FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2040472243> ?p ?o ?g. }

• W2040472243 endingPage "242" @default.
• W2040472243 startingPage "238" @default.
• W2040472243 abstract "Myostatin, also called growth and differentiation factor-8, is a member of the transforming growth factor-β superfamily [1,2]. Myostatin is a secreted protein considered a negative regulator of muscle growth during development and of muscle mass during adulthood [1]. In mouse models, knocking out the myostatin gene, overexpressing proteins neutralizing myostatin, or natural mutations of the myostatin gene cause increased muscle mass [1]. In cattle, naturally occurring myostatin gene mutations leading to inactive protein cause ‘double-muscle cattle’[2]. Recently reported was a child in whom a homozygous myostatin gene mutation that results in reduced production of myostatin protein, was associated with increased muscle bulk and strength [3]. Conversely, mature myostatin protein has been reported increased in muscle tissue of patients with HIV-associated muscle wasting [4], and increased myostatin-precursor protein (MstnPP) mRNA reported in muscle wasting associated with osteoarthritis [5]. Within muscle fibres, myostatin is synthesized as a MstnPP [6,7]. MstnPP and its mRNA, and mature myostatin, are predominantly expressed in skeletal muscle tissue (reviewed in [1]). MstnPP, a 375-amino-acid protein translated from a 3.1 kb mRNA [4], consists of three structural domains: a signal sequence; an N-terminal 28 kDa propeptide, also referred to as latency associated peptide [7]; and a C-terminal 12 kDa mature myostatin peptide [6,7]. Intracellular processing of myostatin from MstnPP has been proposed to occur through furin [6,8]. We recently showed in biopsied sporadic inclusion-body myositis (s-IBM) muscle fibres that both MstnPP and myostatin dimer were significantly increased, and MstnPP was physically associated with amyloid-β precursor protein (AβPP) [9]. Moreover, by light- and electron-microscopic immunocytochemistry, MstnPP/myostatin colocalized with amyloid-β (Aβ)/AβPP [9]. s-IBM is severely progressive, the most common muscle disease of older persons, and there is no successful treatment [10]. Histological hallmarks of s-IBM include: (1) vacuolar degeneration and atrophy of muscle fibres, accompanied by intramuscle-fibre accumulations of ubiquitinated protein aggregates, including Aβ/AβPP; (2) muscle-fibre atrophy; and (3) mononuclear lymphocytic inflammation [10–12]. Recently demonstrated in s-IBM muscle fibres were inhibition of 26S proteasome activity and presence of aggresomes [13]. Accumulation of AβPP/Aβ appears to be an early upstream step in the s-IBM pathogenesis, because: (i) abnormal accumulation of AβPP epitopes appears to precede other abnormalities in IBM muscle fibres [11]; and (ii) several aspects of the s-IBM phenotype, including Aβ accumulation, proteasome inhibition and aggresome formation, were produced in cultured normal human muscle fibres (CHMFs) after long-term overexpression of AβPP in them [13–15]. The latter provides a useful IBM human-muscle tissue-culture model. The aim of the present study was to utilize this model to investigate possible mechanisms responsible for increased MstnPP and myostatin within the biopsied s-IBM muscle fibres. We cultured human muscle fibres from satellite cells obtained from six normal diagnostic muscle biopsies, as described [13–15] and referenced therein. Into well-differentiated 3-week-old cultured muscle fibres we transferred a 3 kb human AβPP-cDNA encoding 751-AβPP using a replication-deficient adenovirus vector at 0.3 × 108 pfu/ml culture medium, as detailed previously [14,15]. In addition, we treated some of the cultures with 1 μm epoxomicin (Biomol Research Laboratories, Plymouth Meeting, PA, USA) [13], an irreversible proteasome inhibitor [16]. Four days after AβPP gene transfer and 24 h after epoxomicin treatment, control and AβPP-overexpressing CHMFs (AβPP+ CHMFs) were processed for light- and electron-microscopic immunocytochemistry, immunoblotting, combined immunoprecipitation/immunoblotting, and reverse transcriptase polymerase chain reaction (RT-PCR), as described [13–15,17]. For all the myostatin studies, we used an anti-myostatin rabbit polyclonal antibody (Chemicon,Temecula, CA, USA), which was shown to be highly specific in our studies and in studies by others [4,9,18]. On immunoblots, this antibody recognizes both a 55 kDa band of MstnPP and a 26 kDa band of mature myostatin dimer, and by immunocytochemistry it recognizes both MstnPP/myostatin [9]. For AβPP/Aβ studies, we used a well-characterized mouse monoclonal 6E10 antibody (Signet, Dedham, MA, USA), diluted 1:100, which morphologically recognizes Aβ/AβPP in both s-IBM muscle fibres and Alzheimer's disease brain [19,20], and by immunoblots recognizes both AβPP and Aβ[19,20]. Single- and double-labelled immunofluorescence was performed as described [13–15], using anti-myostatin antibody diluted 1:100 and 6E10 diluted 1:150. Mouse monoclonal antibody against γ-tubulin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), diluted 1:50, was used to identify aggresomes. Double-labelled gold-immuno-electronmicroscopy was performed on nonproteasome-inhibited AβPP+ CHMFs as previously detailed [13–15], using the same antibodies discussed above. For immunoblotting, cultured muscle fibres were harvested in RIPA buffer, homogenized, and protein concentration measured using the Bradford method [9,13,17]. Denatured 40 μg protein samples were loaded onto 10% NuPage gels, electrophoresed in MOPS-SDS buffer, transferred to nitrocellulose membranes, and immunoprobed overnight with anti-myostatin antibody diluted 1:500. After incubation with appropriate secondary antibody, blots were developed using the enhanced chemiluminescence system. Protein loading was evaluated by actin bands visualized with a mouse monoclonal anti-actin antibody (1:2000) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Quantification of immunoreactivity was performed by densitometric analysis using the Kodak-GelLogic-440 system (Eastman Kodak Company, Rochester, NY, USA). We performed a combined immunoprecipitation-immunoblotting, as described [9,13], to evaluate whether MstnPP physically associates with AβPP/Aβ. In brief, 100 μg of homogenized protein from AβPP+ CHMFs was immunoprecipitated with either 5 μg of 6E10 antibody or anti-myostatin antibody. Immunoprecipitates were incubated with 20 μl of protein-G-Sepharose-4 fast flow (Amersham-Bioscience, Piscataway, NJ, USA), washed, denatured, separated by electrophoresis, transferred to membranes, and immunoprobed with either anti-myostatin or 6E10 antibody, and then with appropriate secondary antibody, and developed. For RT-PCR, total RNA was isolated and processed as described [17]. One microgram of RNA was used in the RT-PCR reaction utilizing the OneStep RT-PCR kit (Qiagen, Valencia, CA, USA), with either primers (i) for MstnPP (Fw-GTGGTACCTCATGCAAAAACTGCAACTCTGT; Rv-ATGGATCCAATCTCATGAGCACCCACAGC), or (ii) for GAPDH [21]. The PCR products were separated on a 2% agarose gel and stained with SYBR Safe DNA gel stain (Invitrogen, Carlsbad, CA, USA). The conditions of the reactions were experimentally checked to ensure that signals were in the linear range of the PCR. Our immunofluorescence results revealed very weak cytoplasmic staining of MstnPP/myostatin within control CHMFs, and much stronger, mainly diffuse, cytoplasmic staining in AβPP+ CHMFs (Figure 1A,B). In 70–80% of the epoxomicin-treated AβPP+ CHMFs, strong MstnPP/myostatin immunoreactivity was present in the form of aggregates, which also contained Aβ/AβPP (Figure 1C,D). Their immuno-colocalization with γ-tubulin indicated that MstnPP/myostatin immunoreactive aggregates were aggresomes [13] (Figure 1E,F). By gold-immuno-electronmicroscopy, within AβPP+ CHMFs, both MstnPP/myostatin and Aβ/AβPP were associated together with 6–10 nm fibrils (Figure 2A) and with amorphous and floccular material (Figure 2B,C). We have previously proposed that 6–10 nm fibrils reflect a β-pleated-sheet amyloid, while amorphous and floccular material may represent preamyloid [22] Additionally, both Aβ/AβPP and MstnPP/myostatin were immunolocalized perinuclearly (Figure 2D). Immunoblots demonstrated that in our AβPP+ CHMFs a 55 kDa MstnPP was increased 2.3-fold (P < 0.01) as compared with control cultures (Figure 3A,B), but according to our semiquantative RT-PCR study there was no significant difference in the amount of MstnPP mRNA between control and AβPP+ CHMFs (Figure 3C,D). Mature myostatin was not apparent on our immunoblots, which is consistent with a recent study demonstrating that in well-differentiated myotubes of C2C12 mouse cell-line, as well as in bovine myotubes, mature myostatin was not detected on immunoblots [8]. Single immunofluorescence of myostatin-precursor protein (MstnPP)/myostatin in control (A) and amyloid-β precursor protein-overexpressing (AβPP+) (B) cultured human muscle fibres shows much stronger, diffuse MstnPP/myostatin immunoreactivity in (B) due to the AβPP overexpression. In AβPP+ fibres, epoxomicin treatment induced strongly MstnPP/myostatin-immunoreactive aggregates (C), which by double-label immunofluorescence colocalize with Aβ/AβPP (D). MstnPP/myostatin immunoreactive aggregates also colocalize with γ-tubulin (E,F) indicating they have features of aggresomes. All ×1100. Double-label gold-immuno-electronmicroscopy in amyloid-β precursor protein-overexpressing (AβPP+) cultured human muscle fibres (CHMFs) show that myostatin-precursor protein (MstnPP)/myostatin (12 nm gold particles) and Aβ/AβPP (6 nm gold particles) are both associated with 6–10 nm filaments and floccular material (A–C). In addition, both MstnPP/myostatin and Aβ/AβPP are present perinuclearly (D). A×61 000; B–D×45 000. (A,B) Immunoblots of myostatin-precursor protein (MstnPP)/myostatin in control, amyloid-β precursor protein-overexpressing (AβPP+) and epoxomicin-treated cultured human muscle fibres (CHMFs). (A) Representative immunoblot of one culture set illustrating increased 55 kDa MstnPP in AβPP+ cultures as compared with controls and epoxomicin-treated cultures. A 26 kDa mature myostatin is not visible on the blot. The actin bands illustrate protein loading. (B) Densitometric analysis of six blots, as in (A). MstnPP was 2.3-fold (P < 0.01) increased in AβPP+ CHMFs. Data are indicated as mean ± standard error. Significance was set at P < 0.05. (C,D) RT-PCR of MstnPP mRNA in control, AβPP+ and epoxomicin-treated CHMFs. (C) Representative agarose gel electrophoresis of products corresponding to MstnPP and GAPDH mRNAs, amplified by RT-PCR method. (D) Densitometric analysis of the PCR bands obtained in six independent experiments, expressed in arbitrary units, indicates that AβPP overexpression did not seem to influence MstnPP mRNA, while epoxomicin treatment decreased MstnPP mRNA 2.5-fold (P < 0.01). GAPDH mRNA was used to normalize the results. (E,F) Combined immunoprecipitation-immunoblot (IP) experiments. (E) IP of AβPP+ CHMFs with anti-MstnPP/myostatin antibody followed by immunoblotting with 6E10 antibody recognizing AβPP, demonstrates a strong 130 kDa band corresponding to AβPP. (F) IP of AβPP+ CHMFs with 6E10 antibody followed by immunoblotting with an antibody against MstnPP/myostatin shows a strong 55 kDa band corresponding to MstnPP. * The primary antibody omitted from the IP reaction; # the primary antibody omitted in the immunoblotting. Both * and # were negative. Proteasome inhibition did not appear to influence the amount of MstnPP (Figure 3A,B), but MstnPP mRNA was decreased 2.5-fold (P < 0.01) as compared with controls (Figure 3C,D). The combined immunoprecipitation-immunoblot procedure showed that, in our IBM culture model, MstnPP and AβPP coimmunoprecipitate (Figure 3E,F), similarly to s-IBM muscle biopsies [9]. The present study provides a novel demonstration in cultured human muscle that overexpression of AβPP/Aβ increases accumulation of MstnPP. Accordingly, this mechanism might be, at least partially, responsible for the increase of MstnPP and of mature myostatin in the biopsied s-IBM muscle fibres [9]. Whether lack of detectable mature myostatin dimer in our model (i) reflects its secretion to the culture medium, or (ii) is related to the sensitivity of our antibody remains to be studied. The mechanism(s) by which AβPP/Aβ increases MstnPP is presently not known. One possibility is that AβPP binding to MstnPP causes its posttranslational modification that lessens its degradation and traffic, resulting in its accumulation. This possibility would accord with the fact that in proteasome-inhibited CHMFs there is no decrease in the total amount of MstnPP despite its significantly decreased mRNA. It is currently not definitely known whether the ubiquitin-proteasome system participates in MstnPP degradation, but in our proteasome-inhibited CHMFs, MstnPP was accumulated in aggresomes, which probably impairs its traffic and degradation. Even though in our study AβPP/Aβ overexpression does not appear to influence MstnPP mRNA, a possible enhancement of MstnPP transcription might be masked by a proteasome inhibition produced by AβPP overexpression [13], and, as we have discussed above, MstnPP mRNA is decreased by proteasome inhibition. Collectively, the data in our current study support the proposal that in s-IBM muscle fibres, abnormalities of AβPP/Aβ precede increased MstnPP/myostatin [9]. As excessive myostatin may contribute to human muscle-fibre atrophy (see above), therapeutically inhibiting MstnPP or myostatin might be beneficial in various neuromuscular disorders associated with atrophy. Our culture system should provide a useful model to study regulatory mechanisms of MstnPP production and its processing in human muscle. And this culture model might be useful in developing new treatment strategies to mitigate myostatin and its effects. Supported by grants (to V.A.) from: the National Institutes of Health (AG16768 Merit Award), The Myositis Association, the Muscular Dystrophy Association, and the Helen Lewis Research Fund. S.W. is on leave from the Department of Anatomy and Neurobiology; A.N. is on leave from the Department of Biochemistry, Medical University of Gdansk, Poland. Maggie Baburyan provided excellent technical assistance in electronmicroscopy." @default.
• W2040472243 created "2016-06-24" @default.
• W2040472243 creator A5000518332 @default.
• W2040472243 creator A5029639156 @default.
• W2040472243 creator A5029701142 @default.
• W2040472243 creator A5051798243 @default.
• W2040472243 creator A5057574174 @default.
• W2040472243 creator A5060033281 @default.
• W2040472243 date "2007-01-22" @default.
• W2040472243 modified "2023-10-12" @default.
• W2040472243 title "Myostatin precursor protein is increased and associates with amyloid-? precursor protein in inclusion-body myositis culture model" @default.
• W2040472243 cites W1517014841 @default.
• W2040472243 cites W175460259 @default.
• W2040472243 cites W1987807233 @default.
• W2040472243 cites W1999554343 @default.
• W2040472243 cites W2005484389 @default.
• W2040472243 cites W2006067401 @default.
• W2040472243 cites W2011968886 @default.
• W2040472243 cites W2019957886 @default.
• W2040472243 cites W2021696285 @default.
• W2040472243 cites W2039709752 @default.
• W2040472243 cites W2064105273 @default.
• W2040472243 cites W2076887965 @default.
• W2040472243 cites W2117660724 @default.
• W2040472243 cites W2137814808 @default.
• W2040472243 cites W2138866489 @default.
• W2040472243 cites W2141923361 @default.
• W2040472243 cites W2158254766 @default.
• W2040472243 cites W2159831275 @default.
• W2040472243 cites W2165868509 @default.
• W2040472243 cites W2166877291 @default.
• W2040472243 cites W79737010 @default.
• W2040472243 doi "https://doi.org/10.1111/j.1365-2990.2006.00821.x" @default.
• W2040472243 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17359364" @default.
• W2040472243 hasPublicationYear "2007" @default.
• W2040472243 type Work @default.
• W2040472243 sameAs 2040472243 @default.
• W2040472243 citedByCount "16" @default.
• W2040472243 countsByYear W20404722432012 @default.
• W2040472243 countsByYear W20404722432015 @default.
• W2040472243 countsByYear W20404722432018 @default.
• W2040472243 crossrefType "journal-article" @default.
• W2040472243 hasAuthorship W2040472243A5000518332 @default.
• W2040472243 hasAuthorship W2040472243A5029639156 @default.
• W2040472243 hasAuthorship W2040472243A5029701142 @default.
• W2040472243 hasAuthorship W2040472243A5051798243 @default.
• W2040472243 hasAuthorship W2040472243A5057574174 @default.
• W2040472243 hasAuthorship W2040472243A5060033281 @default.
• W2040472243 hasConcept C134018914 @default.
• W2040472243 hasConcept C142724271 @default.
• W2040472243 hasConcept C167414201 @default.
• W2040472243 hasConcept C2777327098 @default.
• W2040472243 hasConcept C2777488582 @default.
• W2040472243 hasConcept C2777633098 @default.
• W2040472243 hasConcept C2779134260 @default.
• W2040472243 hasConcept C2781232998 @default.
• W2040472243 hasConcept C31705614 @default.
• W2040472243 hasConcept C502032728 @default.
• W2040472243 hasConcept C71924100 @default.
• W2040472243 hasConceptScore W2040472243C134018914 @default.
• W2040472243 hasConceptScore W2040472243C142724271 @default.
• W2040472243 hasConceptScore W2040472243C167414201 @default.
• W2040472243 hasConceptScore W2040472243C2777327098 @default.
• W2040472243 hasConceptScore W2040472243C2777488582 @default.
• W2040472243 hasConceptScore W2040472243C2777633098 @default.
• W2040472243 hasConceptScore W2040472243C2779134260 @default.
• W2040472243 hasConceptScore W2040472243C2781232998 @default.
• W2040472243 hasConceptScore W2040472243C31705614 @default.
• W2040472243 hasConceptScore W2040472243C502032728 @default.
• W2040472243 hasConceptScore W2040472243C71924100 @default.
• W2040472243 hasIssue "2" @default.
• W2040472243 hasLocation W20404722431 @default.
• W2040472243 hasLocation W20404722432 @default.
• W2040472243 hasOpenAccess W2040472243 @default.
• W2040472243 hasPrimaryLocation W20404722431 @default.
• W2040472243 hasRelatedWork W2122744082 @default.
• W2040472243 hasRelatedWork W2186062199 @default.
• W2040472243 hasRelatedWork W2230943006 @default.
• W2040472243 hasRelatedWork W2461719374 @default.
• W2040472243 hasRelatedWork W2750423117 @default.
• W2040472243 hasRelatedWork W3043348531 @default.
• W2040472243 hasRelatedWork W3093606042 @default.
• W2040472243 hasRelatedWork W3160254567 @default.
• W2040472243 hasRelatedWork W4225493376 @default.
• W2040472243 hasRelatedWork W93957273 @default.
• W2040472243 hasVolume "33" @default.
• W2040472243 isParatext "false" @default.
• W2040472243 isRetracted "false" @default.
• W2040472243 magId "2040472243" @default.
• W2040472243 workType "article" @default.