ABSTRACT
The present study investigates the direct action of 5-methoxytryptophol (5-MTX) in both MC3T3-E1 and RAW264.7 cells and compares it with melatonin (MEL), another 5-methoxyindol known to play a significant role on bone metabolism. We first screened increasing doses of both 5-MTX and MEL to determine their effect on metabolic activity and viability of preosteoblastic MC3T3-E1 cells. The optimal dose was used to determine its effect on differentiation of MC3T3-E1 cells and preosteoclastic RAW264.7 cells. Finally, we investigated the mechanism of action by adding the melatonin receptor antagonist luzindole (LUZ) and detecting the immunostaining of phospho-ERK. In MC3T3-E1 cells, most of the 5-MTX doses reduced slightly the metabolic activity of osteoblasts compared with the control, while MEL only decreased it for the highest dose (2.5 mM). As regards to cytotoxicity, low doses (0.001-0.1 mM) of both indoles showed a protective effect on osteoblasts, while the highest dose of MEL showed a higher cytotoxicity than the 5-MTX one. After 14 days of cell culture, Rankl mRNA levels were decreased, especially for 5-MTX. 5-MTX also induced a higher osteocalcin secretion and mineralization capacity than MEL. In RAW264.7 cells, 5-MTX decreased the number of osteoclast formed and its activity whereas MEL did not affect significantly the number of multinucleated TRAP-positive cells formed and showed a lower activity. Finally, MEL and 5-MTX promoted activation of the ERK1/2 pathway through the phosphorylation of ERK, while LUZ addition suppressed this effect. In conclusion, the present study demonstrates a new role of 5-MTX inhibiting osteoclastogenesis and promoting osteoblast differentiation.
Subject(s)
Indoles/pharmacology , MAP Kinase Signaling System/drug effects , Melatonin/pharmacology , Osteoblasts/drug effects , Osteoclasts/drug effects , Animals , Cell Differentiation/drug effects , Cell Line , Cell Survival/drug effects , Dose-Response Relationship, Drug , Gene Expression Regulation/drug effects , In Vitro Techniques , Mice , RANK Ligand/genetics , Tryptamines/pharmacologyABSTRACT
Cellular migration is a fundamental process linked to diverse pathological states such as diabetes and its complications, atherosclerosis, inflammation, and cancer. The receptor for advanced glycation end products (RAGE) is a multiligand cell surface macromolecule which binds distinct ligands that accumulate in these settings. RAGE-ligand interaction evokes central changes in key biological properties of cells, including proliferation, generation of inflammatory mediators, and migration. Although RAGE-dependent signal transduction is critically dependent on its short cytoplasmic domain, to date the proximate mechanism by which this RAGE domain engages and stimulates cytoplasmic signaling pathways has yet to be identified. Here we show that the RAGE cytoplasmic domain interacts with Diaphanous-1 (Dia-1) both in vitro and in vivo. We employed the human RAGE cytoplasmic domain as "bait" in the yeast two-hybrid assay and identified the formin homology (FH1) domain of Dia-1 as a potential binding partner of this RAGE domain. Immunoprecipitation studies revealed that the RAGE cytoplasmic domain interacts with the FH1 domain of Dia-1. Down-regulation of Dia-1 expression by RNA interference blocks RAGE-mediated activation of Rac-1 and Cdc42 and, in parallel, RAGE ligand-stimulated cellular migration. Taken together, these findings indicate that the interaction of the RAGE cytoplasmic domain with Dia-1 is required to transduce extracellular environmental cues evoked by binding of RAGE ligands to their cell surface receptor, a chief consequence of which is Rac-1 and Cdc42 activation and cellular migration. Because RAGE and Dia-1 are implicated in the regulation of inflammatory, vascular, and transformed cell migration, these findings highlight this interaction as a novel target for therapeutic intervention in inflammation, atherosclerosis, diabetes, and cancer.
