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Apoptosis may determine the release of skeletal alkaline phosphatase activity from human osteoblast-line cells

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Abstract

Although quantitative measurement of skeletal alkaline phosphatase (sALP) activity in serum can provide an index of the rate of bone formation, the metabolic process that determines the release of sALP - from the surface of osteoblasts, into circulation—is unknown. The current studies were intended to examine the hypothesis that the release of sALP from human osteoblasts is a consequence of apoptotic cell death. We measured the release of sALP activity from human osteosarcoma (SaOS-2) cells and normal human bone cells, under basal conditions and in response to agents that increased apoptosis (TNF-α, okadiac acid) and agents that inhibit apoptosis (IGF-I, calpain, and caspase inhibitors). Apoptosis was determined by the presence of nucleosomes (histone-associated DNA) in the cytoplasm of the cells by using a commercial kit. The results of these studies showed that TNF-α and okadiac acid caused dose-and time-dependent increases in apoptosis in the SaOS-2 cells (r=0.78 for doses of TNF-α and r=0.93 for doses of okadiac acid,P<0.005 for each), with associated decreases in cell layer protein (P<0.05 for each) and concomitant increases in the release of sALP activity (e.g., r=0.89 for TNF-α and r=0.75 for okadiac acid,P<0.001 for each). In contrast, caspase and calpain inhibitors reduced apoptosis, increased cell layer protein, and decreased the release of sALP activity (P<0.05 for each). Exposure to IGF-I also decreased apoptosis, in a time- and dose-dependent manner (e.g., r=0.93,P<0.001 for IGF-I doses), with associated proportional effects to increase cell layer protein (P<0.001) and decrease the relase of sALP activity (P<0.001). IGF-I also inhibited the actions of TNF-α and okadiac acid to increase apoptosis and sALP release. The associations between apoptosis and sALP release were not unique to osteosarcoma (i.e., SaOS-2) cells, but also seen with osteoblast-line cells derived from normal human bone. Together, these data demonstrate that the release of sALP activity from human osteoblast-line cellsin vitro is associated with, and may be a consequence of, apoptotic cell death. These findings are consistent with the general hypothesis that the appearance of sALP activity in serum may reflect the turnover of osteoblast-line cells.

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Abbreviations

ALP:

alkaline phosphatase

ANOVA:

analysis of variance

BSA:

bovine serum albumin

calpain inhibitor I:

(Nacetyl-leucyl-leucyl-norleucinal)

calpain inhibitor II:

Nacetyl-leucyl-leucyl-methional

caspase inhibitor:

z-valyl-alanyl-aspartyl(O-methoxy)CH2F

caspase substrate IV:

acetyl-tyrosyl-valyl-alanyl-aspartyl-p-nitroanalide -a colorimetric substrate for caspase activity

DMEM:

Dulbecco’s modified Minimum Essential Medium

DTT:

dithio-threitol

ELISA:

enzyme-linked immunosorbant assay

FCS:

fetal calf serum

IGF-I:

insulin-like growth factor I

PBS:

phosphate-buffered saline

PNPP:

p-nitrophenyl-phosphate (a colorimetric substrate for ALP activity)

SEM:

standard error of the mean

TCA:

trichloroacetic acid

TNF-α:

tumor necrosis factor-alpha

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Authors and Affiliations

  1. Departments of Medicine and Biochemistry, Loma Linda University, 11201 Benton Street, 92357, Loma Linda, California, USA

    J. R. Farley & B. Stilt-Coffing

  2. Jerry L. Pettis Memorial Veterans Medical Center, 11201 Benton Street, 92357, Loma Linda, California, USA

    J. R. Farley & B. Stilt-Coffing

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  1. J. R. Farley
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  2. B. Stilt-Coffing
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Farley, J.R., Stilt-Coffing, B. Apoptosis may determine the release of skeletal alkaline phosphatase activity from human osteoblast-line cells. Calcif Tissue Int 68, 43–52 (2001). https://doi.org/10.1007/BF02685002

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