Anna Teti

Insulin Signaling in Osteoblasts Integrates Bone Remodeling and Energy Metabolism

The broad expression of the insulin receptor suggests that the spectrum of insulin function has not been fully described. A cell type expressing this receptor is the osteoblast, a bone-specific cell favoring glucose metabolism through a hormone, osteocalcin, that becomes active once uncarboxylated. We show here that insulin signaling in osteoblasts is necessary for whole-body glucose homeostasis because it increases osteocalcin activity. To achieve this function insulin signaling in osteoblasts takes advantage of the regulation of osteoclastic bone resorption exerted by osteoblasts. Indeed, since bone resorption occurs at a pH acidic enough to decarboxylate proteins, osteoclasts determine the carboxylation status and function of osteocalcin. Accordingly, increasing or decreasing insulin signaling in osteoblasts promotes or hampers glucose metabolism in a bone resorption-dependent manner in mice and humans. Hence, in a feed-forward loop, insulin signals in osteoblasts activate a hormone, osteocalcin, that promotes glucose metabolism.

Osteoclast-poor human osteopetrosis due to mutations in the gene encoding RANKL

Autosomal recessive osteopetrosis is usually associated with normal or elevated numbers of nonfunctional osteoclasts. Here we report mutations in the gene encoding RANKL (receptor activator of nuclear factor–KB ligand) in six individuals with autosomal recessive osteopetrosis whose bone biopsy specimens lacked osteoclasts. These individuals did not show any obvious defects in immunological parameters and could not be cured by hematopoietic stem cell transplantation; however, exogenous RANKL induced formation of functional osteoclasts from their monocytes, suggesting that they could, theoretically, benefit from exogenous RANKL administration.

Rous sarcoma virus-transformed fibroblasts and cells of monocytic origin display a peculiar dot-like organization of cytoskeletal proteins involved in microfilament-membrane interactions☆

By immunofluorescence and interference reflection microscopy (IRM) we found that F-actin and a group of cytoskeletal proteins involved in microfilament-membrane interaction, including vinculin, alpha-actinin, fimbrin and talin, are specifically organized in discrete dot-like structures corresponding to cell-substratum contact sites in both monocytes and monocyte-derived cells such as macrophages and osteoclasts. These proteins have a precise topological distribution; vinculin and talin form a doughnut-like ring, while actin, fimbrin and alpha-actinin are organized in dots matching the rings. An identical dot-like organization of F-actin and associated cytoskeletal proteins was also detected in malignant fibroblasts transformed by Rous Sarcoma virus (RSV) but not in the corresponding untransformed cells in culture. It is concluded that RSV transformation induces fibroblasts to express a cytoskeletal organization and a pattern of adhesion that are normally found in cells of monocytic origin. We propose that the occurrence of this cytoskeletal organization in RSV-transformed fibroblasts and in monocyte-derived cells may reflect a common ability to migrate across anatomical boundaries.

Chloride channel ClCN7 mutations are responsible for severe recessive, dominant, and intermediate osteopetrosis.

Among 94 osteopetrotic patients presenting with a severe clinical picture and diagnosed early in life, 12 bore mutations in the ClCN7 gene, but only 7 of them had the expected two recessive mutations. The remaining five patients seem to be heterozygous for a ClCN7 mutation, and significant variations were observed in the clinical manifestations of their disease, even within the same family.

Immunocytochemical distribution of extracellular matrix receptors in human osteoclasts: A β3 integrin is colocalized with vinculin and talin in the podosomes of osteoclastoma giant cells

Human osteoclasts (OCLs) obtained from cell suspensions of surgically excised giant cell bone tumors (osteoclastomas) were attached to glass coverslips and analyzed by immunofluorescence with antibodies to integrins and cytoskeletal proteins. It was found that in OCLs (i) podosomes, identified by their F-actin core and by interference reflection microscopy, were predominantly found in a peripheral belt as described in avian OCLs; (ii) each F-actin core was surrounded by a ring of vinculin and talin; (iii) beta 1 integrin was diffuse in the ventral membrane; (iv) beta 3 integrin was distributed in intensely fluorescent rings surrounding F-actin cores; (v) beta 2 integrin was absent; (vi) beta 4 integrin was absent. The macrophages detected in the same coverslips displayed podosomes containing beta 2 but not beta 3, fibroblasts showed adhesion plaques positive for beta 1 and beta 3 but not for beta 2, and platelets were intensely positive for beta 3. These results indicate that OCLs produce an integrin complex that is absent in the monocyte-macrophage lineage.

Do osteocytes contribute to bone mineral homeostasis? Osteocytic osteolysis revisited

Osteocytes are cells buried in the bone matrix. They largely contribute to the regulation of bone remodeling in response to mechanical and microenvironmental changes. Much has been recognized in recent years regarding the role of osteocytes in bone homeostasis, nevertheless their ability to directly contribute to mineral equilibrium has been neglected. In the light of the renewed interest in their biology, we revisited the literature and discuss experimental evidence favoring the hypothesis that osteocytes are able to remove and replace the bone matrix according to the systemic needs of the body. We also reviewed reports against this theory, thus providing current views of what is known so far on the ability of osteocytes to mobilize bone mineral. This re-examination of osteocytic osteolysis might stimulate new interest and open new perspectives in osteocyte biology and in the cellular mechanisms that control bone homeostasis.

Involvement of PLEKHM1 in osteoclastic vesicular transport and osteopetrosis in incisors absent rats and humans

This study illustrates that Plekhm1 is an essential protein for bone resorption, as loss-of-function mutations were found to underlie the osteopetrotic phenotype of the incisors absent rat as well as an intermediate type of human osteopetrosis. Electron and confocal microscopic analysis demonstrated that monocytes from a patient homozygous for the mutation differentiated into osteoclasts normally, but when cultured on dentine discs, the osteoclasts failed to form ruffled borders and showed little evidence of bone resorption. The presence of both RUN and pleckstrin homology domains suggests that Plekhm1 may be linked to small GTPase signaling. We found that Plekhm1 colocalized with Rab7 to late endosomal/lysosomal vesicles in HEK293 and osteoclast-like cells, an effect that was dependent on the prenylation of Rab7. In conclusion, we believe PLEKHM1 to be a novel gene implicated in the development of osteopetrosis, with a putative critical function in vesicular transport in the osteoclast.

Clinical, genetic, and cellular analysis of 49 osteopetrotic patients: implications for diagnosis and treatment

Background: Osteopetrosis, a genetic disease characterised by osteoclast failure, is classified into three forms: infantile malignant autosomal recessive osteopetrosis (ARO), intermediate autosomal recessive osteopetrosis (IRO), and autosomal dominant osteopetrosis (ADO). Methods: We studied 49 patients, 21 with ARO, one with IRO, and 27 with type II ADO (ADO II). Results: Most ARO patients bore known or novel (one case) ATP6i (TCIRG1) gene mutations. Six ADO II patients had no mutations in ClCN7, the only so far recognised gene implicated, suggesting involvement of yet unknown genes. Identical ClCN7 mutations produced differing phenotypes with variable degrees of severity. In ADO II, serum tartrate resistant acid phosphatase was always elevated. Bone alkaline phosphatase (BALP) was generally low, but osteocalcin was high, suggesting perturbed osteoblast differentiation or function. In contrast, BALP was high in ARO patients. Elevated osteoclast surface/bone surface was noted in biopsies from most ARO patients. Cases with high osteoclasts also showed increased osteoblast surface/bone surface. ARO osteoclasts were morphologically normal, with unaltered formation rates, intracellular pH handling, and response to acidification. Their resorption activity was greatly reduced, but not abolished. In control osteoclasts, all resorption activity was abolished by combined inhibition of proton pumping and sodium/proton antiport. Conclusions: These findings provide a rationale for novel therapies targeting pH handling mechanisms in osteoclasts and their microenvironment.

Impaired gastric acidification negatively affects calcium homeostasis and bone mass.

Activation of osteoclasts and their acidification-dependent resorption of bone is thought to maintain proper serum calcium levels. Here we show that osteoclast dysfunction alone does not generally affect calcium homeostasis. Indeed, mice deficient in Src, encoding a tyrosine kinase critical for osteoclast activity, show signs of osteopetrosis, but without hypocalcemia or defects in bone mineralization. Mice deficient in Cckbr, encoding a gastrin receptor that affects acid secretion by parietal cells, have the expected defects in gastric acidification but also secondary hyperparathyroidism and osteoporosis and modest hypocalcemia. These results suggest that alterations in calcium homeostasis can be driven by defects in gastric acidification, especially given that calcium gluconate supplementation fully rescues the phenotype of the Cckbr-mutant mice. Finally, mice deficient in Tcirg1, encoding a subunit of the vacuolar proton pump specifically expressed in both osteoclasts and parietal cells, show hypocalcemia and osteopetrorickets. Although neither Src- nor Cckbr-deficient mice have this latter phenotype, the combined deficiency of both genes results in osteopetrorickets. Thus, we find that osteopetrosis and osteopetrorickets are distinct phenotypes, depending on the site or sites of defective acidification (pages 610–612).

Extracellular protons acidify osteoclasts, reduce cytosolic calcium, and promote expression of cell-matrix attachment structures.

Because metabolic acids stimulate bone resorption in vitro and in vivo, we focused on the cellular events produced by acidosis that might be associated with stimulation of bone remodeling. To this end, we exposed isolated chicken osteoclasts to a metabolic (butyric) acid and observed a fall in both intracellular pH and cytosolic calcium [( Ca2+]i). These phenomena were recapitulated when bone resorptive cells, alkalinized by HCO3 loading, were transferred to a bicarbonate-free environment. The acid-induced decline in osteoclast [Ca2+]i was blocked by either NaCN or Na3VO4, in a Na+-independent fashion, despite the failure of each inhibitor to alter stimulated intracellular acidification. Moreover, K+-induced membrane depolarization also reduced cytosolic calcium in a manner additive to the effect of protons. These findings suggest that osteoclasts adherent to bone lack functional voltage-operated Ca2+ channels, and they reduced [Ca2+]i in response to protons via a membrane residing Ca-ATPase. Most importantly, acidosis enhances formation of podosomes, the contact areas of the osteoclast clear zone, indicating increased adhesion to substrate, an early step in bone resorption. Thus, extracellular acidification of osteoclasts leads to decrements in intracellular pH and calcium, and appears to promote cell-matrix attachment.