Matrix metalloproteinase-19 is expressed by proliferating epithelium but disappears with neoplastic dedifferentiation
Article first published online: 12 DEC 2002
Copyright © 2002 Wiley-Liss, Inc.
International Journal of Cancer
Volume 103, Issue 6, pages 709–716, 1 March 2003
How to Cite
Impola, U., Toriseva, M., Suomela, S., Jeskanen, L., Hieta, N., Jahkola, T., Grenman, R., Kähäri, V.-M. and Saarialho-Kere, U. (2003), Matrix metalloproteinase-19 is expressed by proliferating epithelium but disappears with neoplastic dedifferentiation. Int. J. Cancer, 103: 709–716. doi: 10.1002/ijc.10902
- Issue published online: 3 JAN 2003
- Article first published online: 12 DEC 2002
- Manuscript Accepted: 18 OCT 2002
- Manuscript Revised: 27 SEP 2002
- Manuscript Received: 17 MAY 2002
- Helsinki and Turku University Central Hospital Research Funds
- Academy of Finland
- Finska Läkaresällskapet
- Sigrid Jusélius Foundation
- Cancer Foundation of Finland
- skin cancer;
- wound healing;
- tumor necrosis factor-α
MMP-19 (also designated RASI) is a recently discovered member of a large family of zinc-dependent proteolytic enzymes, most of which have been implicated in cancer growth and metastasis. It differs from the others by its chromosomal location and structure and is expressed by endothelial and vascular smooth muscle cells in vivo. Our aim was to study the putative role of MMP-19 in skin cancer. We also examined its regulation in keratinocyte cultures using quantitative TaqMan RT-PCR. Our results show that MMP-19 can also be detected in stimulated keratinocytes by Northern and Western analyses. In wounds, it was found in keratinocytes outside the migrating area, while in BCC and SCC, it was present in the hyperproliferative (p63-positive), E-cadherin-negative epidermis at the tumor surface but downregulated in invasive cancer islands. Expression was also evident in endothelial cells of neoangiogenic regions and in occasional stromal fibroblasts. Of the 12 tested cytokines/growth factors, only TNF-α and PMA were able to stimulate the expression of MMP-19 mRNA in primary keratinocytes. No MMP-19 mRNA was detected by Northern analysis in cultured HaCaT or A5 cells or in an SCC cell line established from head-and-neck cancer. Our data suggest that, unlike most MMPs, MMP-19 expression in the epidermis is downregulated during transformation and histologic dedifferentiation. © 2002 Wiley-Liss, Inc.
MMPs constitute a family of zinc-dependent proteolytic enzymes which take part in proteolytic degradation of the ECM and BM during cell migration, angiogenesis and proteolytic activation of growth factors, events that are needed in normal tissue remodeling as well as in wound healing and tumor invasion. MMPs can be divided into 6 subgroups: interstitial collagenases (MMP-1, MMP-8 and MMP-13), stromelysins (MMP-3, MMP-10, MMP-11 and MMP-12), matrilysins (MMP-7 and MMP-26), type IV collagenases (MMP-2 and MMP-9), membrane-type MMPs (MMP-14, MMP-15, MMP-16, MMP-17, MMP-24 and MMP-25) and others (MMP-19, MMP-23 and MMP-28).1, 2, 3 Collagenase-1 (MMP-1), stromelysin-2 (MMP-10) and 92 kDa gelatinase (MMP-9) participate in keratinocyte migration during wound repair as well as in cancer cell migration.4, 5 The role of the novel MMPs (from MMP-19 upward) in skin biology has not been well elucidated.
MMP-19, previously referred to as MMP-18,6 is one of the recently cloned members of the MMP family. It differs from the others by its unique chromosomal location (12q14).7 MMP-19 lacks several structural features known to be present in other MMP subclasses, including Asp, Tyr and Gly residues close to the zinc-binding site; the fibronectin-like and the transmembrane domains; as well as the furin-activation sequence, based on which it cannot be included into any of the known subclasses.7 Another recently cloned MMP, epilysin (MMP-28), is structurally most related to MMP-19.3, 8In vitro, MMP-19 is able to degrade many important BM components, e.g., type IV collagen, laminin-1, nidogen, fibronectin, tenascin-C isoform, aggrecan, type I gelatin and cartilage oligomeric matrix protein,9 but does not activate any other latent MMPs.10
By Northern hybridization, MMP-19 was originally detected in placenta, lung, pancreas, liver, ovary, spleen and intestine.7 Independently, it was isolated as an autoantigen from the inflamed synovium of a patient suffering from rheumatoid arthritis.11 MMP-19 has been described in smooth muscle cells of the tunica media of large blood vessels, normal skin and uterine ligaments as well as in endothelial cells of acutely inflamed synovial capillaries.12 It is also detected on the surface of activated peripheral blood mononuclear cells, TH1 lymphocytes and Jurkat T-lymphoma cells.11 MMP-19 has been suggested to play a role in matrix remodeling and in the pathogenesis of rheumatoid arthritis.11, 13 Because of its appearance in normal tissues, it is possible that MMP-19 is important in normal tissue remodeling or activation of secreted and membrane-bound proteins, like growth factors.10
Our aim was to determine whether MMP-19 is induced in the epithelium during remodeling associated with either wound repair or cancer invasion. We show here that MMP-19 can be expressed by proliferating, but not migrating, keratinocytes; is upregulated by TNF-α and PMA in keratinocyte cultures; and, unlike several classical MMPs, disappears from proliferating keratinocytes during neoplastic dedifferentiation.
MATERIAL AND METHODS
Informed consent was obtained from individual subjects for all procedures. The study was approved by the Ethics Committees of the Departments of Dermatology and Plastic Surgery, Helsinki University Central Hospital. Tissue samples included normal skin (n = 4); lichenoid acanthotic dermatitis (n = 3); 6 normally healing wounds;14 venous, decubitus, diabetic, rheumatoid and clinically well-granulating chronic ulcers (n = 20); premalignant tumors such as Bowen's disease (n = 4) and solar keratosis, [malignant epidermal tumors, i.e., BCCs (n = 16; sclerosing subtype n = 7, nodular subtype n = 9) and SCCs, grades I (n = 2), II (n = 14), III (n = 6) and IV (n = 2)]. Formalin-fixed, paraffin-embedded archival specimens were obtained from the Department of Dermatopathology, Skin and Allergy Hospital, University of Helsinki. Diagnoses were confirmed by 2 experienced dermatopathologists.
Immunostaining was performed using the avidin-biotin-peroxidase complex technique (Vectastain ABC Kit; Vector, Burlingame, CA). Diaminobenzidine and aminoethylcarbazole (Ki-67) were used as chromogenic substrates, and Harris hematoxylin was the counterstain, as described in detail previously.15 If needed, sections were pretreated with 10 mg/ml trypsin (type IV collagen) or by antigen retrieval (Ki-67, E-cadherin) as described.16 Rabbit polyclonal antiserum against human MMP-19 (Research Diagnostics, Flanders, NJ) was diluted 1:60 in 1% BSA and reacted overnight at 4°C. A subset of skin samples was also stained with other polyclonal antibodies (PC374; Oncogene, Boston, MA) with identical results. Rabbit antihuman TNF-α (Rockland, Gilbertsville, PA) antibodies were diluted 1:100. Polyclonal anti-Ki-67 antibodies (A047; Dako, Glostrup, Denmark) were used to differentiate proliferating cells from quiescent epithelial cells and diluted 1:200.17 The MAb to p63 (clone 4A4; Neomarkers, Fremont, CA), a keratinocyte stem cell marker, was diluted 1:200. A MAb to type IV collagen (M785, Dako) was used to stain the epithelial basement membrane at 1:75. E-cadherin (HECD-1; Zymed, San Francisco, CA) was diluted 1:60–1:100.18 Controls were performed with mouse preimmune ascites fluid or rabbit preimmune serum.
Human keratinocytes were isolated from normal adult skin obtained from reductive mammoplasties.14 Subcutaneous fat and deep dermis were removed, and the remaining tissue was incubated overnight at 0.25% trypsin in solution A (GIBCO BRL–Life Technologies, Gaithersburg, MD). Following incubation, keratinocytes were scraped from the epidermis with a scalpel and suspended in KGM (GIBCO BRL–Life Technologies), supplemented with 5 ng/ml EGF and 50 μg/ml BPE (supplied by the vendor) and containing 2% decalcified FCS. Keratinocytes were maintained in KGM supplemented with EGF and BPE, and passages 1–5 were used in the experiments. For immunostaining, primary keratinocytes (in both low and high Ca2+ KGM) were also cultured on Lab-Tek chamber slides (plastic or type I collagen-coated) and immunostained using MMP-19 and Ki-67 antibodies as described for tissue samples.
HaCaT cells, an immortalized nontumorigenic human adult epidermal keratinocyte cell line,19 and A5 cells, a ras-transformed benign tumorigenic HaCaT-derived cell line,20 were kindly provided by Dr. N. Fusenig (Deutsche Krebsforschungszentrum, Heidelberg, Germany). HaCaT and A5 cells were cultured in DMEM containing 10% FCS.
The UT-SCC-7 cell line, which forms SCCs in SCID mice,21 was established from metastasis of a cutaneous SCC at the time of operation in the Turku University Central Hospital.22 Cell lines were cultured in DMEM supplemented with 6 mM glutamine, nonessential amino acids and 10% FCS. SCC cells in subcultures 5–10 were homogenous by visual inspection.
Cytokines and growth factors
To study the regulation of MMP-19 expression, equal amounts of primary keratinocytes were plated on 24-well tissue culture plates. Cells that were 70–80% confluent were repeatedly washed with PBS and incubated overnight in KGM without supplements or FCS. Then, keratinocytes were treated with TNF-α (10 ng/ml; Sigma, St. Louis, MO), IL-1β (5 ng/ml; Roche, Mannheim, Germany), TGF-β1 (1–5 ng/ml, Sigma), EGF (10 ng/ml, Sigma), KGF (10 ng/ml, Sigma), HGF (10 ng/ml, Sigma), VEGF (10 ng/ml; R&D Systems, Minneapolis, MN), IGF-I (100 ng/ml, R&D, Systems), IL-10 (10 ng/ml, R&D Systems), IFN-γ (1 ng/ml; Promega, Madison, WI), bFGF (10 ng/ml, Sigma) and PMA (10 ng/ml, Sigma) for 24 hr. All treatments were done in KGM and in KGM with 1.8 μM Ca2+, without supplements or FCS. After 24 hr, total RNA was extracted from the cells. Untreated cells were used as a control.
PCR primers and probes
PCR primers and the MGB probe (CCCGTGGACTACCTG) for MMP-19 were designed using the computer program Primer Express (Applied Biosystems, Foster City, CA). Primers used for amplification were as follows: forward 5′-GCTTCCTACTCCCCATGACAGT-3′ and reverse 5′-GGCTTCTGTAGGTACCC-ATATTGT-3′. The fluorogenic MGB probe (CCCGTGGACTACCTG) contained a reporter dye (FAM) covalently attached at the 5′ end and a quencher dye (TAMRA) covalently attached at the 3′ end. The fluorogenic probe was HPLC-purified. Predeveloped TaqMan assay reagents for endogenous control human GAPDH labeled with VIC reporter dye (Applied Biosystems) were used for amplification of the control gene.
Total cellular RNA from keratinocytes was extracted using the RNeasy Miniprep kit (Qiagen, Chatsworth, CA) following the manufacturer's instructions. RNA was then reverse-transcribed to cDNA with TaqMan reverse transcription reagents (Applied Biosystems) and used as a template in PCR. Real-time quantitative PCRs were performed with the ABI PRISM 7700 Sequence Detector System (Applied Biosystems).14 Reaction conditions were programmed on a power Macintosh (Cupertino, CA) 7200, linked directly to the sequence detector. PCR amplifications were performed in a total volume of 20 μl, containing 5 μl cDNA sample, 10 μl TaqMan Universal PCR Master Mix (Applied Biosystems), 200 nM of each primer and 200 nM of fluorogenic probe. Predeveloped GAPDH endogenous control reagents were used as control genes from the same samples. MicroAmp Optical 96-Well Reaction Plates and Optical Caps (Applied Biosystems) were used in reactions. Reaction conditions were as follows: 2 min at 50°C and 10 min at 94°C, followed by 40 cycles of 15 sec at 94°C and 1 min at 60°C.
Total cellular RNA was isolated from cell cultures using the single-step method.23 Northern blot hybridizations were performed as described previously24 with cDNAs labeled with [α-32P]-dCTP using random priming. A 1.5 kb cDNA was used for MMP-197 detection. [32P]-cDNA/mRNA hybrids were visualized by autoradiography.
To assay the production of MMP-19 by primary epidermal keratinocytes, cell culture media were collected after 24 hr of incubation and concentrated 15-fold with Amicon Ultra Centricon (30,000 MWCO; Millipore, Bedford, MA). Proteins were separated on 8.5% SDS-PAGE gels and transferred to a Hybond ECL filter (Amersham, Arlington Heights, IL). After blocking in PBS containing 5% milk and 0.1% Tween-20, the membrane was incubated with a MAb against human MMP-19 protein (a kind gift from Dr. C. López-Otín, University of Oviedo, Oviedo, Spain) in a final concentration of 2 μg/ml. Specific binding of primary antibody was detected with peroxidase-conjugated secondary antibody (Amersham) diluted 1:1,000 and visualized by ECL (Amersham). Antibody against MMP-1 (AB806; Chemicon, Temecula, CA) was used as a control.
MMP-19 in wounds
In normally healing wounds (6/6), MMP-19 protein was detected in keratinocytes, distal to the migrating edge in areas positive for the proliferation marker Ki-67 (Fig. 1a,a′,b). In chronic wounds (10/20), many more positive keratinocytes were detected away from the migrating front in regions that appeared to colocalize with the proliferative compartment (Fig. 1c,d). In acanthotic, hyperproliferative epithelium, where MMP-19-positive cells were seen in chronic wounds, type IV collagen staining was abnormally faint or absent (Fig. 1e,f). MMP-19-positive endothelial cells were detected in all acute and chronic wounds examined (Fig. 1c). In normal skin (4/4), endothelial cells and occasional fibroblast-like cells and sweat glands were positive for MMP-19 protein (Fig. 1g,h).
Expression of MMP-19 in skin cancer
MMP-19 expression was detected focally in basal keratinocytes already in premalignant lesions such as solar keratosis (4/4) (Fig. 2a) or Bowen's disease (2/4) (Fig. 2c), particularly in areas demonstrating overcrowding of basal cells and disturbances in basal cell polarity. Furthermore, benign hyperproliferative epithelium bordering on or covering malignant tumors was often positive (Fig. 2d,f). In BCC, MMP-19 was expressed by basal keratinocytes at the hyperproliferative surface epithelium of the tumor area but disappeared from BCC islands (9/16) (Fig. 2f,h). No significant differences were detected in the expression pattern between the sclerosing and nodular subtypes. As in wounds, MMP-19-positive keratinocytes were often seen in the same areas as Ki-67 immunostaining but in a greater number of keratinocytes (data not shown). When we stained for p63, a marker for keratinocytes with proliferative capacity,25 better colocalization with MMP-19 was observed (Fig. 2a,b,f,g). However, although abundant expression of p63 could be detected in BCCs and SCCs, MMP-19 protein was not found in invasive cancer islands (Fig. 2f,h). Type IV collagen did not stain normally under MMP-19-positive keratinocytes (Fig. 2d,e).
Like in BCC, MMP-19 was detected in acanthotic, hyperproliferative epithelium bordering on or covering SCCs (18/24), particularly in association with disturbances in basal cell polarity and overcrowdedness (Fig. 3a,b,d). The amount of staining for MMP-19 protein did not increase from well-differentiated to histologically dedifferentiated tumors. Also in SCCs, Ki-67-positive cells were detected in MMP-19-positive regions of epithelium (Fig. 3d,e), while edges of invasive SCC islands were devoid of MMP-19 (Fig. 3a,d,g). In SCCs, MMP-19 was typically upregulated in acanthotic surface epithelium in areas where type IV collagen became abnormal (Fig. 3h,i). In areas of neoangiogenesis, endothelial cells (17/24) also expressed MMP-19 (Fig. 3j).
Since MMP-19 appeared to be induced when the polarity and density of keratinocytes were disturbed, we stained adjacent sections for E-cadherin. E-cadherin staining was abnormal in areas with MMP-19-positive keratinocytes (Figs. 2a′,3m,n).
A subpopulation of primary keratinocytes cultured on Lab-Tek slides demonstrated cytoplasmic MMP-19; mitotic cells, in particular, were often positive (Fig. 4a,d). When wounded cultures were stained, migrating keratinocytes were MMP-19-negative (Fig. 4c), in accordance with our in vivo data.
Regulation of MMP-19 in primary keratinocytes
To study the regulation of MMP-19 expression, we cultured primary keratinocytes in the presence of various growth factors and cytokines and used quantitative real-time RT-PCR for the analysis.
Basal levels of MMP-19 mRNA in unstimulated keratinocytes were quite low, as detected around cycle 31–33. Of the various agents tested, only PMA and TNF-α significantly stimulated expression of MMP-19 mRNA (Fig. 5a). For this reason, a subset of samples was stained for TNF-α, which was detected in keratinocytes adjacent to those expressing MMP-19 (Fig. 3k,l). Induction of cell differentiation with higher Ca concentration did not influence the amount of MMP-19 mRNA in TaqMan analysis (data not shown).
Northern and Western analyses of keratinocytes
In addition to quantitative real-time RT-PCR, induction of MMP-19 mRNA expression by PMA in human primary keratinocytes was detected at low levels with Northern blot analysis (Fig. 5b). A 57 kDa immunoreactive pro-MMP-19 band was detected by Western blot analysis in conditioned medium of PMA-stimulated epidermal keratinocytes, whereas no pro-MMP-19 was detected in conditioned medium of untreated control cells (Fig. 5c).
MMP-19 expression has previously been associated with endothelial and smooth muscle cells of several organs in vivo.12 We show also that cells of epithelial origin are capable of producing MMP-19.26 Unlike several other MMPs, such as MMP-1, MMP-9 and MMP-10,5, 14 MMP-19 is not expressed by migrating keratinocytes in wounds but rather in areas with hyperproliferation and acanthosis. Interestingly, expression of the closely related MMP-28 has also been associated with cell proliferation during epithelial repair.27 Endothelial cells, particularly in neoangiogenic areas of cutaneous wounds and cancer, were also MMP-19-positive.
Cancer invasion involves many MMPs, including MMP-2, MMP-7, MMP-9, MMP-13 and MMP-14.4, 28 The presence of specific MMPs in cancer tissue can even be used as a prognostic marker to predict tumor invasiveness.29 Our results show that MMP-19 is induced in actinic keratosis and Bowen's disease as well as in hyperplastic epithelium bordering SCC and BCC, when the polarity and density of keratinocytes are disturbed, as assessed histologically and by abnormal E-cadherin staining. However, MMP-19 is downregulated in vivo when keratinocytes become malignant and disappears during neoplastic transformation. This was further substantiated when we investigated the expression of MMP-19 mRNA in epidermal keratinocyte-derived cell lines representing different phases of carcinogenesis and varying tumorigenic potentials. MMP-19 mRNA was detected by Northern blot hybridization in primary keratinocytes after stimulation (Fig. 5b) but was not evident in the nontumorigenic keratinocyte cell line HaCaT19 treated with TNF-μ (20 ng/ml), TGF-β1 (5 ng/ml) or IFN-γ (100 U/ml) for 24 hr (data not shown). In addition, no expression of MMP-19 mRNA was detected in A5 cells, a ras-transformed HaCaT cell-derived cell line that forms benign tumors20 or in cutaneous SCC metastasis-derived UT-SCC-7 cells, which form SCCs in SCID mice21 (data not shown). Although immortalized HaCaT cells are nontumorigenic, their MMP expression profile clearly differs from that of primary keratinocytes, e.g., MMP-12 and MMP-13, which are upregulated in the course of epithelial transformation and expressed by HaCaT cells but not by primary keratinocytes.30, 31
While our study was in progress, MMP-19 was shown to be expressed in the epithelium of normal mammary glands and benign mammary tumours.26 In agreement with our data, that report also concluded that MMP-19 expression cannot be associated with tumor progression and may even act in a “protective” manner. Our findings suggest that MMP-19 does not take part in the degradation of the BM and ECM to induce tumor spread but rather participates in normal remodeling of the BM induced after microchanges in BM proteins such as those detected in wounds and Bowen's disease.
As the physiologic substrates of MMP-19 are type IV collagen, laminin-1 and nidogen, it is possible that MMP-19 may be needed to restructure the BM. MMP-19 is coexpressed with type IV collagen in tunica media and with integrin αvβ3 and VEGF-R2 in endothelial cells,12 and in our study MMP-19 was found in the area where the BM was at least partly destroyed when stained with type IV collagen. The presence of MMP-19 in p63-positive epithelium suggests that it is restricted in vivo to areas of keratinocytes with high proliferative potential and absent from cells undergoing terminal differentiation.25
TNF-α and PMA were the only agents that had significant influence on MMP-19 mRNA expression in primary keratinocytes (Fig. 5a). Expression of different MMPs in keratinocytes is generally regulated by signals from the surrounding environment, e.g., growth factors or cytokines and cell–cell and cell–matrix interactions. The promoter of MMP-19 has a TATA box, an AP-1 binding motif and PEA3 as well as TGF-β inhibitory elements.32 AP-1 transcription factors are important in cell proliferation and cellular transformation, and their presence together with the PEA3 site has been reported to confer responsiveness to a variety of growth factors, oncogene products and tumor promoters.33 Interestingly, TNF-α activates AP-1 in keratinocytes.34 MMP-19 mRNA expression in endothelial and smooth muscle cells is upregulated by phorbol ester (TPA), EGF and bFGF;12 but the 2 latter cytokines had no effect on keratinocyte MMP-19 production. TNF-α stimulates the expression of MMP-19 also in fibroblasts (Hieta and Kähäri, unpublished observations).
TNF-α is a macrophage-derived cytokine, which is essential in the inflammatory phase of normal wound healing and can inhibit tumor growth.35 It is also produced by keratinocytes and Langerhans cells36 and by neutrophils in wounds.35 When cultured keratinocytes are treated with TNF-α, proliferation is inhibited but differentiation is promoted.37, 38 TNF-α induces adhesion molecules and apoptosis of keratinocytes.39 In our samples, the localization of MMP-19 did not histologically correlate with apoptosis. Also, the number of TNF-α-stimulated cells was similar to the number of untreated control cells in the 24 hr samples used for TaqMan analysis. In tissue samples, TNF-α protein was expressed in the keratinocytes adjacent to MMP-19-positive cells. The fact that TNF-α induces adhesion molecules and that it stimulates MMP-19 could also suggest that MMP-19 is a reconstructive enzyme in the repair of destroyed BM and that cell adhesion might have an effect on its expression, as shown in myeloid cells40 or by comparing to the presence of E-cadherin. However, caution is needed when extrapolating results from cell culture to tissue in vivo since, based on transgenic mouse studies, many cytokines have different effects on keratinocytes in vivo and in culture.41
In conclusion, we have demonstrated MMP-19 expression in keratinocytes. Unlike most other MMPs, this protein is absent from invasive skin cancer cell nests in vivo and not expressed by transformed epithelial cells in culture, suggesting that it is upregulated in the epithelium early during oncogenesis in a host-response manner to reconstitute normal cell adhesion.
We thank Ms. A. Tallqvist, Ms. M. Potila and Ms. S. Pitkänen for skillful technical assistance.