Elsevier

Bone

Volume 72, March 2015, Pages 43-52
Bone

Original Full Length Article
Serotonin (5-HT) inhibits Factor XIII-A-mediated plasma fibronectin matrix assembly and crosslinking in osteoblast cultures via direct competition with transamidation

https://doi.org/10.1016/j.bone.2014.11.008Get rights and content

Highlights

  • Serotonin competes with Factor XIII-A-mediated crosslinking of plasma FN.

  • Serotonylation of plasma FN inhibits its fibrillogenesis in osteoblast cultures.

  • Serotonin also decreases Factor XIII-A crosslinking/transamidation activity in osteoblast cultures.

  • Serotonin-mediated plasma FN assembly defect affects collagen deposition and mineralization.

Abstract

Serotonin (5-HT) – a monoamine with a variety of physiological functions – has recently emerged as a major regulator of bone mass. 5-HT is synthesized in the brain and the gut, and gut-derived 5-HT contributes to circulating 5-HT levels and is a negative modulator of bone mass and quality. 5-HT's negative effects on the skeleton are considered to be mediated via its receptors and transporter in osteoblasts and osteoclasts; however, 5-HT can also incorporate covalently into proteins via a transglutaminase-mediated serotonylation reaction, which in turn can alter protein function. Plasma fibronectin (pFN) – a major component of the bone extracellular matrix that regulates bone matrix quality in vivo – is a major transglutaminase substrate in bone and in osteoblast cultures. We have recently demonstrated that pFN assembly into osteoblast culture matrix requires a Factor XIII-A (FXIII-A) transglutaminase-mediated crosslinking step that regulates both quantity and quality of type I collagen matrix in vitro. In this study, we show that 5-HT interferes with pFN assembly into the extracellular matrix in osteoblast cultures, which in turn has major consequences on matrix assembly and mineralization. 5-HT treatment of MC3T3-E1 osteoblast cultures dramatically decreased both pFN fibrillogenesis as analyzed by immunofluorescence microscopy and pFN levels in DOC-soluble and DOC-insoluble matrix fractions. This was accompanied by an increase in pFN levels in the culture media. Analysis of the media showed covalent incorporation of 5-HT into pFN. Minor co-localization of pFN with 5-HT was also seen in extracellular fibrils. 5-HT also showed co-localization with FXIII-A on the cell surface and inhibited its transamidation activity directly. 5-HT treatment of osteoblast cultures resulted in a discontinuous pFN matrix and impaired type I collagen deposition, decreased alkaline phosphatase and lysyl oxidase activity, and delayed mineralization of the cultures. Addition of excess exogenous pFN to cultures treated with 5-HT resulted in a significant rescue of pFN fibrillogenesis as well as type I collagen deposition and mineralization. In summary, our study presents a novel mechanism on how increased peripheral extracellular 5-HT levels might contribute to the weakening of bone by directly affecting the stabilization of extracellular matrix networks.

Introduction

Serotonin (5-Hydroxytryptamine, or 5-HT) is a monoamine with diverse biological functions that include participation in vasoconstriction, intestinal movement, modulation of mood and anxiety, as well as regulation of bone mass [1]. 5-HT is generated from tryptophan by the action of tryptophan hydroxylase enzymes (TPH) at two distinct locations: in the central nervous system (brain stem), and in the gut. Five percent of human 5-HT is produced by the brain stem via the action of the Tph2 isoform, and since 5-HT does not cross the blood brain barrier, its actions remain in the central nervous system. The remaining 95% of human 5-HT is synthesized in the gut by enterochromaffin cells via Tph1. Most of this 5-HT pool is stored in the platelets leaving less than 5% 5-HT circulating in the peripheral system [2]. Recent research results show that brain-derived 5-HT supports bone mass accrual [3], as Tph2-knockout mice have decreased bone volume, decreased osteoblast number, lower bone formation rate and increased osteoclastogenesis [4]. Gut-derived 5-HT, on the other hand, has negative effects on bone mass. Elimination of peripheral 5-HT (in Tph1-null mice) causes increased bone mass and increased osteoblast proliferation [1].

Brain 5-HT modulates mood and anxiety, and an increase in the level of 5-HT in the synaptic cleft is known to have an anti-depressant effect. In the central nervous system, more than 90% of 5-HT is taken up into cytosol via the 5-HT transporter (5-HTT) after release to the synaptic cleft [5]. Selective 5-HT re-uptake inhibitors (SSRIs), which block 5-HTT function, are commonly prescribed to patients with depression in order to maintain or increase synaptic 5-HT levels [6]. The effects of SSRIs are not specific to the nervous system, and these modulators of the serotonergic system cause a systemic increase in peripheral (extracellular) 5-HT levels. Mounting evidence shows negative peripheral side effects of SSRIs, which include problems in the gastrointestinal and cardiovascular systems, as well as in bone mass accrual and bone quality [7], [8], [9]. Large-scale cohort studies have demonstrated that patients using SSRIs are at increased risk of fractures and show significantly reduced hip bone mineral density and an increased rate of bone loss compared to non-users [10], [11], [12], [13], [14]. It has also been reported that 5-HTT knockout mice, having increased extracellular 5-HT, are osteopenic [15], [16]. The negative effect of 5-HT on bone mass accrual is considered to be mediated via its receptors and transporter in osteoblasts, and osteoclasts [17], [18], [19], [20]. 5-HT decreases osteoblast proliferation via effecting cyclin D1 expression in a receptor-mediated manner, and elevated cytoplasmic 5-HT levels increase osteoclastogenesis via activation of NF-κB. In addition to these effects, 5-HT can also modulate systems via a mechanism referred to as ‘serotonylation’, where 5-HT incorporates covalently into glutamine residues of proteins via a transglutaminase enzyme-mediated transamidation/monoaminylation reaction (Fig. 1) [21]. This modification has been described to modulate protein and cell function; for example, serotonylation of platelet pro-coagulant proteins facilitates platelet activation and blood clot formation [22], [23], serotonylation of GTPases in pancreatic β-cells modulates insulin secretion [24], and RhoA serotonylation in vascular smooth muscle cells is important for aortic vascular contractility [25].

Transglutaminases (TGs) are a family of calcium-dependent enzymes that catalyze post-translational formation of γ-glutamyl-ε-lysine crosslink (isopeptide bonds) between glutamine (Q) residues and either primary amines (polyamines or monoamines) or lysine (K) residues in a polypeptide or in a protein (Fig. 1A) [26]. TG-mediated crosslinking of proteins occurs in various cellular processes including apoptosis, cytoskeletal organization as well as during extracellular matrix assembly [27], [28]. We have demonstrated that osteoblasts express two TG enzymes, transglutaminase 2 (TG2) and Factor XIII-A (FXIII-A), of which the latter is the main crosslinking enzyme in osteoblast cultures. Our previous work has demonstrated that FXIII-A activity is important for osteoblast matrix deposition and matrix quality and that plasma fibronectin (pFN) is the main FXIII-A substrate in osteoblast culture extracellular matrix [29], [30], [31]. FN is an abundant and multifunctional extracellular matrix glycoprotein which regulates a number of cellular functions including cell adhesion, proliferation, migration and differentiation, and it is found in living systems as two pools — as circulating plasma FN made by hepatocytes, and as cellular FN synthesized by tissue-resident cells [32], [33], [34], [35], [36]. Circulating pFN contributes to 90% of the bone FN matrix, and hepatocyte-specific FN-knockout mice with no circulating pFN have compromised bone matrix integrity and quality [37]. We demonstrated previously that pFN requires a FXIII-A-mediated crosslinking step to form DOC-insoluble matrix and a fibrillar network [38], [39]. In the absence of FXIII-A activity, pFN matrix assembly, type I collagen (COL I) deposition and matrix lysyl oxidase (LOX) activity are decreased, and alkaline phosphatase (ALP) activity is significantly lower, all of which results in delayed mineralization in the osteoblast cultures [38].

In this study, we hypothesized that in addition to its receptor- and transporter-mediated actions on bone cells, 5-HT could also interfere with bone quality via directly inhibiting matrix assembly and stabilization by acting as a competitive inhibitor to the transamidation reaction and protein crosslinking. Indeed, we demonstrate that 5-HT inhibits pFN matrix assembly into a fibrillar network, and that these negative effects of 5-HT on matrix assembly are mediated by direct, covalent binding to pFN and via an interaction with FXIII-A enzyme. The defective pFN assembly leads to decreased COL I deposition, lower lysyl oxidase and alkaline phosphatase levels, ultimately resulting in a decrease in osteoblast culture mineralization. Our work demonstrates a new potential mechanism for how long-term increased peripheral 5-HT levels might weaken bone by directly interfering with the formation of extracellular matrix networks.

Section snippets

Antibodies, proteins and reagents

Rabbit anti-biotin antibody was purchased from Rockland (Gilbertsville, PA, USA), and rabbit anti-fibronectin antibody and normal rabbit IgG were from EMD Millipore (Billerica, MA, USA). Anti-actin antibody, monoclonal anti-vimentin antibody (clone vim 13.2), rabbit anti-serotonin antibody, bovine plasma FN, serotonin hydrochloride, histamine dihydrochloride, dopamine hydrochloride, Thiazolyl Blue Tetrazolium Bromide (MTT), and 3,3′,5,5′-Tetramethylbenzidine (TMB) were obtained from Sigma

5-HT acts as a competitive inhibitor to FXIII-A-mediated crosslinking in vitro and inhibits assembly of pFN in MC3T3-E1 osteoblast cultures

To begin our investigation on whether 5-HT might act as a competitive inhibitor of the transamidation reaction and thus affect matrix stabilization, we examined whether it interferes with FXIII-A-mediated modification of pFN. This was investigated by testing if 5-HT can compete with, and block, FXIII-A-mediated 5-(biotinamido) pentylamine (BPA) incorporation into pFN in vitro. BPA is a biotin-conjugated primary amine, which can covalently incorporate into TG-reactive Q residues of a TG

Discussion

5-HT has emerged as an important regulator of bone mass and bone quality and it exerts negative effects on bone via its transporter and receptors in bone cells. In this study, we show that in addition to its receptor-mediated effects, 5-HT can also incorporate covalently into a bone matrix component – pFN – in a TG enzyme-mediated process referred to as serotonylation. We demonstrate that 5-HT acts as a competitive inhibitor to the FXIII-A-induced pFN crosslinking and insolubilization process,

Acknowledgments

We would like to thank Mrs. Aisha Mousa and Dr. Vamsee D. Myneni for their assistance in this study. This study was supported by grants to MTK from the Canadian Institutes of Health Research (CIHR) (MOP-119403). CC was supported by stipends from the China Scholarship Council.

Conflicts of interest

There are no conflicts of interest between authors.

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