Role of Protein Transamidation in Serotonin-Induced Proliferation and Migration of Pulmonary Artery Smooth Muscle Cells

Rutgers University, Piscataway, New Jersey, USA.
American Journal of Respiratory Cell and Molecular Biology (Impact Factor: 3.99). 04/2011; 44(4):548-55. DOI: 10.1165/rcmb.2010-0078OC
Source: PubMed


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-proliferative response to 5-HT. However, the mechanism underlying the intracellular actions of 5-HT is unknown. We speculated that 5-HTi activates SMC growth by post-translational transamidation of proteins via transglutaminase (TGase) activity, a process referred to as serotonylation. To test this hypothesis, serotonylation of pulmonary artery SMC proteins, and their role in 5-HT-induced proliferative and migratory responses, were assessed. 5-HT caused dose- and 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 serotonylation. Immunoprecipitation assays and HPLC-mass spectral peptide 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.

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    • "Serotonin through 5HT transporter (5HTT) is involved in pulmonary artery SMC and fibroblasts proliferation. Both 5HT transporter (5HTT) and 5HT receptors promote pulmonary artery SMC proliferation and migration, vasoconstriction and local microthrombi [175] [176]. In addition, 5HTT transactivates PDGF receptor in pulmonary artery SMC, indicating crosstalk between 5HT and PDGF pathways, both implicated in the pathogenesis of PH [177]. "
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    ABSTRACT: Pulmonary hypertension (PH) is a sequel of a variety of cardiovascular and systemic diseases. Heterozygous mutation of BMPRII, a member of the TGFβ superfamily is the commonest genetic defect so far identified in PH. Recent advances have contributed a great deal to the understanding of the disease; however, the actual mechanism/s is not yet clear. Endothelial damage is the key underlying feature of PH. The main effects are loss of vascular relaxation response, increased cell proliferation and impaired apoptosis, matrix deposition, obstruction in the small pulmonary arteries, right ventricular hypertrophy; and eventually leading to right heart failure and death. The diagnosis of PH is often made late because of the insidious onset of symptoms, therefore the treatment poses a daunting challenge. Furthermore, depending on the underlying pathology, not all patients respond equally to same therapeutic agents. Current therapy includes a group of drugs mainly involved in improving vascular relaxation (cAMP and cGMP mechanisms) and endothelin receptor blockers alone or in combination. Newer drugs such as guanylate cyclase activators, PDGF blocker, RhoA/Rho kinase blockers have shown encouraging results in animal studies and in a few clinical cases of PH. Other drugs and signaling pathways such as nitrites, PPARγ, ACE2, ghrelin etc. are under investigation. Studies with gene therapy are being actively pursued. This review summarizes the available therapy and the future prospects.
    Cardiovascular & hematological agents in medicinal chemistry 08/2011; 9(3):165-82. DOI:10.2174/187152511797037501
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    • "was shown to promote membrane resealing ( Kawai et al . 2008 ) . When pulmonary artery SMCs were stimulated with serotonin , this induced transamidation of proteins , which could be detected both in cells and their culture supernatant . Three of the four major TG2 substrates were found to be non - muscle myosin heavy chain , filamin B and plakin ( Liu et al . 2010 ) , which are involved in the stabilization of the cytoskeletal network to the cell mem - brane ( Leung et al . 2002 ) . Correspondingly , Factor FXIII may play the role in platelets that TG2 fulfills in SMCs . When stimulated with thrombin or calcium ionophore , which are known to trigger MP generation ( VanWijk et al . 2003 ) , FXIII "
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    ABSTRACT: Transglutaminase 2 (TG2) is a pleiotropic enzyme involved in both intra- and extracellular processes. In the extracellular matrix, TG2 stabilizes the matrix by both covalent cross-linking and disulfide isomerase activity. These functions become especially apparent during matrix remodeling as seen in wound healing, tumor development and vascular remodeling. However, TG2 lacks the signal sequence for a classical secretory mechanism, and the cellular mechanism of TG2 secretion is currently unknown. We developed a green fluorescent TG2 fusion protein to study the hypothesis that TG2 is secreted via microparticles. Characterization of TG2/eGFP, using HEK/293T cells with a low endogenous TG2 expression, showed that cross-linking activity and fibronectin binding were unaffected. Transfection of TG2/eGFP into smooth muscle cells resulted in the formation of microparticles (MPs) enriched in TG2, as detected both by immunofluorescent microscopy and flow cytometry. The fraction of TG2-positive MPs was significantly lower for cross-linking-deficient mutants of TG2, implicating a functional role for TG2 in the formation of MPs. In conclusion, the current data suggest that TG2 is secreted from the cell via microparticles through a process regulated by TG2 cross-linking. Electronic supplementary material The online version of this article (doi:10.1007/s00726-011-1010-3) contains supplementary material, which is available to authorized users.
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    ABSTRACT: Muscle-tendon moment arm magnitudes are essential variables for accurately calculating muscle forces from joint moments. Their measurement requires specialist knowledge and expensive resources. Research has shown that the patellar tendon moment arm length is related to leg anthropometry in children. Here, we asked whether the Achilles tendon moment arm (MA(AT)) can be accurately predicted in pre-pubescent children from surface anthropometry. Age, standing height, mass, foot length, inter-malleolar ankle width, antero-posterior ankle depth, tibial length, lower leg circumference, and distances from the calcaneus to the distal head of the 1st metatarsal and medial malleolus were determined in 49 pre-pubescent children. MA(AT) was calculated at three different ankle positions (neutral, 10° plantarflexion, and 10° dorsiflexion) by differentiating tendon excursion, measured via ultrasonography, with respect to ankle angle change using seven different differentiation techniques. Backwards stepwise regression analyses were performed to identify predictors of MA(AT.) When all variables were included, the regression analysis accounted for a maximum of 49% of MA(AT) variance at the neutral ankle angle when a third-order polynomial was used to differentiate tendon excursion with respect to ankle angle. For this condition, foot length and the distance between calcaneus and 1st metatarsal were the only significant predictors, accounting for 47% of the variance (p<0.05). The absolute error associated with this regression model was 3.8±4.4 mm, which would result in significant error (mean=14.5%) when estimating muscle forces from joint moments. We conclude that MA(AT) cannot be accurately predicted from anthropometric measures in children.
    Journal of Biomechanics 05/2011; 44(10):1839-44. DOI:10.1016/j.jbiomech.2011.03.023 · 2.75 Impact Factor
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