Cellular and molecular alterations of osteoblasts in human disorders of bone formation

Osteogenesis is a complex process characterized sequentially by the commitment of precursor cells, the proliferation of osteoprogenitor cells, the differentiation of pre-osteoblasts into mature osteoblasts and the apposition of a calcified bone matrix. Recent advances in cell and molecular biology have improved our knowledge of the cellular and molecular mechanisms controlling the different steps of bone formation in humans. Using ex vivo/in vitro studies of disorders of bone formation, we showed that the recruitment of osteoprogenitor cells is the most important step controlling the rate of bone formation in both rodents and humans. Accordingly, treatments stimulating osteoblast recruitment were found to increase bone formation in experimental models of osteopenic disorders. Using models of human osteoblastic cells, we identified the profile of phenotypic markers expressed during osteoblast differentiation, and found that hormones and growth factors control osteoblastic cell proliferation and differentiation in a sequential and coordinate manner during osteogenesis in vitro. Our recent evaluation of the phenotypic osteoblast abnormalities induced by genetic mutations in the Gs alpha and FGFR-2 genes led to the characterization of the role of these genes in the alterations of osteoblast proliferation and differentiation in humans. These studies at the histological, cellular and molecular levels provided new insight into the mechanisms that are involved in pathological bone formation in humans. It is expected that further determination of the pathogenic pathways in metabolic and genetic abnormalities in human osteoblasts will help to identify novel target genes and to conceive new therapeutic tools to stimulate bone formation in osteopenic disorders.     

Mechanisms of human hepatocarcinogenesis

The major risk factors and etiological agents responsible for development of hepatocellular carcinoma in humans have been identified and characterized. Among these are chronic infection with hepatitis B virus or hepatitis C virus, exposure to aflatoxin B1, and cirrhosis of any etiology (including alcoholic cirrhosis and cirrhosis associated with genetic liver diseases). Both chronic hepatitis and cirrhosis represent major preneoplastic conditions of the liver as the majority of hepatocellular carcinomas arise in these pathological settings. Hepatocarcinogenesis represents a linear and progressive process in which successively more aberrant monoclonal populations of hepatocytes evolve. Regenerative hepatocytes in focal lesions in the inflamed liver (chronic hepatitis or cirrhosis) give rise to hyperplastic hepatocyte nodules, and these progress to dysplastic nodules, which are thought to be the direct precursor of hepatocellular carcinoma. In most cases, the neoplastic transformation of hepatocytes results from accumulation of genetic damage during the repetitive cellular proliferation that occurs in the injured liver in response to paracrine growth factor and cytokine stimulation. Hepatocellular carcinomas exhibit numerous genetic abnormalities (including chromosomal deletions, rearrangements, aneuploidy, gene amplifications, and mutations), as well as epigenetic alterations (including modulation of DNA methylation). These genetic and epigenetic alterations combine to activate positive mediators of cellular proliferation (including cellular proto-oncogenes and their mitogenic signaling pathways) and inactivate negative mediators of cellular proliferation (including tumor suppressor genes), resulting in cells with autonomous growth potential. However, hepatocellular carcinomas exhibit a high degree of genetic heterogeneity, suggesting that multiple molecular pathways may be involved in the genesis of subsets of hepatocellular neoplasms. Continued investigation of the mechanisms of hepatocarcinogenesis will refine our current understanding of the molecular and cellular basis for neoplastic transformation in liver, enabling the development of effective strategies for prevention and/or more effective treatment of hepatocellular carcinoma.     

Polyostotic fibrous dysplasia in a cynomolgus Macaque (Macaca fascicularis)

A 2.3-y-old female cynomolgus macaque (Macaca fascicularis) presented with a broken right tibia and fibula. Radiographs showed multiple cyst-like defects in all long bones. We suspected that both fractures were pathologic because they occurred through these defects. Ultrasonography, MRI, and dual X-ray absorptiometry revealed that the defects were filled with soft tissue. Grossly, the bones were abnormal in shape, and a gelatinous material filled the defects and the surrounding marrow cavity. Histologically, the gelatinous material was composed of fibrin and cartilage; few normal bone cells were seen. Genetic testing revealed extra material on the short arm of chromosome 8 in all tissues examined, but no copy number alterations of likely clinical significance were observed, and no abnormalities were found that were unique to the lesions. In light of the clinical signs and radiographic and pathologic findings, polyostotic fibrous dysplasia was diagnosed. This report represents the first documented case of fibrous dysplasia in a cynomolgus macaque.     

Disrupted Bone Remodeling Leads to Cochlear Overgrowth and Hearing Loss in a Mouse Model of Fibrous Dysplasia

Normal hearing requires exquisite cooperation between bony and sensorineural structures within the cochlea. For example, the inner ear secretes proteins such as osteoprotegrin (OPG) that can prevent cochlear bone remodeling. Accordingly, diseases that affect bone regulation can also result in hearing loss. Patients with fibrous dysplasia develop trabecular bone overgrowth resulting in hearing loss if the lesions affect the temporal bones. Unfortunately, the mechanisms responsible for this hearing loss, which could be sensorineural and/or conductive, remain unclear. In this study, we used a unique transgenic mouse model of increased G_s G-protein coupled receptor (GPCR) signaling induced by expression of an engineered receptor, Rs1, in osteoblastic cells. These ColI(2.3)⁺/Rs1⁺ mice showed dramatic bone lesions that histologically and radiologically resembled fibrous dysplasia. We found that ColI(2.3)⁺/Rs1⁺ mice showed progressive and severe conductive hearing loss. Ossicular chain impingement increased with the size and number of dysplastic lesions. While sensorineural structures were unaffected, ColI(2.3)⁺/Rs1⁺ cochleae had abnormally high osteoclast activity, together with elevated tartrate resistant acid phosphatase (TRAP) activity and receptor activator of nuclear factor kappa-B ligand (Rankl) mRNA expression. ColI(2.3)⁺/Rs1⁺ cochleae also showed decreased expression of Sclerostin (Sost), an antagonist of the Wnt signaling pathway that normally increases bone formation. The osteocyte canalicular networks of ColI(2.3)⁺/Rs1⁺ cochleae were disrupted and showed abnormal osteocyte morphology. The osteocytes in the ColI(2.3)⁺/Rs1⁺ cochleae showed increased expression of matrix metalloproteinase 13 (MMP-13) and TRAP, both of which can support osteocyte-mediated peri-lacunar remodeling. Thus, while the ossicular chain impingement is sufficient to account for the progressive hearing loss in fibrous dysplasia, the deregulation of bone remodeling extends to the cochlea as well. Our findings suggest that factors regulating bone remodeling, including peri-lacunar remodeling by osteocytes, may be useful targets for treating the bony overgrowths and hearing changes of fibrous dysplasia and other bony pathologies.     

Membrane trafficking in osteoblasts and osteoclasts: new avenues for understanding and treating skeletal diseases

The endocytic and exocytic/secretory pathways are two major intracellular membrane trafficking routes that regulate numerous cellular functions in a variety of cell types. Osteoblasts and osteoclasts, two major bone cells responsible for bone remodeling and homeostasis, are no exceptions. During the past few years, emerging evidence has pinpointed a critical role for endocytic and secretory pathways in osteoblast and osteoclast differentiation and function. The endosomal membrane provides a platform to integrate bone tropic signals of hormones and growth factors in osteoblasts. In osteoclasts, endocytosis, followed by transcytosis, of degraded bone matrix promotes bone resorption. Secretory pathways, especially lysosome secretion, not only participate in bone matrix deposition by osteoblasts and degradation of mineralized bone matrix by osteoclasts; they may also be involved in the coupling of bone resorption and bone formation during bone remodeling. More importantly, mutations in genes encoding regulatory factors within the endocytic and secretory pathways have been identified as causes for bone diseases. Identification of the molecular mechanisms of these genes in bone cells may provide new therapeutic targets for skeletal disorders.     

Monostotic fibrous dysplasia in the temporal bone: A late prehistoric occurrence

Fibrous dysplasia, characterized by benign osteolytic and osteoblastic lesions may involve one or several bones. Recent investigators have suggested that it may be merely a phase of what have previously been thought to be several different bone disease. Isolated fibrous dysplasia in the temporal bone is infrequent. Several reports of this disease have appeared in the literature of paleopathology, but none involved only the temporal bone. Monostotic involvement of the right temporal bone was discovered in the skull of an adult male recovered from an archeological site dating from the Late Mississippian period (A. D. 1,350–A. D. 1,650). It will provide an opportunity for preliminary documentation of the antiquity of this disease in the southeastern portion of the United States.     

Role of connexins and pannexins during ontogeny,regeneration, and pathologies of bone

Electron micrographs revealed the presence of gap junctions in osteoblastic cells over 40 years ago. These intercellular channels formed from connexins are present in bone forming osteoblasts, bone resorbing osteoclasts, and osteocytes (mature osteoblasts embedded in the mineralized bone matrix). More recently, genetic and pharmacologic studies revealed the role of connexins, and in particular Cx43, in the differentiation and function of all bone types. Furthermore, mutations in the gene encoding Cx43 were found to be causally linked to oculodentodigital dysplasia, a condition that results in an abnormal skeleton. Pannexins, molecules with similar structure and single-membrane channel forming potential as connexins when organized as hemichannels, are also expressed in osteoblastic cells. The function of pannexins in bone and cartilage is beginning to be uncovered, but more research is needed to determine the role of pannexins in bone development, adult bone mass and skeletal homeostasis. We describe here the current knowledge on the role of connexins and pannexins on skeletal health and disease.

     

Increased expression of TGF-beta 2 in osteoblasts results in an osteoporosis-like phenotype

The development of the skeleton requires the coordinated activities of bone-forming osteoblasts and bone-resorbing osteoclasts. The activities of these two cell types are likely to be regulated by TGF-beta, which is abundant in bone matrix. We have used transgenic mice to evaluate the role of TGF-beta 2 in bone development and turnover. Osteoblast- specific overexpression of TGF-beta 2 from the osteocalcin promoter resulted in progressive bone loss associated with increases in osteoblastic matrix deposition and osteoclastic bone resorption. This phenotype closely resembles the bone abnormalities seen in human hyperparathyroidism and osteoporosis. Furthermore, a high level of TGF- beta 2 overexpression resulted in defective bone mineralization and severe hypoplasia of the clavicles, a hallmark of the developmental disease cleidocranial dysplasia. Our results suggest that TGF-beta 2 functions as a local positive regulator of bone remodeling and that alterations in TGF-beta 2 synthesis by bone cells, or in their responsiveness to TGF-beta 2, may contribute to the pathogenesis of metabolic bone disease.     

Recql4 haploinsufficiency in mice leads to defects in osteoblast progenitors: Implications for low bone mass phenotype

The cellular and molecular mechanisms that underlie skeletal abnormalities in defective Recql4-related syndromes are poorly understood. Our objective in this study was to explore the function of Recql4 in osteoblast biology both in vitro and in vivo. Immunohistochemistry on adult mouse bone showed Recql4 protein localization in active osteoblasts around growth plate, but not in fully differentiated osteocytes. Consistent with this finding, Recql4 gene expression was high in proliferating mouse osteoblastic MC3T3.E1 cells and decreased as cells progressively lost their proliferation activity during differentiation. Recql4 overexpression in osteoblastic cells exhibited higher proliferation activity, while its depletion impeded cell growth. In addition, bone marrow stromal cells from male Recql4+/- mice had fewer progenitor cells, including osteoprogenitors, indicated by reduced total fibroblast colony forming units (CFU-f) and alkaline phosphatase-positive CFU-f colonies concomitant with reduced bone mass. These findings provide evidence that Recql4 functions as a regulatory protein during osteoprogenitor proliferation, a critical cellular event during skeleton development.     

Coordinate regulation of fibronectin matrix assembly by the plasminogen activator system and vitronectin in human osteosarcoma cells

Background  

Plasminogen activators are known to play a key role in the remodeling of bone matrix which occurs during tumor progression, bone metastasis and bone growth. Dysfunctional remodeling of bone matrix gives rise to the osteoblastic and osteolytic lesions seen in association with metastatic cancers. The molecular mechanisms responsible for the development of these lesions are not well understood. Studies were undertaken to address the role of the plasminogen activator system in the regulation of fibronectin matrix assembly in the osteoblast-like cell line, MG-63.     

Quelle est la signification des anomalies observées en scintigraphie osseuse ?: Retour sur les mécanismes de fixation des bisphosphonates-(99mTc)

Likely, but still controversial, mechanisms of uptake of bisphosphonates-(^99mTc) are recalled: uptake on organic phase, on mineral phase and/or cellular internalization (osteoclasts and osteoblasts). These hypotheses are critically reviewed in the light of recent progress in pathophysiology of malignant (bone metastases, myeloma) and benign (Paget's disease of bone, fibrous dysplasia) disorders at tissular, cellular and molecular levels, from pharmacology of therapeutic bisphosphonates. Such knowledge improvements have been allowed through in vitro studies, animal models and clinical cases in multimodality imaging (radiology, scintigraphy, pathology).     

Disseminated prostate cancer cells can instruct hematopoietic stem and progenitor cells to regulate bone phenotype

Prostate cancer metastases and hematopoietic stem cells (HSC) frequently home to the bone marrow, where they compete to occupy the same HSC niche. We have also shown that under conditions of hematopoietic stress, HSCs secrete the bone morphogenetic proteins (BMP)-2 and BMP-6 that drives osteoblastic differentiation from mesenchymal precursors. As it is not known, we examined whether metastatic prostate cancer cells can alter regulation of normal bone formation by HSCs and hematopoietic progenitor cells (HPC). HSC/HPCs isolated from mice bearing nonmetastatic and metastatic tumor cells were isolated and their ability to influence osteoblastic and osteoclastic differentiation was evaluated. When the animals were inoculated with the LNCaP C4-2B cell line, which produces mixed osteoblastic and osteolytic lesions in bone, HPCs, but not HSCs, were able to induced stromal cells to differentiate down an osteoblastic phenotype. Part of the mechanism responsible for this activity was the production of BMP-2. On the other hand, when the animals were implanted with PC3 cells that exhibits predominantly osteolytic lesions in bone, HSCs derived from these animals were capable of directly differentiating into tartrate-resistant acid phosphatase-positive osteoclasts through an interleukin-6-mediated pathway. These studies for the first time identify HSC/HPCs as novel targets for future therapy involved in the bone abnormalities of prostate cancer.     

The role of SH3BP2 in the pathophysiology of cherubism

Cherubism is a rare bone dysplasia that is characterized by symmetrical bone resorption limited to the jaws. Bone lesions are filled with soft fibrous giant cell-rich tissue that can expand and cause severe facial deformity. The disorder typically begins in children at ages of 2-5 years and the bone resorption and facial swelling continues until puberty; in most cases the lesions regress spontaneously thereafter. Most patients with cherubism have germline mutations in the gene encoding SH3BP2, an adapter protein involved in adaptive and innate immune response signaling. A mouse model carrying a Pro416Arg mutation in SH3BP2 develops osteopenia and expansile lytic lesions in bone and some soft tissue organs. In this review we discuss the genetics of cherubism, the biological functions of SH3BP2 and the analysis of the mouse model. The data suggest that the underlying cause for cherubism is a systemic autoinflammatory response to physiologic challenges despite the localized appearance of bone resorption and fibrous expansion to the jaws in humans.     

New perspectives in the differentiation of bone-forming cells.

Bone formation comprises a complex but ordered sequence of events which involves the proliferation and differentiation of chondrogenic and osteoblastic precursor cells ultimately leading to the formation of a calcified extracellular matrix. This process can be observed in vivo but under these conditions is difficult to study at the molecular level. A number of in vitro models have been developed which recapitulate discrete elements of this process. Using these models, detailed information has been obtained regarding the differentiation of bone forming cells and the molecular biology of the mineralization process. It has been shown that, in vitro, osteoblastic precursor cells can form a mineralized matrix similar to that seen in vivo. This calcification process was shown to consist of three interdependent phases: proliferation, matrix maturation and mineralization. Each of these phases was characterized by the expression of particular genes. Osteoblast precursors have been cloned and consequently shown to be able to differentiate in vitro into a number of other mesenchymal cells, supporting the theory that osteoblasts are derived from multipotent mesenchymal cells. It is possible that markers derived from these models could be used in the future to extend our knowledge of bone formation in vivo.     

Role of connexins and pannexins during ontogeny,regeneration, and pathologies of bone

Electron micrographs revealed the presence of gap junctions in osteoblastic cells over 40 years ago. These intercellular channels formed from connexins are present in bone forming osteoblasts, bone resorbing osteoclasts, and osteocytes (mature osteoblasts embedded in the mineralized bone matrix). More recently, genetic and pharmacologic studies revealed the role of connexins, and in particular Cx43, in the differentiation and function of all bone types. Furthermore, mutations in the gene encoding Cx43 were found to be causally linked to oculodentodigital dysplasia, a condition that results in an abnormal skeleton. Pannexins, molecules with similar structure and single-membrane channel forming potential as connexins when organized as hemichannels, are also expressed in osteoblastic cells. The function of pannexins in bone and cartilage is beginning to be uncovered, but more research is needed to determine the role of pannexins in bone development, adult bone mass and skeletal homeostasis. We describe here the current knowledge on the role of connexins and pannexins on skeletal health and disease.     

Disruption of extracellular matrix structure may cause pseudoachondroplasia phenotypes in the absence of impaired cartilage oligomeric matrix protein secretion

Pseudoachondroplasia and multiple epiphyseal dysplasia are two dominantly inherited chondrodysplasias associated with mutations in cartilage oligomeric matrix protein (COMP). The rarely available patient biopsies show lamellar inclusions in the endoplasmic reticulum. We studied the pathogenesis of these chondrodysplasias by expressing several disease-causing COMP mutations in bovine primary chondrocytes and found that COMP-associated chondrodysplasias are not exclusively storage diseases. Although COMP carrying the mutations D469Delta and D475N was retained within the endoplasmic reticulum, secretion of COMP H587R was only slightly retarded. All pseudoachondroplasia mutations impair cellular viability and cause a disruption of the extracellular matrix formed in alginate culture irrespective of the degree of cellular retention. The mutation D361Y associated with the clinically milder disease multiple epiphyseal dysplasia gave mild retention and limited matrix alterations, but the transfected cells showed normal viability. The effect of mutated COMP on matrix formation and cell-matrix interaction may be a major element in the pathogenesis of COMP-associated chondrodysplasias.