Role of Protein Transamidation in Serotonin-Induced
Proliferation and Migration of Pulmonary Artery
Smooth Muscle Cells
Yinglin Liu1, Lin Wei2, Debra L. Laskin1, and Barry L. Fanburg2
1Rutgers University, Piscataway, New Jersey; and2Tufts Medical Center, Pulmonary Critical Care and Sleep Division, Tupper Research Institute,
Pulmonary hypertension is characterized by elevated pulmonary
artery pressure and pulmonary artery smooth muscle cell (SMC)
proliferation and migration. Clinical and experimental evidence
suggests that serotonin (5-HT) is important in these responses. We
previously demonstrated the participation of the 5-HT transporter
and intracellular 5-HT (5-HTi) in the pulmonary vascular SMC-
the intracellular actions of 5-HT is unknown. We speculated that
5-HTi activates SMC growth by post-translational transamidation of
proteinsvia transglutaminase (TGase) activity, a processreferred to
as serotonylation. To test this hypothesis, serotonylation of pulmo-
nary artery SMC proteins, and their role in 5-HT–induced prolifera-
time-dependent increase in serotonylation of multiple proteins
in both bovine and rat pulmonary artery SMCs. Inhibition of
TGase with dansylcadaverin blocked this activity, as well as SMC-
proliferative and migratory responses to 5-HT. Serotonylation of
proteins also was blocked by 5-HT transporter inhibitors, and was
enhanced by inhibition of monoamine oxidase, an enzyme known
to degrade 5-HTi, indicating that 5-HTi levels regulate serotonyla-
sequencing revealed that a major protein serotonylated by
TGase was fibronectin (FN). 5-HT–stimulated SMC serotonylation
and proliferation were blocked by FN small interfering (si) RNA.
These findings, together with previous observations that FN
expression in the lung strongly correlates with the progression of
pulmonary hypertension in both experimental animals and
humans, suggest an important role of FN serotonylation in the
pathogenesis of this disease.
Keywords: serotonin; transglutaminase; fibronectin; protein serotony-
lation; smooth muscle cell growth
Serotonin (also known as 5-hydroxytryptamine [5-HT]) is one
of the most potent naturally occurring pulmonary vasoconstric-
tors (1). Clinical, experimental, and human genetic data support
a relationship between 5-HT, pulmonary artery remodeling, and
pulmonary hypertension. Our laboratories and others have
demonstrated the importance of both the 5-HT transporter
(5-HTT) and 5-HT receptors in pulmonary vascular smooth
muscle cell (SMC)–proliferative and migratory responses to
5-HT (2–7). We also have shown that 5-HT transport via the
5-HTT triggers activation of platelet-derived growth factor
receptor–b and mitogen-activated protein kinase, which partic-
ipate in proliferation of pulmonary artery SMCs (2, 8). Further-
more, inhibition of intracellular monoamine oxidase (MAO),
which degrades 5-HT, markedly enhances 5-HT–induced pro-
liferation of SMCs (9–11). These data indicate that intracellular
5-HT (5-HTi) is an important signal for SMC proliferation;
however, the mechanism underlying this activity is unknown.
Transglutaminases (TGases), which are abundant in blood
and vascular SMCs (12, 13), catalyze post-translational modifi-
cations of proteins in a calcium-dependent manner (14, 15).
This involves the formation of a covalent bond between the
g-carboxamide group of peptide-bound glutamine residues and
either the amino groups of a primary amine substrate of TGases
(16) or the e-amine group of peptide-bound lysine residues (17).
Modification of TGase substrate proteins by transamidation has
been shown to be important in cell survival, apoptosis, and
cytoskeleton organization (18, 19). The primary amine, 5-HT, is
a TGase substrate. TGase-catalyzed transamidation of proteins
with 5-HT is referred to as serotonylation. Serotonylation of
platelet procoagulant proteins, including fibrinogens and throm-
bospondin, facilitates platelet activation and blood clot forma-
tion (20, 21). Recent studies have shown that serotonylation of
RhoA and smooth muscle b-actin plays an important role in
aortic vascular contractility (22, 23). This 5-HT covalent de-
rivative modification reaction is illustrated in Figure 1.
We speculated that serotonylation of TGase substrate pro-
teins modulates their functions in pulmonary artery SMCs, and
this was investigated in the present studies. 5-HT was found to
initiate serotonylation of several different proteins in bovine
and rat pulmonary artery SMCs, including fibronectin (FN).
Moreover, inhibition of TGase activity reduced both 5-HT–
induced protein serotonylation and proliferation and migration
of SMCs. These data suggest that serotonylation of proteins in
SMCs is important in 5-HT–induced functional responses.
Serotonylation of FN may be particularly relevant, as it has
been reported that both 5-HTT expression and FN accumula-
tion in the lung are directly correlated with the progression of
pulmonary hypertension (24–26).
MATERIALS AND METHODS
Cell Culture and Functional Evaluation
SMCs from bovine and rat pulmonary artery were isolated as pre-
viously described (3, 27). Cells were cultured in Dulbecco’s modified
Eagle’s medium containing 10% FBS, 1% penicillin, and 0.5% strepto-
mycin. Cells from passages 3–9 were used. SMC proliferation was
Serotonylation of pulmonary artery smooth muscle cell
proteins may be important in the development of pulmo-
nary hypertension. More knowledge about this process is
needed and is provided in this article.
(Received in original form February 25, 2010 and in final form May 25, 2010)
This work was supported by National Institute of Health grants HL085260-01
(B.L.F), GM034310, ES004738, CA132624, AR055073, and ES005022 (D.L.L),
and by American Heart Association grant 0725961-H (Y.L.).
Correspondence and requests for reprints should be addressed to Barry
Fanburg, M.D., Tufts Medical Center, Pulmonary, Critical Care and Sleep
Division, 800 Washington Street, #257, Boston, MA 02111. E-mail: BFanburg@
Am J Respir Cell Mol Biol
Originally Published in Press as DOI: 10.1165/rcmb.2010-0078OC on June 17, 2010
Internet address: www.atsjournals.org
Vol 44. pp 548–555, 2011
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Liu, Wei, Laskin, et al.: Serotonylation of Proteins in Pulmonary Artery Smooth Muscle Cells555