Abstract
To investigate the cell biological function of PU.1, a member of the Ets family of transcription factors, a vector capable of expressing the protein was transfected into HT1080 human fibrosarcoma cells. Exogenous expression of PU.1 in HT1080 cells reduced colony-forming efficiency but stimulated cell migration in soft agar, although it did not affect cell growth in adherent culture. Expression of the urokinase-type plasminogen activator (uPA) mRNA, which is known to be correlated with cell migration and invasion, was enhanced in PU.1 transfectants compared with mock transfectants. Run-on analysis demonstrated that uPA transcription was unaffected by PU.1, suggesting that this enhancement mainly occurs at a post-transcriptional level. On the other hand, treatment of HT1080 cells with the synthetic glucocorticoid dexamethasone (DEX; 10(-7) M) significantly reduced uPA gene expression at a transcriptional level. Furthermore, DEX inhibited cell migration in soft agar without affecting cell growth. These negative effects of DEX on uPA expression and cell migration were alleviated by the expression of PU.1 in HT1080 cells, whereas expression of the N-ras oncogene, which is responsible for maintenance of the transformed phenotypes in HT1080 cells, was unaffected by PU.1 expression or DEX treatment in the cells. Our results suggest that expression of PU.1 can stimulate uPA gene expression at the post-transcriptional level, which may subsequently lead to activation of cell motility and/or reduced cell-cell adhesion, but reduces anchorage-independent growth of HT1080 cells.
Highlights
The actin transcription was not reduced by DEX in both cells. These results suggest that the transcription rate of the urokinase-type plasminogen activator (uPA) gene was unaffected by PU. 1 in the absence of DEX and that increase in steadystate level of uPA mRNA by PU.1 is mainly accounted for by post-transcriptional control
We found that exogenous expression of PU.l in HT1080 human fibrosarcoma cells increased cell migration in soft agar
In association with these cell biological changes, expression of the uPA mRNA, which encodes a serine protease correlated with cell migration and invasion, was enhanced in PU. l-transfected HT1080 cells compared with mock transfectants
Summary
A 1.2-kb mouse PU.] full-length cDNA (Klemsz et al, 1990), kindly donated by Dr D. Kabat, was cloned into an eukaryotic expression vector, pLRNL (Hung et al, 1988), downstream of the MuLV promoter. NM-1 and cl-2, were isolated from a human fibrosarcoma cell line, HT1080, purchased from the American Type Culture Collection. Cells were maintained in Dulbecco's modified Eagle minimum essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The cells were transfected with the PU. I expression vector using Lipofectin reagent (Gibco/BRL) and transfectants were selected in the medium containing G418 (1 mg ml-')
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