Metabolic activation stimulates acid secretion and expression of matrix degrading proteases in human osteoblasts

Article (PDF Available)inAnnals of the Rheumatic Diseases 63(1):67-70 · February 2004with21 Reads
DOI: 10.1136/ard.2002.005256 · Source: PubMed
Abstract
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 proteins. 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. Osteoblasts were isolated from trabecular bone and characterised by reverse transcriptase-polymerase chain reaction and immunohistochemistry. Proton secretion was analysed by a cytosensor microphysiometer. 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. 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 degradation.

Figures

EXTENDED REPORT
Metabolic activation stimulates acid secretion and
expression of matrix degrading proteases in human
osteoblasts
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://
www.annrheumdis.com/
supplemental
See end of article for
authors’ affiliations
.......................
Correspondence to:
Dr W K Aicher, Research
Laboratories, Centre for
Orthopaedic Surgery, The
University of Tu¨bingen
Medical School,
Pulvermu¨hlstrasse 5,
D 72070 Tu¨bingen,
Germany;
aicher@uni-tuebingen.de
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
proteins.
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
microphysiometer.
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
degradation.
B
one tissue is constantly in a process of remodelling. For
bone degradation, osteoclasts express matrix degrading
proteases, including different cathepsins
1
and matrix
metalloproteinases (MMPs).
2–4
Further, osteoclasts secrete
protons by a vacuolar (H+) ATPase (vATPase).
5–7
Mutations
of a vATPase subunit cause osteopetrosis
8
and 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.
10–13
Such vATPases
act in intracellular pH regulation as well as in proton
secretion.
13 14
This prompted us to study proton secretion by
osteoblasts.
Parathyroid hormone has been shown to induce proton
secretion by osteoblasts or osteosarcoma cells.
15
We have
shown that synovial fibroblasts secrete protons upon meta-
bolic activation,
16
and fibroblasts isolated from the synovial-
like interface membrane can resorb bone without the help of
osteoclasts.
10 17
These results suggested that activated
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
besides osteoclasts.
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.
19
Consequently, enhanced proton secretion contributes
to degeneration of articular cartilage in rheumatoid arthritis
16
and in other pathological conditions of the musculoskeletal
apparatus as well.
89
Therefore, 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
were characterised by reverse transcriptase-polymerase
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.
16
In 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
67
www.annrheumdis.com
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.
RESULTS
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.
com/supplemental)). Further, time course experiments
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 m mol/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
osteoblasts
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
osteoblast supernatants. Comparably, PMA stimulated
Figure 1 Activation of proton secretion (r
max
/r
eq
in per cent) in osteoblasts upon stimulation by PMA or TNF. (A) Osteoblasts were preincubated for
about 20 minutes in running medium to reach metabolic quiescence characterised by a low spontaneous acidification (r(t) = r
eq
, 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 x
i
(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
68 George,Stein,Mu¨ller, et al
www.annrheumdis.com
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.
DISCUSSION
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 bone
20
and with pericellular acidification through
osteoblasts, T cells, or peritoneal macrophages.
11 15 21
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.
22
In 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
experiments.
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.
25
Although osteolysis by tumours was
shown to require osteoclasts in vivo,
26 27
osteoblasts or
osteosarcomas may represent a considerable proton source
in bone under specific conditions.
28
Consequently, for
physiological bone turnover processes such as growth of
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.
28
As 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.
ACKNOWLEDGEMENTS
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.
Authors’ affiliations
.....................
M George, B Stein, W J Parak, Institute for Applied Physics and Centre
for Nanoscience, Ludwig-Maximilians, University Munich, Munich,
Germany
OMu¨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,
Magdeburg, Germany
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    • "Thus, the increased serum osteoprotegerin levels in humans may reflect a compensatory response against enhanced osteoclastic bone resorption and the resultant loss of bone. In this respect, our data suggest that osteoblast-like cells may also participate in organic matrix degradation during remodeling cycle by producing collagenolytic cathepsin K. Osteoblasts, although their number may be reduced in osteoporotic patients, cover trabeculae and are able to locally decrease extracellular pH [37], a requirement for optimal cathepsin K activity for collagen degradation. It must also be noted that the slight but detectable progressive metabolic acidosis during ageing [38,39] may contribute to bone surface demineralization seen in patients with osteoporosis [34]. "
    [Show abstract] [Hide abstract] ABSTRACT: Healthy bone is a rigid yet living tissue that undergoes continuous remodeling. Osteoclasts resorb bone in the remodeling cycle. They secrete H(+)-ions and proteinases to dissolve bone mineral and degrade organic bone matrix, respectively. One of the main collagenolytic proteinase in osteoclasts is cathepsin K, a member of papain family cysteine proteinases. Recently, it has been shown that osteoblasts may contribute to organic matrix remodeling. We therefore investigated their ability to produce cathepsin K for this action. Trabecular bone samples were collected from patients operated due to a fracture of the femoral neck. Part of the bone was decalcified and the rest was used for cell isolation. Sections from the decalcified bone were immunostained with antibodies against cathepsin K. Isolated cells were characterized for their ability to form mineralized matrix and subsequently analyzed for their cathepsin K production by Western blotting and quantitative RT-PCR. Osteoblasts, bone lining cells and some osteocytes in situ showed cathepsin K immunoreactivity and osteoblast-like cells in vitro produced cathepsin K mRNA and released both 42 kDa pro- and 27 kDa processed cathepsin K to culture media. Osteoblastic cathepsin K may thus contribute to collagenous matrix maintenance and recycling of improperly processed collagen I. Whether osteoblastic cathepsin K synthesis has consequences in diseases characterized by abnormal bone matrix turnover remains to be investigated.
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    • "Conversely, when the ECF calcium is zero, hence the ECF is at a lower concentration than the BECF (0.5 mM/L), the pump –leak system results in a net calcium efflux from bone along the new outward concentration gradient that requires substantial energy expenditure to be maintained. It is speculated that cells are likely to maintain the steady calcium efflux out of bone at calcium-free ECF through two putative cellular mechanisms: (a) the first assumes a return flow of calcium into the BECF at the intact BECF/ECF interface that closes the calcium loop; this might be driven by either the electrochemical gradient (inside negative) generated by the cells [52] or by the unidirectional and vectorial transcellular calcium transport from the plasma side towards the bone mineral facing side [5,20,53,54]; provided the cells are viable, the loop can be maintained and the flux can be measured; although reliable, this pathway would not play a role in plasma calcium homeostasis; (b) the second assumes that an activated proton secretion, associated in part with H- ATPase, might hydrolyze metastable crystals and generate a continuous loss of calcium from the mineral-facing side [55], thus having a putative fast impact on plasma calcium homeostasis [1]. The amount of calcium released maintains a concentration gradient that is detectable by the SIET at the damage site (point sink) that disappears as the cells die. "
    [Show abstract] [Hide abstract] ABSTRACT: The current study tests the hypothesis that basal level and minute-by-minute correction of plasma Ca2+ by outward and inward Ca2+ fluxes from and into an exchangeable ionic pool in bone is controlled by an active partition system without contributions from the bone remodeling system. Direct real-time measurements of Ca2+ fluxes were made using the scanning ion-selective electrode technique (SIET) on living bones maintained ex vivo in physiological conditions. SIET three-dimensional measurements of the local Ca2+ concentration gradient (10 microm spatial resolution) were performed on metatarsal bones of weanling mice after drilling a 100-mum hole through the cortex to expose the internal bone extracellular fluid (BECF) to the bathing solution, whose composition mimicked the extracellular fluid (ECF). Influxes of Ca2+ towards the center of the cortical hole (15.1+/-4.2 pmol cm-2 s-1) were found in the ECF and were reversed to effluxes (7.4+/-2.9 pmol cm-2 s-1) when calcium was depleted from the ECF, mimicking a plasma demand. The reversal from influx to efflux and vice versa was immediate and fluxes in both directions were steady throughout the experimental time (>or=2 h, n=14). Only the efflux was nullified within 10 min by the addition of 10 mM/L Na-Cyanide (n=7), demonstrating its cell dependence. The timeframes of the exchanges and the stability of the Ca2+ fluxes over time suggest the existence of an exchangeable calcium pool in bone. The calcium efflux dependency on viable cells suggests that an active partition system might play a central role in the short-term error correction of plasma calcium without the contribution of bone remodeling.
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