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Journal of Virology, July 2008, p. 6481-6491, Vol. 82, No. 13
0022-538X/08/$08.00+0     doi:10.1128/JVI.00429-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Transcriptional Changes Induced by Bovine Papillomavirus Type 1 in Equine Fibroblasts{triangledown}

Z. Q. Yuan, L. Nicolson, B. Marchetti, E. A. Gault, M. S. Campo, and L. Nasir*

Institute of Comparative Medicine, University of Glasgow Faculty of Veterinary Medicine, Bearsden Road, Glasgow G61 1QH, United Kingdom

Received 27 February 2008/ Accepted 16 April 2008


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ABSTRACT
 
Bovine papillomavirus type 1 (BPV-1) and, less commonly, BPV-2 are associated with the pathogenesis of common equine skin tumors termed sarcoids. In an attempt to understand the mechanisms by which BPV-1 induces sarcoids, we used gene expression profiling as a screening tool to identify candidate genes implicated in disease pathogenesis. Gene expression profiles of equine fibroblasts transformed by BPV-1 experimentally or from explanted tumors were compared with those of control equine fibroblasts to identify genes associated with expression of BPV-1. Analysis of the microarray data identified 81 probe sets that were significantly (P < 0.01) differentially expressed between the BPV-1-transformed and control cell lines. Expression of several deregulated genes, including MMP-1, CXCL5, FRA-1, NKG7, TLR4, and the gene encoding the major histocompatibility complex class I (MHC-I) protein, was confirmed using other BPV-1-transformed cell lines. Furthermore, expression of these genes was examined using a panel of 10 sarcoids. Increased expression of MMP-1, CXCL5, FRA-1, and NKG7 was detected in a subset of tumors, and TLR4 and MHC I showed robust down-regulation in all tumors. Deregulated expression was confirmed at the protein level for MMP-1 and MHC-I. The present report identifies genes modulated by BPV-1 transformation and will help identify the molecular mechanisms involved in disease pathogenesis.


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INTRODUCTION
 
Bovine papillomaviruses (BPVs) infect cattle and cause fibropapillomas or papillomas of epithelia (either cutaneous or mucosal). Papillomas are benign and often regress; however, they occasionally can persist and advance to become malignant tumors (13). Although papillomaviruses are strictly species specific, BPV type 1 (BPV-1) and BPV-2 are implicated in the pathogenesis of equine sarcoids. Sarcoids are locally invasive, fibroblastic skin tumors and represent the most common skin tumor found in equids worldwide (53). BPV-1 and, less commonly, BPV-2 DNA is detected in the majority of sarcoids (reviewed in reference 46) and is believed to be the causative agent. BPV DNA has not been detected in other equine tumors, including melanomas, papillomas, and squamous cell carcinomas (15), although we and others have shown that BPV-1/BPV-2 DNA can be detected in some cases of equine inflammatory skin disease (3, 15, 68). BPV DNA is localized to the fibroblast nuclei (37, 60) and is maintained as a high-copy-number episome, and infection results in the expression of viral genes (2, 10, 15, 49, 67). We have recently shown that BPV-1 transforms primary equine fibroblasts (69).

The clinical presentation of sarcoids varies (reviewed in reference 31). Tumors can exist as single or multiple lesions, and animals with more than 100 tumors are not uncommon. While some lesions can remain quiescent for many years, others undergo rapid growth, especially following trauma (31). Currently little is known about how infection with BPV-1 in fibroblasts contributes to the pathogenesis of sarcoids. Thus, identification of genes whose expression is altered during BPV-1 cell transformation would provide important information on the underlying molecular mechanisms. In the present study we used microarray technology with equine gene chips to analyze gene expression profiles of BPV-1-transformed equine palate fibroblasts and fibroblasts cultured from naturally occurring BPV-1-positive sarcoids (69) to identify genes whose expression may be associated with pathogenesis.


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MATERIALS AND METHODS
 
Cell cultures and tumor samples. Normal EqPalF, BPV-1-transformed S6-1, S6-2, and S6-3 cells and sarcoid cell lines EqS01a, EqS02a, and EqS04b have been described previously (69). Sarcoids were collected from veterinary practices in the United Kingdom or donated by Elaine Marti (University of Berne, Switzerland). They comprised mixed, fibroblastic, and nodular clinical types.

RNA isolation and microarray analysis. RNA was isolated from cells by use of an RNeasy Mini kit (Qiagen, Crawley, United Kingdom), following the supplier's protocol. RNA was used to synthesize double-stranded cDNA by use of a One-Cycle cDNA synthesis kit (Affymetrix, United Kingdom). In vitro transcription (IVT) of cDNA was performed with a GeneChip IVT labeling kit (Affymetrix), according to the instructions of the manufacturer. Briefly, the double-stranded cDNA was transcribed overnight (for 16 h) using biotinylated nucleotides, and the biotinylated cRNA was purified on a spin column. Purified cRNA was quantified using spectrophotometry, and the quality of amplification was assessed using an Experion RNA StdSens analysis kit and an Experion automated electrophoresis system (Bio-Rad Laboratories, Hercules, CA). The cRNA was fragmented using fragmenting buffer in the GeneChip IVT labeling kit, and fragmentation was confirmed by electrophoresis.

The equine-specific Affymetrix GeneChips represented 3,098 probe sets (25). Prewetted chips were loaded with the fragmented, labeled cRNA and hybridized overnight (for 16 h) at 45°C and 60 rpm. Washing and staining of the arrays was performed on a Fluidics Station 400 apparatus (Affymetrix) using the appropriate fluidics scripts (Protocol EukGE-vs4.v2). The arrays were scanned using an Affymetrix GeneChip Scanner 3000 and the data collected and processed with GeneChip operating software (GCOS v1.2.1; Affymetrix). The procedures were performed in four replicate experiments.

Data analysis. Microarray data were analyzed using the rank product technique (12). Genes were classified by using the Gene Ontology database (5).

Real-time qRT-PCR. For real-time quantitative reverse transcription-PCR (qRT-PCR), the primer and probe sets were designed using Primer Express software (Applied Biosystems, United Kingdom). Primer and probe sequences are presented in Table 1. RNA was extracted from cells using an RNeasy Mini kit (Qiagen, Crawley, United Kingdom) or from tissues using an RNeasy fibrous tissue kit (Qiagen) and treated with DNase I (Ambion, United Kingdom). First-strand cDNA synthesis was carried out using a SuperScript III first-strand synthesis system (Invitrogen, United Kingdom). Quantitative gene expression analysis was performed on 100 ng of total cDNA and analyzed using an ABI Prism 7500 sequence detection system (Applied Biosystems). Invitrogen Platinum quantitative PCR SuperMix-UDG with ROX was used in conjunction with the appropriate primers and probe for each reaction. An initial 2 min, 50°C Platinum Taq DNA polymerase activation step and a 2 min, 95°C denaturing step were used, followed by 45 cycles of 15 s at 95°C and 45 s at 60°C. Fluorescence measurements (6-carboxyfluorescein, ROX, and 6-carboxytetramethylrhodamine) were collected for every cycle at 60°C. Reactions were performed in triplicate using four replicate RNA samples. The efficiencies of amplification of all target genes and of the internal reference GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were approximately equal (data not shown). The relative quantification levels or severalfold changes in expression were determined using the comparative CT method (2–{Delta}{Delta}CT) (36).


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  		TABLE 1. Primer and probe sequencesa

Flow cytometry. Flow cytometry for major histocompatibility complex class I (MHC-I) in equine fibroblasts was performed as described in reference 39. Briefly, the cells were permeabilized with 0.1% saponin in phosphate-buffered saline (PBS) for 30 min at room temperature and incubated for 30 min at 41°C with H58A, a monoclonal antibody specific for equine MHC-I (VMRD, Inc.), and a secondary goat anti-mouse immunoglobulin G (Fab-specific) fluorescein isothiocyanate (FITC)-conjugated antibody (Sigma, United Kingdom). All samples were examined in a Beckman Coulter EPICS Elite analyzer equipped with an ion argon laser with 15 mV of excitation at 488 nm. The data were analyzed using Expo 2 software.

Immunofluorescence. Cells were grown on glass coverslips in Dulbecco's modified Eagle's medium-10% fetal bovine serum, and matrix metalloproteinase 1 (MMP-1) was detected with a polyclonal MMP-1 antibody (catalog no. ab38929; Abcam) and FITC-conjugated goat anti-rabbit immunoglobulin G (Sigma). Cells were fixed in 4% formaldehyde in PBS for 30 min and permeabilized for 15 min in PBS containing 0.05% Triton X-100 and 20 mM glycine. Cells were blocked in 1% fetal bovine serum-PBS for 20 min followed by the addition of antibody diluted in blocking buffer and incubation at room temperature for an hour. Both the primary MMP-1 antibody and the FITC-conjugated second antibody were diluted in blocking buffer at 1:100. Cells were washed in blocking buffer and were mounted onto glass slides using Vectashield (Vector Laboratories) followed by confocal microscopy (Leica TCS SP2 microscope).


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RESULTS
 
Identification of genes regulated by BPV-1 in equine fibroblasts. To identify genes whose expression was altered by BPV-1, total RNA was extracted from BPV-1-transformed EqPalFs cells (S6-2 cells), cells from a cell line explanted from an equine sarcoid (EqS01a cells) (69), and control EqPalF parental cells and was used to hybridize to the equine GeneChip comprising 3,098 genes and expressed sequence tags (ESTs) (25). Changes in the level of transcription of each gene/EST were determined by statistical analysis of the differences in mean fluorescence between S6-2 cells, EqS01a cells, and EqPalFs over four replicate experiments. Cutoff values for genes were defined as (a) a level of significance (differential expression) of P < 0.01 and (b) a severalfold change greater than 2.0. Using these criteria, a total of 81 probe sets were modulated in both S6-2 and EqS01a cells (Table 2). Of these, 44 (54%) probe sets were found to be up-regulated and 37 (46%) down-regulated. With a few exceptions, there was good agreement between the results obtained with genes up- or down-regulated in S6-2 and EqS01a cells.


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  		TABLE 2. Genes modulated by BPV-1 in equine fibroblastsa

The differentially expressed genes were assigned to functional categories using Gene Ontology database analysis. For several of the equine ESTs, no homologues were identified by BLAST analysis (December 2007), and these were designated "Unknown." The biological functions of the differentially expressed genes are diverse and include functions in inflammation and immunity, cell adhesion/structural integrity, cell cycle/cell proliferation, apoptosis, and RNA transcription/metabolism. Some genes can straddle two or more groups. Genes with functions not falling into one or more of these five groups were assigned to the "Miscellaneous" group (Table 2).

Here we describe individual genes in these groups with potential relevance to sarcoid pathogenesis and then describe the validation of the microarray data for some of them.

Immunity- and inflammation-related genes. Members of a large subset of genes involved in immune response and inflammation were differentially expressed in S6-2 and EqS01a cells. Within this group, expression of CXC/CC chemokines (CXCL5, GRO2/CXCL2, CXCL6, and CCL7) was significantly increased (Table 2). Chemokines are important for the activation of immune responses and also function in multiple aspects of tumor development and progression (8, 9, 51). Additional up-regulated genes implicated in the inflammatory response included S100A12 (calgranulin C), CGRP (calcitonin gene-related peptide), and NKG7 (natural killer cell group 7). Calgranulin C is a calcium-binding protein, also expressed in skin tumors, which activates the NF-{kappa}B pathway, leading to the synthesis and secretion of proinflammatory mediators (27), CGRP is a neuropeptide released from sensory nerves during inflammation (11), and NKG7 is a cytotoxic-granule membrane protein (42).

Several immune response genes showed down-regulation. These included TLR4 (Toll-like receptor 4), MHC I, IL12B (interleukin-12B [IL-12B]), and C1R (complement component 1, subcomponent r). TLRs are receptors that promote innate immune responses to pathogens (43). MHC-I presents antigenic peptides to cytotoxic T lymphocytes (CTL) and is a pivotal player in the adaptive immune response (16). IL-12 is a multifunctional cytokine which promotes TH1 differentiation, gamma interferon production, proliferation, and the cytolytic activity of NK and CTL cells and is therefore a key regulator of cell-mediated immune responses (17). C1R is a member of the complement system and is involved in clearance of opsonized immune complexes and in immunologic defense (22).

Genes involved in cell adhesion, motility, and integrity. In this group MMP-1 was the most overexpressed gene. MMP-1 encodes MMP-1 (a collagenase with proteolytic activities against interstitial collagen), which promotes cell proliferation, invasion, metastasis, and angiogenesis (18). Another overexpressed gene was CSK (c-src tyrosine kinase). c-SRC is involved in numerous processes, including cell proliferation, survival, cell adhesion, and migration, and is overexpressed in many tumors (28). ALCAM was also up-regulated. ALCAM (activated leukocyte cell adhesion molecule) is expressed in a variety of tissues and cells, including epithelia and fibroblasts, and is implicated in cell adhesion and tumor progression (50).

Genes involved in cell cycle/cell proliferation. Up-regulated genes in this group were FRA-1 (fos-like antigen), CDKN2A/p16INK4A (p16, inhibitor of CDK2), c-KIT (mast cell growth factor), and KNSL (kinesin-like protein 1) (Table 2). FRA-1 is a component of activator protein-1 (AP-1) transcriptional complexes and is implicated in many cancers (65). CDKN2A/p16INK4A is overexpressed in human papillomavirus (HPV)-positive cervical squamous cell carcinomas (SCC) (38). c-KIT encodes a transmembrane tyrosine kinase receptor and is overexpressed in a number of neoplasms (44) as well as in HPV-positive SCCs (38). KNSL1 is a central mitotic spindle protein involved in cytokinesis. While KNSL1 does not appear to have a direct role in cancer, BPV-1 E2 interacts with KNSL2 (66).

Genes involved in apoptosis. Genes encoding inhibitors of apoptosis were both up- and down-regulated in BPV-1-transformed fibroblasts (Table 2). c-IAP-1 (inhibitor of apoptosis 1) and HMGA1 (high mobility group AT-hook 1) (see below) were both up-regulated. c-IAP-1 blocks cell death and is up-regulated in many cancers (58). HMGA1 has many functions (see below), including inhibition of p53-mediated apoptosis (54). Down-regulation of rhoGDI (rho-GDP dissociation inhibitor) and pro-IGF-1a (pro-insulin growth factor 1a), both involved in apoptosis, was also detected (19, 70).

Genes involved in RNA transcription and metabolism. A number of genes involved in transcription regulation and RNA processing/metabolism were deregulated. Two genes were overexpressed: HMGA1 and UPP1 (uridine phosphorylase). HMGA1 is a chromatin protein that binds AT-rich sequences in the minor groove of DNA and is frequently overexpressed in a range of cancer cells (21). The isoform HMG-I(Y) is up-regulated by HPV type 16 (HPV-16) E6 in mouse cells (30). UPP1 is involved in uridine metabolism, and related enzyme activity is elevated in various tumor tissues (29, 34).

Validation of microarray data by real-time qRT-PCR analysis. To validate the differential gene expression profiles obtained by microarray analyses, expression of four up-regulated genes, MMP-1, FRA-1, NKG7, and CXCL5, and two down-regulated genes, TLR and MHC I, was examined by qRT-PCR.

These six genes were chosen for their relevance to the establishment of papillomavirus infection and to cell transformation and therefore for their relevance to sarcoid pathogenesis (see Discussion).

Validations were performed using the original cell lines (S6-2, EqS01a, and EqPalFs) with which the microarray analyses were performed and additional equine cell lines either transformed in vitro by BPV-1 (S6-1 and S6-3) or explanted from sarcoids (EqS02a and EqS04b) (69). Up-regulation of MMP-1, CXCL5, FRA-1, and NKG7 was confirmed by qRT-PCR using the original cell lines (S6-2, EqS01a) (Fig. 1). These genes also showed various degrees of up-regulation in the other cell lines, although no up-regulation of CXCL5 in EqS04b cells was detected. Down-regulation of both TLR4 and MHC I was confirmed in the original and all additional cell lines (Fig. 1). The data demonstrate that the overall results of qRT-PCR were consistent with those of the microarray analyses, although variations between these two types of analyses in the data representing severalfold differences were observed.


Figure 1
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  		FIG. 1. Validation of microarray data. For qRT-PCR, six genes were selected for expression analysis in BPV-1-transformed cells; the data are presented as severalfold changes relative to EqPalF control cell results. GAPDH served as the internal control. Data represent the means of the results obtained from at least three replicate experiments; error bars indicate standard deviations from the means.

Analysis of MMP-1 protein and MHC-I complex. To verify that changes in cellular mRNAs in BPV-1 fibroblasts corresponded to changes in protein expression, cells were analyzed with respect to MMP-1 and MHC-I.

MMP-1. There are no antibodies specific for equine MMP-1; therefore, a cross-reacting human MMP-1 antibody was used. This antibody works well in immunofluorescence but not in Western blot analysis. All BPV-1-transformed cells showed strong green fluorescence (MMP-1) (Fig. 2a) not detected in control cells. These results show that increases in MMP-1 mRNA do indeed correspond to increases in MMP-1 protein.


Figure 2
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  		FIG. 2. Changes in protein levels in BPV-1 cells. (a) EqS01a, EqS04b, and S6-2 cells overexpress MMP-1 compared to control EqPalFs. Images were collected with a confocal microscope. For EqS01a, EqS04b, and EqPalFs, magnification was x20; for S6-2 cells, magnification was x10. (b) S6-2 and EqS01a cells express less MHC-I than control cells. MHC-I expression was assessed by flow cytometry. The forward shift in fluorescence was measured in at least two experiments in duplicate (except for EqS01ap4 cells, which were analyzed only once in duplicate), and mean values are plotted along the y axis; error bars indicate standard deviations.

MHC-I. The antibodies against equine MHC-I did not work well in Western blot analyses, and therefore flow cytometry was used to determine the expression of MHC-I in the cell lines. MHC-I was analyzed using two isolates of S6-2 cells and two different passages of EqS01a cells. Although the forward shift in fluorescence was not very large, it was clear that all the cells had a much reduced amount of MHC-I (Fig. 2b). These results confirm that reduction in MHC-I (heavy chain) RNA indeed results in a down-regulation of MHC-I complex.

Expression of a subset of differentially expressed genes in sarcoid tumors in vivo. Having ascertained that the microarray and qRT-PCR analyses can provide a faithful readout representing protein expression in cells, expression patterns for a panel of 10 BPV-1-positive sarcoid tumors (T1 to T10) were investigated by qRT-PCR (Fig. 3a). MMP-1 expression was up-regulated in all tumors, although there was considerable variation: T4, T6, T7, and T9 showed a relatively modest up-regulation of expression of between 2- and 10-fold; T2, T5, and T8 showed up-regulation of from 70- to over 500-fold; and T3 exhibited a dramatic increase in expression of 9,000-fold. Up-regulation of CXCL5 expression among the tumors was not a consistent finding, and an up-regulation of expression greater than twofold was observed only with T1, T3, and T8. An up-regulation of FRA-1 expression greater than twofold was evident in T5, T8, and T10. NKG7 expression was increased in T4, T7, and T10. MHC I and TLR4 gene expression was significantly (between 40- and 100-fold) down-regulated in all 10 tumors (Fig. 3b).


Figure 3
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  		FIG. 3. Gene expression in sarcoid tumors. Gene expression was studied using a panel of 10 sarcoid tumors (T1 to T10); data are expressed as severalfold changes relative to normal skin tissue control results. Data represent the means of the results obtained from at least three replicate experiments; error bars indicate standard deviations from the means.

Host gene expression patterns were compared with viral gene expression and viral DNA load patterns to elucidate whether specific viral genes were associated with the up- or down-regulation of particular host genes. No correlation was found in either case (data not shown).

No correlation between sarcoid clinical type and host gene expression was evident (data not shown).


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DISCUSSION
 
The mechanisms used by BPV-1 in the pathogenesis of sarcoids are still poorly understood, although certain facets have begun to emerge (46, 47, 69). To advance our knowledge of the impact of BPV-1 on equine cells and understand how BPV-1 infection perturbs host cell gene expression, we have investigated global changes in the transcriptome in equine fibroblasts in response to BPV-1. A total of 81 genes involved in a wide range of functions were deregulated and can be grouped into five specific function categories plus a miscellaneous category and an unknown category. Genes in the last two categories are not discussed further here. It has to be kept in mind that there is a limited number of genes/ESTs on the equine microarray, representing approximately 10% of the total number of equine genes, and that more genes may therefore be deregulated by BPV-1.

Six genes (MMP-1, CXCL5, FRA-1, NKG7, MHC 1, and TLR4) were chosen for validation in our panels of transformed cell lines and sarcoids. In almost all cases, differential expression was confirmed. Furthermore, we confirmed that in the case of MMP-1 and MHC-I, differences in transcription resulted in differences at the protein level. These results give strength to the idea of the use of microarrays for readouts representing changes not only in the transcriptome but also in the proteome of BPV-1-transformed cells. Here, we will discuss the categories of genes modulated by BPV-1, with a particular focus on the genes selected for validation, namely, CXCL5, NKG7, MHC I, MMP-1, TLR4, and FRA-1.

Immunity- and inflammation-related genes. BPV-1 activates an inflammatory response and represses components of both the innate and adaptive immune response. Chemokines are members of a family of small cytokines that play important roles in recruitment of cells needed for the activation of inflammatory/immune responses, and observations identifying their functions in tumor development/progression are beginning to emerge (8, 9, 51). For example, CXCL5 is a strong neutrophil chemoattractant and is implicated in tumor cell proliferation, migration, and invasion (45, 52). Expression of CXCL5 was significantly altered in equine fibroblasts transformed by BPV-1 (Table 2). CXCL5 was up-regulated in all cell lines except one and in 3 out of 10 sarcoids (Fig. 1 and 3a). The up-regulation of CXCL5, and of other chemokines, is likely to modify the tumor microenvironment, facilitating sarcoid growth and invasion, and to contribute to sarcoid recurrence. The variations observed in expression of CXCL5 (and of other genes; see below) in tumors were likely due to the fact that, in contrast to cell lines, sarcoids represent a mixture of fibroblasts, including some that do not harbor BPV DNA. The contribution of additional factors cannot be ruled out.

NKG7 is a cytotoxic-granule membrane protein expressed in activated NK and T cells that regulates the effector function of lymphocytes and neutrophils (42). Up-regulation of NKG7 was variable between the cell lines and not detected in all of the tumors (Fig. 1 and 3a). As with CXCL5 results, this was likely due to the mixed cellularity of sarcoids. To our knowledge, expression of NKG7 in fibroblasts has not been reported before; therefore, its relevance to sarcoids is unclear.

Four genes (TLR4, MHC I, IL12B, and C1R) pivotal in the innate and adaptive immune response to pathogens, including viruses, are noticeably down-regulated by BPV-1. Toll-like receptors are part of the innate immune response and recognize structurally conserved motifs of pathogens, promoting an immune response (43). TLR4 was consistently and robustly down-regulated in all sarcoids and cell lines (Fig. 1 and 3b). Upon interaction with ligand, TLR4 activates NF-{kappa}B signaling, with up-regulation of cytokines and adhesion molecules resulting in pathogen control and clearance (20). TLR4 is the receptor for bacterial lipopolysaccharide; however, there are two known viral proteins that activate TLR4: the fusion protein of respiratory syncytial virus (35), and the Env protein of mouse mammary tumor virus (56). The remarkable down-regulation of TLR4 expression in sarcoids is thought to impair the production of appropriate cytokines and contribute to viral persistence and tumor recurrence. Indeed, approximately 20 to 50% of sarcoids recur following surgical intervention (41, 59). Furthermore, expression of another TLR, TLR9, is inhibited in cervical SCCs and in keratinocytes transformed by HPV-16 E6E7 (26).

MHC-I is a pivotal component of the adaptive immune response, as it presents antigenic peptides to CTL, initiating clearance of infected cells (16). To establish a successful (and persistent) infection, viruses have evolved numerous mechanisms for inhibiting antigen presentation by MHC-I (55, 64). As is the case for BPV-induced tumors in cattle and BPV-transformed bovine cells (4, 6, 40), MHC I transcription is consistently and remarkably down-regulated in all sarcoids and cell lines (Fig. 1 and 3b) and MHC-I complex is underexpressed in cells (Fig. 2b). Although the antibody used may recognize only some MHC-I alleles, the profound down-regulation of MHC I transcripts suggests that, as already shown for bovine and human MHC-I (7, 39), the effect is not restricted by MHC-I. MHC-I reductions in sarcoids are expected to allow BPV infection to become established and the infected cells to escape CTL killing. We have ascertained that the irreversible down-regulation of MHC-I in bovine cells is due to the presence of BPV E5 (6, 39), as is the case in equine cells (B. Marchetti, Z. Q. Yuan, E. A. Gault, L. Nasir, and M. S. Campo, unpublished data).

Cell adhesion and integrity genes. Among members of this group of genes, MMP-1 is the most highly up-regulated gene (Table 1 and Table 2). Its overexpression in tumors and cell lines (Fig. 1 and 3a) is reflected in protein overproduction (Fig. 2a). MMPs represent the most abundant class of extracellular matrix proteins which degrade interstitial collagen, promoting localized invasion and spread of cancer cells to adjacent tissues (18, 48). In concordance with our findings showing up-regulation of MMP-1, collagen turnover is substantially elevated in sarcoids (63). HPV-8 E7 can up-regulate MMP-1, and organotypic cultures of cells transduced with HPV-8 E6 and E7 or E7 alone show changes to the epithelium similar to those seen in sarcoids, including the formation of rete ridges of heterogenous lengths extending into the dermis (1, 23). Given that MMP-1 is associated with the ability not only to degrade the extracellular matrix but also to invade local tissue, it is highly plausible that MMP-1 allows sarcoid cells to invade surrounding tissues. Persistent infection of BPV-1 DNA and its induction of MMP-1 would also contribute to recurrence. It is interesting that the sarcoid T3 showed the highest expression of MMP-1 and CXCL5 (Fig. 1 and 3a). As discussed above, CXCL5 plays a role in migration and invasion (45, 52).

Genes involved in cell cycle/cell proliferation. Several genes involved in cell proliferation were found to be activated in BPV-1 fibroblasts. FRA-1 was consistently activated in our panel of cell lines but not in all tumors. The likely reasons for this have already been discussed. FRA-1 is a component of AP-1, a transcriptional complex implicated in many cancers (65). Interestingly, FRA-1 induces MMP-1 expression (61). The concomitant induced expression of FRA-1 and MMP-1 in sarcoids and sarcoid cells suggests that BPV-1 induces FRA-1 and that this in turn activates MMP-1; however, this requires experimental confirmation.

Although results for these genes have not been validated, genes which merit further analysis are IL12B, CSK, c-IAP-1, and UPP1. IL12B is down-regulated in BPV-1-transformed fibroblasts. IL-12 is a multifunctional cytokine which promotes TH1 differentiation, gamma interferon production, proliferation, and cytolytic activity of NK and CTL cells. IL-12 is therefore a key regulator of cell-mediated immune responses (17). IL-12 underexpression in sarcoids would contribute to an impaired cellular immune response to sarcoid cells.

CSK is up-regulated in BPV-1-transformed fibroblasts. c-SRC has numerous roles in cancer progression (28). BPV E5 induces c-SRC activation (57, 62), and cells with activated c-SRC are more motile and more invasive than control cells (62). c-SRC expression in sarcoids would contribute to invasion and spread.

c-IAP-1 is up-regulated in BPV-1 cells. c-IAP-1 blocks cell death and is up-regulated in many cancer types (58), while attenuation of c-IAP-1 mRNA leads to enhanced tumor cell death (24). Up-regulation of c-IAP-1 would abrogate apoptotic responses in sarcoids. Interestingly, p53 is disabled in sarcoids and sarcoid cells (10, 49, 69).

UPP1 converts the pyrimidine nucleoside uridine into uracil. Enzyme activity is elevated in various tumor tissues (29, 30); it is thought to contribute to the therapeutic efficacy of fluoropyrimidines in cancer patients (14). The increased expression of UPP1 in BPV-1 positive cells suggests that 5-fluorouracil (5-FU) may have therapeutic benefit for horses with sarcoids. Indeed, 5-FU is used in the management of sarcoids, with various degrees of success (32, 33). It is interesting to speculate that different levels of UPP1 may affect sarcoid sensitivity to 5-FU.

In conclusion, this is the first report to document some of the transcriptional changes induced by BPV-1 in equine fibroblasts. The main changes are increases in genes that promote motility and invasion and reduce apoptosis and decreases in genes promoting and shaping the immune response. These changes provide an insight into how BPV-1 contributes to sarcoid pathogenesis, and we propose that sarcoids develop because of immunological failure, lack of apoptosis, and increased cell motility and invasion. Furthermore, our data should lead to the identification of potentially novel targets for therapeutic intervention. For example, inhibition of c-IAP-1 may induce apoptosis in tumor cells or sensitize BPV-1-expressing cells to cytotoxic drug-induced cell death. Similarly, inhibition of MMP-1 may reduce localized infiltration of sarcoid cells.


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ACKNOWLEDGMENTS
 
We are grateful to Iain Morgan for his critical reading of the manuscript. The microarray experiments were performed using the service provided by Alicia Bertone, Ohio State University.

This work was supported by The Horse Trust, United Kingdom, and PetPlan Charitable Trust. M.S.C. is a Fellow of Cancer Research, United Kingdom.


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FOOTNOTES
 
* Corresponding author. Mailing address: Division of Pathological Sciences, Institute of Comparative Medicine, University of Glasgow, Faculty of Veterinary Medicine, Bearsden Road, Glasgow G61 1QH, Scotland, United Kingdom. Phone: 0141 330 5731. Fax: 0141 330 5602. E-mail: l.nasir{at}vet.gla.ac.uk Back

{triangledown} Published ahead of print on 23 April 2008. Back


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Journal of Virology, July 2008, p. 6481-6491, Vol. 82, No. 13
0022-538X/08/$08.00+0     doi:10.1128/JVI.00429-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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