- Email: [email protected]
Cellular uptake of bisphosphonates: Localisation using fluorescently- labelled alendronate
572
Abstracts
B o n e V o l . 17, N o . 6 December 1995:557-596
54
56
ROLE OF OSTEOGENIC PROTEIN-I (BMP-7) IN METABOLIC BONE DISEASES TK Sampath. Creative BioMoleculcs, 45 South St., Hopkinton, MA 01748
EXPRESSION OF INSULIN-LIKE GROWTH FACTOR (IGF) I, TYPE 1 IGF RECEPTORS (R) AND RESPONSIVENESS TO IGF I AND IGF BINDING PROTEIN (IGFBP)-3 OF RAT OSTEOBLASTIC CELLS Ch. Schmid. I. SchlSofer. Ch. Veldman. M. B6ni-Schnatzler, J. Zapf, and E.R, Froesch. Division of Endocrinology and Metabolism, Department of Internal Medicine, University Hospital, CH-8091 ZQdch, Switzedand
Osteogenic Protein-1 (OP-l, also called bone morphogenetic p r o t e i m 7, BMP-7) was originally isolated from bone matrix based on its ability to induce new bone formation in vivo. The predicted amino acid sequence of the corresponding gene suggested that OP-I was a member of the TGF II superfamily of proteins. Precfinical studies in non-human primate models have demonstrated that OP-I is effective in inducing new bone formation and in restoring large segmental defects. Human elinical studies are now in progress to evaluate the efficacy of OP-1 and other related bone morphogenetie proteins in the repair of various orthopedic defects. Evaluation of the direct effects of OP-I on bone remodeling events demonstrate that OP-I i) acts as a potent ehemoattractant and mitogen for osteoblast progenitor cells, ii) stimulates the expression of markers that are characteristic of the osteoblast phenotype, iiil increases the local production of IGF's and their binding proteins, and iv) modulates the action of caleitropie hormones both in vitro and in vivo studies. The local injection of OP-I onto periosteal and endosteal bone surfaces induces new bone formation locally and significantly increases the bone mass. These results suggest that in addition to inducing the differentiation of mesenchymal progenitor cells into a lineage of bone forming ceils, OP-I is capable of acting directly on mature osteoblasts. lmmunolocalization and in situ hybridization studies have shown that OP-1 is expressed predominantly in the kidney in post fetal life, suggesting that OP 1 may have an endocrine role in bone homeostasis. In support of this hypothesis, we have preliminary evidence that OP-1 i s present in the circulation and may provide a therapeutic basis for the use o f OP-1 in the treatment of osteoporosis and various metabolic bone diseases. As such, OP-I could prove to be preferable to current treatment modalities which act to prevent bone resorption, but do not stimulate new bone formation. We have recently determined the crystal structure of OP-1 and we have identified OP-I specific type l and type II receptors. These data provide a structural framework for the rational design of small molecules to mimic OP-I action and form the basis for the development of therapeutic agents for the treatment of osteoporosis. Further, the identification of OP-I promoter sequences allows for the screening of libraries of organic compounds that up regulate OP-I expression and this approach may also serve as a basis to identify novel compounds for the treatment of patients with metabolic bone diseases.
55 T H E SERUM C O N C E N T R A T I O N OF INSULIN-LIKE G R O W T H FACTORBINDING PROTEIN-5 (IGFBP-5) IS RELATED T O V I T A M I N D STATUS IN PATIENTS W I T H OSTEOPOROSIS S.H. S c h a r l a . U.G. L e m o e r t . R. gie~,ler. T.L. L i n k h a r t . a n d S. M o h a n . Abtlg. Knochenstoffwechsel & 13ndokrinologie, Klinik am Kurpark, S c h u s s e n r i e d e r Sir. 5, D - 8 8 3 2 6 A u l e n d o r f , G e r m a n y . Insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs) play an important role in the regulation of bone m e t a b o l i s m . IGFBP-5 e x . ~ b i t s a v e r y h i g h a . f f m i t y to h y d r o x y a p a t i t e a n d is b e l i e v e d to r e g u l a t e t h e s t o r a g e o f IGFs I n t h e b o n e m a t r i x . S e c r e t i o n o f IGFBP-5 is s t i m u l a t e d b y o s t e o t r o p i c h o r m o n e s l i k e progesterone. We therefore tested the hypothesis that patients with osteoporosis have altered serum concenCrarinng of IGFBP-5. M e t h o d s : s e r u m IGFBP-5 ( r a d i o i m m u n o a s s a y using a specific antibody), serum 25-hydroxyvitamin D (25(O1-I)D, I N C S T A R radioimmunoassay), u r I n a r y p y r i d i n i u m c r o s s l i n k s (ELISA), a n d l u m b a r b o n e d e n s i t y (DXA, H o l o g i c ) w a s m e a s u r e d I n 22 c o n s e c u t i v e p a t i e n t s ( a g e 5 6 + 1 5 y e a r s , 13 f e m a l e s , 9 m a l e s ) h o s p i t a l i z e d because of primary osteoporosis (patients with secondary o s t e o p o r o s i s w e r e e x c l u d e d ) . All p a t i e n t s u n d e r w e n t b o n e b i o p s y . W e a l s o m e a s u r e d IGFBP-5 i n 12 " n o r m a l " p a t i e n t s h o s p i t a l i z e d because of disease not related to bone. Results: There was no d i f f e r e n c e I n s e r u m IGFBP-5 l e v e l s b e t w e e n o s t e o p o r o t i c and "normal" subjects (367+60 ng/ml versus 372+60 ng/rnl ; mean+SD). However, we detected a significant positive relationship between IGFBP-5 a n d 2 5 ( O I 1 ) D i n o s t e o p o r o t i c p a t i e n t s ( r = 0 . 4 6 , p < O . 0 5 ) . L o w e r s e r u m l e v e l s o f IGFBP-5 t e n d e d t o b e a s s o c i a t e d w i t h h i g h e r urinary pyridirdttm crossUnks, but the relationship did not reach significance (r=-O.47, p=O.lO; n=13). There was no association of IGFBP-5 w i t h s i g n s o f o s t e o m a l a c i a or high turnover in bone b i o p s i e s . C o n c l u s i o n : V i t a m i n s t a t u s a p p e a r s to m o d u l a t e t h e l e v e l o f t h e o s t e o t r o p i c IGFBP-5, w h i c h a d d s e v i d e n c e to t h e r o l e o f v i t a m i n D d e f i c i e n c y a s r i s k f a c t o r f o r osteoporosi~i. H o w e v e r , IGFBP5 w a s n o t g e n e r a l l y l o w e r i n o s t e o p o r o t i c s u b j e c t s a s c o m p a r e d to subjects without bone disease.
Rat calvadal (RC) bone-derived cells grown in culture were compared to an RC bone-dedvod cell line, PyMS. IGF I stimulated sodium-dependent phosphate transport (Pil") in both ostaoblastic cells in e dose-dependent manner. 0.3 and 4 nM IGF I ware required to achieve half maximal, and 10 and 100 nM IGF I to achieve maximal stimulation of PiT in PyMS and in normal (n) RC cells, respectively. 10 nM IGFBP-3 which inhibits IGF I effects decreased basal P[r in nRC but not in PyMS cells. nRC cells express IGF I mRNA (mainly the 7.5 kb transcript) in vitro and produce IGF I. PyMS cells produce much less, if any IGF I: as measured by radioimmunoassay, less than 0.3 ng IGF I accumulated per day and 10a cells in serum-free medium as compared to 1-2 ng in medium of nRC cells. IGF I mRNA was barely detectable: by Northem blot analysis, its abundance in PyMS was less than 1 % of that observed in nRC cells. nRC cells have been reported to express about 25000 type 1 IGF R per cell. As estimated by lzsI-IGF I competition binding and Scatchard transformation studies, PyMS cells exhibit 5-10 times more specific IGF I binding sites with a similar affinity, of 0,5 nM. As assessed by Northern blot analysis of total and poly(A)÷ RNA, IGF I R mRNA (mainly an 11 kb transcript) was much more abundant in PyMS than in nRC cells. When compared to RNA directly dedved from RC bone, IGF I mRNA levels are greater in nRC cells grown in vitro and much lower in the PyMS line; type 1 IGF R mRNA is less prominent in RNA from the RC tissue than in RNA from both types of cultured cells. Condusions: 1. Stimulation by autocdna IGF I contributes to basal PiT in nRC but not in PyMS cells; IGF I expression by nRC cells grown in culture may be higher than IGF I expression by osteoblasts in vivo. 2. The high sensitivity of PyMS cells towards IGF I is consistent with the finding of insignificant production of IGF I together with particularly prominent expression of type 1 IGF R.
57 LITHIUM STIMULATES CELL PROLIFERATION IN HUMAN BONE CELL CULTURES Klaus Seuwen and Rudolf W&lchli, Preclinical Research, Sandoz AG, CH-4002 Basel, Switzerland. Fluoride stimulates bone formation in humans through an undefined mechanism.' At micromolar concentrations, the anion inhibits inositol monophosphatase (IPase), a key enzyme in the phosphoinositide (PI) cycle (Ganzhorn and Chanal, Biochemistry 29: 6065-71, 1990). As the PI cycle is modulated by many growth and differentiation factors we speculatedthat the effect of fluoride on IPase might be in relation to its anabolic action on bone. We therefore comparedfluoride and lifhium, a potent noncompetitiveinhibitor of IPase, measuring intracellular accumulation of inositol phosphates (IPs) and [3H]thymidine incorporation in human bone derived cells. Fluoride led to a weak accumulation of IPs in intact cells following stimulation of PI turnover with endothelin-l. However, relatively high concentrations (300, 1000 uM) were required for significant effects. At the same high concentrations positive effects of fluoride on cell proliferation were observed in some, but not all experiments. Lithium led to a strong IP buildup at millimolar concentrations, as expected. To our surprise, 3 mM lithium gave rise to clearly significant increases (+100%) of [3H]-thymidine incorporation, measured either in the absence of added growth factors or in the presence of IGF-1 and fetal calf serum. The mitogenic effects of lithium persisted in media containing high concentrationsof free inosifol, rendering unlikely the hypothesis that it acts by depleting the intracellular pool of this metabolite, in order to obtain further support for the hypothesis that inhibition of IPase is at the origin of the stimulated DNA synthesis, a panel of bisphosphonate IPase inhibitors was tested. However, although these compounds proved to be potent inhibitors of IPase activity in vitro, they failed to penetrate into cells and no significant stimulation of [3H]-thymidine incorporation could be observed at nontoxic concentrations. Uthium has been shown in the past to stimulate proliferation of thyroid and pancreatic beta cells. In addition, the ion has been reported to stimulate hematopoiasisand to modulate cytokine expression in bone marrow cultures. Our data suggest that lithium may exert positive effects on bone balance in vivo. They may be of relevance for the finding that patients treated long-term with lithium exhibit a tendency for increased bone mass despite lifhium-induced hyperparathyroidism (Nordenstr6m et al., Metabolism 43: 1563-7, 1994). Our data also support the notion that fluoride acts on bone by modulating PI turnover.
Abstracts
B o n e V o l . 17, N o . 6 December 1995:557-596
54
56
ROLE OF OSTEOGENIC PROTEIN-I (BMP-7) IN METABOLIC BONE DISEASES TK Sampath. Creative BioMoleculcs, 45 South St., Hopkinton, MA 01748
EXPRESSION OF INSULIN-LIKE GROWTH FACTOR (IGF) I, TYPE 1 IGF RECEPTORS (R) AND RESPONSIVENESS TO IGF I AND IGF BINDING PROTEIN (IGFBP)-3 OF RAT OSTEOBLASTIC CELLS Ch. Schmid. I. SchlSofer. Ch. Veldman. M. B6ni-Schnatzler, J. Zapf, and E.R, Froesch. Division of Endocrinology and Metabolism, Department of Internal Medicine, University Hospital, CH-8091 ZQdch, Switzedand
Osteogenic Protein-1 (OP-l, also called bone morphogenetic p r o t e i m 7, BMP-7) was originally isolated from bone matrix based on its ability to induce new bone formation in vivo. The predicted amino acid sequence of the corresponding gene suggested that OP-I was a member of the TGF II superfamily of proteins. Precfinical studies in non-human primate models have demonstrated that OP-I is effective in inducing new bone formation and in restoring large segmental defects. Human elinical studies are now in progress to evaluate the efficacy of OP-1 and other related bone morphogenetie proteins in the repair of various orthopedic defects. Evaluation of the direct effects of OP-I on bone remodeling events demonstrate that OP-I i) acts as a potent ehemoattractant and mitogen for osteoblast progenitor cells, ii) stimulates the expression of markers that are characteristic of the osteoblast phenotype, iiil increases the local production of IGF's and their binding proteins, and iv) modulates the action of caleitropie hormones both in vitro and in vivo studies. The local injection of OP-I onto periosteal and endosteal bone surfaces induces new bone formation locally and significantly increases the bone mass. These results suggest that in addition to inducing the differentiation of mesenchymal progenitor cells into a lineage of bone forming ceils, OP-I is capable of acting directly on mature osteoblasts. lmmunolocalization and in situ hybridization studies have shown that OP-1 is expressed predominantly in the kidney in post fetal life, suggesting that OP 1 may have an endocrine role in bone homeostasis. In support of this hypothesis, we have preliminary evidence that OP-1 i s present in the circulation and may provide a therapeutic basis for the use o f OP-1 in the treatment of osteoporosis and various metabolic bone diseases. As such, OP-I could prove to be preferable to current treatment modalities which act to prevent bone resorption, but do not stimulate new bone formation. We have recently determined the crystal structure of OP-1 and we have identified OP-I specific type l and type II receptors. These data provide a structural framework for the rational design of small molecules to mimic OP-I action and form the basis for the development of therapeutic agents for the treatment of osteoporosis. Further, the identification of OP-I promoter sequences allows for the screening of libraries of organic compounds that up regulate OP-I expression and this approach may also serve as a basis to identify novel compounds for the treatment of patients with metabolic bone diseases.
55 T H E SERUM C O N C E N T R A T I O N OF INSULIN-LIKE G R O W T H FACTORBINDING PROTEIN-5 (IGFBP-5) IS RELATED T O V I T A M I N D STATUS IN PATIENTS W I T H OSTEOPOROSIS S.H. S c h a r l a . U.G. L e m o e r t . R. gie~,ler. T.L. L i n k h a r t . a n d S. M o h a n . Abtlg. Knochenstoffwechsel & 13ndokrinologie, Klinik am Kurpark, S c h u s s e n r i e d e r Sir. 5, D - 8 8 3 2 6 A u l e n d o r f , G e r m a n y . Insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs) play an important role in the regulation of bone m e t a b o l i s m . IGFBP-5 e x . ~ b i t s a v e r y h i g h a . f f m i t y to h y d r o x y a p a t i t e a n d is b e l i e v e d to r e g u l a t e t h e s t o r a g e o f IGFs I n t h e b o n e m a t r i x . S e c r e t i o n o f IGFBP-5 is s t i m u l a t e d b y o s t e o t r o p i c h o r m o n e s l i k e progesterone. We therefore tested the hypothesis that patients with osteoporosis have altered serum concenCrarinng of IGFBP-5. M e t h o d s : s e r u m IGFBP-5 ( r a d i o i m m u n o a s s a y using a specific antibody), serum 25-hydroxyvitamin D (25(O1-I)D, I N C S T A R radioimmunoassay), u r I n a r y p y r i d i n i u m c r o s s l i n k s (ELISA), a n d l u m b a r b o n e d e n s i t y (DXA, H o l o g i c ) w a s m e a s u r e d I n 22 c o n s e c u t i v e p a t i e n t s ( a g e 5 6 + 1 5 y e a r s , 13 f e m a l e s , 9 m a l e s ) h o s p i t a l i z e d because of primary osteoporosis (patients with secondary o s t e o p o r o s i s w e r e e x c l u d e d ) . All p a t i e n t s u n d e r w e n t b o n e b i o p s y . W e a l s o m e a s u r e d IGFBP-5 i n 12 " n o r m a l " p a t i e n t s h o s p i t a l i z e d because of disease not related to bone. Results: There was no d i f f e r e n c e I n s e r u m IGFBP-5 l e v e l s b e t w e e n o s t e o p o r o t i c and "normal" subjects (367+60 ng/ml versus 372+60 ng/rnl ; mean+SD). However, we detected a significant positive relationship between IGFBP-5 a n d 2 5 ( O I 1 ) D i n o s t e o p o r o t i c p a t i e n t s ( r = 0 . 4 6 , p < O . 0 5 ) . L o w e r s e r u m l e v e l s o f IGFBP-5 t e n d e d t o b e a s s o c i a t e d w i t h h i g h e r urinary pyridirdttm crossUnks, but the relationship did not reach significance (r=-O.47, p=O.lO; n=13). There was no association of IGFBP-5 w i t h s i g n s o f o s t e o m a l a c i a or high turnover in bone b i o p s i e s . C o n c l u s i o n : V i t a m i n s t a t u s a p p e a r s to m o d u l a t e t h e l e v e l o f t h e o s t e o t r o p i c IGFBP-5, w h i c h a d d s e v i d e n c e to t h e r o l e o f v i t a m i n D d e f i c i e n c y a s r i s k f a c t o r f o r osteoporosi~i. H o w e v e r , IGFBP5 w a s n o t g e n e r a l l y l o w e r i n o s t e o p o r o t i c s u b j e c t s a s c o m p a r e d to subjects without bone disease.
Rat calvadal (RC) bone-derived cells grown in culture were compared to an RC bone-dedvod cell line, PyMS. IGF I stimulated sodium-dependent phosphate transport (Pil") in both ostaoblastic cells in e dose-dependent manner. 0.3 and 4 nM IGF I ware required to achieve half maximal, and 10 and 100 nM IGF I to achieve maximal stimulation of PiT in PyMS and in normal (n) RC cells, respectively. 10 nM IGFBP-3 which inhibits IGF I effects decreased basal P[r in nRC but not in PyMS cells. nRC cells express IGF I mRNA (mainly the 7.5 kb transcript) in vitro and produce IGF I. PyMS cells produce much less, if any IGF I: as measured by radioimmunoassay, less than 0.3 ng IGF I accumulated per day and 10a cells in serum-free medium as compared to 1-2 ng in medium of nRC cells. IGF I mRNA was barely detectable: by Northem blot analysis, its abundance in PyMS was less than 1 % of that observed in nRC cells. nRC cells have been reported to express about 25000 type 1 IGF R per cell. As estimated by lzsI-IGF I competition binding and Scatchard transformation studies, PyMS cells exhibit 5-10 times more specific IGF I binding sites with a similar affinity, of 0,5 nM. As assessed by Northern blot analysis of total and poly(A)÷ RNA, IGF I R mRNA (mainly an 11 kb transcript) was much more abundant in PyMS than in nRC cells. When compared to RNA directly dedved from RC bone, IGF I mRNA levels are greater in nRC cells grown in vitro and much lower in the PyMS line; type 1 IGF R mRNA is less prominent in RNA from the RC tissue than in RNA from both types of cultured cells. Condusions: 1. Stimulation by autocdna IGF I contributes to basal PiT in nRC but not in PyMS cells; IGF I expression by nRC cells grown in culture may be higher than IGF I expression by osteoblasts in vivo. 2. The high sensitivity of PyMS cells towards IGF I is consistent with the finding of insignificant production of IGF I together with particularly prominent expression of type 1 IGF R.
57 LITHIUM STIMULATES CELL PROLIFERATION IN HUMAN BONE CELL CULTURES Klaus Seuwen and Rudolf W&lchli, Preclinical Research, Sandoz AG, CH-4002 Basel, Switzerland. Fluoride stimulates bone formation in humans through an undefined mechanism.' At micromolar concentrations, the anion inhibits inositol monophosphatase (IPase), a key enzyme in the phosphoinositide (PI) cycle (Ganzhorn and Chanal, Biochemistry 29: 6065-71, 1990). As the PI cycle is modulated by many growth and differentiation factors we speculatedthat the effect of fluoride on IPase might be in relation to its anabolic action on bone. We therefore comparedfluoride and lifhium, a potent noncompetitiveinhibitor of IPase, measuring intracellular accumulation of inositol phosphates (IPs) and [3H]thymidine incorporation in human bone derived cells. Fluoride led to a weak accumulation of IPs in intact cells following stimulation of PI turnover with endothelin-l. However, relatively high concentrations (300, 1000 uM) were required for significant effects. At the same high concentrations positive effects of fluoride on cell proliferation were observed in some, but not all experiments. Lithium led to a strong IP buildup at millimolar concentrations, as expected. To our surprise, 3 mM lithium gave rise to clearly significant increases (+100%) of [3H]-thymidine incorporation, measured either in the absence of added growth factors or in the presence of IGF-1 and fetal calf serum. The mitogenic effects of lithium persisted in media containing high concentrationsof free inosifol, rendering unlikely the hypothesis that it acts by depleting the intracellular pool of this metabolite, in order to obtain further support for the hypothesis that inhibition of IPase is at the origin of the stimulated DNA synthesis, a panel of bisphosphonate IPase inhibitors was tested. However, although these compounds proved to be potent inhibitors of IPase activity in vitro, they failed to penetrate into cells and no significant stimulation of [3H]-thymidine incorporation could be observed at nontoxic concentrations. Uthium has been shown in the past to stimulate proliferation of thyroid and pancreatic beta cells. In addition, the ion has been reported to stimulate hematopoiasisand to modulate cytokine expression in bone marrow cultures. Our data suggest that lithium may exert positive effects on bone balance in vivo. They may be of relevance for the finding that patients treated long-term with lithium exhibit a tendency for increased bone mass despite lifhium-induced hyperparathyroidism (Nordenstr6m et al., Metabolism 43: 1563-7, 1994). Our data also support the notion that fluoride acts on bone by modulating PI turnover.