FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2076695692> ?p ?o ?g. }

• W2076695692 endingPage "15455" @default.
• W2076695692 startingPage "15449" @default.
• W2076695692 abstract "RAW 264.7 macrophages express nonmuscle myosin heavy chain II-A as the only significant nonmuscle myosin heavy chain isoform, with expression of nonmuscle myosin heavy chain II-B and II-C low or absent. Treatment of the cells with sodium butyrate, an inhibitor of histone deacetylase, led to the dose-dependent induction of nonmuscle myosin heavy chain II-C. Trichostatin A, another inhibitor of histone deacetylase, also induced nonmuscle myosin heavy chain II-C. Induction of nonmuscle myosin heavy chain II-C in response to these histone deacetylase inhibitors was attenuated by mithramycin, an inhibitor of Sp1 binding to GC-rich DNA sequences. Bacterial lipopolysaccharide alone had no effect on basal nonmuscle myosin heavy chain II-C expression, but attenuated butyrate-mediated induction of nonmuscle myosin heavy chain II-C. The effects of lipopolysaccharide were mimicked by the nitric oxide donors sodium nitroprusside and spermine NONOate, suggesting a role for nitric oxide in the lipopolysaccharide-mediated down-regulation of nonmuscle myosin heavy chain II-C induction. This was supported by experiments with the inducible nitric-oxide synthase inhibitor 1400W, which partially blocked the lipopolysaccharide-mediated attenuation of nonmuscle myosin heavy chain induction. 8-Bromo-cGMP had no effect on nonmuscle myosin heavy chain induction, consistent with a cGMP-independent mechanism for nitric oxide-mediated inhibition of nonmuscle myosin heavy chain II-C induction. RAW 264.7 macrophages express nonmuscle myosin heavy chain II-A as the only significant nonmuscle myosin heavy chain isoform, with expression of nonmuscle myosin heavy chain II-B and II-C low or absent. Treatment of the cells with sodium butyrate, an inhibitor of histone deacetylase, led to the dose-dependent induction of nonmuscle myosin heavy chain II-C. Trichostatin A, another inhibitor of histone deacetylase, also induced nonmuscle myosin heavy chain II-C. Induction of nonmuscle myosin heavy chain II-C in response to these histone deacetylase inhibitors was attenuated by mithramycin, an inhibitor of Sp1 binding to GC-rich DNA sequences. Bacterial lipopolysaccharide alone had no effect on basal nonmuscle myosin heavy chain II-C expression, but attenuated butyrate-mediated induction of nonmuscle myosin heavy chain II-C. The effects of lipopolysaccharide were mimicked by the nitric oxide donors sodium nitroprusside and spermine NONOate, suggesting a role for nitric oxide in the lipopolysaccharide-mediated down-regulation of nonmuscle myosin heavy chain II-C induction. This was supported by experiments with the inducible nitric-oxide synthase inhibitor 1400W, which partially blocked the lipopolysaccharide-mediated attenuation of nonmuscle myosin heavy chain induction. 8-Bromo-cGMP had no effect on nonmuscle myosin heavy chain induction, consistent with a cGMP-independent mechanism for nitric oxide-mediated inhibition of nonmuscle myosin heavy chain II-C induction. nonmuscle myosin heavy chain lipopolysaccharide trichostatin A, (4,6-dimethyl-7-[p-dimethylaminophenyl]-7-oxohepta-2,4-dienohydroxamic acid) nitric oxide N-(3-aminomethyl)benzylacetamidine N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine inducible nitric-oxide synthase histone deacetylase reverse transcriptase Vertebrate nonmuscle myosin II represents a branch of the myosin superfamily, closely related to skeletal and smooth muscle myosin II isoforms. This ubiquitously distributed class of myosins has been shown to be present in all vertebrate cells including smooth muscle, cardiac muscle, and skeletal muscle cells where, similar to the more highly expressed isoforms of myosin II, they consist of a hexamer containing two heavy chains (200 kDa) and two pairs of light chains (20 and 17 kDa). Two isoforms of the nonmuscle myosin heavy chain (NMHC),1 termed NMHC II-A and II-B, were identified several years ago (1Katsuragawa Y. Yanagisawa M. Inoue A. Masaki T. Eur. J. Biochem. 1989; 184: 611-616Crossref PubMed Scopus (113) Google Scholar, 2Shohet R.V. Conti M.A. Kawamoto S. Preston Y.A. Brill D.A. Adelstein R.S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7726-7730Crossref PubMed Scopus (102) Google Scholar, 3Takahashi M. Kawamoto S. Adelstein R.S. J. Biol. Chem. 1992; 267: 17864-17871Abstract Full Text PDF PubMed Google Scholar). The genes encoding these myosin heavy chains are located on different chromosomes (4Simons M. Wang M. McBride O.W. Kawamoto S. Yamakawa K. Gdula D. Adelstein R.S. Weir L. Circ. Res. 1991; 69: 530-539Crossref PubMed Scopus (216) Google Scholar) and the tissue distributions of the isoforms differ (4Simons M. Wang M. McBride O.W. Kawamoto S. Yamakawa K. Gdula D. Adelstein R.S. Weir L. Circ. Res. 1991; 69: 530-539Crossref PubMed Scopus (216) Google Scholar, 5Kawamoto S. Adelstein R.S. J. Cell Biol. 1991; 112: 915-924Crossref PubMed Scopus (175) Google Scholar), although there is considerable overlap, with most cells and tissues expressing both isoforms to a greater or lesser degree. Recently, completion of the sequencing of the human genome revealed the presence of a third nonmuscle myosin, NMHC II-C, which again, has a distinct chromosomal location compared with the other two isoforms (6Berg J.S. Powell B.C. Cheney R.E. Mol. Biol. Cell. 2001; 12: 780-794Crossref PubMed Scopus (616) Google Scholar).In hematopoietic cells, NMHC II-A is believed to be the predominant NMHC isoform; for example, in the rat basophilic leukemia RBL-2H3 cell line, NMHC II-B was shown to be absent (7Choi O.H. Park C.S. Itoh K. Adelstein R.S. Beaven M.A. J. Muscle Res. Cell Motil. 1996; 17: 69-77Crossref PubMed Scopus (29) Google Scholar). We were, therefore, interested in whether NMHC II-C is expressed in hematopoietic cells. Here we demonstrate that expression of NMHC II-C is low or absent in undifferentiated macrophage RAW 264.7 cells. However, treatment of the cells with the histone deacetylase (HDAC) inhibitors sodium butyrate or trichostatin A, which have been shown to lead to differentiation of a wide range of cell types, results in induction of NMHC II-C. Moreover, induction of NMHC II-C is attenuated by bacterial lipopolysaccharide in an NO-dependent but cGMP-independent fashion.EXPERIMENTAL PROCEDURESRAW 264.7 cells were obtained from ATCC (Manassas, VA) and were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Sodium butyrate, trichostatin A, mithramycin, and lipopolysaccharide (Escherichia coli serotype 0127:B8) were purchased from Sigma. Sodium nitroprusside, 1400W, 8-bromo-cGMP, and spermine NONOate were from Calbiochem (La Jolla, CA). Antibodies to sequences near the carboxyl-terminal end of human NMHC II-C (RQVFRLEEGVASDEEAEE) and near the amino-terminal of mouse NMHC II-C (VTMSVSGRKVASRPGP) were produced in rabbits and affinity purified against the corresponding peptides. An antibody to poly(ADP-ribose) polymerase was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). A monoclonal antibody to iNOS was obtained from Transduction Laboratories (Lexington, KY). Nitrate and nitrite were measured using a kit from Roche Molecular Biochemicals (Indianapolis, IN).Immunoblotting and ImmunoprecipitationCells were washed twice with phosphate-buffered saline containing 5 mm EDTA and 1 mm orthovanadate, lysed by addition of lysis buffer, and the cells were scraped from the plate with a plastic scraper. Lysis buffer consisted of 25 mm Hepes, pH 7.5, 0.3 mNaCl, 1.5 mm MgCl2, 0.1% Triton X-100, 1 mm sodium orthovanadate, 20 mmβ-glycerophosphate, 0.2 mm EDTA, 0.5 mmdithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 20 μm leupeptin, 0.15 units/ml aprotinin at 4 °C. Cell lysates were transferred to a microcentrifuge tube and Triton-insoluble material pelleted at 14,000 × g for 10 min. After taking an aliquot for protein determination (8Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213377) Google Scholar) to permit equal protein loading of gel lanes, samples were heated in SDS sample buffer (70 °C, 10 min) and subjected to SDS-PAGE (NuPAGE, Invitrogen). Samples were transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore, Bedford, MA) and immunoblotted using standard methods. Bound secondary antibody was detected using luminol blotting reagents (Santa Cruz Biotechnology) and the signal captured on Biomax MS film (Eastman Kodak, Rochester, NY). Films were scanned using a laser densitometer (Amersham Biosciences) and bands were quantified using ImageQuant software. For immunoprecipitation, following centrifugation of lysates and determination of protein content, aliquots of the lysates were rocked for 1 h with the COOH-terminal NMHC II-C antibody. Protein A/G-agarose (Santa Cruz Biotechnology) was then added, and the mixture was rocked overnight at 4 °C. The agarose beads were washed 4 times with ice-cold phosphate-buffered saline, SDS sample buffer was added, and the samples were subjected to SDS-PAGE and Western blotting as described above.RT-PCRTotal RNA was isolated from cells using Trizol (Invitrogen). Reverse transcription was carried out on 2 μg of RNA using Superscript II and random primers (Invitrogen). PCR was then carried out using Platinum PCR Supermix (Invitrogen), with the cycle number adjusted to keep product formation in the linear range. For HPRT, 25 cycles were used and for NMHC II-C, 35 cycles. The primers for the NMHC II-C PCR were: 5 prime, 5′-gccgaacgaagtttctcag-3′; 3 prime, 5′ tcattggggtgtggcagg-3′. The product is a 618-bp fragment corresponding to the last 205 amino acids and the termination codon.RESULTSWe first investigated whether NMHC II-C was expressed in RAW 264.7 macrophages. Immunoblotting of RAW 264.7 cell lysate with an antibody specific for NMHC II-C demonstrated the myosin isoform to be present at a very low concentration in the macrophages (Fig.1A). In contrast, NMHC II-C was expressed more abundantly in PC12 and COS cells. Treatment of the RAW 264.7 cells with sodium butyrate for 24 h led to the dose-dependent induction of NMHC II-C expression (Fig.1B). A time course of NMHC II-C induction using 10 mm butyrate is shown in Fig. 1C; significant induction of NMHC II-C was detectable by 10 h of stimulation. Induction of NMHC II-C was reversible, because removal of butyrate after 3 days of induction led to a decline in NMHC II-C levels (Fig.1D). To confirm the identity of the 225-kDa band identified by the antibody to the COOH terminus of NMHC II-C, immunoblotting was performed with a second antibody that recognizes a different epitope, close to the amino terminus of NMHC II-C (see “Experimental Procedures”). A similar induction of the 225-kDa band was observed (Fig. 1E), supporting the identity of the induced band as NMHC II-C.Analysis of NMHC II-C mRNA expression by RT-PCR demonstrated that the mRNA level was very low in untreated cells, but was increased at 6 h and maximal at 10–16 h butyrate treatment (Fig.2A). RT-PCR of HPRT mRNA was used as a control, showing that similar mRNA amounts were used. Induction of NMHC II-C mRNA was not dependent on de novoprotein synthesis, because cycloheximide in the presence of butyrate did not inhibit induction (Fig. 2B, lanes 5 and6). In fact, cycloheximide alone induced NMHC II-C mRNA expression (lanes 3 and 4) and appeared to enhance butyrate-mediated induction (lanes 5 and6). Cycloheximide was, however, effective at preventing induction of NMHC II-C protein expression, as demonstrated by immunoblot analysis (Fig. 2C).Figure 2Induction of NMHC II-C mRNA in response to butyrate.A, time course of induction of NMHC II-C mRNA. Total RNA was subjected to reverse transcription and the cDNA used for PCR using primers specific for NMHC II-C (top) or for the housekeeping gene HPRT (bottom). The left lanes contain DNA markers. B, effect of cycloheximide on induction of NMHC II-C mRNA. Cells were treated with cycloheximide, 3.6 or 36 μm, for 30 min and then incubated with or without sodium butyrate, 10 mm, for 16 h before preparation of RNA and RT-PCR as above. C, effect of cycloheximide on induction of NMHC II-C protein. Cells were incubated with or without cycloheximide, 36 μm, for 30 min before incubating in the presence or absence of butyrate, 10 mm, for 24 h. Cells were then lysed and lysates were subjected to immunoblotting for NMHC II-C. Note the absence of signal in lane 4 compared with lane 2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Sodium butyrate has been shown to inhibit HDAC and the resultant histone hyperacetylation and chromatin rearrangement have been proposed as the mechanism by which butyrate activates transcription (9Kruh J. Mol. Cell. Biochem. 1982; 42: 65-82PubMed Google Scholar). To assess the role of HDAC inhibition in NMHC II-C induction, cells were treated with trichostatin A (TSA), which is also known to inhibit HDAC. TSA stimulation led to the induction of NMHC II-C and the extent of induction was similar to that obtained with butyrate (Fig.3).Figure 3Treatment of RAW 264.7 cells with trichostatin A induces NMHC II-C.A, time course of NMHC II-C induction in response to TSA, 100 ng/ml (lanes 1–5). Induction in response to 10 mm butyrate for 24 h is shown in lane 6. B, dose response for induction of NMHC II-C in response to TSA. Cells were incubated for 24 h with the indicated concentrations of TSA before lysis and immunoblotting for NMHC II-C.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine whether the effects of butyrate on induction of NMHC II-C were specific for RAW 264.7 cells, the RBL-2H3 rat mast cell line and HeLa cells were stimulated with butyrate. Non-stimulated RBL-2H3 cells do not express NMHC II-B (7Choi O.H. Park C.S. Itoh K. Adelstein R.S. Beaven M.A. J. Muscle Res. Cell Motil. 1996; 17: 69-77Crossref PubMed Scopus (29) Google Scholar) and NMHC II-C is also undetectable (Fig.4A). However, treatment with butyrate led to a robust induction of expression of NMHC II-C (Fig.4A). Expression of NMHC II-C is also absent in non-stimulated HeLa cells (Fig. 4B). Extended treatment for 72 h with high dose butyrate (10 mm) led to a very modest induction of NMHC II-C, much lower than that found in the hemopoietic macrophage and mast cell lines. A second slower migrating band was also recognized by the COOH terminus NMHC II-C antibody (Fig.4B); however, immunoprecipitation of NMHC II-C with the carboxyl terminus antibody followed by immunoblotting of the immunoprecipitate with the mouse NH2 terminus antibody showed only a single band induced by butyrate at 225,000, suggesting that the higher molecular weight band is probably nonspecific (Fig. 4C).Figure 4Butyrate induces NMHC II-C in RBL-2H3 but only marginally in HeLa cells. RBL-2H3 (A) and HeLa cells (B) were treated with the indicated concentrations of butyrate for 72 h before preparation of cell lysates and immunoblotting for NMHC II-C. An equal loading of lysate from RAW 264.7 macrophages treated with butyrate (10 mm) was loaded in the right-hand lane for comparison. For RBL-2H3 cells, detached cells were collected by centrifugation, washed, and lysed after pooling with attached cells. An additional band migrating more slowly than NMHC II-C was observed in the HeLa experiments. C, immunoprecipitation of lysates prepared from HeLa cells treated with or without butyrate, 10 mm, for 72 h was performed using the carboxyl terminus NMHC II-C antibody. Immunoprecipitated proteins were then subjected to SDS-PAGE followed by immunoblotting with a second NMHC II-C antibody to an amino terminus epitope. A single band at 225 kDa was observed, consistent with the slower migrating band in panel B being nonspecific.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The transcription factor Sp1 has been implicated in butyrate-mediated gene regulation (10Nakano K. Mizuno T. Sowa Y. Orita T. Yoshino T. Okuyama Y. Fujita T. Ohtani-Fujita N. Matsukawa Y. Tokino T. Yamagishi H. Oka T. Nomura H. Sakai T. J. Biol. Chem. 1997; 272: 22199-22206Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar, 11Wang S. Wang W. Wesley R.A. Danner R.L. J. Biol. Chem. 1999; 274: 33190-33193Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 12Walker G.E. Wilson E.M. Powell D. Oh Y. Endocrinology. 2001; 142: 3817-3827Crossref PubMed Scopus (84) Google Scholar, 13Yang J. Kawai Y. Hanson R.W. Arinze I.J. J. Biol. Chem. 2001; 276: 25742-25752Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). To address the possible role of Sp1 in butyrate-mediated induction of NMHC II-C, RAW 264.7 cells were preincubated with the antibiotic mithramycin, which binds to GC-rich DNA and inhibits Sp1 binding (14Blume S.W. Snyder R.C. Ray R. Thomas S. Koller C.A. Miller D.M. J. Clin. Invest. 1991; 88: 1613-1621Crossref PubMed Scopus (296) Google Scholar). Mithramycin inhibited induction of NMHC II-C in a dose-dependent manner (Fig.5, A and B), consistent with a role for Sp1 in butyrate-mediated NMHC II-C induction. A similar mithramycin-mediated inhibition was observed when TSA was used to induce NMHC II-C (results not shown). Measurement of NMHC II-C mRNA by RT-PCR demonstrated a similar pattern of inhibition by mithramycin (Fig. 5C), indicating that the inhibitor was acting at the transcriptional level.Figure 5NMHC II-C induction is attenuated by the Sp1 inhibitor mithramycin.A, cells were pretreated with mithramycin, 10 nm to 10 μm, for 30 min before incubation with sodium butyrate, 10 mm, for 20 h. B, cells were pretreated with mithramycin, 1 μm, for 30 min before incubation with sodium butyrate, 2 or 10 mm, for 20 h. Cell lysates were then prepared and subjected to immunoblotting for NMHC II-C. C, cells were treated with mithramycin for 30 min followed by sodium butyrate for 16 h before preparation of RNA and RT-PCR using primers for NMHC II-C (top) or HPRT (bottom).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Butyrate has been shown to modulate the effects of bacterial LPS on macrophages and endothelial cells, inhibiting iNOS expression (15Chakravortty D. Koide N. Kato Y. Sugiyama T. Mu M.M. Yoshida T. Yokochi T. J. Endotoxin. Res. 2000; 6: 243-247Crossref PubMed Scopus (64) Google Scholar), but enhancing expression of interleukin-8 (16Fusunyan R.D. Quinn J.J. Ohno Y. MacDermott R.P. Sanderson I.R. Pediatr. Res. 1998; 43: 84-90Crossref PubMed Scopus (72) Google Scholar) and alkaline phosphatase (17Fukushima K. Sasaki I. Takahashi K. Naito H. Ogawa H. Sato S. Matsuno S. Digestion. 1998; 59: 683-688Crossref PubMed Scopus (15) Google Scholar). It was, therefore, of interest to determine whether there was any interaction between butyrate and LPS in modulating NMHC II-C isoform expression. Treatment of naive RAW 264.7 cells with LPS induced expression of iNOS as expected (Fig.6A, lower blot,lane 2), but had no effect on expression of NMHC II-C (upper blot). However, when LPS was added in combination with butyrate, 2 or 10 mm, induction of NMHC II-C (upper blot, lanes 4 and 6) was attenuated. The effects of LPS on NMHC II-C induction are summarized in Fig. 6B. As reported previously (15Chakravortty D. Koide N. Kato Y. Sugiyama T. Mu M.M. Yoshida T. Yokochi T. J. Endotoxin. Res. 2000; 6: 243-247Crossref PubMed Scopus (64) Google Scholar), butyrate attenuated iNOS induction in response to LPS (Fig. 6A, lanes 4 and 6, lower blot).Figure 6LPS attenuates NMHC II-C induction.Cells were treated ± butyrate, 2 or 10 mm, in the presence or absence of LPS (1 μg/ml) for 24 h. Detached cells were collected by centrifugation and lysed after pooling with attached cells. Cell lysates were then subjected to immunoblotting for NMHC II-C and iNOS (A). B, quantification of the effect of LPS on butyrate-mediated NMHC II-C induction. Films obtained in three experiments were scanned and the band intensities were normalized to induction in response to 10 mm butyrate. Results are expressed as mean ± S.E. *, p < 0.05versus butyrate stimulation in the absence of LPS.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The inhibition of NMHC II-C induction in response to LPS raised the possibility that the NO produced in response to the induction of iNOS played a role in regulation of NMHC II-C expression. This was confirmed by induction of NMHC II-C with butyrate in the presence or absence of nitric oxide donors. Sodium nitroprusside, 0.25 mm (Fig.7A), and spermine NONOate, 0.2 mm (Fig. 7B), attenuated NMHC II-C expression in response to butyrate. The results are summarized in Fig. 7C. Conversely, the inhibition of butyrate-mediated NMHC II-C induction by LPS was attenuated by inhibition of iNOS with the specific inhibitor 1400W (Fig. 8). Inhibition of iNOS was confirmed by measurement of the stable products of NO breakdown, nitrate and nitrite; 1400W inhibited nitrite + nitrate production in response to LPS by 90% (results not shown).Figure 7NMHC II-C induction is inhibited by NOdonors. Cells were treated ± butyrate, 2 or 10 mm, in the presence or absence of sodium nitroprusside, 0.25 mm (A), or spermine NONOate, 0.2 mm (B), for 24 h. Detached cells were collected by centrifugation and lysed after pooling with attached cells. Cell lysates were then subjected to immunoblotting for NMHC II-C. C, quantification of the effect of NO donors on butyrate-mediated NMHC II-C induction. Results are expressed as mean ± S.E. for four experiments. *, p < 0.05versus butyrate stimulation in the absence of NO donor.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 8iNOS inhibition blocks the LPS-mediated inhibition of NMHC II-C induction. Cells were pretreated with the iNOS inhibitor 1400W, 10 μm, for 30 min before incubation ± LPS, 1 μg/ml, and butyrate, 2 mm(lanes 2–5) or 10 mm (lanes 6–9) for 20 h. Cell lysates were then prepared and immunoblotted for NMHC II-C (A). B, quantification of the effects of LPS and iNOS inhibition on butyrate-mediated NMHC II-C induction. Results are expressed as mean ± S.E. from four experiments. *,p < 0.05 versus butyrate stimulation in the absence of LPS. §, p < 0.05 versusbutyrate + LPS.View Large Image Figure ViewerDownload Hi-res image Download (PPT)NO mediates its effects on vasodilation via activation of guanylate cyclase and elevation of cGMP, but it can also act in a cGMP-independent fashion. To address the role of cGMP in the inhibitory action of NO, induction of NMHC II-C with butyrate was performed in the presence or absence of a cell-permeable cGMP analog. 8-Bromo-cGMP, 0.1 to 1 mm, had no effect on NMHC II-C induction in response to butyrate (Fig. 9), arguing against a role for cGMP elevation in the attenuation of NMHC II-C induction by NO and LPS.Figure 98-Bromo-cGMP does not inhibit butyrate-mediated NMHC II-C induction. Cells were pretreated with the indicated concentration of 8-bromo-cGMP for 30 min before incubation with butyrate, 10 mm, for 20 h. Cell lysates were then prepared and immunoblotted for NMHC II-C.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONNMHC-II isoforms differ in their tissue distributions, indicating that expression of the genes is regulated in a cell-specific manner. Evidence has been obtained previously for transcriptional regulation of both NMHC II-A and II-B expression. A 100-bp region in intron 1 ofMYH9, the gene encoding human NMHC II-A, located 23 kb downstream from the transcriptional start site, has been shown to activate transcription in a cell-type and differentiation-specific manner (18Beohar N. Kawamoto S. J. Biol. Chem. 1998; 273: 9168-9178Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). This region contains a binding site for Sp1 or Sp3, a site for USF1 or USF2, and a novel site (18Beohar N. Kawamoto S. J. Biol. Chem. 1998; 273: 9168-9178Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Induction of NMHC II-A was demonstrated during differentiation of HL-60 myeloid cells and U-937 promonocytic cells to a more monocytic phenotype (19Toothaker L.E. Gonzalez D.A. Tung N. Lemons R.S. Le Beau M.M. Arnaout M.A. Clayton L.K. Tenen D.G. Blood. 1991; 78: 1826-1833Crossref PubMed Google Scholar). For NMHC II-B, the homeobox protein Hex has been demonstrated to induce transcription in a cAMP-response element-dependent manner, although Hex binds to a specific homeodomain-binding sequence rather than cAMP-response element (20Sekiguchi K. Kurabayashi M. Oyama Y. Aihara Y. Tanaka T. Sakamoto H. Hoshino Y. Kanda T. Yokoyama T. Shimomura Y. Iijima H. Ohyama Y. Nagai R. Circ. Res. 2001; 88: 52-58Crossref PubMed Scopus (38) Google Scholar). NMHC II-B is also down-regulated in cells transformed by a variety of oncogenes (21Yam J.W. Zheng J.Y. Hsiao W.L. Biochem. Biophys. Res. Commun. 1999; 266: 472-480Crossref PubMed Scopus (12) Google Scholar).Here we demonstrate using mouse macrophages that expression of NMHC II-C can be regulated by a physiologically relevant stimulant, sodium butyrate. In contrast, this treatment had no effect on the expression of II-A, which is abundant in these cells. NMHC II-B, which is not expressed in these cells, similar to RBL-2H3 cells, was only minimally up-regulated (data not shown). Butyrate is produced in large quantities by bacterial fermentation of fiber and its intracolonic concentration is typically in the 5–15 mm range (22Topping D.L. Clifton P.M. Physiol. Rev. 2001; 81: 1031-1064Crossref PubMed Scopus (2220) Google Scholar). Stimulation with this short chain fatty acid has been shown to lead to differentiation in a wide range of cell types and, in many cases, can also cause apoptosis. A diverse range of gene products have been shown to be induced by butyrate, including the G-protein Gαi2 (13Yang J. Kawai Y. Hanson R.W. Arinze I.J. J. Biol. Chem. 2001; 276: 25742-25752Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), cell-cycle related proteins such as p21Waf1 (10Nakano K. Mizuno T. Sowa Y. Orita T. Yoshino T. Okuyama Y. Fujita T. Ohtani-Fujita N. Matsukawa Y. Tokino T. Yamagishi H. Oka T. Nomura H. Sakai T. J. Biol. Chem. 1997; 272: 22199-22206Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar, 23Litvak D.A. Evers B.M. Hwang K.O. Hellmich M.R. Ko T.C. Townsend Jr., C.M. Surgery. 1998; 124: 161-169Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) and p27Kip1 (23Litvak D.A. Evers B.M. Hwang K.O. Hellmich M.R. Ko T.C. Townsend Jr., C.M. Surgery. 1998; 124: 161-169Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), γ-globin (24Zhang J.W. Raich N. Enver T. Anagnou N.P. Stamatoyannopoulos G. Dev. Genet. 1990; 11: 168-174Crossref PubMed Scopus (12) Google Scholar), and alkaline phosphatase (25Koyama H. Ono T. J. Cell. Physiol. 1976; 88: 49-56Crossref PubMed Scopus (35) Google Scholar). Induction of NMHC II-C was found both in hematopoietic cell lines, the macrophage line RAW 264.7 and basophilic leukemia line RBL-2H3, and in non-hematopoietic HeLa fibroblasts. However, HeLa cells were much less sensitive to butyrate than the hematopoietic lines.The induction of NMHC II-C mRNA expression does not requirede novo protein synthesis, because cycloheximide, at concentrations that abolished induction of NMHC II-C protein expression, tended to increase butyrate-mediated NMHC II-C mRNA expression. Cycloheximide alone was also able to induce increased amounts of NMHC II-C mRNA. The induction of specific mRNAs in response to cycloheximide treatment is often interpreted to indicate regulation of mRNA expression by a labile regulatory protein (26Chesnokov V.N. Mertvetsov N.P. Biokhimiya. 1990; 55: 1276-1278PubMed Google Scholar). However, other mechanisms by which cycloheximide can act include stabilization of mRNA (27Ikeda K. Lu C. Weir E.C. Mangin M. Broadus A.E. J. Biol. Chem. 1990; 265: 5398-5402Abstract Full Text PDF PubMed Google Scholar, 28Penhoat A. Jaillard C. Begeot M. Durand P. Saez J.M. Mol. Cell. Endocrinol. 1996; 121: 57-63Crossref PubMed Scopus (14) Google Scholar) and direct transcriptional activation (29Edwards D.R. Mahadevan L.C. EMBO J. 1992; 11: 2415-2424Crossref PubMed Scopus (278) Google Scholar, 30Cesari M. Heliot L. Meplan C. Pabion M. Khochbin S. Biochem. J. 1998; 336: 619-624Crossref PubMed Scopus (5) Google Scholar). Cooperation between cycloheximide and TSA in inducing histone H1 mRNA was proposed to involve a cycloheximide-mediated rearrangement of chromatin leading to general transcriptional de-repression (30Cesari M. Heliot L. Meplan C. Pabion M. Khochbin S. Biochem. J. 1998; 336: 619-624Crossref PubMed Scopus (5) Google Scholar). H" @default.
• W2076695692 created "2016-06-24" @default.
• W2076695692 creator A5002243330 @default.
• W2076695692 creator A5016373230 @default.
• W2076695692 creator A5039168531 @default.
• W2076695692 date "2003-04-01" @default.
• W2076695692 modified "2023-10-15" @default.
• W2076695692 title "Induction of Nonmuscle Myosin Heavy Chain II-C by Butyrate in RAW 264.7 Mouse Macrophages" @default.
• W2076695692 cites W1506486937 @default.
• W2076695692 cites W1803425892 @default.
• W2076695692 cites W1877854700 @default.
• W2076695692 cites W1967720431 @default.
• W2076695692 cites W1972483048 @default.
• W2076695692 cites W1975261636 @default.
• W2076695692 cites W1977523193 @default.
• W2076695692 cites W1978060272 @default.
• W2076695692 cites W1981333036 @default.
• W2076695692 cites W1981429363 @default.
• W2076695692 cites W1987486890 @default.
• W2076695692 cites W1992475307 @default.
• W2076695692 cites W2001434884 @default.
• W2076695692 cites W2004188995 @default.
• W2076695692 cites W2007210699 @default.
• W2076695692 cites W2015892667 @default.
• W2076695692 cites W2022870221 @default.
• W2076695692 cites W2033247222 @default.
• W2076695692 cites W2036797046 @default.
• W2076695692 cites W2053510126 @default.
• W2076695692 cites W2056738734 @default.
• W2076695692 cites W2087669636 @default.
• W2076695692 cites W2108989813 @default.
• W2076695692 cites W2110808326 @default.
• W2076695692 cites W2113210499 @default.
• W2076695692 cites W2116665363 @default.
• W2076695692 cites W2123687793 @default.
• W2076695692 cites W2127016760 @default.
• W2076695692 cites W2136328348 @default.
• W2076695692 cites W2149724080 @default.
• W2076695692 cites W2152049181 @default.
• W2076695692 cites W2153686053 @default.
• W2076695692 cites W216009453 @default.
• W2076695692 cites W2169671660 @default.
• W2076695692 cites W2170138004 @default.
• W2076695692 cites W2470299685 @default.
• W2076695692 cites W36502835 @default.
• W2076695692 cites W4293247451 @default.
• W2076695692 cites W92508582 @default.
• W2076695692 cites W96591720 @default.
• W2076695692 doi "https://doi.org/10.1074/jbc.m210145200" @default.
• W2076695692 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12598534" @default.
• W2076695692 hasPublicationYear "2003" @default.
• W2076695692 type Work @default.
• W2076695692 sameAs 2076695692 @default.
• W2076695692 citedByCount "24" @default.
• W2076695692 countsByYear W20766956922012 @default.
• W2076695692 countsByYear W20766956922020 @default.
• W2076695692 crossrefType "journal-article" @default.
• W2076695692 hasAuthorship W2076695692A5002243330 @default.
• W2076695692 hasAuthorship W2076695692A5016373230 @default.
• W2076695692 hasAuthorship W2076695692A5039168531 @default.
• W2076695692 hasBestOaLocation W20766956921 @default.
• W2076695692 hasConcept C100544194 @default.
• W2076695692 hasConcept C104317684 @default.
• W2076695692 hasConcept C121332964 @default.
• W2076695692 hasConcept C1276947 @default.
• W2076695692 hasConcept C153911025 @default.
• W2076695692 hasConcept C185592680 @default.
• W2076695692 hasConcept C199185054 @default.
• W2076695692 hasConcept C2776979534 @default.
• W2076695692 hasConcept C55493867 @default.
• W2076695692 hasConcept C6997183 @default.
• W2076695692 hasConcept C84613547 @default.
• W2076695692 hasConcept C86803240 @default.
• W2076695692 hasConcept C95444343 @default.
• W2076695692 hasConceptScore W2076695692C100544194 @default.
• W2076695692 hasConceptScore W2076695692C104317684 @default.
• W2076695692 hasConceptScore W2076695692C121332964 @default.
• W2076695692 hasConceptScore W2076695692C1276947 @default.
• W2076695692 hasConceptScore W2076695692C153911025 @default.
• W2076695692 hasConceptScore W2076695692C185592680 @default.
• W2076695692 hasConceptScore W2076695692C199185054 @default.
• W2076695692 hasConceptScore W2076695692C2776979534 @default.
• W2076695692 hasConceptScore W2076695692C55493867 @default.
• W2076695692 hasConceptScore W2076695692C6997183 @default.
• W2076695692 hasConceptScore W2076695692C84613547 @default.
• W2076695692 hasConceptScore W2076695692C86803240 @default.
• W2076695692 hasConceptScore W2076695692C95444343 @default.
• W2076695692 hasIssue "17" @default.
• W2076695692 hasLocation W20766956921 @default.
• W2076695692 hasOpenAccess W2076695692 @default.
• W2076695692 hasPrimaryLocation W20766956921 @default.
• W2076695692 hasRelatedWork W1511469829 @default.
• W2076695692 hasRelatedWork W1965805109 @default.
• W2076695692 hasRelatedWork W2072735043 @default.
• W2076695692 hasRelatedWork W2267531623 @default.
• W2076695692 hasRelatedWork W2416640367 @default.
• W2076695692 hasRelatedWork W2888803714 @default.
• W2076695692 hasRelatedWork W3101976153 @default.

• next