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STRO-1+ a cells towards the osteoblast, chondrocyte and adipocyte lineages
Bone Vol. 27, No. 4, Supplement October 2000:1 S-54S
0 41 THE ROLE OF PTHrP ON CHONDROCYTE DIFFERENTIATION IN THE POST-EMBRYONIC GROWTH PLATE C. Farquharson, B. Houston, E. Seawright and D. Jefferies Bone Biology. Group, Division of Integrative Biology, Roslin Institute, Roslin, Scotland. The pace of growth plate chondroc~nte (GPC) differentiation is under local control, and in embryonic cartilage PTHrP is a major negative regulator. Doubts exist, however, on the exact growth plate distribution of PTHrP, Indian hedgehog (Ihh) and their receptors It is also unclear if the PTHrP-Ihh autoregulatory loop exists in the post-embryonic growth plate and therefore we have attempted to clarify the role of PTHrP in chondrocyte differentiation within the chick growth plate. PTHrP immunostaining was present within epiphyseal and proliferating chondrocytes but the strongest-staining was observed within the cells of the hypertrophic zone To further characterise gene expression, GPC's were fractionated into 5 distinct maturational phenotypes and semiquantitative RT-PCR indicated that PTHrP, Ihh and their receptors were expressed in all fractions. The PTH/PTHrP receptor (PTHR) was upregulated in the early differentiating chondroc)¢es. Functional PTHR's were demonstrated by chondrocytes of all fractions responding to PTHrP ('~ [3H]-thymidine incoJrporation and cAMP production and ,L alkaline phosphatase activity). The paracrine control of GPC differentiation from epiphyseal chondrocyte (EC) derived PTHrP was suggested by 2 observations. Firstly, PTHrP expression by EC's was higher than by GPC's and no evidence was obtained to suggest that EC's responded (unlike GPC's) to exogenous PTHrP. Secondly, GPC hypertrophy was inhibited when epiphyseai cartilage remained attached to growth plate explants. The resulting morphology was similar to that observed when explants (without epiphyseal cartilage) were cultured with exogenous PTHrP. A similar inhibition of GPC differentiation was noted in co-cultures of GPC's and EC's. These results suggest that the components of the PTHrP/Ihh regulatory loop are all present within the post-embryonic growth plate However, the paracrine control of GPC differentiation by EC derived PT|~P cannot be ruled out
0 43 RECIPROCAL DIFFERENTIATION OF TRIPOTENTIAL CLONAL HUMAN MARROW STROMAL F/STRO-I+ A CELLS TOWARDS THE OSTEOBLAST, CHONDROCYTE A N D A D I P O C Y T E L I N E A G E S . S Ahdjoudj, F Lasmoles, B O Oyajobi, A Lomri, P Delannoy, PJ Marie. 1 N S E R M U349, Cell and M o l e c u l a r B i o l o g y o f Bone, Lariboisi6re Hospital, Paris, France. W e assessed the differentiation o f i m m o r t a l i z e d clonal F / S T R O - I + A h u m a n fetal bone m a r r o w stromal ceils in response to d e x a m e t h a s o n e (Dex), indomethacin/insulin (Indo/Ins) and linoleic acid (LA). Under basal conditions, F / S T R O - I + A cells e x p r e s s e d m R N A s for markers o f the osteoblast lineage [CBFA1, osteocalcin (OC), alkaline p h o s p h a t a s e (ALP), type 1 collagen), o f the c h o n d r o c y t e lineage (aggrecan, types l i B , I X and X collagen), and o f the adipocyte lineage (PPAR?, C/EBPct, aP2, G 3 P D H , lipoprotein lipase, leptin). T r e a t m e n t with Dex increased C B F A 1 , O C and ALP m R N A and protein levels. E x p o s u r e to L A enhanced expression o f adipocytic genes and c y t o p l a s m i c triglycerides accumulation, and s u p p r e s s e d the D e x - i n d u c e d stimulation o f osteoblast m a r k e r genes. Indo/Ins stimulated the synthesis o f a g g r e c a n and types I X and X collagen m R N A levels and cartilaginous proteins, and suppressed both basal and Dexp r o m o t e d expression o f osteoblast markers. Conversely, stimulation o f o s t e o b l a s t o g e n e s i s by Dex s u p p r e s s e d both basal and I n d o / l n s - s t i m u l a t e d c h o n d r o c y t e genes. The d a t a s h o w that the clonal h u m a n fetal bone m a r r o w stromal F / S T R O - I + A cell line is a lineage-unrestricted c o m m o n p r o g e n i t o r that expresses tripotential adipocyte, o s t e o b l a s t or c h o n d r o c y t e characteristics, and that differentiation t o w a r d s one p a t h w a y restricts e x p r e s s i o n o f other lineage-specific genes. The data also p r o v i d e novel evidence :for a dual reciprocal regulation o f osteoblast/chondroblast and osteoblast/adipocyte differentiation o f F / S T R O - I + A h u m a n bone m a r r o w stromal cells
Abstracts
23S
0 42 ON EXPOSURE TO ANOREXIA NERVOSA, THE TEMPORAL VARIATION IN AXIAL AND APPENDICULAR SKELETAL DEVELOPMENT PREDISPOSES TO SITE-SPECIFIC DEFICITS IN BONE SIZE AND DENSITY: E Seeman, MK Karlsson, Y Duan. Dept. Medicine, University of Melbourne, Austin & Repatriation Medical Centre, Melbourne Australia Skeletal development is heterogeneous. Throughout growth, bone size is more maturationally advanced than the mineral being accrued within its periosteal envelope; before puberty, appendicular growth is more rapid than axial growth; during puberty, appendicular growth slows and axial growth accelerates. We studied women with differing age of onset of anorexia nervosa (AN) to determine whether this temporal heterogeneity in growth predisposed to the development of deficits in bone size and volumetric bone mineral density (vBMD) which varied by site and severity depending on the age at which AN occurred. Bone size and vBMD of the third lumbar vertebra and femoral neck were measured using dual x-ray absorptiometry in 210 women aged 21 years (range 12-40) with AN. Results were expressed as Z scores (SDs, mean + sem). Bone width depended on the age of onset of AN; when the onset of AN occurred: (i) before 15 yrs of age, deficits in vertebral body and femoral neck width did not differ (-0.77 + 0.27SD and - 0.55 + 0.17SD respectively), (ii) between 15-19 yrs of age, deficits in vertebral body width (- 0.95 + 0.16SD) were three times the deficits in femoral neck width (- 0.28 + 0.14SD) (p < 0.05 comparing the deficits), (iii) after 19 yrs of age, deficits in vertebral body width (0.49 + 0.26SD, p = 0.05) were half that in women with earlier onset of AN. No deficit in bone width was observed at the femoral neck. Deficits in vBMD at the vertebra and femoral neck were independent of the age of onset of AN, but increased as the duration of AN increased, being about 0.5SD lower at the vertebra than femoral neck. We infer that the maturational development of a region at the time of exposure to disease, and disease duration, determine the site, magnitude and type of trait deficit in anorexia nervosa. Bone fragility due to reduced bone size and reduced vBMD in adulthood is partly established during growth.
0 44 THE INFLUENCES OF GROWTH HORMONE AND GONADAL HORMONE ON RADIAL BONE GROWTH, CORTICAL THICKNESS, AND BONE STRENGTH. B-T Kimj. L Mosekilde2, XZ Zhang j, L Tornvig2, JS Thomsen2, E Seemank IDept. Medicine, Austin & Repatriation Medical Centre, Melbourne, Australia, and 2Dept. Cell Biology, Institute of Anatomy, University of Aarhus, Aarhus Denmark. We have reported the effects of prepubertal gonadectomy (Gx) at on bone mineral density (BMD) in growth hormone mptete (GH+) and deficient (GH-) rats. To examine the structural and blomechanical basis of the changes, we measured periosteal and endocortical diameters, cortical thickness (CT), cortical area (CA) and bending strength of the femoral diaphysis in 200 male and female GH- Fischer dwarf rats and GH+ Sprague-Dawelyrats during 8 months. In GH+ nonGx females, CA increased due to periosteal apposition ~d endocortical contraction. In GH+ nonGx males, CA increased because of periosteal apposition with no endoconical contraction. Gx in GH+ females increased periostealdiameter but endocorticalcontractiondecreased relative to GH+ nonGx so that CT was less but CA was similar (the bone was bigger) with no fall in bending strength. Gx in GH+ males reduced periosteal expansion with no change in endocortical diameter; CT, CA and bending strength decreased. GH- nonGx females had reduced periosteal expansion and reduced endocortical contraction; CT, CA and bending strength decreased relative to GH+ nonGx and all other groups. GH- nonGx males had reduced periosteal expansion, no change in endocortical diameter and reduced bending strength relative to GH+ nonGx. In the presence of GH-, Gx in females did not increased periosteal expansion but endoconical contraction still occurred; CT and CA was greater than GH- nonGx rats but remained lower than GH+ Gx and non Gx controls. Bending strength was greater than GH- nonGx but less than GH+ groups. GH- Gx in males had reduced periosteal diameter but endocortical contraction occurred, (not seen in GH+ males); CT remained unchanged but CA was reduced (the bone was smaller); bending strength was not reduced relative to GH- nonGx but was lower than GH+ groups We infer that females have lower bending strength than males because they have lower bone size and mass. Increased bone size following Gx in females compensates for the loss of cortical thickness and strength is not reduced. Gx in males results in reduced size, mass and strength but less so in the presence of GH deficiency, where cortical thickness is maintained by endocortical contraction partly offsetting the effects of reduced bone size.
0 41 THE ROLE OF PTHrP ON CHONDROCYTE DIFFERENTIATION IN THE POST-EMBRYONIC GROWTH PLATE C. Farquharson, B. Houston, E. Seawright and D. Jefferies Bone Biology. Group, Division of Integrative Biology, Roslin Institute, Roslin, Scotland. The pace of growth plate chondroc~nte (GPC) differentiation is under local control, and in embryonic cartilage PTHrP is a major negative regulator. Doubts exist, however, on the exact growth plate distribution of PTHrP, Indian hedgehog (Ihh) and their receptors It is also unclear if the PTHrP-Ihh autoregulatory loop exists in the post-embryonic growth plate and therefore we have attempted to clarify the role of PTHrP in chondrocyte differentiation within the chick growth plate. PTHrP immunostaining was present within epiphyseal and proliferating chondrocytes but the strongest-staining was observed within the cells of the hypertrophic zone To further characterise gene expression, GPC's were fractionated into 5 distinct maturational phenotypes and semiquantitative RT-PCR indicated that PTHrP, Ihh and their receptors were expressed in all fractions. The PTH/PTHrP receptor (PTHR) was upregulated in the early differentiating chondroc)¢es. Functional PTHR's were demonstrated by chondrocytes of all fractions responding to PTHrP ('~ [3H]-thymidine incoJrporation and cAMP production and ,L alkaline phosphatase activity). The paracrine control of GPC differentiation from epiphyseal chondrocyte (EC) derived PTHrP was suggested by 2 observations. Firstly, PTHrP expression by EC's was higher than by GPC's and no evidence was obtained to suggest that EC's responded (unlike GPC's) to exogenous PTHrP. Secondly, GPC hypertrophy was inhibited when epiphyseai cartilage remained attached to growth plate explants. The resulting morphology was similar to that observed when explants (without epiphyseal cartilage) were cultured with exogenous PTHrP. A similar inhibition of GPC differentiation was noted in co-cultures of GPC's and EC's. These results suggest that the components of the PTHrP/Ihh regulatory loop are all present within the post-embryonic growth plate However, the paracrine control of GPC differentiation by EC derived PT|~P cannot be ruled out
0 43 RECIPROCAL DIFFERENTIATION OF TRIPOTENTIAL CLONAL HUMAN MARROW STROMAL F/STRO-I+ A CELLS TOWARDS THE OSTEOBLAST, CHONDROCYTE A N D A D I P O C Y T E L I N E A G E S . S Ahdjoudj, F Lasmoles, B O Oyajobi, A Lomri, P Delannoy, PJ Marie. 1 N S E R M U349, Cell and M o l e c u l a r B i o l o g y o f Bone, Lariboisi6re Hospital, Paris, France. W e assessed the differentiation o f i m m o r t a l i z e d clonal F / S T R O - I + A h u m a n fetal bone m a r r o w stromal ceils in response to d e x a m e t h a s o n e (Dex), indomethacin/insulin (Indo/Ins) and linoleic acid (LA). Under basal conditions, F / S T R O - I + A cells e x p r e s s e d m R N A s for markers o f the osteoblast lineage [CBFA1, osteocalcin (OC), alkaline p h o s p h a t a s e (ALP), type 1 collagen), o f the c h o n d r o c y t e lineage (aggrecan, types l i B , I X and X collagen), and o f the adipocyte lineage (PPAR?, C/EBPct, aP2, G 3 P D H , lipoprotein lipase, leptin). T r e a t m e n t with Dex increased C B F A 1 , O C and ALP m R N A and protein levels. E x p o s u r e to L A enhanced expression o f adipocytic genes and c y t o p l a s m i c triglycerides accumulation, and s u p p r e s s e d the D e x - i n d u c e d stimulation o f osteoblast m a r k e r genes. Indo/Ins stimulated the synthesis o f a g g r e c a n and types I X and X collagen m R N A levels and cartilaginous proteins, and suppressed both basal and Dexp r o m o t e d expression o f osteoblast markers. Conversely, stimulation o f o s t e o b l a s t o g e n e s i s by Dex s u p p r e s s e d both basal and I n d o / l n s - s t i m u l a t e d c h o n d r o c y t e genes. The d a t a s h o w that the clonal h u m a n fetal bone m a r r o w stromal F / S T R O - I + A cell line is a lineage-unrestricted c o m m o n p r o g e n i t o r that expresses tripotential adipocyte, o s t e o b l a s t or c h o n d r o c y t e characteristics, and that differentiation t o w a r d s one p a t h w a y restricts e x p r e s s i o n o f other lineage-specific genes. The data also p r o v i d e novel evidence :for a dual reciprocal regulation o f osteoblast/chondroblast and osteoblast/adipocyte differentiation o f F / S T R O - I + A h u m a n bone m a r r o w stromal cells
Abstracts
23S
0 42 ON EXPOSURE TO ANOREXIA NERVOSA, THE TEMPORAL VARIATION IN AXIAL AND APPENDICULAR SKELETAL DEVELOPMENT PREDISPOSES TO SITE-SPECIFIC DEFICITS IN BONE SIZE AND DENSITY: E Seeman, MK Karlsson, Y Duan. Dept. Medicine, University of Melbourne, Austin & Repatriation Medical Centre, Melbourne Australia Skeletal development is heterogeneous. Throughout growth, bone size is more maturationally advanced than the mineral being accrued within its periosteal envelope; before puberty, appendicular growth is more rapid than axial growth; during puberty, appendicular growth slows and axial growth accelerates. We studied women with differing age of onset of anorexia nervosa (AN) to determine whether this temporal heterogeneity in growth predisposed to the development of deficits in bone size and volumetric bone mineral density (vBMD) which varied by site and severity depending on the age at which AN occurred. Bone size and vBMD of the third lumbar vertebra and femoral neck were measured using dual x-ray absorptiometry in 210 women aged 21 years (range 12-40) with AN. Results were expressed as Z scores (SDs, mean + sem). Bone width depended on the age of onset of AN; when the onset of AN occurred: (i) before 15 yrs of age, deficits in vertebral body and femoral neck width did not differ (-0.77 + 0.27SD and - 0.55 + 0.17SD respectively), (ii) between 15-19 yrs of age, deficits in vertebral body width (- 0.95 + 0.16SD) were three times the deficits in femoral neck width (- 0.28 + 0.14SD) (p < 0.05 comparing the deficits), (iii) after 19 yrs of age, deficits in vertebral body width (0.49 + 0.26SD, p = 0.05) were half that in women with earlier onset of AN. No deficit in bone width was observed at the femoral neck. Deficits in vBMD at the vertebra and femoral neck were independent of the age of onset of AN, but increased as the duration of AN increased, being about 0.5SD lower at the vertebra than femoral neck. We infer that the maturational development of a region at the time of exposure to disease, and disease duration, determine the site, magnitude and type of trait deficit in anorexia nervosa. Bone fragility due to reduced bone size and reduced vBMD in adulthood is partly established during growth.
0 44 THE INFLUENCES OF GROWTH HORMONE AND GONADAL HORMONE ON RADIAL BONE GROWTH, CORTICAL THICKNESS, AND BONE STRENGTH. B-T Kimj. L Mosekilde2, XZ Zhang j, L Tornvig2, JS Thomsen2, E Seemank IDept. Medicine, Austin & Repatriation Medical Centre, Melbourne, Australia, and 2Dept. Cell Biology, Institute of Anatomy, University of Aarhus, Aarhus Denmark. We have reported the effects of prepubertal gonadectomy (Gx) at on bone mineral density (BMD) in growth hormone mptete (GH+) and deficient (GH-) rats. To examine the structural and blomechanical basis of the changes, we measured periosteal and endocortical diameters, cortical thickness (CT), cortical area (CA) and bending strength of the femoral diaphysis in 200 male and female GH- Fischer dwarf rats and GH+ Sprague-Dawelyrats during 8 months. In GH+ nonGx females, CA increased due to periosteal apposition ~d endocortical contraction. In GH+ nonGx males, CA increased because of periosteal apposition with no endoconical contraction. Gx in GH+ females increased periostealdiameter but endocorticalcontractiondecreased relative to GH+ nonGx so that CT was less but CA was similar (the bone was bigger) with no fall in bending strength. Gx in GH+ males reduced periosteal expansion with no change in endocortical diameter; CT, CA and bending strength decreased. GH- nonGx females had reduced periosteal expansion and reduced endocortical contraction; CT, CA and bending strength decreased relative to GH+ nonGx and all other groups. GH- nonGx males had reduced periosteal expansion, no change in endocortical diameter and reduced bending strength relative to GH+ nonGx. In the presence of GH-, Gx in females did not increased periosteal expansion but endoconical contraction still occurred; CT and CA was greater than GH- nonGx rats but remained lower than GH+ Gx and non Gx controls. Bending strength was greater than GH- nonGx but less than GH+ groups. GH- Gx in males had reduced periosteal diameter but endocortical contraction occurred, (not seen in GH+ males); CT remained unchanged but CA was reduced (the bone was smaller); bending strength was not reduced relative to GH- nonGx but was lower than GH+ groups We infer that females have lower bending strength than males because they have lower bone size and mass. Increased bone size following Gx in females compensates for the loss of cortical thickness and strength is not reduced. Gx in males results in reduced size, mass and strength but less so in the presence of GH deficiency, where cortical thickness is maintained by endocortical contraction partly offsetting the effects of reduced bone size.