Correction of the mineralization defect in hyp mice treated with protease inhibitors CA074 and pepstatin

Abstract

Increased expression of several osteoblastic proteases and MEPE (a bone matrix protein) occurs in X-linked hypophosphatemic rickets (hyp). This is associated with an increased release of a protease-resistant MEPE peptide (ASARM peptide), a potent inhibitor of mineralization. Cathepsin B cleaves MEPE releasing ASARM peptide and hyp osteoblast/osteocyte cells hypersecrete cathepsin D, an activator of cathepsin B. Our aims were to determine whether cathepsin inhibitors correct the mineralization defect in vivo and whether hyp-bone ASARM peptide levels are reduced after protease treatment. Normal littermates and hyp mice (n = 6) were injected intraperitoneally once a day for 4 weeks with pepstatin, CAO74 or vehicle. Animals were then sacrificed and bones plus serum removed for comprehensive analysis. All hyp mice groups (treated and untreated) remained hypophosphatemic with serum 1,25 vitamin D3 inappropriately normal. Serum PTH was significantly elevated in all hyp mice groups relative to normal mice (P = 0.0017). Untreated hyp mice had six-fold elevated levels of serum alkaline-phosphatase and twofold elevated levels of ASARM peptides relative to normal mice (P < 0.001). In contrast, serum alkaline phosphatase and serum ASARM peptides were significantly reduced (normalized) in hyp mice treated with CA074 or pepstatin. Serum FGF23 levels remained high in all hyp animal groups (P < 0.0001). Hyp mice treated with protease inhibitors showed dramatic reductions in unmineralized osteoid (femurs) compared to control hyp mice (Goldner staining). Also, hyp animals treated with protease inhibitors showed marked and significant improvements in growth plate width (42%), osteoid thickness (40%) and cortical area (40%) (P < 0.002). The mineralization apposition rate, bone formation rate and mineralization surface were normalized by protease-treatment. High-resolution pQCT mineral histomorphometry measurements and uCT also confirmed a marked mineralization improvement. Finally, the growth plate and cortical bone of hyp femurs contained a massive accumulation of osteoblast-derived ASARM peptide(s) that was reduced in hyp animals treated with CA074 or pepstatin. This study confirms in vivo administration of cathepsin inhibitors improves bone mineralization in hyp mice. This may be due to a protease inhibitor mediated decrease in proteolytic degradation of the extracellular matrix and a reduced release of ASARM peptides (potent mineralization inhibitors).

Phosphorylated acidic serine–aspartate-rich MEPE-associated motif peptide from matrix extracellular phosphoglycoprotein inhibits phosphate regulating gene with homologies to endopeptidases on the X-chromosome enzyme activity

“…2) and non-phosphorylated ASARM peptide (data not shown) had no effect on PHEX activity at micromolar levels. The inhibitory effects of MEPE and ASARM-PO 4 on PHEX enzyme activity are consistent with previously reported surface plasmon resonance binding studies (Rowe et al 2006).…”

“…Recently, the importance of proteolytic processing in Hyp has been shown by the improved bone mineralization following the in vivo administration of cathepsin inhibitors (Rowe et al 2006). Secondly, we confirm the ability of the ASARM-PO 4 peptide to regulate mineralization.…”

Role of Cathepsin D in U18666A-induced Neuronal Cell Death

“…Although pepstatin A acts as a general inhibitor of aspartic peptidases, it remains the most potent inhibitor of cathepsin D activity known so far (100). Intraperitoneal administration of pepstatin A has been shown to be beneficial for experimental colitis and bone mineralization (101,102), but it needs to be determined whether intracerebroventricular administration of the inhibitor can mitigate NPC pathology. Given the evidence that selective cathepsin B inhibitor can reduce brain A␤ levels/ deposition and improve cognitive behavioral deficits in animal models of Alzheimer disease (72,103), it is likely that the availability of rather selective and potent cathepsin D inhibitors that can cross the blood-brain barrier may provide a new therapeutic opportunity in the treatment of NPC pathology.…”

Endocrine functions of bone in mineral metabolism regulation

“…In Hyp mice, the elevated proteolytic activity is associated with the increased production of an ASARM peptide from MEPE and DMP1, which has the ability to inhibit mineralization (84) and may represent Minhibin (83). Moreover, the administration of cathepsin inhibitors improves bone mineralization in Hyp mice, without correcting FGF23 expression or hypophosphatemia, indicating the separate regulation of hypophosphatemia and mineralization by PHEX (85).…”