1 Institute of Virology, University of Cologne, Fürst-Pückler-Str. 56, 50935 Cologne, Germany
2 Department of Dermatology, University of Cologne, Joseph-Stelzmann-Str. 9, 50924 Cologne, Germany
Correspondence
Sigrun Smola-Hess
s.smola{at}uni-koeln.de
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ABSTRACT |
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Present address: Paul Hartmann AG, PO Box 1420, D-89504 Heidenheim, Germany.
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MAIN TEXT |
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Malignant tumour cells have acquired mechanisms to degrade basement membrane structures and invade the underlying tissue. Tumour invasion finally determines the prognosis of the disease. Production of the matrix-degrading type IV collagenase matrix metalloproteinase (MMP)-9 and activation of MMP-2 have been correlated to metastatic disease progression (Azzam et al., 1993; Liotta et al., 1980
; Nuovo et al., 1995
). These so-called gelatinases are produced by the tumour cells and by surrounding stromal cells (Okada et al., 1995
). In contrast to these enzymes, collagenase-2 (MMP-8) may have an opposite role in carcinogenesis. Loss of MMP-8 even conferred increased skin tumour susceptibility to male mice (Balbin et al., 2003
). MMP-9 is regulated by many cytokines at the transcriptional level. We have recently shown that cervical carcinoma cells do not constitutively produce MMP-9; however, this protease can be induced by CD40 ligand (Smola-Hess et al., 2001
). In HPV16-transgenic mice, MMP-9 is mainly localized in haematopoietic cells within the tumour stroma (Coussens et al., 2000
). Pro-MMP-2, the zymogen of MMP-2, is constitutively produced in most cell types (reviewed by Ries & Petrides, 1995
). Total MMP activity in the tissue is regulated by complex post-translational mechanisms like proteolytic conversion and activation of the zymogen, interaction with extracellular matrix components and regulation by tissue inhibitors of metalloproteinases (TIMPs) (Goldberg et al., 1989
). MMP-2 activation is of particular interest for tumour cell invasion. This MMP has been extensively studied in pre-neoplastic and malignant lesions of the uterine cervix. It could be shown that MMP-2 expression and activation gradually increased during progression and correlated with poor prognosis (Brummer et al., 2002
; Davidson et al., 1999
; Gaiotto et al., 2004
; Sheu et al., 2003
). However, MMP-2, produced by the tumour cells or surrounding stromal cells, may be recruited to the surface membrane of tumour cells via membrane-type (MT) MMPs and it has been shown that MT MMPs concentrate MMP-2 at the front of tumour invasion (Mitra et al., 2003
; Sato et al., 1994
, 1996
). Active MMP-2 is also associated with the membrane fraction of human cervical cancer cells (Chattopadhyay et al., 2001
). Six MT MMPs were identified and, except for MT-4 MMP, all have the capacity to cleave MMP-2 (reviewed by Hernandez-Barrantes et al., 2002
; Seiki et al., 2003
). Pro-MT MMPs are activated by intracellular subtilisin-type serine proteases before they reach the cell surface (Pei & Weiss, 1995
). In addition, inactive MT MMPs may also be present on the cell surface. MT-1 MMP (MMP-14), which plays a major role in invasive activity of neoplastic cells (Sabeh et al., 2004
), has a high affinity for TIMP-2/pro-MMP-2 complexes. Pro-MMP-2 in these ternary complexes is activated by neighbouring free MT-1 MMP molecules not engaged in TIMP-2 or TIMP-2/pro-MMP-2 binding (Butler et al., 1998
; Itoh et al., 2001
; Strongin et al., 1995
).
MT-1 MMP expression has recently been detected in cervical cancer specimens in vivo and invasiveness has been correlated with high-level expression of MT-1 MMP (Gilles et al., 1996; Sheu et al., 2003
). However, it remains unclear whether MT-1 MMP is constitutively produced in HPV-positive cells or induced by environmental stimuli comparable to the MMP-9 expression pattern (Smola-Hess et al., 2001
). In this study, we first analysed MT-1 MMP expression in HPV-transformed keratinocytes and cervical carcinoma cell lines in vitro and then investigated the impact of HPV on MT-1 MMP expression in HaCaT cells, an HPV-negative cell line, and in primary human keratinocytes.
To study MT-1 MMP expression, the following cell lines were analysed: SiHa, CaSki (both HPV16-positive), the HPV18-positive SCC lines C4-1 and SW756 and adenocarcinoma cell line HeLa, as well as keratinocytes transformed in vitro with HPV16 (HPKIA; Durst et al., 1987) or HPV18 (K5-1). Total RNA was extracted from monolayer cell cultures using the RNAzol B (WAK-Chemie Medical) according to the manufacturer's instructions. Total RNA (20 µg) of each sample was analysed by Northern blot hybridization with a random-primed 32P-labelled cDNA probe for MT-1 MMP (kindly provided by Dr H. Sato, Knazawa University, School of Medicine, Knazawa, Japan) and subsequently with an 18S rRNA probe. All investigated cell lines expressed the 4·5 kb MT-1 MMP transcript constitutively (Fig. 1
) except the adenocarcinoma cell line HeLa, which has been shown to be negative for MT-1 MMP (Okada et al., 1995
). These data demonstrated that MT-1 MMP expression is constitutive, at least in the HPV-positive SCCs, and, unlike pro-MMP-9, does not require further exogenous activation. Interestingly, MT-1 MMP was also strongly expressed in non-malignant keratinocytes transformed with genital high-risk HPV. This suggested that the virus itself might contribute to MT-1 MMP expression, even in non-malignant keratinocytes.
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Mock-transfected clones expressed only low levels of MT-1 MMP mRNA, while for HaCaT clones expressing the HPV16 E7 gene product much stronger hybridization signals were observed, slightly varying among the clones analysed (Fig. 2a, upper left panel). To test MT-1 MMP protein expression levels in the E7 transfectants, we prepared membranes from all cell clones by ultracentrifugation on a 38 % sucrose cushion (model L565; Beckman Instruments) for 1 h at 100 000 g. MT-1 MMP protein was detected by Western blot analysis with anti MT-1 MMP mAb (2 µg ml1; Calbiochem) followed by detection with peroxidase-labelled goat anti-mouse Ab and the ECL substrate reaction (Amersham Pharmacia Biotech) following the manufacturer's instructions (Fig. 2b
). MT-1 MMP protein was expressed at higher levels in the HPV16 E7-transfected clones than in the mock-transfected controls, corresponding to the mRNA expression pattern. Furthermore, MT-1 MMP mRNA and protein were not only upregulated in HPV16 E7-expressing but also in HPV8 E7-expressing HaCaT clones (Fig. 2a and b
, right panels).
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This finding is interesting since it might explain high MT-1 MMP expression not only in carcinomas of the uterine cervix but also in papillomas, dysplastic skin and SCCs of mice transgenic for the early genes of HPV8 under the control of the cytokeratin 14 promoter (H. J. Pfister & C. Mauch, unpublished observations). Thus, the E7 protein from high-risk HPVs was able to induce MT-1 MMP in the context of the whole virus (HPKIA cells), the entire HPV early region (HPV8-transgenic mice) and in a p53-mutated keratinocyte cell line, as well as in primary human keratinocytes.
We next studied whether MT-1 MMP expression in the E7-transfected HaCaT clones led to activation of pro-MMP-2. HaCaT wild-type, mock and stably E7-transfected HaCaT keratinocytes were seeded in 24-well plates at a density of 3x105 cells per well in DMEM/10 % fetal calf serum. After 20 h, cells were washed twice with serum-free DMEM and incubated for 24 h in 200 µl serum-free DMEM. To assess constitutive versus inducible MMP activity in zymograms, the cells were also stimulated with different concentrations of TNF- in serum-free medium. This inflammatory cytokine is a product of activated macrophages, which are present in the infiltrate of cervical carcinoma in vivo. TNF-
is known to be a potent inducer of gelatinase activity in many different cell types. For zymograms, 20 µl of the conditioned media was separated by 10 % SDS-PAGE containing 1 mg gelatin (Sigma) ml1. The gels were washed in 2·5 % Triton X-100 for 30 min, followed by incubation at 37 °C for 48 h in 50 mM Tris/HCl pH 8·0 and 5 mM CaCl2 and were finally stained with Coomassie blue R250 (Merck) to reveal bands with gelatinolytic activity.
As shown in Fig. 3 HaCaT control cells and all transfected HaCaT clones constitutively produced the 72 kDa pro-form of MMP-2. Addition of increasing doses of TNF-
gradually induced the 92 kDa form of MMP-9 but had only marginal effects on MMP-2 activation. A striking difference between HPV E7 and mock transfectants was noted concerning MMP-2 activation. Neither HaCaT wild-type cells nor mock transfectants produced significant amounts of activated MMP-2 (Fig. 3
, upper panel). In contrast, in supernatants of HaCaT cells expressing HPV16 E7 (Fig. 3
, middle panel) and also HPV8 E7 (Fig. 3
, lower panel) the processed 62 and 59 kDa forms of MMP-2 were present, indicating proteolytic activation. Thus, MMP-2 was constitutively activated in the E7-expressing keratinocytes and did not require further stimulation by pro-inflammatory cytokines. TNF-
only slightly enhanced E7-mediated pro-MMP-2 activation at the highest concentrations.
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What could be the role of MT-1 MMP upregulation in HPV-positive keratinocytes? Obviously, MT-1 MMP expression plays a major role in pro-MMP-2 activation and therefore provides a basis for the cells to acquire an invasive phenotype. However, this is not the only function of MT-1 MMP. Upregulation of this protease may also have a physiological role during the viral life cycle by promoting cellular proliferation. Recently, a list of novel substrates for MT-1 MMP has been reported, which includes MT-1 MMP itself, plasminogen, chemokines, cytokines and a sheddase activity for the transmembrane mucin MUC1, playing a role in tumour progression (Tam et al., 2004; Thathiah & Carson, 2004
; for review see Sternlicht & Werb, 2001
). Plasminogen induces selective proteolysis of insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4) and promotes autocrine IGF-II bioavailability in human carcinoma cells (Remacle-Bonnet et al., 1997
). IGF can promote keratinocyte proliferation, a prerequisite for HPV replication (Aaronson et al., 1990
). It has also been shown that MT-1 MMP cleaves CD44 and the
2 chain of laminin 5, all associated with cell migration (Kajita et al., 2001
; Koshikawa et al., 2000
), thus enhancing the autonomous locomotion of cells. In addition, MT-1 MMP seems to enhance angiogenesis through upregulation of vascular endothelial growth factor expression (Sounni et al., 2002
), which is observed in pre-malignant stages of HPV-induced intraepithelial lesions and in HPV-associated cancer (Dobbs et al., 1997
; Guidi et al., 1995
; Obermair et al., 1997
; Smith-McCune et al., 1997
).
In summary, our data demonstrate that the E7 proteins of the high-risk HPV types 16 and 8 lead to the induction of MT-1 MMP, possibly explaining high MT-1 MMP expression in HPV8-transgenic mice (unpublished). This E7-mediated mechanism may profoundly alter the phenotype of infected keratinocytes, providing a basis for cellular proliferation, angiogenesis and also, later in malignant progression, for invasion of HPV-positive tumour cells.
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ACKNOWLEDGEMENTS |
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Received 24 August 2004;
accepted 17 January 2005.
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