ABSTRACT
Epidermal growth factor (EGF) expresses mitogenic activity by a mechanism that requires the EGF receptor (EGFR). We report that murine embryonic fibroblasts (MEFs) proliferate in response to EGF only when these cells express the urokinase receptor (uPAR). EGFR expression was equivalent in uPAR-/- and uPAR+/+ MEFs. In response to EGF, these cells demonstrated equivalent overall EGFR tyrosine phosphorylation and ERK/MAP kinase activation; however, phosphorylation of Tyr-845 in the EGFR, which has been implicated in cell growth, was substantially decreased in uPAR-/- MEFs. STAT5b activation also was decreased. As Tyr-845 is a c-Src target, we overexpressed c-Src in uPAR-/- MEFs and rescued EGF mitogenic activity. Rescue also was achieved by expressing murine but not human uPAR, suggesting a role for autocrine uPAR cell-signaling. In MDA-MB 231 breast cancer cells, EGF mitogenic activity was blocked by uPAR gene silencing, with antibodies that block uPA-binding to uPAR, and with a synthetic peptide that disrupts uPAR-dependent cell signaling. Again, c-Src overexpression rescued the mitogenic activity of EGF. We conclude that uPAR-dependent cell-signaling may prime cells to proliferate in response to EGF by promoting Tyr-845 phosphorylation and STAT5b activation. The importance of this pathway depends on the c-Src level in the cell.
Subject(s)
Breast Neoplasms/pathology , Cell Proliferation/drug effects , Epidermal Growth Factor/pharmacology , Receptors, Cell Surface/physiology , Animals , Autocrine Communication , Breast Neoplasms/metabolism , Cells, Cultured , Embryo, Mammalian/cytology , Embryo, Mammalian/drug effects , Embryo, Mammalian/metabolism , ErbB Receptors/metabolism , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/metabolism , Humans , Mice , Mice, Knockout , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation , Plasminogen/metabolism , Protein Binding , Proto-Oncogene Proteins pp60(c-src)/metabolism , RNA, Small Interfering/pharmacology , Receptors, Urokinase Plasminogen Activator , STAT5 Transcription Factor/metabolism , Tyrosine/metabolism , Urokinase-Type Plasminogen Activator/metabolismABSTRACT
One- or two-gram doses of ceftriaxone were administered intravenously to 30 patients before cataract extraction. With the 1-g dose, mean aqueous humor concentrations of 0.93 and 0.88 microgram/mL were found at approximately 2 and 12 hours after administration, respectively. With the 2-g dose, a mean level of 2.47 micrograms/mL was observed at two hours; levels of more than 2 micrograms/mL were found in two patients 13 hours after administration. Both the 1- and 2-g doses thus produce aqueous humor levels many times higher than the minimum inhibitory concentration of ceftriaxone for 90% of most Enterobacteriaceae, excluding Pseudomonas. Concentrations adequate for Staphylococcus aureus and Staphylococcus epidermidis were not, however, obtained.
