u-0126 and monorden

Sensorineural hearing deficiencies result from the loss of auditory hair cells. This hearing loss is permanent in humans and mammals because hair cells are not spontaneously replaced. In other animals such as birds, this is not the case. Damage to the avian cochlea evokes proliferation of supporting cells and the generation of functionally competent replacement hair cells. Signal transduction pathways are clinically useful as potential therapeutic targets, so there is significant interest in identifying the key signal transduction pathways that regulate the formation of replacement hair cells. In a previous study from our lab, we showed that forskolin (FSK) treatment induces auditory supporting cell proliferation and formation of replacement hair cells in the absence of sound or aminoglycoside treatment. Here, we show that FSK-induced supporting cell proliferation is mediated by cell-specific accumulation of cyclic adenosine monophosphate (cAMP) in avian supporting cells and the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. By a combination of immunostaining and pharmacological analyses, we show that FSK treatment increases cAMP levels in avian auditory supporting cells and that several ERK MAP inhibitors effectively block FSK-induced supporting cell proliferation. Next, we demonstrate by Western blotting and immunostaining analyses the expression of several ERK MAPK signaling molecules in the avian auditory epithelium and the cell-specific expression of B-Raf in avian auditory supporting cells. Collectively, these data suggest that FSK-induced supporting cell proliferation in the avian auditory epithelium is mediated by increases of cAMP levels in supporting cells and the cell-specific expression of the ERK MAPK family member B-Raf in supporting cells.

Topics: Animals; Antimetabolites; Apigenin; Birds; Bromodeoxyuridine; Butadienes; Cell Division; Colforsin; Cyclic AMP; Enzyme Inhibitors; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Hair Cells, Auditory; Macrolides; MAP Kinase Signaling System; Myosin VIIa; Myosins; Nitriles; Organ Culture Techniques; Organ of Corti; Proto-Oncogene Proteins B-raf

Osteoclast differentiation is a multi-step process that involves cell proliferation, commitment, and fusion. Some adhesion molecules, including integrin alphavbeta3, have been shown to have roles in osteoclast fusion. In the course of studying with pharmacologic agents known to inhibit protein tyrosine kinases of the Src family, we found that radicicol increased cell fusion during receptor activator of nuclear factor kappaB ligand (RANKL)-driven differentiation of osteoclasts at concentrations far below the ones shown to inhibit its targets in previous studies. Treatments of low doses of radicicol to RAW 264.7 cells that undergo osteoclastic differentiation in the presence of RANKL enhanced the RANKL-induced gene expression of integrin beta3 without any effect on the expression of integrin alphav, which was constitutively high. The cell surface level of integrin alphavbeta3 complexes was consequently augmented by radicicol. In addition, sustained ERK and MEK activation was observed in cells treated with both radicicol and RANKL. More importantly, modulation of ERK activity by the MEK inhibitor U0126 or the gene transduction of a constitutively active form of MEK resulted in a suppression and increment, respectively, of integrin beta3 induction by RANKL. Our data indicate that sustained ERK activity is associated with integrin beta3 induction and subsequent cell surface expression of the alphavbeta3 integrin complex, which may contribute to cell fusion during RANKL-directed osteoclastogenesis.

Topics: Animals; Bone Resorption; Butadienes; Carrier Proteins; Cell Differentiation; Cell Fusion; Cell Line; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gene Expression Regulation; Integrin alphaVbeta3; Integrin beta3; Lactones; Macrolides; MAP Kinase Kinase 1; Membrane Fusion; Membrane Glycoproteins; Mice; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Nitriles; Osteoclasts; Protein Serine-Threonine Kinases; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B