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 microphysiometer, 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 induction experiments cells were stimulated with tumour necrosis factor α (TNF; 1–100 ng/ml) or phorbol myristate acetate (PMA; 0.1 ng/ml to 10 μg/ml). To block Na⁺/H⁺ transmembrane proton pumps or vacuolar-type H⁺ ATPases (vATPases), Amiloride (0.5 and 1 mmol/l) or Bafilomycin A1 (1 and 2 µmol/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 osteoblast 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 parathyroid 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 passages) 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)).

TNFα stimulates proton secretion in osteoblasts

We next tested TNFα for its induction of proton secretion by osteoblasts. Early passage osteoblasts responded to TNFα at higher concentrations (10–100 ng/ml) with a proton secretion of 20–35% above the equilibrium level. Low concentrations 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 µmol/l, 2 µmol/l), and Amiloride, a blocker of Na⁺/H⁺ exchange ATPases at a higher concentration (500 µmol/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 acidification 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 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 bone²⁰ 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 macrophages. 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 represent 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 considerably 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.