Potential role of proprotein convertase SKI-1 in the mineralization of primary bone.
The biochemical mechanism controlling nucleation of mineral crystals in developing bone, along with the growth and propagation of these crystals once formed, remains poorly understood. To define the nucleation mechanism, a proteomics analysis was begun on isolated biomineralization foci (BMF), sites of initial crystal nucleation in osteoblastic cell cultures and in primary bone. Comparative analyses of the protein profile for mineralized BMF with that for total osteoblast cultures revealed the latter were enriched in several proteins including BAG-75 and BSP, as well as fragments of each. When 12 protease inhibitors were added separately to UMR 106-01 osteoblastic cultures, only the serine protease inhibitor 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) blocked cleavage of BAG-75 and BSP, and prevented mineral crystal nucleation within BMF. Consideration of the specificities of the inhibitors tested and the fact that AEBSF inhibition was not dependent upon inclusion of FBS in the culture media indicated that mineral nucleation does not require serine protease plasmin, thrombin, kallikrein, urokinase, C1s or furin. In contrast, SKI-1 (S1P or site-1) is a membrane-bound serine protease inhibitable by AEBSF. We show here for the first time that mineralizing UMR 106 cells express a 98-kDa active, soluble form of SKI-1 within BMF. In contrast, nonmineralizing UMR cells appear to differentially process SKI-1 into smaller immunoreactive fragments (<35 kDa). These findings suggest that SKI-1 plays a direct or indirect role in assembly of functional nucleation complexes containing BAG-75 and BSP and their fragments, thus facilitating initial mineral nucleation within these biomineralization foci.
Gorski
Jeff P
JP
Department of Oral Biology, School of Dentistry, University of Missouri at Kansas City, Kansas City, Mo. 64108, USA. gorskij@umkc.edu
Huffman
Nichole T
NT
Cui
Chaoying
C
Henderson
Ellen P
EP
Midura
Ronald J
RJ
Seidah
Nabil G
NG
D000818:Animals; D001842:Bone and Bones / Q000187:drug effects / Q000201:enzymology*; D002113:Calcification, Physiologic; D015151:Immunoblotting; D051379:Mice; D008954:Models, Biological; D010006:Osteoblasts / Q000187:drug effects / Q000201:enzymology; D043484:Proprotein Convertases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism*; D013450:Sulfones / Q000494:pharmacology
D013450:Sulfones; C002010:4-(2-aminoethyl)benzenesulfonylfluoride; D043484:Proprotein Convertases; D012697:Serine Endopeptidases; C115575:membrane-bound transcription factor peptidase, site 1