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Transglutaminase 2 is secreted from smooth muscle cells by transamidation-dependent microparticle formation.

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.

SUBMITTER: van den Akker J

PROVIDER: S-EPMC3266506 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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Transglutaminase 2 is secreted from smooth muscle cells by transamidation-dependent microparticle formation.

van den Akker Jeroen J van Weert Angela A Afink Gijs G Bakker Erik N T P EN van der Pol Edwin E Böing Anita N AN Nieuwland Rienk R VanBavel Ed E

Amino acids 20110810 2-3

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. ...[more]

PMID: 21830119

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Project description:Arterial calcification is a phenotype of vascular repair in atherosclerosis, diabetes, hyperphosphatemic renal failure, and aging. Arterial calcification is modulated by transition of arterial smooth muscle cells (SMCs) from contractile to chondro-osseous differentiation programmed in response to increases in P(i), bone morphogenetic protein-2, and certain other stimuli. Transglutaminase (TG)2 release modulates tissue repair, partly by transamidation-catalyzed covalent crosslinking of extracellular matrix substrates. TG2 regulates cultured SMC differentiation, resistance artery remodeling to vasoconstriction, and atherosclerotic lesion size. Here, TG2 expression was required for the majority of TG activity in mouse and human aortic SMCs. TG2(-/-) SMCs lost the capacity for P(i) donor-induced formation of multicellular bone-like nodules and for increased expression of the type III sodium-dependent P(i) cotransporter Pit-1 and certain osteoblast and chondrocyte genes (tissue-nonspecific alkaline phosphatase, the osteoblast master transcription factor runx2, and chondrocyte-restricted aggrecan), and for P(i) donor- and bone morphogenetic protein-2-induced calcification. Uniquely in TG2(-/-) SMCs, P(i) donor treatment increased expression of the physiological SMC chondro-osseous differentiation and calcification inhibitors osteoprotegerin, matrix Gla protein, and osteopontin. Conversely, TG2(-/-) SMCs, unlike wild-type SMCs, failed to maintain contractile differentiation on laminin. Exogenous catalytically active TG2 augmented calcification by TG2(-/-) SMC in response to P(i) donor treatment. TG2 expression also drove P(i)-stimulated calcification of mouse aortic ring organ cultures, which was suppressed by the TG2 catalytic site-specific inhibitor Boc-DON-Gln-Ile-Val-OMe (10 micromol/L). Our results suggest that TG2 release in injured arteries is critical for programming chondro-osseous SMC differentiation and calcification in response to increased P(i) and bone morphogenetic protein-2.

Project description:BackgroundSmooth muscle cells (SMCs), which make up the medial layer of arteries, are key cell types involved in cardiovascular diseases (CVD), the leading cause of mortality and morbidity worldwide. In response to microenvironment alterations, SMCs dedifferentiate from a "contractile" to a "synthetic" phenotype characterized by an increased proliferation, migration, production of extracellular matrix (ECM) components, and decreased expression of SMC-specific contractile markers. These phenotypic changes result in vascular remodeling and contribute to the pathogenesis of CVD, including coronary artery disease (CAD), stroke, hypertension, and aortic aneurysms. Here, we aim to identify the genetic variants that regulate ECM secretion in SMCs and predict the causal proteins associated with vascular disease-related loci identified in genome-wide association studies (GWAS).MethodsUsing human aortic SMCs from 123 multi-ancestry healthy heart transplant donors, we collected the serum-free media in which the cells were cultured for 24 hours and conducted Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic analysis of the conditioned media.ResultsWe measured the abundance of 270 ECM and related proteins. Next, we performed protein quantitative trait locus mapping (pQTL) and identified 20 loci associated with secreted protein abundance in SMCs. We functionally annotated these loci using a colocalization approach. This approach prioritized the genetic variant rs6739323-A at the 2p22.3 locus, which is associated with lower expression of LTBP1 in SMCs and atherosclerosis-prone areas of the aorta, and increased risk for SMC calcification. We found that LTBP1 expression is abundant in SMCs, and its expression at mRNA and protein levels was reduced in unstable and advanced atherosclerotic plaque lesions.ConclusionsOur results unravel the SMC proteome signature associated with vascular disorders, which may help identify potential therapeutic targets to accelerate the pathway to translation.

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Transglutaminase 2 is secreted from smooth muscle cells by transamidation-dependent microparticle formation.

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Transglutaminase 2 is secreted from smooth muscle cells by transamidation-dependent microparticle formation.

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