Osteopontin Gene Regulation by Oscillatory Fluid Flow via Intracellular Calcium Mobilization and Activation of Mitogen-activated Protein Kinase in MC3T3–E1 Osteoblasts
2001; Elsevier BV; Volume: 276; Issue: 16 Linguagem: Inglês
10.1074/jbc.m009846200
ISSN1083-351X
AutoresJun You, Gwendolen C. Reilly, Xuechu Zhen, Clare E. Yellowley, Qian Chen, Henry J. Donahue, Christopher R. Jacobs,
Tópico(s)Alkaline Phosphatase Research Studies
ResumoRecently fluid flow has been shown to be a potent physical stimulus in the regulation of bone cell metabolism. However, most investigators have applied steady or pulsing flow profiles rather than oscillatory fluid flow, which occurs in vivo because of mechanical loading. Here oscillatory fluid flow was demonstrated to be a potentially important physical signal for loading-induced changes in bone cell metabolism. We selected three well known biological response variables including intracellular calcium (Ca2+i), mitogen-activated protein kinase (MAPK) activity, and osteopontin (OPN) mRNA levels to examine the response of MC3T3–E1 osteoblastic cells to oscillatory fluid flow with shear stresses ranging from 2 to −2 Newtons/m2 at 1 Hz, which is in the range expected to occur during routine physical activities. Our results showed that within 1 min, oscillatory flow induced cell Ca2+i mobilization, whereas two MAPKs (ERK and p38) were activated over a 2-h time frame. However, there was no activation of JNK. Furthermore 2 h of oscillatory fluid flow increased steady-state OPN mRNA expression levels by approximately 4-fold, 24 h after exposure to fluid flow. The presence of both ERK and p38 inhibitors and thapsigargin completely abolished the effect of oscillatory flow on steady-state OPN mRNA levels. In addition, experiments using a variety of pharmacological agents suggest that oscillatory flow induces Ca2+i mobilization via the L-type voltage-operated calcium channel and the inositol 1,4,5-trisphosphate pathway. Recently fluid flow has been shown to be a potent physical stimulus in the regulation of bone cell metabolism. However, most investigators have applied steady or pulsing flow profiles rather than oscillatory fluid flow, which occurs in vivo because of mechanical loading. Here oscillatory fluid flow was demonstrated to be a potentially important physical signal for loading-induced changes in bone cell metabolism. We selected three well known biological response variables including intracellular calcium (Ca2+i), mitogen-activated protein kinase (MAPK) activity, and osteopontin (OPN) mRNA levels to examine the response of MC3T3–E1 osteoblastic cells to oscillatory fluid flow with shear stresses ranging from 2 to −2 Newtons/m2 at 1 Hz, which is in the range expected to occur during routine physical activities. Our results showed that within 1 min, oscillatory flow induced cell Ca2+i mobilization, whereas two MAPKs (ERK and p38) were activated over a 2-h time frame. However, there was no activation of JNK. Furthermore 2 h of oscillatory fluid flow increased steady-state OPN mRNA expression levels by approximately 4-fold, 24 h after exposure to fluid flow. The presence of both ERK and p38 inhibitors and thapsigargin completely abolished the effect of oscillatory flow on steady-state OPN mRNA levels. In addition, experiments using a variety of pharmacological agents suggest that oscillatory flow induces Ca2+i mobilization via the L-type voltage-operated calcium channel and the inositol 1,4,5-trisphosphate pathway. Mechanical loading plays an important role in regulating bone metabolism. Increased mechanical loading increases bone formation and decreases bone resorption (1Morey E.R. Baylink D.J. Science.. 1978; 201: 1138-1141Google Scholar). The absence of mechanical stimulation causes reduced bone matrix protein production, mineral content, and bone formation, as well as an increase in bone resorption (2Sessions N.D. Halloran B.P. Bikle D.D. Wronski T.J. Cone C.M. Morey-Holton E. Am. J. Physiol... 1989; 257: E606-E610Google Scholar). However, the mechanism by which bone cells sense and respond to their physical environment is still poorly understood. In this study we examine a novel physical stimulus and loading-induced oscillatory fluid flow and demonstrate that when applied to cultured osteoblastic cells at levels expected to occur in vivo it regulates mRNA levels for an important bone matrix protein, osteopontin (OPN).1 Furthermore, this regulation occurs via an increase in intracellular calcium (Ca2+i) and mitogen-activated protein kinases (MAPKs). The sensitivity of bone tissue to mechanical loading has been proposed to involve a variety of cellular biophysical signals including loading-induced electric fields, matrix strain, and fluid flow. The latter effect of loading, originally described by Piekarski et al. (3Piekarski K. Munro M. Nature.. 1977; 269: 80-82Google Scholar), has recently been proposed to directly regulate bone cell metabolism in vivo (4Weinbaum S. Cowin S.C. Zeng Y.A. J. Biomech... 1994; 27: 339-360Google Scholar, 5Cowin S.C. Weinbaum S. Zeng Y. J. Biomech... 1995; 28: 1281-1297Google Scholar). Furthermore, relative to other loading-induced biophysical signals applied to cells in vitro, fluid flow appears to be significantly more potent at physiological levels (6McLeod K.J. Donahue H.J. Levin P.E. Rubin C.T. Brighton C.T. Pollack S.R. Electromagnetics in Biology and Medicine.San Francisco Press. 1991; : 111-115Google Scholar, 7Owan I. Burr D.B. Turner C.H. Qiu J. Tu Y. Onyia J.E. Duncan R.L. Am. J. Physiol... 1997; 42: C810-C815Google Scholar, 8Smalt R. Mitchell T. Howard R.L. Chambers T.J. Am. J. Physiol... 1997; 273: E751-E758Google Scholar, 9You J. Yellowley C.E. Donahue H.J. Zhang Y. Chen Q. Jacobs C.R. J. Biomech. Eng... 2000; 122: 387-393Google Scholar, 10Hung C.T. Allen F.D. Pollack S.R. Brighton C.T. J. Biomech... 1996; 29: 1403-1409Google Scholar). The origin of loading-induced fluid flow is a consequence of the fact that a significant component of bone tissue is unbound fluid. Bone tissue contains an extracellular fluid compartment that has been demonstrated to communicate with the vascular compartment, and mechanical loading has been shown to enhance fluid exchange between the two spaces (11Knothe Tate M.L. Niederer P. Knothe U. Bone.. 1998; 22: 107-117Google Scholar). When bone is exposed to mechanical loading fluid in the matrix is pressurized and tends to flow into haversian canals. As loading is removed (e.g. during the gait cycle) the pressure gradients, and consequently the direction of fluid flow, are reversed resulting in a flow-time history experienced by the cells that is oscillatory in nature. In vitro experiments have shown fluid flow to have a number of effects on bone cells including Ca2+i mobilization (12Hung C.T. Pollack S.R. Reilly T.M. Brighton C.T. Clin. Orthop... 1995; 313: 256-269Google Scholar), production of nitric oxide and prostaglandin E2 (8Smalt R. Mitchell T. Howard R.L. Chambers T.J. Am. J. Physiol... 1997; 273: E751-E758Google Scholar, 13Ajubi N.E. Klein-Nulend J. Alblas M.J. Burger E.H. Nijweide P.J. Am. J. Physiol... 1999; 276: E171-E178Google Scholar), and regulation of the expression of genes for OPN, Cyclooxygenase-2, and c-Fos (9You J. Yellowley C.E. Donahue H.J. Zhang Y. Chen Q. Jacobs C.R. J. Biomech. Eng... 2000; 122: 387-393Google Scholar, 14Chen N.X. Ryder K.D. Pavalko F.M. Turner C.H. Burr D.B. Qiu J. Duncan R.L. Am. J. Physiol... 2000; 278: C989-C997Google Scholar, 15Pavalko F.M. Chen N.X. Turner C.H. Burr D.B. Atkinson S. Hsieh Y.F. Qiu J. Duncan R.L. Am. J. Physiol... 1998; 275: C1591-C1601Google Scholar). However, it is important to note that only one study to date utilized a reversing flow profile and found significantly different results when contrasted with nonreversing flow (16Jacobs C.R. Yellowley C.E. Davis B.R. Zhou Z. Cimbala J.M. Donahue H.J. J. Biomech... 1998; 31: 969-976Google Scholar). Thus, the aim of this study is to detail important aspects of the biochemical response pathway including immediate, intermediate, and long term effects of oscillatory fluid flow on bone cells, as well as on their inter-relationships. To achieve this goal, we first investigated three well known biological osteogenic response variables. Ca2+i, a known second messenger transducing extracellular signals to the cell interior, was our immediate response variable. Activity of MAPKs is important for regulating cell differentiation and apoptosis by transmitting extracellular signals to the nucleus (17Seger R. Krebs E.G. FASEB J... 1995; 9: 726-735Google Scholar, 18Kyriakis J.M. Banerjee P. Nikolakaki E. Dai T. Rubie E.A. Ahmad M.F. Avruch J. Woodgett J.R. Nature.. 1994; 369: 156-160Google Scholar) and was our intermediate response variable. OPN is characterized as one of the predominant noncollagenous proteins that accumulate in the extracellular matrix of bone (19Denhardt D.T. Guo X. FASEB J... 1993; 7: 1475-1482Google Scholar, 20Gerstenfeld L.C. Uporova T. Ashkar S. Salih E. Gotoh Y. McKee M.D. Nanci A. Glimcher M.J. Ann. N. Y. Acad. Sci... 1995; 760: 67-82Google Scholar) and is also believed to be an important factor associated with bone remodeling caused by mechanical stress in vivo(21Terai K. Takano-Yamamoto T. Ohba Y. Hiura K. Sugimoto M. Sato M. Kawahata H. Inaguma N. Kitamura Y. Nomura S. J. Bone Miner. Res... 1999; 14: 839-849Google Scholar). Recently, strong evidence suggests that OPN is an important factor in loading induced bone cell metabolism (22Toma C.D. Ashkar S. Gray M.L. Schaffer J.L. Gerstenfeld L.C. J. Bone Miner. Res... 1997; 12: 1626-1636Google Scholar, 23Harter L.V. Hruska K.A. Duncan R.L. Endocrinology.. 1995; 136: 528-535Google Scholar, 24Kubota T. Yamauchi M. Onozaki J. Sato S. Suzuki Y. Sodek J. Arch. Oral Biol... 1993; 38: 23-30Google Scholar). Furthermore, the role of osteopontin in extracellular matrix is more than structural. It has been shown to be involved in regulating bone cell attachment, osteoclast function, and mineralization, suggesting a central role in both the initiation and regulation of bone remodeling (25Reinholt F.P. Hultenby K. Oldberg A. Heinegard D. Proc. Natl. Acad. Sci. U. S. A... 1990; 87: 4473-4475Google Scholar, 26Giachelli C.M. Steitz S. Matrix Biol... 2000; 19: 615-622Google Scholar). Therefore, we quantified steady-state OPN mRNA levels as a long term response to oscillatory flow. Recently MAPK family members including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAP kinase have been shown to be important signaling components linking mechanical stimuli to cellular responses, including cell growth, differentiation, and metabolic regulation, in endothelial cells, smooth muscle cells, and myocytes (27Jo H. Sipos K. Go Y.M. Law R. Rong J. McDonald J.M. J. Biol. Chem... 1997; 272: 1395-1401Google Scholar, 28Yan C. Takahashi M. Okuda M. Lee J.D. Berk B.C. J. Biol. Chem... 1999; 274: 143-150Google Scholar, 29Li C. Hu Y. Mayr M. Xu Q. J. Biol. Chem... 1999; 274: 25273-25280Google Scholar, 30Liang F. Gardner D.G. J. Clin. Invest... 1999; 104: 1603-1612Google Scholar). However, the role of MAPKs in bone cell mechanotransduction has not been determined. Moreover the role of Ca2+i in osteogenic gene transcription is unclear, especially in the case of oscillatory fluid flow. Therefore, the second goal of this study is to elucidate the roles of Ca2+i and the three major MAPKs in bone cell osteopontin gene expression induced by oscillatory flow. Finally, the mechanism responsible for fluid-flow-induced Ca2+i mobilization has not been fully established, particularly for the oscillatory flow profiles expected to occur in vivo. Yellowley et al. (31Yellowley C.E. Jacobs C.R. Donahue H.J. J. Cell. Physiol... 1999; 180: 402-408Google Scholar) demonstrated that the steady flow-induced Ca2+i responses in bovine articular chondrocytes involved both influx of external Ca2+ and release of internal Ca2+ from IP3-sensitive stores and that the mechanism is G-protein-activated. Similar results were observed in bone cells stimulated by steady fluid flow (14Chen N.X. Ryder K.D. Pavalko F.M. Turner C.H. Burr D.B. Qiu J. Duncan R.L. Am. J. Physiol... 2000; 278: C989-C997Google Scholar, 32Hung C.T. Allen F.D. Pollack S.R. Brighton C.T. J. Biomech... 1996; 29: 1411-1417Google Scholar). However there is evidence to suggest that steady and oscillatory fluid flow may have different biophysical effects on bone cells (16Jacobs C.R. Yellowley C.E. Davis B.R. Zhou Z. Cimbala J.M. Donahue H.J. J. Biomech... 1998; 31: 969-976Google Scholar). Therefore, the third goal of this study is to elucidate the mechanism contributing to oscillatory flow-induced Ca2+i mobilization in bone cells. Steady flow (32Hung C.T. Allen F.D. Pollack S.R. Brighton C.T. J. Biomech... 1996; 29: 1411-1417Google Scholar), substrate stretch (33Walker L.M. Publicover S.J. Preston M.R. Said Ahmed M.A. El Haj A.J. J. Cell. Biochem... 2000; 79: 648-661Google Scholar), and whole bone loading experiments (34Rawlinson S.C. Pitsillides A.A. Lanyon L.E. Bone.. 1996; 19: 609-614Google Scholar) suggest that either stretch-activated mechanosensitive channels and/or L-type voltage-operated calcium channels (L-type VOCCs) may be involved. Additionally, it is not known whether the involvement of the IP3-sensitive stores is as important in the response to oscillatory fluid flow or whether other internal pathways (the ryanodine-sensitive pathway) may be involved in Ca2+i mobilization. The mouse osteoblastic cell line MC3T3–E1 was cultured in minimal essential medium (MEM-α; Life Technologies, Inc.) containing 10% fetal bovine serum (FBS; Hyclone, Logan, UT), 1% penicillin and streptomycin (Life Technologies, Inc.) and maintained in a humidified incubator at 37 °C with 5% CO2. All cells were subcultured on glass slides for 2 days prior to experiments, with the exception of cells cultured for Ca2+i studies, for which quartz slides were used, for UV transparency. 3 × 105 cells were seeded on the glass slides (75 × 38 × 1.0 mm), and 0.85 × 105 cells were seeded on the quartz slides (76 × 26 × 1.6 mm). There are no significant differences observed in the behavior of MC3T3–E1 cells grown on normal glass slides versus quartz slides. 2Unpublished data. It is important to note that under these conditions the cells had not reached confluency nor did the medium (which did not include ascorbic acid or β-glycerophosphate) or time in culture (2 days) support differentiation or mineralization (35McCauley L.K. Koh A.J. Beecher C.A. Cui Y. Decker J.D. Franceschi R.T. J. Bone Miner. Res... 1995; 10: 1243-1255Google Scholar). Cells were exposed to oscillatory fluid flow in MEM-α and 2% FBS for calcium imaging experiments, and in MEM-α and 10% FBS for long term 2-h experiments. Two different parallel plate flow chamber sizes were utilized. Larger chambers with a rectangular fluid volume of 56 × 24 × 0.28 mm were employed for long term flow to accommodate the larger glass slides. This size of slide was necessary to obtain adequate amounts of cell protein and mRNA. The smaller chamber design, fluid volume 38 × 10 × 0.28 mm, was employed in the calcium imaging studies where total cell number is not an issue. The oscillatory flow device was described in our previous study (16Jacobs C.R. Yellowley C.E. Davis B.R. Zhou Z. Cimbala J.M. Donahue H.J. J. Biomech... 1998; 31: 969-976Google Scholar). Briefly, a Hamilton glass syringe was mounted in a small servopneumatic loading frame (EnduraTec, Eden Prairie, MN). The flow rate was monitored with an ultrasonic flowmeter with a 100-Hz frequency response (Transonic Systems Inc., Ithaca, NY). Intracellular calcium ion concentration ([Ca2+]i) was quantified with the fluorescent dye fura-2. fura-2 exhibits a shift in absorption when bound to Ca2+ such that the emission intensity when illuminated with ultraviolet light increases with calcium concentration at a wavelength of 340 nm and decreases with calcium concentration at 380 nm. The ratio of light intensity between the two wavelengths corresponds to calcium concentration. A calibration curve of intensity ratio and calcium concentration was obtained using fura-2 in buffered calcium standards supplied by the manufacturer (Molecular Probes, Inc., Eugene, OR). Preconfluent (80%) cells were washed with MEM-α and 2% FBS at 37 °C, incubated with 10 μm fura-2-acetoxymethyl ester (Molecular Probes, Inc., Eugene, OR) solution for 30 min at 37 °C, then washed again with fresh MEM-α and 2% FBS prior to experiments. Cell ensembles were illuminated at wavelengths of 340 and 380 nm in turn. Emitted light was passed through a 510-nm interference filter and detected with an intensifier charge coupled device camera (International Ltd., Sterling, VA). Images were recorded, one every 2 s, and analyzed using image analysis software (Metafluor; Universal Imaging, West Chester, PA). Basal [Ca2+]i was sampled for 3 min and followed by 3 min of oscillatory fluid flow (peak shear stress 2n/m2, 1 Hz). There are three major MAPKs, p38 MAPK, ERK, and JNK. 100 μg of lysate protein from either control or flowed cells was immunoprecipitated with anti-p38 MAPK, anti-ERK1/2, or anti-JNK antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight. Following addition of 15 μl of protein A/G for 2 h, the immunocomplex was collected by centrifugation, and the kinase reaction was then conducted in a kinase reaction buffer containing substrates myelin basic protein (for p38 MAPK or ERK) or c-Jun glutathione S-transferase (for JNK) in the presence of [γ-32P]ATP as described before (36Zhen X. Uryu K. Wang H.Y. Friedman E. Mol. Pharmacol... 1998; 54: 453-458Google Scholar). The reaction mix was subjected to SDS polyacrylamide gel electrophoresis, and phosphorylation of substrates was determined by autoradiography. The steady-state osteopontin mRNA level was quantified by quantitative real time reverse transcription polymerase chain reaction (QRT RT-PCR) (9You J. Yellowley C.E. Donahue H.J. Zhang Y. Chen Q. Jacobs C.R. J. Biomech. Eng... 2000; 122: 387-393Google Scholar). Briefly, this technique is based on the detection of a fluorescent signal produced by an OPN-specific oligonucleotide probe during PCR primer extension (Prism 7700 sequence detection system; Applied Biosystems, Frost City, CA). The RNeasy mini kit (Qiagen Inc., Valencia, CA) was used to extract total RNA after lysis and homogenization with the QIAshredder mini column system (Qiagen Inc., Valencia, CA). Mouse osteopontin cDNA primers and probes were designed using sequence data from Miyazaki et al. (37Miyazaki Y. Setoguchi M. Yoshida S. Higuchi Y. Akizuki S. Yamamoto S. J. Biol. Chem... 1990; 265: 14432-14438Google Scholar) (GenBankTM accession numberX51834) and the QRT RT-PCR probe/primer design software Primer Express (version 1.0; Applied Biosystems, Frost City, CA). The fluorogenic oligonucleotide probe for mouse osteopontin was 5′-CGG TGA AAG TGA CTG ATT CTG GCA GCT C-3′ (Synthetic Genetics, San Diego, CA). The forward and reverse PCR primers were 5′-GGC ATT GCC TCC TCC CTC-3′, and 5′-GCA GGC TGT AAA GCT TCT CC-3′, respectively. These sequences were synthesized, and PCR conditions were optimized with respect to concentrations of Mg2+, probe, and both primers. Relative changes in the levels of OPN mRNA and 18 S rRNA were quantified 24 h after mechanical stimulation. The following series of pharmacological agents was used to examine the mechanism of calcium mobilization: thapsigargin (50 nm), gadolinium chloride (10 μm), nifedipine (20 μm), ryanodine (1 and 20 μm), 2-aminoethoxydiphenyl borate (2APB; 100 mm), U73122 and U73343 (4 or 5 μm). Thapsigargin is an inhibitor of the ATP-dependent Ca2+ pump of intracellular Ca2+ stores that causes Ca2+ discharge (38Thastrup O. Cullen P.J. Drobak B.K. Hanley M.R. Dawson A.P. Proc. Natl. Acad. Sci. U. S. A... 1990; 87: 2466-2470Google Scholar) and was used to empty the intracellular calcium stores. Gadolinium chloride (10 μm) (Aldrich) is a putative stretch-activated channel blocker (39Hamill O.P. McBride Jr., D.W. Pharmacol. Rev... 1996; 48: 231-252Google Scholar). Nifedipine is a blocker of the L-type VOCC (40Ferrante J. Triggle D.J. Biochem. Pharmacol... 1990; 39: 1267-1270Google Scholar). Ryanodine, which affects ryanodine-sensitive channels in intracellular calcium stores, was used in two concentrations, 1 μm, which is expected to hold the channel open, and 20 μm, which is expected to block the channel (41Meissner G. J. Biol. Chem... 1986; 261: 6300-6306Google Scholar, 42Hasselbach W. Migala A. FEBS Lett... 1987; 221: 119-123Google Scholar). U73122 inhibits the action of phospholipase C and possibly phospholipase A2 and thereby the production of IP3. Thus, it inhibits the release of calcium through IP3-sensitive intracellular calcium stores (14Chen N.X. Ryder K.D. Pavalko F.M. Turner C.H. Burr D.B. Qiu J. Duncan R.L. Am. J. Physiol... 2000; 278: C989-C997Google Scholar, 43Bleasdale J.E. Thakur N.R. Gremban R.S. Bundy G.L. Fitzpatrick F.A. Smith R.J. Bunting S. J. Pharmacol. Exp. Ther... 1990; 255: 756-768Google Scholar).U73343, an isoform of U73122 that does not inhibit IP3production, was used as a control. 2APB is a specific inhibitor of the IP3 receptor and does not affect ryanodine-sensitive or membrane calcium channels (44Maruyama T. Kanaji T. Nakade S. Kanno T. Mikoshiba K. J. Biochem. ( Tokyo ).. 1997; 122: 498-505Google Scholar, 45Hamada T. Liou S.Y. Fukushima T. Maruyama T. Watanabe S. Mikoshiba K. Ishida N. Neurosci. Lett... 1999; 263: 125-128Google Scholar). Cells were pretreated with medium containing the required drug for 30–60 min prior to flow, and the drug remained present during the flow experiments. Nifedipine, ryanodine, and 2APB were dissolved in 100% ethanol to give a final concentration of ethanol in the flow medium of 0.1% (v/v), and vehicle controls were conducted with the same concentration of ethanol. Thapsigargin, U73122, and U73343 were dissolved in Me2SO to give a final concentration of Me2SO in the flow medium of 0.0032, 0.17, and 0.17% (v/v), respectively. Gadolinium chloride was directly dissolved in medium. Thapsigargin and gadolinium chloride were also used in long term flow experiments to examine the role of [Ca2+]i in downstream responses. For the MAPK investigations, cells were incubated with MAPK inhibitor for 2 h before the fluid flow experiments were performed. The p38 inhibitor SB203580 (10 μm) or the ERK inhibitor PD98059 (10 μm; Calbiochem-Novabiochem) was also present in the flow medium. All pharmacological agents were from Sigma unless indicated. We used a numerical procedure from mechanical analysis, known as Rainflow cycle counting, to identify calcium oscillations (46Jacobs C.R. Yellowley C.E. Nelson D.V. Donahue H.J. Comput. Methods Biomech. Biomed. Eng... 2000; 3: 31-40Google Scholar). Briefly, this technique identifies complete cycles or oscillations in the time history data even when they are superimposed upon each other and therefore can be used to distinguish and quantify [Ca2+]i responses from background noise. We defined a response as an oscillation in [Ca2+]i at least 2-fold greater than that of the average baseline level of nontreated cells. Baseline [Ca2+]i data were recorded for each slide for 3 min prior to the application of oscillatory fluid flow. Data were expressed as mean ± S.E. To compare observations from no flow and flow responses a two-sample Student's t test was used in which sample variance was not assumed to be equal. To compare observations from more than two groups, a one-way analysis of variance was employed followed by a Bonferroni selected pairs multiple comparisons test (Instat; GraphPad Software Inc., San Diego, CA).p < 0.05 was considered statistically significant. For calcium experiments all controls were combined as no effect of vehicles was found (one-way analysis of variance). Typical cell Ca2+iresponses are shown in Fig.1 A. The fraction of MC3T3–E1 cells responding with an increase in Ca2+i to oscillatory fluid flow (peak shear stress 2 n/m2, 1 Hz) is shown in Fig. 1 B. The data were obtained from six individual experiments (slides) and a total of 334 cells. Within 30 s of starting oscillatory flow, 59.1 ± 4.6% of cells increased [Ca2+]i, which was significantly different from no flow periods (8.9 ± 1.6%). However the responding cell [Ca2+]i amplitudes (65.5 ± 17.5 nm) for flow periods were not statistically different from those for no flow periods (86.5 ± 18.3 nm). The time courses of activation of three major MAPKs in MC3T3–E1 cells are shown in Fig.2. At each time point cells from two slides were combined to yield sufficient protein for the MAPK activity assay. In the absence of flow there was minimal p38, ERK1/2, and JNK activity. However, dramatic responses for p38 and ERK1/2 activities were observed beginning 15 min after applying oscillatory flow. p38 activity reached a maximum at 30 min and returned to initial levels 90 min after the onset of oscillatory flow (Fig. 2). ERK1/2 activity reached a maximum at 60 min and returned to its pre-flow value at 90 min. In contrast, there was no change in JNK activity during a 90-min flow period, indicating a selective activation of p38/ERKs in response to flow. The long time frame biological response, steady-state OPN mRNA level, was quantified in response to oscillatory fluid flow at 1 Hz, resulting in a wall shear stress of 2 n/m2, utilizing QRT RT-PCR. The cells that experienced oscillatory flow or no flow for 2 h were then incubated for an additional 24 h prior to collection. Our results show oscillatory fluid flow increased steady-state osteopontin mRNA levels by 3.96 ± 0.76-fold over no flow control (see Fig. 3; p < 0.05). To assess the role of Ca2+i in the OPN mRNA response to oscillatory fluid flow, cells were subjected to oscillatory fluid flow in the presence of 50 nm thapsigargin. Interestingly thapsigargin completely blocked the oscillatory flow effect on steady-state OPN mRNA levels (0.93 ± 0.08 × no flow levels), which were not statistically different from those for no flow period (Fig. 3). However, gadolinium chloride (GdCl3; 10 μm) did not attenuate the flow effect on steady-state OPN mRNA levels (4.19 ± 0.21 × no flow levels), which were not statistically different from those for flow control case. Based on the MAPK activation results, two MAPK inhibitors were employed to block the activity of ERK1/2 and p38. Cells were exposed to 10 μm of the p38 inhibitor SB203580 (SB) for 2 h prior to and for the duration of oscillatory fluid flow. SB reduced the effect of fluid flow on steady-state OPN mRNA levels to 1.61 ± 0.50 × no flow levels. Similar results of ERK1/2 inhibitor PD98059 (PD; 10 μm) were obtained with a reduction of steady-state OPN mRNA to 1.76 ± 0.21 × no flow levels. Moreover the presence of both inhibitors (SB + PD) completely abolished the effect (0.84 ± 0.05 × no flow levels; not statistically different). Those results suggest that activation of p38 MAPK and ERKs is synergistically involved in flow-mediated OPN expression. The number of cells that responded to oscillatory fluid flow with a change in intracellular calcium in the presence of GdCl3 was not significantly different from control (62.2 ± 3.4%; see Fig.4 A). In contrast to the results for GdCl3, nifedipine, an L-type VOCC blocker, did reduce the number of cells responding to flow. The number of cells responding in the presence of nifedipine (29.3 ± 13.6%) was as low as in the no flow case, and the mean increase in [Ca2+]i over baseline (35.6 ± 2.9 nm) was lower than in flow controls. On application of thapsigargin, which emptied intracellular stores, there was a significant (p < 0.05) decrease in the percentage of cells responding to oscillatory flow. U73122, which inhibits production of IP3 via the phospholipase C pathway and does not affect membrane channels, reduced the number of cells responding to 18.0 ± 9.0%, compared with 48.0 ± 9.5% in the control group. 2APB, which acts directly on IP3receptors (45Hamada T. Liou S.Y. Fukushima T. Maruyama T. Watanabe S. Mikoshiba K. Ishida N. Neurosci. Lett... 1999; 263: 125-128Google Scholar) rather than on IP3 catalysis, blocked the response completely. Ryanodine at 1 μm had a small but statistically significant effect on the number of cells responding, which was reduced to 41.6 ± 9.9%. It also had a small effect on the mean increase in [Ca2+]i over baseline, which was reduced to 37.4 ± 5.5 nm. At 20 μm, at which concentration the ryanodine-sensitive channel should have been blocked, there was a very small and not statistically significant reduction in the number of cells responding, to 55.1 ± 10.4%. Although there were some differences in the mean response amplitudes (Fig. 4 B), these were not found to be statistically significant. Some drug-treated cells (nifedipine and ryanodine) showed higher responses in the no flow period compared with controls (data not shown), because the drugs caused increased spontaneous calcium oscillations and increased drift in the baseline levels. Although a large number of in vitro studies have been aimed at discovering the regulatory effect of mechanical loading in bone adaptation, little consensus can be found in the literature regarding the appropriate biophysical signals. For example, bone cells have been shown to respond with metabolic changes to deformation induced by stretching of the substrate to which they are attached (22Toma C.D. Ashkar S. Gray M.L. Schaffer J.L. Gerstenfeld L.C. J. Bone Miner. Res... 1997; 12: 1626-1636Google Scholar,47Rodan G.A. Bourret L.A. Harvey A. Mensi T. Science.. 1975; 189: 467-469Google Scholar, 48Buckley M.J. Banes A.J. Levin L.G. Sumpio B.E. Sato M. Jordan R. Gilbert J. Link G.W. Tran Son Tay R. Bone Miner... 1988; 4: 225-236Google Scholar, 49Brighton C.T. Strafford B. Gross S.B. Leatherwood D.F. Williams J.L. Pollack S.R. J. Bone Jt. Surg. Am... 1991; 73: 320-331Google Scholar). However, these studies employed either hyperphysiologic levels of strain or systems known to induce mechanical effects other than pure strain (50Brown T.D. J. Biomech... 2000; 33: 3-14Google Scholar). More recent studies have suggested that bone cells are more responsive to the fluid flow induced by mechanical strain than directly to the strain in the tissue (7Owan I. Burr D.B. Turner C.H. Qiu J.
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