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Agonists for peroxisome proliferator activated receptor increase osteogenesis in vitro.
Bone Vol. 27, No. 4, Supplement October 2000:1 S-54S
P 21 CORTISOL DECREASES HEPATOCYTE GROWTH FACTOR LEVELS IN H U M A N OSTEOBLAST-LIKE CELLS
Skrtic S. Ohlsson C Research Center for Endocrinology and Metabolism, Sahlgrenska University' Hospital, SE-413 45 GOteborg, Sweden. Osteoporosis is a well-known side effect of long-term treatment with glucocorticoids. The hepatocyte growth factor (HGF) receptor is expressed by human osteoclasts and osteoblasts and mouse osteoblasts also express HGF, indicating that HGF may regulate bone metabolism. Because HGF could be a candidate factor in the local paracrine signaling between osteoblasts and osteoclasts in bone, we decided to studv if human osteoblasts secrete HGF and if glucocorticoids regulate the expression of HGF HGF was easily detectable in the culture medium from human osteoblast-like cells (hOB) The HGF protein released into the culture medium was increased with increasing confluency. Hydrocortisone decreased the amount of HGF released into the culture medium from hOB in a dosedependent manner with a m~ximal effect at 10-6M. Time-course studies revealed that hydrocortisone decreased the amount of HGF released into the culture medium significantly after l 6 h of stimulation (65+2 % of control culture). This effect of hydrocortisone was maximal after 24 h of stimulation (52_+8 % of control culture). In conclusion HGF is produced by primaD' cultured hOB cells. Fmlhernmre, the amount of HGF released into the culture medium is decreased by glucocorticoids. The biological significance of this finding remains to be demonstrated.
P 23 MECHANICAL PROPERTIES OF OSTEOBLASTS SUBJECTED TO CYCLICAL STRAIN - A N ATOMIC FORCE MICROSCOPY STUDY ROMY THOMAS, MALAV SHROFF, JIM TRIFFITTLBONE RESEARCH LABORATORY, NUFFIELD ORTH. CENTRE, OXFORD, 0)(3 7LD, GUILLAUME CHARRAS, MICHAEL HORTON DEPT. OF MEDICINE, UCL, LONDON, WCtE 6JF
Numerous studies have documented the effects of cyclical physiological strains on osteoblast-like cells. To date however, no study has addressed the relationship between cyclical strain, cytoskeletal rearrangement and mechanical properties of the osteoblast. Further the effects of hyper-physiological cyclical strains on the osteoblast cytoskeletal adaptive response are yet to be determined. In order to test the hypothesis that the mechanical properties of the osteoblast cell membrane may" be altered in response to cyclical strain stimuli, an atomic force microscope was used to study the membrane stiffness characteristics of an MG-63 osteoblastic cell line. Cells were seeded at a density of I x 105 on flexible polyurethane wells attached to a computer controlled cell stretching device. Cells were subjected to cyclical strain of 200,000 microstrain for one hour and cell membrane mechanical property measurements made 24 hours later. Mechanical properties of cells were evaluated (as a relationship between external force and the membrane indentation depth) for 5 different locations on each cell. Measurements for 10 cells each from strained and unstrained groups were obtained. No significant change in cell membrane mechanical property parameters between groups was detected 24 hours following a strain stimulus. Possible reasons are discussed. The feasibility of evaluating the micro-mechanical properties of osteoblast-like cells using an atomic force microscope is demonstrated Studies utilising difl'erent strain regimens with mechanical property, assessments at different time points from initiation of strain are underway.
Abstracts
37S
P 22 A G O N I S T S FOR P E R O X I S O M E P R O L I F E R A T O R A C T I V A T E D R E C E P T O R INCREASE OSTEOGENESIS I N VITRO.
K.Still and A.M. Scutt. Dept of Human Metabolism and Clinical Biochemist~', University of Sheffield, UK. A number of substances, in particular prostaglandin E2 (PGE2), are known which stimulate bone formation when administered to adult animals. However. its use in the clinic is prevented by its profound side effects. There are currently no bone anabolic agents on the market except for fluoride, which increases bone mineral densiB' by an unknown mechanism but paradoxically is also associated with an increase in fracture rate Hence there is an urgent need for new bone anabolic drugs. The half-life of PGE2 in vivo is known to be less than 2 seconds. It is also known that PGE series prostaglandins undergo a dehydration reaction in the presence of serum albumin to form prostanoids of the A series. Therefore, we considered the possibility, that some of the effects of PGE2 may be caused by its degradation product, PGA2. We have subsequently shown that PGA2 shows positive activi~' in a number of in vitro assays normally indicative of bone anabolic activity, namely the CFU-f assay, non-adherent stromaI precursor assay and the calvarial collagen synthesis assay PGA~ & A2 are known to bind to the nuclear receptors PPAR c~, /3 and 8 We investigated the effects of a number of PPAR agunists with known specificity' on colony formation using the CFU-f assay. Initial studies show that these drugs are indeed anabolic In particular, drugs which are mainly agonists for PPAR{5 caused an anabolic response similar to that of PGE2, suggesting a role for PPAR8 in bone formation.
P 24 OSCILLATING FLUID FLOW INCREASES CALCIUM AND ANNEX1N V M R N A EXPRESSION 1N BONE CELLS CE Yellowlev, Y Zhang, CR Jacobs and HJ Donahue MRL, Department of Orthopaedics and Rehabilitation, Penn State College of Medicine, Hershey, Pennsylvania 17033, U S A Mechanical loading plays a dominant role in the establishment and maintenance of structurally competent bone. However, the mechanisms by which physical signals exert biological effects are unknown. We examined the effects of oscillating fluid flow on intracellular calcium concentration ([Ca2+]i) as well as annexin V (AnxV) mRNA expression and cell protein localization in bone cells. Anx V, an abundant Ca2+-depundent phospholipid binding protein in hone (1), has been proposed to act as a mechanosensitive ion channel (2) and may play a role in mechanotransduction. MG63, human osteosarcoma cells, were mounted in a parallel plate flow chamber and subjected to a IHz sinusoidally oscillating fluid flow (20dynes/cm 2 peak shear stress). [Ca2+]i was quantified using Fura2AM Quantitative real time reverse transcriptase PCR and imlnunocytochemistt2¢ were used to quantify changes in AnxV expression and cellular distribution respectively. Oscillating fluid flow stimulated an immediate, transient increase in [Ca2-]i AnxV mRNA levels increased 1.7fold in response to 2hrs of flow and a 24hr incubation period. Uniform preflow AnxV distribution was unaffected by 2hrs of flow. The Ca2+i response to flow in MG63 suggests that fluid flow-induced mobilization of Ca2*i may contribute to the mechanism by which mechanical loads are transduced by bone cells. AnxV mRNA levels were increased by fluid flow suggesting that it may be a mechanically regulated proteth Further experiments are planned to determine a possible role l\n AnxV in the mechanotransduction pathway 1 Mohiti J et al. (199S), Molecular Membrane Biology, 12:321-329. 2 Mobasheri A. et al (1998), Histology and Histopathology, 13(3):893-910
P 21 CORTISOL DECREASES HEPATOCYTE GROWTH FACTOR LEVELS IN H U M A N OSTEOBLAST-LIKE CELLS
Skrtic S. Ohlsson C Research Center for Endocrinology and Metabolism, Sahlgrenska University' Hospital, SE-413 45 GOteborg, Sweden. Osteoporosis is a well-known side effect of long-term treatment with glucocorticoids. The hepatocyte growth factor (HGF) receptor is expressed by human osteoclasts and osteoblasts and mouse osteoblasts also express HGF, indicating that HGF may regulate bone metabolism. Because HGF could be a candidate factor in the local paracrine signaling between osteoblasts and osteoclasts in bone, we decided to studv if human osteoblasts secrete HGF and if glucocorticoids regulate the expression of HGF HGF was easily detectable in the culture medium from human osteoblast-like cells (hOB) The HGF protein released into the culture medium was increased with increasing confluency. Hydrocortisone decreased the amount of HGF released into the culture medium from hOB in a dosedependent manner with a m~ximal effect at 10-6M. Time-course studies revealed that hydrocortisone decreased the amount of HGF released into the culture medium significantly after l 6 h of stimulation (65+2 % of control culture). This effect of hydrocortisone was maximal after 24 h of stimulation (52_+8 % of control culture). In conclusion HGF is produced by primaD' cultured hOB cells. Fmlhernmre, the amount of HGF released into the culture medium is decreased by glucocorticoids. The biological significance of this finding remains to be demonstrated.
P 23 MECHANICAL PROPERTIES OF OSTEOBLASTS SUBJECTED TO CYCLICAL STRAIN - A N ATOMIC FORCE MICROSCOPY STUDY ROMY THOMAS, MALAV SHROFF, JIM TRIFFITTLBONE RESEARCH LABORATORY, NUFFIELD ORTH. CENTRE, OXFORD, 0)(3 7LD, GUILLAUME CHARRAS, MICHAEL HORTON DEPT. OF MEDICINE, UCL, LONDON, WCtE 6JF
Numerous studies have documented the effects of cyclical physiological strains on osteoblast-like cells. To date however, no study has addressed the relationship between cyclical strain, cytoskeletal rearrangement and mechanical properties of the osteoblast. Further the effects of hyper-physiological cyclical strains on the osteoblast cytoskeletal adaptive response are yet to be determined. In order to test the hypothesis that the mechanical properties of the osteoblast cell membrane may" be altered in response to cyclical strain stimuli, an atomic force microscope was used to study the membrane stiffness characteristics of an MG-63 osteoblastic cell line. Cells were seeded at a density of I x 105 on flexible polyurethane wells attached to a computer controlled cell stretching device. Cells were subjected to cyclical strain of 200,000 microstrain for one hour and cell membrane mechanical property measurements made 24 hours later. Mechanical properties of cells were evaluated (as a relationship between external force and the membrane indentation depth) for 5 different locations on each cell. Measurements for 10 cells each from strained and unstrained groups were obtained. No significant change in cell membrane mechanical property parameters between groups was detected 24 hours following a strain stimulus. Possible reasons are discussed. The feasibility of evaluating the micro-mechanical properties of osteoblast-like cells using an atomic force microscope is demonstrated Studies utilising difl'erent strain regimens with mechanical property, assessments at different time points from initiation of strain are underway.
Abstracts
37S
P 22 A G O N I S T S FOR P E R O X I S O M E P R O L I F E R A T O R A C T I V A T E D R E C E P T O R INCREASE OSTEOGENESIS I N VITRO.
K.Still and A.M. Scutt. Dept of Human Metabolism and Clinical Biochemist~', University of Sheffield, UK. A number of substances, in particular prostaglandin E2 (PGE2), are known which stimulate bone formation when administered to adult animals. However. its use in the clinic is prevented by its profound side effects. There are currently no bone anabolic agents on the market except for fluoride, which increases bone mineral densiB' by an unknown mechanism but paradoxically is also associated with an increase in fracture rate Hence there is an urgent need for new bone anabolic drugs. The half-life of PGE2 in vivo is known to be less than 2 seconds. It is also known that PGE series prostaglandins undergo a dehydration reaction in the presence of serum albumin to form prostanoids of the A series. Therefore, we considered the possibility, that some of the effects of PGE2 may be caused by its degradation product, PGA2. We have subsequently shown that PGA2 shows positive activi~' in a number of in vitro assays normally indicative of bone anabolic activity, namely the CFU-f assay, non-adherent stromaI precursor assay and the calvarial collagen synthesis assay PGA~ & A2 are known to bind to the nuclear receptors PPAR c~, /3 and 8 We investigated the effects of a number of PPAR agunists with known specificity' on colony formation using the CFU-f assay. Initial studies show that these drugs are indeed anabolic In particular, drugs which are mainly agonists for PPAR{5 caused an anabolic response similar to that of PGE2, suggesting a role for PPAR8 in bone formation.
P 24 OSCILLATING FLUID FLOW INCREASES CALCIUM AND ANNEX1N V M R N A EXPRESSION 1N BONE CELLS CE Yellowlev, Y Zhang, CR Jacobs and HJ Donahue MRL, Department of Orthopaedics and Rehabilitation, Penn State College of Medicine, Hershey, Pennsylvania 17033, U S A Mechanical loading plays a dominant role in the establishment and maintenance of structurally competent bone. However, the mechanisms by which physical signals exert biological effects are unknown. We examined the effects of oscillating fluid flow on intracellular calcium concentration ([Ca2+]i) as well as annexin V (AnxV) mRNA expression and cell protein localization in bone cells. Anx V, an abundant Ca2+-depundent phospholipid binding protein in hone (1), has been proposed to act as a mechanosensitive ion channel (2) and may play a role in mechanotransduction. MG63, human osteosarcoma cells, were mounted in a parallel plate flow chamber and subjected to a IHz sinusoidally oscillating fluid flow (20dynes/cm 2 peak shear stress). [Ca2+]i was quantified using Fura2AM Quantitative real time reverse transcriptase PCR and imlnunocytochemistt2¢ were used to quantify changes in AnxV expression and cellular distribution respectively. Oscillating fluid flow stimulated an immediate, transient increase in [Ca2-]i AnxV mRNA levels increased 1.7fold in response to 2hrs of flow and a 24hr incubation period. Uniform preflow AnxV distribution was unaffected by 2hrs of flow. The Ca2+i response to flow in MG63 suggests that fluid flow-induced mobilization of Ca2*i may contribute to the mechanism by which mechanical loads are transduced by bone cells. AnxV mRNA levels were increased by fluid flow suggesting that it may be a mechanically regulated proteth Further experiments are planned to determine a possible role l\n AnxV in the mechanotransduction pathway 1 Mohiti J et al. (199S), Molecular Membrane Biology, 12:321-329. 2 Mobasheri A. et al (1998), Histology and Histopathology, 13(3):893-910