The Epstein-Barr virus encoded LMP1 oncoprotein modulates cell adhesion via regulation of activin A/TGFβ and β1 integrin signalling

Approximately 20% of global cancer incidence is causally linked to an infectious agent. EpsteinBarr virus (EBV) accounts for around 1% of all virus-associated cancers and is associated with nasopharyngeal carcinoma (NPC). Latent membrane protein 1 (LMP1), the major oncoprotein encoded by EBV, behaves as a constitutively active tumour necrosis factor (TNF) receptor activating a variety of signalling pathways, including the three classic MAPKs (ERK-MAPK, p38 MAPK and JNK/SAPK). The present study identifes novel signalling properties for this integral membrane protein via the induction and secretion of activin A and TGFβ1, which are both required for LMP1’s ability to induce the expression of the extracellular matrix protein, fbronectin. However, it is evident that LMP1 is unable to activate the classic Smad-dependent TGFβ signalling pathway, but rather elicits its efects through the non-Smad arm of TGFβ signalling. In addition, there is a requirement for JNK/SAPK signalling in LMP1-mediated fbronectin induction. LMP1 also induces the expression and activation of the major fbronectin receptor, α5β1 integrin, an efect that is accompanied by increased focal adhesion formation and turnover. Taken together, these fndings support the putative role for LMP1 in the pathogenesis of NPC by contributing to the metastatic potential of epithelial cells

Lower proportions of the full-length isoform expressed from the CD40 risk allele in monocytes and dendritic cells.

<p>Association of rs1883832 genotype (CC, TC, TT) with the proportion of full-length isoform of CD40 (%FL) expressed in <i>in vitro</i> differentiated dendritic cells (DC1, DC2) from healthy controls (n = 49). Molar ratios of isoforms were quantitated by RT-PCR and amplification of a region spanning CD40 exon 4–10, followed by electrophoretic separation and fluorescent detection (Bioanalyzer, Agilent); p values by Mann-Whitney test.</p

CD40 protein is under- expressed in B lymphocytes of MS patients.

<p>B lymphocyte subsets from healthy controls (n = 86) and MS patients (n = 24) were identified by flow cytometry and CD40 expression determined relative to an isotype control (relative fluorescence intensity; RFI). Surface levels of CD40 were compared in total B-lymphocytes (A), B lymphocytes from rs1883832 CC individuals (B; n = 49 healthy controls, n = 12 MS patients), naïve B lymphocytes (C), classical memory B lymphocytes (D) and IgM memory B lymphocytes (E). P-values were determined using Mann—Whitney test.</p

CD40 expression is higher in differentiated dendritic cell subsets.

<p>CD40 expression was determined in freshly purified immune cell subsets (B cells, monocytes) or <i>in vitro</i> differentiated dendritic cells (DC1, DC2) from healthy controls. Gene expression by RT-PCR (A; n = 49) and relative fluorescence intensity (RFI) by flow cytometry (B; n = 41) are shown; *significantly different from monocytes and DCs; **significantly different from B cells and monocytes (A) or from monocytes (B); p < 0.05 by Mann-Whitney test.</p

Genotype dependent CD40 protein expression in peripheral B-lymphocyte subsets of MS patients and healthy controls.

<p>B lymphocyte subsets from healthy controls (n = 86) and MS patients (n = 21) were identified by flow cytometry (A) and CD40 expression determined relative to an isotype control (relative fluorescence intensity; RFI). Regulatory B cells were identified as CD19+CD38hiCD24hi (data not shown) (B). Association of rs1883832 genotype with CD40 expression in healthy controls (CC = 49, CT = 27, TT = 10) was examined in total B lymphocytes (C), naïve B-lymphocytes (D) and classical memory B-lymphocytes (E), and in total B lymphocytes (F), naïve B lymphocytes (G) and classical B lymphocytes (H) of MS patients (CC = 12, CT = 7, TT = 2). p values were determined by Mann-Whitney U test comparison of each group.</p

CD40 mRNA expression in peripheral blood immune cell subsets.

<p>CD40 mRNA expression was determined by RT-PCR in freshly purified immune cell subsets or <i>in vitro</i> differentiated subsets (Th1, Th2, Th17; differentiated from fresh CD4CD45RA) from healthy controls (n = 3, or n = 2 for pDC).</p

Proportions of the full-length isoform expressed from the CD40 risk allele in whole blood.

<p>Association of rs1883832 genotype (CC, TC, TT) with the proportion of full-length isoform of CD40 (%FL) expressed in whole blood from healthy controls (A; n = 38) and MS (B; n = 32). Molar ratios of isoforms were quantitated by RT-PCR and amplification of a region spanning CD40 exon 4–10, followed by electrophoretic separation and fluorescent detection (Bioanalyzer, Agilent). Trends were observed for CC > CT in controls (p < 0.13) and for CT > TT in MS, (p < 0.056); p values by Mann-Whitney test.</p

Results of the association testing for variants identified in <i>MERTK</i>.

<p>Results of the association testing for variants identified in <i>MERTK</i>.</p

SNPs within <i>MERTK</i> define both risk and protective haplotypes associated with MS susceptibility.

<p>28 SNPs within the <i>MERTK</i> gene form a single block of very high LD (D'>0.99, LOD≥2). The five most frequent haplotypes (population frequency >1%) are shown in this schematic, along with the <i>p</i>-value of association of each haplotype with MS susceptibility as determined using a Chi-square test. Arrowhead indicates the haplotype-tagging allele of rs13414207 in haplotype 5. The alleles presented for rs17174870 are inferred from data obtained from sequencing the opposite strand but are presented as CT to maintain consistency with the remainder of the study.</p

The haplotype structure of variants identified in <i>MERTK</i>.

<p>The 52 variants in <i>MERTK</i> genotyped for association with MS susceptibility fall into 4 separate blocks. Haplotype blocks are connected with thick lines if connections are observed in >10% samples and thin lines if connections are observed in >1% samples. A schematic of the <i>MERTK</i> gene is shown underneath indicating the relationship of the blocks to the physical structure of <i>MERTK</i>. The haplotypes coloured in red are significantly associated with MS susceptibility (p<0.05) and the haplotype coloured in blue is associated with protection (p<0.05). The <i>p</i>-value of association was determined using a Chi-square test. #This variant represents a tri-nucleotide in-del (T/- = TGG; -)</p