We have previously reported that epidermal growth factor (EGF) stimulates branching morphogenesis of the fetal mouse submandibular gland (SMG) (M. Kashimata and E. W. Gresik, 1997, Dev. Dyn. 208, 149-161) and that the EGF receptor (EGFR) is localized principally, if not exclusively, on the epithelial components of the fetal SMG (E. W. Gresik, M. Kashimata, Y. Kadoya, R. Mathews, N. Minami, and S. Yamashina, 1997, J. Histochem. Cytochem. 45, 1651-1657). The EGFR is a receptor tyrosine kinase, and after binding of its ligand, it triggers several intracellular signaling cascades, among them the one activating the mitogen-activated protein kinases (MAPK) ERK-1/2. Here we investigated whether EGF utilizes the ERK-1/2 signaling cascade to stimulate branching morphogenesis in the fetal mouse SMG. SMG rudiments were collected as matched pairs at E14, E16, and E18 (E0 = day of vaginal plug); placed into wells of defined medium (BGJb); and exposed to EGF for 5 or 30 min or to medium alone (controls). By Western blotting we found that EGF induced the appearance of multiple bands of phosphotyrosine-containing proteins, including bands at 170 kDa and 44 kDa/42 kDa, presumably corresponding to the phosphorylated forms of EGFR and ERK-1/2, respectively. Other blots showed the specific appearance of the phosphorylated EGFR and of phospho-ERK-1/2 in response to EGF. Immunohistochemical staining for phosphotyrosine increased at the plasma membrane after EGF stimulation for 5 or 30 min. Diffuse cytoplasmic staining for MEK-1/2 (the MAPK kinase that activates ERK-1/2) increased near the cell membrane after EGF stimulation. Phospho-ERK-1/2 was localized in the nuclei of a few epithelial cells after EGF for 5 min, but in the nuclei of many cells after EGF for 30 min. PD98059, an inhibitor of phosphorylation and activation of MEK-1/2, by itself inhibited branching morphogenesis and, furthermore, decreased the stimulatory effect of EGF on branching. Western blots confirmed that this inhibitor blocked phosphorylation of ERK-1/2 in fetal SMGs exposed to EGF. These results show that components of the ERK-1/2 signaling cascade are present in epithelial cells of the fetal SMG, that they are activated by EGF, and that inhibition of this cascade perturbs branching morphogenesis. However, EGF did not cause phosphorylation of two other MAPKs, SAPK/JNK or p38MAPK, in fetal SMGs. These results imply that the ERK-1/2 signaling is responsible, at least in part, for the stimulatory effect of EGF on branching morphogenesis of the fetal mouse SMG.

BackgroundTo improve the clinical outcome of patients who suffered ischemic stroke, cerebral ischemia-reperfusion (I/R) injury is one of the major concerns that should be conquered. Inflammatory reactions are considered a major contributor to brain injury following cerebral ischemia, and I/R exacerbates these reactions. The aim of this study was to investigate the possible ameliorative effects of progranulin (PGRN) against I/R injury in mice.MethodsIn vivo I/R was induced in four-week-old male ddY mice by 2 h of MCAO (middle cerebral artery occlusion) followed by 22 h of reperfusion. We evaluate expression of PGRN in I/R brain, efficacy of recombinant-PGRN (r-PGRN) treatment and its therapeutic time-window on I/R injury. Two hours after MCAO, 1.0 ng of r-PRGN or PBS was administered via intracerebroventricular. We assess neutrophil infiltration, expression of tumor necrosis factor (TNF)-α, matrix metalloproteinase-9 (MMP-9) and phosphorylation of nuclear factor-κB (NF-κB) by immunofluorescense staining and Western blotting. We also investigate neutrophil chemotaxis and intercellular adhesion molecule-1 (ICAM-1) expression in vitro inflammation models using isolated neutrophils and endothelial cells.ResultsWe found that expression of PGRN was decreased in the I/R mouse brain. r-PGRN treatment at 2 h after MCAO resulted in a reduction in the infarct volume and decreased brain swelling; this led to an improvement in neurological scores and to a reduction of mortality rate at 24 h and 7 d after MCAO, respectively. Immunohistochemistry, Western blotting, and gelatin zymography also confirmed that r-PGRN treatment suppressed neutrophil recruitment into the I/R brain, and this led to a reduction of NF-κB and MMP-9 activation. In the in vitro inflammation models, PGRN suppressed both the neutrophil chemotaxis and ICAM-1 expression caused by TNF-α in endothelial cells.ConclusionsPGRN exerted ameliorative effects against I/R-induced inflammation, and these effects may be due to the inhibition of neutrophil recruitment into the I/R brain.

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