Overexpression of matrix metalloproteinase 1 in dermal fibroblasts from DNA repair-deficient/cancer-prone xeroderma pigmentosum group C patients

Abstract

Xeroderma pigmentosum (XP) is a rare, recessively inherited genetic disease characterized by skin cancer proneness and premature aging in photoexposed area. The disease results from defective nucleotide excision repair of ultraviolet (UV)-induced DNA lesions. Reconstruction of group C (XP-C) skin in vitro previously suggested that patients' dermal fibroblasts might be involved in promoting skin cancer development, as they elicited microinvasions of both control and XP-C keratinocytes within dermal equivalents. Here we show that in the absence of UV exposure XP-C fibroblasts exhibit aged-like features such as an elongated and dendritic shape. We analysed the repertoire of expression of matrix metalloproteinases (MMPs) involved in skin aging and cancer. All XP-C fibroblasts tested in this study overexpressed specifically and significantly MMP1. MMP1 expression was also found increased in the dermis of XP-C skin sections suggesting the active contribution of XP-C mesenchymal cells to skin aging and exacerbated carcinogenesis. Increased MMP1 expression in cultured XP-C fibroblasts resulted from MMP1 mRNA accumulation and enhanced transcriptional activity of the MMP1 gene promoter. Deletion analysis revealed the essential role of AP-1 activation in constitutive MMP1 overexpression in XP-C primary fibroblasts. In parallel, levels of reactive oxygen species and FOSB DNA-binding activity were found increased in XP-C fibroblasts. Altogether, these observations suggest that beyond its role in nucleotide excision repair the XPC protein may be important in cell metabolism and fate in the absence of UV.

Introduction

Exposure to solar light is important in skin photoaging and in the development of basal and squamous cell carcinomas (BCC and SCC), the prevailing types of human cancers (Kraemer, 1997; DePinho, 2000). Solar radiation contains UVB wavelengths (280–315 nm) that induce DNA lesions called cyclobutane pyrimidine dimers (CPD) and pyrimidine 6-4 pyrimidone photoproducts (6-4 PP), at bipyrimidine DNA sequences. Both lesions are mutagenic and may provoke tumoral onset. Xeroderma pigmentosum (XP) is a rare (about 1 out of 500 000 newborns) genetic disease inherited as autosomal and recessive traits. XP patients present a high photosensitivity, premature skin aging and a proneness (2000 × ) to BCC and SCC in photoexposed skin areas. Most XP patients fall within one of the seven genetic groups of complementation (XP-A–XP-G) connected to a mutation in one of the ‘XP genes’ (XPAXPG) involved in nucleotide excision repair (NER). NER is the most versatile repair mechanism dedicated to the removal of bulky DNA adducts including CPD and 6-4 PP (Bootsma et al., 1995). In XP patients, the steady-state behavior as well as the responses of XP cells to ultraviolet (UV) light within the natural, three-dimensional architecture of the skin have received little investigations. Previously, our organotypic skin reconstructions (Bernerd et al., 2001, 2005) revealed that in the absence of exogenous stress XP-C fibroblasts induce epidermal invasions of control and XP-C keratinocytes within the dermal compartment of skin. These observations suggested that dermal fibroblasts could also contribute to the dramatic predisposition of XP-C patients to invasive skin carcinoma (SCC).

Matrix metalloproteinases (MMPs) form a family of metalloendopeptidases responsible for the proteolysis of extracellular matrix (ECM) components upon various biological processes including development, wound healing, aging and carcinogenesis (Egeblad and Werb, 2002; Kerkela et al., 2002; Kerkela and Saarialho-Kere, 2003; Comoglio and Trusolino, 2005). Interestingly, high levels of MMP1 and MMP3 expression have been found at the invasive front of SCC, as well as in surrounding stromal cells (Tsukifuji et al., 1999; Egeblad and Werb, 2002). Activity of MMPs has also been shown to be involved in the release of a range of regulatory proteins associated with ECM components such as growth factors and their receptors, cytokines and chemokines (Vu and Werb, 2000; Sternlicht and Werb, 2001; Stamenkovic, 2003).

Here, we investigated expression of MMPs in XP-C fibroblasts and report that levels of MMP1 protein and mRNA are significantly increased in XP-C compared to control fibroblasts resulting from enhanced activity of the MMP1 gene promoter. These differences were observed along with significant accumulation of reactive oxygen species (ROS), in the absence of external genotoxic stress, suggesting that the function of the XPC protein extends beyond NER. Thus, the absence of XPC could also indirectly contribute to tumoral invasion in XP patients.

Results

XP-C primary fibroblasts exhibit an aged-like phenotype

The phenotype of XP-C fibroblasts was studied in the three-dimensional environment of a collagen I dermal equivalent (three and six independent strains of either control (non-XP) or XP-C primary dermal fibroblasts, respectively). At 24 h after the beginning of the contraction, dermal equivalents containing XP-C fibroblasts were systematically smaller (about 11%) than those containing control fibroblasts (data not shown). To assess the morphology of XP-C fibroblasts, sections of control and XP-C dermal equivalents were immunolabelled for vimentin, a marker for adult mesenchymal cells, and for β1-integrin, a subunit of the membrane receptor participating to the anchorage of fibroblasts to collagen I fibers. XP-C fibroblasts were about twice as long as control fibroblasts (control: 54 μm; XP-C: 102 μm) and also exhibited a higher dendricity (Figure 1).

Figure 1
figure1

Phenotype of xeroderma pigmentosum group C (XP-C) fibroblasts embedded in a dermal equivalent. Confocal analysis of 30 μm cryosections of control and XP-C dermal equivalents immunolabelled for vimentin or β1-integrin. Note that XP-C fibroblasts are much larger and more dendritic than control fibroblasts. Vim, vimentin; β1-int, β1-integrin.

Increased MMP1 expression in XP-C fibroblasts

The secretion of MMP1, 2, 3, 7, 9 and 13 was measured by enzyme-linked immunosorbent assay (ELISA) in culture supernatants of control and XP-C fibroblasts cultured either as monolayers (Figure 2a, left panel) or in dermal equivalents (Figure 2a, right panel). In both culture conditions, only MMP1, 2 and 3 could reliably be detected. Levels of MMP2 and MMP3 secreted by XP-C fibroblasts monolayers were similar to those of control fibroblasts, whereas MMP1 secretion was significantly increased (7.3–fold, P<0.025) in culture supernatants of XP-C fibroblasts (Figure 2a, left panel). Importantly, MMP1 increase was not related to the age of donors (P>0.05, Pearson's correlation test). Secretion of MMP1 (2.6-fold) and, in a lesser extent, of MMP3 (2.1-fold) but not of MMP2, was also found substantially increased in culture supernatants of XP-C dermal equivalents compared to controls (Figure 2a, right panel). Western blot analysis of proteins present in culture supernatants (Figure 2b, upper panel) confirmed increased MMP1 secretion by XP-C compared to control fibroblasts (monolayers and dermal equivalents). Accordingly, zymography analyses indicated higher MMP1 activity in XP-C supernatants (Figure 2b, lower panel). Western blot analysis using total protein extracts from monolayer cultures showed that the level of intracellular MMP1 was also significantly increased (about 3.1-fold, P<0.025) in XP-C compared to controls (Figure 2c). Importantly, immunolabeling of dermal equivalents and skin sections of XP-C patients (when available, n=2) also indicated higher MMP1 staining in XP-C than in controls (Figure 2d).

Figure 2
figure2

Increased production of matrix metalloproteinase 1 (MMP1) in xeroderma pigmentosum group C (XP-C) fibroblasts. (a) Enzyme-linked immunosorbent assay (ELISA) analysis of secreted MMPs in supernatants of control (wild-type (WT): FMD, HF29 and HF84) and xeroderma pigmentosum group C (XP-C) fibroblasts cultured as monolayers (n=3: XP148, XP433, XP521; n=6: XP148, XP188, XP373, XP424, XP433, XP521) or in dermal equivalents (n=4: XP148, XP188, XP373 and XP521). In both culture conditions, XP-C fibroblasts secrete more MMP1 than control fibroblasts (**P<0.025). (b) Upper panel: western blot analysis of secreted MMPs in supernatants of control (WT) and XP-C fibroblasts cultured as indicated. Lower panel: casein gel zymography of control (WT) and XP-C supernatants. Note increased digestion of the substrate by culture supernatants of XP-C fibroblasts. (c) Upper panel: western blot analysis of intracellular protein extracts from control and XP-C fibroblasts. Lower panel: blot quantification of intracellular MMP1. For each strain, MMP1 level was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) level. Data are represented as mean±s.e.m. of three independent blots and are plotted as fold induction over FMD (control fibroblast strain used as a reference). The mean amounts of MMP1 in WT and XP-C fibroblasts strains are illustrated as a thin and a large dotted line, respectively. The threefold increase of MMP1 in XP-C fibroblasts is significant, **P<0.025. (d) Immunolabeling of MMP1 on 5 μm paraffin sections of control (HF29) and XP-C (XP148 and XP373) dermal equivalents (DE), and of control human skin (HS29) and XP-C skin (XP148) ex vivo, as indicated. The negative control performed by omitting the anti-MMP1 antibody (no 1st mab) illustrates specificity of the labeling. Bar: 150 μm.

Amount of MMP1, MMP2 and MMP3 mRNAs was measured by real-time quantitative PCR on RNA from control and XP-C fibroblasts. Only MMP1 mRNA was significantly increased (11.5-fold, P<0.05) in XP-C fibroblasts (Figure 3a) compared to controls. As the MMP1 inhibitor TIMP1 can neutralize MMP1 activity, amount of TIMP1 mRNA was also assessed but no difference was observed between control and XP-C fibroblasts (Figure 3a).

Figure 3
figure3

Increased transcription of the matrix metalloproteinase 1 (MMP1) gene promoter in xeroderma pigmentosum group C (XP-C) fibroblasts depends on the proximal AP-1 sequence. (a) mRNA levels of MMP1, 2, 3 and TIMP1 in control (wild-type (WT): FMD, HF29 and HF84) and XP-C (XP148, XP433 and XP521) fibroblasts measured by real-time quantitative PCR. Amount of MMP1 mRNA is significantly higher in XP-C than in control fibroblasts, *P<0.05. (b) Relative transcriptional activity of the full-length (4.4 kb) MMP1 promoter (pCollLUC −4400) and (c) of the proximal 136 bp 5′-regulatory region spanning from nucleotide −73 to +63 of the MMP1 gene (pCollCAT-73). As illustrated, full-length and short MMP1 promoter constructs contain four and one AP-1-binding sequences, respectively. Experiments were repeated at least two times in triplicates. Data are represented as the mean±s.d. of triplicates. The mean transcriptional activities in WT and XP-C fibroblasts strains are indicated as a thin and a large dotted line, respectively. *P<0.05; a.u., arbitrary units.

Role of activator protein-1 pathway in increased MMP1 transcription in XP-C fibroblasts

Whether increased amount of MMP1 mRNA in XP-C fibroblasts resulted from enhanced transcriptional activity was assessed by transient transfection reporter assay using a plasmid construct harboring 4400 bp of the 5′-regulatory region of the human MMP1 gene (PCollLUC-4400; Angel et al., 1987; Rutter et al., 1997). Transcriptional activity of the MMP1 reporter construct was found about five times higher (P<0.05) in XP-C compared to control fibroblasts (Figure 3b).

The proximal AP-1 responsive element (position −73 to −67) has been shown to be essential for both basal and AP-1-activated transcription of the MMP1 gene (Angel et al., 1987; White and Brinckerhoff, 1995). To assess the specific contribution of this AP-1 sequence, the activity of a shorter reporter construct, spanning from nucleotide −73 bp to +63 of the 5′-regulatory sequence of the MMP1 gene (Angel et al., 1987; Stein et al., 1989) was measured, indicating a 2.2-fold significant (P<0.05) increase in the transcriptional activity in XP-C compared to control fibroblasts (Figure 3c).

AP-1 is a complex transcription factor composed of heterodimer combinations of members of the JUN and FOS families (Karin et al., 1997) that bind to the AP-1 cognate sequence TGA(G/C)TCA to activate or repress transcription. Qualitative and quantitative AP-1 DNA-binding activity was measured in nuclear extracts prepared from primary control and XP-C fibroblasts. Among members of the AP-1 family tested (c-JUN, JUNB, JUND, c-FOS, FOSB, FRA-1 and FRA-2), only FOSB isoforms exhibited a substantially higher functional binding activity (1.4-fold) to the AP-1 DNA sequence in nuclear extracts from XP-C compared to control fibroblasts (Figure 4a). The amount of FOSB2 protein, measured by western blot, was increased in XP-C fibroblasts nuclear extracts (Figure 4b). Surprisingly, functional binding and nuclear expression of phosphorylated c-JUN (P-c-JUN) were slightly decreased in XP-C fibroblasts.

Figure 4
figure4

FOSB DNA binding is enhanced in xeroderma pigmentosum group C (XP-C) fibroblasts. (a) DNA-binding activity of AP-1 members analysed in nuclear extracts from primary control (wild-type (WT): FMD, HF29 and HF84) and XP-C (XP148, XP373 and XP521) fibroblasts. For each strain, relative DNA-binding activity was expressed as fold induction over FMD (control fibroblast strain used as a reference). Data are represented as mean±s.e.m. of the values of two independent experiments performed on two sets of independent nuclear extracts. Note that among all members of the AP-1 family, only FOSB showed a substantial increased binding activity. (b) Western blot analysis of phospho-c-JUN (P-c-JUN Ser63), FOSB and ETS1 in nuclear extracts from control and XP-C fibroblasts. Note that the FOSB2 isoform of FOSB appears increased in nuclear extracts from XP-C compared to control fibroblasts whereas P-c-JUN appears decreased. a.u., arbitrary units.

Accumulation of ROS in XP-C primary fibroblasts

As the increase of MMP1 gene transcription in XP-C fibroblasts was observed in the absence of UV exposure, we hypothesized that it could result from deregulated cellular metabolism. Accumulation of ROS was measured by flow cytometry using the CM-H2DCFDA probe. Figure 5 indicates a 1.5-fold significant (P<0.05) increase in ROS accumulation in XP-C compared to control fibroblasts. Interestingly, treatment of XP148 cells using the antioxidant substance epigallocatechin-O-gallate (EGCG) resulted in the decrease of both ROS and secreted MMP1 (Supplementary data 1).

Figure 5
figure5

Increased production of intracellular reactive oxygen species (ROS) in xeroderma pigmentosum group C (XP-C) fibroblasts. Control (wild-type, WT) and XP-C fibroblasts were stained with CM-H2DCFDA and analysed by flow cytometry. ROS levels are expressed as relative fluorescence units. Data are represented as mean±m.d. of two independent experiments (see also Supplementary data 1 ).

Discussion

We present here one of the very few studies relying on the use of several independent human primary XP-C cells.

Our previous observations suggested that XP-C fibroblasts promote development of carcinoma-like structures ex vivo (Bernerd et al., 2001, 2005). Present data further indicate that XP-C primary fibroblasts exhibit increased length and dendricity, some features highly reminiscent of chronically and photoaged skin in individuals from the general population (Fligiel et al., 2003; Varani et al., 2004; Shin et al., 2005), though none of the XP-C cells studied here have a history of exposure to UV light.

In addition, MMP1 expression and secretion were significantly higher in XP-C than in control primary fibroblasts suggesting its contribution in collagen I remodeling and microinvasive propensity of keratinocytes (Bernerd et al., 2001, 2005). Our study had to be limited to six primary strains of XP-C fibroblasts due to the extreme rarity of the disease. Whether MMP1 overexpression is a common, general and specific phenotypic trait of the XP-C complementation group remains to determine, although MMP1 overexpression was observed in all the XP-C strains studied here. In contrast, as suggested in other studies (Khorramizadeh et al., 1999), MMP1 level was not different in one fetal XP-C fibroblast strain (8-month fetus) compared to control postnatal fibroblasts (data not shown). In the general population, MMP1 expression increases with skin aging (Fisher et al., 1996). In XP-C patients, MMP1 overexpression could thus be a worsening actor toward exacerbated premature skin aging and tumor susceptibility.

MMP1 overexpression in XP-C fibroblasts results from higher rates of MMP1 gene transcription and accumulation of MMP1 mRNA. In contrast, TIMP1 mRNA amount was not increased in XP-C fibroblasts compared to controls. Transcription of the MMP1 gene was shown to be enhanced in response to the UV challenge (Stein et al., 1989), most notably through the activation of AP-1 transcriptional factors (Angel et al., 1987). A reporter plasmid containing solely the proximal AP-1 sequence of the human MMP1 gene was sufficient for driving increased MMP1 transcription in XP-C fibroblasts. Among members of the AP-1 family, FOSB isoforms (including FOSB and FOSB2) were the only ones presenting substantially increased binding to the AP-1 cognate sequence in nuclear extracts from XP-C fibroblasts. In contrast, DNA-binding and protein expression of P-c-JUN appeared both slightly decreased. These observations could be connected to the capacity of the FOSB2 isoform to form functional homodimers (Jorissen et al., 2007). Alternatively, under some stress circumstances related to acidification, FOSB has been shown to functionally heterodimerize with MafG in DNA-binding complexes (Shimokawa et al., 2005). Whether P-c-JUN decrease and FOSB increase in XP-C fibroblasts are connected or independent events remains to be determined. Interestingly, high FOSB expression has been significantly associated with MMP1 overexpression in breast carcinomas (Milde-Langosch et al., 2004). In addition, FOSB mRNA has been reported to increase upon early contraction of dermal equivalents containing human primary foreskin fibroblasts (Rosenfeldt et al., 1998) and also upon mechanical stress of bone (Inoue et al., 2004). In contrast, the level of ETS1, an other transcription factor that could also be potentially involved in the regulation of MMP1 gene expression, was found very similar in control and XP-C fibroblasts nuclear extracts (Figure 4). Taken together, these observations suggest that higher contractile properties and MMP1 overexpression observed in XP-C dermal equivalents could also be linked to increased FOSB binding. This does not exclude, however, the contribution of other transcription factors in upregulation of endogenous MMP1 gene expression in XP-C fibroblasts.

All observations reported here were obtained in the absence of exogenous stress such as UV irradiation suggesting that AP-1-dependent MMP1 transcription could be linked to an endogenous metabolic stress. The amount of ROS accumulation indicated higher (about 1.5) level of ROS in XP-C compared to control fibroblasts. These observations are in good agreement with previous reports indicating a lower level of enzymes such as superoxide dismutase (Nishigori et al., 1989) or catalase in XP compared to non-XP fibroblasts (Vuillaume et al., 1992). Activity of these enzymes counteracts ROS accumulation, but their decrease contributes to aging, genetic instability and cancer development (Chung et al., 2001; Radisky et al., 2005; Cat et al., 2006). Interestingly, recent investigations in HL60 cells have indicated that depletion of glutathione, a potent ROS scavenger, strongly elicits FOSB gene transcription in response to hydrogen peroxide (H2O2) pro-oxidant stress (Fratelli et al., 2005). Diminished catalase activity and increased H2O2 concentration have also been reported in chronically and photoaged skin fibroblasts isolated from the general population. In wild-type (WT) fibroblasts, decreased catalase activity could be associated to the activation of c-JUN N-terminal kinase and P38 stress pathways, leading to increased phosphorylation of c-JUN, and consequently, increased in MMPs expression (Brenneisen et al., 1997; Wenk et al., 1999; Chung et al., 2000; Rhie et al., 2001; Shin et al., 2005). In contrast, FOSB expression upon chronological and photo-induced skin aging in the general population has not been determined. Our data, however, suggest that chronically and photoaged fibroblasts may share similarities with non-photoexposed primary fibroblasts isolated from XP-C patients. This hypothesis is also supported by our previous investigations that revealed (1) higher sensitivity of XP-C compared to control cells toward the ROS-inducing UVA irradiation (Otto et al., 1999, 2) 50% reduced rates of S-phase cells in primary XP-C keratinocytes absence of exogenous stress such as UV suggesting metabolism-induced impairment of cell cycle (Arnaudeau-Begard et al., 2003). In addition, reports by Shimizu et al. (2003) and D'Errico et al. (2006) evidenced a role of XPC in the elimination of ROS-induced DNA adducts. The absence of the functional XPC protein in XP-C fibroblasts may thus explain ROS accumulation and hence activation of stress pathways including the AP-1 pathway.

Relevance of the increase in MMP1 expression in cultured XP-C fibroblasts was supported by indirect immunolabeling of nonlesional XP-C skin in situ. Numerous studies reported overexpression of MMPs during neoplasiogenesis and metastasis (Egeblad and Werb, 2002; Mueller and Fusenig, 2004). MMP1 has been shown to be mainly involved in the invasive/metastatic behavior of tumor cells through their interaction with the stroma (Benbow et al., 1999; Brinckerhoff et al., 2000; Airola and Fusenig, 2001). In the general population, BCCs account for approximately 75% and SCCs for 25% of non-melanocytic skin tumors. BCCs virtually never lead to metastasis whereas SCCs do at a significant rate (10−2). In the case of BCCs, MMP1 seems to be overexpressed solely in stromal cells surrounding the tumor (Monhian et al., 2005). In the case of SCCs, high amount of MMP1 mRNA has been detected in both the tumor and surrounding stromal cells, a pattern that could contribute to the significant metastatic potential of SCCs compared to BCCs (Gray et al., 1992; Tsukifuji et al., 1999). Importantly, the ratio of BCCs to SCCs is inverted in XP patients (about 25% BCCs and 75% SCCs) (Kraemer, 1997). Our data suggest that MMP1 overexpression in XP-C dermal fibroblasts could be determinant in promoting SCC development in these patients. This hypothesis will be addressed by long-term grafting of organotypic XP-C skin cultures on the mouse (Del Rio et al., 2002).

In conclusion, our data indicate that absence of the XPC protein in primary patients fibroblasts not only impacts NER but may also be accompanied by MMP1 overexpression, an event documented in skin aging and carcinogenesis. In the absence of any curative treatment of the disease, identification of molecular targets such as the MMP1 gene and/or upstream activating pathways might help develop innovative pharmacological approaches to skin cancer prevention and treatment.

Materials and methods

Patients and skin biopsies

Control human skin was obtained from mammary plastic surgery. All XP-C patients presented a marked photosensitivity and were unrelated (Table 1). XP-C skin biopsies from non-photoexposed sites (buttock) were obtained with the patients' or parents' informed consent in accordance with bioethical rules.

Table 1 Patients and cell characteristics

Human control and XP fibroblasts were isolated and cultured in Dulbecco's modified Eagle's medium (Invitrogen, Cergy Pontoise, France) supplemented with 10% fetal bovine serum (D Dutscher, Brumath, France) and 1% antibiotics (penicillin/streptomycin), at 37 °C in a 10% CO2 atmosphere. All experiments were carried out using cells at passage 5–9.

Determination of XP complementation group

XP fibroblasts were infected by retroviral particles expressing WT, XPC or other WT XP genes, and the complementation groups were determined as described (Frechet et al., 2006).

Dermal equivalent

Dermal equivalents were prepared as described initially (Asselineau et al., 1985) using 106 human dermal fibroblasts (WT or XP-C) in 5 mg/ml native bovine type I collagen (Symatese Biomateriaux, Chaponost, France). Mean perimeter of dermal equivalents was measured 24 h after the beginning of the incubation at 37 °C, 5% CO2 atmosphere.

Immunolabelings and confocal microscopy analysis

Dermal equivalents were embedded in Tissue Tek (Sakura Finetek, Zoeterwoude, the Netherlands) and frozen in liquid nitrogen. Immunofluorescence analysis was performed on 30 μm vertical cryosections, as previously described (Bernerd and Asselineau, 1997). Sections were incubated with the first antibody for 1 h 30 min and then with the secondary antibody for 1 h. Antibodies were diluted in phosphate-buffered saline (PBS) as follows: vimentin: 1/10 (clone V9; Monosan, Uden, the Netherlands); β1-integrin: 1/50 (clone K20; Immunotech, Villepinte, France); rabbit anti-mouse immunoglobulin G fluorescein isothiocyanate-conjugate: 1/100 (Dako, Trappes, France). Nuclei were counterstained using propidium iodide (5 μg/ml; Sigma-Aldrich, Saint Quentin Fallavier, France). Stacks of confocal images were collected using a Zeiss LSM 510 laser scanning confocal microscope under a 20 × 0.75 NA apochromat plan objectif. Z-projection of slices and image analysis were performed using Zeiss LSM Image examiner software.

MMP1 labelings were carried out on 5 μm paraffin sections of control and XP-C skin or dermal equivalents. Endogenous peroxidases were quenched using 3% H2O2 for 10 min. Sections were then incubated overnight at 4 °C with anti-MMP1 antibody (clone 36665; R&D Systems, Lille, France) at a 1/200 dilution in Zymed Diluent Reagent (Invitrogen). MMP1 labelings were revealed using Envision+ anti-mouse secondary antibody coupled to peroxidase (Dako) for 45 min and liquid diaminobenzidine substrate (Dako) for 10 min. Sections were counterstained with Mayer's haemalaun before mounting.

Detection of soluble MMP by enzyme-linked immunoassay

Culture supernatants were collected after 4 days of culture. The amount of secreted human MMP was analysed by ELISA (Biotrak Kit, Amersham Biosciences, Freiburg, Germany) according to the manufacturer's instructions.

Western blot analysis

Culture supernatants were concentrated using Vivaspin columns (Vivascience, Sartorius Group, Hannover, Germany). For analysis of intracellular proteins, fibroblasts were lysed in radioimmunoprecipitation assay buffer. Protein concentration was estimated using Bradford reagent (Bio-Rad, Marnes La Coquette, France). Proteins (50 μg for MMPs, 15 μg for nuclear extracts analysis) were separated by electrophoresis on 10% SDS–polyacrylamide gels, and transferred onto a polyvinyl difluoride membrane (Amersham Biosciences). Membranes were saturated with 5% nonfat dried milk in PBS/0.1 % Tween (Sigma, St Louis, MO, USA) and probed using monoclonal antibodies at the indicated dilutions: MMP1: 1/400 (clone 36665; R&D Systems), MMP2: 1/1000 (clone 42-5D11; MP Biochemicals, Illkirch, France), MMP3:1/100 (clone 55-2A4; Calbiochem, Merck KGaA, Darmstadt, Germany), P-c-Jun (P-S63): 1/5000 (clone Y172; Epitomics, Burlingame, CA, USA), Ets1: 1/400 (clone N-276; Santa Cruz Biotechnology, Santa Cruz, CA, USA), FosB: 1/1000 (clone 5G4; Cell Signaling, Danvers, MA, USA; glyceraldehyde-3-phosphate dehydrogenase (GAPDH): 1/5000 (clone 9484; Abcam, Cambridge, UK). Blots were revealed using electrochemiluminescence reagents (Amersham Biosciences). Quantification was performed using GeneGnome device and GeneTools software (Syngene, Synoptics Ltd, Cambridge, UK).

Zymography

Precast 10% zymogram blue casein gels from Novex (Invitrogen) were used to study MMP1 proteolytic activity. After gel electrophoresis, gels were washed twice in Zymogram Renaturing buffer (Bio-Rad), and incubated for 72 h at 37 °C in a Zymogram-Developing buffer (Bio-Rad). Caseinolytic proteins were identified as clear bands on a dark background.

Real-time quantitative PCR

Total RNA was extracted using the RNeasy Fibrous Tissue Kit (Qiagen, Courtaboeuf, France). RNA concentration and quality analysis were performed using the 2100 Bioanalyzer (Agilent Technologies, Massy, France). First-strand cDNA synthesis was performed from 1 μg total RNA, 1 h at 42 °C, using the Advantage RT-for-PCR Kit (Clontech, Montigny-le-Bretonneux, France).

Real-time quantitative PCR was performed using LightCycler (LC) system 2.0 (Roche Diagnostics, Meylan, France) in LightCycler capillary, using 0.4 pM primer oligonucleotides, 4 mM MgCl2 and LightCycler FastStart-DNA Master SYBR Green I mixture. Primers and PCR conditions are described in Table 2. Template concentrations were deduced from a standard curve using the fit points method of the LightCycler software. Specificity of each amplification was assessed by systematically performing a melting curve analysis. Control PCR were run on GAPDH, β2-microglobulin (β2-M) and ribosomal protein L13a (RPL13aA) cDNAs and analysed using GeNorm program to eliminate the worst-scoring housekeeping gene (Vandesompele et al., 2002).

Table 2 Oligonucleotides used in quantitative reverse-transcription PCR experiments

Transient transfection

Plasmids

pCollLUC-4400 and pCollCAT-73 consist of 4400 and 136 bp subfragments of the 5′-regulatory region of the human MMP1 gene inserted upstream of either the luciferase (LUC) (Rutter et al., 1997) or the chloramphenicol acetyl transferase (CAT) reporter gene (Collier et al., 1988; Frisch et al., 1990). pCollLUC-4400 and pCollCAT-73 were transfected along with the internal control plasmids phR-TK and pRSV-β-galactosidase (pRSV-βgal), respectively.

Transfection assay

A total of 500 000 cells were transfected with a mixture of either 1.5 μg of pCollLUC-4400 and 0.5 μg of phR-TK or 2.5 μg of pCollCAT-73 and 0.5 μg of pRSV-βgal using the Nucleofector System (Amaxa Biosystems, Köln, Germany). Protein extracts were prepared 48 h after transfection and analysed using either the dual-luciferase reporter assay system (pCollLUC-4400; Promega, Charbonnières, France) or CAT ELISA and β-Gal ELISA assay kits (pCollCAT-73; Roche Diagnostics).

Nuclear extracts and AP-1 DNA-binding assay

Nuclear extracts were prepared from fibroblasts monolayers using the Nuclear Extract Kit (Active Motif, Rivensart, Belgium). To verify specificity of extraction, 15 μg of either nuclear or cytoplasmic extracts were analysed by western blotting using anti-ATM (dilution 1/1000; clone 2C1; GeneTex, San Antonio, TX, USA) as a nuclear marker and anti-NFκB p50 (dilution 1/500; clone E-10; Santa Cruz Biotechnology) as a cytoplasmic marker (data not shown). AP-1 DNA-binding assays were performed using the TransAM AP-1 Family Kit (Active Motif).

Measurement of intracellular ROS

Fibroblasts were incubated with 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA; 5 μM) in Hank's buffered salt solution (Invitrogen) for 30 min at 37 °C before the fluorescent intensity resulting from the oxidation of H2DCF by ROS was measured using a cytometer (CellQuest software; Calibur; BD Biosciences) at the excitation and emission wavelengths of 485 and 530 nm, respectively.

EGCG (Sigma-Aldrich) treatment of fibroblasts was carried out at 15 μM for 3 days in the presence of 1% fetal calf serum.

Statistical analysis

Representation

For the box plot representation (Figures 2 and 3), the line in the middle of the box represents the median. The box extends from the 25th percentile to the 75th percentile. The lines emerging from the box extend to the upper and lower adjacent values.

Statistical test

Significance of the differences between experimental values measured in XP-C and in control fibroblasts was assessed using the Mann–Whitney U-test. *P<0.05; **P<0.025.

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Acknowledgements

Dr Asselineau and Dr J Leclaire are gratefully acknowledged for continuous support and encouragements. We are indebted to V Marty, C Pierrard and Dr C Marionnet for their expert help. We are also grateful to Dr A Jalil for confocal microscopy. We thank Professor P Herrlich and Professor CE Brinckerhoff for DNA reporter constructs. Many thanks to F Duvigneau for editing English usage in the article. TM gratefully acknowledges fundings from the Association pour la Recherche sur le Cancer (no. 3590), the Fondation de l'Avenir, the Société Française de Dermatologie, the Association Française contre les Myopathies. AS thanks the association ‘Les enfants de la Lune’ for its support.

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Correspondence to F Bernerd or T Magnaldo.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

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Fréchet, M., Warrick, E., Vioux, C. et al. Overexpression of matrix metalloproteinase 1 in dermal fibroblasts from DNA repair-deficient/cancer-prone xeroderma pigmentosum group C patients. Oncogene 27, 5223–5232 (2008). https://doi.org/10.1038/onc.2008.153

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Keywords

  • xeroderma pigmentosum
  • skin
  • cancer
  • MMP1
  • AP-1
  • ROS

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