5-HT prompt TPO mRNA transcription via H3Q5 serotonylation

Abstract Background: 5-HT (serotonin) is considered as a novel cell growth factor. Our previous studies have shown that 5-HT is a pleiotropic growth factor for CD34+ hematopoietic stem cells and megakaryocytes. However, the effect of 5-HT on thrombopoietin (TPO) production remains unexplored. Methods: Q-PCR, Western Blot and ELISA were used in the TPO study. Flow Cytometry or Immunofluorescence microscope were used in the megakaryocytes study. The relationship between 5-HT and TPO was studied in traumatic mouse, Tph1-depleted mouse and Tph1-/- mouse model. Results: Traumatic mouse model showed that both 5-HT and TPO were increased, but the rise in TPO lagged that of 5-HT. Subsequently, we established a 5-HT-depleted mouse model using LX1606, an inhibitor of tryptophan hydroxylase 1 (Tph1), the rate-limiting enzyme in 5-HT synthesis. After treatment with LX1606, both 5-HT and TPO were significantly reduced. Tph1 KO mice exhibited a ~95% reduction in circulating 5-HT levels, leading to a significant decrease in plasma TPO levels. Subsequently, we assessed bone marrow megakaryocytes in Tph1 KO and 5-HT-depleted mice and found a slight decrease in the number of megakaryocytes in the bone marrow of Tph1 knockout mice compared to wild-type mice, although this difference did not reach statistical significance. In-vitro study, 5-HT significantly promoted TPO mRNA and protein expression. Following 5-HT treatment of hepatic cells, a marked increase in the gene expression levels of serotonin transporter (SERT) and transglutaminases 2 (TGM2) was observed. Given the significant role of TGM2 in mediating serotonylation, we hypothesized that serotonylation may play a crucial role in 5-HT-mediated regulation of TPO mRNA expression. Subsequently, Western blot analysis of H3Q5ser revealed that 5-HT significantly enhanced H3Q5ser levels in a concentration-dependent manner. Conclusion: This study reveals a novel mechanism for 5-HT-driven thrombopoiesis. 5-HT was transported into hepatocytes via SERT, where it was catalyzed by TGM2 to induce serotonylation of histone H3Q5, thereby facilitating transcriptional activation of the TPO gene.