Plasma Membrane Factor XIIIA Transglutaminase Activity Regulates Osteoblast Matrix Secretion and Deposition by Affecting Microtubule Dynamics

Abstract

Transglutaminase activity, arising potentially from transglutaminase 2 (TG2) and Factor XIIIA (FXIIIA), has been linked to osteoblast differentiation where it is required for type I collagen and fibronectin matrix deposition. In this study we have used an irreversible TG-inhibitor to ‘block –and-track’ enzyme(s) targeted during osteoblast differentiation. We show that the irreversible TG-inhibitor is highly potent in inhibiting osteoblast differentiation and mineralization and reduces secretion of both fibronectin and type I collagen and their release from the cell surface. Tracking of the dansyl probe by Western blotting and immunofluorescence microscopy demonstrated that the inhibitor targets plasma membrane-associated FXIIIA. TG2 appears not to contribute to crosslinking activity on the osteoblast surface. Inhibition of FXIIIA with NC9 resulted in defective secretory vesicle delivery to the plasma membrane which was attributable to a disorganized microtubule network and decreased microtubule association with the plasma membrane. NC9 inhibition of FXIIIA resulted in destabilization of microtubules as assessed by cellular Glu-tubulin levels. Furthermore, NC9 blocked modification of Glu-tubulin into 150 kDa high-molecular weight Glu-tubulin form which was specifically localized to the plasma membrane. FXIIIA enzyme and its crosslinking activity were colocalized with plasma membrane-associated tubulin, and thus, it appears that FXIIIA crosslinking activity is directed towards stabilizing the interaction of microtubules with the plasma membrane. Our work provides the first mechanistic cues as to how transglutaminase activity could affect protein secretion and matrix deposition in osteoblasts and suggests a novel function for plasma membrane FXIIIA in microtubule dynamics.

Low extracellular sodium promotes adipogenic commitment of human mesenchymal stromal cells: a novel mechanism for chronic hyponatremia-induced bone loss

“…Several soluble factors, including adipokines and cytokines, exert a regulatory effect on the activity of different bone marrow cell populations, suppressing or promoting haematopoiesis, osteogenesis, and bone resorption [35,36] The rearrangement of MTs, the major component of the cytoskeleton, is involved in the regulation of differentiation processes and predispose cells to acquire a functional mature phenotype [37][38][39]. The analysis of MTs organization by immunofluorescence microscopy showed a strong rearrangement of tubulin in hMSC maintained for 7 days at low extracellular [Na ?…”

“…of 115 mM and became more evident at 90 mM, with partial disruption of MTs. This event, even allowing osteoblast differentiation [37], can affect cell function, in particular the secretion of matrix molecules [38]. As a consequence bone quality might be impaired, contributing to the bone fragility observed in hyponatremic patients [6].…”

Tissue transglutaminase (TG2) activity regulates osteoblast differentiation and mineralization in the SAOS-2 cell line

“…However, Al-Jallad et al (16) reported that FXIIIA could play a major role in MC3T3-E1 cell differentiation and collagen I matrix formation, and TG2 crosslinking activity did not contribute to osteoblast differentiation (16,24). However, in the current study, although we did not assay the expression of FXIIIA, we did find that TG2 levels continued to increase steadily during osteoblast differentiation.…”

Cellular Functions of Tissue Transglutaminase

“…Inhibition of endogenous TG activity in preosteoblast cultures with cystamine resulted in complete abrogation of mineralization, attributable to reduced ECM accumulation and an arrested state of osteoblast differentiation (Al-Jallad et al, 2006); however, recent evidence indicated that FXIIIA rather than TG2 acted as the major regulator of ECM deposition (Al-Jallad et al, 2011). Finally, TG2-induced osteoblast-like transformation of phenotypically plastic cells, such as vascular smooth muscle cells (Faverman et al, 2008), suggested that TG2 may be critical for vascular calcification (Johnson et al, 2008a).…”