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Biomedical subjects

A Teti

Publications and source records attributed to A Teti.

At least 19 recordsLinked to original sources
PubMed1992

Protein kinase C affects microfilaments, bone resorption, and [Ca2+]o sensing in cultured osteoclasts.

The effects of protein kinase C (PKC) in the control of osteoclast activity are still unknown. We investigated the role of the enzyme in the control of microfilament organization, podosome assembly, bone resorption, and extracellular Ca2+ sensing in chicken and rabbit osteoclasts treated with agents known to affect PKC activity. Cells were treated for 20 min with a PKC activator [phorbol 12-myristate 13-acetate (PMA)], a PKC inhibitor (staurosporine), a protein kinase A (PKA) inhibitor (H-9), a guanosine 3',5'-cyclic monophosphate-dependent protein kinase-PKA-PKC inhibitor (H-7), or with the inactive phorbol, 4 alpha-phorbol, to examine microfilaments by decoration with rhodamine-phalloidin. In PMA-treated osteoclasts, the number of microfilament-containing adhesion structures (podosomes) per cell decreased. However, enlarged microfilamentous cores in podosomes and stress fiber-like filaments, otherwise absent in controls, appeared. Whereas H-7 induced increase of the number of podosomes, staurosporine, H-9, and 4 alpha-phorbol failed to change microfilament organization. Chicken osteoclasts received also long-term treatment with the agents in the presence of [3H]proline-prelabeled chicken or rat bone particles to measure bone resorption. PMA, as well as staurosporine and H-7, stimulated the resorbing activity, whereas cells were insensitive to H-9 and 4 alpha-phorbol. Measurement of cytosolic free calcium concentration in PMA-treated fura-2-loaded single osteoclasts demonstrated a synergistic effect of PKC activation on the inhibitory extracellular calcium concentration-sensing mechanism, which was, by contrast, blocked by H-7, staurosporine, and H-9 and was insensitive to 4 alpha-phorbol. These results indicate that PKC regulates osteoclast activity inducing both morphological and functional modifications.

Actin Cytoskeleton↗
PubMed1992

Regulation of cellular functions by extracellular matrix.

Multicellular organisms are formed by specialized cells assembled in tissues. Individual cells contact and interact with other cells and with the extracellular matrix--a network of secreted proteins and carbohydrates that fills the intercellular spaces. The extracellular matrix helps cells to bind together and regulates a number of cellular functions, such as adhesion, migration, proliferation, and differentiation. It is formed by macromolecules, locally secreted by resident cells. The two main classes of macromolecules are polysaccharide glycosaminoglycans, usually covalently linked to proteins in the form of proteoglycans, and fibrous proteins of two functional types, structural (collagen, elastin) and adhesive (fibronectin, laminin, vitronectin, etc.). Receptors for extracellular matrix macromolecules are present in virtually all of the cells studied. They belong to the superfamily of integrins, alpha beta heterodimers, which, in most cases, recognize the Arg-Gly-Asp sequence of extracellular matrix proteins. On the exterior side of the cell, integrins link an extracellular matrix macromolecule, whereas in the cytosol, they bind the cytoskeleton, thereby forming a membrane bridge between extracellular and intracellular fibers. This structure enables the cell to adhere to the substratum. Similar to hormone- or growth factor-receptor binding, the interaction of the integrin with its specific ligand induces immediate signal transduction and influences cellular activities.

Animals↗
PubMed1992

Cells from human bone giant cell tumors show a [Ca2+]o-sensing.

Giant cells from a human giant cell tumor of bone, showing several osteoclast features were tested for their capability of detecting the [Ca2+]o by a receptor like [Ca2+]o sensing. We found that cultured cells responded to elevation of [Ca2+]o, obtained adding 4 mM Ca2+ to the 2 mM Ca2+ containing buffer, by a transient increase of [Ca2+]i. Proliferative cells induced to differentiate by treatment with 10(-8) M 1,25 dihydroxyvitamin D3, were upregulated in their capability of responding to elevated [Ca2+]o. In fact, in this circumstance, the peak of [Ca2+]o-induced [Ca2+]i rise was increased compared to untreated cells. This suggests that 1,25 dihydroxyvitamin D3 induces a more efficient regulation of osteoclast activity.

Bone Neoplasms↗
PubMed1992

Protein kinase C-dependent phosphorylation regulates osteoclast calcium-sensing.

Osteoclasts display a membrane Ca(2+)-sensing mechanism capable of detecting the extracellular calcium concentration ([Ca2+]o), and to induce increase of [Ca2+]i and inhibition of bone resorption. The ultimate result of the stimulation of such sensing is probably the activation of protein kinase C (PKC). To demonstrate whether PKC plays a role in the control of the osteoclast activity, we treated rabbit single osteoclasts with agents known to activate or to inhibit the enzyme. We measured [Ca2+]i in single fura 2-loaded single cells and found that activation of PKC by phorbol esters doubled the [Ca2+]o-induced [Ca2+]i elevation, whereas inhibition of the enzyme by H7, staurosporine or sphingosine, completely blocked the ability of the cell to respond to elevated [Ca2+]i. By contrast, a control inactive agent, 4Aphorbol, failed to modify the cellular response to elevated [Ca2+]o. We conclude that PKC plays a synergistic role in the regulation of osteoclast Ca(2+)-sensing. Since we have previously demonstrated that activation of PKA up-regulates the Ca(2+)-sensing as well, we hypothesize that such mechanism is positively fed-back by both PKA and PKC-dependent threonine/serine phosphorylations.

Animals↗
PubMed1992

Functional and biochemical characterization of osteoclast-like cells derived from giant cell tumours of bone.

Cells harvested from human giant cell tumours of bone were characterized on the basis of morphological features, proliferative capacity, total(AP) and tartrate resistant acid phosphatase (TRAP) activity, and hormonal response. Culture were formed by mononucleated and multinucleated cells. Mononucleated cells showed fibroblastic morphology, whereas multinucleated cells showed osteoclastic phenotype. We conclude that in these cultures mature osteoclasts and their mononuclear precursors are present.

Bone Neoplasms↗
PubMed1992

Integrin expression and adhesion property of osteoclast-like cells from giant cell tumours of bone.

Cells cultured from human giant cell tumours of bone were used to study interactions with different extracellular matrix proteins as Collagen, Fibronectin, Osteocalcin, Thrombospondin and Bone Sialoprotein II. Cells were capable of recognizing these substrata; beta 3 integrin subunit was distributed in focal adhesions, together with beta 1 on BSPII, FN, and in presence of serum, whereas and presented a diffuse organization onto the other substrate. beta 1 alone was expressed over collagen coated coverslips.

Bone Neoplasms↗
PubMed1991

Recognition of osteopontin and related peptides by an alpha v beta 3 integrin stimulates immediate cell signals in osteoclasts.

We have investigated the nature of immediate cell signals produced by occupancy of the chicken osteoclast alpha v beta 3 integrin. Synthetic osteopontin and peptides from the osteopontin and bone sialoprotein sequences containing Arg-Gly-Asp stimulated immediate reductions in osteoclast cytosolic Ca2+. The changes in cytosolic Ca2+ required the Arg-Gly-Asp sequence and were blocked by a monoclonal antibody to the alpha v beta 3 integrin, LM609. Osteoclast stimulation by the proteins through the integrin did not require immobilization since soluble peptides produced changes in cytosolic Ca2+ and inhibited osteoclast binding to bone particles and bone resorption. The decrease in cytosolic Ca2+ stimulated by osteopontin and related peptides appeared to be due to activation of a plasma membrane Ca(2+)-ATPase by calmodulin. Thus, the data suggest that ligand binding to the osteoclast alpha v beta 3 integrin results in calmodulin-dependent reduction in cytosolic Ca2+ which participates in regulation of osteoclast function.

Amino Acid Sequence↗
PubMed1991

Osteoblast-osteoclast relationships in bone resorption: osteoblasts enhance osteoclast activity in a serum-free co-culture system.

Osteoblast-osteoclast relationships in bone resorption are unclear. We investigated whether osteoblasts constitutively influence osteoclast activity. We employed a serum-free co-culture system in which chicken osteoclasts and chick calvaria or, alternatively, isolated chick osteoblasts were cultured in two different compartments separated by a 0.45 micron porous membrane permeable to soluble molecules. Osteoclastic bone resorption, evaluated by release of 3H-proline from prelabeled bone fragments, was significantly enhanced by bone cells resident in the calvaria, as well as by isolated osteoblasts. Stimulation was specific, since periosteal cells, or skin fibroblasts, failed to mimic osteoblast activity. Conditioned medium from osteoblast cultures stimulated osteoclast function in a similar manner, indicating that paracrine signals, capable of crossing the porous membrane separating the two compartments, are released by the bone forming cells.

Alkaline Phosphatase↗
PubMed1991

Parathyroid hormone binding to cultured avian osteoclasts.

Parathyroid hormone (PTH) increases serum calcium concentration via a controversial cellular mechanism. We investigated whether PTH binds avian osteoclasts. Isolated hypocalcaemic hen osteoclasts were incubated with [125I]--bovine PTH (1-84). Specific binding of the hormone to the cells, which reached the equilibrium within 60 min, was observed. Half maximal binding was reached by 10 min. Binding was competitively inhibited by increasing doses of unlabeled PTH, and was about 55% displaced by adding, at the equilibrium, 10(-6) M unlabeled PTH. Autoradiography demonstrated specific label on the osteoclast. The cellular mechanism activated by the hormone remains to be elucidated.

Animals↗
PubMed1991

Clear zone in osteoclast function: role of podosomes in regulation of bone-resorbing activity.

The adhesion of osteoclasts to the bone matrix is mandatory for bone resorption. Contact of the osteoclast with bone surface induces, in fact, cell polarization and organization of the resorbing apparatus, the so-called "ruffled border." Cell-matrix interaction in osteoclasts is a complex phenomenon resulting from formation of the "clear zone," a cytoplasmic area presenting the adhering plasma membrane, or "sealing membrane." The sealing membrane surrounds the ruffled border and seals the resorbing compartment, namely the extracellular space in which bone resorption takes place. Adhesion at this level occurs via specialized discrete structures, the "podosomes." Podosomes present most of the protein commonly found in focal adhesions, but with a peculiar organization. They are dynamic elements suitable for regulation, according with the functional demand of the cell. Their assembly increases during bone resorption and is regulated by the cytosolic free calcium concentration and the activity of protein kinase C.

Animals↗
PubMed1991

Effects of calcium-phosphate-based materials on proliferation and alkaline phosphatase activity of newborn rat periosteal cells in vitro.

The effects of dental materials, intended for bone substitution, on cell growth and alkaline phosphatase activity of newborn rat periosteal cells have been studied in vitro. Confluent periosteal cells were exposed to three apatite-based materials (400 micrograms/mL) with different physico-chemical properties. The materials were a beta-tricalcium phosphate with a microporous granular structure obtained by sinterization (Synthograft, Johnson & Johnson, East Windsor, NY), a 40-60-mesh microporous durapatite ceramic (Periograf, Sterling Drug, Inc., Rensselaer, NY), and a 1-2-mm-diameter hydroxyapatite ceramic (Osprovit, Feldmuhle Aktiengeselschaft, Plochingen, Germany) with macropores larger than 100 microns. Cell proliferation and alkaline phosphatase activity were assessed by incorporation of 3H-thymidine into trichloroacetic-acid-precipitable material and by a fluorimetric method, respectively. Cell viability and compatibility with the materials were determined by morphology in phase-contrast microscopy. Periosteal cells showed increased proliferation following exposure to Synthograft, but were unaffected by Osprovit, whereas Periograf caused significantly reduced cell growth. Alkaline phosphatase activity was unaffected by Osprovit, but was decreased by both Synthograft and Periograf. The results indicated a differential response of periosteal cells to bone-substituting materials with heterogeneous physico-chemical characteristics.

Alkaline Phosphatase↗
PubMed1990

Osteoclast cytosolic calcium, regulated by voltage-gated calcium channels and extracellular calcium, controls podosome assembly and bone resorption.

The mechanisms of Ca2+ entry and their effects on cell function were investigated in cultured chicken osteoclasts and putative osteoclasts produced by fusion of mononuclear cell precursors. Voltage-gated Ca2+ channels (VGCC) were detected by the effects of membrane depolarization with K+, BAY K 8644, and dihydropyridine antagonists. K+ produced dose-dependent increases of cytosolic calcium ([Ca2+]i) in osteoclasts on glass coverslips. Half-maximal effects were achieved at 70 mM K+. The effects of K+ were completely inhibited by dihydropyridine derivative Ca2+ channel blocking agents. BAY K 8644 (5 X 10(-6) M), a VGCC agonist, stimulated Ca2+ entry which was inhibited by nicardipine. VGCCs were inactivated by the attachment of osteoclasts to bone, indicating a rapid phenotypic change in Ca2+ entry mechanisms associated with adhesion of osteoclasts to their resorption substrate. Increasing extracellular Ca2+ ([Ca2+]e) induced Ca2+ release from intracellular stores and Ca2+ influx. The Ca2+ release was blocked by dantrolene (10(-5) M), and the influx by La3+. The effects of [Ca2+]e on [Ca2+]i suggests the presence of a Ca2+ receptor on the osteoclast cell membrane that could be coupled to mechanisms regulating cell function. Expression of the [Ca2+]e effect on [Ca2+]i was similar in the presence or absence of bone matrix substrate. Each of the mechanisms producing increases in [Ca2+]i, (membrane depolarization, BAY K 8644, and [Ca2+]e) reduced expression of the osteoclast-specific adhesion structure, the podosome. The decrease in podosome expression was mirrored by a 50% decrease in bone resorptive activity. Thus, stimulated increases of osteoclast [Ca2+]i lead to cytoskeletal changes affecting cell adhesion and decreasing bone resorptive activity.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1990

Osteoclast bone resorption is enhanced in the presence of osteoblasts.

Bone resorption activity by osteoclasts has been evaluated in a co-culture system in which osteoclasts have been plated in the presence of osteoblasts. The system prevents cell-cell contact but permits diffusion of molecules through the pores of a millipore membrane that separates the two compartments in which the two cell types have been plated. Results demonstrated that osteoblasts exert a stimulatory effect over osteoclast bone resorption due to soluble molecules capable of passing through the membrane pores. The effect is specific since periosteal cells, which do not express osteoblastic characteristics, fail to induce changes in the osteoclast activity. PTH does not affect osteoblast-mediated enhancement of bone resorption, indicating that the stimulatory effect that the hormone exert in vivo occurs via a different cellular system.

Animals↗
PubMed1990

The role of protein kinase C in the osteoclast activity.

Isolated chicken osteoclasts in culture have been treated with 100 nM PMA for 20 minutes, and processed for the decoration of the microfilaments with fluorescent phalloidin. Results demonstrated that this phorbol ester, which activates the protein kinase C, induces the assembly of microfilaments in stress-fibers, and enlarges the microfilamentous core of podosomes. This results indicate that the protein kinase C mediates specific arrangement of microfilaments in osteoclasts. The substratum for protein kinase C-mediated phosphorylation is however still unknown.

Actin Cytoskeleton↗
PubMed1990

Cytosolic free calcium dependent regulation of osteoclast bone resorbing activity.

Osteoclasts are sensitive to KCl-induced depolarization and to increased extracellular calcium concentration, and respond to these treatments with cytosolic calcium increase. In this study we evaluated the possibility that these experimental conditions could affect osteoclast bone resorption. We found that, incubating osteoclasts with 3H-proline previously labeled bone particles the resorbing activity was inhibited by both depolarization and extracellular calcium concentration increase. The released radioactivity was, in fact, 48% and 52% respectively compared to the untreated cultures. These data demonstrated that cytosolic calcium increase is one of the messengers of the pathway that inhibits, in this condition, bone resorption. Furthermore, as in parathyroid cells, extracellular calcium acts with a negative direct feedback mechanism that controls osteoclast activity.

Animals↗
PubMed1990

Osteoblast-osteoclast interaction in bone resorption. Preliminary results.

Osteoclastic bone resorption has been evaluated in vitro by release of tritiated collagen fragments from 3H-proline prelabeled bone particles incubated for 48 hours in presence of avian isolated osteoclasts. Cells were co-incubated with periosteum-free chick calvarial fragments by interposition of 0.4 micron millipore membrane transwells, in presence or absence of 10(-8) M 1.34 bovine parathyroid hormone (PTH). Results demonstrated that i) calvaria exert a stimulating effect over osteoclastic bone resorption which was 1.8 fold enhanced with respect to controls (p less than 0.001). ii) the stimulating effect is exerted by calvarium-derived soluble molecules capable of crossing the 0.4 micron millipore membrane interposed between calvarial fragments and osteoclasts, iii) in this experimental system no further enhancement of calvarial stimulating effect is operated by PTH treatment.

Animals↗
PubMed1989

Control of cytosolic free calcium in rat and chicken osteoclasts. The role of extracellular calcium and calcitonin.

Single cell [Ca2+], studies were performed in chicken and rat osteoclasts loaded with fura-2 and exposed to a variety of treatments. Under resting conditions, basal [Ca2+]i, was 79.2 +/- 47.3 and 84.3 +/- 65.7 nM (averages +/- S.D.; n = 141 and 126) in the osteoclasts of the two species, respectively. Basal [Ca2+]i was stable in all rat and in approximately 80% of chicken osteoclasts. In the remaining 20%, spontaneous, irregular [Ca2+], fluctuations were observed (amplitude range: 50-200 nm over basal values). Increase of [Ca2+]o over the concentration of the Krebs-Ringer incubation medium (2 mM) induced rises of [Ca2+] in almost all cells investigated. [Ca2+] rises were already appreciable with 0.5 mM [Ca2+]o additions and reached high values with 4 mM additions: 390 +/- 113 and 364 +/- 214 nM [Ca2+], in rat and chicken osteoclasts, respectively (n = 122 and 101). Qualitatively, the responses to [Ca2+]o additions consisted of discrete [Ca2+]i transients, biphasic (an initial spike followed by a plateau), or monophasic (either the spike or the plateau). In a few chicken osteoclasts, the [Ca2+]i increase occurring after [Ca2+]o addition consisted of multiple, irregular fluctuations, similar to those observed in 20% of these cells under resting conditions. In individual osteoclasts subsequently exposed to multiple [Ca2+]o increase pulses, the type of the [Ca2+]i transient (mono- or biphasic) was maintained, and the size was dependent on the magnitude of the [Ca2+]o additions. Effects similar to those of [Ca2+]o were induced by the addition of Cd2+ or Ba2+ (but not La3+ or Mg2+) into the medium. The Cd2+ effect was maintained in part even in a Ca2+-free medium. Of various hormones and factors, parathormone, 1,25-dihydroxyvitamin D3, and prostaglandin E2 were inactive. In contrast, calcitonin was active in rat osteoclasts (which express numerous receptors). [Ca2+]i increases were small (19 +/- 17.9 nM; n = 21) when the hormone was administered alone; they were synergistic (severalfold potentiation) when the hormone was administered before or after [Ca2+]o. The [Ca2+]i effects of calcitonin were mimicked by 8Br-cAMP (31 +/- 26 nM; n = 12) when the nucleotide was administered alone; marked synergism when it was administered in combination with [Ca2+]o. This paper demonstrates for the first time that changes of [Ca2+]i are induced in osteoclasts by treatments with [Ca2+]o and calcitonin and can therefore be involved in intracellular mediation of the physiological effects of these two extracellular signals.

Animals↗