Activation mechanisms of the urokinase-type plasminogen activator promoter by hepatocyte growth factor/scatter factor.

Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic effector inducing invasion and metastasis of tumor cells that express the Met tyrosine kinase receptor. One of the effectors of HGF/SF is the urokinase-type plasminogen activator, a serine protease that facilitates tumor progression and metastasis by controlling the synthesis of the extracellular matrix degrading plasmin. Stimulation of NIH 3T3 cells that were stably transfected with the human Met receptor (NIH 3T3-Methum) with HGF/SF induced a trans-activation of the urokinase promoter and urokinase secretion. Induction of the urokinase promoter by HGF/SF via the Met receptor was blocked by co-expression of a dominant-negative Grb2 and Sos1 expression construct. Further, the expression of the catalytically inactive mutants of Ha-Ras, RhoA, c-Raf, and Erk2 or addition of the Mek1-specific inhibitor PD 098059 abrogated the stimulation of the urokinase promoter by HGF/SF. A sequence residing between -2109 and -1870 base pairs (bp) was critical for stimulation of the urokinase gene by HGF/SF. Mobility shift assays with oligonucleotides spanning an AP-1 site at -1880 bp or a combined PEA3/AP-1 site at -1967 bp showed binding of nuclear factors from NIH 3T3-Methum cells. Expression of an expression plasmid that inhibits DNA binding of AP-1 proteins (A-Fos) abrogated inducible and basal activation of the urokinase promoter. Nuclear extract from unstimulated NIH 3T3-Methum cells contained more JunD and showed a stronger JunD supershift with the AP-1 oligonucleotides, compared with HGF/SF-stimulated cells. Consistent with the levels of JunD expression being functionally important for basal expression of the urokinase promoter, we found that overexpression of wild type JunD inhibited the induction of the urokinase promoter by HGF/SF. These data suggest that the induction of urokinase by HGF/SF is regulated by a Grb2/Sos1/Ha-Ras/c-Raf/RhoA/Mek1/Erk2/c-++ +Jun-dependent mitogen-activated protein kinase pathway.     

Autoregulation of fos: the dyad symmetry element as the major target of repression.

Fos and Jun co-operatively repress the fos promoter. Removal of all putative Fos/Jun binding sites from the fos promoter neither obliterates the repression by Fos/Jun in transient cotransfection experiments in NIH3T3 cells nor the turn-off kinetics of serum-induced fos expression in stably transfected NIH3T3 cells. The dyad symmetry element (DSE) suffices to subject a promoter to this type of repression. However, one of the putative Fos/Jun binding sites (-292 to -299 and thus located immediately adjacent to the DSE), determines the very low level of basal expression.     

Coordinated induction of two unrelated glucose-regulated protein genes by a calcium ionophore: human BiP/GRP78 and GAPDH

The induction of human BiP/GRP78 and GAPDH protein genes by the calcium ionophore A23187 was determined. Steady-state levels of mRNA for both the glucose starvation-responsive BiP/GRP78 gene and the glucose-responsive GAPDH gene were dramatically induced in a variety of human cells. There is a homologous palindromatic sequence GCCGTTAACGGC in the active promoter region of the two genes that is known to be required for the induction of mammalian BiP/GRP78 by A23187. The evidence confirms in general the function of this element in the regulation of calcium-associated gene activity.