PubMed · 2465469
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1989-03-04. Fibronectins and vitronectin.. https://pubmed.ncbi.nlm.nih.gov/2465469/
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The ability of prosthetic wear debris to induce pro-inflammatory responses in macrophages is widely appreciated, but little is known about the molecular mechanisms involved in particle recognition. Specifically, the nature of the cell surface receptors that interact with wear debris is poorly understood. Elucidating the identities of these receptors and how they interact with different types of wear debris are critical to understanding how wear debris initiates periprosthetic osteolysis. We examined the involvement of opsonization, complement receptor 3 (CR3), and scavenger receptor A (SRA), in responses to polymethylmethacrylate (PMMA) and titanium wear particles. Serum dependence of pro-inflammatory responses to PMMA and titanium was tested, and serum proteins that adhered to these two types of particles were identified. Several serum proteins, including known opsonins such as C3bi and fibronectin, adhered to PMMA but not titanium, and serum was required for pro-inflammatory signaling induced by PMMA, but not by titanium. Phagocytosis of PMMA and titanium by macrophages was demonstrated by flow cytometry. Blocking CR3 specifically inhibited phagocytosis of PMMA by macrophages, whereas blocking SRA specifically inhibited titanium uptake. Direct involvement of CR3 and SRA in cell-particle interaction was assessed by expression of these receptors in nonphagocytic HEK293 cells. CR3 specifically induced cell binding to PMMA particles and adhesion to PMMA-coated plates, while SRA specifically induced binding to titanium particles and adhesion to titanium-coated plates. Taken together, these results suggest involvement of opsonization, complement, and integrin receptors, including CR3 and fibronectin receptors, in PMMA action, and an involvement of scavenger receptors in responses to titanium.
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Domain 10 of type III fibronectin (10FNIII) is known to play a pivotal role in the mechanical interactions between cell surface integrins and the extracellular matrix. Recent molecular dynamics simulations have predicted that 10FNIII, when exposed to a stretching force, unfolds along two pathways, each with a distinct, mechanically stable intermediate. Here, we use single-molecule force spectroscopy combined with protein engineering to test these predictions by probing the mechanical unfolding pathway of 10FNIII. Stretching single polyproteins containing the 10FNIII module resulted in sawtooth patterns where 10FNIII was seen unfolding in two consecutive steps. The native state unfolded at 100(+/-20) pN, elongating (10)FNIII by 12(+/-2) nm and reaching a clearly marked intermediate that unfolded at 50(+/-20) pN. Unfolding of the intermediate completed the elongation of the molecule by extending another 19(+/-2) nm. Site-directed mutagenesis of residues in the A and B beta-strands (E9P and L19P) resulted in sawtooth patterns with all-or-none unfolding events that elongated the molecule by 19(+/-2) nm. In contrast, mutating residues in the G beta-strand gave results that were dependent on amino acid position. The mutation I88P in the middle of the G beta-strand resulted in native like unfolding sawtooth patterns showing an intact intermediate state. The mutation Y92P, which is near the end of G beta-strand, produced sawtooth patterns with all-or-none unfolding events that lengthened the molecule by 17(+/-2) nm. These results are consistent with the view that 10FNIII can unfold in two different ways. Along one pathway, the detachment of the A and B beta-strands from the body of the folded module constitute the first unfolding event, followed by the unfolding of the remaining beta-sandwich structure. Along the second pathway, the detachment of the G beta-strands is involved in the first unfolding event. These results are in excellent agreement with the sequence of events predicted by molecular dynamics simulations of the 10FNIII module.