Melatonin promotes self-renewal of nestin-positive pancreatic stem cells through activation of the MT2/ERK/SMAD/nestin axis

Although melatonin has been shown to exhibit a wide variety of biological functions, its effects on promotion of self-renewal in pancreatic stem cells remain unknown. In this study, we incubated murine pancreatic stem cells (PSCs) with various concentrations of melatonin (0.01, 0.1, 1, 10 or 100 mu M) to screen for the optimum culture medium for increasing cell proliferation. We found that 10 mu M melatonin can significantly increase proliferation and enhance expression of a stem cell marker, nestin, in PSCs via melatonin receptor 2 (MT2). Thus, we used 10 mu M melatonin to study the melatonin-mediated molecular mechanisms of cell proliferation in PSCs. We applied extracellular signal-regulated kinase (ERK) pathway inhibitor SCH772984 and transforming growth factor beta (TGF-beta) pathway inhibitor SB431542, along with interfering RNAs siERK1, siERK2, siSmad2, siSmad3, siSmad4 and siNestin, to melatonin-treated PSCs to research the roles of these genes in self-renewal. The results revealed a novel molecular mechanism by which melatonin promotes self-renewal of PSCs: a chain reaction in the MT2/ERK/SMAD/nestin axis promoted the aforementioned self-renewal as well as inhibited differentiation. In addition, upregulation of nestin created a positive feedback loop in the regulation of the transforming growth factor beta 1 (TGF-beta 1)/SMADs pathway by promoting expression of Smad4. Conversely, knockdown of nestin significantly suppressed the proliferative effect in melatonin-treated PSCs. These are all novel mechanisms through which the ERK pathway cooperatively crosstalks with the SMAD pathway to regulate nestin expression, thereby enhancing self-renewal in PSCs.