Metabolic activation stimulates acid secretion and
expression of matrix degrading proteases in human
M George, B Stein, O Mu ¨ller, M Weis-Klemm, T Pap, W J Parak, W K Aicher
Web extra table W1 and
figs W1–W4 can be seen
on the web site at http://
See end of article for
Dr W K Aicher, Research
Laboratories, Centre for
Orthopaedic Surgery, The
University of Tu ¨bingen
Pulvermu ¨hlstrasse 5,
D 72070 Tu ¨bingen,
Accepted 12 June 2003
Ann Rheum Dis 2004;63:67–70. doi: 10.1136/ard.2002.005256
Background: Both cellular and matrix components of healthy bone are permanently renewed in a
balanced homoeostasis. Osteoclastic bone resorption involves the expression of vacuolar-type ATPase
proton pumps (vATPase) on the outer cell membrane and the secretion of matrix degrading proteases.
Osteoblasts modulate the deposition of bone mineral components and secrete extracellular matrix
Objectives: To investigate the ability of osteoblasts and osteosarcoma to secrete acid and express matrix
degrading proteases upon metabolic activation. To examine also the potential contribution of vATPases to
proton secretion expressed on osteoblasts.
Methods: Osteoblasts were isolated from trabecular bone and characterised by reverse transcriptase-
polymerase chain reaction and immunohistochemistry. Proton secretion was analysed by a cytosensor
Results: Osteoblasts not only express matrix degrading proteases upon stimulation with tumour necrosis
factor or with phorbol ester but they also secrete protons upon activation. Proton secretion by osteoblasts is
associated partially with proton pump ATPases.
Conclusion: These data suggest that, in addition to monocyte derived osteoclasts, cytokine activated
mesenchymal osteoblasts and osteosarcoma cells may contribute to the acidic milieu required for bone
metalloproteinases (MMPs).2–4Further, osteoclasts secrete
protons by a vacuolar (H+) ATPase (vATPase).5–7Mutations
of a vATPase subunit cause osteopetrosis8and deletion of a
vATPase subunit results in osteosclerosis.9
Previously the vATPase activity was associated with
osteoclasts only. But recently, vATPases have been described
in a variety of other cell lineages as well.10–13Such vATPases
act in intracellular pH regulation as well as in proton
secretion.13 14This prompted us to study proton secretion by
Parathyroid hormone has been shown to induce proton
secretion by osteoblasts or osteosarcoma cells.15We have
shown that synovial fibroblasts secrete protons upon meta-
bolic activation,16and fibroblasts isolated from the synovial-
like interface membrane can resorb bone without the help of
mesenchymal cells secrete considerable amounts of protons
and thereby contribute to bone degradation under certain
conditions. As osteoblasts are closely related to fibroblasts,18
we investigated osteoblasts as a second proton source in bone
Protons have an important role in collagen degradation of
demineralised bone areas or in cartilage as an acidic milieu is
a prerequisite for optimal enzymatic activity of collagenolytic
enzymes such as cathepsins, and also for solubilisation of
collagen fibres before enzymatic degradation by collage-
nases.19Consequently, enhanced proton secretion contributes
one tissue is constantly in a process of remodelling. For
bone degradation, osteoclasts express matrix degrading
proteases, including different cathepsins1and matrix
to degeneration of articular cartilage in rheumatoid arthritis16
and in other pathological conditions of the musculoskeletal
apparatus as well.8 9Therefore, we investigated mechanisms
of proton secretion in human osteoblasts.
MATERIALS AND METHODS
Preparation of osteoblasts
Osteoblasts were isolated from trabecular bone and expanded
in Dulbecco’s modified Eagle’s medium enriched with 20%
fetal calf serum and antibiotics. Osteosarcoma lines SAOS-2
and MG-63 (ATCC) served as osteoblast controls. The cells
chain reaction (RT-PCR; table W1 (available at http://www.
annrheumdis.com/supplemental)) and immunohistochemistry.
Secretion of MMPs was evaluated by enzyme linked
immunosorbent assay (ELISA).
Cytosensor microphysiometer analysis
Proton secretion was analysed by a cytosensor microphysi-
ometer, and pericellular acidification was measured as
described previously.16In brief, proton release of the cells
after stimulation increases the acidification rate (fig W1
(http://www.annrheumdis.com/supplemental)). For induc-
tion experiments cells were stimulated with tumour necrosis
... ............ ............. ............ ............. ...........
Abbreviations: BMP, bone morphogenic protein; MMP, matrix
metalloproteinase; PMA, phorbol myristate acetate; RT-PCR, reverse
transcriptase-polymerase chain reaction; TNFa, tumour necrosis factor a
factor a (TNF; 1–100 ng/ml) or phorbol myristate acetate
(PMA; 0.1 ng/ml to 10 mg/ml). To block Na+/H+transmem-
brane proton pumps or vacuolar-type H+ATPases (vATPases),
Amiloride (0.5 and 1 mmol/l) or Bafilomycin A1 (1 and
2 mmol/l; Calbiochem, Bad Soden, Germany) were added to
the osteoblasts, and proton secretion was recorded.
Functional characterisation of the osteoblasts
To characterise the cells under study, the expression of
osteocalcin was detected by immunohistochemistry (fig W2A
(http://www.annrheumdis.com/supplemental)), and osteo-
blast associated genes, including alkaline phosphatase, bone
morphogenic protein (BMP)-2, BMP-4, osteocalcin, and
osteopontin, were detected by RT-PCR analysis, confirming
that the cells under investigation were osteoblast-like cells
(fig W2B (http://www.annrheumdis.com/supplemental)).
Proton secretion by osteoblasts
Phorbol ester (PMA) activates protein kinase C and para-
thyroid hormone induced pericellular acidification in SAOS-2
osteosarcoma by a protein kinase C dependent pathway.15
Consequently, we tested osteoblasts and osteosarcoma cells
for proton secretion upon activation with PMA. In early
passage osteoblasts (passage 4–8), PMA induced a dose
dependent acidification, and 35% proton secretion above
equilibrium was reached (fig W3A (http://www.annrheumdis.
com/supplemental)). After long term culture (10–12 pass-
ages) osteoblasts responded with lower sensitivity to PMA
induced proton secretion (fig W3A (http://www.annrheumdis.
showed that proton secretion was activated immediately
after addition of PMA, reaching the plateau as early as
10 minutes after induction (fig 1A). PMA-induced proton
secretion in SAOS-2 osteosarcoma cells confirmed that
mesenchymal cells secrete protons upon metabolic activation,
and a proton secretion above 40% was induced with PMA
(fig W3A (http://www.annrheumdis.com/supplemental)).
TNFa stimulates proton secretion in osteoblasts
We next tested TNFa for its induction of proton secretion by
osteoblasts. Early passage osteoblasts responded to TNFa at
higher concentrations (10–100 ng/ml) with a proton secre-
tion of 20–35% above the equilibrium level. Low concentra-
tions of TNF (1–2 ng/ml) induced an acidification rate of
about 20% above the equilibrium level. The TNF-induced
proton secretion was reduced in late passage osteoblasts
(fig W3B ((http://www.annrheumdis.com/supplemental)).
Further, time course experiments showed that the TNF
response was delayed to some extent (fig 1B) in comparison
with the PMA induced acidification (fig 1A), and the
maximal proton secretion was reached 20 minutes after
induction (fig 1B).
As different mechanisms may contribute to pericellular
acidification, we used Bafilomycin A1, a specific vATPase
blocker (1 mmol/l, 2 mmol/l), and Amiloride, a blocker of Na+/
H+exchange ATPases at a higher concentration (500 mmol/l,
1 mmol/l), to reduce the proton secretion. Both compounds
reduced proton secretion by 5 and 12% or by 10 and 12%,
respectively (fig 2). This indicates that the involvement of
proton pump ATPases of either the vacuolar type (vATPase)
or Na+/H+exchange ATPases works across the extracellular
membrane of osteoblasts, thus contributing to the acidifica-
tion reported here.
Expression of proteolytic enzymes in activated
To test whether activated osteoblasts may contribute to
degradation of extracellular matrix proteins, the expression
of proteolytic enzymes was investigated. In osteoblasts
mRNA encoding MMP-1 was enhanced by addition of TNF
(p(0.17) and PMA (p(0.17) about 25-fold (fig W4A (http://
www.annrheumdis.com/supplemental)). MMP-3 encoding
mRNA was stimulated by TNF (p(0.136) and PMA
(p(0.05) on average fivefold as determined by real time
quantitative RT-PCR (fig W4A). Further, TNF augmented
MMP-1 (p(0.13) and MMP-3 (p(0.02) concentrations in
about 20 minutes in running medium to reach metabolic quiescence characterised by a low spontaneous acidification (r(t)=req, white zone). After
15 minutes of preincubation the fluctuations of the acidification value drop below 5%. Then cells are activated with PMA (grey zone), and the metabolic
responses are recorded as a function of time. Each data point represents the mean value of n individual measurements of xi(n=5–10) and the error
bars represent the normalised standard deviations. Addition of 1 mg/ml PMA induced an immediate increase in acidification, reaching a plateau at
about 115% after 20 minutes. Addition of 5 mg/ml PMA induced a higher response reaching more than 120% acidification. Addition of 10 mg/ml
PMA induced an immediate acidification response and a plateau was not observed after 40 minutes of induction. (B) Osteoblasts were activated with
TNF (grey zone) as described above (see (A)). Addition of 20 ng/ml TNF induced a slow increase in acidification reaching about 115% after
20 minutes. Addition of 50 ng/ml TNF induced a response reaching more than 120% acidification. Addition of 100 ng/ml TNFa at first reduced the
acidification rate below the equilibrium level, then an acidification response was recorded reaching about 125% after 20 minutes
Activation of proton secretion (rmax/reqin per cent) in osteoblasts upon stimulation by PMA or TNF. (A) Osteoblasts were preincubated for
68George, Stein, Mu ¨ller, et al
MMP-1 (p(0.096) as well as MMP-3 secretion (p(0.27) (fig
W4B (http://www.annrheumdis.com/supplemental)). These
results indicate that cells of the osteoblast lineage may
contribute to degradation of the extracellular matrix upon
activation by both proton and protease production.
Previously, we showed that fibroblasts may secrete protons
upon metabolic activation. To study osteoblast associated
proton secretion we activated the cells by addition of PMA, as
this compound has previously been associated with proton
secretion in bone20and with pericellular acidification through
Interestingly, an overall trend towards a lower acidification
response was noted in late passage osteoblasts in comparison
with early passage cells or SAOS-2 in osteosarcoma cells.
However, detection of osteoblast marker genes supports our
suggestion that besides osteoclasts the osteoblasts may
secrete protons upon activation. Addition of TNF was used
as a physiological activator of the osteoblasts. TNF is a
prominent product of monocytic cells and, in addition, TNF
activates osteoclast resorptive activity.22In our experiments,
TNF activated proton secretion in human normal osteoblasts.
Activated osteoblasts express different matrix MMPs.23 24
The activation of proton secretion in the presence of
enhanced MMP activity may result in a catabolic situation.
Our data support these findings as expression of MMP-1 and
MMP-3 were up regulated in osteoblasts. However, owing to
variations in gene induction in the individual samples the
statistical significance was not high. Nevertheless, induction
of MMP-1 and MMP-3 were recorded in all individual
The resorptive capacity of cells in bone is principally
associated with osteoclasts or tumour associated macro-
phages. Proton secretion by individual osteoblast cells is
probably rather small as they lack ruffled membrane areas
equipped with proton pumps. Of note, osteoblasts outnumber
osteoclasts in bone. The lifespan of osteoclasts and their
differentiation capabilities are limited at least in vitro in
comparison with osteoblasts. In addition, osteoclasts are
rather sensitive to apoptosis inducing signals in comparison
with osteoblasts.25Although osteolysis by tumours was
shown to require osteoclasts in vivo,26 27osteoblasts or
osteosarcomas may represent a considerable proton source
in bone under specific conditions.28
physiological bone turnover processes such as growth of
macrophages.11 15 21
bone, wound healing, or load adaptation, osteoclasts repre-
sent the primary cells for mineral and protein matrix
degradation. In chronic inflammatory processes, osteoporosis
of the elderly, cancer, or under other pathological conditions
the slow resorbing but apoptosis resistant osteoblast may
contribute to osteolysis.
The specific contribution of different cellular proton
sources for pericellular acidification by osteoblasts remains
to be investigated. Enhanced glycolysis may account for
pericellular acidification. This may explain in part the
observation that early passage osteoblasts show higher
proton secretion than the same cells at later passages or
SAOS-2 cells. However, as cells are serum starved before
cytosensor microphysiometry, glycolysis is probably consid-
erably reduced under these conditions. This indicates that
additional proton sources are active on osteoblasts. Such
proton sources include Na+/H+ion exchange ATPases or
vATPases.28As we found mRNA encoding the 116 kDa
subunit of the vATPase in osteoblasts (not shown) and a
reduced pericellular acidification upon addition of the
specific proton pump blocker Bafilomycin A1 and Amiloride
to the cells, our data suggest that specific cell membrane
associated ATPases participate in proton extrusion and in
pericellular acidification by osteoblasts or osteosarcoma cells.
Therefore, mesenchymal cells may contribute directly to
tissue degradation using different proton sources, including
vATPase and H+ion exchange ATPase activities and matrix
degrading proteases. To the best of our knowledge, this is the
first report of mechanisms of pericellular acidification by
proton pumps on osteoblasts or osteosarcoma cells.
We thank Professor HE Gaub for experimental advice and providing
the equipment, Angelika Kardinal and Andrea Mast for excellent
technical support, and Professors Giehl, Martini, and Sell for suitable
tissue samples. This project was supported by DFG grants Ai 16/10–1,
Ai 16/14–1, and Pa 698/3–1, by the Schuler Foundation (University of
Tu ¨bingen) and, in part, by institutional funding.
M George, B Stein, W J Parak, Institute for Applied Physics and Centre
for Nanoscience, Ludwig-Maximilians, University Munich, Munich,
O Mu ¨ller, M Weis-Klemm, W K Aicher, Centre for Orthopaedic
Surgery, University of Tu ¨bingen Medical School, Tu ¨bingen, Germany
T Pap, Department of Internal Medicine, University of Magdeburg,
1 Blair HC, Sidonio RF, Friedberg RC, Khan NN, Dong SS. Proteinase
expression during differentiation of human osteoclasts in vitro. J Cell Biochem
2 Beedles KE, Sharpe PT, Wagner EF, Grigoriadis AE. A putative role for c-Fos
in the pathophysiology of Paget’s disease. J Bone Miner Res 1999;14:221–8.
3 Sato T, del Carmen Ovejero M, Hou P, Heegaard AM, Kumegawa M,
Foged NT, et al. Identification of the membrane-type matrix metalloproteinase
MT1-MMP in osteoclasts. J Cell Sci 1997;110(Pt 5):589–96.
4 Sato T, Foged NT, Delaisse JM. The migration of purified osteoclasts through
collagen is inhibited by matrix metalloproteinase inhibitors. J Bone Miner Res
5 Lee BS, Holliday LS, Krits I, Gluck SL. Vacuolar H+-ATPase activity and
expression in mouse bone marrow cultures. J Bone Miner Res
6 Teitelbaum SL, Lam J, Takeshita S, Barker JE, Kanagawa O, Ross FP. Bone
resorption by osteoclasts. Osteoclasts, integrins, and osteoporosis TNF-alpha
induces osteoclastogenesis by direct stimulation of macrophages exposed to
permissive levels of RANK ligand. Science 2000;289:1504–8.
7 Rousselle AV, Heymann D. Osteoclastic acidification pathways during bone
resorption. Bone 2002;30:533–540.
8 Kornak U, Schulz A, Friedrich W, Uhlhaas S, Kremens B, Voit T, et al.
Mutations in the a3 subunit of the vacuolar H(+)-ATPase cause infantile
malignant osteopetrosis. Hum Mol Genet 2000;9:2059–63.
9 Scimeca JC, Franchi A, Trojani C, Parrinello H, Grosgeorge J, Robert C, et al.
The gene encoding the mouse homologue of the human osteoclast-specific
116-kDa V-ATPase subunit bears a deletion in osteosclerotic (oc/oc) mutants.
Bafilomycin A1 or Amiloride. Osteoblast-like cells were incubated in
medium containing 1 mM and 2 mM Bafilomycin A 1 or 500 mM and
10000 mM Amiloride. Pericellular acidification was recorded by
cytosensor microphysiometer. Each data point represents the mean of
two individual measurements and the error bars represent the
normalised standard deviations. Both the vATPase proton pump blocker,
Bafilomycin A1, and the blocker of Na+/H+transmembrane exchange
pathways, Amiloride, reduced proton secretion by 5–12%.
Blocking proton secretion by osteoblasts upon addition of
Metabolic activation in human osteoblasts 69
10 Otsu S, Pap T, Shigeyama Y, Aicher WK, Pap G, Gay RE, et al. Identification Download full-text
of a novel splice variant of an osteoclast-like v-ATPase beta-1 subunit in
activated fibroblasts [abstract]. Arthritis Rheum 2000;43(suppl):S511.
11 Heinemann T, Bulwin GC, Randall J, Schnieders B, Sandhoff K, Volk HD, et al.
Genomic organization of the gene coding for TIRC7, a novel membrane
protein essential for T cell activation. Genomics 1999;57:398–406.
12 Karet F, Finberg K, Nelson R, Nayir A, Mocan H, Sanjad SA, et al. Mutations
in the gene encoding B1 subunit of H+-ATPase cause renal tubular acidosis
with sensorineural deafness. Nat Genet 1999;21:84–90.
13 Smith A, Skaug J, Choate K, Nayir A, Bakkaloglu A, Ozen S, et al. Mutations
in ATP6N1B, encoding a new kidney vacuolar proton pump 116-kD subunit,
cause recessive distal renal tubular acidosis with preserved hearing. Nat
14 Dorup J, Scholz H, Maunsbach AB, Kaissling B. Effects of inhibition of the
vacuolar-type proton pump on nephron ultrastructure and acidification in the
isolated perfused rat kidney. Exp Nephrol 1995;3:180–92.
15 Barrett MG, Belinsky GS, Tashjian AH. A new action of parathyroid hormone.
Receptor-mediated stimulation of extracellular acidification in human
osteoblast-like SaOS-2 cells. J Biol Chem 1997;272:26346–53.
16 Parak WJ, Dannohl S, George M, Schuler MK, Schaumburger J, Gaub HE,
et al. Metabolic activation stimulates acid production in synovial fibroblasts.
J Rheumatol 2000;27:2312–22.
17 Pap T, Claus A, Ohtsu S, Hummel KM, Schwartz P, Drynda S, et al.
Osteoclast-independent bone resorption by fibroblast-like cells. Arthitis Res
18 Ducy P, Schinke T, Karsenty G. The osteoblast: a sophisticated fibroblast under
central surveillance. Science 2000;289:1501–4.
19 Miller EJ. Chemistry of the collagens and their distribution. In: Piez KA,
Reddi AH, eds. Extracellular matrix biochemistry. New York: Elsevier Science,
20 Belinsky GS, Tashjian AH. Direct mesurement of hormone-induced
acidification in intact bone. J Bone Miner Res 2000;15:550–6.
21 Nordstrom T, Grinstein S, Brisseau GF, Manolson MF, Rotstein OD.
Protein kinase C activation accelerates proton extrusion by vacuolar-type
H(+)-ATPases in murine peritoneal macrophages. FEBS Lett
22 Kobayashi K, Takahashi N, Jimi E, Udagawa N, Takami M, Kotake S, et al.
Tumor necrosis factor alpha stimulates osteoclast differentiation by a
mechanism independent of the ODF/RANKL-RANK interaction. J Exp Med
23 Varghese S, Rydziel S, Canalis E. Basic fibroblast growth factor stimulates
collagenase-3 promoter activity in osteoblasts through an activator protein-1-
binding site. Endocrinology 2000;141:2185–91.
24 Rifas L, Fausto A, Scott MJ, Avioli LV, Welgus HG. Expression of
metalloproteinases and tissue inhibitors of metalloproteinases in human
osteoblast-like cells: differentiation is associated with repression of
metalloproteinase biosynthesis. Endocrinology 1994;134:213–21.
25 Hughes DE, Dai A, Tiffee JC, Li HH, Mundy GR, Boyce BF. Estrogen promotes
apoptosis of murine osteoclasts mediated by TGF-beta. Nat Med
26 Clohisy DR, Ogilvie CM, Carpenter RJ, Ramnaraine ML. Localized, tumor-
associated osteolysis involves the recruitment and activation of osteoclasts.
J Orthop Res 1996;14:2–6.
27 Clohisy DR, Ramnaraine ML. Osteoclasts are required for bone tumors to
grow and destroy bone. J Orthop Res 1998;16:660–6.
28 Mobasheri A, Golding S, Pagakis SN, Corkey K, Pocock AE, Fermor B, et al.
Expression of cation exchanger NHE and anion exchanger AE
isoforms in primary human bone-derived osteoblasts. Cell Biol Int
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