PTHrP increases transcriptional activity of the integrin subunit alpha5

Abstract

Increasing evidence is emerging highlighting the role of parathyroid hormone-related protein (PTHrP) during metastasis by regulating cell adhesion. The current study demonstrated that modulation of PTHrP expression by PTHrP overexpression and small interfering RNA-induced silencing resulted in changes in cell adhesion and integrin expression. RNA interference of endogenous PTHrP caused a significant reduction in cell adhesion of a breast cancer cell line to collagen type I, fibronectin and laminin (P<0.05) and of a colon cancer cell to collagen type I and fibronectin (P<0.05). Overexpression of PTHrP induced a significant increase in cell adhesion of colon (P<0.0001) and breast (P<0.05) cancer cells to the same extracellular matrix proteins. These PTHrP-mediated effects were attributed to changes in integrin expression as the differences in adhesion profile correlated with the integrin expression profile. In an attempt to elucidate the mechanism whereby PTHrP regulates integrin expression, promoter activity of the integrin alpha5 subunit was analysed and significant increases in transcriptional activity were observed in PTHrP overexpressing cells (P<0.0001), which was dependent on nuclear localisation. These results indicate that modulation of cell adhesion is a normal physiological action of PTHrP, mediated by increasing integrin gene transcription.

Figure 1

Relative PTHrP gene expression in MDAMB231 and HT29 cells stably transfected with constructs encoding for either wild-type or NLS-mutated PTHrP, expressed relative to vector control cells (P<0.0001).

Figure 2

PTHrP knockdown. (A) Endogenous PTHrP expression in MGLVA1 ascites, MDAMB231 and HT29 cell lines, expressed relative to MDAMB231 cells. (B) The effect of increasing concentrations of three siRNA homologous to different regions of the PTHrP gene on gene expression in MGLVA1 ascites, 24 h after transfection and relative to GAPDH siRNA (P<0.0001). (C) PTHrP and β-actin protein expression in MDAMB231 and HT29 cells 5 days after transfection with 800 pM of either scrambled control or Tgt19 siRNA. (D) Percentage decrease in PTHrP protein expression in MDAMB231 and HT29 cells transfected with Tgt19 siRNA, as determined by densitometry analysis and expressed relative to scrambled control siRNA. (E) The effect of Tgt19 siRNA transfection on OAS and STAT gene expression in MDAMB231 and HT29 cells, expressed relative to scrambled control siRNA.

Figure 3

Cell adhesion of PTHrP overexpressing (A) MDAMB231 and (B) HT29 cells to the ECM proteins collagen type I, fibronectin and laminin, compared with vector control cells (^*P<0.05, ^**P<0.001 and ^***P<0.0001).

Figure 4

Adhesion of (A) MDAMB231 and (B) HT29 cells to collagen type I, fibronectin and laminin five days after transient transfection with either Tgt19 siRNA or scrambled control (^*P<0.05, ^**P<0.0001).

Figure 5

Integrin subunit gene (MDAMB231 and HT29, A and B, respectively, P<0.0001) and cell surface expression (MDAMB231 and HT29, C and D, respectively, P<0.0001) in cells expressing either wild-type or NLS-mutant PTHrP, compared with vector-control cells.

Figure 6

Integrin subunit gene (A, P<0.0001) and cell surface expression (B, P<0.0001) in cells transiently transfected with either scrambled control or PTHrP siRNA, results expressed relative to scrambled control.

Figure 7

Integrin knockdown. (A) Effect of increasing concentrations of either α₅ or α₆ siRNA on respective gene expression in MDAMB231 cells, 24 h following transfection and expressed relative to α₅ scrambled control siRNA (P<0.0001). (B) Integrin cell surface expression in MDAMB231 cells transiently transfected with 5 nM of α₅ or α₆ siRNA after 3 days, compared with α₅-scrambled control siRNA (^*P<0.001, P<0.0001). (C) Adhesion of MDAMB231 cells to ECM, 3 days after transfection with 5 nM of either α₅ or α₆ siRNA, compared with scrambled control siRNA (P<0.0001). (D) Effect of PTHrP overexpression on α₅ cell surface expression in HT29 and MDAMB231 cell lines, 3 days following transfection with 5 nM of either α₅ or α₆ siRNA (P<0.0001). (E) Cell adhesion of HT29 and MDAMB231 cells to fibronectin 3 days after transient transfection with α₅ siRNA, compared with scrambled control-transfected cells (P<0.0001). (F) Cell adhesion of HT29 and MDAMB231 cells to laminin following 3 days after transient transfection with α₆ siRNA, compared with scrambled control-transfected cells (P<0.0001).

Figure 8

α₅ promoter activity in stably transfected MDAMB231 and HT29 cells (vector, sense, NLS-mutated), as measured by luciferase assay (P<0.0001).