Human Mesenchymal Stromal Cells Derived from Different Tissues Show Similar Profiles of c-ErbB Receptor Family Expression at the mRNA and Protein Levels

Abstract

The c-ErbB receptor family is a fundamental cell signaling system that regulates cell proliferation, motility, apoptosis, differentiation, and other key cellular functions. Overexpressed and mutated in some tumors, c-ErbB receptors play a pivotal role in their progression but are also present in many non-malignant cells, including those that are promising from the point of view of regenerative medicine, such as mesenchymal stromal cells (MSCs). The role of c-ErbB receptors in these cells is not clearly understood, and the data on their expression are sporadic. Therefore, the systemic characterization of c-ErbB receptor family expression in MSCs from a wide range of tissues is of high priority. Here, using RT-qPCR and Western blotting analysis, we evaluated the c-ErbB receptors expression pattern at the mRNA and protein levels in human MSCs isolated from six different tissues. We found that MSCs possess considerable EGFR and HER2 mRNA levels comparable to those in some malignant cells while showing trace HER3 and HER4 expression. However, EGFR but not HER2 was detected in MSCs at the protein level. We also show that the absence of HER2 protein is not associated with its rapid lysosomal degradation. We conclude that c-ErbB signaling in human MSCs is exclusively mediated by EGFR.

Keywords: c-ErbB receptors; human mesenchymal stromal cells; lysosomal degradation; mRNA and protein expression.

Figure 1

Heatmap with hierarchical clustering representing relative expression of EGFR, HER2, HER3, and HER4 genes in six lines of human malignant cells (yellow) and seven types of human mesenchymal stromal cells (green), derived from different tissues. (a) Relative gene expression was obtained using real-time qPCR and calculated with the 2^−ΔΔCt method normalized against the geometric mean of the three reference genes (YWHAZ, POP4, and EIF2B1) and HeLa (for EGFR, HER2, and HER3) or MCF-7 (for HER4) expression levels. The meaning in each heatmap cell is the median of the log₂-transformed relative gene expression values of the biological samples of the indicated cell type. The heatmap cell color corresponds to median value and changes from light (the lowest relative gene expression value) to deep purple (the highest relative gene expression value). A white color of the cell and NA abbreviation indicates that no expression was registered. Hierarchical clustering was performed using the pheatmap R package (package ver. 1.0.12, R ver. 4.2.3). (b) Quantification cycles (Cq) values of EGFR, HER2, HER3, and HER4 receptors in malignant (yellow) and mesenchymal stromal cells (MSCs, green). Data are presented as median and range. Biological samples with no detected expression are not shown.

Figure 2

EGFR but not HER2 protein expression is detected in human MSCs. (a) Total cell lysates of the human malignant cells and MSCs of different origin were separated by SDS-PAGE and processed for Western blotting. Blots were stained with anti-EGFR antibodies (upper line) and anti-HER2 antibodies (middle line). Nitrocellulose membrane was stained with Ponceau S solution for total protein and used as the loading control (lower line). (b) The Western blots were quantified and normalized against SK-BR-3. The densitometry data are presented as the means ± s.e. (c,d)—immunofluorescence of EGFR (c) and HER2 (d) in HeLa and enMSC 2804 cells. HeLa, enMSC 2804, and SK-BR-3 were fixed, permeabilized, and stained with anti-EGFR or anti-HER2 antibodies followed by GAR-Alexa Fluor 488 secondary antibodies staining (green). The cell nuclei were visualized using Hoechst 33285 (blue). Cell borders are highlighted in white. Scale bar: 20 µm.

Figure 3

The absence of HER2 expression at the protein level is not associated with its rapid degradation via the lysosomal pathway. Three different types of the human MSCs (MSCWJ-1, ECL 2455, and enMSC 2804) were subjected to 24 h treatment with lysosomal inhibitors Chloroquine (Chlq, C = 5 µM; 50 µM), or Bafilomycin A1 (BafA, 100 nM). Total cell lysates of these cells and the malignant SK-BR-3 cell line (served as HER2-positive control) were separated by SDS-PAGE and processed for Western blotting. Blots were stained with anti-EGFR, anti-HER2, anti-p62 (Abcam Inc., USA), anti-LC3A/B, and anti-alpha tubulin antibodies. The nitrocellulose membrane was stained with Ponceau S solution for total protein analysis and used as a loading control. The EGFR and HER2 Western blots were quantified and normalized against each line control cells (Ctrl) or SK-BR-3 in the case of HER2. Relative optical densities are presented as the means ± s.e of three independent experiments.