title: Effects of diastereoisomers of 1,25-dihydroxyvitamin D3-26,23-lactone on alkaline phosphatase and collagen synthesis in osteoblastic cells.
abstract: The effects of the four diastereoisomers of 1,25-dihydroxyvitamin D3-26,23-lactone (1,25-(OH)2D3-26,23-lactone) on alkaline phosphatase (AP) activity and collagen and noncollagen protein synthesis were examined in cultures of the osteoblastic clone MC3T3-E1 cell line. The four lactone diastereoisomers had little effect on the protein and DNA content of the cells. The 23(S),25(S)- and 23(R),25(R)-1,25-(OH)2D3-26,23-lactones increased AP activity in a linear dose-dependent fashion. Maximal effects were observed at 100 and 1000 pg/ml, respectively. In contrast, the naturally occurring 23(S),25(R)-, 1,25-(OH)2D3-26,23-lactone and the 23(R),25(S)-1,25-(OH)2D3-26,23-lactone showed biphasic stimulatory effects on AP activity. At both 80 and 10,000 pg/ml, they stimulated maximum increases in alkaline phosphatase activity. At 80 pg/ml the 23(S),25(R)- and 23(R),25(S)-isomers stimulated an increase in collagen synthesis, while at 10,000 pg/ml these isomers and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) did not. Moreover, these two isomers (at 10,000 pg/ml) plus insulin or dexamethasone had an additive effect on AP activity, but not at 80 pg/ml. At 80 pg/ml but not at 10,000 pg/ml, the 23(S),25(R)-isomer had an additive effect on AP activity with the simultaneous addition of 25-hydroxyvitamin D3. Relative to 1,25-(OH)2D3, the binding affinities of 23(S),25(S)-, 23(R),25(R)-, 23(S),25(R)- and 23(R),25(S)-1,25-(OH)2D3-26,23-lactones were calculated to be 1/13.0, 1/131.8, 1/805.2, and 1/1083.3, respectively. No metabolites could be detected in the medium when [1-3H]23(S),25(R)-1,25-(OH)2D3-26,23-lactone (the naturally occurring diastereoisomer) was added to the cultures. However, the stimulative effects of 1,25-(OH)2D3 and the 23(S),25(R)-isomer at both concentrations were completely abolished by L-1-tosyl-amido-2-phenylethyl chloromethyl ketone. These results indicate that 1,25-(OH)2D3-lactone has a stimulative effect on osteoblastic cell functions in vitro. The naturally occurring 23(S),25(R)-1,25-(OH)2D3-lactone acts biphasically and may act on bone metabolism in vivo, possibly through a 1,25-(OH)2D3-receptor-mediated pathway.
authors.affiliation: Department of Biochemistry, Teijin Institute for Bio-Medical Research, Tokyo, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Ishizuka
authors.forename: M
authors.initials: M
authors.lastname: Kiyoki
authors.forename: N
authors.initials: N
authors.lastname: Kurihara
authors.forename: Y
authors.initials: Y
authors.lastname: Hakeda
authors.forename: K
authors.initials: K
authors.lastname: Ikeda
authors.forename: M
authors.initials: M
authors.lastname: Kumegawa
authors.forename: A W
authors.initials: AW
authors.lastname: Norman
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism*; D000818:Animals; D002117:Calcitriol / Q000031:analogs & derivatives* / Q000097:blood / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D003094:Collagen / Q000096:biosynthesis*; D003600:Cytosol / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism*; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid / Q000378:metabolism; D013237:Stereoisomerism; D013329:Structure-Activity Relationship
chemical_list: D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; C026947:1,25-dihydroxyvitamin D3-26,23-lactone; D003094:Collagen; D000469:Alkaline Phosphatase; D002117:Calcitriol