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Influence of hydrostatic pressure on expression of heat shock protein 70 and matrix synthesis in chondrocytes
R. Hayashi and C. Balny (Editors), High Pressure Bioscience and Biotechnology 9 1996 Elsevier Science B.V. All rights reserved.
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Influence of hydrostatic pressure on expression of heat shock protein 70 and matrix synthesis in chondrocytes K. TAKAHASHI, T. KUBO, Y. ARAI, Y. HIRASAWA, J. IMANISHI*, K. KOBAYASHI* and M. TAKIGAWA* * Departments of Orthopaedic Surgery and *Microbiology, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602, Japan. * *Department of Biochemistry and Molecular Dentistry, Okayama University Dental School, Shikada-cho, Okayama 700, Japan.
Abstract To investigate the influence of hydrostatic pressure (HP) on the expressions of heat shock protein 70 (HSP70), and to know the relationship between HSP70 expression and matrix synthesis, we subjected chondrocyte-like cells to HP. HSP70 were enhanced after exposure to 10 to 50 MPa of HP. 35S sulfate incorporation into the cultured cells increased after exposure to 5 MPa of HP and decreased after 50 MPa of HP.
1. I N T R O D U C T I O N Mechanical stress is known to have an important role in the control of matrix synthesis in cartilage. Certain levels of hydrostatic pressure (HP) can stimulate the synthesis of matrix by chondrocytes and can maintain matrix metabolism. However, some levels of HP can depress the turnover of matrix and may lead to cartilage degradation (1). On the other hand, heat shock protein (HSP) is produced by cells after the application of various stresses. It was reported that active synthesis of 70-kDa HSP (HSP70), correlated with the severity of osteoarthritis (OA) (2). In this study, we exposed chondrocyte-like cells to HP and examined the relationship between HSP70 expression and matrix synthesis. 2. M A T E R I A L S AND M E T H O D S
Cell culture HCS-2/8 cells (3) were seeded in plastic petri dishes in Dulbecco's modified Eagle's medium (DMEM) contains 10% fetal bovine serum The cells were maintained at 37~ in a humidified atmosphere of 5% CO2. Pressure application After reached confluence, HCS-2/8 cells were exposed to HP ranging from 1 MPa to 50 MPa. In all assays, duration of HP exposure was set to be 2 hours. The petri dishes were placed in a teflon pouch, which was filled with DMEM. The pouch was then placed in a
80 stainless steel pressurization vessel (inside size, ~65 m m X 9 0 mm), equipped with an oil pressure apparatus (Type KP5B, Hikari Koatsu, Hiroshima, Japan). Temperature was maintained at 37~
35s sulfate incorporation assay for proteoglycan synthesis Control cells and cells after HP exposure (5 and 50 MPa) were labeled for 2 hours by 3 5S sulfate. 35S sulfate incorporation was measured by using a scintillation counter, and was normalized by the total protein concentration. The statistical significance of results was evaluated by using the Student's t-test.
Northern blotting Total RNA was extracted from the control and HP (1, 5, 10, and 50 MPa) exposed cells between 30 minutes and 24 hours after HP exposure. Northern blotting was performed according to the method described previously (4). RNA loading was examined by probing for 15-actin mRNA.
Western blotting Control cells and cells exposed to 1, 5, 10, and 50 Mpa of HP. The cells were harvested at 4 and 8 hours after HP exposure and then sonicated. HSP70 was detected in Western blotting as previously described (4), and a monoclonal antibody (Amersham) was used as the primary antibody.
3. RESULTS
Effects of liP on proteoglycan synthesis in HCS-2/8 cells Exposure to 50 MPa of HP resulted significant (p<0.001) decreases of 35S sulfate incorporation, i.e., 63.5+1.77% (mean+SD, n=18) of the levels in control cells (under atmospheric pressure, 100+5.52%, n=17 ). However, 35S sulfate incorporation increased when the cells were exposed to 5 MPa of liP (109.6__+8.17%, n=17, p<0.001) (Fig.l).
Fig. 1. Effects of HP exposure on 3 5S sulfate incorporation. The figures are expressed in % to the levels of control cells. Results are mean___SD. C: control under atmospheric pressure.* " p<0.001.
81 HP-induced HSP70 in HCS-2/8 cells HSP70 mRNA were detected by Northern blotting in the cells grown under atmospheric pressure. After exposure to HP for 2 hours ranging from 1 MPa to 50 MPa, pressuredependent elevation in HSP70 mRNA was observed at 4 and 8 hours after the release of HP (Fig. 2). At 12 and 24 hours after the release of HP, HSP70 mRNA levels were close to that under atmospheric pressure. Western blotting demonstrated that HSP70 in cells exposed to 50 MPa of HP increased 4 and 8 hours after the release of HE compared with that in the control cells (Fig. 3).
Fig. 3.
Induction of HSP70 in HCS-2/8 cells after HP exposure. HSP70 in cells at 4 hours after HP exposure was detected by Western blotting. C: control under atmospheric pressure.
4. D I S C U S S I O N Maximum contact pressures in the human hips during a walk are reported to range from 3
82 WlPa to 10 MPa (5). and the oressure level can rise to almost 20 Mpa during some activities (6). HP is considered to rise at the same order. Our study showed that the rate of proteoglycan synthesis increased after giving 5 MPa of HP. and markedly decreased after giving 50 MPa of HP. These results indicate that HP at physiologic magnitudes can increase the proteoglycan synthesis rate in chondrocytes, while HP at unphysiologic magnitude decrease the proteoglycan synthesis rate in chondrocytes. We previously demonstrated that chondrocytes from OA tissue expressed HSP70, and the level elevated as severity of OA increased (2). HSP is produced by cells which received various stresses, such as heat shock, heavy metals, ethyl alcohol, sulfhydryl reagents, amino acid analogs, viral infections, and oxygen deprivation (7). Analysis of HSP expression will provide many indications of cell responses to mechanical stress. After exposure to HP at unphysiologic level (50MPa), HSP70 expression was markedly enhanced immediately after the HP exposure. Such a rapid elevation of HSP70 mRNA level was not observed in the cells receiving HP at lower magnitudes. These results indicate 50 MPa of HP is excessively strong for chondrocytes and such a high magnitude of HP affectes chondrocytes adversely. High HP also promotes dissociation, unfolding, and misassembly of oligomeric proteins (8). Major roles of HSP70 are to bind and refold partially denatured or misfolded proteins, and to dissociate abnormally aggregated proteins (7). It is possible that HSP70 is induced in cells immediately after HP exposure in order to bind denatured or misfolded proteins which are generated in the cells exposed to 50 MPa of HP as in the cells exposed to heat shock. In conclusion, This study demonstrates that HP at physiologic level increase proteoglycan synthesis, and excessively strong HP evokes enhancement of HSP70 expression along with the decrease in proteoglycan synthesis. These results indicate HP may be a important modulator of the metabolic activities of chondrocvtes and HSP70 could be a charasteristic indicator of an abnormal and harmful state of chondrocytes which receive excessively high HP in articular cartilage. 5. REFERENCES
1 A. C. Hall, J. E G. Urban, K. A. Gehl: J Orthop Res 9:1-10, 1991. 2 T. Kubo, C. A. Towle, H. J. Mankin, B. J. Treadwell: Arthritis Rheum 28:1140-1145, 1985. 3 M. Takigawa, K. Tajima, H-O. Pan, M. Emoto, A. Kinoshita, E Suzuki, Y. Takano, Y. Moil: Cancer Res 49:3996-4002, 1989. 4 K. Kobayashi, E. Ohgitani, Y. Tanaka, M. Kita, J. Imanishi: Microbiol Immunol 38:321325, 1994. 5 N.Y. E Afohe, P. D. Byers, W. C. Hutton: J Bone Joint Surg 69B:536-541, 1987. 6 W. A. Hodge, R. S. Fijan, K. L. Carlson, R. G. Burgess, W. H. Harris, R. W. Mann: Proc Nath Acad Sci USA 83:2879-2883, 1986. 7 R. I. Morimoto, A. Tissieres, C. Georgopoulos: Cold Spring Harbor Laboratory Press, 1450, 1990. 8 M. Gross, R. Jaenicke: Eur J Biochem 221:617-630, 1994.
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Influence of hydrostatic pressure on expression of heat shock protein 70 and matrix synthesis in chondrocytes K. TAKAHASHI, T. KUBO, Y. ARAI, Y. HIRASAWA, J. IMANISHI*, K. KOBAYASHI* and M. TAKIGAWA* * Departments of Orthopaedic Surgery and *Microbiology, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602, Japan. * *Department of Biochemistry and Molecular Dentistry, Okayama University Dental School, Shikada-cho, Okayama 700, Japan.
Abstract To investigate the influence of hydrostatic pressure (HP) on the expressions of heat shock protein 70 (HSP70), and to know the relationship between HSP70 expression and matrix synthesis, we subjected chondrocyte-like cells to HP. HSP70 were enhanced after exposure to 10 to 50 MPa of HP. 35S sulfate incorporation into the cultured cells increased after exposure to 5 MPa of HP and decreased after 50 MPa of HP.
1. I N T R O D U C T I O N Mechanical stress is known to have an important role in the control of matrix synthesis in cartilage. Certain levels of hydrostatic pressure (HP) can stimulate the synthesis of matrix by chondrocytes and can maintain matrix metabolism. However, some levels of HP can depress the turnover of matrix and may lead to cartilage degradation (1). On the other hand, heat shock protein (HSP) is produced by cells after the application of various stresses. It was reported that active synthesis of 70-kDa HSP (HSP70), correlated with the severity of osteoarthritis (OA) (2). In this study, we exposed chondrocyte-like cells to HP and examined the relationship between HSP70 expression and matrix synthesis. 2. M A T E R I A L S AND M E T H O D S
Cell culture HCS-2/8 cells (3) were seeded in plastic petri dishes in Dulbecco's modified Eagle's medium (DMEM) contains 10% fetal bovine serum The cells were maintained at 37~ in a humidified atmosphere of 5% CO2. Pressure application After reached confluence, HCS-2/8 cells were exposed to HP ranging from 1 MPa to 50 MPa. In all assays, duration of HP exposure was set to be 2 hours. The petri dishes were placed in a teflon pouch, which was filled with DMEM. The pouch was then placed in a
80 stainless steel pressurization vessel (inside size, ~65 m m X 9 0 mm), equipped with an oil pressure apparatus (Type KP5B, Hikari Koatsu, Hiroshima, Japan). Temperature was maintained at 37~
35s sulfate incorporation assay for proteoglycan synthesis Control cells and cells after HP exposure (5 and 50 MPa) were labeled for 2 hours by 3 5S sulfate. 35S sulfate incorporation was measured by using a scintillation counter, and was normalized by the total protein concentration. The statistical significance of results was evaluated by using the Student's t-test.
Northern blotting Total RNA was extracted from the control and HP (1, 5, 10, and 50 MPa) exposed cells between 30 minutes and 24 hours after HP exposure. Northern blotting was performed according to the method described previously (4). RNA loading was examined by probing for 15-actin mRNA.
Western blotting Control cells and cells exposed to 1, 5, 10, and 50 Mpa of HP. The cells were harvested at 4 and 8 hours after HP exposure and then sonicated. HSP70 was detected in Western blotting as previously described (4), and a monoclonal antibody (Amersham) was used as the primary antibody.
3. RESULTS
Effects of liP on proteoglycan synthesis in HCS-2/8 cells Exposure to 50 MPa of HP resulted significant (p<0.001) decreases of 35S sulfate incorporation, i.e., 63.5+1.77% (mean+SD, n=18) of the levels in control cells (under atmospheric pressure, 100+5.52%, n=17 ). However, 35S sulfate incorporation increased when the cells were exposed to 5 MPa of liP (109.6__+8.17%, n=17, p<0.001) (Fig.l).
Fig. 1. Effects of HP exposure on 3 5S sulfate incorporation. The figures are expressed in % to the levels of control cells. Results are mean___SD. C: control under atmospheric pressure.* " p<0.001.
81 HP-induced HSP70 in HCS-2/8 cells HSP70 mRNA were detected by Northern blotting in the cells grown under atmospheric pressure. After exposure to HP for 2 hours ranging from 1 MPa to 50 MPa, pressuredependent elevation in HSP70 mRNA was observed at 4 and 8 hours after the release of HP (Fig. 2). At 12 and 24 hours after the release of HP, HSP70 mRNA levels were close to that under atmospheric pressure. Western blotting demonstrated that HSP70 in cells exposed to 50 MPa of HP increased 4 and 8 hours after the release of HE compared with that in the control cells (Fig. 3).
Fig. 3.
Induction of HSP70 in HCS-2/8 cells after HP exposure. HSP70 in cells at 4 hours after HP exposure was detected by Western blotting. C: control under atmospheric pressure.
4. D I S C U S S I O N Maximum contact pressures in the human hips during a walk are reported to range from 3
82 WlPa to 10 MPa (5). and the oressure level can rise to almost 20 Mpa during some activities (6). HP is considered to rise at the same order. Our study showed that the rate of proteoglycan synthesis increased after giving 5 MPa of HP. and markedly decreased after giving 50 MPa of HP. These results indicate that HP at physiologic magnitudes can increase the proteoglycan synthesis rate in chondrocytes, while HP at unphysiologic magnitude decrease the proteoglycan synthesis rate in chondrocytes. We previously demonstrated that chondrocytes from OA tissue expressed HSP70, and the level elevated as severity of OA increased (2). HSP is produced by cells which received various stresses, such as heat shock, heavy metals, ethyl alcohol, sulfhydryl reagents, amino acid analogs, viral infections, and oxygen deprivation (7). Analysis of HSP expression will provide many indications of cell responses to mechanical stress. After exposure to HP at unphysiologic level (50MPa), HSP70 expression was markedly enhanced immediately after the HP exposure. Such a rapid elevation of HSP70 mRNA level was not observed in the cells receiving HP at lower magnitudes. These results indicate 50 MPa of HP is excessively strong for chondrocytes and such a high magnitude of HP affectes chondrocytes adversely. High HP also promotes dissociation, unfolding, and misassembly of oligomeric proteins (8). Major roles of HSP70 are to bind and refold partially denatured or misfolded proteins, and to dissociate abnormally aggregated proteins (7). It is possible that HSP70 is induced in cells immediately after HP exposure in order to bind denatured or misfolded proteins which are generated in the cells exposed to 50 MPa of HP as in the cells exposed to heat shock. In conclusion, This study demonstrates that HP at physiologic level increase proteoglycan synthesis, and excessively strong HP evokes enhancement of HSP70 expression along with the decrease in proteoglycan synthesis. These results indicate HP may be a important modulator of the metabolic activities of chondrocvtes and HSP70 could be a charasteristic indicator of an abnormal and harmful state of chondrocytes which receive excessively high HP in articular cartilage. 5. REFERENCES
1 A. C. Hall, J. E G. Urban, K. A. Gehl: J Orthop Res 9:1-10, 1991. 2 T. Kubo, C. A. Towle, H. J. Mankin, B. J. Treadwell: Arthritis Rheum 28:1140-1145, 1985. 3 M. Takigawa, K. Tajima, H-O. Pan, M. Emoto, A. Kinoshita, E Suzuki, Y. Takano, Y. Moil: Cancer Res 49:3996-4002, 1989. 4 K. Kobayashi, E. Ohgitani, Y. Tanaka, M. Kita, J. Imanishi: Microbiol Immunol 38:321325, 1994. 5 N.Y. E Afohe, P. D. Byers, W. C. Hutton: J Bone Joint Surg 69B:536-541, 1987. 6 W. A. Hodge, R. S. Fijan, K. L. Carlson, R. G. Burgess, W. H. Harris, R. W. Mann: Proc Nath Acad Sci USA 83:2879-2883, 1986. 7 R. I. Morimoto, A. Tissieres, C. Georgopoulos: Cold Spring Harbor Laboratory Press, 1450, 1990. 8 M. Gross, R. Jaenicke: Eur J Biochem 221:617-630, 1994.