Gene expression profile reveals abnormalities of multiple signaling pathways in mesenchymal stem cell derived from patients with systemic lupus erythematosus

Abstract

We aimed to compare bone-marrow-derived mesenchymal stem cells (BMMSCs) between systemic lupus erythematosus (SLE) and normal controls by means of cDNA microarray, immunohistochemistry, immunofluorescence, and immunoblotting. Our results showed there were a total of 1, 905 genes which were differentially expressed by BMMSCs derived from SLE patients, of which, 652 genes were upregulated and 1, 253 were downregulated. Gene ontology (GO) analysis showed that the majority of these genes were related to cell cycle and protein binding. Pathway analysis exhibited that differentially regulated signal pathways involved actin cytoskeleton, focal adhesion, tight junction, and TGF-β pathway. The high protein level of BMP-5 and low expression of Id-1 indicated that there might be dysregulation in BMP/TGF-β signaling pathway. The expression of Id-1 in SLE BMMSCs was reversely correlated with serum TNF-α levels. The protein level of cyclin E decreased in the cell cycling regulation pathway. Moreover, the MAPK signaling pathway was activated in BMMSCs from SLE patients via phosphorylation of ERK1/2 and SAPK/JNK. The actin distribution pattern of BMMSCs from SLE patients was also found disordered. Our results suggested that there were distinguished differences of BMMSCs between SLE patients and normal controls.

Figure 1

Genes differentially expressed in BMMSCs between SLE patients and normal controls. Genes were shown by the ratio of hybridization intensity between normal control and SLE BMMSCs. The ratio ≥2 or ≤0.5 was considered significant. Genes highly expressed in BMMSCs from normal controls were highlighted in green, while those highly expressed in BMMSCs from SLE patients were highlighted in red.

Figure 2

Classification of the differentially expressed genes according to the pathway and GO analysis by MAS software. (a) The top 10 statistically significant (P < 0.001) pathways. (b) The top 10 statistically significant (P < 0.001) molecular functions by GO analysis. The numbers indicated the differentially expressed genes in the specific pathway or function.

Figure 3

Actin distribution patterns in BMMSCs from SLE patients or the normal controls. (a, b) Actin filaments were stained with Alexa Flour 594 conjucted phalloidin (red) and nuclei were counterstained with DAPI (blue). Cells (white arrows) were observed by fluorescence microscope. (a): BMMSCs from normal controls, (b): BMMSCs from SLE patients. (magnification ×200) (c, d) Actin filaments was stained with phalloidin-FITC and observed under confocal microscopy. (c): BMMSC from one normal control, (d): BMMSC from one SLE patient (Bar = 20).

Figure 4

mRNA and protein levels of cyclin D and cyclin E in BMMSCs from SLE patients. (a) Immunoblotting analysis of cyclin D and cyclin E. (b) qRT-PCR studies of the expression of cyclin D and cyclin E2 in BMMSCs from SLE and normal controls (n = 5, P > 0.05). Results were shown as mean ± SEM, each performed with triplicate samples. (c) Quantity analysis showed low protein level of cyclin E in cells from SLE patients, **P < 0.01 by Student's t-test. SLE: systemic lupus erythematosus, Nor: normal controls.

Figure 5

Immunohistochemical detection of BMP-5 protein and mRNA levels of Id-1, Id-2, Id-3 in BMMSCs. (a) magnification ×100, (b) magnification ×200. The left pictures are isotype controls, the right are BMP-5 immunohistochemical staining. (c) qRT-PCR studies of the expression of Id-1, Id-2, and Id-3 in BMMSCs from SLE patients and normal controls (n = 10). *P < 0.05 by nonparametric test of SPSS 16.0 software. SLE: systemic lupus erythematosus, Nor: normal controls.

Figure 6

MAPK pathway in BMMSCs from SLE patients. Immunoblotting showed the phosphorylation of JNK and ERK1/2 was higher in BMMSCs from SLE patients. Results were shown as mean ± SEM. *P < 0.05 (SLE versus normal controls by Student's t-test). SLE: systemic lupus erythematosus, Nor: normal controls.

Figure 7

The correlation of Id-1 mRNA expression with serum TNF-α levels in SLE patients. (a, b) Id-1 mRNA level was not correlated with antinuclear antibodies (ANA) levels and SLE disease activity index (SLEDAI) score of SLE patients. (c) The serum level of TNF-α in SLE patients (n = 10) were higher than that of normal controls (n = 20). **P < 0.01 versus normal controls by Student's t-test. (d) Id-1 mRNA expression was reversely correlated with serum TNF-α level of SLE patients (P = 0.01, r ² = 0.55). SLE: systemic lupus erythematosus, Nor: normal controls.