MT1-MMP modulates the mechanosensitivity of osteocytes.
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.
- SourceAvailable from: Aviral Vatsa[Show abstract] [Hide abstract]
ABSTRACT: The human skeleton is a miracle of engineering, combining both toughness and light weight. It does so because bones possess cellular mechanisms wherein external mechanical loads are sensed. These mechanical loads are transformed into biological signals, which ultimately direct bone formation and/or bone resorption. Osteocytes, since they are ubiquitous in the mineralized matrix, are the cells that sense mechanical loads and transduce the mechanical signals into a chemical response. The osteocytes then release signaling molecules, which orchestrate the recruitment and activity of osteoblasts or osteoclasts, resulting in the adaptation of bone mass and structure. In this review, we highlight current insights in bone adaptation to external mechanical loading, with an emphasis on how a mechanical load placed on whole bones is translated and amplified into a mechanical signal that is subsequently sensed by the osteocytes. This article is part of a Special Issue entitled Osteocyte.Bone 10/2012; · 4.46 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Zebrafish keratocytes collectively migrate rapidly when established in explant cultures but little is known about the signals that initiate motility or the signal transduction pathways that result in an epithelial to mesenchymal transition. Matrix metalloproteinases (MMPs) are strong candidates for playing a role in this regulation and have previously not been analysed in this wound healing model system. Results presented here document a rapid and dramatic rise in MMP14a, MMP2, MMP9 and MMP13a mRNA levels over time. In a motility assay, a broad-spectrum MMP inhibitor and an inhibitor specific for MMP2 and MMP9 significantly decrease cell migration in a dose dependent manner but treatment with an MMP13 specific inhibitor significantly increases cell sheet area. Immunofluorescence staining with an antibody specific for the catalytic domain of MMP14 indicates that activated MMP14 protein is highly expressed on cells at the leading edge of a sheet compared with follower cells in the centre of the sheet, and is augmented further in leader cells that are stretched, thus likely in the process of detaching from the cell sheet. These data are consistent with a model in which active MMP14 at the leading edge of cell sheets in explant cultures triggers activation of MMP2 and/or MMP9, thus creating promigratory signal(s) that outweigh the inhibitory role of targets cleaved by MMP13. Taken together, these data suggest that MMPs play an important but complex role in regulating the collective cell migration of zebrafish keratocytes and provide support for the relevance of using zebrafish as a model for human disease.Cell Biology International Reports. 12/2013; 20(2).
- [Show abstract] [Hide abstract]
ABSTRACT: Matrix metalloproteinases (MMPs) are extracellular matrix (ECM) degrading enzymes and have complex and specific regulation networks. This includes activation interactions, where one MMP family member activates another. ECM degradation and MMP activation can be initiated by several different stimuli including changes in ECM mechanical properties or intracellular contractility. These mechanical stimuli are known enhancers of metastatic potential. MMP-14 facilitates local ECM degradation and is well known as a major mediator of cell migration, angiogenesis and invasion. Recently, function blocking antibodies have been developed to specifically block MMP-14, providing a useful tool for research as well as therapeutic applications. Here we utilize a selective MMP-14 function blocking antibody to delineate the role of MMP-14 as an activator of other MMPs in response to changes in cellular contractility and ECM stiffness. Inhibition using function blocking antibodies reveals that MMP-14 activates soluble MMPs like MMP-2 and -9 under various mechanical stimuli in the pancreatic cancer cell line, Panc-1. In addition, inhibition of MMP-14 abates Panc-1 cell extension into 3D gels to levels seen with non-specific pan-MMP inhibitors at higher concentrations. This strengthens the case for MMP function blocking antibodies as more potent and specific MMP inhibition therapeutics.Biochemical and Biophysical Research Communications 05/2014; · 2.28 Impact Factor
Mechanical stimulation of
osteocytes and regulation of
The studies described in this thesis were carried out at the section Oral Cell
Biology of the Academic Centre Dentistry Amsterdam (ACTA), University of
Amsterdam and VU University Amsterdam, Research Institute MOVE,
Amsterdam, The Netherlands. The work of R.N. Kulkarni was supported by a
grant from the University of Amsterdam.
Printing of this thesis has been supported by:
Nederlandse Vereniging voor Calcium- en Botstofwisseling (NVCB)
Netherlands Institute for Dental Sciences (IOT)
© Rishikesh Nandkumar Kulkarni, 2012. All rights reserved.
No part of this book may be reproduced, stored in retrievable system, or
transmitted in any form or by any means, mechanical, photo-copying, recording,
or otherwise, without the prior written permission of the holder of copyright.
Printed by Ipskamp drukkers B.V., Enschede, The Netherlands (2012)
Cover design by Vinodhkumar R and Rishikesh Kulkarni
Mechanical stimulation of
osteocytes and regulation of
ter verkrijging van de graad Doctor aan
de Vrije Universiteit Amsterdam,
op gezag van de rector magnificus
prof.dr. L.M. Bouter,
in het openbaar te verdedigen
ten overstaan van de promotiecommissie
van de Faculteit der Tandheelkunde
op maandag 4 juni 2012 om 15.45 uur
in het auditorium van de universiteit,
De Boelelaan 1105
Rishikesh Nandkumar Kulkarni
geboren te Parali, India
promotoren: prof.dr. J. Klein Nulend
prof.dr. V. Everts
copromotor: dr. A.D. Bakker
Dedicated to my mother
Sou. Rajani Kulkarni
Shri. Nandkumar Kulkarni
CHAPTER 1: General Introduction 9
CHAPTER 2: Inhibition of Osteoclastogenesis by Mechanically Loaded 19
Osteocytes: Involvement of MEPE
CHAPTER 3: MT1-MMP Modulates the Mechanosensitivity of Osteocytes 41
CHAPTER 4: Mechanical Loading Prevents the Stimulating Effect of 61
IL-1β on Osteocyte-Modulated Osteoclastogenesis
CHAPTER 5: IL-6 Stimulates Osteocytes to Produce Factors that Increase 81
Osteoblast Differentiation but Not Osteocyte-Mediated
CHAPTER 6: Mechano-response of Osteocytes In Situ in an Ex Vivo 109
Mechanical Loading Model of Murine Fibula
CHAPTER 7: General Discussion 127
GENERAL SUMMARY 139
ALGEMENE SAMENVATTING 143
THANK YOU, DHANYAWAD, DANK JULLIE WEL 148
CURRICULUM VITAE 151
Bone is a living tissue that is remodeled throughout life. This is accomplished by
so-called basic multicellular units (BMUs), consisting of osteoclasts and
osteoblasts acting in a coordinated fashion to resorb existing bone and form
new bone. Remodeling allows bone tissue to adapt its internal structure and
mass to mechanical demands to ensure maximal strength with minimal bone
mass1-4. The osteoclasts start to dig a tunnel through compact bone, and the
osteoblasts refill the tunnel. The alignment of secondary osteons along the
dominant loading direction suggests that remodeling is guided by mechanical
strain, indicating that mechanical adaptation occurs throughout life at each
Osteocytes, the bone mechanosensors, are very sensitive to
mechanical strain applied to intact bone tissue7-11. Computational models have
shown that cells lying at the surface of bone, such as osteoblasts and bone
lining cells, would be less sensitive to changes in the loading pattern than the
osteocytes, embedded within the calcified matrix12,13. Importantly, targeted
ablation of osteocytes in mice disturbs the adaptation of bone to mechanical
loading11. Burger et al.5 has proposed that alignment during remodeling occurs
as a result of different canalicular flow patterns around the tip of the cutting
cone and reversal zone during loading. Low canalicular flow around the tip of
the cutting cone reduces nitric oxide (NO) production by local osteocytes
thereby causing their apoptosis. Studies in growing bone report that osteocyte
apoptosis is associated with osteoclastic resorption14,15. Osteocyte apoptosis
attracts osteoclasts, leading to further excavation of bone in the direction of
loading. At the transition between cutting cone and reversal zone, enhanced
canalicular flow stimulates osteocytes to release NO, which induces osteoclast
retraction from the bone surface. Together a treadmill exists of attaching and
detaching osteoclasts in the tip and the periphery of the cutting cone, and the
digging of a tunnel in the direction of mechanical loading. It has been shown in
cell culture experiments, that osteocytes produce high levels of NO in response
to mechanical loading16-19, and to a localized mechanical loading on the single
osteocyte level20. NO is a short-lived highly reactive free radical involved in
several biological processes, including the regulation of bone metabolism. NO
mediates the inhibition of osteoclast activity after mechanical loading of bone21,
and adaptive bone formation in vivo22. It is largely unknown which signaling
molecules, either dependent or independent of NO, are produced by
mechanically stimulated osteocytes that regulate osteoclastogenesis.
This thesis focuses on the understanding of how mechanically
stimulated osteocytes regulate osteoclastogenesis. The aim was to identify
signaling molecules produced by mechanically stimulated osteocytes that affect
osteoclastogenesis. Since mechanosensation is a vital phenomenon for survival
of osteocytes and eventually in the inhibition of osteoclastogenesis, we also
aimed to identify molecules that affect osteocyte mechanosensitivity.
Osteoclasts are multinucleated cells that arise from haemopoietic cells
of the monocyte/macrophage lineage23,24. Osteoclast formation in vivo is
thought to be induced by direct cell–cell contact of pre-osteoblastic/stromal cells
with monocyte/macrophage osteoclast precursors23,25. Osteoblast lineage cells
secrete key molecules responsible for osteoclast differentiation, i.e.
macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear
factor kappa-B ligand (RANKL). RANKL binds to its receptor, receptor activator
of nuclear factor kappa B (RANK), on the surface of osteoclast precursors to
stimulate these precursors to commit to the osteoclastic phenotype26. Besides
the membrane-bound form, RANKL also exist in a soluble form. RANKL activity
is negatively regulated by osteoprotegerin (OPG), which competes with RANK
as a soluble receptor.
In the absence of mechanical loading, stasis of interstitial fluid occurs,
leading to a lack of fluid shear stress in bone2. Tatsumi et al.11 have shown that
osteocytes produce pro-osteoclastogenic signals in the absence of mechanical
loading leading to stimulation of bone resorption. Recently, it has been reported
that mice lacking RANKL specifically in osteocytes have a severe osteopetrotic
phenotype indicating that osteocytes are the major source of RANKL in bone
remodeling in vivo27. Alternatively, mechanically loaded osteocytes produce
factors that inhibit osteoclastogenesis and osteoclast recruitment, while under
disuse conditions osteocytes decrease the production of osteoclast-inhibiting
One likely candidate produced by mechanically loaded osteocytes to
inhibit osteoclastogenesis is matrix extracellular phosphoglycoprotein (MEPE).
We investigated whether mechanical loading of osteocytes affects osteocyte-
stimulated osteoclastogenesis by involvement of MEPE (Chapter 2).
Osteocytes express membrane-type matrix metalloproteinase-1 (MT1-
MMP)28, which is a membrane-anchored metalloproteinase that mediates
pericellular proteolysis of a wide range of extracellular matrix proteins including
type-I collagen. MT1-MMP is a transmembrane molecule with its extracellular
domain associated with extracellular matrix molecules, while its intracellular
domain is a modulator of cell signalling and activates molecules such as src and
Akt29. MT1-MMP deficiency leads to the loss of formation of osteocyte
processes28, and also affects bone mass. Therefore, a reduction in
mechanosensitivity of MT1-MMP–deficient osteocytes might be expected. We
aimed to investigate whether MT1-MMP plays a role in the osteocyte response
to mechanical loading (Chapter 3).
The upregulation of NO production by osteocytes in response to
mechanical stimulation is inhibited by interleukin-1β (IL-1β)30. Inflammatory
diseases such as rheumatoid arthritis are often accompanied by higher plasma
and synovial fluid levels of IL-1β31,32, and increased osteoclastic bone
resorption33. NO mediates the osteoclast inhibition by mechanically stimulated
osteocytes21. Since IL-1β alters the osteocyte response to mechanical loading,
we investigated the effect of IL-1β on osteocyte-modulated osteoclastogenesis
in the presence or absence of mechanical loading of osteocytes (Chapter 4).
Interleukin-6 (IL-6) levels in serum of rheumatoid arthritis patients are
higher compared to healthy individuals34. It is still under debate whether IL-6 is
an anabolic or catabolic cytokine in bone mass regulation. It is yet unknown
whether IL-6 affects the osteocyte response to mechanical loading. We aimed
to investigate whether IL-6 alters the production of signaling molecules by
mechanically stimulated osteocytes, and whether IL-6 alters the communication
between osteocytes with osteoclasts and osteoblasts (Chapter 5).
Several molecules including NO have been shown to modulate the
activity of osteoclasts and osteoblasts based on in vitro experiments with
osteocytes cultured on flat, stiff substrates coated with collagen type I21,35,36.
However, osteocytes in situ employ a largely different subset of integrins for
anchorage to their extracellular matrix37,38 than osteocytes seeded on collagen
type I in vitro. Moreover, osteocytes on flat substrates need stronger
mechanical stimuli in order to respond with the production of NO than a non-
adherent osteocyte39. It is still an enigma how osteocytes in situ transduce the
minute mechanical stimuli that occur as a result of physical activity into a strong
chemical response. Therefore a three-dimensional (3D) system to study the
response of osteocytes in situ to mechanical loading is needed. We developed
an ex vivo fibular loading model to study the response of osteocytes in situ to
mechanical loading (Chapter 6).
In summary, this thesis aims to address the following scientific questions:
1. Does mechanical loading of osteocytes affect osteocyte-stimulated
osteoclastogenesis by involvement of MEPE (Chapter 2)?
2. Does MT1-MMP play a role in the osteocyte response to mechanical
loading (Chapter 3)?
3. Does IL-1β affect osteocyte-modulated osteoclastogenesis in the presence
or absence of mechanical loading of osteocytes (Chapter 4)?
4. Does IL-6 alter the mechano-response of osteocytes, and the
communication of osteocytes with osteoblasts and/or osteoclasts (Chapter
5. Do osteocytes in situ in an ex vivo mechanical loading model of murine
fibulae respond to mechanical stimulation (Chapter 6)?
1. Burger EH, Klein-Nulend J, Van der Plas A, Nijweide PJ. Function of
osteocytes in bone – Their role in mechanotransduction. J Nutr
2. Burger EH, Klein-Nulend J. Mechanotransduction in bone - role of the
lacunocanalicular network. FASEB J 1999;13:S101–S112.
3. Parfitt AM. Osteonal and hemi-osteonal remodeling: the spatial and
temporal framework for signal traffic in adult human bone. J Cell
Frost HM. Tetracycline-based histological analysis of bone remodeling.
Calcif Tissue Res 1969;3:211–237.
Burger EH, Klein-Nulend J, Smit TH. Strain-derived canalicular fluid
flow regulates osteoclast activity in a remodelling osteon--a proposal. J
Smit TH, Burger EH, Huyghe JM. A case for strain-induced fluid flow as
a regulator of BMU-coupling and osteonal alignment. J Bone Miner Res
7. Dallas SL, Zaman G, Pead MJ, Lanyon LE. Early strain-related
changes in cultured embryonic chick tibiotarsi parallel those associated
with adaptive modeling in vivo. J Bone Miner Res 1993;8:251–259.
8. Lean JM, Jagger CJ, Chambers TJ, Chow JW. Increased insulin-like
growth factor I mRNA expression in rat osteocytes in response to
mechanical stimulation. Am J Physiol 1995;268:E318–E327.
9. Forwood MR, Kelly WL, Worth NF. Localization of prostaglandin
endoperoxidase H synthase (PGHS)-1 and PGHS-2 in bone following
mechanical loading in vivo. Anat Rec 1998;252:580–586.
10. Terai K, Takano-Yamamoto T, Ohba Y, Hiura K, Sugimoto M, Sato
M, Kawahata H, Inaguma N, Kitamura Y, Nomura S. Role of
osteopontin in bone remodeling caused by mechanical stress. J Bone
Miner Res 1999;14:839–849.
11. Tatsumi S, Ishi K, Amizuka N, Li M, Kobayashi T, Kohno K, Ito M,
Takeshita S, Ikeda K. Targeted ablation of osteocytes induces
osteoporosis with defective mechanotransduction.
12. Mullender MG, Huiskes R. Proposal for the regulatory mechanism of
Wolff’s law. J Orthop Res 1995;13:503–512.
13. Weinbaum S, Cowin SC, Zeng Y. A model for the excitation of
osteocytes by mechanical loading-induced bone fluid shear stresses. J
14. Bronckers AL, Goei W, Luo G, Karsenty G, D'Souza RN, Lyaruu
DM, Burger EH (1996) DNA fragmentation during bone formation in
neonatal rodents assessed by transferase-mediated end labeling. J
Bone Miner Res 11:1281-1291.
15. Noble BS, Stevens H, Loveridge N, Reeve J. Identification of apoptotic
changes in osteocytes in normal and pathological human bone. Bone
16. Klein-Nulend J, van der Plas A, Semeins CM, Ajubi NE, Frangos JA,
Nijweide PJ, Burger EH. Sensitivity of osteocytes to biomechanical
stress in vitro. FASEB J 1995;9:441-445.
17. Bacabac RG, Smit TH, Van Loon JJ, Doulabi BZ, Helder M, Klein-
Nulend J. Bone cell responses to high-frequency vibration stress: does
the nucleus oscillate within the cytoplasm? FASEB J 2006;20:858-64.
18. Klein-Nulend J, Semeins CM, Ajubi NE, Nijweide PJ, Burger EH.
Pulsating fluid flow increases nitric oxide (NO) synthesis by osteocytes
but periosteal fibroblasts—correlation with prostaglandin upregulation.
Biochem Biophys Res Commun 1995;217:640-648.
19. Bakker AD, Soejima K, Klein-Nulend J, Burger EH. The production of
nitric oxide and prostaglandin E(2) by primary bone cells is shear stress
dependent. J Biomech 2001;34:671-677.
20. Vatsa A, Mizuno D, Smit TH, Schmidt CF, MacKintosh FC, Klein-
Nulend J. Bio imaging of intracellular nitric oxide production in single
bone cells after mechanical stimulation. J Bone Miner Res
21. Tan SD, de Vries TJ, Kuijpers-Jagtman AM, Semeins CM, Everts V,
Klein-Nulend J. Osteocytes subjected to fluid flow inhibit osteoclast
formation and bone resorption. Bone 2007;41:745-751.
22. Fox SW, Chambers TJ, Chow JW. Nitric oxide is an early mediator of
the increase in bone formation by mechanical stimulation. Am J Physiol
23. Lerner U. New molecules in the tumor necrosis factor ligand and
receptor superfamilies with importance for physiological and
pathological bone resorption. Crit Rev Oral Biol Med 2004;15:64-81.