Role of abnormal neutral endopeptidase-like activities inHyp mouse bone cells in renal phosphate transport

Abstract

2002.-We investigated whether the absence of Phex (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) in the Hyp mouse affects the expression and activity of neprilysin (NEP) and of endothelin-converting enzyme-like endopeptidase (ECEL1/DINE) in bone marrow stromal cells (BMSC) and osteoblasts (Ob). Total NEP-like activity was higher in Ob than in BMSC regardless of genotype, and Hyp cells showed higher activities than normal. Conditioned media (CM) from Hyp BMSC and Ob inhibited inorganic phosphate (Pi) uptake by mouse proximal tubule cells, and incubating Hyp Ob with phosphoramidon prevented the production of the inhibitor of renal P i uptake. A linear relationship was observed between the NEP-like activity of Hyp and normal cells and the inhibition of Pi uptake. NEP and ECEL1/DINE mRNA levels were higher in Hyp cells than in normal cells, and in situ hybridization of ECEL1/DINE confirmed higher levels of expression in the Hyp mouse than in normal cells. In conclusion, we observed a correlation between the inhibition of Pi uptake by CM from Hyp cells and elevated NEP-like activities.X-linked hypophosphatemia; Phex; putative inhibitor of renal phosphate uptake RENAL PHOSPHATE WASTING in human X-linked hypophosphatemia (XLH) is attributable to a specific reduction of phosphate (P i ) reabsorption in the proximal tubule (reviewed in Ref. 37). Phenotypic characteristics of XLH involve paradoxically low 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] levels in response to hypophosphatemia, osteomalacia, rickets, and lower limb deformities that sometimes require surgery. The murine Hyp model reproduces these characteristics (15,18,48,49) and has been extensively used to study the cause of hypophosphatemia. In recent years it has become evident that the observed hypophosphatemia is not due to an intrinsic renal cell defect but results from humoral mediation (25). This hypothesis was first proposed by Meyer et al. (30) following the observation that parabiosis between a normal and a Hyp mouse results in progressive hypophosphatemia in the normal animal in association with a diminished renal reabsorption of phosphate. Kidney transplantation from normal to Hyp mice and from Hyp to normal mice and measurements of P i uptake by immortalized cell cultures from the renal proximal tubule of normal and Hyp mice also argued in favor of a humoral mediation (32,33). More recently, we demonstrated the direct intervention of a humoral factor, present in Hyp mice, on P i uptake inhibition by primary mouse proximal tubule cells in vitro, and we also showed that Hyp osteoblasts could produce and/or modify this factor (25).The PHEX/Phex (human/mouse) gene (phosphateregulating gene with homologies to endopeptidases on the X chromosome) has been shown to be mutated in XLH patients (16) and in Hyp mice (9). Because PHEX/ Phex codes for a putative neutral endopeptidase, it was suggested that the enzyme could either inactivate an inhibitor of phosphate reabsorption or hydrolyze a propeptide that stimulates phosph...

MEPE has the properties of an osteoblastic phosphatonin and minhibin

“…Thus, sequestration and protection of MEPE and perhaps other matrix proteins by PHEX likely occurs on the extracellular osteoblast surface. Cathepsin D, neprilysin and ECEL-1/DINE proteases are markedly up-regulated in Hyp mice osteoblasts and bone marrow stromal cells (BMSC) [17,34]. The reported massive up-regulation of MEPE, the excess protease expression and the lack of functional PHEX would collectively increase the levels of MEPE ASARM peptide dramatically in Hyp (Fig.…”

“…The OBPTN(s) (osteoblastic in provenance) have the capacity to inhibit renal phosphate handling and mineralization. We would propose that in the Hyp osteoblast, the documented overexpression of cathepsins, ECEL-1/DINE proteases [17,34] MEPE [2,3,29] and lack of functional PHEX [33] results in increased ASARM peptide (minhibin) and also an excess of MEPE-specific phosphate inhibitor peptides [OB-phosphatonin(s)] (Fig. 2).…”

“…Cathepsin B is expressed in the osteoblast together with PHEX and MEPE [1,2,16,26,28,29,50,61,71,75,86]. NEP, ECEL-1/DINE and cathepsin D and MEPE are up-regulated in Hyp osteoblasts that have defective PHEX [2,3,17,29,34]. PHEX protects MEPE from cathepsin B specific cleavage [29] (possibly by sequestration on the cell surface) and prevents release of the ASARM peptide.…”

“…Evidence suggests, however, that other factors may also play a role in this complex bone-renal pathway. The mineralization and phosphate-inhibiting modalities of conditioned media derived from Hyp osteoblasts and normal osteoblast cell lines stably transfected with PHEX [17,51,59,80,101], the confirmation of intrinsic Hyp osteoblast mineralization defects [18,[20][21][22][23][67][68][69]102,103], in osteoblasts/bone indicates that there may be osteoblastic phosphatonins (OB-PTN) and extra-osseous phosphatonins (EO-PTN) molecules and pathways. Both OB-PTN and EO-PTN pathways may be contiguous, overlapping or separate.…”

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

“…The motif also binds to PHEX with high affinity and may interfere with PHEX activity [69]. Of note, PHEX protects MEPE from cathepsin proteolysis [32], and hyp osteoblasts overexpress proteases (cathepsin D, NEP, ECEL1) and MEPE [1,2,7,16,21,31,32,[40][41][42]46,51,67,68,74,77]. Since the ASARM motif is resistant to proteolysis, overproduction of MEPE protein in the presence of excess proteases would be expected to generate large amounts of biologically active protease-resistant ASARM peptide(s) [67][68][69]71].…”