1Department of Orthopaedics and 2Department of Nutrition, The University of Tokushima School of Medicine, 3Division of Genetic Information, Institute for Genome Research, The University of Tokushima, Tokushima, Japan
Submitted 22 November 2004 ; accepted in final form 7 April 2005
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
sciatic neurectomy; Gpnmb family; C2C12 cells; NIH-3T3 cells; osteoactivin-transgenic mice
To address this issue, we previously examined the expression of 26,000 genes in rat gastrocnemius muscle atrophied by denervation or spaceflight using an Affymetrix DNA microarray analytical system (25). We found that both conditions remarkably increased the expression of osteoactivin, a functionally unknown type I membrane glycoprotein, in skeletal muscle. Besides being an EST gene (AA818039
[GenBank]
), the osteoactivin gene was only a transcript, whose expression was upregulated more than eightfold by spaceflight and denervation (25).
Osteoactivin is a rat homolog of Gpnmb family, which was originally reported to be highly expressed in human melanoma cells (36). Recently, osteoactivin transcripts have been reported to increase in various diseases associated with fibrosis, such as osteopetrosis and liver cirrhosis (26, 30). Osteoactivin has several interesting binding motifs for extracellular matrix proteins: a heparin-binding motif and an RGD motif for an integrin-binding site (33). On the basis of these findings, we hypothesize that highly expressed osteoactivin might interact with infiltrated cells, such as fibroblasts, leading to the degeneration or regeneration of extracellular matrix in denervated skeletal muscle.
In this study, we examined pathophysiological roles of osteoactivin in the mouse skeletal muscle atrophied by denervation. Immunohistochemical analysis revealed that denervation increased the expression of osteoactivin in muscle fiber membrane, whereas matrix metalloproteinase 3 (MMP-3) and MMP-9 were highly expressed in neighboring fibroblasts. Overexpression of osteoactivin or treatment with its recombinant proteins increased collagen type I, MMP-3, and MMP-9 in mouse NIH-3T3 fibroblasts, but not in mouse C2C12 myoblasts. Moreover, these matrix-related genes were more expressed in fibroblasts infiltrated into denervated skeletal muscles of osteoactivin-transgenic mice, compared with those in wild-type mice. Our present results suggest that the interaction between osteoactivin and fibroblasts stimulates expression of these matrix proteins and that osteoactivin is a key protein for regulating fibroblast functions in the skeletal muscle atrophied by denervation.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
For the denervation procedure, the dorsal skin of the right thigh was cut and the posterior muscles were divided to show the sciatic nerve. Chronic denervation was caused by removal of a sciatic nerve section of 5 mm in length (23). In the tail suspension procedure, a piece of tape was attached on the tail, and this tape was connected to a swivel tied to a horizontal bar at the top of the cage (37). Control animals were prepared in a parallel procedure. The hindlimb skeletal muscles, such as gastrocnemius, soleus, and tibialis anterior muscles, were isolated at the indicated times. After the wet weight was measured, the skeletal muscles were immediately frozen in chilled isopentane and liquid nitrogen and were stored at 80°C until analysis.
Cell culture. Mouse myoblastic C2C12 cells were purchased from Dainippon Pharmaceutical (Osaka, Japan). Mouse fibroblastic NIH-3T3 cells were a kind gift from Dr. N. Harada of The University of Tokushima School of Medicine, Tokushima, Japan. The cells were maintained and proliferated at 37°C with 5% CO2-95% air in Dulbecco's modified Eagle's medium, supplemented with 10% fetal calf serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. The first to third passages of cells from the purchased ones were used for the following experiments.
To examine the effects of growth factors on osteoactivin expression, C2C12 cells were treated with 5 ng/ml recombinant human basic fibroblast growth factor (bFGF) (Kaken Pharmaceutical, Tokyo, Japan) in the absence or presence of 300 µg/ml heparin (Sigma, St. Louis, MO), 25 ng/ml platelet-derived growth factor (PDGF) purified from human platelets (Sigma), and 2.512.5 ng/ml recombinant human MMP-3 (Sigma), respectively.
Transfection of osteoactivin. We constructed an expression vector for V5/His-tagged mouse osteoactivin by using reverse transcription-polymerase chain reaction (RT-PCR) and cloning techniques as described previously (4, 13). Total RNA was extracted from NIH-3T3 cells with an acid guanidinium thiocyanate-phenol-chloroform mixture (Isogen; Nippon Gene, Tokyo, Japan) according to the standard protocol (5). First-strand cDNAs were reverse transcribed at 37°C for 50 min from 1 µg of the extracted total RNA with oligo-dT15 primer and SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA). After the initial denaturation at 94°C for 2 min, second-strand synthesis and DNA amplification with Pfx DNA polymerase (Invitrogen) and the osteoactivin primer set (5'-CACCATGGAAAGTCTCTGCGGGGTC-3' and 5'-GAGTGTCCTTGGCTTGTCCTGGAGC-3') were accomplished through 30 cycles of the following incubations: 15 s at 94°C, 30 s at 60°C, 90 s at 68°C, with the use of a thermal cycler (MJ Research, Watertown, MA). The PCR products were sequenced and cloned into an expression vector pcDNA3.1/V5-His (Invitrogen). For overexpression of osteoactivin in vitro, NIH-3T3 or C2C12 cells were transfected with 2 µg/60-mm dish of the purified plasmid containing osteoactivin (pcDNA3.1/V5-His-tagged osteoactivin) by using FuGene6 (Roche Diagnostics, Mannheim, Germany), according to the method of Hellgren et al. (12).
Generation of osteoactivin-transgenic mice.
To express osteoactivin effectively in vivo, the BamHI/PmeI fragment of pcDNA3.1/V5-His-tagged osteoactivin was subcloned into an expression vector containing the cytomegalovirus immediate early enhancer chicken -globin hybrid promoter (31). It provided a transgene cassette composed of the cytomegalovirus promoter and rabbit
-globin exons 2 and 3 linked to V5-His-tagged osteoactivin cDNA. The V5-His-tagged rat osteoactivin cDNA construct was injected into fertilized BDF1 eggs for the production of transgenic mice (Japan SLC). After the most expressed osteoactivin transgenics were back-crossed into BDF1 mice, three strains of heterozygous transgenic mice were generated. Expression of osteoactivin was determined by RT-PCR for osteoactivin and immunobotting for V5 as described below. We maintained the mice under specific pathogen-free conditions, and some of them at 612 wk of age were used for experiments.
Treatment with recombinant osteoactivin protein. cDNA encoding mouse osteoactivin (AA22355) truncated the transmembrane domain was amplified by RT-PCR with the following primer set: 5'-TCCATAAGATTAGCGGATCCTACCTG-3' and 5'-TCAGACCGCTTCTGCGTTCTGATTTA-3'. The cDNA product was cloned into pBAD/myc-His vector (Invitrogen). The recombinant protein was produced in Escherichia coli and purified with a Ni-NTA resin agarose (Invitrogen), according to the standard protocol (29). NIH-3T3 cells with 60% of confluence were treated with 25 nM (final concentration) of the recombinant osteoactivin or LacZ (control) and cultured for the indicated times. In some experiments, 100 µM RGDS peptide, an integrin inhibitor (Sigma), or 300 µg/ml heparin was simultaneously added to NIH-3T3 cells. After being washed with phosphate-buffered saline (PBS), cells were suspended in 50 mM Tris·HCl buffer, pH 7.5, containing 150 mM NaCl, 1% Triton X-100, and 1 tablet of 25-ml protease inhibitor cocktails (Roche Diagnostics).
Preparation of an anti-osteoactivin antibody. Because an anti-osteoactivin antibody was not commercially available, an antiserum against recombinant mouse osteoactivin peptide (AA527575) fused to glutathione-S-transferase (GST) was raised in rabbits. Briefly, the GST-fused osteoactivin peptide was expressed in E. coli and purified as described previously (34). Each of two rabbits was immunized with 300 µg of the purified GST-fused osteoactivin peptide in Freund's complete adjuvant (1:1) and monthly boosted. Six months later, blood was collected from the central artery of the ear, and the IgG fraction of the antiserum was prepared by the method of Steinbuch and Audran (35). We further subjected the IgG fraction to affinity column chromatography with HiTrap NHS (Amersham Biosciences, Piscataway, NJ) conjugated with the recombinant osteoactivin peptide (AA527575). The bound antibody was eluted with 1 mM HCl, immediately neutralized, and stored at 4°C.
Immunoblot analysis.
Immunoblot analysis was performed as described previously (13). The whole cell and skeletal muscle extracts (40 µg protein/lane) were subjected to SDS-6, 8, 10, or 12% PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 4% skim milk and then incubated with primary antibodies for 1 h at 25°C. The primary antibodies used for the analysis were as follows: polyclonal anti-osteoactivin, polyclonal anti-MMP-2 (Sigma), monoclonal anti-MMP-3 (R&D Systems, Minneapolis, MN), polyclonal anti-MMP-9 (Sigma), polyclonal anti-collagen type I (Santa Cruz Biotechnology), monoclonal anti-V5 (Invitrogen), or anti--actin antibody (Oncogene Research Products, San Diego, CA). The bound antibodies were detected with the use of the suitable secondary antibodies and the enhanced chemiluminescence system (Amersham Biosciences). Signals were quantitated by densitometric analysis. We used
-actin as an internal standard protein because the denervation did not change the amount of
-actin. Protein concentrations were determined by Lowry's method with bovine serum albumin as a standard (19).
Histochemical analysis. To examine the localization of osteoactivin or MMP-3 in the denervated gastrocnemius muscle, immunohistochemical analysis was performed. Sections (5 µm) were fixed in ice-cold acetone for 10 min. After being rinsed with PBS three times, the sections were incubated with a 1:100 dilution of monoclonal anti-mouse MMP-3 antibody or polyclonal anti-mouse MMP-9 antibody (Sigma) and a 1:40 dilution of polyclonal anti-osteoactivin antibody at 4°C for 18 h. After being washed with PBS, the sections were incubated with a 1:600 dilution of secondary Alexa 488-conjugated anti-mouse IgG (Molecular Probes, Eugene, OR) and Alexa 568-conjugated anti-rabbit IgG (Molecular Probes) for 1 h at room temperature in the dark. For detection of vimentin, the fixed sections were incubated with a 1:200 dilution of monoclonal anti-human vimentin antibody labeled with Cy3 (Sigma) at 4°C for 18 h. In some cases, the sections were further incubated with a 1:1,000 dilution of Hoechst-33342 (Dojindo, Kumamoto, Japan) for 10 min at room temperature in the dark after being washed. The samples were then rinsed with PBS three times and treated with Vectashield (Vector Laboratories, Burlingame, CA). Target proteins were detected and visualized with fluorescence microscopy. The sections were counterstained with hematoxylin and eosin (HE). The cross-sectional area of myofibers was measured with the use of WinRoof software (version 5, Mitani, Fukui, Japan).
Semiquantitative or real-time RT-PCR. To measure the level of mRNA, semiquantitative RT-PCR was performed as described previously (13). After the synthesis of first-strand cDNAs from mRNAs, second strand synthesis and amplification of target genes were performed as described above. In this case, the PCR buffer contained two sets of primers to amplify the target gene cDNA and the internal standard glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA simultaneously. The sense and antisense primers used in this study are shown in Table 1. The amplification was terminated 15 min later at 72°C when PCR products were linearly amplified. The PCR products were separated by electrophoresis in an 8% PAGE and detected with a highly sensitive nucleic acid staining reagent (TaKaRa, Tokyo, Japan). The staining intensities of the target bands and internal standard gene cDNAs were estimated with an image analyzer (FMBIO II, TaKaRa), and the intensity ratio of a target gene cDNA to the internal standard gene cDNA was calculated. We used GAPDH as an internal standard gene because the denervation did not change the level of GAPDH mRNA (data not shown).
|
Statistical analysis. All data were statistically evaluated by ANOVA with SPSS software (version 6.1; SPSS, Tokyo, Japan) and were expressed as means ± SD, n = 36. One-way ANOVA was used to determine the significant effects of denervation or osteoactivin on the measured variables. Individual differences between groups were assessed with Duncan's multiple-range test. Differences were considered significant at P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Denervation-mediated expression of matrix-related genes. Because denervation has been reported to induce expression of matrix-related genes, including MMPs (16), we examined expression of MMPs in the gastrocnemius muscle of denervated mice. On day 10 after denervation, when osteoactivin protein reached the peak value, the amounts of MMP-2, -3, -9, and -14 transcripts were significantly increased in the muscle atrophied by denervation (Fig. 2A). Among them, the expression of MMP-3 mRNA was most significantly increased. Consistent with the increase in levels of MMP-3 and MMP-9 transcripts, denervation significantly accumulated the MMP-3 and MMP-9 proteins in gastrocnemius muscle (Fig. 2B). Increase in MMP-3 proteins by denervation was bigger than that of MMP-9.
|
|
Effects of growth factors on expression of osteoactivin protein in C2C12 cells.
Our anti-mouse osteoactivin antibody detected two bands of 68- and 98-kDa molecular masses, which corresponded to nonglycosylated and glycosylated osteoactivins, respectively (28), in immunoblot analysis (Fig. 4A). Because only glycosylated osteoactivin responded to denervation and treatment with bFGF or PDGF, we showed the immunoblotting results of glycosylated osteoactivin below.
|
Involvement of osteoactivin in expression of collagen type I, MMP-3, and MMP-9 in fibroblasts. To elucidate the involvement of osteoactivin in high expression of matrix-related genes in denervated skeletal muscle, we overexpressed osteoactivin in mouse C2C12 myoblastic cells. We did not detect any increase in MMP expression in osteoactivin-overexpressing C2C12 cells (Fig. 5A). In addition to myoblastic cells, we transfected osteoactivin into various cells types (data not shown), such as mouse MC3T3-E1 osteoblast-like cells and NIH-3T3 fibroblasts. Only overexpression of osteoactivin in NIH-3T3 cells significantly induced expression of MMP-3 (Fig. 5A). In this case, osteoactivin did not increase the amount of collagen type I, MMP-2, or MMP-9 protein (Fig. 5A).
|
Osteoactivin has two binding motifs, heparin-binding and integrin-binding motifs, in the extracellular domain (33). When two potential inhibitors for these binding motifs, heparin and RGDS peptide, were added to NIH-3T3 cells treated with recombinant osteoactivin, the former prevented osteoactivin-mediated expression of MMP-3, but the latter did not (Fig. 5C). Heparin also decreased levels of collagen type I and MMP-9 in NIH-3T3 cells treated with recombinant osteoactivin (data not shown). Simultaneous treatment with heparin and LacZ (control protein) did not affect the amount of MMP-3 protein (data not shown).
Involvement of osteoactivin in expression of MMP-3 and MMP-9 in Schwann cells. Rich et al. (28) have recently demonstrated that osteoactivin was involved in expression of MMPs in human glioma cells. We also observed the nerve with Wallerian degeneration in the denervated skeletal muscle. Denervation caused axon and myelin breakdown in the distal neuronal segment and induced expression of osteoactivin, MMP-3, and MMP-9 in the segment (Fig. 6, and data not shown). MMP-3 and MMP-9 were highly expressed in neural cells of the segment. In contrast, denervation induced osteoactivin in Schwann-like cells (Fig. 6), which were stained with an anti-S-100 antibody (data not shown).
|
|
Recently, several ubiquitin ligases, such as atrogin-1 (MAFbx-1) and MuRF-1, have been reported to be responsible for denervation-mediated muscle atrophy (1). To further elucidate the effects of osteoactivin on muscle proteolysis caused by denervation, we measured amounts of atrogin-1 transcripts in gastrocnemius muscle of denervated osteoactivin-transgenic mice. Denervation stimulated expression of atrogin-1 in gastrocnemius muscle of wild-type mice about fourfold (Fig. 7E). However, overexpression of osteoactivin did not change the denervation-mediated expression of atrogin-1 (Fig. 7E).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To elucidate whether osteoactivin influences denervation-induced myolysis, we have measured the mass (wet weight and cross-sectional area) of hindlimb skeletal muscles and expression of atrogin-1, a muscle atrophy-related gene (1), in gastrocnemius muscle of osteoactivin-transgenic mice. In osteoactivin-transgenic mice, denervation significantly increased wet weights of gastrocnemius and soleus muscles compared with those in wild-type mice, whereas the cross-sectional area of gastrocnemius muscle was not changed. In addition, denervation significantly increased expression of atrogin-1, as described by Bodine et al. (1). However, in vivo overexpression of osteoactivin did not affect this denervation-mediated induction of atrogin-1. These findings suggest that in vivo overexpression of osteoactivin might not change denervation-mediated muscle atrophy, but increase volumes of interstitial spaces between muscle fibers.
In osteoactivin-transgenic mice without denervation, high expression of matrix-related genes was not observed in hindlimb skeletal muscle. Only in denervated skeletal muscle did osteoactivin stimulate expression of MMP-3 in myofibroblasts. Denervation-mediated activation of myofibroblasts might be multistep reactions and require other factors, such as inducers for infiltration of fibroblasts into the skeletal muscles.
Treatment with recombinant osteoactivin increased the amounts of MMP-3, MMP-9, and collagen type I in NIH-3T3 fibroblasts. MMP-3 has been reported to regulate growth and development of tissues by selective degradation of insulin-like growth factor-I (IGF-1)/IGF-1-binding protein complexes (9). Secreted MMP-9 is also involved in the migration and myotube formation of myoblastic cells (17). Furthermore, targeted disruption of the MMP-3 gene in mice caused a delay in wound healing due to a failure in fibroblast contraction (2, 3). Synthetic MMP inhibitors undergoing clinical trials had reversible musculoskeletal toxicity as the main side effect (14). On the basis of these findings, an osteoactivin-mediated increase in MMPs in skeletal muscle might be useful for regeneration or degeneration in the denervated skeletal muscle, leading to compensation for the loss of muscle volume or protection of muscle fibers against injury after denervation.
Recombinant osteoactivin truncated the COOH-terminal domain (intracellular domain) was used in this study, because a full-length osteoactivin expressed in E. coli was quite insoluble (data not shown). In addition, overexpression of the truncated osteoactivin had effects on fibroblasts similar to those of full-length osteoactivin (data not shown). Therefore, we focused on the extracellular domain of osteoactivin as a functional site. Osteoactivin has two functional motifs in the extracellular domain: a heparin-binding motif and an RGD motif for an integrin-binding site (33). In fibroblasts treated with recombinant osteoactivin, an integrin inhibitor did not suppress expression of collagen type I, MMP-3, or MMP-9, whereas heparin significantly canceled osteoactivin-mediated expression of these proteins. These findings strongly suggest that osteoactivin affects fibroblasts via a heparin-binding motif, but not via an integrin-binding motif. Fibroblasts highly expressing heparan sulfate proteoglycans have been reported to be proliferated near denervated synaptic sites in the skeletal muscle (10). The interaction between osteoactivin in myocytes and heparan sulfate proteoglycan on the surface of infiltrated fibroblasts might play an important role in regulating functions of fibroblasts in the interstitial spaces of the denervated skeletal muscle. Identifying a proteoglycan (receptor) specifically binding to osteoactivin is the next and ongoing important subject.
Because osteoactivin has been reported to be involved in expression of MMPs in human glioma cells (28), we also examined the expression of osteoactivin and MMPs in neurectomized neurons. Osteoactivin was overexpressed in Schwann-like cells in neurons with Wallerian degeneration, whereas MMP-3 and MMP-9 were present in neuronal cells in the neurectomized sciatic nerve. In neurons, osteoactivin expressed in Schwann-like cells might induce expression of MMP-3 in neuronal cells. Upregulation of MMPs, such as MMP-2 and MMP-9, during denervation has been reported to enable axonal regrowth by removal of inhibitory factors from the basal lamina of Schwann cells (8). Therefore, it is likely that in the nerve system, osteoactivin might function as an activator for connective cells and facilitate the regeneration of neurons by secretion of MMP-3 and MMP-9 from axons in the vicinity of Schwann cells.
Expression of osteoactivin has been reported to be up-regulated in osteoblasts of rats with osteopetrosis or in hepatocytes of rats with liver cirrhosis. However, the factors that trigger expression of osteoactivin are little known. In skeletal muscles and neurons during denervation, the expression of bFGF and PDGF has been reported to be upregulated (11, 27). In this study, we found that bFGF and PDGF significantly induced expression of osteoactivin in skeletal muscle cells. Heparin, an antagonist of bFGF, significantly inhibited bFGF-mediated expression of osteoactivin protein, strongly suggesting that in denervated skeletal muscle, expression of osteoactivin might be regulated by these denervation-derived growth factors, especially bFGF. These results were supported by the study of Dell'Era et al. (7) that in macroarray analysis of bFGF-transformed endothelial cells, expression of osteoactivin was remarkably upregulated. Recently, it has been reported that in the liver of bFGF-deficient mice, liver fibrosis caused by exposure to chronic carbon tetrachloride was remarkably decreased (38). Osteoactivin might play an important role in fibrosis in such diseases. Further examinations are required to elucidate this hypothesis.
In general, unloading, such as spaceflight, tail suspension, and denervation, preferentially affects slow-twitch skeletal muscles more than fast-twitch skeletal muscles. For example, the regulation of skeletal muscle genes by denervation contrasted between the two types of skeletal muscles, although denervation caused an increase in UCP3 mRNA levels in both types of muscles (15). Denervation also caused slow to fast transformation in rat soleus muscle (20). Our present finding that soleus muscle sustained a much prolonged increase in the level of osteoactivin, compared with gastrocnemius muscle, was consistent with these previous reports, although the detailed mechanism of distinct osteoactivin expression in soleus and gastrocnemius muscles are unknown at this present. However, histochemical analysis of osteoactivin in soleus muscle showed a pattern similar to that in gastrocnemius muscle, except for the expression period (data not shown), indicating that osteoactivin expressed in soleus and gastrocnemius muscle has similar physiological roles.
This study provides the first evidence that osteoactivin is a novel activator of fibroblasts in denervated skeletal muscle. Development of agents for regulating the osteoactivin function is ongoing and will be an important step to further elucidate the physiological function of fibroblasts for regeneration or degeneration in extracellular matrix after denervation.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Bullard KM, Mudgett JS, Scheuenstuhl H, Hunt TK, and Banda MJ. Stromelysin-1-deficient fibroblasts display impaired contraction in vitro. J Surg Res 84: 3134, 1999.[CrossRef][ISI][Medline]
3. Bullard KM, Lund L, Mudgett JS, Werb Z, Mellin JN, Hunt TK, Murphy B, Ronan J, and Banda MJ. Impaired wound contraction in stromelysin-1 deficient mice. Ann Surg 230: 260265, 1999.[CrossRef][ISI][Medline]
4. Cheng C and Shuman S. Recombinogenic flap ligation pathway for intrinsic repair of topoisomerase IB-induced double-strand breaks. Mol Cell Biol 20: 80598068, 2000.
5. Chomczynski P and Mackey K. Modification of the TRI reagent procedure for isolation of RNA from polysaccharide- and proteoglycan-rich sources. Biotechniques 19: 942945, 1995.[ISI][Medline]
6. Connor EA and McMahan UJ. Cell accumulation in the junctional region of denervated muscle. J Cell Biol 104: 109120, 1987.[Abstract]
7. Dell'Era P, Coco L, Ronca R, Sennino B, and Presta M. Gene expression profile in fibroblast growth factor 2-transformed endothelial cells. Oncogene 21: 24332440, 2002.[CrossRef][ISI][Medline]
8. Ferguson TA and Muir D. MMP-2 and MMP-9 increase the neurite-promoting potential of Schwann cell basal laminae and are upregulated in degenerated nerve. Mol Cell Neurosci 16: 157167, 2000.[CrossRef][ISI][Medline]
9. Fowlkes JL, Serra DM, Bunn RC, Thrailkill KM, Enghild JJ, and Nagase H. Regulation of insulin-like growth factor (IGF)-I action by matrix metalloproteinase-3 involves selective disruption of IGF-I/IGF-binding protein-3 complexes. Endocrinology 145: 620626, 2004.
10. Gatchalian CL, Schachner M, and Sanes JR. Fibroblasts that proliferate near denervated synaptic sites in skeletal muscle synthesize the adhesive molecules tenascin (J1), N-CAM, fibronectin, and a heparan sulfate proteoglycan. J Cell Biol 108: 18731890, 1989.[Abstract]
11. Grothe C, Meisinger C, and Claus P. In vivo expression and localization of the fibroblast growth factor system in the intact and lesioned rat peripheral nerve and spinal ganglia. J Comp Neurol 434: 342357, 2001.[CrossRef][ISI][Medline]
12. Hellgren I, Drvota V, Pieper R, Enoksson S, Blomberg P, Islam KB, and Sylven C. Highly efficient cell-mediated gene transfer using non-viral vectors and FuGene6: in vitro and in vivo studies. Cell Mol Life Sci 57: 13261333, 2000.[ISI][Medline]
13. Ikemoto M, Nikawa T, Takeda S, Watanabe C, Kitano T, Baldwin KM, Izumi R, Nonaka I, Towatari T, Teshima S, Rokutan K, and Kishi K. Space shuttle flight (STS-90) enhances degradation of rat myosin heavy chain in association with activation of ubiquitin-proteasome pathway. FASEB J 15: 12791281, 2001.
14. Jabs T, Tschope M, Colling C, Hahlbrock K, and Scheel D. Elicitor-stimulated ion fluxes and O2 from the oxidative burst are essential components in triggering defense gene activation and phytoalexin synthesis in parsley. Proc Natl Acad Sci USA 94: 48004805, 1997.
15. Kontani Y, Wang Z, Furuyama T, Sato Y, Mori N, and Yamashita H. Effects of aging and denervation on the expression of uncoupling proteins in slow- and fast-twitch muscles of rats. J Biochem (Tokyo) 132: 309315, 2002.[Abstract]
16. Kherif S, Dehaupas M, Lafuma C, Fardeau M, and Alameddine HS. Matrix metalloproteinases MMP-2 and MMP-9 in denervated muscle and injured nerve. Neuropathol Appl Neurobiol 24: 309319, 1998.[CrossRef][ISI][Medline]
17. Lewis MP, Tippett HL, Sinanan AC, Morgan MJ, and Hunt NP. Gelatinase-B (matrix metalloproteinase-9; MMP-9) secretion is involved in the migratory phase of human and murine muscle cell cultures. J Muscle Res Cell Motil 21: 223233, 2000.[CrossRef][ISI][Medline]
18. Li Y, Foster W, Deasy BM, Chan Y, Prisk V, Tang Y, Cummins J, and Huard J. Transforming growth factor-1 induces the differentiation of myogenic cells into fibrotic cells in injured skeletal muscle: a key event in muscle fibrogenesis. Am J Pathol 164: 10071019, 2004.
19. Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265275, 1951.
20. Midrio M, Danieli-Betto D, Megighian A, Velussi C, Catani C, and Carraro U. Slow-to-fast transformation of denervated soleus muscle of the rat, in the presence of an antifibrillatory drug. Pflügers Arch 420: 446450, 1992.[CrossRef][ISI][Medline]
21. Murray MA and Robbins N. Cell proliferation in denervated muscle: time course, distribution and relation to disuse. Neuroscience 7: 18171822, 1982.[CrossRef][ISI][Medline]
22. Murray MA and Robbins N. Cell proliferation in denervated muscle: identity and origin of dividing cells. Neuroscience 7: 18231833, 1982.[CrossRef][ISI][Medline]
23. Musacchia XJ, Steffen JM, and Fell RD. Disuse atrophy of skeletal muscle: animal models. Exerc Sport Sci Rev 16: 6187, 1988.[Medline]
24. Newman DR, Li CM, Simmons R, Khosla J, and Sannes PL. Heparin affects signaling pathways stimulated by fibroblast growth factor-1 and -2 in type II cells. Am J Physiol Lung Cell Mol Physiol 287: L191L200, 2004.
25. Nikawa T, Ishidoh K, Hirasaka K, Ishihara I, Ikemoto M, Kano M, Kominami E, Nonaka I, Ogawa T, Adams GR, Baldwin KM, Yasui N, Kishi K, and Takeda S. Skeletal muscle gene expression in space-flown rats. FASEB J 18: 522524, 2004.
26. Onaga M, Ido A, Hasuike S, Uto H, Moriuchi A, Nagata K, Hori T, Hayashi K, and Tsubouchi H. Osteoactivin expressed during cirrhosis development in rats fed a choline-deficient, L-amino acid-defined diet, accelerates motility of hepatoma cells. J Hepatol 39: 779785, 2003.[CrossRef][ISI][Medline]
27. Oya T, Zhao YL, Takagawa K, Kawaguchi M, Shirakawa K, Yamauchi T, and Sasahara M. Platelet-derived growth factor- expression induced after rat peripheral nerve injuries. Glia 38: 303312, 2002.[CrossRef][ISI][Medline]
28. Rich JN, Shi Q, Hjelmeland M, Cummings TJ, Kuan CT, Bigner DD, Counter CM, and Wang XF. Bone-related genes expressed in advanced malignancies induce invasion and metastasis in a genetically defined human cancer model. J Biol Chem 278: 1595115957, 2003.
29. Rosado-Ruiz T, Antommattei-Perez FM, Cadilla CL, and Lopez-Garriga J. Expression and purification of recombinant hemoglobin I from Lucina pectinata. J Protein Chem 20: 311315, 2001.[CrossRef][ISI][Medline]
30. Safadi FF, Xu J, Smock SL, Rico MC, Owen TA, and Popoff SN. Cloning and characterization of osteoactivin, a novel cDNA expressed in osteoblasts. J Cell Biochem 84: 1226, 2001.[CrossRef][ISI][Medline]
31. Sakai K and Miyazaki J. A transgenic mouse line that retains Cre recombinase acitivity in mature oocytes irrespective of the Cre transgene transmission. Biochem Biophys Res Commun 237: 318324, 1997.[CrossRef][ISI][Medline]
32. Salonen V, Lehto M, Kalimo M, Penttinen R, and Aro H. Changes in intramuscular collagen and fibronectin in denervation atrophy. Muscle Nerve 8: 125131, 1985.[CrossRef][ISI][Medline]
33. Shikano S, Bonkobara M, Zukas PK, and Ariizumi K. Molecular cloning of a dendritic cell-associated transmembrane protein, DC-HIL, that promotes RGD-dependent adhesion of endothelial cells through recognition of heparan sulfate proteoglycans. J Biol Chem 276: 81258134, 2001.
34. Smith DB and Johnson KS. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67: 3140, 1988.[CrossRef][ISI][Medline]
35. Steinbuch M and Audran R. The isolation of IgG from mammalian sera with the aid of caprylic acid. Arch Biochem Biophys 134: 279284, 1969.[CrossRef][ISI][Medline]
36. Weterman MA, Ajubi N, van Dinter IM, Degen WG, van Muijen GN, Ruitter DJ, and Bloemers HP. Nmb, a novel gene, is expressed in low-metastatic human melanoma cell lines and xenografts. Int J Cancer 60: 7381, 1995.[ISI][Medline]
37. Wronski TJ and Morey-Holton ER. Skeletal response to simulated weightlessness: a comparison of suspension techniques. Aviat Space Environ Med 58: 6368, 1987.[ISI][Medline]
38. Yu C, Wang F, Jin C, Huang X, Miller DL, Basilico C, and McKeehan WL. Role of fibroblast growth factor type 1 and 2 in carbon tetrachloride-induced hepatic injury and fibrogenesis. Am J Pathol 163: 16531662, 2003.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |