MT1-MMP modulates the mechanosensitivity of osteocytes

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Abstract

Membrane-type matrix metalloproteinase-1 (MT1-MMP) is expressed by mechanosensitive osteocytes and affects bone mass. The extracellular domain of MT1-MMP is connected to extracellular matrix, while its intracellular domain is a strong modulator of cell signaling. In theory MT1-MMP could thus transduce mechanical stimuli into a chemical response. We hypothesized that MT1-MMP plays a role in the osteocyte response to mechanical stimuli.

MT1-MMP-positive and knockdown (siRNA) MLO-Y4 osteocytes were mechanically stimulated with a pulsating fluid flow (PFF). Focal adhesions were visualized by paxillin immunostaining. Osteocyte number, number of empty lacunae, and osteocyte morphology were measured in long bones of MT1-MMP+/+ and MT1-MMP−/− mice.

PFF decreased MT1-MMP mRNA and protein expression in MLO-Y4 osteocytes, suggesting that mechanical loading may affect pericellular matrix remodeling by osteocytes. MT1-MMP knockdown enhanced NO production and c-jun and c-fos mRNA expression in response to PFF, concomitantly with an increased number and size of focal adhesions, indicating that MT1-MMP knockdown osteocytes have an increased sensitivity to mechanical loading. Osteocytes in MT1-MMP−/− bone were more elongated and followed the principle loading direction, suggesting that they might sense mechanical loading. This was supported by a lower number of empty lacunae in MT1-MMP−/− bone, as osteocytes lacking mechanical stimuli tend to undergo apoptosis.

In conclusion, mechanical stimulation decreased MT1-MMP expression by MLO-Y4 osteocytes, and MT1-MMP knockdown increased the osteocyte response to mechanical stimulation, demonstrating a novel and unexpected role for MT1-MMP in mechanosensing.

Highlights

► MT1-MMP knockdown increased the osteocyte response to mechanical stimulation. ► MT1-MMP knockdown increased the number and size of focal adhesions in osteocytes. ► MT1-MMP−/− mice had a lower number of empty lacunae and a higher osteocyte density. ► Mechanical stimulation decreased MT1-MMP expression by osteocytes. ► Our results show a novel and unexpected role for MT1-MMP in mechanosensing.

Introduction

Osteocytes play a pivotal role in the adaptive response of bone to mechanical loading, by sensing the mechanical loads and orchestrating the activity of osteoblasts that produce bone and osteoclasts that degrade bone [1], [2]. Osteocytes are stellate shaped cells, and by far the most abundant bone cell type; vastly outnumbering osteoblasts and osteoclasts combined. The osteocyte cell bodies are situated in lacunae within the hard mineralized matrix of bone. From the cell bodies, long slender processes (50–60 per cell) radiate out and connect to the surrounding osteocytes, to the cells lining the bone surface, as well as to the extraosseous space [3], [4]. These cell processes allow rapid communication between osteocytes. The osteocyte cell processes also allow mechanosensing, as mechanical loading of bone results in matrix deformations [5] that drive a flow of interstitial fluid, specifically around the osteocyte cell processes [1]. This flow of fluid can act as a mechanical signal for the osteocytes [6].

Osteocytes express MT1-MMP (or MMP-14), which is a membrane-anchored metalloproteinase that mediates pericellular proteolysis of a wide range of extracellular matrix proteins including type-I collagen [7], [8]. MT1-MMP is also involved in shedding of CD44 from cell surface [9]. CD44 is highly expressed in osteocytes and has a role in adhesion, invasion and/or cell–cell signaling [10]. Therefore MT1-MMP deficiency leads to the loss of formation of osteocyte processes [11], and a reduction in mechanosensitivity of MT1-MMP deficient osteocytes in vivo might be expected.

MT1-MMP may affect the intrinsic sensitivity of osteocytes, as MT1-MMP-deficient myeloid progenitors and mouse lung endothelial cells (MLEC) have larger focal adhesions [12]. Focal adhesions are prime candidate structures for converting extracellular mechanical signals such as fluid shear stress into a biological response of osteocytes [13], [14]. In addition, MT1-MMP is a transmembrane molecule, with its extracellular domain connected to extracellular matrix molecules, while its intracellular domain is a strong modulator of cell signaling and activates molecules such as src and Akt [15]. These two latter molecules are both known to be activated by mechanical stimuli [14]. This puts MT1-MMP in an ideal position to transduce mechanical perturbations occurring in the extracellular environment into an intracellular mechanical response. Therefore, we aimed to investigate whether MT1-MMP plays a role in the osteocyte response to mechanical loading.

To study whether MT1-MMP plays a role in the osteocyte response to mechanical loading we investigated the effect of MT1-MMP deficiency on nitric oxide (NO) production, as well as on cox-2, c-jun, and c-fos mRNA expression by MLO-Y4 osteocytes in response to mechanical loading. We studied NO production as a parameter of osteocyte mechanosensitivity, since NO is an important regulator of the response of bone to mechanical stress [16]. Furthermore, osteocyte number, number of empty lacunae, and osteocyte morphology in long bones of MT1-MMP+/+ and MT1-MMP−/− mice were studied as features of osteocyte mechanosensitivity in vivo.

Section snippets

siRNA mediated ablation of MT1-MMP expression

A pre-designed siRNA oligonucleotide sequence (AGAACUUCGUGUUGCCUGAtt) against murine MT1-MMP and a pre-designed non-silencing (scrambled) siRNA oligonucleotide sequence as a negative control were used for transfection (Applied Biosystems, Foster, CA, USA). Transfection was performed by electroporation using the Microporator Pipetype Electroporation System (Digital Bio, Hopkinton, MA, USA); MLO-Y4 osteocytes (kindly provided by Dr. Bonewald, San Antonio, TX, USA) [17] were resuspended in T-cell

Mechanosensitivity increases in MLO-Y4 osteocytes in the absence of MT1-MMP

To determine the role of MT1-MMP in the mechanosensitivity of osteocytes, MT1-MMP was knocked down in MLO-Y4 osteocytes. Introduction of siRNA directed against MT1-MMP decreased MT1-MMP protein levels by 70% compared to scrambled siRNA-transfected osteocytes (Fig. 1A), demonstrating the efficacy of the siRNA. MT1-MMP knockdown did not alter the stimulating effect of PFF on Cox-2 expression by MLO-Y4 osteocytes (data not shown). PFF increased NO production by 3.5-fold compared to static control

Discussion

In this study we show that, in addition to the widely known role of MT1-MMP as proteinase, MT1-MMP may play a role in cellular mechanosensing. We found that osteocytes in which MT1-MMP was downregulated have an increased mechanosensitivity in response to mechanical loading. Not only NO production was increased, but also gene expression of c-jun and c-fos was upregulated. This indicates that MT1-MMP knockdown increased the response of osteocytes to mechanical stimuli. The increase in osteocyte

Acknowledgments

The work of Rishikesh N. Kulkarni was supported by a Grant from the University of Amsterdam. The Research Institute MOVE of the VU University Amsterdam supported the work of Astrid D. Bakker. The authors thank Kenn Holmbeck (NIDCR, Bethesda, WA) for supplying MT1-MMP+/+ and MT1-MMP−/− mice, and Antonius L.J.J. Bronckers and Behrouz Zandieh Doulabi (ACTA, Amsterdam, NL) for helpful discussions. The authors also thank Marion A. van Duin for excellent technical assistance.

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