- Email: [email protected]
Oncogenous hypophosphataemic osteomalacia: effects on phosphate transport and vitamin D metabolism in cultured human kidney cells
Bone Vol. 16, No. 6 June 1995:6794i95
ELSEVIER
ABSTRACTS FROM THE BONE AND TOOTH SOCIETY AUTUMN MEETING, DECEMBER 16, 1994 Venue." University College London Medical School, The Middlesex Hospital, London, UK. Local Organiser: Dr. Sheelagh Farrow, Department of Medicine, University College London Medical School, The Middlesex Hospital, London, U K . O1. Longitudinal changes of bone mineral density in p r i m a r y hyperparathyroidism CY G u o , W Thomas*, R Eastell
a i m e d at the i m m u n o p u r i f i c a t i o n of the factor(s) involved in defective p h o s p h a t e h a n d l i n g , c a n n o w potentially be devised.
Department of Human Metabolism and Clinical Biochemistry and *Department of Surgery, University of Sheffield, Sheffield
0 3 . A relationship between a polymorphism in the first intron of c o l l a g e n I a l p h a I a n d p o s t m e n o p a u s a l osteopomsis SPA Grant, SH Ralston
P r i m a r y h y p e r p a r a t h y r o i d i s m (PHI>T) is a k n o w n risk factor for p o s t m e n o p a u s a l o s t e o p o r o s i s . It is u n c e r t a i n w h e t h e r it is associated with accelerated b o n e loss, a n d w h e t h e r b o n e loss is r e v e r s e d to a s i m i l a r e x t e n t b y s u r g e r y a n d h o r m o n e r e p l a c e m e n t t h e r a p y (HRT). To e x a m i n e this, w e s t u d i e d 36 p o s t m e n o p a u s a l w o m e n w i t h PHPT, ages 48 to 80 years (mean, 63.4). Patients w e r e treated b y s u r g e r y (n, 20), HRT (n, 5), or given no t r e a t m e n t (n, 11). BMD w a s m e a s u r e d at baseline a n d after 18 to 30 m o n t h s (mean, 23) at total b o d y (TB), l u m b a r spine (LS) and femoral n e c k (FN), b y DXA ( L u n a r DPX). W e c o m p a r e d these results w i t h BMD m e a s u r e d in 36 a g e - m a t c h e d healthy w o m e n over 24 m o n t h s . At baseline, BMD w a s decreased at all 3 skeletal sites in p a t i e n t s w h e n c o m p a r e d w i t h controls (p<0.001). The d e c r e a s e in BMD, e x p r e s s e d as Z scores, w a s similar at all 3 skeletal sites. W h e n c o m p a r e d to the rate of c h a n g e of BMD in the control g r o u p , b o n e gain w a s significant at all 3 skeletal sites after s u r g e r y a n d HRT, a n d b o n e loss w a s significant at FN, but not at TB a n d LS, in u n t r e a t e d patients. We c o n c l u d e that p o s t m e n o p a u s a l w o m e n w i t h P H I ~ h a v e accelerated b o n e loss at the f e m o r a l n e c k a n d t h a t b o t h s u r g e r y a n d HRT result in similar increases in BMD at all sites m e a s u r e d .
Department of Medicine and Therapeutics, Polwarth Building, University of Aberdeen, Aberdeen B a c k g r o u n d : Genetic factors are k n o w n to p l a y a role in the p a t h o g e n e s i s of o s t e o p o r o s i s . In this s t u d y , w e looked for evidence of p o l y m o r p h i s m s in the transcriptional control region of the c o l l a g e n I a l p h a 1 g e n e (COLIA1) in p a t i e n t s with p o s t m e n o p a u s a l osteoporosis. M e t h o d s : P o l y m e r a s e c h a i n r e a c t i o n / Single s t r a n d e d c o n f o r m a t i o n a l p o l y m o r p h i s m ( P C R / S S C P ) w a s u s e d , in c o m b i n a t i o n w i t h D N A s e q u e n c i n g , to identify p o l y m o r p h i s m s in the t r a n s c r i p t i o n a l control regions of COLIA] (the p r o m o t e r a n d first intron). Following characterisation, rapid screening was facilitated by PCR site-directed mutagenesis near the p o l y m o r p h i c region to g e n e r a t e a restriction site in the case of the rarer allele. Results: In the first intron, a G to T p o l y m o r p h i s m at the first b a s e of a c o n s e n s u s s e q u e n c e for a n S p l b i n d i n g m o t i f (KGGGCGGRRY) h a d a f r e q u e n c y of 4 heterozygotes out of 20 n o r m a l c o n t r o l s (20%) a n d 13 h e t e r o z y g o t e s o u t of 27 o s t e o p o r o s i s p a t i e n t s (48%). This difference w a s significant at p<0.05. Discussion: These preliminary data show a significant correlation b e t w e e n a p o l y m o r p h i c SpI b i n d i n g motif within the first intron of COLIAI a n d osteoporotic fracture a l t h o u g h larger n u m b e r s of p a t i e n t s a n d controls will need to be screened to confirn this effect a n d to s t u d y its relationship with bone density.
0 2 . Oncogenoua hypophoaphataemic oateomalacia: effects on phosphate transport and vitamin D metabolism in cultured human kidney celia PSN Rowe, M H e w i s o n , FJ Cockerill, JN G o u l d i n g , A O n g , JLH O ' R i o r d a n
Supported by a grant from TENOVUS, Scotland
Department of Medicine, University College London Medical School, Middlesex Hospital, London WIN 8AA O n c o g e n i c h y p o p h o s p h a t a e m i c osteomalacia (OHO) is a t u m o u r acquired s y n d r o m e that results in low levels of s e r u m p h o s p h a t e a n d 1,25-(OH)2D 3. The a i m of this s t u d y w a s to characterise t u m o u r d e r i v e d c o m p o n e n t s r e s p o n s i b l e for the d e f e c t i v e phosphate handling, and vitamin D metabolism. Transformed h u m a n k i d n e y cells (CL8), w e r e f o u n d to express k t - h y d r o x y l a s e in defined s e r u m free m e d i u m (100 fmoles/hr/106cells), a n d also N a + d e p e n d e n t p h o s p h a t e c o - t r a n s p o r t ( N a / P i ) (Snmol P i / m g p r o t e i n / 1 0 min). These cells w e r e exposed to conditioned media f r o m a n O H O t u m o u r (TCM), a n d also skin fibroblasts from the s a m e patient. TCM increased lc~-hydroxylase activity b y 230%, a n d Inhibited N a / P i b y 24%. In a s e c o n d s t u d y pre a n d post o p e r a t i v e s e r u m f r o m the s a m e O H O patient w a s u s e d to raise p o l y c l o n a l a n t i s e r u m to t u m o u r d e r i v e d p r o d u c t s ( p r e - o p antiserum and post-op antiserum respectively). Western a n a l y s i s r e v e a l e d the p r e s e n c e of t w o p r o t e i n s (-48-68 KDa) w h e n p r e - o p a n t i s e r u m w a s u s e d to screen t u m o u r d e r i v e d media. These p r o t e i n s w e r e not detected in the skin fibroblast m e d i a , a n d w e r e not f o u n d w h e n p r e p a r a t i o n s w e r e screened w i t h p o s t - o p a n t i s e r u m . In a d d i t i o n t h e t u m o u r - i n d u c e d c h a n g e s in t i s s u e c u l t u r e w e r e i n h i b i t e d w h e n cells w e r e p r e i n c u b a t e d w i t h p r e b u t n o t p o s t O H O a n t i s e r u m . These studies p r o v i d e a d d i t i o n a l s u p p o r t for a circulating c o m p o n e n t a f f e c t i n g p h o s p h a t e h a n d l i n g , a n d v i t a m i n D m e t a b o l i s m in O H O . F u r t h e r m o r e the t u m o u r factor m a y well be associated w i t h the p r o t e i n s d e t e c t e d b y the p r e - o p antisera. Strategies © 1995 by Elsevier Science Inc.
0 4 . Monoclonal antibodies to 131 integrin block bone resorption MA H o r t o n , P Lakkakorpi*, K V/i/in/inen*, MA Helfrich**
ICRF, London, *Department of Anatomy, University of Oulu, Oulu, Finland and **Department of Medicine and Therapeutics, University of Aberdeen, Aberdeen Tl~e i n v o l v e m e n t of ctvl~3 integrin in osteoclast (OC) a d h e s i o n to, a n d r e s o r p t i o n of, b o n e is well established. OCs also express t w o 131 integrins, ct2131a n d av131. Recognition of collagen b y OCs involves I~1 integrins, b u t their role in b o n e resorption has not b e e n a n a l y s e d . The effect of a d h e s i o n b l o c k i n g m a b s a g a i n s t chick (CSAT), m o u s e (9EG7) a n d h u m a n (mab 13) 131 (up to 5 0 , g / m l ) o n r e s o r p t i o n w a s assessed in a 'pit' assay a n d the d i s t r i b u t i o n of 131 e x a m i n e d u s i n g confocal microscopy. 131 m a b s blocked O C adhesion (up to 75%, p<0.03 ) a n d resorption b y u p to 93% (p<0.001); a s i m i l a r d e g r e e of inhibition w a s seen with echistatin a n d 133 mabs. 131 integrins were a b u n d a n t in basolateral a n d ruffled b o r d e r m e m b r a n e s , b u t u n d e t e c t a b l e in the sealing z o n e of r e s o r b i n g OCs. W e c o n c l u d e t h a t 131 i n t e g r i n s a r e i n v o l v e d in t h e p r o c e s s of b o n e r e s o r p t i o n b y OCs, t h o u g h p r o b a b l y not in the m a i n t e n a n c e of the ion i m p e r m e a b l e tight seal b e t w e e n the O C a n d b o n e matrix. W e are c u r r e n t l y investigating if the inhibitory effects occur at early stages of O C d e v e l o p m e n t , initial interaction w i t h b o n e matrix or via actions on r e g u l a t o r y osteoblastic cells w h i c h also express 131 integrins. 679
8756-3282/95/$9.50 8756-3282(95)00087-T
ELSEVIER
ABSTRACTS FROM THE BONE AND TOOTH SOCIETY AUTUMN MEETING, DECEMBER 16, 1994 Venue." University College London Medical School, The Middlesex Hospital, London, UK. Local Organiser: Dr. Sheelagh Farrow, Department of Medicine, University College London Medical School, The Middlesex Hospital, London, U K . O1. Longitudinal changes of bone mineral density in p r i m a r y hyperparathyroidism CY G u o , W Thomas*, R Eastell
a i m e d at the i m m u n o p u r i f i c a t i o n of the factor(s) involved in defective p h o s p h a t e h a n d l i n g , c a n n o w potentially be devised.
Department of Human Metabolism and Clinical Biochemistry and *Department of Surgery, University of Sheffield, Sheffield
0 3 . A relationship between a polymorphism in the first intron of c o l l a g e n I a l p h a I a n d p o s t m e n o p a u s a l osteopomsis SPA Grant, SH Ralston
P r i m a r y h y p e r p a r a t h y r o i d i s m (PHI>T) is a k n o w n risk factor for p o s t m e n o p a u s a l o s t e o p o r o s i s . It is u n c e r t a i n w h e t h e r it is associated with accelerated b o n e loss, a n d w h e t h e r b o n e loss is r e v e r s e d to a s i m i l a r e x t e n t b y s u r g e r y a n d h o r m o n e r e p l a c e m e n t t h e r a p y (HRT). To e x a m i n e this, w e s t u d i e d 36 p o s t m e n o p a u s a l w o m e n w i t h PHPT, ages 48 to 80 years (mean, 63.4). Patients w e r e treated b y s u r g e r y (n, 20), HRT (n, 5), or given no t r e a t m e n t (n, 11). BMD w a s m e a s u r e d at baseline a n d after 18 to 30 m o n t h s (mean, 23) at total b o d y (TB), l u m b a r spine (LS) and femoral n e c k (FN), b y DXA ( L u n a r DPX). W e c o m p a r e d these results w i t h BMD m e a s u r e d in 36 a g e - m a t c h e d healthy w o m e n over 24 m o n t h s . At baseline, BMD w a s decreased at all 3 skeletal sites in p a t i e n t s w h e n c o m p a r e d w i t h controls (p<0.001). The d e c r e a s e in BMD, e x p r e s s e d as Z scores, w a s similar at all 3 skeletal sites. W h e n c o m p a r e d to the rate of c h a n g e of BMD in the control g r o u p , b o n e gain w a s significant at all 3 skeletal sites after s u r g e r y a n d HRT, a n d b o n e loss w a s significant at FN, but not at TB a n d LS, in u n t r e a t e d patients. We c o n c l u d e that p o s t m e n o p a u s a l w o m e n w i t h P H I ~ h a v e accelerated b o n e loss at the f e m o r a l n e c k a n d t h a t b o t h s u r g e r y a n d HRT result in similar increases in BMD at all sites m e a s u r e d .
Department of Medicine and Therapeutics, Polwarth Building, University of Aberdeen, Aberdeen B a c k g r o u n d : Genetic factors are k n o w n to p l a y a role in the p a t h o g e n e s i s of o s t e o p o r o s i s . In this s t u d y , w e looked for evidence of p o l y m o r p h i s m s in the transcriptional control region of the c o l l a g e n I a l p h a 1 g e n e (COLIA1) in p a t i e n t s with p o s t m e n o p a u s a l osteoporosis. M e t h o d s : P o l y m e r a s e c h a i n r e a c t i o n / Single s t r a n d e d c o n f o r m a t i o n a l p o l y m o r p h i s m ( P C R / S S C P ) w a s u s e d , in c o m b i n a t i o n w i t h D N A s e q u e n c i n g , to identify p o l y m o r p h i s m s in the t r a n s c r i p t i o n a l control regions of COLIA] (the p r o m o t e r a n d first intron). Following characterisation, rapid screening was facilitated by PCR site-directed mutagenesis near the p o l y m o r p h i c region to g e n e r a t e a restriction site in the case of the rarer allele. Results: In the first intron, a G to T p o l y m o r p h i s m at the first b a s e of a c o n s e n s u s s e q u e n c e for a n S p l b i n d i n g m o t i f (KGGGCGGRRY) h a d a f r e q u e n c y of 4 heterozygotes out of 20 n o r m a l c o n t r o l s (20%) a n d 13 h e t e r o z y g o t e s o u t of 27 o s t e o p o r o s i s p a t i e n t s (48%). This difference w a s significant at p<0.05. Discussion: These preliminary data show a significant correlation b e t w e e n a p o l y m o r p h i c SpI b i n d i n g motif within the first intron of COLIAI a n d osteoporotic fracture a l t h o u g h larger n u m b e r s of p a t i e n t s a n d controls will need to be screened to confirn this effect a n d to s t u d y its relationship with bone density.
0 2 . Oncogenoua hypophoaphataemic oateomalacia: effects on phosphate transport and vitamin D metabolism in cultured human kidney celia PSN Rowe, M H e w i s o n , FJ Cockerill, JN G o u l d i n g , A O n g , JLH O ' R i o r d a n
Supported by a grant from TENOVUS, Scotland
Department of Medicine, University College London Medical School, Middlesex Hospital, London WIN 8AA O n c o g e n i c h y p o p h o s p h a t a e m i c osteomalacia (OHO) is a t u m o u r acquired s y n d r o m e that results in low levels of s e r u m p h o s p h a t e a n d 1,25-(OH)2D 3. The a i m of this s t u d y w a s to characterise t u m o u r d e r i v e d c o m p o n e n t s r e s p o n s i b l e for the d e f e c t i v e phosphate handling, and vitamin D metabolism. Transformed h u m a n k i d n e y cells (CL8), w e r e f o u n d to express k t - h y d r o x y l a s e in defined s e r u m free m e d i u m (100 fmoles/hr/106cells), a n d also N a + d e p e n d e n t p h o s p h a t e c o - t r a n s p o r t ( N a / P i ) (Snmol P i / m g p r o t e i n / 1 0 min). These cells w e r e exposed to conditioned media f r o m a n O H O t u m o u r (TCM), a n d also skin fibroblasts from the s a m e patient. TCM increased lc~-hydroxylase activity b y 230%, a n d Inhibited N a / P i b y 24%. In a s e c o n d s t u d y pre a n d post o p e r a t i v e s e r u m f r o m the s a m e O H O patient w a s u s e d to raise p o l y c l o n a l a n t i s e r u m to t u m o u r d e r i v e d p r o d u c t s ( p r e - o p antiserum and post-op antiserum respectively). Western a n a l y s i s r e v e a l e d the p r e s e n c e of t w o p r o t e i n s (-48-68 KDa) w h e n p r e - o p a n t i s e r u m w a s u s e d to screen t u m o u r d e r i v e d media. These p r o t e i n s w e r e not detected in the skin fibroblast m e d i a , a n d w e r e not f o u n d w h e n p r e p a r a t i o n s w e r e screened w i t h p o s t - o p a n t i s e r u m . In a d d i t i o n t h e t u m o u r - i n d u c e d c h a n g e s in t i s s u e c u l t u r e w e r e i n h i b i t e d w h e n cells w e r e p r e i n c u b a t e d w i t h p r e b u t n o t p o s t O H O a n t i s e r u m . These studies p r o v i d e a d d i t i o n a l s u p p o r t for a circulating c o m p o n e n t a f f e c t i n g p h o s p h a t e h a n d l i n g , a n d v i t a m i n D m e t a b o l i s m in O H O . F u r t h e r m o r e the t u m o u r factor m a y well be associated w i t h the p r o t e i n s d e t e c t e d b y the p r e - o p antisera. Strategies © 1995 by Elsevier Science Inc.
0 4 . Monoclonal antibodies to 131 integrin block bone resorption MA H o r t o n , P Lakkakorpi*, K V/i/in/inen*, MA Helfrich**
ICRF, London, *Department of Anatomy, University of Oulu, Oulu, Finland and **Department of Medicine and Therapeutics, University of Aberdeen, Aberdeen Tl~e i n v o l v e m e n t of ctvl~3 integrin in osteoclast (OC) a d h e s i o n to, a n d r e s o r p t i o n of, b o n e is well established. OCs also express t w o 131 integrins, ct2131a n d av131. Recognition of collagen b y OCs involves I~1 integrins, b u t their role in b o n e resorption has not b e e n a n a l y s e d . The effect of a d h e s i o n b l o c k i n g m a b s a g a i n s t chick (CSAT), m o u s e (9EG7) a n d h u m a n (mab 13) 131 (up to 5 0 , g / m l ) o n r e s o r p t i o n w a s assessed in a 'pit' assay a n d the d i s t r i b u t i o n of 131 e x a m i n e d u s i n g confocal microscopy. 131 m a b s blocked O C adhesion (up to 75%, p<0.03 ) a n d resorption b y u p to 93% (p<0.001); a s i m i l a r d e g r e e of inhibition w a s seen with echistatin a n d 133 mabs. 131 integrins were a b u n d a n t in basolateral a n d ruffled b o r d e r m e m b r a n e s , b u t u n d e t e c t a b l e in the sealing z o n e of r e s o r b i n g OCs. W e c o n c l u d e t h a t 131 i n t e g r i n s a r e i n v o l v e d in t h e p r o c e s s of b o n e r e s o r p t i o n b y OCs, t h o u g h p r o b a b l y not in the m a i n t e n a n c e of the ion i m p e r m e a b l e tight seal b e t w e e n the O C a n d b o n e matrix. W e are c u r r e n t l y investigating if the inhibitory effects occur at early stages of O C d e v e l o p m e n t , initial interaction w i t h b o n e matrix or via actions on r e g u l a t o r y osteoblastic cells w h i c h also express 131 integrins. 679
8756-3282/95/$9.50 8756-3282(95)00087-T