EXPRESSION OF 5-HT
RECEPTORS AND NEURONAL MARKER MAP2, GFAP AND TAU ON HUMAN MEGAKARYOCYTES
AND MEGAKARYOCYTIC CELL LINES
Yang M, Li K, Yuen PMP, Pong NH, Wong R, Chik
KW, Li CK, Shing MK, Fok TF
Department of Paediatrics, The Prince of Wales
Hospital, The Chinese University of Hong Kong, Hong Kong
Objective:
Serotonin (5-Hydroxytryptamine, 5-HT) is a monoamine with a molecular
weight of 176.2 dalton which can affect the functions of many cells
including platelets. It has been reported that serotonin has mitogenic
effects on smooth muscle cells and fibroblasts. Recently, serotonin has
been recognized not only as a neurotransmitter and vasoactive agent, but
also as a growth factor which may be representative of a class of molecules
promoting cell growth by interacting with G protein coupled receptors. Our
previous studies have shown that serotonin significantly stimulates
megakaryocyte colony formation with maximum stimulation at 100 nM and
ketanserin, a 5-HT2 receptor antagonist, blocks the mitogenic effect of
serotonin on megakaryocytopoiesis.
Methods:
In this study, we investigated the expression of
5-HT2A, 2B and 2C receptors, and other neuronal marker on megakaryocytic
lineage and the effect of serotonin on anti-apoptosis in megakaryocytes
using RT-PCR, flowcytometry, immunofluorescence staining, annexin V and
caspase-3 assay.
Results:
We found that: (1) Using RT-PCR method, 5-HT2A
receptors were identified on the four megakaryocytic cell lines: Meg-01,
Dami, CHRF-288-11 and M-07e. 5-HT2B receptors were also detected on Meg-01,
Dami and M-07e but not on CHRF-288-11 cells; (2) Using flow cytometry with
monoclonal antibodies, 5-HT2A, 2B and 2C receptors were identified on all
four megakaryocytic cell lines. It was not detected on the control cells K562;
(3) The expression of the three receptors were further confirmed on human
bone marrow megakaryocytes by using immunofluorescence staining; (4)
Furthermore, the anti-apoptotic effect of serotonin was studied in M-07e
cells by annexin V and active caspase-3 assay using flow cytometry. Our
preliminary results demonstrated that the apoptotic effect of
thrombospondin-1 (TSP-1) (2.5 g/ml) was abolished by serotonin
(100 nM) as well as TPO (50 ng/ml) in the culture of M-07e cells; and (5)
The neuronal marker MAP2, GFAP and Tau were also identified on human bone
marrow megakaryocytes and human megakaryocytic cell lines (Meg-01, Dami and
M-07e cells).
Conclusions:
This study demonstrated that megakaryocytic lineage expresses 5-HT2A, 2B
and 2C receptors and serotonin has anti-apoptotic effect on megakaryocytic
cell line in vitro.