Depletion of αV integrins from osteosarcoma cells by intracellular antibody expression induces bone differentiation marker genes and suppresses gelatinase (MMP-2) synthesis
- 作者：Koistinen ; Pekka ; Pulli ; Timo ; Uitto ; Veli-Jukka ; Nissinen ; Liisa ; Hyypiä ; Timo ; Heino ; Jyrki
- 关键词：Osteosarcoma ; Integrins ; Matrix metalloproteinases ; Single-chain antibody ; Intracellular antibody ; ALP ; Alkaline phosphatase ; DMEM ; Dulbecco's modification of Eagle's medium ; EtBr ; Ethidium bromide ; FCS ; Fetal calf serum ; FN ; fibronectin ; GAPDH ; Glycerald
- 刊名：Matrix Biology
- 出版年：1999
- 期刊代码：64_0945053x
- 类别：imm
- 出版时间：June, 1999
- 卷：18
- 期：3
- 页码：239-251
- 文件大小：1.09 M
摘要
Integrin heterodimers sharing the common αV subunit are receptors for adhesion glycoproteins such as vitronectin and fibronectin. They are suggested to play an essential role in cell anchoring, differentiation, and survival. Here, we describe the construction of an expression plasmid coding for an intracellular single-chain antibody against αV integrin subunit. Saos-2 osteosarcoma cells transfected with this DNA construct showed an approximately 70-100%decrease in the cell surface expression of αVβ3 and αVβ5 integrins as shown by flow cytometry. Intracellular antibody expression had no effect on the mRNA levels of αV integrin. Pulse chase experiments of metabolically labeled integrins showed that the translation of precursor αV integrin subunit was not affected. However, the maturation of αV integrins as glycoproteins was slow suggesting that the transport from endoplasmic reticulum to Golgi complex was partially prevented. Depletion of αV integrins from Saos-2 cells led to a decreased ability to spread on fibronectin and vitronectin. Furthermore, the expression of osteoblast differentiation marker genes, alkaline phosphatase and osteopontin, was induced and concomitantly the expression of matrix metalloproteinase-2 decreased. Thus, αV integrins seem to be important regulators of osteosarcoma cell phenotypes. Our data also indicate that the expression of intracellular antibodies is an effective strategy to study the significance of specific integrins for cell phenotype and differentiation.