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Calcium (Ca) stimulates dna synthesis and sodium-dependent phosphate (NadPi) transport in rat osteoblastic cells
Bone Vol. 17, No. 6 December 1995:557-596
Abstracts
575
66
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PROSTAGLANDIN E2 INDUCES C-FOS mRNA EXPRESSION IN THE OSTEOBLASTIC CELL LINE MC3T3-E1 USING THE SERUM RESPONSIVE ELEMENT. F. Varga, H. Glantschnio. M. RumDler. K. Klaushofer. Ludwig BoltzmannI Inst'tute of Osteology, 4Lth Med. Dept., Hanusch Hospital, Heinrich-Collin Str. 30, A-1140 Vienna, Austria.
FLUORIDE (F) STIMULATES SODIUM-DEPENDENT (Nad) ALANINE BUT NOT PHOSPHATE TRANSPORT IN HUMAN OSTEOBLASTIC CELLS Ch. M. Veldman, I. Schl/~pfer, Ch. Schmid. Metabolic Unit, Department of Medicine, University Hospital, CH-8091 ZiJrich, Switzerland
Prostaglandins (PGs), particularly PGE2, are produced by bone cells and have powerful effects on bone metabolism (L.G. Raisz et al Osteoporosis Int ,Suppl 1:$136-140, 1993). PGE2 acts through Gprotein coupled receptors and activates the Ca~/PKC-DAG pathways (P.V. Halushka, et al., Annual Rev Pharmacol Toxicol, 29, 213, 1989). In bone cells, PGE2 was found to activate also the cAMP-dependent PKA pathway (O. Kozawa, et al. , Exp Cell Res, 198, 130, 1992). Among the multiple cellular actions of PGE2 on osteoblasts, most importantly for skeletal development, it induces c-fos gene expression (M. Fang, et al., Endocrinology, 131, 2113, 1992). The c-fos promoter region contains several regulatory elements including binding sites for the c-AMP responsive element binding protein (CREB), and for the serum responsive factors (SRF). We used gel mobility shift assay to investigate which signal transduction pathway is used by PGE2 to enhance c-fos expression. MC3T3-E1 cells were seeded at a density of 50.000 cells/cm 2 and cultured for 4 or 8 days in otMEM supplemented with 5% FCS. After 3 hours preculture in ctMEM supplemented with 0.1% BSA cells were treated with PGE2 (2x10 -6 M) for 10, 30 and 60 min. c-fos expression was studied by northern blotting. For gel mobility shift assay nuclear proteins were extracted from PGE2 treated (1 hour) and untreated cells. 32p labelled oligonucleotides of CRE and SRE were incubated with the nuclear protein extracts for 20 min and subjected to agarose gel electrophoresis. We found that in MC3T3-E1 cells PGE2 is a potent inducer of c-fos mRNA expression. The effect was significant already after 10 min, lasting for 1 hour with a peak at 30 min. This stimulation was even stronger than the effect of 10 I.tg/ml EGF. Gel mobility shift assay revealed a significant retardation of the SRE by the nuclear extracts of PGE2 treated cells but not of the CRE. We therefore conclude that PGE2 stimulates c-fos gene expression in osteoblasts via the serum responsive elements of the c-fos promoter rather than via the cAMP responsive element.
F increases trabecular bone mass. Specific effects of F on bone cells in vitro appear to depend on the origin species (chick, rat or human). Our aim was to look for effects of F on Nad alanine and phosphate transport in a well defined human osteoblastic cell line, SAOS-2. 2 x l0 s cells were plated per dish in serum-containing medium. 3d later, they were exposed to test agents in serum-free medium for 24 h. Nad alanine transport was measured as the difference of '4C-alanine uptake in a Na- buffer and a choline-buffer, respectively. NaF at 100 laM increased Nad alan±he transport 2.0_+0.1-fold (mean+SEM of n=27; 9 exp. in triplicate) in SAOS2 cells after 24 h by increasing the Vm~x(24.4_+0.3 pmoI alanine/dish x 10 rain in F-treated cells versus 12.7_+0.4 pmol alanine/dish x 10 min in control cells) but not the K~I (47.7+7.2 ltM in F-treated cells versus 57.3+7.3 r-tM in control cells) of the Naa alanine transport system. The effect of F was dose-dependent and a significant response to F was seen at concentrations as low as 0.3 laM. Under the same conditions F had no effect on Naa phosphate transport. An increase in Nad alanine transport was observed after 0.5 h F also stimulated Nad alanine transport in normal rat osteoblastlike cells. Insulin-like growth factor (IGF) l at 10 ruM also increased Nad alan±he transport in SAOS-2 cells, however, its effect was less selective because it also increased Nad phosphate transport, l0 nM Genistein, a tyrosine kinase blocker did not block the effect of F but that of IGF I on Naa alanine transport. In conclusion, F stimulates Nad alanine transport in human osteoblastic cells in a dose-dependent manner.
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CALCIUM (Ca) STIMULATES DNA SYNTHESIS AND SODIUMDEPENDENT PHOSPHATE (NadPi) TRANSPORT IN RAT OSTEOBLASTIC CELLS Ch M. Veldman, I. Schla.pfer, Ch Schmid. Metabolic Unit, Department of Medicine, University Hospital, CH-8091 Zt~rich, Switzerland
STAR VOLUME OF COMPLETED TRABECULAR WALLS A. V e s t e r b y and K. Meldqaard. D e p a r t m e n t of Forensic Medicine, U n i v e r s i t y of Aarhus, A a r h u s B o n e and M i n e r a l ~ e s e a r c h Group, D K - 8 0 0 0 A a r h u s C, Denmark.
"
A rat bone-derived cell line, PyMS was used to study effects of extraceIlular Ca on DNA synthesis and NaePi transport in rat osteoblastic cells 2 x l0 ~ cells were plated per dish in serum-containing medium 3d later, they were exposed to test agents in serum-free medium (containing 03 mM Ca) for 24 h Addition of Ca stimulated SH-thymidine incorporation into DNA in a dose-dependent manner, up to 5.2+l.8-fold (mean+SEM of n=15; 5 exp in triplicate) at 1 mM (final concentration of t 3 raM) Na,LPi transport was measured as the difference of S2PO4 uptake in a Nabuffer and a choline-buffer, respectively Addition of l mM CaCI2 (to yield a final concentration o i l 3 mM Ca) increased NadPi transport from 158_+4 (control, [0 3 mM Ca]) to 185+2 (1 2-fold) within 2 h and up to 232_+3 pnaol Pi/dish x 10 rain (I 5±0.3-fold (mean±SEM of n=21; 7 exp. in triplicate)) after 24 h The effect of Ca was dose-dependent and a significant response to Ca was seen when the concentration was raised by 0 1 mM (from 0.3 to 0 4 mM Ca) Under the same conditions Ca had neither an effect on the Na-dependent alan±he transport after 2 and 24 h nor on protein content Addition of I mM Ca stimulated alkaline phosphatase activity up to 25±0.5-fold (mean+SEM of n=6; 2 exp. in triplicate). Insulin-like growth factor (IGF) I also stimulated NadPi transport and DNA synthesis, and the effects of I0 nM IGF I (maximally effective) and Ca were additive lO0 nM wortmannin, a phospatidylinositol-3 (PI-3) kinase blocker decreased basal and blocked Ca-stimulated DNA synthesis and did not affect basal and partly inhibited Ca-stimulated NadPi transport, l0 n/vl genistein, a tyrosine kinase blocker did not block basal and Ca- but IGF Istimulated DNA synthesis and Na~Pi transport. We conclude that IGF I and Ca use different intracellular signalling pathways to increase DNA synthesis and NadPi transport in rat osteoblastic cells
To e v a l u a t e b o n e b a l a n c e the w a l l t h i c k n e s s of c o m p l e t e d p a c k e t s o r t r a b e c u l a r o s t e o n e s is o n e of the m o s t i m p o r t a n t p a r a m e t e r s . D i f f e r e n t m e t h o d s for m e a s u r i n g the m e a n w a l l t h i c k n e s s h a v e b e e n used: C a l c u l a t e d from four e q u i d i s t a n t l y s p a c e d m e a s u r e m e n t s of e a c h p r o f i l e ( p r o f i l e referent), r a n d o m s a m p l i n g m e a s u r e m e n t s ( s u r f a c e referent), or s i m p l y b y c o u n t i n g the n u m b e r of l a m e l l a e w i t h i n e a c h c o m p l e t e d packet. T h e s e m e t h o d s h a v e in R o m e s t u d i e s n o t b e e n a b l e to s h o w d i f f e r e n c e s b e t w e e n n o r m a l i n d i v i d u a l s and patients with prlmaryosteporosis, possibly because of t h e i n s e n s i v l t y of the m e t h o d s to d i s c l o s e small d i f f e r e n c e s in the a m o u n t of b o n e formed. The s t a r v o l u m e of a n o b j e c t is the m e a n v o l u m e of the o b j e c t seen from a r a n d o m s a m p l e d p o i n t in all p o s s i b l e and r a n d o m d i r e c t i o n s . The p a r a m e t e r is a n a b s o l u t e 3-D e s t i m a t e (mm3), it is m o d e l free and unbiased. The m a r r o w s p a c e star v o l u m e has s h o w n to be v e r y s e n s i t i v e to d i s c l o s e c h a n g e s of t r a b e c u l a r b o n e s t r u c t u r e in g r o u p s of i n d i v i d u a l s w i t h s m a l l and i n s i g n i f i c a n t d i f f e r e n c e s in the f r a c t i o n a l a m o u n t of b o n e volume. P r e l i m i n a r y s t u d i e s i n d i c a t e t h a t t h i s p a r a m e t e r c a n b e a p p l i e d to c o m p l e t e d p a c k e t s as well, and it m a y be e v e n m o r e s e n s i t i v e t h a n the p a r a m e t e r s u s e d u n t i l n o w in d e m o n s t r a t i n g d i f f e r e n c e s in the a m o u n t of b o n e f o r m e d b y the osteoblasts.
Abstracts
575
66
68
PROSTAGLANDIN E2 INDUCES C-FOS mRNA EXPRESSION IN THE OSTEOBLASTIC CELL LINE MC3T3-E1 USING THE SERUM RESPONSIVE ELEMENT. F. Varga, H. Glantschnio. M. RumDler. K. Klaushofer. Ludwig BoltzmannI Inst'tute of Osteology, 4Lth Med. Dept., Hanusch Hospital, Heinrich-Collin Str. 30, A-1140 Vienna, Austria.
FLUORIDE (F) STIMULATES SODIUM-DEPENDENT (Nad) ALANINE BUT NOT PHOSPHATE TRANSPORT IN HUMAN OSTEOBLASTIC CELLS Ch. M. Veldman, I. Schl/~pfer, Ch. Schmid. Metabolic Unit, Department of Medicine, University Hospital, CH-8091 ZiJrich, Switzerland
Prostaglandins (PGs), particularly PGE2, are produced by bone cells and have powerful effects on bone metabolism (L.G. Raisz et al Osteoporosis Int ,Suppl 1:$136-140, 1993). PGE2 acts through Gprotein coupled receptors and activates the Ca~/PKC-DAG pathways (P.V. Halushka, et al., Annual Rev Pharmacol Toxicol, 29, 213, 1989). In bone cells, PGE2 was found to activate also the cAMP-dependent PKA pathway (O. Kozawa, et al. , Exp Cell Res, 198, 130, 1992). Among the multiple cellular actions of PGE2 on osteoblasts, most importantly for skeletal development, it induces c-fos gene expression (M. Fang, et al., Endocrinology, 131, 2113, 1992). The c-fos promoter region contains several regulatory elements including binding sites for the c-AMP responsive element binding protein (CREB), and for the serum responsive factors (SRF). We used gel mobility shift assay to investigate which signal transduction pathway is used by PGE2 to enhance c-fos expression. MC3T3-E1 cells were seeded at a density of 50.000 cells/cm 2 and cultured for 4 or 8 days in otMEM supplemented with 5% FCS. After 3 hours preculture in ctMEM supplemented with 0.1% BSA cells were treated with PGE2 (2x10 -6 M) for 10, 30 and 60 min. c-fos expression was studied by northern blotting. For gel mobility shift assay nuclear proteins were extracted from PGE2 treated (1 hour) and untreated cells. 32p labelled oligonucleotides of CRE and SRE were incubated with the nuclear protein extracts for 20 min and subjected to agarose gel electrophoresis. We found that in MC3T3-E1 cells PGE2 is a potent inducer of c-fos mRNA expression. The effect was significant already after 10 min, lasting for 1 hour with a peak at 30 min. This stimulation was even stronger than the effect of 10 I.tg/ml EGF. Gel mobility shift assay revealed a significant retardation of the SRE by the nuclear extracts of PGE2 treated cells but not of the CRE. We therefore conclude that PGE2 stimulates c-fos gene expression in osteoblasts via the serum responsive elements of the c-fos promoter rather than via the cAMP responsive element.
F increases trabecular bone mass. Specific effects of F on bone cells in vitro appear to depend on the origin species (chick, rat or human). Our aim was to look for effects of F on Nad alanine and phosphate transport in a well defined human osteoblastic cell line, SAOS-2. 2 x l0 s cells were plated per dish in serum-containing medium. 3d later, they were exposed to test agents in serum-free medium for 24 h. Nad alanine transport was measured as the difference of '4C-alanine uptake in a Na- buffer and a choline-buffer, respectively. NaF at 100 laM increased Nad alan±he transport 2.0_+0.1-fold (mean+SEM of n=27; 9 exp. in triplicate) in SAOS2 cells after 24 h by increasing the Vm~x(24.4_+0.3 pmoI alanine/dish x 10 rain in F-treated cells versus 12.7_+0.4 pmol alanine/dish x 10 min in control cells) but not the K~I (47.7+7.2 ltM in F-treated cells versus 57.3+7.3 r-tM in control cells) of the Naa alanine transport system. The effect of F was dose-dependent and a significant response to F was seen at concentrations as low as 0.3 laM. Under the same conditions F had no effect on Naa phosphate transport. An increase in Nad alanine transport was observed after 0.5 h F also stimulated Nad alanine transport in normal rat osteoblastlike cells. Insulin-like growth factor (IGF) l at 10 ruM also increased Nad alan±he transport in SAOS-2 cells, however, its effect was less selective because it also increased Nad phosphate transport, l0 nM Genistein, a tyrosine kinase blocker did not block the effect of F but that of IGF I on Naa alanine transport. In conclusion, F stimulates Nad alanine transport in human osteoblastic cells in a dose-dependent manner.
67
69
CALCIUM (Ca) STIMULATES DNA SYNTHESIS AND SODIUMDEPENDENT PHOSPHATE (NadPi) TRANSPORT IN RAT OSTEOBLASTIC CELLS Ch M. Veldman, I. Schla.pfer, Ch Schmid. Metabolic Unit, Department of Medicine, University Hospital, CH-8091 Zt~rich, Switzerland
STAR VOLUME OF COMPLETED TRABECULAR WALLS A. V e s t e r b y and K. Meldqaard. D e p a r t m e n t of Forensic Medicine, U n i v e r s i t y of Aarhus, A a r h u s B o n e and M i n e r a l ~ e s e a r c h Group, D K - 8 0 0 0 A a r h u s C, Denmark.
"
A rat bone-derived cell line, PyMS was used to study effects of extraceIlular Ca on DNA synthesis and NaePi transport in rat osteoblastic cells 2 x l0 ~ cells were plated per dish in serum-containing medium 3d later, they were exposed to test agents in serum-free medium (containing 03 mM Ca) for 24 h Addition of Ca stimulated SH-thymidine incorporation into DNA in a dose-dependent manner, up to 5.2+l.8-fold (mean+SEM of n=15; 5 exp in triplicate) at 1 mM (final concentration of t 3 raM) Na,LPi transport was measured as the difference of S2PO4 uptake in a Nabuffer and a choline-buffer, respectively Addition of l mM CaCI2 (to yield a final concentration o i l 3 mM Ca) increased NadPi transport from 158_+4 (control, [0 3 mM Ca]) to 185+2 (1 2-fold) within 2 h and up to 232_+3 pnaol Pi/dish x 10 rain (I 5±0.3-fold (mean±SEM of n=21; 7 exp. in triplicate)) after 24 h The effect of Ca was dose-dependent and a significant response to Ca was seen when the concentration was raised by 0 1 mM (from 0.3 to 0 4 mM Ca) Under the same conditions Ca had neither an effect on the Na-dependent alan±he transport after 2 and 24 h nor on protein content Addition of I mM Ca stimulated alkaline phosphatase activity up to 25±0.5-fold (mean+SEM of n=6; 2 exp. in triplicate). Insulin-like growth factor (IGF) I also stimulated NadPi transport and DNA synthesis, and the effects of I0 nM IGF I (maximally effective) and Ca were additive lO0 nM wortmannin, a phospatidylinositol-3 (PI-3) kinase blocker decreased basal and blocked Ca-stimulated DNA synthesis and did not affect basal and partly inhibited Ca-stimulated NadPi transport, l0 n/vl genistein, a tyrosine kinase blocker did not block basal and Ca- but IGF Istimulated DNA synthesis and Na~Pi transport. We conclude that IGF I and Ca use different intracellular signalling pathways to increase DNA synthesis and NadPi transport in rat osteoblastic cells
To e v a l u a t e b o n e b a l a n c e the w a l l t h i c k n e s s of c o m p l e t e d p a c k e t s o r t r a b e c u l a r o s t e o n e s is o n e of the m o s t i m p o r t a n t p a r a m e t e r s . D i f f e r e n t m e t h o d s for m e a s u r i n g the m e a n w a l l t h i c k n e s s h a v e b e e n used: C a l c u l a t e d from four e q u i d i s t a n t l y s p a c e d m e a s u r e m e n t s of e a c h p r o f i l e ( p r o f i l e referent), r a n d o m s a m p l i n g m e a s u r e m e n t s ( s u r f a c e referent), or s i m p l y b y c o u n t i n g the n u m b e r of l a m e l l a e w i t h i n e a c h c o m p l e t e d packet. T h e s e m e t h o d s h a v e in R o m e s t u d i e s n o t b e e n a b l e to s h o w d i f f e r e n c e s b e t w e e n n o r m a l i n d i v i d u a l s and patients with prlmaryosteporosis, possibly because of t h e i n s e n s i v l t y of the m e t h o d s to d i s c l o s e small d i f f e r e n c e s in the a m o u n t of b o n e formed. The s t a r v o l u m e of a n o b j e c t is the m e a n v o l u m e of the o b j e c t seen from a r a n d o m s a m p l e d p o i n t in all p o s s i b l e and r a n d o m d i r e c t i o n s . The p a r a m e t e r is a n a b s o l u t e 3-D e s t i m a t e (mm3), it is m o d e l free and unbiased. The m a r r o w s p a c e star v o l u m e has s h o w n to be v e r y s e n s i t i v e to d i s c l o s e c h a n g e s of t r a b e c u l a r b o n e s t r u c t u r e in g r o u p s of i n d i v i d u a l s w i t h s m a l l and i n s i g n i f i c a n t d i f f e r e n c e s in the f r a c t i o n a l a m o u n t of b o n e volume. P r e l i m i n a r y s t u d i e s i n d i c a t e t h a t t h i s p a r a m e t e r c a n b e a p p l i e d to c o m p l e t e d p a c k e t s as well, and it m a y be e v e n m o r e s e n s i t i v e t h a n the p a r a m e t e r s u s e d u n t i l n o w in d e m o n s t r a t i n g d i f f e r e n c e s in the a m o u n t of b o n e f o r m e d b y the osteoblasts.