Dephosphorylation of osteopontin and bone sialoprotein by osteoclastic tartrate-resistant acid phosphatase. Modulation of osteoclast adhesion in vitro.

S U M M A R YBecause the development and activity of osteoclasts in bone remodeling is critically dependent on cell-cell and cell-matrix interactions, we used laser confocal microscopy to study the response of osteoclasts to lipopolysaccharide (LPS; 10 g/ml), parathyroid hormone (PTH; 10 Ϫ 8 M), and bisphosphonates (BPs; 1-25 M clodronate or 0.1-2.5 M risedronate) in cultured neonatal calvaria. Following treatment with LPS or PTH ( Ͻ 48 hr), osteopontin (OPN) and the ␣ v ␤ 3 integrin were found colocalized with the actin ring in the sealing zone of actively resorbing osteoclasts. In contrast, non-resorbing osteoclasts in BPtreated cultures showed morphological abnormalities, including retraction of pseudopods and vacuolization of cytoplasm. In the combined presence of LPS and BP, bone-resorbing osteoclasts were smaller and the sealing zone diffuse, reflecting reduced actin, OPN, and ␤ 3 integrin staining. Depth analyses of calvaria showed that the area of resorbed bone was filled with proliferating osteoblastic cells that stained for alkaline phosphatase, collagen type I, and bone sialoprotein, regardless of the presence of BPs. These studies show that confocal microscopy of neonatal calvaria in culture can be used to assess the cytological relationships between osteoclasts and osteoblastic cells in response to agents that regulate bone remodeling in situ, avoiding systemic effects that can compromise in vivo studies and artifacts associated with studies of isolated osteoclasts. T he modeling and remodeling that occurs throughout the growth and development of bones requires a close coordination between the activities of the boneforming osteoblasts and the bone-resorbing osteoclasts. Loss of cooperativity between these cells can result in the development of an abnormal skeleton and, in the adult, decreased (osteoporosis) or increased (osteopetrosis) bone mass. Regulation of bone remodeling involves systemic control by hormones such as parathyroid hormone (PTH) and 1,25 di-hydroxyvitamin D3 (vitamin D3), and cytokines displaying autocrine and paracrine functions that provide local communication between osteoblasts and osteoclasts. Remodeling of adult bone occurs continuously as a requirement of functional adaptation and for calcium homeostasis. In response to decreased serum calcium, PTH and vitamin D3 restore calcium levels through their effects on the intestine, kidney, and bone. Both PTH and vitamin D3 increase bone resorption by osteoclasts to release calcium through the dissolution of the hydroxyapatite crystals. However, these effects are largely mediated by osteoblastic cells, which express cell surface and steroid receptors for the PTH and vitamin D3, respectively. The activity of osteoclasts is also targeted directly and indirectly by inflammatory mediators during fracture repair and in the formation of bone metastases, as well as in response to bacterial infection. Lipopolysaccharides (LPS) produced by bacteria are potent stimulators of osteoclastic resorption, functioning through

Section: Discussionmentioning

confidence: 99%

Osteoclast Responses to Lipopolysaccharide, Parathyroid Hormone and Bisphosphonates in Neonatal Murine Calvaria Analyzed by Laser Scanning Confocal Microscopy

Suzuki

Takeyama

Kikuchi

et al. 2005

“…However, the actual role(s) of this enzyme remains to be explored. In osteoclasts, TRAP has been suggested to generate free radicals and to directly participate in bone resorption (Hayman and Cox 1994), to dephosphorylate osteopontin and bone sialoprotein, and to serve as an osteoclast detachment factor (Ek-Rylander et al 1994). TRAP is also suggested to serve as a protein tyrosine phosphatase (Halleen et al 1998) and to modulate intracellular vesicular transport (Hollberg et al 2002).…”

Section: Discussionmentioning

confidence: 99%

Eccentric Localization of Osteocytes Expressing Enzymatic Activities, Protein, and mRNA Signals for Type 5 Tartrate-resistant Acid Phosphatase (TRAP)

Nakano

Toyosawa

Takano

2004

Enzymatic activity of type 5 tartrate-resistant acid phosphatase (TRAP) has been regarded as one of the reliable markers for osteoclasts and their precursors. The presence of TRAP activity in osteocytes near the bone resorbing surface has also been pointed out in some reports. However, the significance of TRAP reactions in osteocytes remains controversial and, in fact, there is no agreement as to whether the histochemical enzyme reactions in osteocytes represent the TRAP enzyme generated by the respective osteocytes or is a mere diffusion artifact of the reaction products derived from the nearby osteoclasts. Current histochemical, immunohistochemical, and in situ hybridization studies of rat and canine bones confirmed TRAP enzyme activity, TRAP immunoreactivity, and the expression of Trap mRNA signals in osteocytes located close to the bone-resorbing surface. TRAP/Trap- positive osteocytes thus identified were confined to the areas no further than 200 microm from the bone-resorbing surface and showed apparent upregulation of TRAP/Trap expression toward the active osteoclasts. Spatial and temporal patterns of TRAP/Trap expression in the osteocytes should serve as a valuable parameter for further analyses of biological interactions between the osteocytes and the osteoclasts associated with bone remodeling.

“…In bone, TRAP has been ascribed a role in bone resorption on the basis of its localization to osteoclasts (Andersson and Marks 1989;Ek-Rylander et al 1991;Yaziji et al 1995) and its ability to dephosphorylate bone matrix phosphoproteins such as bone sialoprotein and osteopontin (OPN) (Ek-Rylander et al 1994). OPN is known as an anchor for the binding of osteoclasts to the bone via integrin receptors (Reinholt et al 1990a,b;Flores et al 1992), suggesting that one potential physiological function of TRAP is to regulate osteoclast attachment to or mobility on bone.…”

Section: Figure 10mentioning

confidence: 99%

“…A role for TRAP in the resorption of bone and in cartilage breakdown in vivo is indi-cated by the skeletal alterations observed in TRAP knockout mice (Hayman et al 1996). TRAP is a phosphoprotein phosphatase acting on phosphoproteins such as bone sialoprotein, osteopontin and osteonectin present in the bone matrix (Ek-Rylander et al 1994;Andersson and Ek-Rylander 1995;Ljusberg et al 1999). The role of PAP in other tissues is less well understood.…”

mentioning

confidence: 99%

Expression and Distribution of Tartrate-resistant Purple Acid Phosphatase in the Rat Nervous System

Lång

Schultzberg

Andersson

2001

S U M M A R Y Tartrate-resistant purple acid phosphatase (TRAP) of osteoclasts and certain cells of the monocyte-macrophage lineage belongs to the family of purple acid phosphatases (PAPs). We provide here evidence for TRAP/PAP expression in the central and peripheral nervous systems in the rat. TRAP/PAP protein was partially purified and characterized from the trigeminal ganglion, brain, and spinal cord. The TRAP activity (U/mg tissue) in these tissues was about 10-20 times lower than in bone. Reducing agents, e.g. ascorbate and ferric iron, increased the TRAP activity from the neural tissues (nTRAP) and addition of oxidizing agents completely inactivated both bone and nTRAP. The IC 50 for three known oxyanion inhibitors of TRAP/PAP was similar for bone and nTRAP with the same rank order of potency (molybdate Ͼ tungstate Ͼ phosphate). This indicates that the redox-sensitive binuclear iron center characteristic of mammalian PAPs is present also in nTRAP. Western blots of partially purified nTRAP revealed a band with the expected size of 35 kD. The expression of TRAP in the trigeminal ganglion, brain, and spinal cord was confirmed at the mRNA level by RT-PCR. In situ hybridization histochemistry demonstrated TRAP mRNA expression in small ganglion cells of the trigeminal ganglion, in ␣ -motor neurons of the ventral spinal cord, and in Purkinje cells of the cerebellum. TRAP-like immunoreactivity was encountered in the cytoplasm of neuronal cell bodies in specific areas of both the central and the peripheral nervous system. Together, the data demonstrate that active TRAP/PAP is expressed in certain parts of the rat nervous system.