Fas/CD95 is associated with glucocorticoid-induced osteocyte apoptosis.

Prolonged use of glucocorticoids is associated with decreased bone formation, increased resorption and osteonecrosis, through direct and indirect effects on the activity and viability of bone effector cells, osteoblasts and osteoclasts, and osteocytes. This study has investigated molecular pathways implicated in Dexamethasone-induced apoptosis of osteocytes, using a cell line and primary chicken cells. MLO-Y4 osteocytes were pre-treated with several bisphosphonates representing a range of anti-resorptive activities and conformation/structure relationships, and were subsequently challenged with Dexamethasone. Apoptotic cells were detected at various times after treatment using morphological and biochemical criteria. Dex was shown to induce apoptosis associated with the Fas/CD95 death receptor and in a caspase 8 dependent manner. The apoptotic response was inhibited by all variants of the BP molecules, including those with reduced anti-resorptive activity, indicating that Dex-induced apoptosis is independent of anti-osteoclastic activity. Dex-induced apoptosis was associated with a transient increase in phosphorylated ERK 1/2 and was blocked by the ERK inhibitor UO126. In addition, both UO126 and BPs decreased localization of Fas to the cell membrane. ERK activation by PMA did not induce death or Fas upregulation, suggesting that Fas may be important for the induction of apoptosis and the existence of an additional factor activated by Dex which enables the cooperation between the Dex-activated ERK and Fas pathways, during apoptosis of osteocytes. Furthermore, upregulation of death and Fas was not accompanied by upregulation of FasL, pointing to the possible existence of FasL-independent Fas-associated death in these cells.

Expression of serotonin receptors in bone.

The 5-hydroxytryptamine (5-HT) receptors 5-HT(2A), 5-HT(2B), and 5-HT(2C) belong to a subfamily of serotonin receptors. Amino acid and mRNA sequences of these receptors have been published for several species including man. The 5-HT(2) receptors have been reported to act on nervous, muscle, and endothelial tissues. Here we report the presence of 5-HT(2B) receptor in fetal chicken bone cells. 5-HT(2B) receptor mRNA expression was demonstrated in osteocytes, osteoblasts, and periosteal fibroblasts, a population containing osteoblast precursor cells. Pharmacological studies using several agonists and antagonists showed that occupancy of the 5-HT(2B) receptor stimulates the proliferation of periosteal fibroblasts. Activity of the 5-HT(2A) receptor could however not be excluded. mRNA for both receptors was shown to be equally present in adult mouse osteoblasts. Osteocytes, which showed the highest expression of 5-HT(2B) receptor mRNA in chicken, and to a lesser extent osteoblasts, are considered to be mechanosensor cells involved in the adaptation of bone to its mechanical usage. Nitric oxide is one of the signaling molecules that is released upon mechanical stimulation of osteocytes and osteoblasts. The serotonin analog alpha-methyl-5-HT, which preferentially binds to 5-HT(2) receptors, decreased nitric oxide release by mechanically stimulated mouse osteoblasts. These results demonstrate that serotonin is involved in bone metabolism and its mechanoregulation.

Differential stimulation of prostaglandin G/H synthase-2 in osteocytes and other osteogenic cells by pulsating fluid flow.

Mechanical stress produces flow of fluid in the osteocytic lacunar-canalicular network, which is likely the physiological signal for the adaptive response of bone. We compared the induction of prostaglandin G/H synthase-2 (PGHS-2) by pulsating fluid flow (PFF) and serum in osteocytes, osteoblasts, and periosteal fibroblasts, isolated from 18-day-old fetal chicken calvariae. A serum-deprived mixed population of primarily osteocytes and osteoblasts responded to serum with a two- to threefold induction of PGHS-2 mRNA. Serum stimulated PGHS-2-derived PGE(2) release from osteoblasts and osteocytes but not from periosteal fibroblasts as NS-398, a PGHS-2 blocker, inhibited PGE(2) release from osteocytes and osteoblasts with 65%, but not that from periosteal fibroblasts. On the other hand PFF (0.7 Pa, 5 Hz) stimulated (3 fold) PGHS-2 mRNA only in OCY. The related PGE(2) response could be completely inhibited by NS-398. We conclude that osteocytes have a higher intrinsic sensitivity for loading-derived fluid flow than osteoblasts or periosteal fibroblasts.

A frame-shift mutation in the type I parathyroid hormone (PTH)/PTH-related peptide receptor causing Blomstrand lethal osteochondrodysplasia.

Blomstrand osteochondrodysplasia (BOCD) is a rare lethal skeletal dysplasia characterized by accelerated endochondral and intramembranous ossification. Comparison of the characteristics of BOCD with type I PTH/PTH-related peptide (PTHrP) receptor-ablated mice reveals striking similarities that are most prominent in the growth plate. In both cases, the growth plate is reduced in size due to a strongly diminished zone of resting cartilage and the near absence of columnar arrangement of proliferating chondrocytes. This overall similarity suggested that an inactivating mutation of the PTH/PTHrP receptor might be the underlying genetic defect causing BOCD. Indeed, inactivating mutations of the PTH/PTHrP receptor have been recently identified in two cases of BOCD. We describe here a novel inactivating mutation in the PTH/PTHrP receptor. Sequence analysis of all coding exons of the type I PTH/ PTHrP receptor gene and complementary DNA of a case with BOCD identified a homozygous point mutation in exon EL2 in which one nucleotide (G at position 1122) was absent. The mutation was inherited from both parents, supporting the autosomal recessive nature of the disease. The missense mutation resulted in a shift in the open reading frame, leading to a truncated protein that completely diverged from the wild-type sequence after amino acid 364. The mutant receptor, therefore, lacked transmembrane domains 5, 6, and 7; the connecting intra- and extracellular loops; and the cytoplasmic tail. Functional analysis of the mutant receptor in COS-7 cells and of dermal fibroblasts obtained from the case proved that the mutation was indeed inactivating. Neither the transiently transfected COS-7 cells nor the dermal fibroblasts responded to a challenge with PTH or PTHrP with a rise in intracellular cAMP levels, in sharp contrast to control cells. Our results provide further evidence that BOCD is caused by inactivating mutations of the type I PTH/PTHrP receptor and underscore the importance of this receptor in mammalian skeletal development.

Signal transduction pathways involved in fluid flow-induced PGE2 production by cultured osteocytes.

To maintain its structural competence, the skeleton adapts to changes in its mechanical environment. Osteocytes are generally considered the bone mechanosensory cells that translate mechanical signals into biochemical, bone metabolism-regulating stimuli necessary for the adaptive process. Prostaglandins are an important part of this mechanobiochemical signaling. We investigated the signal transduction pathways in osteocytes through which mechanical stress generates an acute release of prostaglandin E2 (PGE2). Isolated chicken osteocytes were subjected to 10 min of pulsating fluid flow (PFF; 0.7 +/- 0.03 Pa at 5 Hz), and PGE2 release was measured. Blockers of Ca2+ entry into the cell or Ca2+ release from internal stores markedly inhibited the PFF-induced PGE2 release, as did disruption of the actin cytoskeleton by cytochalasin B. Specific inhibitors of Ca2+-activated phospholipase C, protein kinase C, and phospholipase A2 also decreased PFF-induced PGE2 release. These results are consistent with the hypothesis that PFF raises intracellular Ca2+ by an enhanced entry through mechanosensitive ion channels in combination with Ca2+- and inositol trisphosphate (the product of phospholipase C)-induced Ca2+ release from intracellular stores. Ca2+ and protein kinase C then stimulate phospholipase A2 activity, arachidonic acid production, and ultimately PGE2 release.

The role of osteoblast density and endogenous interleukin-6 production in osteoclast formation from the hemopoietic stem cell line FDCP-MIX C2GM in coculture with primary osteoblasts.

Osteoclast formation from the hemopoietic stem cell line FDCP-mix C2GM was shown to be strongly dependent on osteoblast density. In cocultures of C2GM cells with fetal mouse osteoblasts seeded at high density (i.e., 2.5 x 10(4) cells/cm2), we found a significantly lower osteoclast formation compared with cocultures with osteoblasts seeded at low density (i.e., 1 x 10(4) cells/cm2). The differentiation state of osteoblasts in high-density cultures resembled more than that of osteoblasts in low-density cultures, the differentiation state of mature osteoblasts, since the cells in the former cultures showed higher alkaline phosphatase (APase) activity than the cells in the latter cultures, and nodules were formed in high-density cultures but not in low-density cultures. Endogenous interleukin-6 (IL-6) production was found to be significantly lower in high-density cultures, which may partly explain the impaired osteoclast formation in high-density cocultures. Addition of IL-6 to the high-density cocultures indeed restored osteoclast formation. There appeared to be no overt difference in IL-6 receptor mRNA expression between high-density and low-density cultures. In conclusion, this paper suggests that mature, highly differentiated osteoblasts are not directly involved in osteoclastogenesis. In contrast, osteoblast-like cells lacking mature osteoblast markers induce osteoclast formation. Whether these low-density osteoblast-like cells represent an immature differentiation state or the lining cell phenotype is unclear.

Stem cell factor stimulates chicken osteoclast activity in vitro.

Stem cell factor (SCF) is a polypeptide growth factor active on multiple cell types, mainly of hematopoietic origin. We studied the effects of avian SCF on the differentiation of chicken osteoclasts from their putative progenitors as well as on the bone-resorbing activity of terminally differentiated osteoclasts. Osteoclast formation was analyzed in long-term cocultures of osteoblasts and nonadherent, osteoclast-depleted bone marrow cells. Osteoclast activity was studied in short-term (48 h) cultures of bone marrow cell populations enriched for osteoclasts, on dentine slices. SCF strongly enhanced osteoclast differentiation. The IL-6-related chicken myelomonocytic growth factor (cMGF) had a similar effect, and the effects of SCF and cMGF were additive. SCF, but not cMGF, also stimulated the bone-resorbing activity of existing osteoclasts. As osteoblasts have been found to regulate osteoclast activity and formation, chicken osteoblasts were tested for their ability to express and secrete SCF. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that osteoblasts express SCF mRNA and that parathyroid hormone increases expression levels about fourfold. SCF did not accumulate in the culture medium, but remained cell (osteoblasts) surface associated.

Immunocytochemical demonstration of extracellular matrix proteins in isolated osteocytes.

Cultures of isolated osteocytes may offer an appropriate system to study osteocyte function, since isolated osteocytes in culture behave very much like osteocytes in vivo. In this paper we studied the capacity of osteocytes to change their surrounding extracellular matrix by production of matrix proteins. With an immunocytochemical method we determined the presence of collagen type I, fibronectin, osteocalcin, osteopontin and osteonectin in cultures of isolated chicken osteocytes, osteoblasts and periosteal fibroblasts. In osteoblast and periosteal fibroblast cultures, large extracellular networks of collagen type I and fibronectin were formed, but in osteocyte populations, extracellular threads of collagen or fibronectin were only rarely found. The percentage of cells positive for osteocalcin, osteonectin and osteopontin in the Golgi apparatus, on the other hand, was highest in the osteocyte population. These results show that osteocytes have the ability to alter the composition of their surrounding extracellular matrix by producing matrix proteins. We suggest this property is of importance for the regulation of the calcification of the bone matrix immediately surrounding the cells. More importantly, as osteocytes depend for their role as mechanosensor cells on their interaction with matrix proteins, the adaptation of the surrounding matrix offers a way to regulate their response to mechanical loading.

Pulsating fluid flow increases prostaglandin production by cultured chicken osteocytes--a cytoskeleton-dependent process.

It has been postulated that the transduction of mechanical stress signals to bone cells occurs via loading-dependent flow of interstitial fluid through the lacuno-canalicular network of bone. We have shown earlier that chicken osteocytes release enhanced amounts of prostaglandin E2 after 1 h treatment with pulsating fluid flow (PFF, 0.5 +/- 0.02 Pa, 5 Hz). Here we study the acute response to PFF on three cell populations derived from fetal chick calvariae, namely periosteal fibroblasts (PF), an osteoblast and osteocyte containing population (OBmix), and osteocytes (OCY), and the involvement of the actin-cytoskeleton in this process. All three cell populations rapidly (OCY: within 5 min, OBmix, PF: within 10 min) increased their release of prostaglandins E2 and I2 in response to PFF, but the response by OCY was 2-4 times higher than that by OBmix or PF. Disruption of the actin-cytoskeleton by cytochalasin B completely abolished the response. We conclude that osteocytes are more sensitive to fluid shear stress than immature bone cells, and that the actin-cytoskeleton is involved in the response to fluid flow.

Adhesive properties of isolated chick osteocytes in vitro.

Different functions have been proposed for osteocytes over time, but it is now generally accepted that their most important task lies in the sensing of strain caused by mechanical loading on bone. The fact that mechanical strain can be sensed as deformation of the extracellular matrix or as fluid shear stress along the cell, in the space between cell membrane and extracellular matrix, requires that osteocytes have close (specialized) contact with the bone matrix. We studied to which extracellular matrix proteins isolated chicken osteocytes adhere and whether this adhesion is mediated by specific cell adhesion receptors called integrins. The adhesive properties of the osteocytes were compared with that of osteoblasts. Osteocytes (and osteoblasts) adhere to the same substrates (i.e., collagen types I and II, collagen fibers, osteopontin, osteonectin, fibronectin, fibrinogen, thrombospondin, and laminin). Cell spreading varied between substrates, from all cells rounded on thrombospondin to all cells fully spread out on osteopontin, osteonectin, vitronectin, fibronectin, fibrinogen, and laminin. The percentage of osteocytes adhered was equivalent to that of osteoblasts adhered on all substrates except osteopontin and vitronectin, where osteocytes adhered less. The adhesion of osteocytes and osteoblasts to osteopontin, osteonectin, vitronectin, and fibrinogen was strongly inhibited, and to fibronectin and laminin moderately, by an RGD peptide. No RGD inhibition was found on collagen. An antibody against chicken integrin alpha v beta 3, the monoclonal antibody (MAb) 23C6, did not interfere with the adhesion of osteocytes and osteoblasts to matrix proteins, whereas an MAb against chicken integrin subunit beta 1 (CSAT) strongly inhibited adhesion to all substrates. Labeling with osteocyte-specific MAbs (OB7.3, OB37.4, and OB37.11) also did not hinder the adhesion of osteocytes to collagen type I, vitronectin, and osteopontin. Adhesion sites on osteocytes were small compared with the large adhesion plaques of osteoblasts, as demonstrated by interference reflection microscopy and immunocytochemically by staining for vinculin. Osteocyte adhesion is analogous to osteoblast adhesion with regard to the range of extracellular matrix proteins to which they adhere. The adhesion is mediated by the integrin subunit beta 1, but other integrins or nonintegrin adhesion receptors are also involved. Osteocytes make contact with the extracellular matrix via small attachment points which colocalize with vinculin. This connection between the bone matrix and the cytoskeleton may be important for osteocytic sensing of mechanical strain, as it supplies a transduction route of extracellular (mechanical) signals into intracellular messages.

Pulsating fluid flow increases nitric oxide (NO) synthesis by osteocytes but not periosteal fibroblasts--correlation with prostaglandin upregulation.

Osteocytes are extremely sensitive to fluid shear stress, a phenomenon that may be related to mechanical adaptation of bone (FASEB J 9:441,1995). Here we examined the effect of pulsating fluid flow (PFF, 0.5 +/- 0.02 Pa, 5 Hz, 0.4 Pa/sec) on the release of NO, in relation with upregulation of prostaglandin E2 (PGE2). Chicken calvarial osteocytes, but not periosteal fibroblasts, as well as mouse calvarial cells responded to PFF with a rapid and transient 2 to 3-fold stimulation of NO release. The effect was maximal after 5 min and leveled off thereafter. PFF also stimulated PGE2 release. This effect was significant after 10 min and continued throughout 60 min PFF treatment. Inhibition of NO release by NG-monomethyl-L-arginine prevented the effect of PFF on NO as well as PGE2 release. These results suggest that NO is a mediator of mechanical effects in bone, leading to enhanced PGE2 release. They further strengthen the hypothesis that fluid flow through the osteocyte canalicular network provides the physical stimulus for mechanosensation in bone.

Tetrodotoxin-sensitive fast Na+ current in embryonic chicken osteoclasts.

A voltage-dependent, fast, transient inward current was characterized in embryonic chicken osteoclasts using the permeabilized patch configuration of the patch-clamp technique. The current was activated by depolarizations to higher than -28 +/- 4 mV from a holding potential of -80 mV. It peaked within 1-1.5 ms, and inactivated within 3.3-6.9 ms. The 50% inactivation voltage was -59 +/- 6 mV with a steepness factor of 0.11 +/- 0.06. The current disappeared with the removal of extracellular Na+ and was reversibly blocked by tetrodotoxin (K0.5 < 15 nM) but not by verapamil (< or = 100 microM). We conclude that this new current in embryonic chicken osteoclasts is a sodium current known from excitable cells.

Function of osteocytes in bone--their role in mechanotransduction.

Although osteocytes are by far the most abundant cell type of bone, they are least understood in terms of function and regulation. Previous studies have concentrated on their possible role as mobilizers of bone calcium, via the process of osteocytic osteolysis. Currently, however, their possible involvement in mechanical adaptation, the process whereby bone tissue maintains maximal functional strength with minimal bone mass, is discussed. We have recently obtained experimental evidence that osteocytes are the mechanosensory cells of bone, involved in the transduction of mechanical loads into biochemical signals. Our results support the hypothesis that flow of fluid through the lacunar-canalicular system as a result of loading provides the physical signal that activates the cells.

Induction of osteoclast characteristics in cultured avian blood monocytes; modulation by osteoblasts and 1,25-(OH)2 vitamin D3.

It has been established, that the osteoclast is derived from the haemopoietic stem cell, but its exact lineage is still controversial. It is sometimes suggested, that osteoclasts and monocytes/macrophages are related cells. It has also been suggested that osteoclast differentiation is regulated by osteoblasts and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). In the present paper we addressed the question whether avian monocytes can differentiate into osteoclasts in vitro, using an array of immunocytochemical, enzyme cytochemical and function markers. We have also determined the effects of osteoblasts, osteoblast conditioned medium and 1,25-(OH)2D3 on the expression of osteoclastic features on monocytes during culture. Monocytes developed tartrate resistant acid phosphatase (TRAcP) enzyme activity and antigens for all anti-osteoclast antibodies tested, during culture. However, they did not acquire the ability to resorb dentine and still showed phagocytosis of latex spheres. This indicates that the monocytes developed into cells resembling osteoclasts but lacking their function while retaining the function of macrophages. Osteoblast conditioned medium stimulated TRAcP enzyme activity and proliferation of monocytes in cultures. Addition of osteoblasts or osteoblast conditioned medium to monocyte cultures on dentine in the presence or absence of 1,25-(OH)2D3 did not result in the generation of genuine osteoclasts, nor in pit formation. 1,25-(OH)2D3 appeared to be cytotoxic to the avian monocytes in concentrations considered optimal for mouse osteoclast formation. These results suggest that avian monocytes do not readily differentiate into osteoclasts under in vitro conditions that stimulate osteoclast differentiation from bone marrow derived haemopoietic cells. Furthermore, labelling with anti-osteoclast antibodies and TRAcP as osteoclast-markers should be used only with great caution in the identification of osteoclasts formed in vitro.

Extracellular adenosine triphosphate. A shock to hemopoietic cells.

Many or perhaps all cell types in the body possess extracellular binding sites for nucleosides and nucleotides, the purinoceptors. The binding sites that favor adenosine triphosphate (ATP) have been classified as P2-purinoceptors. One subclass of the P2-purinoceptors is the P2z-purinoceptor that mediates the permeabilizing effect of ATP4- (the fully ionized form of adenosine triphosphate). In the presence of millimolar concentrations of ATP4-, this receptor, which was found on all cells of hemopoietic origin but not on cells of stromal origin, renders the sensitive cells permeable for molecules up to 1 kD. This property has been used to eliminate cells of hemopoietic origin from mixed populations. Skeletal- and blood-forming tissues have a complex cellular composition of predominantly stromal and hemopoietic cells. The 2 cell types influence each others' formation, differentiation, and activities in a largely unclarified manner. Rigorous separation would help the study of the properties and potencies of the 2 cell types and their mutual interactions. A short treatment of cell populations isolated from bone, bone marrow, or cartilage with 2 mM adenosine triphosphate and 1 mM of cytotoxic, but not permeant, potassium thiocyanate obliterates all cells of hemopoietic origin, including macrophages, osteoclasts, and their progenitors from these populations.

Sensitivity of osteocytes to biomechanical stress in vitro.

It has been known for more than a century that bone tissue adapts to functional stress by changes in structure and mass. However, the mechanism by which stress is translated into cellular activities of bone formation and resorption is unknown. We studied the response of isolated osteocytes derived from embryonic chicken calvariae to intermittent hydrostatic compression as well as pulsating fluid flow, and compared their response to osteoblasts and periosteal fibroblasts. Osteocytes, but not osteoblasts or periosteal fibroblasts, reacted to 1 h pulsating fluid flow with a sustained release of prostaglandin E2. Intermittent hydrostatic compression stimulated prostaglandin production to a lesser extent: after 6 and 24 h in osteocytes and after 6 h in osteoblasts. These data provide evidence that osteocytes are the most mechanosensitive cells in bone involved in the transduction of mechanical stress into a biological response. The results support the hypothesis that stress on bone causes fluid flow in the lacunar-canalicular system, which stimulates the osteocytes to produce factors that regulate bone metabolism.

Cell membrane stretch in osteoclasts triggers a self-reinforcing Ca2+ entry pathway.

Many cell types respond to mechanical membrane perturbation with intracellular Ca2+ responses. Stretch-activated (SA) ion channels may be involved in such responses. We studied the occurrence as well as the underlying mechanisms of cell membrane stretch-evoked responses in fetal chicken osteoclasts using separate and simultaneous patch-clamp and Ca2+ imaging measurements. In the present paper, evidence is presented showing that such responses involve a self-reinforcing mechanism including SA channel activity, Ca(2+)-activated K+ (KCa) channel activity, membrane potential changes and local and general intracellular Ca2+ ([Ca2+]i) increases. The model we propose is that during membrane stretch, both SA channels and KCa channels open at membrane potential values near the resting membrane potential. SA channel characterization showed that these SA channels are permeable to Ca2+. During membrane stretch, Ca2+ influx through SA channels and hyperpolarization due to KCa channel activity serve as positive feedback, leading ultimately to a Ca2+ wave and cell membrane hyperpolarization. This self-reinforcing mechanism is turned off upon SA channel closure after cessation of membrane stretch. We suggest that this Ca2+ entry mechanism plays a role in regulation of osteoclast activity.

Effect of therapeutic ultrasound on endochondral ossification.

The effect of therapeutic doses of ultrasound was tested on endochondral ossification of in vitro developing metatarsal long bone rudiments of 16- and 17-day-old fetal mice. Bone growth, calcification and resorption following exposure to several doses of pulse-wave (PW) or continuous-wave (CW) ultrasound were examined. PW was applied at intensities between 0.1 W cm-2 and 0.77 W cm-2 (Isatp) and CW intensities were 0.1 W cm-2 or 0.5 W cm-2 (Isata). After 1 week of culture, the metatarsal long bone rudiments were fixed and paraffin sections were prepared for histological evaluation and for measurement of the relative contribution of the various cartilage zones to the total bone length. In contrast to treatment with CW ultrasound, treatment of 16-day-old metatarsal long bone rudiments with PW ultrasound resulted after 4 days of culture in significantly increased longitudinal growth. Histology revealed a significant increased length of the proliferative zone, whereas the length of the hypertrophic cartilage zone was unaltered. This might indicate that proliferation of the cartilage cells is stimulated without influence on cell differentiation.

Time-lapse microcinematography of osteocytes.

We succeeded in the isolation of osteocytes from parietal bones of 16-day-old chick embryos. Isolated osteocytes showed a typical stellate morphology. More than 95% of these cells reacted with the osteocyte-specific antibody OB 7.3. In culture osteocytes formed gap junctions with each other, as could be established by ACAS. Sixteen-millimeter time-lapse microcinematography of the cells also demonstrated the formation of intercellular connections and gap junctions, and portrayed the interaction between osteocytes and osteoclasts: osteocytes seemed to inhibit osteoclast activity. This cinematography also showed the ability of osteocytes to proliferate after they had been disconnected from each other. Thereafter these cells redifferentiated into osteoblasts that became embedded in bone matrix produced by themselves. These findings suggest that osteocytes might be involved in bone formation during remodeling.