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Secreted frizzled-related proteins are expressed by osteoblasts.
Bone Vol. 27, No. 4, Supplement October 2000:1 S-54S
Abstracts
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MALE AND FEMALE BONE CEI ,I ~ DIFFER IN IHEIR ATP-INDUCED Ca~+ RESPONSE FOLLOWING EXPOSURE TO HIGH SHEAR
R E G U L A T I O N OF 2 5 - H Y D R O X Y V I T A M I N D3-1H Y D R O X Y L A S E (CYPI) EXPRESSION BY P A R A T H Y R O I D H O R M O N E IN KIDNEY A N D BONE CELLS.
P L Hoodf S. R Bolsover,~g S. Susv¢$o2and L E Lanyort2 'Dept Physio~, ~,~versi(vCo~ge 1~u~ ~ ( IK : [Xept VeteruJxyBasic &'terr~ Rokttl VeterinaryCollege,Lo,~kra, t ]K Constitutive release of ATP by primary bone cell populations is modulated by fluid-flow induced shear stresses(l). Interstitial fluid flow has been suggested to modulate bone remodellingIz) and has been shown to elicit increases in the concentration of intracellular free calcium [Ca2+]i(3).In this study the calcium transients evoked by fluid flow and by exogenous application of ATP +/- a prior exposure to shear stress were compared in both male and female rat osteoblasts. Osteoblasts were extracted from 6 male and 6 female Sprague-Dawley rats and cultured seperately. Cells were loaded with the calcium indicator dye Fura-2 and placed in a parallel plate flow chamber which was mounted on the stage of an inverted microscope and attached to an alternating flow system. Both male and female cells showed a significant although similar increase in [Ca2+]i in response to I0uM ATP unidirectionally flowed into the chamber [35.1+6.3 and 38.8+49nM]. In separate experiments cells were exposed to high shear stress applied with the flow direction alternating at 0.1Hz in ATP free media followed immediately by the unidirectional application of 10uM ATP at a low shear stress. Both male and female derived cells showed a significant increase over basal [Ca2"]i in response to alternating fluid flow [103.8+5 and 106.5+8.4nM], again there was no difference in the response between the sexes. However, when 10uM ATP was applied immediately after shear stress the male increase to ATP was significantly larger than the corresponding female increase [141.3+5.7vs121.3+4.9nM, p<0.001], although both ATP responses were larger than in the absence of prior shear stress.These results show that both male and female derived bone cells modify their responses to ATP following exposure to high induced shear stress and that this modification is significantly different between the sexes.
Xuihui Gao, Prem Dwivedi, Howard Morris#, John Omdahl* and Brian May. Department of Biochemistry,University of Adelaide; #Department of Clinical Biochemistry, IMVS, Adelaide; *Department of Biochemistry, University of New Mexico, Albuquerque, USA. Vitamm D is critical for many processes including calcium homeostasis, bone formation and cell differentiation. The vitarmn D 1hydroxylase (CYP I ) dictates levels of biologically active 1,25dihydroxyvitamin D, through activation of vitamin D The major site of CYPI synthesis is the kidney although there are other extra-renal sites of synthesis. The regulation of the gene for CYPI in the kidney is under the hormonal control of parathyroid hormone. Renal CYP1 is induced by parathyroid hormone in response to low levels of serum calcium. In turn, increased CYP 1 levels restore serum calcium levels through the synthesis and action of 1,25-dihydroxyvitamm D We are investigating the molecular mechanism of parathyroid hormone action. Using CYP 1 promoter-luciferase constructs we observe induction of promoter activity in transiently transfected kidney cells following treatment with parathyroid hormone. In contrast, no hormone reduction of these constructs is observed in osteoblasts even with exogenously expressed parathyroid hormone receptor. Substantial levels of basal expression of the promoter constructs were observed in both cell types although basal expression in the osteoblast cells was higher than that in kidney cells. Parathyroid hormone induction of the promoter in kidney cells was completely inhibited by the protein kinase A inhibitor, H89 but basal expression was unaffected by the inhibitor. Deletion analysis of promoter constructs localised the basal and hormone responsive regions. Using site directed mutagenesis we have identified CYP1 promoter sites that contribute to basal and hormone induced expression.
References (1) Bowler WB. et al. 1998 Bone and Tooth Society Abstract 014 (2) Bergula AP. et al. 1999 Bone Vol 24 pp 171-177 (3) Hung C.T. et al. 1995 Clinical Orthopaedics and Related Research Vol 3 I3 pp 258-269
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S E C R E T E D F R I Z Z L E D - R E L A T E D PROTEINS ARE EXPRESSED BY OSTEOBLASTS.
EFFECTS IN VITRO M1NERALISATION ON 1,25D AND PTH INDUCTION OF 25 HYDROXYVITAMIN D-24HYDROXYLASE IN UMR106 CELLS S Iida 1, A Turner 2, HA Morris 1, BK May3. ~ o n of Clinical Biochemistry, Institute of Medical and Veterinary Science, Departments of Physiology2 and Biochemistrys, University of Adelaide, Adelaide 5000, South Australia, Australia Vitamin D homeostasis is controlled by two enz~anes, 25 hydroxyvitamln D-l-hydroxylase, and 25 hydroxyvitarnin D-24-hydroxylase (CYP24). The role of these enzymes is well known in kidney, but is unknown in bone. CYP24 rnRNA levels are increased by 1,25 dihydroxyvitamm D (1,25D) and increased further by combined 1,25D and PTH treatment in the osteoblast-like cell UMR106. We now report the effects of these treatments on CYP24 enz~a'ne activity in UMRI06 cells during proliferation and following induction ofmmeralisation. UMR 106-06 cells were cultured in DMEM medium containing 10% FCS and plated at subconfluencT before incubating for 24 hours in serum-free medium containing either vehicle or 1,25D ( 10-11-10-7 M) with or without PTH (10" 7 M) Cultunng cells in growth medium containing ascorbate-2-phosphate (100uM) and K2HPO4 (5mM) for 9 days induced mineralisation (1). Cells were then treated with hormones as for proliferating cells. Cellular CYP24 activity was assayed by incubating with 3H-25(OH)2Ds for 6 hours tbllo~ng which vitamin D metabolites were extracted from cells and separated by HPLC where authentic fractions for 3H-24,25(OH)2D3 were quantified by scintillation counting. Mineralisation was identified by nodule formation after treatment for 7 days. CYP24 activity was increased in mineralising cells compared with proliferating cells in the absence of 1,25D (P<0.001). 1,25D increased CYP24 activity in a dose dependent manner in both proliferating and mineralising cells (P<0.001) and incubation with PTH further stimulated activity at 10"TM 1,25D(P<0.025). 1,25D and PTH act synergistically to stimulate CYP24 activity in UMR 106 cells This effect is greater during mineralisation than during proliferation. These findings suggest that local vitamin D metabolism plays an important role during bone development. (1) Stanford CM, et at. J Biol Chem 1995;270:9420-9428.
Karl Hausler, Nicole Hol~vood, Jan Elliott, Jack Martin and Matthew Gillespie St. Vincent's Institute of Medical Research, 9 Princes Street, Fitzroy 3065. Osteoblasts are responsible fe,r bone formation as well as bone resorption via their interactions with osteoclastic progenitors. We have examined the gene expression profile of osteoblastic cells at different stages of differentiation, and by their capacity" to support osteoclast formation using a rnRNA profiling technique of differential displ~' of mRNA. Differentially expressed mRNA species were confirmed by RT-PCR, and their expression profiles determined against a variety, of osteoblastic cell lines. By this approach we identified that sFRP-I was highly expressed in the tsJ2 compared w4th the tsJ14 cell line. sFRP-I is a member of secreted proteins that contains homology to the cysteine rich ligand-binding domain of the frizzled family of transmembrane receptors, and is an antagonist to the Wnt - Frizzled signal transduction pathway. Frizzled receptors and their ligand binding partners (Wnts) are developmentally important signalling molecules involved in cell proliferation, morphology, motility and fate. sFRP-1 was expressed by relatively immature osteoblasts and its expression was upregulated in response to IL-I1 or dexmnethasone. however PTH or 1,25 (O1-1)2 vitamin D3 did not effect its expression. In situ hybridization established that sFRP-1 was expressed by hypertrophic chondrocytes, but not by cells of the transitional or proliferative zones. In murine co-cultures of osteoblastic cells with haematopoietic cells, a time dependent enhancement of sFRP-I expression was noted, and that this process was dependent upon cellcell contact. Combined these data suggest that sFRP-I, and the WntFrizzled signalling pathway, may be important in the development of cells in the bone, and cell to cell signalling.
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Abstracts
P5
P6
MALE AND FEMALE BONE CEI ,I ~ DIFFER IN IHEIR ATP-INDUCED Ca~+ RESPONSE FOLLOWING EXPOSURE TO HIGH SHEAR
R E G U L A T I O N OF 2 5 - H Y D R O X Y V I T A M I N D3-1H Y D R O X Y L A S E (CYPI) EXPRESSION BY P A R A T H Y R O I D H O R M O N E IN KIDNEY A N D BONE CELLS.
P L Hoodf S. R Bolsover,~g S. Susv¢$o2and L E Lanyort2 'Dept Physio~, ~,~versi(vCo~ge 1~u~ ~ ( IK : [Xept VeteruJxyBasic &'terr~ Rokttl VeterinaryCollege,Lo,~kra, t ]K Constitutive release of ATP by primary bone cell populations is modulated by fluid-flow induced shear stresses(l). Interstitial fluid flow has been suggested to modulate bone remodellingIz) and has been shown to elicit increases in the concentration of intracellular free calcium [Ca2+]i(3).In this study the calcium transients evoked by fluid flow and by exogenous application of ATP +/- a prior exposure to shear stress were compared in both male and female rat osteoblasts. Osteoblasts were extracted from 6 male and 6 female Sprague-Dawley rats and cultured seperately. Cells were loaded with the calcium indicator dye Fura-2 and placed in a parallel plate flow chamber which was mounted on the stage of an inverted microscope and attached to an alternating flow system. Both male and female cells showed a significant although similar increase in [Ca2+]i in response to I0uM ATP unidirectionally flowed into the chamber [35.1+6.3 and 38.8+49nM]. In separate experiments cells were exposed to high shear stress applied with the flow direction alternating at 0.1Hz in ATP free media followed immediately by the unidirectional application of 10uM ATP at a low shear stress. Both male and female derived cells showed a significant increase over basal [Ca2"]i in response to alternating fluid flow [103.8+5 and 106.5+8.4nM], again there was no difference in the response between the sexes. However, when 10uM ATP was applied immediately after shear stress the male increase to ATP was significantly larger than the corresponding female increase [141.3+5.7vs121.3+4.9nM, p<0.001], although both ATP responses were larger than in the absence of prior shear stress.These results show that both male and female derived bone cells modify their responses to ATP following exposure to high induced shear stress and that this modification is significantly different between the sexes.
Xuihui Gao, Prem Dwivedi, Howard Morris#, John Omdahl* and Brian May. Department of Biochemistry,University of Adelaide; #Department of Clinical Biochemistry, IMVS, Adelaide; *Department of Biochemistry, University of New Mexico, Albuquerque, USA. Vitamm D is critical for many processes including calcium homeostasis, bone formation and cell differentiation. The vitarmn D 1hydroxylase (CYP I ) dictates levels of biologically active 1,25dihydroxyvitamin D, through activation of vitamin D The major site of CYPI synthesis is the kidney although there are other extra-renal sites of synthesis. The regulation of the gene for CYPI in the kidney is under the hormonal control of parathyroid hormone. Renal CYP1 is induced by parathyroid hormone in response to low levels of serum calcium. In turn, increased CYP 1 levels restore serum calcium levels through the synthesis and action of 1,25-dihydroxyvitamm D We are investigating the molecular mechanism of parathyroid hormone action. Using CYP 1 promoter-luciferase constructs we observe induction of promoter activity in transiently transfected kidney cells following treatment with parathyroid hormone. In contrast, no hormone reduction of these constructs is observed in osteoblasts even with exogenously expressed parathyroid hormone receptor. Substantial levels of basal expression of the promoter constructs were observed in both cell types although basal expression in the osteoblast cells was higher than that in kidney cells. Parathyroid hormone induction of the promoter in kidney cells was completely inhibited by the protein kinase A inhibitor, H89 but basal expression was unaffected by the inhibitor. Deletion analysis of promoter constructs localised the basal and hormone responsive regions. Using site directed mutagenesis we have identified CYP1 promoter sites that contribute to basal and hormone induced expression.
References (1) Bowler WB. et al. 1998 Bone and Tooth Society Abstract 014 (2) Bergula AP. et al. 1999 Bone Vol 24 pp 171-177 (3) Hung C.T. et al. 1995 Clinical Orthopaedics and Related Research Vol 3 I3 pp 258-269
P7
P8
S E C R E T E D F R I Z Z L E D - R E L A T E D PROTEINS ARE EXPRESSED BY OSTEOBLASTS.
EFFECTS IN VITRO M1NERALISATION ON 1,25D AND PTH INDUCTION OF 25 HYDROXYVITAMIN D-24HYDROXYLASE IN UMR106 CELLS S Iida 1, A Turner 2, HA Morris 1, BK May3. ~ o n of Clinical Biochemistry, Institute of Medical and Veterinary Science, Departments of Physiology2 and Biochemistrys, University of Adelaide, Adelaide 5000, South Australia, Australia Vitamin D homeostasis is controlled by two enz~anes, 25 hydroxyvitamln D-l-hydroxylase, and 25 hydroxyvitarnin D-24-hydroxylase (CYP24). The role of these enzymes is well known in kidney, but is unknown in bone. CYP24 rnRNA levels are increased by 1,25 dihydroxyvitamm D (1,25D) and increased further by combined 1,25D and PTH treatment in the osteoblast-like cell UMR106. We now report the effects of these treatments on CYP24 enz~a'ne activity in UMRI06 cells during proliferation and following induction ofmmeralisation. UMR 106-06 cells were cultured in DMEM medium containing 10% FCS and plated at subconfluencT before incubating for 24 hours in serum-free medium containing either vehicle or 1,25D ( 10-11-10-7 M) with or without PTH (10" 7 M) Cultunng cells in growth medium containing ascorbate-2-phosphate (100uM) and K2HPO4 (5mM) for 9 days induced mineralisation (1). Cells were then treated with hormones as for proliferating cells. Cellular CYP24 activity was assayed by incubating with 3H-25(OH)2Ds for 6 hours tbllo~ng which vitamin D metabolites were extracted from cells and separated by HPLC where authentic fractions for 3H-24,25(OH)2D3 were quantified by scintillation counting. Mineralisation was identified by nodule formation after treatment for 7 days. CYP24 activity was increased in mineralising cells compared with proliferating cells in the absence of 1,25D (P<0.001). 1,25D increased CYP24 activity in a dose dependent manner in both proliferating and mineralising cells (P<0.001) and incubation with PTH further stimulated activity at 10"TM 1,25D(P<0.025). 1,25D and PTH act synergistically to stimulate CYP24 activity in UMR 106 cells This effect is greater during mineralisation than during proliferation. These findings suggest that local vitamin D metabolism plays an important role during bone development. (1) Stanford CM, et at. J Biol Chem 1995;270:9420-9428.
Karl Hausler, Nicole Hol~vood, Jan Elliott, Jack Martin and Matthew Gillespie St. Vincent's Institute of Medical Research, 9 Princes Street, Fitzroy 3065. Osteoblasts are responsible fe,r bone formation as well as bone resorption via their interactions with osteoclastic progenitors. We have examined the gene expression profile of osteoblastic cells at different stages of differentiation, and by their capacity" to support osteoclast formation using a rnRNA profiling technique of differential displ~' of mRNA. Differentially expressed mRNA species were confirmed by RT-PCR, and their expression profiles determined against a variety, of osteoblastic cell lines. By this approach we identified that sFRP-I was highly expressed in the tsJ2 compared w4th the tsJ14 cell line. sFRP-I is a member of secreted proteins that contains homology to the cysteine rich ligand-binding domain of the frizzled family of transmembrane receptors, and is an antagonist to the Wnt - Frizzled signal transduction pathway. Frizzled receptors and their ligand binding partners (Wnts) are developmentally important signalling molecules involved in cell proliferation, morphology, motility and fate. sFRP-1 was expressed by relatively immature osteoblasts and its expression was upregulated in response to IL-I1 or dexmnethasone. however PTH or 1,25 (O1-1)2 vitamin D3 did not effect its expression. In situ hybridization established that sFRP-1 was expressed by hypertrophic chondrocytes, but not by cells of the transitional or proliferative zones. In murine co-cultures of osteoblastic cells with haematopoietic cells, a time dependent enhancement of sFRP-I expression was noted, and that this process was dependent upon cellcell contact. Combined these data suggest that sFRP-I, and the WntFrizzled signalling pathway, may be important in the development of cells in the bone, and cell to cell signalling.
33S