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. 2007 Jan-Mar;1(1):43-51.
Epub 2007 Jan 27.

Elevated cell invasion is induced by hypoxia in a human pituitary adenoma cell line

Daizo Yoshida  1 , Akira Teramoto
Affiliations

Elevated cell invasion is induced by hypoxia in a human pituitary adenoma cell line

Daizo Yoshida et al. Cell Adh Migr. 2007 Jan-Mar.

Abstract

Pituitary adenoma tissues are hypovascular, and have a lower partial oxygen pressure compared with neighboring normal organs. In this study, we investigated whether hypoxia influences the cell invasiveness of the human pituitary adenoma cell line, HP-75. HP-75 cells were exposed to hypoxic (1-10% oxygen) or normoxic (21% oxygen) conditions for 24 hours. Gelatin and reverse zymogram assays were used to determine the enzyme activities of matrix metalloproteinases (MMP) and tissue inhibitor of metalloproteinases (TIMP). Cell adhesion and Matrigel cell invasion were examined with a Boiden chamber. Finally, the mRNA gene expression profiles of cells exposed to hypoxia or normoxia were examined by cDNA microarray and confirmed with real-time RT-PCR and flow cytometry. The gelatin and reverse zymograms revealed that the activities of MMP and TIMP were not significantly altered by hypoxia. Matrigel cell invasion and cell adhesion to Matrigel or collagen type IV were increased by hypoxia (3.8- and 4.8-fold, respectively). The cDNA microarray analysis revealed that laminin beta2 chain mRNA was specifically up-regulated under hypoxic conditions (4.96-fold). Finally, real-time RT-PCR and flow cytometry verified the elevated expression of laminin beta2 chain at the mRNA and protein levels under hypoxic conditions. RNA interference with siRNA targeting laminin beta2 inhibited Matrigel invasion and adhesion to collagen type IV in a dose-dependent manner. Collectively, these results suggested that hypoxia (1% oxygen) enhanced the cell invasion properties of a pituitary adenoma cell line in association with elevated expression of laminin beta2 and enhanced binding to collagen type IV.

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Figures

Figure 1
Figure 1
The activities of MMPs and TIMPs, and cell invasion through Mtrigel are examined in hypoxic condition. Left; Gelatin and reverse zymography reveal that the activities of MMP-2, MMP-9 and TIMP-1 are not affected by incubation of HP-75 cells under hypoxic conditions throughout 1–21% oxygen. Right; In contrast, cell invasion through Matrigel is significantly elevated in cells incubated with 1% oxygen versus those incubated with physiologically normal levels of 21% oxygen.
Figure 2
Figure 2
Cell adhesion to Matrigel, collagen type I or IV, fibronectin, vitornection are compared between 1% and 21% oxygen. (Top) Cell adhesion is increased under hypoxic versus normoxic conditions, respectively for Matrigel (p < 0.05) and for collagen type IV (p < 0.01). (Botton) There are significant elevated cell adhesion to collagen type IV at 1% oxygen.
Figure 3A
Figure 3A
Expressions of genes in hypoxia at 1% are compared with those at 21% oxygen by cDNA micro array analysis. Genes relating to cell adhesion molecules. Of the examined expression of laminin β2 (an ECM gene) is significantly elevated by hypoxia, showing a 4.96-fold elevation in cells after exposure at 1% oxygen.
Figure 3B
Figure 3B
Expressions of genes in hypoxia at 1% are compared with those at 21% oxygen by cDNA micro array analysis. Cell signaling intermediates. In particular, genes for matrix metalloproteinase (MMP) family are not altered at 1% oxygen.
Figure 4
Figure 4
To verify the results obtained by cDNA microarray analysis, real-time RT-PCR and flowcytometric nalysis are performed. (Top) The up-regulation of laminin β2 under hypoxic conditions is verified by real-time RT-PCR, which reveals that cells incubated at 1% oxygen shows a significant increase in laminin β2 versus those incubated under normoxic conditions. However, incubation of cells at 3, 5, or 10% oxygen does not induce significant up-regulation of laminin β2. (Bottom) Flow cytometric analysis reveals that expression of laminin β2 on the cell surface is elevated following incubation of cells under hypoxic (1%) conditions versus normoxia demonstrates that expression of laminin β2 on the cell surface is significantly increased by incubation of cells only with at 1% oxygen.
Figure 5
Figure 5
Laminin β2 mRNA is interfered by siRNA method and confirmed by real-time RT-PCR and Western blotting. (Top) Real-time RT-PCR shows a significant decrease in expression of laminin β2 mRNA by siRNA in a dose-dependent manner versus the control (25nM, p < 0.005; 50nM, p < 0.005; 100nM, p < 0.001), meanwhile the cells transfected Lamin A/C siRNA, or scramble siRNA does not. (Bottom) Western blotting analysis indicates a significant decrease in laminin β2 expression in a dose-dependent manner versus the control, compared with the amounts of β-actin as internal standard.
Figure 6
Figure 6
Cell invasion and adhesion are investigated when laminin β2 mRNA was interfered. Similarly, cell invasion through Matrigel and cell adhesion to collagen type IV are significantly reduced in a dose-dependent manner.
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