Department of Microbiology-Immunology, Northwestern University Medical School, 303 E. Chicago Avenue, Chicago, IL 60611, USA1
Author for correspondence: Richard Longnecker. Fax +1 312 503 1339. e-mail r-longnecker{at}northwestern.edu
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Abstract |
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Previous studies using in vitro models of EBV latent infections in B lymphocytes established that LMP2A blocks normal B-cell receptor (BCR) signal transduction by the association with Src family protein tyrosine kinases (PTKs) and the Syk PTK (Burkhardt et al., 1992 ; Fruehling & Longnecker, 1997
; Fruehling et al., 1998
; Miller et al., 1994
, 1995
). In vivo studies using transgenic mice that express LMP2A in B lymphocytes have indicated that LMP2A not only blocks BCR signal transduction but also provides a BCR-like signal that allows for B-cell development and survival in the absence of normal BCR signals (Caldwell et al., 1998
, 2000
). Very little is known in regard to the effects of LMP2A on epithelial biology. A recent study indicates that LMP2A is phosphorylated upon adhesion of epithelial cells expressing LMP2A to extracellular matrixes, which suggests that LMP2A may be important in altering normal epithelial signal transduction (Scholle et al., 1999
). Signal transduction in epithelial cells uses a similar repertoire of signal-transducing proteins as is used in normal BCR signal transduction. Thus, to determine if LMP2A may also provide an inappropriate developmental or survival signal in epithelial cells which would result in the alteration of normal epithelial biology, transgenic mice with LMP2A expression directed to the basal layer of the epidermis were constructed using a keratin 14 (K14) promoter construct. A total of six murine K14LMP2A transgenic lines were analysed, all which had no alteration in normal epithelial differentiation despite abundant LMP2A expression in the basal layer of the epidermis.
In order to drive expression of LMP2A to differentiating epithelia, a previously described K14 cassette was obtained from E. Fuchs (University of Chicago, USA). The K14 construct containing the K14 promoter, K14 polyA tail and betaglobin splice site is shown in Fig. 1(A). This expression construct was previously used to construct multiple transgenic lines which resulted in the expression of the transgene in the basal layer of the epidermis (Guo et al., 1993
; Turksen et al., 1992
; Vassar et al., 1992
). A previously described chimeric LMP2A gene consisting of both cDNA and genomic sequences (Caldwell et al., 1998
, 2000
) was placed downstream of the K14 promoter in the expression plasmid. The transgene was excised from the parental vector and used for injections into mouse embryos. Twenty-six mice were produced. At 3 weeks of age, DNA was prepared from mice tail snips, digested with BamHI, subjected to gel electrophoresis in 0·8% agarose, and transferred to Gene Screen Plus (NEN Life Science) as previously described (Caldwell et al., 2000
). Southern blot analysis of genomic tail DNA using an LMP2A-specific probe identified eight founder mice (Fig. 1B
). Seven of the eight lines contained the unit length 5100 bp band resulting from head to tail concatemers of the transgene (Fig. 1B
, lines 101, 102, 103, 104, 105, 107 and 108). Line 106 lacked this band and was not analysed further. The additional bands in each line which hybridize to the LMP2A-specific probe probably represent fusion bands of the LMP2A transgene with murine DNA sequences or rearranged LMP2A transgenes. The transgenic mice were bred into a CD1 wild-type background purchased from Jackson Laboratories. Transgenic line 108 did not breed and therefore was not analysed. Lines 101105 and 107 were analysed as described below.
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To determine if the expression of LMP2A altered the normal morphology and/or differentiation of the epithelium, tail and tongue sections were analysed from 6-week-old K14LMP2A transgenic mice and littermate controls. Fig. 2(A) shows a schematic representation (left) of the four stages of epithelial development and the expression of the differentiation markers associated with each stage lined up with a representative photomicrograph (right) of an H&E-stained tail section from a wild-type mouse control for reference purposes. H&E-stained paraffin-embedded sections were prepared by placing mouse tail or tongue samples in buffered Formalde-Fresh (Fisher), embedding in paraffin, and staining 56 µm sections with H&E. As observed in Fig. 2(B)
(tail sections) or Fig. 2(C)
(tongue sections), there was no apparent change in thickness or epithelial differentiation in the LMP2A transgenic lines 101, 102 and 107 when compared to littermate controls. This result was also observed in the 103, 104 and 105 K14LMP2A transgenic lines (data not shown).
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LMP1, EBNA1, BARTs, EBERs and LMP2A are the EBV latency-associated genes most consistently detected in NPC tumour biopsies (for review, Raab-Traub, 1992 ; Cruchley et al., 1997
; Rickinson & Kieff, 1996
). Work previously done on LMP1 has shown it to have transforming effects in rodent fibroblasts, growth-altering effects in B lymphocytes, and it is absolutely required for transformation of primary B lymphocytes with EBV (for review, Kieff, 1996
; Longnecker, 1998
). In transgenic mice, LMP1 induces epithelial hyperplasia and aberrant expression of keratin when expressed in the epithelium and induces lymphoproliferation when expressed in lymphocytes (Kulwichit et al., 1998
; Wilson et al., 1990
). There is little information in regard to the effects of EBNA1 on normal epithelial biology. Transgenic murine studies have indicated that EBNA1 may predispose lymphocytes to malignant transformation (Wilson et al., 1996
). Despite these observations, EBNA1 does not dramatically alter B-lymphocyte phenotype in vitro. The exact role of the BARTs and EBERs in EBV-mediated transformation is yet to be determined. Results with LMP2A transgenic mice with lymphocyte-directed expression indicated a putative role for LMP2A in EBV latency and transformation that was not previously appreciated. In these studies, LMP2A was shown to alter normal B-cell development and provide a survival signal to B lymphocytes (Caldwell et al., 1998
, 2000
). In this study, we investigated the effect of LMP2A expression on normal epithelial differentiation also utilizing a transgenic approach.
Work in vitro has shown that LMP2A specifically binds to and regulates the activity of the Src family PTKs and the Syk PTK. The Syk PTK accumulates in most haematopoietic cell types, including B cells, mast cells, platelets and immature T cells (for review, Weiss & Littman, 1994 ). Zap-70, a related PTK, is expressed only in T cells (for review, Weiss & Littman, 1994
). There is no indication that either of these PTKs are expressed in epithelial-derived cell lines. In contrast, at least four Src family PTKs (Thomas & Brugge, 1997
), two of which have been shown to bind LMP2A, are expressed in epithelial cells (Burkhardt et al., 1992
). Src has been shown to be activated following integrin engagement following attachment of epithelial cells to a fibronectin matrix (Kaplan et al., 1995
). Other signal transduction cascades present in epithelial cells in which Src family kinases are important include signalling induced by the cadherins and receptor protein tyrosine kinases. In addition to the role of Src family PTKs in epithelial signal transduction, there are additional signalling proteins, many of which are shared with B-lymphocyte signal transduction, which may be affected by the expression of LMP2A. Despite the central role of Src family PTKs in normal epithelial signal transduction and the ability of LMP2A to alter normal Src family PTK function in B lymphocytes, there was no observable effect of LMP2A expression on epithelial cells in the K14LMP2A transgenic mice.
Given the dramatic effects that LMP2A can have on B-lymphocyte differentiation and survival, it is surprising that LMP2A appears to have little effect on epithelial differentiation despite the importance of Src family PTK signalling in epithelial cells. LMP2A is phosphorylated by Csk upon adhesion to extracellular matrixes in cell lines grown in tissue culture. Since LMP2A is consistently detected in most NPC samples, it is likely that LMP2A will have a role in the development of EBV-associated epithelial malignancies. LMP2A may not be the primary initiating event, but may act in concert with other viral proteins such as EBNA1 and LMP1, which are also typically expressed in NPC biopsies. Alternatively, the alteration of a specific cell protein may be required for LMP2A-specific effects in epithelial cells. Future studies utilizing the LMP2A transgenic mice constructed in this study will entail the mating of the LMP2A transgenic lines with transgenic lines expressing other EBV proteins expressed in NPC to determine if they may act in concert. In addition, studies to determine if LMP2A may have more subtle effects on epithelial cells, which may be evident by analysing wound healing and chemical carcinogenesis in the LMP2A transgenic mice, will be pursued. These studies may shed light on the role of LMP2A in EBV-associated epithelial cancers.
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References |
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Received 19 April 2000;
accepted 26 May 2000.