Endocrine Abstracts

The physiological and neuroendocrine functions of the pineal gland hormone, melatonin and its therapeutic potential critically depend on the understanding of its target sites and its mechanism of action. There is evidence that melatonin promoted osteoblast differentiation and mineralization in MC3T3-E1 (mouse preosteoblast) and rat osteoblast-like osteosarcoma 17/2.8 cells. Our group set up an experimental model of mouse osteoblast culture. As a marker specific for osteoblast we used Cbfa1 expression. Cbfa1 is an osteoblast-specific transcription factor positively regulating osteoblast differentiation and function. Its expression and/or transcription activity are thus potential targets for bone anabolic therapies.

Objective: The aim of this study was to assess the effect of melatonin treatment on mouse osteoblast in culture by studying the Cbfa1 gene expression, the major regulator of the osteoblast phenotype, and induction of mineralization, expression of differentiated osteoblasts.

Material and methods: Mouse osteoblasts were seeded for RNA extraction or von Kossa specific staining for mineralization and treated with melatonin (10⁻¹¹–10⁻⁶ M). After incubation period cells were harvested and RNA extracted. 1 μg RNA was reverse transcribed to single stranded cDNA and then specifically amplified for Cbfa1. Cells were also stained specifically for alkaline phosphatase and mineralization.

Results: Melatonin reduced Cbfa1 expression in both experiments. As regards mineralization, groups treated with melatonin showed high mineralization in an early development stage when normally mineralization does not occurs (as could be seen in control sample).

Conclusion: The von Kossa positive staining showed that melatonin promotes mineralization in osteoblasts in early development stages probably by its stimulatory effect on BSP, ALP and osteocalcin gene expression. The slight inhibition in Cbfa1 expression needs further studies in order to see if this effect is correlated with the precocious fully differentiation of osteoblasts (mineralizing cells).