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Response of rat osteoclasts to small shifts in extracellular pH: evidence for an “on-off switch”?
Bone Vol. 16, No. 6 June 1 9 9 5 : 6 7 9 ~ 9 5
Abstracts
i m m u n o r e a c t i v i t y w e r e identified u s i n g the A P A A P p r o c e d u r e in the p r e s e n c e of l e v a m i s o l e to inhibit e n d o g e n o u s , alkaline p h o s p h a t a s e activity. Bcl-2 protein w a s localised specifically and r e p r o d u c i b l y to osteoblasts actively e n g a g e d in b o n e formation. Using a combination histochemistry and immunocytochemisty it w a s s h o w n t h a t the cells expressing Bcl-2 also expressed high levels of a l k a l i n e p h o s p h a t a s e activity. This represents the first d e m o n s t r a t i o n t h a t t h e p r o d u c t of a g e n e i n v o l v e d in the r e g u l a t i o n of P C D is e x p r e s s e d in a d u l t h u m a n b o n e a n d s t r o n g l y s u g g e s t s t h a t a p o p t o s i s is an i m p o r t a n t m e c h a n i s m for r e g u l a t i n g the size a n d ultimate fate of the osteoblast population.
0 1 0 . R e s p o n s e of rat osteoclasts to small shifts in extracellular pH: e v i d e n c e for an "on-off switch"?. M Spowage*, TR Arnett
Department of Anatomy and Developmental Biology, University College London, London WCIE and *Boots Pharmaceuticals Research Department, Nottingham NG2 3AA P r e v i o u s s t u d i e s s h o w e d that in c u l t u r e m e d i a buffered with HEPES alone, b o n e r e s o r p t i o n b y d i s a g g r e g a t e d rat osteoclasts (OCs) i n c r e a s e d s t e a d i l y as p H w a s r e d u c e d f r o m 7.4 to 6.8. Further e x p e r i m e n t s u s i n g C O 2 / H C O 3 buffered media suggested that rat O C s m a y be h i g h l y sensitive to p H c h a n g e s within a n a r r o w e r range, closer to physiological norms. To examine this possibility in m o r e detail, w e c u l t u r e d O C - c o n t a i n i n g mixed b o n e cell p o p u l a t i o n s f r o m neonatal rat b o n e s on 5 m m bovine cortical b o n e discs in s t a n d a r d M i n i m u m Essential M e d i u m (with Earle's salts a n d 10% FCS) modified b y small a m o u n t s of HCI or N a O H . R e s o r p t i o n pits f o r m e d on b o n e wafers w e r e assessed b y reflected light m i c r o s c o p y after c o u n t i n g tartrateresistant acid p h o s p h a t a s e positive multinucleate OCs. N a O H 10 m e q / l NaOH 5 meq/l Control HCI 5 mecl/l HC1 10 m e q / l HCI 15 m e q / l
final p H 7.420 7.371 7.283 7.214 7.078 6.848
[HCO3"J(mM) 26.1 22.5 18.0 14.4 10.1 5.7
pits/slice 0 0 0.60i-0.40 3.40_+0.25* 3.80_+0.66* 1.80_+0.38
pils/OC 0 0 0.05_+0.03 0.26_+0.03 0.45_+0.12 0.22_+0.06
Resorption values s h o w n are m e a n s + SEM (n=5) for 26 h low d e n s i t y cultures; PCO2 w a s 41 m m Hg; *p<0.02 vs. control. The data s h o w p r o f o u n d s t i m u l a t i o n of pit formation associated with a p H d r o p of only 0.069 unit; resorption in this experiment w a s abolished a b o v e p H 7.283. This effect w a s h i g h l y reproducible. The results are c o n s i s t e n t w i t h the possibility that very small shifts in e x t r a c e l l u l a r p H in vivo c o u l d m o d u l a t e m a m m a l i a n OC activity.
Oll.
M a t r i x m e t a l l o p r o t e i n a s e expression in human bone : in
situ i m m u n o l o c a l i s a t i o n studies S Bord, A H o m e r , R Wolman*, C-C H u a n g , J C o m p s t o n
Department of Medicine, University of Cambridge, Cambridge CB2 2QQ and *Royal National Orthopaedic Hospital, Stanmore, Middlesex Matrix m e t a l l o p r o t e i n a s e s ( M M P s ) are a family of e n z y m e s r e s p o n s i b l e for e x t r a c e l l u l a r matrix d e g r a d a t i o n ; their role in h u m a n b o n e r e m o d e l l i n g has yet to be established. We h a v e p r e v i o u s l y s h o w n constitutive a n d inducible synthesis of MMPs b y h u m a n o s t e o b l a s t s in vitro a n d are n o w investigating t h e presence a n d localisation of MMPs a n d TIMP in h u m a n bone in situ u s i n g h e t e r o t o p i c b o n e as a m o d e l of rapid turnover. Snap frozen or cold e m b e d d e d bone samples w e r e sectioned a n d e x p r e s s i o n of c o l l a g e n a s e , s t r o m e l y s i n , gelatinases A a n d B, T1MP a n d o s t e o c a l c i n a s s e s s e d u s i n g p o l y c l o n a l a n t i b o d i e s , whilst ALP and TRAP were determined by enzyme h i s t o c h e m i s t r y . M o n o c l o n a l a n t i b o d i e s to TRAP, CD68, CD29 a n d CD4 further a i d e d cell identification. S t r o n g c o l l a g e n a s e e x p r e s s i o n w a s s h o w n b y active cells i n v o l v e d w i t h m a t r i x f o r m a t i o n T h e s e cells also s h o w e d osteocaIcin a n d A L P positivity. [n a d d i t i o n c o l l a g e n a s e w a s I o c a l i s e d o n r e s o r b e d e d g e s of the w o v e n b o n e w h e r e m u l t i n u c l e a t e d T R A P a n d CD68 positive cells indicated the p r e s e n c e of o s t e o c l a s t s . Cells s t a i n i n g for g e l a t i n a s e B a n d s t r o m e l y s i n a p p e a r e d a l o n g the r e s o r b i n g edges, with a s t r o n g
signal in n e w l y i n c o r p o r a t e d osteocytes. A lower expression of gelatinase A w a s largely confined to lining cells a n d new osteoid a n d w a s n o t detected in osteocytes. A L P w a s most m a r k e d in areas of n e w b o n e f o r m a t i o n whilst TRAP activity was highest in the calcifying cartilage exhibiting e n d o c h o n d r a l ossification a n d also in the large a n d n u m e r o u s osteocytes w h e r e CD68 w a s not present. O u r results d e m o n s t r a t e for the first time the presence of MMPs in h u m a n b o n e in situ a n d s u g g e s t t h a t MMPs p l a y a role in a c t i v a t i o n a n d in c o l l a g e n d e g r a d a t i o n a s s o c i a t e d w i t h resorption.
681
Abstracts
i m m u n o r e a c t i v i t y w e r e identified u s i n g the A P A A P p r o c e d u r e in the p r e s e n c e of l e v a m i s o l e to inhibit e n d o g e n o u s , alkaline p h o s p h a t a s e activity. Bcl-2 protein w a s localised specifically and r e p r o d u c i b l y to osteoblasts actively e n g a g e d in b o n e formation. Using a combination histochemistry and immunocytochemisty it w a s s h o w n t h a t the cells expressing Bcl-2 also expressed high levels of a l k a l i n e p h o s p h a t a s e activity. This represents the first d e m o n s t r a t i o n t h a t t h e p r o d u c t of a g e n e i n v o l v e d in the r e g u l a t i o n of P C D is e x p r e s s e d in a d u l t h u m a n b o n e a n d s t r o n g l y s u g g e s t s t h a t a p o p t o s i s is an i m p o r t a n t m e c h a n i s m for r e g u l a t i n g the size a n d ultimate fate of the osteoblast population.
0 1 0 . R e s p o n s e of rat osteoclasts to small shifts in extracellular pH: e v i d e n c e for an "on-off switch"?. M Spowage*, TR Arnett
Department of Anatomy and Developmental Biology, University College London, London WCIE and *Boots Pharmaceuticals Research Department, Nottingham NG2 3AA P r e v i o u s s t u d i e s s h o w e d that in c u l t u r e m e d i a buffered with HEPES alone, b o n e r e s o r p t i o n b y d i s a g g r e g a t e d rat osteoclasts (OCs) i n c r e a s e d s t e a d i l y as p H w a s r e d u c e d f r o m 7.4 to 6.8. Further e x p e r i m e n t s u s i n g C O 2 / H C O 3 buffered media suggested that rat O C s m a y be h i g h l y sensitive to p H c h a n g e s within a n a r r o w e r range, closer to physiological norms. To examine this possibility in m o r e detail, w e c u l t u r e d O C - c o n t a i n i n g mixed b o n e cell p o p u l a t i o n s f r o m neonatal rat b o n e s on 5 m m bovine cortical b o n e discs in s t a n d a r d M i n i m u m Essential M e d i u m (with Earle's salts a n d 10% FCS) modified b y small a m o u n t s of HCI or N a O H . R e s o r p t i o n pits f o r m e d on b o n e wafers w e r e assessed b y reflected light m i c r o s c o p y after c o u n t i n g tartrateresistant acid p h o s p h a t a s e positive multinucleate OCs. N a O H 10 m e q / l NaOH 5 meq/l Control HCI 5 mecl/l HC1 10 m e q / l HCI 15 m e q / l
final p H 7.420 7.371 7.283 7.214 7.078 6.848
[HCO3"J(mM) 26.1 22.5 18.0 14.4 10.1 5.7
pits/slice 0 0 0.60i-0.40 3.40_+0.25* 3.80_+0.66* 1.80_+0.38
pils/OC 0 0 0.05_+0.03 0.26_+0.03 0.45_+0.12 0.22_+0.06
Resorption values s h o w n are m e a n s + SEM (n=5) for 26 h low d e n s i t y cultures; PCO2 w a s 41 m m Hg; *p<0.02 vs. control. The data s h o w p r o f o u n d s t i m u l a t i o n of pit formation associated with a p H d r o p of only 0.069 unit; resorption in this experiment w a s abolished a b o v e p H 7.283. This effect w a s h i g h l y reproducible. The results are c o n s i s t e n t w i t h the possibility that very small shifts in e x t r a c e l l u l a r p H in vivo c o u l d m o d u l a t e m a m m a l i a n OC activity.
Oll.
M a t r i x m e t a l l o p r o t e i n a s e expression in human bone : in
situ i m m u n o l o c a l i s a t i o n studies S Bord, A H o m e r , R Wolman*, C-C H u a n g , J C o m p s t o n
Department of Medicine, University of Cambridge, Cambridge CB2 2QQ and *Royal National Orthopaedic Hospital, Stanmore, Middlesex Matrix m e t a l l o p r o t e i n a s e s ( M M P s ) are a family of e n z y m e s r e s p o n s i b l e for e x t r a c e l l u l a r matrix d e g r a d a t i o n ; their role in h u m a n b o n e r e m o d e l l i n g has yet to be established. We h a v e p r e v i o u s l y s h o w n constitutive a n d inducible synthesis of MMPs b y h u m a n o s t e o b l a s t s in vitro a n d are n o w investigating t h e presence a n d localisation of MMPs a n d TIMP in h u m a n bone in situ u s i n g h e t e r o t o p i c b o n e as a m o d e l of rapid turnover. Snap frozen or cold e m b e d d e d bone samples w e r e sectioned a n d e x p r e s s i o n of c o l l a g e n a s e , s t r o m e l y s i n , gelatinases A a n d B, T1MP a n d o s t e o c a l c i n a s s e s s e d u s i n g p o l y c l o n a l a n t i b o d i e s , whilst ALP and TRAP were determined by enzyme h i s t o c h e m i s t r y . M o n o c l o n a l a n t i b o d i e s to TRAP, CD68, CD29 a n d CD4 further a i d e d cell identification. S t r o n g c o l l a g e n a s e e x p r e s s i o n w a s s h o w n b y active cells i n v o l v e d w i t h m a t r i x f o r m a t i o n T h e s e cells also s h o w e d osteocaIcin a n d A L P positivity. [n a d d i t i o n c o l l a g e n a s e w a s I o c a l i s e d o n r e s o r b e d e d g e s of the w o v e n b o n e w h e r e m u l t i n u c l e a t e d T R A P a n d CD68 positive cells indicated the p r e s e n c e of o s t e o c l a s t s . Cells s t a i n i n g for g e l a t i n a s e B a n d s t r o m e l y s i n a p p e a r e d a l o n g the r e s o r b i n g edges, with a s t r o n g
signal in n e w l y i n c o r p o r a t e d osteocytes. A lower expression of gelatinase A w a s largely confined to lining cells a n d new osteoid a n d w a s n o t detected in osteocytes. A L P w a s most m a r k e d in areas of n e w b o n e f o r m a t i o n whilst TRAP activity was highest in the calcifying cartilage exhibiting e n d o c h o n d r a l ossification a n d also in the large a n d n u m e r o u s osteocytes w h e r e CD68 w a s not present. O u r results d e m o n s t r a t e for the first time the presence of MMPs in h u m a n b o n e in situ a n d s u g g e s t t h a t MMPs p l a y a role in a c t i v a t i o n a n d in c o l l a g e n d e g r a d a t i o n a s s o c i a t e d w i t h resorption.
681