Serotonin stimulated parathyroid hormone related protein induction in the mammary epithelia by transglutaminase-dependent serotonylation

Mammary-derived serotonin [5HT] has been implicated in breast-to-bone communication during lactation by increasing parathyroid hormone related-peptide [PTHrP] in the mammary gland. It is well-established that PTHrP acts on the bone to liberate calcium for the milk during lactation; however, the mechanism of 5HT’s regulation of PTHrP has not been fully elucidated. Recently, serotonylation, has been shown to be involved in a variety of physiological processes. Therefore, we investigated whether serotonylation is involved in 5HT’s regulation of PTHrP in the mammary gland. Using lactogenic differentiated mouse mammary epithelial cells, we studied the effect of increased intracellular 5HT using the antidepressant, fluoxetine [FLX], or 5-hydroxytryptophan ([5HTP] 5HT precursor) with or without transglutaminase inhibition on PTHrP induction and activity and the potential serotonylation target protein, RhoA. Treatment with FLX or 5HTP significantly increased intracellular 5HT concentration and subsequently increased PTHrP gene expression which was reduced with transglutaminase inhibition. Further, we demonstrated that transglutaminase becomes more active with lactogenic differentiation and with 5HTP or FLX treatment. We examined RhoA, Rac1, and Rab4 as potential serotonylation target proteins and have concluded RhoA is likely a serotonylation target protein. Our data suggest that 5HT regulates PTHrP induction in part through the process of serotonylation during lactation.