Original Full Length ArticleSerotonin (5-HT) inhibits Factor XIII-A-mediated plasma fibronectin matrix assembly and crosslinking in osteoblast cultures via direct competition with transamidation
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.
References (73)
- et al.
Lrp5 controls bone formation by inhibiting serotonin synthesis in the duodenum
Cell
(2008) - et al.
A serotonin-dependent mechanism explains the leptin regulation of bone mass, appetite, and energy expenditure
Cell
(2009) - et al.
Mechanisms of action of selective serotonin reuptake inhibitors in the treatment of psychiatric disorders
Eur Neuropsychopharmacol
(1999) - et al.
Neurotransmitter action in osteoblasts: expression of a functional system for serotonin receptor activation and reuptake
Bone
(2001) - et al.
Expression of serotonin receptors in bone
J Biol Chem
(2001) - et al.
Serotonin transporter and receptor expression in osteocytic MLO-Y4 cells
Bone
(2006) - et al.
Serotonylation of small GTPases is a signal transduction pathway that triggers platelet alpha-granule release
Cell
(2003) Coated-platelets: an emerging component of the procoagulant response
J Thromb Haemost
(2005)- et al.
Transglutaminase-dependent RhoA activation and depletion by serotonin in vascular smooth muscle cells
J Biol Chem
(2007) - et al.
Transglutaminase activity regulates osteoblast differentiation and matrix mineralization in MC3T3-E1 osteoblast cultures
Matrix Biol
(2006)
Transglutaminase activity arising from Factor XIIIA is required for stabilization and conversion of plasma fibronectin into matrix in osteoblast cultures
Bone
Factor XIII-A transglutaminase acts as a switch between preadipocyte proliferation and differentiation
Blood
Identification of osteogenic purmorphamine derivatives
Mol Cells
Fibronectin binds and enhances the activity of bone morphogenetic protein 1
J Biol Chem
Cellular fibronectin binds to lysyl oxidase with high affinity and is critical for its proteolytic activation
J Biol Chem
Serotonergic dystrophy induced by excess serotonin
Mol Cell Neurosci
Cross-linking of osteopontin by tissue transglutaminase increases its collagen binding properties
J Biol Chem
Bone histomorphometric and biomechanical abnormalities in mice homozygous for deletion of the dopamine transporter gene
Bone
Fibronectin and fibrin provide a provisional matrix for epidermal cell migration during wound reepithelialization
J Investig Dermatol
A major fraction of fibronectin present in the extracellular matrix of tissues is plasma-derived
J Biol Chem
Megakaryocyte-matrix interaction within bone marrow: new roles for fibronectin and factor XIII-A
Blood
A quantitative analysis of the incorporation of fibulin-1 into extracellular matrix indicates that fibronectin assembly is required
Matrix Biol
Cross-linking of collagen and fibronectin by factor XIIIa. Localization of participating glutaminyl residues to a tryptic fragment of fibronectin
J Biol Chem
Cross-linking of cold-insoluble globulin by fibrin-stabilizing factor
J Biol Chem
Update in serotonin and bone
J Clin Endocrinol Metab
The two faces of serotonin in bone biology
J Cell Biol
Serotonin synthesis, release and reuptake in terminals: a mathematical model
Theor Biol Med Model
SSRI antidepressant medications: adverse effects and tolerability
Prim Care Companion J Clin Psychiatry
Use of selective serotonin reuptake inhibitors and risk of fracture: a systematic review and meta-analysis
J Bone Miner Res
Minireview: a skeleton in serotonin's closet?
Endocrinology
Skeletal effects of serotonin (5-hydroxytryptamine) transporter inhibition: evidence from clinical studies
J Musculoskelet Neuronal Interact
Effect of selective serotonin reuptake inhibitors on the risk of fracture
Arch Intern Med
Use of antidepressants and rates of hip bone loss in older women: the study of osteoporotic fractures
Arch Intern Med
Selective serotonin reuptake inhibitors and other antidepressants and risk of fracture
Calcif Tissue Int
Antidepressant use and 10-year incident fracture risk: the population-based Canadian Multicentre Osteoporosis Study (CaMoS)
Osteoporos Int
Inhibition of the serotonin (5-hydroxytryptamine) transporter reduces bone accrual during growth
Endocrinology
Cited by (24)
Arecanut (Areca catechu L.) seed polyphenol improves osteoporosis via gut-serotonin mediated Wnt/β-catenin pathway in ovariectomized rats
2021, Journal of Functional FoodsCitation Excerpt :The liver showed fatty degeneration of liver cells, necrosis of some liver cells, infiltration of inflammatory cells and haemorrhage in OVX and the ACP maintained the normal states of the liver, indicated the pathological changes which may induce the liver metabolic disorder have occurred in the liver of OVX-rats. Previous studies have declared that the accrual peripheral serotonin is a key inhibitor of bone formation (Cui & Kaartinen, 2015; Ducy & Karsenty, 2010; Yadav et al., 2008). Inflammatory bowel disease patients always have been found the decreasing of bone mass due to the increased 5-HT (Lavoie et al., 2019) indicated that the gut-derived 5-HT influenced (Yano et al., 2015) could inhibit bone formation (Yadav et al., 2010).
Factor XIIIa inhibitors as potential novel drugs for venous thromboembolism
2020, European Journal of Medicinal ChemistryCitation Excerpt :The primary amine-containing inhibitors afford a nucleophilic attack at the acyl-enzyme intermediate during the catalysis cycle resulting in FXIIIa inhibition. Along these lines, multiple endogenous monoamines including serotonin (5-HT) 5, dopamine 6, and histamine 7 (Fig. 4) were shown to competitively inhibit FXIIIa-mediated crosslinking reactions [69]. Particularly, 5-HT was shown to act as a competitive inhibitor of FXIIIa-mediated crosslinking of plasma fibronectin in both an in vitro trans-glutamination activity assay and in MC3T3-E1 osteoblast cultures over the concentration range of 20–500 μM [69].
Assessment of expression and specific activities of transglutaminases TG1, TG2, and FXIII-A during osteoclastogenesis
2020, Analytical BiochemistryCitation Excerpt :The presence of TGs in bone and bone cells has been well documented via both in vitro and in vivo studies [14–16]. While the original work has mostly focused on osteoblasts [17–25], latest work by us and others have revealed their regulatory roles in osteoclastogenesis [26–29]. The work of Raghu et al. showed that FXIII-A deficiency resulted in reduced osteoclastogenesis in vivo and in vitro [27].
Mineralization-inhibiting effects of transglutaminase-crosslinked polymeric osteopontin
2017, BoneCitation Excerpt :The two enzymes appear to have distinct functions and different substrates [47]. Factor XIII-A is upregulated during MC3T3-E1 osteoblast differentiation, and contributes to the majority of the TG activity associated with differentiating osteoblast cells where it is involved in collagen type I and fibronectin assembly, and plasma fibronectin stabilization during early extracellular matrix assembly [43,44,48,49]. TG2 is constitutively expressed in osteoblasts, and in addition to its TG enzymatic activity, TG2 can also acts as a GTPase/ATPase, protein kinase, and as an adaptor protein, and its activity and function is regulated by the local Ca2 + concentration, GTP and ATP concentrations, and proteolysis by enzymes such as MT1-MMP [35,50–63].
Crosstalk between the Neuroendocrine System and Bone Homeostasis
2024, Endocrine ReviewsEmerging roles of nerve-bone axis in modulating skeletal system
2024, Medicinal Research Reviews