The serotonergic system plays a significant regulatory role in osteoblast differentiation and proliferation. Serotonin (five-hydroxytryptamine or 5HT) may promote or inhibit osteoblast proliferation depending upon the serotonin receptor isoforms expressed by the cell. Classically, 5HT receptor 1B (5HTR1B) reduces osteoblast proliferation by inhibiting phosphorylation of the cAMP response element binding protein (CREB) transcription factor. Conversely, 5HT receptor 2A (5HTR2A) activation positively influences osteoblast proliferation through extracellular signal-regulated kinase (ERK) signaling. Despite the serotonergic system being well characterized in normal osteoblasts, minimal information concerning the influence of serotonin on malignant osteoblasts is reported. The objectives of our study were to elucidate the expression, function, and potential of therapeutic targeting of these two receptors in a canine osteosarcoma cell line (COS) and primary normal canine osteoblasts (CnOb).
Equal levels of htr1b (gene) and 5HTR1B (protein) expression were found in normal and malignant osteoblasts. Treatment with serotonin enhanced viability of osteosarcoma cells but not normal osteoblasts. Challenge with the 5HTR1B agonist anpirtoline did not change cell viability. Rather, incubation with the specific receptor antagonist SB224289 caused reduction in osteoblast viability and osteosarcoma cells were more sensitive to this inhibitory effect. Investigation of this inhibitory activity showed 5HTR1B antagonism induces capase-3/7 activation in malignant cells. Evaluation of phosphorylated levels of CREB and ERK revealed abnormal 5HTR1B signaling in COS.
Htr2a (gene) and 5HTR2A (protein) expression was observed in COS and CnOb, with osteosarcoma cells demonstrating five-fold greater receptor expression than normal osteoblasts. Antagonism of 5HTR2A with the inhibitor ritanserin attenuated viability in neoplastic and normal cells. COS appeared more sensitive to the inhibitory activity of ritanserin than did CnOb. 5HTR2A blockade induced increased activity of caspase-3/7 in COS, thus promoting apoptosis in osteosarcoma cells. Discordant patterns of CREB and ERK phosphorylation were found between malignant and normal osteoblasts following 5HTR2A pharmacologic manipulation, suggesting aberrant receptor signaling in neoplastic cells.
Cumulatively, our findings demonstrate that functional aspects of the serotonergic signaling system are present in a canine osteosarcoma cell line. 5HTR1B and 5HTR2A could plausibly have roles in the genesis and progression of osteosarcoma. Importantly, both serotonin receptors appear to be novel therapeutic targets. Future investigations into the relationship between canine osteosarcoma and other serotonergic signaling components may highlight previously unknown aspects of osteosarcoma biology.