N1E-115 mouse neuroblastoma cells express MT1 melatonin receptors and produce neurites in response to melatonin.

Melatonin, a pineal hormone that induces sleep, has become a popular over-the-counter drug. The cellular effects of melatonin, however, are only beginning to be studied. We have recently shown that stimulation of the MT1 melatonin receptor induces rapid and dramatic cytoskeletal rearrangements in transformed non-neuronal cells (Witt-Enderby et al., Cell. Motil. Cytoskel. 46 (2000) 28). These cytoskeletal changes result in the formation of structures that closely resemble neurites. In this work, we show that the N1E-115 mouse neuroblastoma cell line rapidly responds to melatonin stimulation and forms neurites within 24 h. We also demonstrate that these cells readily bind 2-[125I]iodomelatonin at levels consistent with what is noted for native tissues (B(max)=3.43+/-1.56 fmol/mg protein; K(d)=240 pM). Western analysis shows that these cells possess and express melatonin receptors of the MT1 subtype. Treatment with pertussis toxin eliminates neurite formation whereas treatment with the MT2 subtype-specific activator, BMNEP, does not induce neurite formation. We have previously shown that increases in MEK 1/2 and ERK 1/2 phosphorylation are correlated with the shape changes in transformed CHO cells. Western analysis of the MEK/ERK signaling pathway in N1E-115 cells shows that this pathway is most likely maximally and constitutively stimulated. This may account for the spontaneous production of neurites noted for this cell line after long culture periods. The results of this work show that melatonin receptor stimulation in a neuronal cell type results in the formation of neurites and that the receptors responsible for melatonin-induced neurite formation in N1E-115 cells are most likely of the MT1 subtype.

Melatonin induction of filamentous structures in non-neuronal cells that is dependent on expression of the human mt1 melatonin receptor.

Melatonin has gained recent popularity as a treatment for insomnia and other sleep disorders; however, its cellular effects are unknown. We report the effects of melatonin on the cellular morphology of Chinese hamster ovary (CHO) cells transformed to express the human melatonin receptors, mt1 and MT2. Our results show that melatonin exerts a strong influence on cellular shape and cytoskeletal organization in a receptor-dependent and possibly subtype-selective manner. The cell shape change that we see after a 5-h treatment of these non-neuronal cells with a pharmacological concentration of melatonin consists of the formation of long filamentous outgrowths that are reminiscent of the neurite processes produced by differentiating nerve cells. This morphological change occurs exclusively in cells expressing the mt1 receptor. We find that the microtubule and microfilament organization within these outgrowths is similar to that of neurites. Microtubules are required for the shape change to occur as Colcemid added in combination with melatonin completely blocks outgrowth formation. We demonstrate that the number of cells showing the altered cell shape is dependent on melatonin concentration, constant exposure to melatonin and that outgrowth frequencies increase when protein kinase A (PKA) is inhibited. Concomitant melatonin-dependent increases in MEK 1/2 and ERK 1/2 phosphorylation are noted in mt1-CHO cells only. The production of filamentous outgrowths is dependent on the translation of new protein but not the transcription of new mRNA. Outgrowth number is not controlled by centrosomes but is instead controlled by the polymerization state of the actin cytoskeleton. The results of this work show that the organization of the cytoskeleton is affected by processes specifically mediated or regulated by the mt1 receptor and may represent a novel alternative mechanism for the stimulation of process formation.