1. ¹Department of Pathology, University of Pittsburgh, and Veteran's Affairs Medical Center, Pittsburgh, PA, USA
2. ²Department of Cell Biology and Physiology, University of Pittsburgh, and Veteran's Affairs Medical Center, Pittsburgh, PA, USA
3. ³Department of Medicine, University of Pittsburgh, and Veteran's Affairs Medical Center, Pittsburgh, PA, USA
4. ⁴Istituto di Tecnologie Biomediche, CNR, Segrate, Italy
5. ⁵Istituto Clinico Humanitas, IRCCS, Rozzano, Italy
6. ⁶Division of Blood and Marrow Transplantation, Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA

Correspondence: Professor HC Blair, MD, Department of Pathology, University of Pittsburgh, 705 Scaife Hall, Pittsburgh, PA 15261, USA. E-mail: hcblair@imap.pitt.edu

Received 26 October 2008; Revised 24 April 2009; Accepted 26 April 2009; Published online 22 June 2009.

Abstract

In vitro differentiated monocytes were used to characterize the cellular defect in a type of osteopetrosis with minimally functional osteoclasts, in which defects associated with common causes of osteopetrosis were excluded by gene sequencing. Monocytes from the blood of a 28-year-old patient were differentiated in media with RANKL and CSF-1. Cell fusion, acid compartments within cells, and tartrate resistant acid phosphatase (TRAP) activity were normal. However, the osteoclasts made abnormally small pits on the dentine. Phalloidin labeling showed that the cell attachments lacked the peripheral ring structure that supports lacunar resorption. Instead, the osteoclasts had clusters of podosomes near the center of cell attachments. Antibody to the v3 integrin pair or to the C-terminal of 3 did not label podosomes, but antibody to v labeled them. Western blots using antibody to the N-terminal of 3 showed a protein of reduced size. Integrins 1 and 5 were upregulated, but, in contrast to observations in 3 defects, 2 had not increased. The -GTP exchange protein Vav3, a key attachment organizing protein, did not localize normally with peripheral attachment structures. Vav3 forms of 70 kD and 90 kD were identified on western blots. However, the proteins 3 integrin, Vav3, Plekhm1, and Src, implicated in attachment defects, had normal exon sequences. In this new type of osteopetrosis, the integrin-organizing complex is dysfunctional, and at least two attachment proteins may be partially degraded.