The process of protein serotonylation was previously demonstrated in platelets, where it was shown that transamidation of surface procoagulant proteins, including fibrinogen and thrombospondin, facilitates activation and blood clot formation (20
). Serotonylation of proteins also has been reported to modulate insulin secretion in pancreatic β cells (33
). Moreover, Guilluy and colleagues (34
) found that transamidation of RhoA by 5-HT led to its activation and depletion in aortic SMCs. Recent studies have demonstrated that serotonylation of contractile and cytoskeleton proteins, including 42-kD smooth muscle actin, is important in isometric contraction of 5-HT–biotin–treated aortic smooth muscle (22
Cellular internalization of 5-HT through the 5-HTT leads to SMC signaling and proliferation (11
). The present studies demonstrate that protein serotonylation participates in the proliferative and migratory response of SMCs to 5-HT. Thus, we found that 5-HT induces serotonylation of several 220- to approximately 240-kD proteins; moreover, TGase inhibition blocks this effect. We also detected approximately 65-kD serotonylated proteins in bovine, but not in rat, SMCs. Antibody neutralization studies demonstrated that the 220- to 240-kD protein(s), but not the approximately 65-kD one(s), specifically bound the 5-HT antibody. It is likely that bovine SMCs express some endogenous BSA precursor protein (69 kD), which reacts with anti–5-HT–BSA antibody (36
). Our findings that serotonylation is blocked by 5-HTT inhibitors support a role of 5-HTi in this process. 5-HT, at a concentration of 1 μmol/L, was used in these studies because our previous results showed that this concentration of 5-HT is actively transported by the 5-HTT, whereas higher concentrations enter the cell by diffusion (9
). We also found that serotonylation of proteins is increased in the presence of a MAO inhibitor, which prevents degradation of internalized 5-HT. These data indicate that serotonylation of proteins is regulated by the activity of the 5-HTT, MAO, and TGases. Whereas 5-HTT mediates uptake of 5-HT into the cells, both MAO and TGase control 5-HTi metabolism of 5-HT.
The importance of protein serotonylation in the pulmonary artery SMC-proliferative and migratory responses to 5-HT were also investigated. Our findings, that inhibition of TGase blocked these responses, support the concept that this enzymatic process is key to the biologic activity of 5-HT in SMCs.
MS/MS peptide sequencing revealed that the major proteins modified by serotonylation include nonmuscle myosin heavy chain IIB (229 kD), plakin (195 kD), FN (257 kD), and filamin B (278 kD). FN is known to participate in SMC proliferation (34
). Immunoprecipitation techniques confirmed that FN was specifically serotonylated by 5-HT. The finding that FN siRNA blocked the proliferative effects of 5-HT on SMCs provides additional support for a role of this protein in SMC function. However, at the present time, it is not clear whether other serotonylated proteins also participate in these mitogenic effects. We demonstrated that serotonylated FN (Figure 5B) and other proteins (data not shown) are present in cell culture medium when SMCs are incubated with 5-HT. We therefore suspected that serotonylated FNs may be released into medium upon cellular interaction with 5-HT. This could be analogous to the process by which it gains access to intercellular matrix and interacts with α5β1-, αIIb
β3-, α4β1-, and α4β7-integrins, leading to differentiation, proliferation, migration, and apoptosis (37
). The intact FN molecule has four glutamine residue sites sensitive to tissue TGases. The glutamine residues at positions 3 and 4 of the FN amino terminus have been identified as the transglutamination sites for plasma and tissue TGase (42
In the hypertensive pulmonary vessel wall, vascular SMCs acquire a proliferative, synthetic phenotype, and migrate into the subendothelium (43
). It has been reported that increased expression of FN not only contributes to the migration of vascular SMCs, but also induces the transformation of these cells into a proliferative phenotype (38
). Furthermore, FN deposition in lung is highly correlated with the severity of pulmonary hypertension in rodents with chronic hypoxia and in patients with pulmonary hypertensive (24
). Recent studies demonstrate that interactions between αIIbβ3-integrin and 5-HTT regulate 5-HT transport and platelet aggregation in mice and humans, indicating cross-talk between 5-HTT signaling and FN/integrin signaling (47
Our studies provide evidence of crosslinking of cellular FN and FN fragments in culture medium from 5-HT–treated SMCs. FN crosslinking with other ECM components is a critical step in ECM assembly, and numerous reports have demonstrated that some FN fragments resulting from enzymatic degradation are potent inducers of cell migration (48
). Conformational change of the FN molecule may expose certain hidden glutamine residues important in the formation of covalent bonds between FN and the
-amino group of a lysine residue in fibrin. Serotonylated modification of FN may cause a conformational change in the FN that subsequently triggers FN crosslinking with ECM proteins, leading to SMC growth.
In summary, the present studies demonstrate that tissue TGases use 5-HTi to modify substrate proteins (in particular, FN) post-translationally, and that this is important in regulating 5-HT–induced proliferation and migration of pulmonary artery SMCs. Furthermore, this action may play an important role in the development of pulmonary hypertension.