Use of a Phage Display Technique to Identify Potential Osteoblast Binding Sites Within Osteoclast Lacunae

Sheu, Tzong‐Jen; Schwarz, Edward M.; O’Keefe, Regis J.; Rosier, Randy N.; Puzas, J. Edward

doi:10.1359/jbmr.2002.17.5.915

Cited by 46 publications

(51 citation statements)

References 31 publications

(22 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…GST-TRIP and TRAP were incubated for 1 h, and any proteins associated with the GST-TRIP were extracted from the reaction mixture by incubation with glutathione-coated beads. The proteins were analyzed by FarWestern analysis utilizing a phage clone with high affinity for TRAP (27). In lane A of Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In this procedure, two concentrations of TRAP and control proteins (bovine serum albumin, 5 ug) were loaded in a 10% SDS-PAGE gel and electrophoresed. They were then transferred to polyvinylidene difluoride membranes (PerkinElmer Life Sciences) and incubated with 10 10 M13 phage particles from a GPC4 phage (Clone 5), which have been shown to have high affinity and high specificity for TRAP (27). The membrane was washed in PBS with 0.5% (v/v) Tween 20 four times.…”

Section: Methodsmentioning

confidence: 99%

“…The lacunae have been shown to contain substantial amounts of TRAP (27). After 7-10 days of culture, only osteoblasts residing within the lacunae had differentiated to the point of producing histochemically detectable alkaline phosphatase (Fig.…”

Section: Fig 2 Specificity and Affinity Of Trap Binding To Trip-1mentioning

confidence: 99%

“…bovine serum albumin, BSA) were incubated with the GST-TRIP-1 fusion protein, and the molecules associating with the GST-TRIP-1 were extracted by exposure to glutathione beads. The "pulled-down" proteins were separated on a denaturing gel, and the level of TRAP was measured with Far Western analysis (27). Lane A shows that GST has no affinity for TRAP.…”

Section: Fig 2 Specificity and Affinity Of Trap Binding To Trip-1mentioning

confidence: 99%

See 3 more Smart Citations

A Phage Display Technique Identifies a Novel Regulator of Cell Differentiation

Sheu¹,

Schwarz²,

Martinez³

et al. 2003

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The formation of new bone during the process of bone remodeling occurs almost exclusively at sites of prior bone resorption. In an attempt to discover what regulatory pathways are utilized by osteoblasts to effect this site-specific formation event we probed components of an active bone resorption surface with an osteoblast phage expression library. In these experiments primary cultures of rat osteoblasts were used to construct a phage display library in T7 phage. Tartrate-resistant acid phosphatase (type V) (TRAP) was used as the bait in a biopanning procedure. 40 phage clones with very high affinity for TRAP were sequenced, and of the clones with multiple consensus sequences we identified a regulatory protein that modulates osteoblast differentiation. This protein is the TGF␤ receptor-interacting protein (TRIP-1). Our data demonstrate that TRAP activation of TRIP-1 evokes a TGF␤-like differentiation process. Specifically, TRIP-1 activation increases the activity and expression of osteoblast alkaline phosphatase, osteoprotegerin, collagen, and Runx2. Moreover, we show that TRAP interacts with TRIP intracellularly, that activation of the TGF␤ type II receptor by TRIP-1 occurs in the presence of TRAP and that the differentiation process is mediated through the Smad2/3 pathway. A final experiment demonstrates that osteoblasts, when cultured in osteoclast lacunae containing TRAP, rapidly and specifically differentiate into a mature bone-forming phenotype. We hypothesize that binding to TRAP may be one mechanism by which the full osteoblast phenotype is expressed during the process of bone remodeling.The formation of new bone during the process of bone remodeling occurs almost exclusively at sites of prior bone resorption. As there are 1-2 million active remodeling sites in an adult skeleton at any point in time (1), this spatial localization of formation plays a key role in maintaining skeletal architecture. Aberrant or disorganized formation could lead to architectural changes that would weaken skeletal structure.The observation that osteoclastic bone resorption precedes osteoblastic bone formation has been a well known characteristic of the cell activity in the basal metabolic unit (BMU) described by Frost (2), nearly 40 years ago. In a BMU, osteoclastic activation is followed by a reversal phase in which the osteoclast either migrates to another bony site or dies by apoptosis. Osteoblast bone formation then proceeds directly on the reversal line and if the two activities are matched in amount, a constant skeletal mass is preserved. Activation of osteoprogenitor cells in the immediate area of a resorption event has been attributed to the release of growth factors from the bone during osteoclast activity. This process, first termed coupling by Harris and Heany in 1969 (3, 4), accounts for the temporal sequence of resorption preceding formation. However, what has not been appreciated is that the formation occurs at the immediate sites were osteoclast activity occurred.In work performed by Gray, Jones, and Boyde (5,...

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Fig 2 Specificity and Affinity Of Trap Binding To Trip-1mentioning

confidence: 99%

Section: Fig 2 Specificity and Affinity Of Trap Binding To Trip-1mentioning

confidence: 99%

See 2 more Smart Citations

A Phage Display Technique Identifies a Novel Regulator of Cell Differentiation

Sheu¹,

Schwarz²,

Martinez³

et al. 2003

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Only recently, a more direct anabolic role of osteoclasts in stimulating bone formation has been indicated, in which osteoclasts secrete anabolic growth factors that mediate osteoblast chemotaxis, proliferation, differentiation, and mineralization (Koh et al 2005;Martin and Sims 2005;Henriksen et al 2006;Karsdal et al 2007). Some of the osteoclast-secreted factors that may enhance osteoblast activity include TGF-b (Robinson et al 1996), IGF-1 (Hayden et al 1995), TRAP (tartrate-resistant acid phosphatase) (Sheu et al 2002(Sheu et al ,2003, and BMPs (Anderson et al 2000;Dhanyamraju et al 2003;Garimella et al 2006;McCullough et al 2007). BMPs are members of the TGF-b superfamily that have highly conserved seven-cysteine repeats in their carboxy terminus (Wozney 1992).…”

mentioning

confidence: 99%

Expression and Synthesis of Bone Morphogenetic Proteins by Osteoclasts: A Possible Path to Anabolic Bone Remodeling

Garimella

Tague

Zhang

et al. 2008

J Histochem Cytochem.

View full text Add to dashboard Cite

Skeletal remodeling is a finely orchestrated process coupling bone formation to bone resorption. The dynamics of coupling is regulated by the microenvironment at the bone remodeling site, which in turn is influenced by the intercellular communication between cells like osteoclasts and osteoblasts. Understanding the dynamics of coupling is important in devising new therapeutic approaches to the treatment of skeletal diseases characterized by disturbances in the bone remodeling process. In this study, we report the localization of bone morphogenetic proteins (BMPs) in osteoclasts generated from primary cocultures of bone marrow cells from mouse femur and tibia with mouse calvarial osteoblasts, using immunocytochemistry and in situ hybridization. Positive staining was seen in osteoclasts for BMP-2, -4, -6, and -7. Real-time PCR was used to quantitatively confirm the expression of transcripts for BMP-2, BMP-4, and BMP-6 mRNA in murine osteoclasts. Finally, the presence of BMP-2, -4, -6, and-7 proteins was confirmed in osteoclast lysates by Western blotting. Overall, our data suggest a possible direct role for osteoclasts in promoting bone formation via expression and synthesis of BMPs, which then would play an important role in promoting the recruitment, proliferation, and differentiation of osteoblasts at bone resorption sites.

show abstract

Carbonate substituted hydroxyapatite: Resorption by osteoclasts modifies the osteoblastic response

Spence

Patel

Brooks

et al. 2008

J Biomedical Materials Res

View full text Add to dashboard Cite

Carbonate substitution within the hydroxyapatite (HA) lattice improves osteoconduction, although the mechanism by which this occurs is unclear. Discs of dense, sintered, phase-pure HA and AB-type carbonate substituted hydroxyapatite (CHA) were cultured for 21 days with human CD14+ cells in the presence of macrophage-colony stimulating factor (M-CSF) and soluble receptor activator of nuclear factor (NF)-kappaB (sRANKL), during which time osteoclasts developed and resorbed the ceramic surface. Discs were then seeded with human osteoblasts (HOBs), and proliferation and collagen synthesis were measured. On some discs, the conditioned proteinaceous layer left behind by the osteoclasts was preserved. Proliferation of HOBs was increased on resorbed compared to control (unresorbed) surfaces on both materials, provided this osteoclast-conditioned layer was left intact. Collagen synthesis by HOBs was increased on previously resorbed surfaces compared to unresorbed surfaces. This effect was seen on both materials but was seen at an earlier time point on CHA. The results suggest that osteoclasts can condition synthetic bioceramic surfaces and alter the responses of osteoblasts that subsequently populate them. Carbonate substitution may enhance osteoconduction indirectly via effects on enhanced bioresorption.

show abstract

Use of a Phage Display Technique to Identify Potential Osteoblast Binding Sites Within Osteoclast Lacunae

Cited by 46 publications

References 31 publications

A Phage Display Technique Identifies a Novel Regulator of Cell Differentiation

A Phage Display Technique Identifies a Novel Regulator of Cell Differentiation

Expression and Synthesis of Bone Morphogenetic Proteins by Osteoclasts: A Possible Path to Anabolic Bone Remodeling

Carbonate substituted hydroxyapatite: Resorption by osteoclasts modifies the osteoblastic response

Contact Info

Product

Resources

About