Fig 7. Osteoclasts downregulate the mannose receptor-dependent recapture mechanism that targets HYAL1 to lysosomes in macrophages.

(A) Osteoclasts derived from RAW264.7 cells were treated for 24 h with 15 mg/mL of mannan (Man) or 5 mM of Man-6-P, and the intracellular amount of HYAL1 was subsequently visualized using western blotting. The graph shows the quantifications of 52 kDa precursor (grey squares) and 48 kDa mature (white squares) HYAL1 forms in n = 3 independent experiments (mean ± SD). (B) Osteoclasts (Ost) and their RAW264.7 precursor macrophages (Mac) were incubated for 5, 10 or 30 min with iodinated rhHYAL1. When indicated, 10 mg/mL of mannan (Man) were added on the cells 5 min prior to the assay and 20 mg/mL during the pulse period. After washing, the proteins bound to the surface were stripped prior to cell lysis and counting of the internalized radioactivity. Three independent experiments were quantified (mean ± SD). (C) After a 2 h incubation at 37°C with rhHYAL1 (± leupeptin [Leu]/pepstatin A [Pepst A]) followed by a 15 min chase, the amount of rhHYAL1 endocytosed by macrophages (Mac) and osteoclasts (Ost) was detected using renatured protein zymography. Of note, only low amounts of proteins are loaded on the gel in these assays; therefore, the endogenous protein is below detection level. One representative experiment is shown (total independent experiments n = 3). (D) The mRNA expression levels of the mannose receptor and CI-MPR were measured by qPCR in macrophages and osteoclasts. The ΔCT values obtained in 3 independent experiments were averaged and used to calculate fold changes in osteoclasts relative to precursor macrophages (2^-ΔΔCT).