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Abstract The expression of the urokinase-type plasminogen activator, which plays a crucial role in tissue remodeling by controlling the synthesis of the broadly acting plasmin serine protease, is regulated by several tyrosine kinases. Since the actions of these tyrosine kinases is dependent on the activation of ras proteins, we undertook a study to identify signaling events downstream of ras responsible for the stimulation of urokinase promoter activity. Transient expression of an activated c-Ha- ras in OVCAR-3 cells, which do not harbor the mutated oncogene, led to a dose-dependent trans-activation of the urokinase promoter.

A sequence residing between −2109 and −1964 was critical for the stimulation of the urokinase promoter by c-Ha- ras. Sam broadcaster pro 20147. Mutation of an AP-1 and a PEA3 site at −1967 and −1973, respectively, or the co-expression of a transactivation domain-lacking c- jun substantially impaired the ability of c-Ha- ras to stimulate urokinase promoter activity.

The induction of the urokinase promoter by ras was completely blocked by expression of a dominant negative c- raf expression vector and substantially reduced in cells made to co-express a catalytically inactive mitogen-activated protein kinase kinase. Further, the expression of an ERK1/ERK2-inactivating phosphatase (CL100) abrogated the stimulation of the urokinase promoter by c-Ha- ras. These data argue for a role of a mitogen-activated protein kinase-dependent signaling pathway in the regulation of urokinase promoter activity by ras. Bukvi dlya raspechatki na printere.

INTRODUCTION The urokinase-type plasminogen activator has been implicated in a variety of physiological and pathological processes including prostatic involution, cytotrophoblast implantation, and tumor cell invasion, all of which require extensive extracellular matrix proteolysis(,, ). Urokinase facilitates this process by mediating the conversion of the abundant zymogen, plasminogen, into the widely acting serine protease plasmin, the latter which degrades several extracellular matrix components including laminin, fibronectin, and possibly type IV collagen(, ). Transcription of the urokinase gene gives rise to a 2.5-kilobase mRNA(), which is subsequently translated into a single chain 50-kDa proenzyme (, ) and secreted into the extracellular space. The steady state level of urokinase mRNA is determined at two levels. Firstly, the expression of the gene is regulated by 2.1 kilobases of the 5′-flanking sequence().

Secondly, recent studies have indicated that 3′-untranslated sequences play a role in determining the stability of the urokinase mRNA(). Several studies have indicated that urokinase expression is regulated by growth factors (HGF/SF, EGF), which bind to transmembrane receptor protein tyrosine kinases, and by non-receptor protein tyrosine kinases including v- src and v- yes(,, ). One of the early events in this pathway involves the coupling of the ligand-activated receptor protein tyrosine kinase to ras through Grb2.sos or Shc Grb2.sos, this increasing the exchange of GTP for GDP and culminating in an activated ras(, ). The pivotal role of ras in mediating the signal generated by tyrosine kinases has been deduced from several experimental observations. First and foremost, inhibitory ras mutant proteins () block the stimulation of growth and transformation of NIH 3T3 cells by oncogenic tyrosine kinases. Secondly, revertant clones isolated from activated ras-transformed NIH 3T3 cells are resistant to retransformation by protein tyrosine kinase-encoding oncogenes(). Thirdly, ras, like protein tyrosine kinases, elevates urokinase mRNA levels in chick embryo fibroblasts().

However, the events connecting the ras signal to altered gene expression are still not entirely clear. Certainly, the involvement of c- raf, mitogen-activated protein kinase kinase (MAPKK also referred to as MEK) and mitogen-activated protein kinases (MAPK), which form successive tiers in a ras-dependent signal transduction pathway, has been suggested from several reports. C- raf is a protein serine/threonine kinase which acts as a cytoplasmic signal transducer downstream of ras() and is an immediate upstream activator of MAPKK (MEK1)(, ).