Skeletal alkaline phosphatase activity is primarily released from human osteoblasts in an insoluble form, and the net release is inhibited by calcium and skeletal growth factors.
Skeletal alkaline phosphatase (ALP) is anchored to membrane inositol-phosphate on the outer surface of osteoblasts. Although skeletal ALP activity in serum is, essentially, all in an anchorless (soluble) form, in vitro studies indicate that ALP can be released in either an anchorless, soluble form (e.g., by a phospholipase) or an anchor-intact, insoluble form (e.g., by vesicle exocytosis). The current studies were intended to define the contributions of each of these putative processes of ALP release and to assess the significance of regulation by calcium (Ca) and skeletal effectors. ALP activity was measured in serum-free medium from replicate cultures of human osteosarcoma (SaOS-2) cells and normal human bone cells. Temperature-sensitive phase distribution (in Triton X-114) allowed separation of soluble from insoluble ALP activity. Our studies revealed that most of the ALP activity released from SaOS-2 cells was in an insoluble form (78% +/- 8%), a percentage that was constant between 2 and 96 hours. A similar result was seen for normal human bone cells. Calcium had a negative, biphasic dose-dependent effect on net release of ALP activity: r = -0.85, P < 0.001 at 24 hours, with KIapparent values for biphasic inhibition of 20 and 300 mumol/l Ca. Of the skeletal effectors tested, insulin-like growth factor-II (IGF-II) had the greatest effect, decreasing the net release of ALP activity in a dose-dependent manner (r = -0.82, P < 0.005). Neither Ca nor IGF-II affected the distribution of soluble/insoluble ALP activity by more than 9%. IGF-II had no effect on extracellular ALP stability, but the addition of Ca to Ca-free cultures resulted in parallel losses of extracellular ALP activity and ALP immunoreactive protein (P < 0.001 for each). A similar effect was seen when Ca was added to Ca-free, cell-free, conditioned medium, but not when Ca was added to purified ALP, which is consistent with the general hypothesis that a Ca-dependent protease might be present in the cell-conditioned medium. Together, these data suggest that most of the ALP activity released from osteoblasts is insoluble (and, presumably, anchorless), net release of ALP activity is negatively regulated by Ca and skeletal growth factors, the effect of Ca may reflect Ca-dependent protease activity, and an exogenous (e.g., serum) phospholipase may be responsible for releasing ALP from its insoluble anchor.
Anh
D J
DJ
Departments of Medicine and Biochemistry, Loma Linda University, and Jerry L. Pettis Memorial Veterans Medical Center, 11201 Benton Street, Loma Linda, CA 92357, USA.
Dimai
H P
HP
Hall
S L
SL
Farley
J R
JR
D000469:Alkaline Phosphatase / Q000097:blood / Q000187:drug effects* / Q000378:metabolism*; D001842:Bone and Bones / Q000166:cytology / Q000201:enzymology*; D002118:Calcium / Q000008:administration & dosage / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005346:Fibroblast Growth Factors / Q000494:pharmacology; D006133:Growth Substances / Q000494:pharmacology*; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D007335:Insulin-Like Growth Factor II / Q000494:pharmacology; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D010281:Parathyroid Hormone / Q000008:administration & dosage / Q000494:pharmacology; D011506:Proteins / Q000494:pharmacology*; D012995:Solubility; D013997:Time Factors; D014407:Tumor Cells, Cultured
D006133:Growth Substances; C035170:IGF2 protein, human; D010281:Parathyroid Hormone; D011506:Proteins; D005346:Fibroblast Growth Factors; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D000469:Alkaline Phosphatase; D002118:Calcium