ABSTRACT
Recently the transcriptional up-regulation of human beta-defensin 2 (HBD-2) by lipopolysaccharide (LPS) was found to be associated with NF-kappaB binding site. Although the general mechanisms of NF-kappaB activation by LPS stimulation are well understood, less is known about the signal transduction pathway leading to LPS-induced NF-kappaB activation in human corneal epithelial (HCE) cells. The aim of this study was to investigate the intracellular signals involved in LPS-induced HBD-2 mRNA expression in HCE cells. Pretreatments of inhibitors for NF-kappaB, protein tyrosine kinase, p38 mitogen activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK) attenuated the LPS-induced NF-kappaB DNA binding activity and HBD-2 mRNA expression. Furthermore, pretreatments with inhibitors for protein kinase C (PKC), phosphatidylcholine-phospholipase C, phosphatidylinositol-phospholipase C, or phosphatidate phosphohydrolase prevented LPS-induced HBD-2 mRNA expression and HBD-2 prmoter-driven luciferase activity. However, the increased expression of HBD-2 mRNA and the increased DNA binding activity of NF-kappaB induced by LPS were not changed by the blockage of extracellular signal-regulated kinase (ERK) and of addition of antioxidants. Forskolin, a protein kinase A (PKA) agonist did not induce HBD-2 mRNA expression. These data demonstrate that LPS-induced HBD-2 mRNA expression via NF-kappaB is, at least in part, dependent on PKC, p38 MAPK, JNK, and protein tyrosine kinase status, but appears to be independent on PKA, ERK and ROS in HCE cells. Taken together, there may be more than one signaling pathways that lead to LPS-induced up-regulation of HBD-2 mRNA expression in HCE cells.
Subject(s)
Humans , Antioxidants , Binding Sites , Colforsin , Cyclic AMP-Dependent Protein Kinases , DNA , Epithelial Cells , JNK Mitogen-Activated Protein Kinases , Luciferases , NF-kappa B , p38 Mitogen-Activated Protein Kinases , Phosphatidate Phosphatase , Phosphotransferases , Protein Kinase C , Protein Kinases , Protein-Tyrosine Kinases , RNA, Messenger , Signal Transduction , Up-RegulationABSTRACT
The effect of epinephrine on phosphatidate phosphohydrolase [PAP] activity of isolated human hepatocytes was studied. Epineprine inhibited the enzyme activity progressively at concentrations above 0.1 micro M, reaching a maximum inhibition of 64.5% at 100 micro concentration. Inclusion of alprenolol, a Beta- receptor blocker, in the incubation mixture abolished the inhibitory effect of epinephrine on PAP, whereas the alpha-receptor antagonist phentolamine, or agonist phenylephrine, did not significantly change the hormone's effect. Addition of dibutyryl-cAMP or aminophylline [a cAMP phosphodiesterase inhibitor] to the incubation mixture together with epinephrine caused further enzyme inhibitism reaching 65.6% and 63.7%, respectively, compared to 49% inhibition caused by epinephrine alone under the same conditions. Dibutyryl-cAMP alone also inhibited PAP activity [51%]. The results suggested that epinephrine affects human hepatocyte PAP activity through beta- adrenoceptor activation and cAMP is involved in the mechanism by which PAP activity is altered