Cedric Minkin

Bone acid phosphatase: Tartrate-resistant acid phosphatase as a marker of osteoclast function

SummaryOrgan cultures of newborn mouse calvaria were used to test the hypothesis that tartrate-resistant acid phosphatase might serve as a biochemical marker for osteoclast function. When bone resorption was stimulatedin vitro with either parathyroid hormone or 1,25(OH)2D3, there was a significant increase in both tartrate-resistant and tartrate-sensitive acid phosphatase activity in the medium relative to cultured controls. Tartrate-resistant activity was localized histochemically primarily over the osteoclast and appeared as three distinct activity bands when electrophoresed on polyacrylamide gels. The tartrate-sensitive activity was found primarily associated with bone cells other than the osteoclast using histochemical techniques, and was resolved into five bands on polyacrylamide gels. The results obtained from biochemical assays, histochemical observations, and polyacrylamide gel electrophoresis suggest that bone resorptionin vitro results in the release of tartrate-resistant acid phosphatase from osteoclasts and tartrate-sensitive acid phosphatase from other bone cells as well as osteoclasts. Tartrate-resistant acid phosphatases of bone may be suitable biochemical probes for osteoclast function, but it will be necessary to achieve further purification in order to develop analytical methods with sufficient sensitivity and specificity (e.g., immunochemical) to ensure precise localization and quantitation.

Nonparathyroid Humoral Hypercalcemia in Patients with Neoplastic Diseases

Abstract Eleven patients had hypercalcemia and hypophosphatemia associated with various nonparathyroid neoplasms in the absence of bony metastases (pseudohyperparathyroidism). In nine patients surgical ablation or other antitumor therapy resulted in remission of the biochemical abnormalities. Parathyroid hormone was undetectable both in peripheral blood and in tumor tissue from all patients when assessed with multiple immunoassays that detect not only intact parathyroid hormone but proparathyroid hormone and biologically active and inactive parathyroid hormone fragments in addition. However, extracts of tissues caused active calcium resorption from bone in vitro. The chemical nature of the presumed humoral substance (or substances) is unknown. The findings suggest that in many patients with neoplasms and clinical and biochemical features typical of pseudohyperparathyroidism some humoral substance other than parathyroid hormone must be responsible for hypercalcemia and other features of their disease. (N E...

Bone Marrow Mesenchymal Stem Cells Provide an Alternate Pathway of Osteoclast Activation and Bone Destruction by Cancer Cells

The bone is the third most common site of cancer metastasis. To invade the bone, tumor cells produce osteoclast-activating factors that increase bone resorption by osteoclasts. Here we report that human neuroblastoma cells that form osteolytic lesions in vivo do not produce osteoclast-activating factors but rather stimulate osteoclast activity in the presence of human bone marrow mesenchymal stem cells. This alternative pathway of osteoclast activation involves a nonadhesive interaction between neuroblastoma cells and bone marrow mesenchymal stem cells. Stimulated bone marrow mesenchymal stem cells express markedly increased levels of interleukin-6, which is then responsible for osteoclast activation. This report describes a critical role of bone marrow mesenchymal stem cells in bone destruction in cancer.

Osteoclast Molecular Phenotyping by Random cDNA Sequencing

The osteoclast is a cell type that is highly specialized for its bone resorption function. In order to decipher the numerous biochemical functions of osteoclasts, a description of the gene expression profile of osteoclasts would be beneficial. We have sought to identify genes that are highly expressed in osteoclasts by partially sequencing 194 randomly chosen cDNA clones from a representative rabbit osteoclast cDNA library. Comparison to nucleic acid and protein sequence databases indicates that 135 of these cDNAs are identical to or homologous to known mammalian genes. Reverse transcription-polymerase chain reaction (RT-PCR) assays with microisolated osteoclasts were used to verify the osteoclast expression of some of these genes. Fifty-nine cDNAs, including two abundantly expressed species, have no significant similarity to the sequence databases and likely represent novel genes. The most abundant of the osteoclast expressed genes encode cofilin and the vacuolar H(+)-ATPase 16 kd subunit. Each were represented at a frequency of 4.1% of the clones in the library (95% confidence interval = 2.4-6.6%). The high expression of these gene products is consistent with the high motility of osteoclasts and their very active hydrogen ion secretion. Other abundantly expressed sequences include beta-actin (95% C.I. = 2.0-6.0%), creatine kinase B (95% C.I. = 1.2-4.9%), c-fms and ribosomal protein L18 (95% C.I. = 0.8-4.3%), and cathepsin-OC2, cyclophilin, delta-aminolevulinate synthetase, 16S mitochondrial rRNA, and two novel gene sequences (95% C.I. = 0.5-3.6%).

Inhibition of parathyroid hormone stimulated bone resorptionin vitro by the antibiotic mithramycin

Mithramycin, an antibiotic which blocks DNA-dependent RNA metabolism, has been demonstrated to be hypocalcaemicin vivo. It has been suggested that this effect is obtained by an inhibition of bone resorption. We have tested this antibiotic for its effects on bone resorptionin vitro using new-born mouse calvaria and this report presents evidence that mithramycin not only inhibits bone resorptionin vitro but also inhibits the formation of bone and is cytotoxic at all concentrations which appear to be effective at inhibiting resorption. The effectiveness of this antibiotic at inhibiting resorption appears to be related to the degree of bone resorption activity present when it is introduced to the system. Morphological observations also raised the question as to whether or not this agent may cause abnormalities in the normal processes of bone cell modulation resulting in the formation of cartilage.RésuméLa mithramycine, qui est un antibiotique bloquant le métabolisme de lARN, lié à lADN, possède des propriétés hypocalcémiquesin vivo. Cette action pourrait être en rapport avec une inhibition de la résorption osseuse. La résorption osseuse de cet antibiotique a été testéein vitro en utilisant des calottes craniennes de souris nouveau-nés. Il semble que la mithramycine, non seulement inhibe la résorption osseusein vitro, mais inhibe aussi la formation osseuse et se révèle cytotoxique à toutes le concentrati ons qui inhibent la résorption osseuse. Lefficacité de lantibiotique comme inhibiteur de résorption parait liée au degré dactivité de résorption observée lorsquon lintroduit dans ce système. Des observations morphologiques posent le problème des anomalies provoquées par cet agent dans le processus normal de différenciation de cellules osseuses au cours de la formation du cartilage.ZusammenfassungMithramycin, ein Antibioticum, welches den DNS-abhängigen RNS-Stoffwechsel blockiert, wirktin vivo hypocalcämisch. Es wurde vermutet, daß diese Wirkung durch eine Hemmung der Knochenresorption erzielt wurde. Wir haben dieses Antibioticum auf seine Wirkungen auf die Knochenresorptionin vitro geprüft und verwendeten zu diesem Zwecke Schädeldächer von neugeborenen Mäusen. Die vorliegende Arbeit enthält den Beweis, daß Mithramycin nicht nur die Knochenresorptionin vitro, sondern auch die Knochenbildung hemmt und daß es in allen Konzentrationen, welche die Resorptionshemmung bewirken, cytotoxisch ist. Die Wirksamkeit dieses Antibioticums als Resorptionshemmer scheint im Zusammenhang zu stehen mit dem Ausmaß der Knochenresorptions-Aktivität zum Zeitpunkt, da das Antibioticum in das System eingeführt wird. Morphologische Beobachtungen ließen auch die Frage aufkommen, ob Mithramycin Abnormalitäten im normalen Ablauf der Knochenzell-Verwandlungen, welche zur Bildung von Knorpel führen, verursachen könne oder nicht.

Plasminogen Activator System in Osteoclasts

To determine which genes of the plasminogen activator (PA) system were expressed in osteoclasts, RNA extracted from microisolated mouse osteoclasts was used as template for reverse transcribed polymerase chain reaction (RT‐PCR) with gene‐specific primer pairs. Using this approach, the expression of RNAs for tissue‐type plasminogen activator, urokinase‐type plasminogen activator, plasminogen activator inhibitor‐1, plasminogen activator inhibitor‐2, protease nexin, and urokinase receptor isoform 1 (uPAR1) were detected in mouse osteoclasts. The expression of uPAR RNA in osteoclasts was confirmed by in situ hybridization with a uPAR1 probe. RNA encoding the uPAR isoform 2 was not detected in mouse osteoclasts, but a novel unspliced uPAR RNA variant was detected in these cells. The novel uPAR variant and uPAR1 RNA were also detected in mouse calvarial osteoblasts, kidney, muscle, and the mouse macrophage cell line J774A.1 by RT‐PCR. The presence of RNAs for most of the components of the PA system in osteoclasts suggests that it may have a functional role in this cell type.

Hypercalcemia in Reticulum Cell Sarcoma Without Hyperparathyroidism or Skeletal Metastases

Abstract A patient with reticulum cell sarcoma and hypercalcemia had a remission of the hypercalcemia after localized radiation therapy. The tumor and hypercalcemia recurred 2 years later. Examinat...

Bone remodeling in W/Wv mast cell deficient mice

Strong experimental evidence exists for a relationship between mast cells and bone disease, but the role of mast cells in the regulation of bone remodeling is unknown. In order to address this question, mast cell deficient mice (W/Wv) were paired with their mast cell sufficient (+/+) littermates and evaluated for differences in response to an induced cycle of bone remodeling. This was achieved using a tooth egression protocol, in which a synchronous cycle of bone remodeling was induced in the mandibular buccal alveolar periosteum by extraction of the opposing dentition. Quantitative histomorphometric changes during the activation, resorption, reversal, and formation phases of bone remodeling were documented using standard techniques. Most cell deficient mutants exhibited the following defects in response to an induced cycle of bone remodeling: (a) the onset of the remodeling cycle was delayed by a prolonged activation phase, (b) the duration and extent of the active formation phase was decreased, and (c) the amount of new bone matrix synthesized was diminished while mineralization rates were found to be normal. These results suggest that mast cells and their mediators provide a paracrine mechanism which influences the recruitment of osteoclast and osteoblast progenitors and their participation in bone remodeling. Nonetheless, since bone remodeling occurs in mast cell deficient mice, albeit less efficiently, this mechanism is most likely one of several redundant mechanisms that provide for adequate skeletal homeostasis.

Effects of parathyroid hormone and calcitonin on adenylate cyclase in murine mononuclear phagocytes

Abstract Peritoneal mononuclear phagocytes elicited by thioglycollate demonstrate responsiveness to parathyroid hormone (PTH) and calcitonin (CT) which differs from that seen in the normal resident population. PTH causes a twofold stimulation of adenylate cyclase activity in elicited cells but inhibits this activity in resident cells. CT causes a greater stimulation of adenylate cyclase in elicited than in resident cells. Both CT and PTH cause an increase in cyclic AMP accumulation in cultures of elicited mononuclear phagocytes. These results indicate that cells of the mononuclear phagocyte lineage have functional receptors for both PTH and CT. This is the first biochemical evidence to support the hypothesis that mononuclear phagocytes are precursors of the bone resorbing osteoclast.

Osteoclasts, mononuclear phagocytes, and physiological bone resorption

ConclusionsThe concerns expressed here, as well as those noted by others in recent reviews, underscore the confusion that has entered the literature regarding the role of osteoclasts, as distinct from that of mononuclear phagocytes, in bone resorption. This confusion could be resolved if the following suggestions are adopted. First, the term “resorption” or “bone resorption” should be reserved for describing a process of bone removal in which osteoclasts are the primary function cell type. Second, the term osteoclast should be used to define a bone-resorbing cell characterized by the criteria presented earlier. Third, until unambiguous evidence is provided, it must not be assumed that cells of the mononuclear phagocyte series, such as peripheral blood monocytes or elicited peritoneal macrophages, are osteoclast precursors.