to elevated processing of ErbB4 and promotion of cell survival. Analogous mechanisms may be active also in inflammed tissue and may also concern other proteolytically processed transmembrane proteins.
hemagglutinin tag peptide
tumor necrosis factor alpha converting enzyme
The authors wish to express their gratitude to Dr. Markku Salmivirta who passed away by the time of this research project. The authors thank Mrs. Taina Kalevo-Mattila for excellent technical assistance. This work was supported by The Finnish Academy, Sigrid Juselius Foundation, Finnish Cancer Foundations, K. A. Johansson's Foundation, Jenny and Antti Wihuri Foundation, Paulo Foundation and Turku University Foundation.
- Yu WH, Woessner JF: Heparan sulfate proteoglycans as extracellular docking molecules for matrilysin (matrix metalloproteinase 7). J Biol Chem. 2000, 275: 4183-4191. 10.1074/jbc.275.6.4183.View ArticlePubMed
- Scholefield Z, Yates EA, Wayne G, Amour A, McDowell W, Turnbull JE: Heparan sulfate regulates amyloid precursor protein processing by BACE1, the Alzheimer's beta-secretase. J Cell Biol. 2003, 163: 97-107. 10.1083/jcb.200303059.PubMed CentralView ArticlePubMed
- Killar L, White J, Black R, Peschon J: Adamalysins. A family of metzincins including TNF-alpha converting enzyme (TACE). Ann N Y Acad Sci. 1999, 878: 442-452. 10.1111/j.1749-6632.1999.tb07701.x.View ArticlePubMed
- Ni CY, Murphy MP, Golde TE, Carpenter G: gamma-Secretase cleavage and nuclear localization of ErbB-4 receptor tyrosine kinase. Science. 2001, 294: 2179-2181. 10.1126/science.1065412.View ArticlePubMed
- Komuro A, Nagai M, Navin NE, Sudol M: WW domain-containing protein YAP associates with ErbB-4 and acts as a co-transcriptional activator for the carboxyl-terminal fragment of ErbB-4 that translocates to the nucleus. J Biol Chem. 2003, 278: 33334-33341. 10.1074/jbc.M305597200.View ArticlePubMed
- Rio C, Buxbaum JD, Peschon JJ, Corfas G: Tumor necrosis factor-alpha-converting enzyme is required for cleavage of erbB4/HER4. J Biol Chem. 2000, 275: 10379-10387. 10.1074/jbc.275.14.10379.View ArticlePubMed
- Yarden Y, Sliwkowski MX: Untangling the ErbB signalling network. Nat Rev Mol Cel Biol. 2001, 2: 127-137. 10.1038/35052073.View Article
- Määttä JA, Sundvall M, Junttila TT, Peri L, Isola J, Egeblad M, Elenius K: Proteolytic cleavage and phosphorylation of tumor-associated ErbB4 isoform promote ligand-independent survival and cancer cell growth. Mol Biol Cell. 2006, 17: 67-79. 10.1091/mbc.E05-05-0402.PubMed CentralView ArticlePubMed
- Elenius K, Corfas G, Paul S, Choi CJ, Rio C, Plowman GD, Klagsbrun M: A novel juxtamembrane domain isoform of HER4/ErbB4. Isoform-specific tissue distribution and differential processing in response to phorbol ester. J Biol Chem. 1997, 272: 26716-26768. 10.1074/jbc.272.42.26761.View Article
- Elenius K, Choi CJ, Paul S, Santiestevan E, Nishi E, Klagsbrun M: Characterization of a naturally occurring ErbB4 isoform that does not bind or activate phosphatidyl inositol 3-kinase. Oncogene. 1999, 18: 2607-2615. 10.1038/sj.onc.1202612.View ArticlePubMed
- de Mestre AM, Rao S, Hornby JR, Soe-Htwe T, Khachigian LM, Hulett MD: Early growth response gene 1 (EGR1) regulates heparanase gene transcription in tumor cells. J Biol Chem. 2005, 280: 35136-35147. 10.1074/jbc.M503414200.View ArticlePubMed
- Vlodavsky I, Eldor A, Haimovitz-Friedman A, Matzner Y, Ishai-Michaeli R, Lider O, Naparstek Y, Cohen IR, Fuks Z: Expression of heparanase by platelets and circulating cells of the immune system: possible involvement in diapedesis and extravasation. Invasion Metastasis. 1992, 12: 112-127.PubMed
- Chen G, Wang D, Vikramadithyan R, Yagyu H, Saxena U, Pillarisetti S, Goldberg IJ: Inflammatory cytokines and fatty acids regulate endothelial cell heparanase expression. Biochemistry. 2004, 43: 4971-4977. 10.1021/bi0356552.View ArticlePubMed
- Gohji K, Okamoto M, Kitazawa S, Toyoshima M, Dong I, Katsuoka Y, Nakajima M: Heparanase protein and gene expression in bladder cancer. J Urol. 2001, 166: 1286-1290. 10.1016/S0022-5347(05)65754-0.View ArticlePubMed
- Maxhimer JB, Quiros RM, Stewart R, Dowlatshahi K, Gattuso P, Fan M, Prinz RA, Xu X: Heparanase-1 expression is associated with the metastatic potential of breast cancer. Surgery. 2002, 132: 326-333. 10.1067/msy.2002.125719.View ArticlePubMed
- Mikami S, Ohashi K, Usui Y, Nemoto T, Katsube K, Yanagishita M, Nakajima M, Nakamura K, Koike M: Loss of syndecan-1 and increased expression of heparanase in invasive esophageal carcinomas. Jpn J Cancer Res. 2001, 92: 1062-1073.View ArticlePubMed
- Koliopanos A, Friess H, Kleeff J, Shi X, Liao Q, Pecker I, Vlodavsky I, Zimmermann A, Buchler M: Heparanase expression in primary and metastatic pancreatic cancer. Cancer Res. 2001, 61: 4655-4659.PubMed
- Junttila TT, Sundvall M, Lundin M, Lundin J, Tanner M, Härkönen P, Joensuu H, Isola J, Elenius KE: Cleavable ErbB4 isoform in estrogen receptor-regulated growth of breast cancer cells. Cancer Res. 2005, 65: 1384-1393. 10.1158/0008-5472.CAN-04-3150.View ArticlePubMed
- Gschwind A, Hart S, Fischer OM, Ullrich A: TACE cleavage of proamphiregulin regulates GPCR-induced proliferation and motility of cancer cells. EMBO J. 2003, 22: 2411-2421. 10.1093/emboj/cdg231.PubMed CentralView ArticlePubMed
- Sundvall M, Peri L, Määttä JA, Tvorogov D, Paatero I, Savisalo M, Silvennoinen O, Yarden Y, Elenius K: Differential nuclear localization and kinase activity of alternative ErbB4 intracellular domains. Oncogene. 2007, 26: 6905-6914. 10.1038/sj.onc.1210501.View ArticlePubMed
- Schlöndorff J, Becherer JD, Blobel CP: Intracellular maturation and localization of the tumour necrosis factor alpha convertase (TACE). Biochem J. 2000, 347: 131-138. 10.1042/0264-6021:3470131.PubMed CentralView ArticlePubMed
- Vecchi M, Baulida J, Carpenter G: Selective Cleavage of the Heregulin Receptor ErbB-4 by Protein Kinase C Activation. J Biol Chem. 1996, 271: 18989-18995. 10.1074/jbc.271.31.18989.View ArticlePubMed
- Sundvall M, Korhonen A, Paatero I, Gaudio E, Melino G, Croce CM, Aqeilan RI, Elenius K: Isoform-specific monoubiquitination, endocytosis, and degradation of alternatively spliced ErbB4 isoforms. Proc Natl Acad Sci USA. 2008, 105: 4162-4167. 10.1073/pnas.0708333105.PubMed CentralView ArticlePubMed
- Holladay LA, Savage CR, Cohen S, Puett D: Conformation and unfolding thermodynamics of epidermal growth factor and derivatives. Biochemistry. 1976, 15: 2624-2633. 10.1021/bi00657a023.View ArticlePubMed
- Hovingh P, Linker A: The enzymatic degradation of heparin and heparin sulphate. J Biol Chem. 1970, 245: 6170-6175.PubMed
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.