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

Abstract

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. Introduction:Serotonin (5-hydroxytryptamine [5HT]), an established monoamine and neurotransmitter, is synthesized from L-tryptophan in a 2-step conversion requiring a hydroxylation step and a decarboxylation step. The hydroxylation is performed by the rate-limiting enzyme, tryptophan hydroxylase [TPH], producing 5-hydroxytryptophan [5HTP], which is decarboxylated by aromatic Lamino acid decarboxylase producing 5HT (1). 5HT is then released into the blood and stored by platelets, which lack the TPH1 enzyme. Degradation of 5HT occurs through oxidation by monoamine oxidase into 5-hydroxyindolacetic acid which is excreted in the urine. In humans and in mice, there are two TPH enzymes: TPH2 which converts L-tryptophan to 5HTP in the central nervous system and TPH1 which converts L-tryptophan to 5HTP in the periphery (2-4). 5HT and TPH1/2 are unable to cross the blood brain barrier, resulting in compartmentalization of 5HT (2,5). While 5HT is well-known as a central neurotransmitter altering behavior and mood, over 95% of 5HT is produced in the periphery in the gut enterochromaffin cells. During lactation, it has been has demonstrated that the mammary gland contributes approximately 50% of circulating 5HT (6,7).Mammary-derived 5HT is important in regulating maternal calcium homeostasis and breast-tobone communication via the synthesis and secretion of the parathyroid hormone related protein [PTHrP] in the mammary gland during lactation (8-10). PTHrP is secreted from the mammary epithelial cells and then acts on the bone to liberate calcium for milk synthe...