Ultrasound Stimulates NF-kB Activation and iNOS Expression Via the Ras/Raf/MEK/ERK Signaling Pathway in Cultured Preosteoblasts
Abstract
It has been demonstrated that ultrasound stimulation accelerates the healing of fractures in animal models and in clinical applications where surgery is not involved. Nitric oxide serves as a critical early mediator in the formation of bone induced by mechanical forces. Our investigation revealed that the expression of inducible nitric oxide synthase mediated by ultrasound was reduced by a Ras inhibitor (manumycin A), a Raf-1 inhibitor (GW5074), a MEK inhibitor (PD98059), an NF-kB inhibitor (PDTC), and an IkB protease inhibitor (TPCK). Ultrasound-induced activation of Ras was inhibited by manumycin A. The phosphorylation of Raf-1 at Ser338 in response to ultrasound was inhibited by both manumycin A and GW5074. Ultrasound-induced activation of MEK and ERK was inhibited by manumycin A, GW5074, and PD98059. Stimulation of preosteoblasts with ultrasound led to the activation of IkB kinase a/b, IkBa phosphorylation, p65 phosphorylation at Ser276, and the movement of p65 and p50 from the cytoplasm to the nucleus, as well as kB-luciferase activity. The ultrasound-mediated increase in IKK a/b, IkBa, and p65 phosphorylation, kB-luciferase activity, and the binding of p65 and p50 to the NF-kB element were all inhibited by manumycin A, GW5074, and PD98059. These findings suggest that ultrasound increases the expression of iNOS in preosteoblasts through the Ras/Raf-1/MEK/ERK/IKKab and NF-kB signaling pathways.
Introduction
The process of fracture healing is a complex physiological event involving the coordinated participation of various cell types. Among the different methods used to influence fracture healing, ultrasound stands out due to its non-invasive nature and ease of application. Low intensity levels of ultrasound are employed to accelerate fracture healing and are considered to be neither thermal nor destructive. Studies have shown that low-intensity ultrasound accelerates fracture healing in animal models and in clinical settings where surgery is not performed.
Bone is a dynamic tissue that undergoes remodeling in response to mechanical loads from its external environment. While the enhancement of fracture healing by ultrasound is well-established, the fundamental mechanism of the mechanotransduction pathway involved in cellular responses to ultrasound remains largely unknown. It has been demonstrated that exposure to low-intensity ultrasound pulses increases the release of nitric oxide and prostaglandin, both of which are necessary for bone formation induced by mechanical stimuli. However, the mechanisms by which osteoblasts detect ultrasound stress and transmit this signal across the cell membrane to activate signaling pathways involved in bone metabolism, such as the induction of inducible nitric oxide synthase and the release of nitric oxide, are still not well understood.
Nitric oxide is a highly reactive nitrogen radical that plays a role in numerous biological processes, including the regulation of vascular tone, platelet and leukocyte adhesion, neurotransmission, and the mediation of excessive vasodilation and cytotoxic actions of macrophages against microbes and tumor cells. Its production is regulated by a family of enzymes known as nitric oxide synthases, which oxidize the guanidine portion of L-arginine, resulting in the equimolar production of nitric oxide and L-citrulline. Two main classes of NOS have been identified based on their expression and regulation. The constitutive form, found in neurons (nNOS) or endothelial cells (eNOS), is a calcium-dependent enzyme. The inducible form (iNOS), on the other hand, present in macrophages and other cells, is regulated at the transcriptional level in response to lipopolysaccharide or certain proinflammatory cytokines and does not require calcium for its activity.
Ras has been found to interact with multiple effector systems to trigger distinct physiological and pathological responses, such as cell proliferation and the release of proinflammatory mediators. An important group of Ras effectors is the MAPK family. The classic Ras-mediated pathway involves the binding of Raf-1 and subsequent phosphorylation of Raf-1 at Ser338 by various kinases, which in turn activates ERKs, and consequently phosphorylates many target proteins, including transcription factors and protein kinases. A role for Ras in the induction of iNOS has been suggested in many cell types. However, the role of the Ras/Raf-1/MEK/ERK pathway in iNOS expression induced by ultrasound has not been investigated in preosteoblasts. This study aimed to determine the role of the Ras/Raf-1/MEK/ERK pathway in NF-kB activation and iNOS expression mediated by ultrasound in cultured preosteoblasts. Our hypothesis is that ultrasound might activate the Ras/Raf-1/MEK/ERK pathway, which subsequently induces IkB kinase ab and NF-kB activation, ultimately leading to iNOS expression and nitric oxide production in preosteoblasts.
Experimental Procedures Materials
Protein A/G beads, anti-mouse and anti-rabbit IgG-conjugated horseradish peroxidase, rabbit polyclonal antibodies specific for IkBa, p-IkBa, IKKa/b, p65, p50, p-ERK, ERK, Raf-1, p-MEK1/2, and MEK1/2 were obtained from Santa Cruz Biotechnology. Rabbit polyclonal antibody specific for Raf-1 phosphorylated at Ser338 was purchased from Cell Signaling and Neuroscience. A Ras dominant negative mutant (RasN17) and a Ras activity assay kit were purchased from Upstate Biotechnology. PDTC, TPCK, and PD98059 were obtained from Calbiochem. The iNOS promoter construct (piNOS-Luc) was provided by Dr. E.A. Ratovitski. The NF-kB luciferase plasmid was purchased from Stratagene. The IKKa(KM) and IKKb(KM) mutants were provided by Dr. H. Nakano. The ERK2 dominant negative mutant was provided by Dr. M. Cobb. The pSV-b-galactosidase vector and luciferase assay kit were purchased from Promega. All other chemicals were obtained from Sigma–Aldrich.
Cell cultures
A murine preosteoblastic cell line MC3T3-E1 was obtained from Riken Cell Bank. Cells were grown on plastic cell culture dishes in an atmosphere of 95% air and 5% CO2 with a-MEM, which was supplemented with 20 mM HEPES, 10% heat-inactivated fetal bovine serum, 2 mM glutamine, penicillin (100 U/ml), and streptomycin (100 mg/ml), with the pH adjusted to 7.6. The culture medium was changed twice per week.
Ultrasound treatment
MC3T3-E1 cells were cultured in six-well plates for 24 hours and then subjected to ultrasound treatment. A UV-sterilized unfocused circular transducer was immersed vertically into each culture well, positioned to just contact the surface of the culture medium. The transducer generated a low intensity pulsed ultrasound signal that has been clinically proven to enhance fracture healing. The driving signal consisted of a 1.5 MHz sinusoidal ultrasound carrier wave, amplitude modulated with a 1 kHz pulse, and a pulse width of 200 msec, resulting in a 20% duty cycle. The Spatial Average Time Average Intensity was 30 mW/cm2 with a temporal average power of 117 mW and an Effective Radiating Area of 3.88 cm2. The exposure time was 20 minutes for all cultures, which is the FDA-approved treatment time for bone healing. The distance between the transducer and the cells was approximately 5 mm. Control samples were prepared in the same manner but without exposure to ultrasound. Cells were harvested at 5, 15, 30, and 60 minutes after ultrasound stimulation. To confirm the downstream and signaling pathways following ultrasound treatment, preosteoblasts were pretreated with various inhibitors or DMSO as a vehicle control for 30 minutes before ultrasound stimulation was applied.
Assay of nitric oxide
MC3T3-E1 cells were exposed to ultrasound in six-well plates. The production of nitric oxide was determined by measuring the levels of its stable metabolite, nitrite, in the culture medium. Aliquots of the sample were mixed with Griess reagent and incubated at twenty-five degrees Celsius for ten minutes in a ninety-six-well plate. The absorbance was then measured at a wavelength of 550 nanometers using a microplate reader.
Western blot analysis
Cellular lysates were prepared following established procedures. The proteins were separated using SDS–PAGE and transferred onto Immobilon polyvinyldifluoride membranes. The membranes were blocked with a four percent bovine serum albumin solution for one hour at room temperature and subsequently probed with rabbit anti-mouse antibodies against inducible nitric oxide synthase at a dilution of 1:1000 for one hour at room temperature. After three washing steps, the membranes were incubated with a donkey anti-rabbit peroxidase-conjugated secondary antibody at a dilution of 1:1000 for one hour at room temperature. The protein bands were visualized using enhanced chemiluminescence and exposed to Kodak X-OMAT LS film. Quantitative data were obtained through densitometric analysis using a computing densitometer and ImageQuant software.
Ras activity assay
Ras activity was measured using a commercially available Ras activity assay kit, following the manufacturer’s instructions. Briefly, the cells were washed twice with ice-cold phosphate-buffered saline and then lysed in magnesium lysis buffer. The lysates were then centrifuged. Equal volumes of the lysates were incubated with five milligrams of the Ras-binding domain of Raf-1 at four degrees Celsius overnight. The beads were subsequently washed three times with magnesium lysis buffer. The bound Ras proteins were then solubilized in 2× Laemmli sample buffer and quantitatively detected by Western blotting using ten percent SDS–PAGE with a mouse monoclonal Ras antibody and the ECL system, followed by densitometry of the corresponding bands using scientific imaging systems.
Transfection and reporter gene assay
Preosteoblasts were co-transfected with 0.5 micrograms of the inducible nitric oxide synthase promoter plasmid and 0.5 micrograms of a beta-galactosidase expression vector. Preosteoblasts were grown to seventy percent confluency in six-well plates and transfected on the following day using Lipofectamine 2000. The DNA was premixed with OPTI-MEM, and Lipofectamine 2000 was also premixed with OPTI-MEM for five minutes, respectively. The resulting mixture was incubated for twenty-five minutes at room temperature and then added to each well. After twenty-four hours of incubation, transfection was complete, and the cells were incubated with the indicated agents. The media were removed twenty-four hours after ultrasound stimulation, and the cells were washed once with cold phosphate-buffered saline. To prepare lysates, one hundred microliters of reporter lysis buffer was added to each well, and the cells were scraped from the dishes. The supernatant was collected after centrifugation at 13,000 revolutions per minute for thirty seconds. Aliquots of the cell lysates containing equal amounts of protein were placed into the wells of an opaque black ninety-six-well microplate. An equal volume of luciferase substrate was added to all samples, and luminescence was measured using a microplate luminometer. The measured luciferase activity was normalized to the transfection efficiency, which was monitored by the co-transfected beta-galactosidase expression vector. In experiments involving dominant-negative mutants, cells were co-transfected with the reporter plasmid, the beta-galactosidase plasmid, and either the Ras or ERK mutant or the empty vector.
Chromatin immunoprecipitation assay
Chromatin immunoprecipitation analysis was performed following established procedures. DNA immunoprecipitated using either an anti-p65 or an anti-p50 antibody was purified. The DNA was then extracted using phenol–chloroform. The purified DNA pellet was subjected to polymerase chain reaction. The polymerase chain reaction products were then separated by electrophoresis on a 1.5% agarose gel and visualized under ultraviolet light. Specific primer sequences were used to amplify a region across the inducible nitric oxide synthase promoter.
Statistics
All values are presented as means with their standard errors. The statistical significance of differences between the experimental groups and the control groups was determined using Student’s t-test. A difference was considered statistically significant if the calculated P-value was less than 0.05.
Results
Ras is involved in ultrasound-induced inducible nitric oxide synthase expression
It has been previously shown that the application of pulsed low-intensity ultrasound increased the release of nitric oxide by up-regulating the expression of inducible nitric oxide synthase, which is recognized as important for bone formation induced by mechanical stimuli. To investigate whether Ras might play a role in mediating the ultrasound-induced expression of inducible nitric oxide synthase, manumycin A, an inhibitor of Ras, was employed. The results indicated that pretreatment of preosteoblasts with manumycin A inhibited the ultrasound-induced expression of inducible nitric oxide synthase in a manner dependent on the concentration of the inhibitor. Furthermore, manumycin A also reduced the production of nitric oxide induced by ultrasound, as determined by the Griess reaction. Subsequently, the activity of Ras in response to ultrasound stimulation was directly measured. The data revealed that exposure of preosteoblasts to ultrasound for a duration of twenty minutes led to a time-dependent increase in Ras activity. This was assessed by immunoblotting samples for Ras that were immunoprecipitated from cellular lysates using the Ras-binding domain of Raf-1. Maximal activation of Ras was observed between five and fifteen minutes after the initiation of stimulation with ultrasound. The ultrasound-induced increase in Ras activity was significantly inhibited by pretreating the cells for thirty minutes with manumycin A in a concentration-dependent manner. Taken together, these findings strongly suggest that the activation of Ras is involved in the ultrasound-induced expression of inducible nitric oxide synthase in preosteoblasts.
US promotes Raf-1 activation in preosteoblasts
To investigate whether Raf-1, a target protein of Ras, might play a critical role in the expression of inducible nitric oxide synthase induced by ultrasound, the Raf-1 inhibitor, GW 5074, was utilized. The results showed that pretreatment of preosteoblast cells with GW5074 inhibited the ultrasound-induced expression of inducible nitric oxide synthase and the production of nitric oxide in a concentration-dependent manner. Raf-1 associates with Ras-GTP and subsequently undergoes additional modifications, such as phosphorylation at Ser338, to become active. The activated Raf-1 then initiates a sequential activation of downstream molecules. Therefore, the phosphorylation of Raf-1 at Ser338 is a crucial step in its activation. Next, the phosphorylation of Raf-1 at Ser338 in preosteoblasts following ultrasound stimulation was further examined using an antibody specific for phospho-Raf-1 at Ser338. When cells were exposed to ultrasound for twenty minutes, the phosphorylation of Raf-1 at Ser338 increased at ten minutes and reached a peak between ten and forty-five minutes. Additionally, the ultrasound-induced phosphorylation of Raf-1 at Ser338 was inhibited by treatment with manumycin A and GW5074. These findings indicate that Raf-1 is a downstream molecule of Ras and is involved in the ultrasound-mediated expression of inducible nitric oxide synthase and the production of nitric oxide.
The signaling pathways of MEK and ERK are involved in the potentiating action of ultrasound stimulation
Next, we aimed to determine whether ultrasound is capable of activating MEK, a significant downstream target of Raf-1, which has been shown to induce gene expression. The role of MEK in the ultrasound-induced expression of inducible nitric oxide synthase was tested using the specific MEK inhibitor, PD98059. The results demonstrated that the ultrasound-induced expression of inducible nitric oxide synthase was markedly reduced by pretreating cells with PD98059 in a concentration-dependent manner. Pretreatment of cells with PD98059 also inhibited the production of nitric oxide induced by ultrasound. To directly confirm the crucial role of MEK in the expression of inducible nitric oxide synthase, the phosphorylation of MEK1/2 in response to ultrasound was determined. Exposure to ultrasound for twenty minutes induced the phosphorylation of MEK1/2 in a time-dependent manner. The protein level of MEK1/2 was not affected by ultrasound stimulation. Pretreatment of cells with manumycin A, GW5074, and PD98059 inhibited the ultrasound-induced activation of MEK1/2. The Ras inhibitor (manumycin A) and the Raf-1 inhibitor (GW5074) were more effective than the MEK inhibitor (PD98059) in inhibiting the ultrasound-induced phosphorylation of MEK. Subsequently, we examined whether the activation of ERK is involved in the increase of inducible nitric oxide synthase expression caused by ultrasound stimulation. Following exposure to ultrasound for twenty minutes, ERK phosphorylation increased at ten minutes, reaching its maximum between fifteen and forty-five minutes. The ultrasound-induced ERK phosphorylation was significantly inhibited by pretreating the cells for thirty minutes with manumycin A, GW5074, and PD98059. Taken together, these results indicated that the Ras/Raf-1/MEK/ERK pathway is involved in the ultrasound-induced expression of inducible nitric oxide synthase.
Involvement of NF-kB in ultrasound-induced inducible nitric oxide synthase expression
The activation of NF-kB has been reported to be necessary for the ultrasound-induced expression of COX-2 in human chondrocytes. To investigate whether NF-kB activation is involved in the signal transduction pathway leading to the expression of inducible nitric oxide synthase caused by ultrasound, the NF-kB inhibitor pyrrolidine dithiocarbamate was used. The results showed that pyrrolidine dithiocarbamate inhibited the enhancement of inducible nitric oxide synthase expression induced by ultrasound. Furthermore, pretreatment of preosteoblasts with an IkB protease inhibitor also antagonized the potentiating action of ultrasound. Treatment of cells with pyrrolidine dithiocarbamate and the IkB protease inhibitor also reduced the ultrasound-induced production of nitric oxide. It has been reported that the NF-kB element is important for the activation of the inducible nitric oxide synthase gene. NF-kB activation was further evaluated by analyzing the movement of NF-kB from the cytoplasm to the nucleus, as well as by chromatin immunoprecipitation assay. Stimulation of cells with ultrasound resulted in a marked movement of p65 and p50 NF-kB from the cytoplasm to the nucleus. The in vivo recruitment of p65 and p50 to the inducible nitric oxide synthase promoter was assessed by chromatin immunoprecipitation assay. The in vivo binding of p65 and p50 to the NF-kB element of the inducible nitric oxide synthase promoter occurred as early as ten minutes and was sustained up to sixty minutes after ultrasound stimulation. The binding of p65 and p50 to the NF-kB element induced by ultrasound stimulation was reduced by manumycin A, GW5074, and PD98059. To further confirm the involvement of the NF-kB element in the ultrasound-induced expression of inducible nitric oxide synthase, transient transfection was performed using kB promoter-luciferase constructs. Preosteoblasts exposed to ultrasound showed a 3.1-fold increase in kB promoter activity. This increase in kB activity induced by ultrasound was antagonized by manumycin A, GW5074, and PD98059. These results suggest that NF-kB activation is necessary for the ultrasound-induced expression of inducible nitric oxide synthase in preosteoblasts.
US causes an increase in IKK a/b phosphorylation, IkBa phosphorylation, and p65 phosphorylation
The upstream molecules involved in the ultrasound-induced activation of NF-kB were further examined. Stimulation of cells with ultrasound induced the phosphorylation of IKKa/b in a time-dependent manner. Transfection with either an IKKa or an IKKb mutant significantly inhibited the ultrasound-induced production of nitric oxide. These data suggest that the activation of IKKa/b is involved in the ultrasound-induced expression of inducible nitric oxide synthase in preosteoblasts. Stimulation of preosteoblasts with ultrasound also caused the phosphorylation of IkBa and the phosphorylation of p65 at Ser276 in a time-dependent manner. Pretreatment of cells with manumycin A, GW5074, and PD98059 reduced the ultrasound-induced phosphorylation of IKKa/b, IkBa, and p65.
Increase of inducible nitric oxide synthase promoter activity by ultrasound stimulation
To further investigate the pathways involved in the ultrasound-induced expression of inducible nitric oxide synthase, transient transfection was performed using the inducible nitric oxide synthase promoter-luciferase construct, which contains the mouse inducible nitric oxide synthase promoter fused to the luciferase reporter gene. Exposure to ultrasound led to a 3.1-fold increase in inducible nitric oxide synthase promoter activity in preosteoblasts. The increase in inducible nitric oxide synthase activity induced by ultrasound stimulation was antagonized by manumycin A, GW5074, PD98059, pyrrolidine dithiocarbamate, and the IkB protease inhibitor.
Discussion
Bone cells possess mechanisms to detect various physical forces and convert these signals to adapt to their surrounding environment. The non-invasive nature of ultrasound offers significant advantages in practical applications. Although ultrasound is used clinically to treat fracture repair, the molecular mechanisms by which it affects cell functions, such as protein metabolism, are largely unknown. Previous findings indicate that exposure to ultrasound transiently increases the expression of integrins on the cell membrane and leads to the expression of inducible nitric oxide synthase and the synthesis of nitric oxide. However, the signaling pathway by which ultrasound stimulation influences inducible nitric oxide synthase expression and nitric oxide production has remained mostly unclear. In this study, a new mechanism has been identified through which ultrasound can stimulate inducible nitric oxide synthase expression in cultured preosteoblasts via the sequential activation of Ras, Raf-1, MEK, ERK, IKKa/b, and NF-kB. These results provide the first detailed characterization of the ultrasound signaling pathway that stimulates inducible nitric oxide synthase expression through the activation of Ras.
Ras proteins are part of a larger family of small GTPases known to play a crucial role in signaling pathways that lead to cell proliferation, differentiation, and transformation. Several studies have suggested that Ras might be critically involved in the induction of inducible nitric oxide synthase expression. In the present study, it was shown that manumycin A, an inhibitor of Ras farnesyl transferase, inhibited the ultrasound-induced increase in inducible nitric oxide synthase expression, nitric oxide production, and inducible nitric oxide synthase promoter-luciferase activity in preosteoblasts. Furthermore, a dominant-negative mutant of Ras also reduced the ultrasound-induced increase in inducible nitric oxide synthase promoter-luciferase activity. Additionally, ultrasound stimulation increased the kinase activity of Ras. Pretreatment of cells with manumycin A reduced the ultrasound-increased Ras activity. These findings suggest that the activation of Ras might be involved in the inducible nitric oxide synthase expression mediated by ultrasound. Ras, an oncogenic protein, plays a critical role in the induction of inducible nitric oxide synthase protein. Ras can activate a number of signaling pathways, including the Raf-1/MEK/ERK pathway and the phosphatidylinositol 3-kinase/Akt/NF-kB pathway. In RAW 264.7 macrophages, lipopolysaccharide induces the expression of the tumor necrosis factor gene through the Ras/Raf-1/MEK/ERK pathway. In murine fibroblasts, the transcription of cyclooxygenase-2 induced by oncogenes and growth factors requires Ras-dependent Raf-2/MAPKK/ERK activation. In this study, it was first observed that exposure of preosteoblasts to ultrasound caused sequential activations of Ras, Raf-1, MEK, and ERK, and that manumycin A, GW5074, and PD98059 all inhibited ultrasound-induced ERK activation and inducible nitric oxide synthase expression. Moreover, a Ras or ERK2 mutant also reduced the ultrasound-induced increase in inducible nitric oxide synthase promoter activity. These results suggested that the Ras/Raf-1/MEK/ERK signaling pathway is important for the ultrasound-induced expression of inducible nitric oxide synthase.
In both mice and humans, the promoter region of the inducible nitric oxide synthase gene contains a binding site for many transcription factors, including NF-kB, in its 5′ region. Furthermore, NF-kB has been shown to control the induced transcription of the inducible nitric oxide synthase gene. The results of this study demonstrate that NF-kB activation contributes to the ultrasound-induced expression of inducible nitric oxide synthase in cultured preosteoblasts, and that inhibitors of the NF-kB-dependent signaling pathway, including pyrrolidine dithiocarbamate or an IkB protease inhibitor, inhibited the ultrasound-induced expression of inducible nitric oxide synthase and the production of nitric oxide. In its inactive state, NF-kB is typically held in the cytoplasm by the inhibitor protein IkB. Upon stimulation, such as by tumor necrosis factor-alpha, IkB proteins become phosphorylated by the multisubunit IKK complex, which subsequently targets IkB for ubiquitination, and these phosphorylated IkB proteins are then degraded by the 26S proteasome. Finally, the liberated NF-kB moves to the nucleus, where it activates the responsive gene. In the present study, it was found that treating preosteoblasts with ultrasound resulted in increased phosphorylation of IKKa/b, the movement of p65 and p50 from the cytoplasm to the nucleus, and the binding of p65 and p50 to the NF-kB element on the inducible nitric oxide synthase promoter. Using transient transfection with kB-luciferase as an indicator of NF-kB activity, it was also found that ultrasound induced an increase in NF-kB activity. The IKKs can be stimulated by various proinflammatory stimuli, including interleukin-1 beta, peptidoglycan, and thrombin. These extracellular signals activate the IKK complex, which is composed of catalytic subunits (IKKa and IKKb) and a regulatory subunit. This kinase complex, in turn, phosphorylates IkBa at specific serine residues, signaling its degradation via ubiquitination. The released NF-kB then translocates into the nucleus, where it promotes NF-kB-dependent transcription. The findings of our experiments showed that pretreatment of preosteoblasts with manumycin A, GW5074, and PD98059 antagonized the ultrasound-induced increase in the phosphorylation of IKKa/b, IkBa, and p65. Based on these findings, it is suggested that the Ras/Raf-1/MEK/ERK pathway is involved in the ultrasound-induced activation of IKKa/b. Primary osteoblasts were also used from the calvaria bone of fetal mice to confirm the results observed in MC3T3-E1 cells. Manumycin A, GW5074, PD98059, pyrrolidine dithiocarbamate, and the IkB protease inhibitor, as well as mutants of Ras, ERK2, IKKa, and IKKb, also reduced the ultrasound-increased nitric oxide production and inducible nitric oxide synthase promoter activity in primary cultured osteoblasts. In addition, ultrasound stimulation also increased Ras activity and the phosphorylation of Raf-1, MEK, ERK, IKK, IkBa, and p65 in primary osteoblasts. Therefore, the same signaling pathways may be involved in primary cultured osteoblasts.
In conclusion, the signaling pathway involved in the ultrasound-induced expression of inducible nitric oxide synthase in cultured preosteoblasts has been investigated. Ultrasound increases inducible nitric oxide synthase expression by activating Ras, Raf-1, MEK, ERK, and IKKab, which enhances the binding of p65 and p50 to the NF-kB site, resulting in the transactivation of inducible nitric oxide synthase production.