Mechanical Strain Differentially Regulates Endothelial Nitric-oxide Synthase and Receptor Activator of Nuclear κB Ligand Expression via ERK1/2 MAPK
2003; Elsevier BV; Volume: 278; Issue: 36 Linguagem: Inglês
10.1074/jbc.m302822200
ISSN1083-351X
AutoresJanet Rubin, Tamara C. Murphy, Li Zhu, Eileen Roy, Mark S. Nanes, Xian Fan,
Tópico(s)TGF-β signaling in diseases
ResumoExercise promotes positive bone remodeling through controlling cellular processes in bone. Nitric oxide (NO), generated from endothelial nitric-oxide synthase (eNOS), prevents resorption, whereas receptor activator of nuclear κB ligand (RANKL) promotes resorption through regulating osteoclast activity. Here we show that mechanical strain differentially regulates eNOS and RANKL expression from osteoprogenitor stromal cells in a magnitude-dependent fashion. Strain (0.25–2%) induction of eNOS expression was magnitude-dependent, reaching a plateau at 218 ± 36% of control eNOS. This was accompanied by increases in eNOS protein and a doubling of NO production. Concurrently, 0.25% strain inhibited RANKL expression with increasing response up to 1% strain (44 ± 3% of control RANKL). These differential responses to mechanical input were blocked when an ERK1/2 inhibitor was present during strain application. Inhibition of NO generation did not prevent strain-activated ERK1/2. To confirm the role of ERK1/2, cells were treated with an adenovirus encoding a constitutively activated MEK; Ad.caMEK significantly increased eNOS expression and NO production by more than 4-fold and decreased RANKL expression by half. In contrast, inhibition of strain-activated c-Jun kinase failed to prevent strain effects on either eNOS or RANKL. Our data suggest that physiologic levels of mechanical strain utilize ERK1/2 kinase to coordinately regulate eNOS and RANKL in a manner leading to positive bone remodeling. Exercise promotes positive bone remodeling through controlling cellular processes in bone. Nitric oxide (NO), generated from endothelial nitric-oxide synthase (eNOS), prevents resorption, whereas receptor activator of nuclear κB ligand (RANKL) promotes resorption through regulating osteoclast activity. Here we show that mechanical strain differentially regulates eNOS and RANKL expression from osteoprogenitor stromal cells in a magnitude-dependent fashion. Strain (0.25–2%) induction of eNOS expression was magnitude-dependent, reaching a plateau at 218 ± 36% of control eNOS. This was accompanied by increases in eNOS protein and a doubling of NO production. Concurrently, 0.25% strain inhibited RANKL expression with increasing response up to 1% strain (44 ± 3% of control RANKL). These differential responses to mechanical input were blocked when an ERK1/2 inhibitor was present during strain application. Inhibition of NO generation did not prevent strain-activated ERK1/2. To confirm the role of ERK1/2, cells were treated with an adenovirus encoding a constitutively activated MEK; Ad.caMEK significantly increased eNOS expression and NO production by more than 4-fold and decreased RANKL expression by half. In contrast, inhibition of strain-activated c-Jun kinase failed to prevent strain effects on either eNOS or RANKL. Our data suggest that physiologic levels of mechanical strain utilize ERK1/2 kinase to coordinately regulate eNOS and RANKL in a manner leading to positive bone remodeling. The capacity of bone to remodel to meet functional structural demands was recognized by Wolff in 1892 as the "law of bone transformation" (1Wolff J. Verlag August Hirschwald. 1892; Google Scholar). Studies in humans (2Bennell K.L. Malcolm S.A. Khan K.M. Thomas S.A. Reid S.J. Brukner P.D. Ebeling P.R. Wark J.D. Bone. 1997; 20: 477-484Crossref PubMed Scopus (183) Google Scholar, 3Judex S. Gross T.S. Zernicke R.F. J. Bone Miner. Res. 1997; 12: 1737-1745Crossref PubMed Scopus (189) Google Scholar) and animals (4Rubin C. Xu G. Judex S. FASEB J. 2001; 15: 2225-2229Crossref PubMed Scopus (248) Google Scholar, 5Watanuki M. Sakai A. Sakata T. Tsurukami H. Miwa M. Uchida Y. Watanabe K. Ikeda K. Nakamura T. J. Bone Miner. 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Studying cellular response to mechanical strain in vitro, we have previously shown that murine osteoprogenitor cells respond to the application of strain by decreasing the expression of receptor activator of NF-κB ligand (RANKL) 1The abbreviations used are: RANKL, receptor activator of nuclear κB ligand; NOS, nitric-oxide synthase; eNOS, endothelial NOS; iNOS, inflammatory NOS; nNOS, neuronal NOS; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; JNKi, JNK inhibitor; ERKi, ERK inhibitor; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MOI, multiplicity of infection; l-NAME, N ω–nitro-l-arginine methyl ester. mRNA (13Rubin J. Murphy T. Nanes M.S. Fan X. Am. J. Physiol. 2000; 278: C1126-C1132Crossref PubMed Google Scholar). RANKL is the dominant molecule controlling osteoclastogenesis (14Fan X. Fan D. Gewant H. Royce C. Nanes M. Rubin J. Am. J. Physiol. Endocrinol. 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Since the diminished expression of RANKL by bone cells is inextricably linked to a repression of osteoclast formation, we wondered whether other "proformative" events might be associated with signals initiated by mechanical factors. In this work, we show that endothelial nitric oxide synthase (eNOS) is regulated by strain in a divergent fashion; strain induces the expression of eNOS at the same time that this mechanical input decreases expression of RANKL. eNOS, which generates nitric oxide (NO), appears to promote an anabolic picture in bone (24Koyama A. Otsuka E. Inoue A. Hirose S. Hagiwara H. Eur. J. Pharmacol. 2000; 391: 225-231Crossref PubMed Scopus (34) Google Scholar, 25Ralston S.H. Br. J. Rheumatol. 1997; 36: 831-838Crossref PubMed Scopus (96) Google Scholar). NO has been shown to have an inhibitory effect on both osteoclast formation and activation (26Collin-Osdoby P. Rothe L. Bekker S. Anderson F. Osdoby P. J. Bone Miner. 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We will show in this work that induction of eNOS expression, similarly to mechanical regulation of RANKL, also requires activation of ERK1/2 kinase. To confirm the role of ERK1/2 in these mechanically controlled events, we utilize an adenovirus causing constitutive ERK1/2 activity and show both that eNOS is up-regulated and that RANKL is down-regulated in the presence of an activated MAPK signaling system. Materials and Reagents—Antibodies to total ERK1/2 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and those to phosphorylated ERK1/2 were from New England Biolabs (Beverly, MA). ERK1/2 inhibitor PD98059 was obtained from Calbiochem, as was the JNK inhibitor (JNKi; SP600125). Fetal bovine serum was from Hyclone (Logan, UT). Other chemicals and supplies were purchased from Sigma. Cell Culture—To generate primary stromal cell cultures, murine marrow cells collected from the tibiae and femurs of 3–5-week-old male C57BL/6 mice were plated in 6-well plates at 1.6 × 106 cells/cm2 as previously published (17Rubin J. Murphy T. Fan X. Goldschmidt M. Taylor W. J. Bone Miner. Res. 2002; 17: 1452-1460Crossref PubMed Scopus (116) Google Scholar). After 60 min, nonadherent cells containing the stromal elements were transferred to Bioflex collagen I-coated plates (Flexcell Corp., McKeesport, PA) in α-minimal essential medium plus 10% fetal bovine serum. The next day nonadherent cells were discarded; adherent stromal cells were cultured with 10 nm 1,25-dihydroxyvitamin D added to stimulate RANKL expression on day 4. Strain regimens were applied on day 6. For experiments where lysates were made for Western analyses, inhibitors were added 30 min prior to strain, and the experiment was stopped as indicated. When the end point was to measure mRNA species, total mRNA was isolated 24 h after beginning strain induction. Application of Mechanical Strain—Uniform equibiaxial mechanical strain was generated using a Flexcell Bioflex instrument (Flexcell Corp., McKeesport, PA) as previously described (13Rubin J. Murphy T. Nanes M.S. Fan X. Am. J. Physiol. 2000; 278: C1126-C1132Crossref PubMed Google Scholar). Strain magnitudes were as noted from 0.25 to 2%, with strain frequency fixed at 10 cycles/min (0.17 Hz). Similar plates containing control cultures were kept in the same incubator but were not subjected to strain regimens. Measurement of NO—A fluorometric assay was used to measure nitrite in samples using the reagent 2,3-diaminonaphthalene with comparison with a NaNO2 standard curve (0–10 μm) as described previously (31Misko T.P. Schilling R.J. Salvemini D. Moore W.M. Currie M.G. Anal. Biochem. 1993; 214: 11-16Crossref PubMed Scopus (961) Google Scholar, 32Kleinhenz D. Fan X. Rubin J. Hart C. Free Radic. Biol. Med. 2003; 34: 856-861Crossref PubMed Scopus (38) Google Scholar). Briefly, 100 μl of standards and samples were added to microtiter 96-well plates (DYNEX Technologies, Inc.) and mixed with 10 μl of fresh 2,3-diaminonaphthalene (prepared in 0.62 m HCl) for 10 min at room temperature. The reactions were terminated with 5 μl of 2.8 n NaOH. Formation of the 2,3-diaminonaphthotriazole end product was measured using an LB 50 plate reader (PerkinElmer Life Sciences) with excitation at 360 nm and emission at 440 nm. All standards and samples were measured in triplicate. Western Blot Analysis—For NOS expression, proteins were extracted from stromal cells in boiling lysis buffer (10 mm Tris, pH 7.4, 1% SDS, and 1 mm sodium orthovanadate). Cell lysates were boiled for an additional 5 min and passed three times through a 26-gauge needle. After centrifugation at 16,000 × g for 5 min to remove insoluble material, protein concentrations in the supernatant were determined using the Bio-Rad DC protein assay kit. Samples containing 200 μg of total protein were electrophoresed through a 7.5% SDS-PAGE and transferred to a 0.45-μm polyvinylidene difluoride membrane. Membranes were immersed in blocking buffer containing TBS with 0.1% Tween 20 (TBST) and 5% nonfat milk overnight at 4 °C. The eNOS and inflammatory NOS (iNOS) isoforms were identified using respective polyclonal antibodies (1:1000; Transduction Laboratories, Lexington, KY); membranes were washed three times with TBST and then incubated with second antibody conjugated with horseradish peroxidase (1:1500). The proteins were detected by ECL plus chemiluminescence kit (Amersham Biosciences). Real Time PCR to Assess mRNA Species—Analysis of eNOS, RANKL, and 18 S mRNA was performed as in Refs. 17Rubin J. Murphy T. Fan X. Goldschmidt M. Taylor W. J. Bone Miner. Res. 2002; 17: 1452-1460Crossref PubMed Scopus (116) Google Scholar and 33Fan X. Roy E. Zhu L. Murphy T.C. Kozlowski M. Nanes M.S. Rubin J. J. Biol. Chem. 2003; 278: 10232-10238Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar using the iCycler (Bio-Rad). Briefly, reverse transcription of 0.5 μg of total RNA treated with DNase I was performed with random decamers (Ambion, Austin, TX) and superscript II reverse transcriptase (Invitrogen). For real time PCR, amplification reactions were performed in 25 μl containing primers at 0.5 μm and dNTPs (0.2 mm each) in PCR buffer and 0.03 units of Taq polymerase (Invitrogen) along with SYBR-green (Molecular Probes, Inc., Eugene, OR) at 1:150,000. Aliquots of cDNA were diluted 10–10,000-fold for 18 S and 5–625-fold for eNOS, iNOS, and RANKL to generate relative standard curves with which sample cDNA was compared (34Johnson M.R. Wang K. Smith J.B. Heslin M.J. Diasio R.B. Anal. Biochem. 2000; 278: 175-184Crossref PubMed Scopus (324) Google Scholar). For eNOS, forward and reverse primers were 5′-AAC CAG CGT CCT GCA AAC C-3′ and 5′-CAA TGT GAG TCC GAA AAT GTC C-3′, respectively, creating an amplicon of 133 bp. For iNOS, forward primer 5′-GCA TGG ACC AGT ATA AGG CAA GCA-3′ and reverse primer 5′-GCT TCT GGT CGA TGT CAT GAG CAA-3′ amplified a 222-base pair amplicon (35Riancho J.A. Salas E. Zarrabeitia M.T. Olmos J.M. Amado J.A. Fernandez-Luna J.L. Gonzalez-Macias J. J. Bone Miner. Res. 1995; 10: 439-446Crossref PubMed Scopus (157) Google Scholar). RANKL primers were reported in Ref. 17Rubin J. Murphy T. Fan X. Goldschmidt M. Taylor W. J. Bone Miner. Res. 2002; 17: 1452-1460Crossref PubMed Scopus (116) Google Scholar. Standards and samples were run in triplicate. Dilution curves showed that PCR efficiency was more than 90% for all species including 18 S. Adenovirus Preparation—The adenovirus carrying the constitutively activated mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) (Ad.caMEK) was generated in our laboratory beginning with the pMCL-MKK1 plasmid carrying the caMEK sequence generously provided by Dr. Natalie Ahn (University of Colorado). As published (36Mansour S.J. Matten W.T. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Vande Woude G.F. Ahn N.G. Science. 1994; 265: 966-970Crossref PubMed Scopus (1260) Google Scholar), the MEK was constitutively activated by removal of the coding sequence for amino acids 32–51 and alteration of the nucleotides encoding two serine regulatory sites at 218 and 221 to glutamic and aspartic acid, respectively. The caMEK sequence was removed with XbaI and HindIII and inserted into the pShuttle vector as per the Stratagene protocol (Stratagene, La Jolla, CA). The pShuttle now carrying the caMEK sequence was linearized with PmeI and co-transformed with pAdEasy in recA-proficient bacteria. Small kanamycin resistant colonies were selected and digested with PacI. After confirmation of appropriate band patterns, the viral DNA was used to transform recA-deficient bacteria. Colonies containing the desired gene as analyzed by PCR were selected, and the DNA was linearized with PacI and purified before calcium phosphate transfection of HEK293 cells. After transduction, HEK293 cell layers were overlaid with agarose and assessed for viral plaque formation at 10 days. Viruses were eluted from plaques and amplified in HEK293 cells; infected cells were subjected to three freeze/thaw cycles, and the supernatants were collected for use in experiments. For virus applications, viral lysates were added with 10 μg/ml LipofectAMINE in serum-free medium at the indicated MOIs, with an equal volume of media containing 20% fetal bovine serum added at 5 h. Cellular responses were studied at 24 h. Assessment of MAPKs—Western blotting for MAPK phosphorylation was performed as detailed in Ref. 17Rubin J. Murphy T. Fan X. Goldschmidt M. Taylor W. J. Bone Miner. Res. 2002; 17: 1452-1460Crossref PubMed Scopus (116) Google Scholar. Briefly, the supernatants of cell lysates were concentrated, and 10 μg of protein was chromatographed and transferred to polyvinylidene difluoride membrane. Primary and secondary antibody incubation was followed by measurement of chemiluminescence (ECL-plus; Amersham Biosciences). Assessment of JNK and ERK Activation—To visualize the ability of the Ad.caMEK to activate ERK1/2, we utilized Western analysis to see the 42- and 44-kDa phosphorylated bands as in Ref. 17Rubin J. Murphy T. Fan X. Goldschmidt M. Taylor W. J. Bone Miner. Res. 2002; 17: 1452-1460Crossref PubMed Scopus (116) Google Scholar. To assess c-Jun N-terminal protein kinase (JNK) activity, a c-Jun phosphorylation assay was utilized as previously; briefly, soluble cell lysates in cold Triton lysis buffer with proteinase inhibitors were incubated with 2.5 μg of agarose-bound glutathione S-transferase-c-Jun-(1–79) (Calbiochem) in a total volume of 400 μl of Triton lysis buffer overnight at 4 °C on rotating platform (17Rubin J. Murphy T. Fan X. Goldschmidt M. Taylor W. J. Bone Miner. Res. 2002; 17: 1452-1460Crossref PubMed Scopus (116) Google Scholar, 37Whitmarsh A.J. Davis R.J. Methods Enzymol. 2001; 332: 319-336Crossref PubMed Scopus (41) Google Scholar). Washed agarose beads were mixed with 100 μm ATP and 5 μCi of [γ-32P]ATP, and reactions were carried out at 30 °C for 30 min. Phosphoproteins were separated on a 15% SDS-PAGE gel, and phosphorylated glutathione S-transferase-c-Jun-(1–79) was visualized at ∼37 kDa. Statistical Analysis—Results are expressed as the mean ± S.E. Statistical significance was evaluated by Dunnett or Bonferroni's post hoc one-way analysis of variance (GraphPad Prism). Mechanical Strain Induces eNOS Expression in Murine Stromal Cells—Bone cells express multiple forms of NOS, with eNOS being the most prominent isoform. iNOS and neuronal NOS (nNOS) have both been described in bone during growth and fracture healing but are clearly present in lower amounts than eNOS in bone cells themselves (38Helfrich M.H. Evans D.E. Grabowski P.S. Pollock J.S. Ohshima H. Ralston S.H. J. Bone Miner. Res. 1997; 12: 1108-1115Crossref PubMed Scopus (152) Google Scholar, 39MacPherson H. Noble B.S. Ralston S.H. Bone. 1999; 24: 179-185Crossref PubMed Scopus (79) Google Scholar). Using Western blotting to identify species in C57BL/6 murine stromal cells, we were able to identify eNOS protein in samples where 200 μg of lysate protein was loaded. To assess whether the NOS might be residing in the membrane component, we also separated membrane protein and found that similar loading of 200 μg of membrane protein did not alter the amount of eNOS reacting with the antibody. In cells exposed to 2% magnitude mechanical strain overnight, eNOS increased, as shown in a representative blot (Fig. 1a). iNOS was not visible in Western blots from these lysates, despite successful blotting of an assay positive control for the iNOS species. Because of sensitivity in assessing amounts of eNOS protein, we confirmed the strain inductive response using a sensitive real time PCR assay for eNOS mRNA. This assay was designed to recognize only eNOS, and not iNOS or nNOS, on the basis of primer specificity. In Fig. 1b, compiled from three separate experiments where cells were exposed to 24 h of 2% strain, eNOS mRNA significantly increased by more than 2-fold. Using primers specific for murine iNOS, we demonstrated a lack of response to strain of this NOS isoform; we performed real time PCR on control and cells strained for 24 h. Grouped real time PCR data from three experiments showed no significant difference in levels of iNOS mRNA between control (100 ± 10%) and strained cells (90 ± 25%). These data ruled out a contribution from iNOS to the strain-induced increase in nitric oxide described below. Gene Expression Is Differentially Sensitive to Strain Magnitude—The response of eNOS was in the opposite direction to the strain-induced decrease in RANKL gene expression, which we had explored previously (17Rubin J. Murphy T. Fan X. Goldschmidt M. Taylor W. J. Bone Miner. Res. 2002; 17: 1452-1460Crossref PubMed Scopus (116) Google Scholar). To understand better the differences in mechanical regulation of these two genes, eNOS and RANKL, we performed a series of experiments where strain magnitude was varied. Our current strain instrumentation allowed application of uniform strain as low as 0.25%, and we studied four different strain regimens up to 2% magnitude. At least three separate experiments were performed at each strain magnitude and compiled in Fig. 2. For each sample, eNOS, RANKL, and 18 S were amplified from samples subjected to reverse transcription with random decamers as described under "Experimental Procedures." The data was expressed as percentage of target control mRNA compared with 18 S in the sample. eNOS mRNA responded to 0.25% strain, rising to 120%, and continued to increase, reaching a plateau at 161 ± 28% compared with eNOS mRNA measured in unstrained cells (p < 0.001), a rise comparable with the previous series shown in Fig. 1b. RANKL also responded to application of 0.25% strain, reaching a nadir by 1% strain, as shown in Fig. 2. This result can be compared with previous results where strains less than or equal to 1% were not effective when dosed for only 6 h (17Rubin J. Murphy T. Fan X. Goldschmidt M. Taylor W. J. Bone Miner. Res. 2002; 17: 1452-1460Crossref PubMed Scopus (116) Google Scholar). In the experiments presented here, application of strain for 24 h allowed us to generate reproducible mechanical effects on both eNOS and RANKL gene expression with strains significantly lower than 1%. Results showing that strain caused a divergent response of these two genes at all magnitudes studied suggested that mechanical regulation of eNOS and RANKL share a proximal signaling pathway. NO Release Is Stimulated by Strain and Reflects Increases in eNOS—We next measured nitric oxide production from murine cells during strain. NO did not rise immediately, in contrast to the immediate activation in response to fluid shear in cellular NOS of both osteocytes and osteoblasts (40Klein-Nulend J. Helfrich M.H. Sterck J.G. MacPherson H. Joldersma M. Ralston S.H. Semeins C.M. Burger E.H. Biochem. Biophys. Res. Commun. 1998; 250: 108-114Crossref PubMed Scopus (162) Google Scholar, 41Smalt R. Mitchell F.T. Howard R.L. Chambers T.J. Adv. Exp. Med. Biol. 1997; 433: 311-314Crossref PubMed Scopus (41) Google Scholar). Even at 60 min, as measured by a more sensitive assay (31Misko T.P. Schilling R.J. Salvemini D. Moore W.M. Currie M.G. Anal. Biochem. 1993; 214: 11-16Crossref PubMed Scopus (961) Google Scholar, 32Kleinhenz D. Fan X. Rubin J. Hart C. Free Radic. Biol. Med. 2003; 34: 856-861Crossref PubMed Scopus (38) Google Scholar) than used in the latter studies, NO was not significantly different from that secreted into media by unstrained cell cultures (Fig. 3a). However, NO was significantly increased by 2-fold after 24 h of strain, as shown in Fig. 3b. Furthermore, the strain induction of NO required the activity of endogenous NOS, as the competitive inhibitor, l-NAME, blocked the effect of strain on NO production, shown in Fig. 3c. Because iNOS was not affected by the strain protocol (see Fig. 1c), we inferred that the increased NO resulted from increased gene and protein expression of the endothelial form of NOS. Inhibition of ERK Activation Indicates That ERK Has Primary Effects on Both eNOS and RANKL—We had previously shown that straining cells at magnitudes less than 2% caused activation of both ERK1/2 and JNK (17Rubin J. Murphy T. Fan X. Goldschmidt M. Taylor W. J. Bone Miner. Res. 2002; 17: 1452-1460Crossref PubMed Scopus (116) Google Scholar). Here we set out to prove that strain induction of eNOS expression is blocked by inhibition of ERK1/2 activation. The effect of overnight strain in this series of five experiments was to increase eNOS expression to 195 ± 26% of the unstrained control, as expected (Fig. 4a). Treatment with ERKi during the strain protocol prevented any strain-induced increase in eNOS as shown in the second gray bar in Fig. 4a, which shows an insignificant difference between the unstrained and strained cultures in the presence of ERKi. Exposure of unstrained controls overnight to an efficacious concentration of ERK inhibitor (ERKi) unexpectedly increased eNOS expression to 136 ± 8% of unstrained control levels, suggesting that basal ERK1/2 activity operates some regulatory control over eNOS expression in bone cells. However, when ERK1/2 was inhibited, application of strain failed to cause further increases in eNOS mRNA levels. The effect of strain on control cells in this series of experiments was to diminish RANKL expression to 58 ± 4% of control (Fig. 4b). The strain inhibition was ablated in cultures where ERK1/2 activation was blocked by treatment with ERKi; RANKL expression was not significantly different from that in unstrained cells treated with ERKi. However, as shown in Fig. 4, when primary stromal cultures were exposed to the ERKi for 16 h, RANKL expression increased significantly to 144 ± 25% of that of control stromal cells. Compared with our previous experiments, ERKi applied for only 6 h during the straining protocol was shown to prevent strain-induced decrements in RANKL mRNA expression but did not affect basal RANKL expression (17Rubin J. Murphy T. Fan X. Goldschmidt M. Taylor W. J. Bone Miner. Res. 2002; 17: 1452-1460Crossref PubMed Scopus (116) Google Scholar). That a longer exposure to ERKi raised RANKL mRNA expression suggests that RANKL expression is affected by ERK1/2 even in the absence of mechanical input, indicating that ERK activation may tonically limit RANKL expression. In sum, inhibition of ERK activation blocks strain effects on both eNOS and RANKL. We showed, in Fig. 3b, that NO did not rise until 24 h later, and this rise was preceded by an increase in eNOS expression and protein. Since nitric-oxide synthase is known to be activated acutely by strain (40Klein-Nulend J. Helfrich M.H. Sterck J.G. MacPherson H. Joldersma M. Ralston S.H. Semeins C.M. Burger E.H. Biochem. Biophys. Res. Commun. 1998; 250: 108-114Crossref PubMed Scopus (162) Google Scholar), and Jessop et al. (42Jessop H.L. Rawlinson S.C. Pitsillides A.A. Lanyon L.E. Bone. 2002; 31: 186-194Crossref PubMed Scopus (134) Google Scholar) have suggested that of the activation of ERK1/2 in osteoblast-like cells may be dependent on immediate NO production, we considered whether ERK1/2 activation required rapid NO generation during strain. Cells were cultured overnight with l-NAME and then subjected to strain regimen for 10 min. ERK1/2 activation was measured as shown in Fig. 4c. Strain activation of ERK1/2 was not altered in cells where endogenous NO production was prevented by the competitive inhibitor, l-NAME. This indicated that NO does not have an acute role in strain activation of ERK1/2. Constitutive Activation of MEK Reproduces the Strain Effect on Both Genes—To further understand the relationship between ERK activation and its divergent regulation of eNOS and RANKL, we generated an adenovirus to deliver a constitutively activated MEK to cells. The use of adenovirus to transfer the gene was necessitated because these primary stromal cells are not very susceptible to infection by retro- and adeno-associated viruses as well as to liposo
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