The effect of a Mycoplasma hyorhinis protein (p37) on gene expression in mouse fibroblasts

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Life Sciences, Department of Botany, Faculty of Science, Technology and Engineering, La Trobe University, Bundoora.The p37 protein is a lipoprotein associated with the plasma membrane of Mycoplasma hyorhinis. Cell lines treated with the purified p37 protein exhibit reduced heterotypic contact inhibition of locomotion in the Abercrombie confronted explant invasion assay. In addition p37-treated and p37-transformed cells show increased migration through transwell chambers and increased invasivity through a Matrigel matrix. However, the molecular mechanisms underlying these responses are not known. This thesis identifies and characterizes the changes in gene expression of the NIH3T3 (mouse) fibroblast cell line in response to added p37 protein. A microarray analysis of p37-treated and untreated NIH3T3 cells identified many genes that were up or downregulated. The most strongly up-regulated genes encoded cytokines and acute phase proteins which play a major role in the inflammatory response. Quantitative PCR was used to determine the temporal expression pattern of the p37-induced gene expression over 24hr. The cytokine interleukin 6 (Il6) was identified as a primary response gene. Possible explanations for this response are discussed. The Il6 protein, via the Il6 receptor, activates STAT3 transcription of secondary response genes. Unexpectedly, inhibition of the Il6 receptor and of STAT3 activation increased the potency of p37-induced gene expression. Inhibition of the Toll-like receptor 4 inhibited p37-induced gene expression in NIH3T3 cells. NIH3T3 cells were transfected with the p37 gene. The transfected cells lifted readily from the tissue culture plate and were smaller than the wild type NIH3T3 cells. Expression levels of eight of the twenty genes selected based on the microarray data were higher in p37-transfected cells than p37-treated NIH3T3 cells. p37-treated NIH3T3 cells exhibited increased migration rates in wound healing assays and rapid activation of RhoA. Mutating the four amino acids required for thiamine pyrophosphate binding by p37 reduced the capacity of the protein to up-regulate gene expression. Angptl4 was an exception, the mutated p37 protein further stimulated expression of the gene. The removal of the 20 C-terminal amino acids of p37 greatly reduced the proteins capacity for XVI stimulating gene expression. However, the stimulation of Angptl4 was again the exception and induction was only affected when 40 C-terminal amino acids were removed from p37. Finally, the amino acid sequence of p37 was compared with proteins from other mycoplasma species. Several homologues were observed

Gene expression at different time points (2, 4, 8, 12 and 24 hours) following p37 (5 μg ml^-1) addition to NIH3T3 fibroblasts.

<p>Fold change (E^-ΔΔCt) of mRNA expression levels of NIH3T3 fibroblasts treated with 5 μg ml^-1 p37 at 2, 4, 8, 12 or 24 hours. The 25 μg ml^-1 p37 for 24 hours results are presented for comparison. Significant differences between treated and untreated cells were calculated by ANOVA analysis (p-values ≤0.05 are shown in bold).</p><p>Gene expression at different time points (2, 4, 8, 12 and 24 hours) following p37 (5 μg ml^-1) addition to NIH3T3 fibroblasts.</p

IL6R inhibition effect on p37-induced gene expression in NIH3T3 fibroblasts.

<p>Quantitative PCR analysis of NIH3T3 fibroblasts treated with 25 μg ml^-1 p37 for 24 hours (black) or pre-treated with 0.1 μg ml^-1 IL6R<i>i</i> for an hour prior to 25 μg ml^-1 p37 treatment for 24 hours (grey). Significant differences between p37 + IL6R<i>i</i> treatment and p37 treatment was calculated using ANOVA analysis (+p<0.05, ++p<0.01, +++p<0.001).</p

Genes identified in the inflammatory response and autoimmunity RT² Profiler Array.

<p>Fold change (E^-ΔΔCt) of mRNA expression levels of NIH3T3 fibroblasts treated with 100 μM S31-201 or 25 μg ml^-1 p37 and 100 μM S31-201 over 24 hours. Significant differences between treated and untreated cells were calculated by ANOVA analysis (p-values ≤0.05 are shown in bold).</p><p>Genes identified in the inflammatory response and autoimmunity RT² Profiler Array.</p

The <i>Mycoplasma hyorhinis</i> p37 Protein Rapidly Induces Genes in Fibroblasts Associated with Inflammation and Cancer

<div><p>The p37 protein at the surface of <i>Mycoplasma hyorhinis</i> cells forms part of a high-affinity transport system and has been found associated with animal and human cancers. Here we show in NIH3T3 fibroblasts, p37 rapidly induces the expression of genes implicated in inflammation and cancer progression. This gene activation was principally via the Tlr4 receptor. Activity was lost from p37 when the C-terminal 20 amino acids were removed or the four amino acids specific for the hydrogen bonding of thiamine pyrophosphate had been replaced by valine. Blocking the IL6 receptor or inhibiting STAT3 signalling resulted in increased p37-induced gene expression. Since cancer associated fibroblasts support growth, invasion and metastasis via their ability to regulate tumour-related inflammation, the rapid induction in fibroblasts of pro-inflammatory genes by p37 might be expected to influence cancer development.</p></div

Purification of the p37 protein using Ni-affinity chromatography.

<p>Purified p37 was separated by 12% SDS-PAGE and stained with Coomassie blue <b>(A).</b> The purified protein was transferred to polyvinylidene fluoride membranes and probed with the T7-Tag monoclonal antibody and the goat anti-mouse IgG Horseradish Peroxide (HRP) conjugate (<b>B</b>). The molecular weight standards (MW) are in kilo Daltons (kDa) and indicated on the left of the figure. The purified p37 protein ran to the position of approximately 52 kDa. The p37 protein is predicted to be 43.5 kDa with an additional 8.5 kDa as a result of the 6x His Tag and Xpress Epitope of the pRSET A reading frame. The identity of the purified p37 protein was further confirmed using protein sequencing.</p

The effect of 0.5 μM VIPER and CP7 on p37-induced gene expression in NIH3T3 fibroblasts.

<p>Quantitative PCR (qPCR) analysis of NIH3T3 fibroblasts treated with 25 μg ml^-1 p37 for 24 hours (black) or pre-treated for 2 hours with VIPER (grey) or the control peptide CP7 (white) prior to 25 μg ml^-1 p37-treatment for 24 hours. Significant differences between CP7 or VIPER+p37 treatments and p37 treatment were calculated using ANOVA analysis (+p≤0.05, ++p≤0.01, +++p≤0.001).</p

Gene expression of NIH3T3 fibroblasts treated with different concentrations of purified p37 (0.5, 1, 5 and 25 μg ml^-1) for 24 hours.

<p>Fold change (E^-ΔΔCt) of mRNA expression levels of NIH3T3 fibroblasts treated with 0.5, 1, 5 or 25 μg ml^-1 p37 for 24 hours. Significant differences between treated and untreated cells were calculated by ANOVA analysis (p-values ≤0.05 are shown in bold).</p><p>Gene expression of NIH3T3 fibroblasts treated with different concentrations of purified p37 (0.5, 1, 5 and 25 μg ml^-1) for 24 hours.</p