Mechanisms of Osteoclast Dysfunction in Human Osteopetrosis: Abnormal Osteoclastogenesis and Lack of Osteoclast-Specific Adhesion Structures

Abstract

Osteoclasts from a patient affected by osteopetrosis were examined in vivo and in vitro. Iliac crest biopsy revealed an osteosclerotic pattern, with prominent numbers of osteoclasts noted for hypernuclearity and incomplete adherence to the bone surface. A population comprising tartrate-resistant acid phosphatase (TRAP)-positive, multinucleated and mononuclear cells, and alkaline phosphatase-positive stromal fibroblasts was obtained in vitro from bone marrow. Mononuclear TRAP-positive precursors spontaneously fused in culture to form giant osteoclast-like cells. These cells expressed the osteoclast marker MMP-9 and calcitonin receptor, and lacked the macrophage marker, Fc receptor. Expression and distribution of c-src, c-fms, and CD68, and response to steroid hormones relevant to osteoclast differentiation and function were apparently normal, whereas cell retraction in response to calcitonin was impaired. TRAP-positive multinucleated cells did not form osteoclast-specific adhesion structures (clear zone, podosomes, or actin rings). Bone resorption rate was severely reduced in vitro. Focal adhesions and stress fibers were observed en lieu of podosomes and actin rings. Adhesion structures contained low levels of immunoreactive vitronectin receptor, most of this integrin being retained in cytoplasmic vesicles. These data provide the first characterization of abnormal differentiation and function of human osteopetrotic osteoclastlike

Genotype-Phenotype Relationship in Human ATP6i-Dependent Autosomal Recessive Osteopetrosis

“…Membrane distribution of the vitronectin ␣V␤3 integrin receptor was unremarkable ( Figure 10, C and D). Thus, at variance with previous observations by us and others, 27,28 and similar to what observed by Flanagan and colleagues 29 in patients affected by malignant osteopetrosis of unknown genetic origin, no obvious alteration of the adhesion and polarization patterns is observed in our ATP6i-dependent patients. Consistently, in the osteopetrotic osteoclasts PYK2 and c-Src appeared normally distributed at the paramarginal area overlapping the actin ring ( Figure 10, E and F) where these tyrosine kinases are supposed to contribute to integrin-mediated osteoclast adhesion and outside-in signaling.…”

“…Nevertheless, relative to another case of osteopetrosis investigated in our laboratory, 27 where the ruffled borders of the osteoclasts were lacking and the adhesion properties altered, the osteoclasts of our ATP6i-positive patients showed no obvious morphological alterations by routine histology. Holliday and colleagues 12 and Lee and colleagues 13 have demonstrated that the mouse osteoclast V-ATPase interacts with microfilaments during bone resorption and proposed that proton transport by this pump is associated with reorganization of the actin cytoskeleton during osteoclast activation.…”

“…Long-term cultures resulted in the overgrowth of a stromal-like cell population, both in the patient and in the normal controls. However, histochemical staining revealed the presence of TRAP-positive giant cells only in the patient ( Figure 8B), whereas, as already described, 27 in the cultures of controls no TRAPpositive cells or only TRAP-positive putative mononuclear osteoclast precursors were observed (not shown). Despite the large numbers of osteoclasts in the bone marrow culture of the patient, only small pits could be detected ( Figure 8C), thus indicating a failure in the resorbing function of the giant cells.…”

Osteoclast diseases

“…A number of centres have described the successful generation of osteoclasts from cord blood (Cournot et al, 1993), peripheral blood (Flanagan et al, 2000;Helfrich and Gerritsen, 2001), or bone marrow (Teti et al, 1999) obtained from patients with "osteoclast-rich" osteopetrosis. In all studies, osteoclasts formed, but were defective in bone resorption.…”

Osteoclast-Derived Serum Tartrate-Resistant Acid Phosphatase 5b in Albers-Schönberg Disease (Type II Autosomal Dominant Osteopetrosis)

“…A histologic evaluation of bone biopsies from ADO2 patients reveals a marked increase in the number and size of osteoclasts, which demonstrate no signs of active bone resorption (5,14,15 ). Ultrastructural examinations have shown that osteoclasts from ADO2 patients have no ruffled borders, but they do have normal clear zones and numerous lysosomal vacuoles containing dense substances (16 ).…”