Ezrin silencing remodulates the expression of Phosphoinositide-specific Phospholipase C enzymes in human osteosarcoma cell lines

Abstract

Ezrin, a protein belonging to the Ezrin, radixin and moesin (ERM) family, was engaged in the metastatic spread of osteosarcoma. The Protein 4.1, Ezrin, radixin, moesin (FERM) domain of Ezrin binds the membrane Phosphatydil inositol (4,5) bisphosphate (PIP2), a crucial molecule belonging to the Phosphoinositide (PI) signal transduction pathway. The cytoskeleton cross-linker function of Ezrin largely depends on membrane PIP2 levels, and thus upon the activity of related enzymes belonging to the PI-specific phospholipase C (PI-PLC) family. Based on the role of Ezrin in tumour progression and metastasis, we silenced the expression of Vil2 (OMIM *123900), the gene which codifies for Ezrin, in cultured human osteosarcoma 143B and Hs888 cell lines. After Ezrin silencing, the growth rate of both cell lines was significantly reduced and morphogical changes were observed. We also observed moderate variations both of selected PI-PLC enzymes within the cell and of expression of the corresponding PLC genes. In 143B cell line the transcription of PLCB1 decreased, of PLCG2 increased and of PLCE differed in a time-dependent manner. In Hs888, the expression of PLCB1 and of PLCD4 significantly increased, of PLCE moderately increased in a time dependent manner; the expression of PLCG2 was up-regulated. These observations indicate that Ezrin silencing affects the transcription of selected PLC genes, suggesting that Ezrin might influence the expression regulation of PI-PLC enzymes.

Fig. 1

Effectiveness of Ezrin silencing. a Istogram of mRNA transcript concentrations after 0, 24 and 48 h from Ezrin silencing in 143B (gray) and Hs888 (black) cell lines. b Gel electrophoresis from Western blot of Ezrin protein in 143B and Hs888 cell lines compared to actin protein loading control. Growth curve after Ezrin silencing (left) effect of Ezrin siRNA on cell morphology (right) in 143B cells: the growth of silenced cells is significantly slowed with respect to untreated cells (p < 0,5). Bar errors are indicated. N = untrasfected control 143B cells; C = metafectamine transfected control 143B cells; Ezrin siRNA transfected 143B cells. II a and b - morphological changes in 143B cells (40X, contrast-phase microscopy). Irregular outline of the plasma membrane, reduced intercellular adhesion and cytoplasm micro-vacuolization. Growth curve after Ezrin silencing (left) effect of Ezrin siRNA on cell morphology (right) in Hs888 cells: the growth of silenced cells is slowed with respect to untreated cells. Bar errors are indicated. N = untrasfected control Hs888 cells; C = metafectamine transfected control Hs888 cells; Ezrin siRNA transfectedHs888 cells. III c and d - morphological changes in Hs888 cells (20, contrast-phase microscopy). Quantitative reduction of cells displaying substantially well-preserved structure

Fig. 2

Real-time results after Ezrin silencing. Istograms of the transcript concentrations of PLC genes after 0, 24 and 48 h from Ezrin silencing in 143B (gray) and Hs888 (black) cell lines

Fig. 3

Partial co-localization of Ezrin and PI-PLC ε Fluorescence immunocytochemistry of Ezrin (green) and PI-PLC ε (red) in 143B (upper) and Hs888 (lower) cell lines. Diaminophenyl indole (DAPI, blue) counterstain for nuclei (60X)

Fig. 4

Immunofluorescence analyses. a Localization of PI-PLC β1 and PI-PLC γ2 in 143B cell line. LEFT. Fluorescence immunocytochemistry of PI-PLC β1 (red) in 143B (upper) and in 143B cells after Ezrin silencing (lower). Diaminophenyl indole (DAPI, blue) counterstain for nuclei (60X). RIGHT. Fluorescence immunocytochemistry of PI-PLC γ2 (red) in 143B (upper) and in 143B cells after Ezrin silencing (lower). Diaminophenyl indole (DAPI, blue) counterstain for nuclei (60X). b Localization of PI-PLC β1 and PI-PLC γ2 in Hs888 cell line LEFT. Fluorescence immunocytochemistry of PI-PLC β1 (red) in Hs888 (upper) and in Hs888 cells after Ezrin silencing (lower). Diaminophenyl indole (DAPI, blue) counterstain for nuclei (60X). RIGHT. Fluorescence immunocytochemistry of PI-PLC γ2 (red) in Hs888 (upper) and in Hs888 cells after Ezrin silencing (lower). Diaminophenyl indole (DAPI, blue) counterstain for nuclei (60X)