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1 Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita 12, Nishi 6, Kita-ku, Sapporo 060-0812, Japan
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Department of Biomembrane and Biofunctional Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan
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Centre for Instrumental Analysis, Hokkaido University, Sapporo, Japan
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Laboratory of Molecular and Cellular Pathology, Graduate School of Medicine, Hokkaido University, Sapporo, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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Tenascin-X (TNX) is the largest member of the tenascin family. Initially, a gene that overlaps its 3' genomic portion with the steroid 21-hydroxylase gene in the opposite direction was discovered in the class III region of the major histocompatibility complex (MHC) (Morel et al. 1989; Matsumoto et al. 1992a, 1992b; Bristow et al. 1993). TNX was also discovered independently as a new ECM glycoprotein localized on collagen fibrils and was named flexilin (Lethias et al. 1996). TNX is predominantly present in connective tissues of the heart and skeletal muscle as well as in the dermis of the skin (Matsumoto et al. 1994; Burch et al. 1995). In humans, TNX deficiency has been shown to cause a distinct, recessive form of Ehlers-Danlos syndrome (EDS), which is a genetically heterogeneous group of heritable connective-tissue disorders (Burch et al. 1997; Schalkwijk et al. 2001). TNX-deficient patients showed typical skin hyperextensibility, joint hypermobility and easy bruising. TNX-deficient (TNX–/–) mice generated by a gene-targeting strategy also showed progressive skin hyperextensibility, similar to that in patients with EDS. Biochemical analysis revealed that the density of collagen fibrils in the skin was reduced due to the decrease in collagen I deposition in the extracellular space (Mao et al. 2002). Mao et al. (2002) suggests that TNX is involved in the regulation of collagen synthesis and fibrillogenesis. Recently, Minamitani et al. (2004) described that the expression levels of type VI collagen as well as collagen fibril-associated molecules such as type XII and type VI collagens, decorin, lumican and fibromodulin are affected by TNX deficiency. We found that the TNX–/– mice showed significant increases in degree of tumour invasion and metastasis due to increased levels of activity of matrix metalloproteinases (MMPs), especially MMP-2 and MMP-9 (Matsumoto et al. 2001). Recently, we showed that induction of MMP-2 by TNX deficiency is mediated through the c-Jun N-terminal kinase and protein tyrosine kinase phosphorylation pathways (Matsumoto et al. 2004).
MMPs are involved in the degradation of many different components of the extracellular matrix (Stetler-Stevenson et al. 1993). Besides tumour invasion and metastasis, MMPs are thought to be involved in morphogenesis such as involution after lactation (Sympson et al. 1994) and also in branching of the lung (Ganser et al. 1991) and adipocyte differentiation (Alexander et al. 2001; Bouloumiéet al. 2001). Indeed, dramatic changes in ECM components have been shown to be necessary for adipose tissue remodeling (Zangani et al. 1999). Since the ECM is always in close association with cells throughout the process of differentiation, alterations in the types and levels of ECM components induced by MMPs would influence differentiation. Thus, we speculated that adipogenesis and/or lipogenesis are affected by increased MMP activities owing to TNX deficiency.
The aim of the present study was to determine whether TNX deficiency impairs the normal regulation of lipogenesis in the skin. In consequence, TNX deficiency showed increased expression levels of genes involved in lipogenesis, such as sterol response element-binding protein (SREBP), fatty acid synthase (FAS) and stearoyl CoA desturase, resulting in an increased amount of triglyceride and altered composition of fatty acids. These findings suggest that TNX plays a distinct role in lipogenesis.
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Results |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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We have proved that MMP activities are increased in the skin of TNX–/– mice. Augmentation of the expression of MMPs results in rapid ECM remodeling both in physiological and pathological situations (Shapiro 1998). To determine whether TNX deficiency caused the anatomical abnormality, skin from the backs of TNX–/– mice was examined in comparison with that of wild-type mice. TNX is abundant throughout the stroma of the epidermis and dermis as well as subcutaneous tissues and muscle in the skin (Fig. 1A). Histochemical studies revealed that the thickness of adipose tissue beneath the dermis in TNX–/– mice is clearly increased compared with that in wild-type mice (Fig. 1Ba and 1Bc). We selected 27 regions of adipose tissue lying just beneath the dermis from three male TNX–/– mice and three weight-matched male wild-type mice at one month of age and analysed the thicknesses of adipose tissues using NIH Image software (Fig. 1Ba,Bc,C). The mean thicknesses of adipose tissues of wild-type and TNX–/– mice were 52.4 ± 14.4 µm and 107.9 ± 22.3 µm (mean ± SE; n = 27 regions for each genotype of mice), respectively. The difference between the adipose tissue thickness in wild-type mice and that in TNX–/– mice was statistically significant (P < 0.01). Lipid accumulation in adipocytes was also examined by staining with Oil Red O. As shown in Fig. 1Bb and 1Bd, there was considerably more lipid accumulation in adipocytes from TNX–/– mice than in adipocytes from wild-type mice. These results indicate that there was an increase in lipid accumulation within adipocytes from TNX–/– mice.
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Next, the composition of increased lipids was determined by HPTLC analyses (Fig. 2A). Total lipids were extracted from subcutaneous adipose tissues and separated into simple lipids and compound lipids such as phospholipids with different solvent systems by HPTLC. As for simple lipids (Fig. 2A), a significant increase in the level of triglyceride was found in subcutaneous tissue from TNX–/– mice compared with that in subcutaneous tissue from wild-type mice. Measurement of each spot of triglyceride by using a spot scanner showed that subcutaneous tissue from TNX–/– mice contains twice the level of triglyceride compared to subcutaneous tissue from wild-type mice. On the other hand, there was no difference between the amounts or types of phospholipids in wild-type and TNX–/– mice (Fig. 2B). Glycolipids such as ganglioside were extracted from total lipids by two methods as described in Experimental procedures. HPTLC analysis of glycolipids showed that content of NeuAc(
2-3)Gal(ß1-4)Glcß1-cer (GM3) was slightly increased (1.6 fold) in subcutaneous tissue from TNX–/– mice compared with that in subcutaneous tissue from wild-type mice (Fig. 2C,D).
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An increase in the amount of triglyceride in the skin of TNX–/– mice was naturally accompanied by an increase in the amount of triglyceride-associated fatty acids. Thus, we examined the composition of fatty acids that constitute triglyceride in order to determine whether the composition of increased fatty acids in the skin of TNX–/– mice is the same as that in the skin of wild-type mice. Following the separation of triglyceride by HPTLC, the fatty acids in triglyceride were eluted from the plates and then determined by GC-MS analysis. Figure 3A,B shows representative chromatographs of eluates obtained from HPTLC. Next, the kind of fatty acid comprising each peak was identified by MS. Most of the fatty acids present in the subcutaneous tissue of the mouse skin was eluted within the retention time of 20 min. These major fatty acids included myristic acid (C14 : 0), palmitoic acid (C16 : 0), palmitoleic acid (C16 : 1), stearic acid (C18 : 0), oleic acid (C18 : 1), and linoleic acid (C18 : 2). Based on the percentage of the area of the peak containing each fatty acid with respect to total area of the six major fatty acids, the relative proportion of each fatty acid was calculated (Fig. 3C). Interestingly, the level of the saturated fatty acid palmitoic acid (C16 : 0) was significantly decreased and the levels of the unsaturated fatty acids palmitoleic acid (C16 : 1) and oleic acid (C18 : 1) were significantly increased in the skin of TNX–/– mice compared with the levels in the skin of wild-type mice (P < 0.05). The distribution patterns of other major fatty acids such as myristic acid (C14 : 0), stearic acid (C18 : 0) and linoleic acid (C18 : 2) in wild-type and TNX–/– mice were almost the same. These results indicated that not only accumulation of triglyceride but also alterations of the composition of triglyceride-associated fatty acids, particularly decrease in a saturated fatty acid and an increase in unsaturated fatty acids, occurred in the subcutaneous tissues of TNX–/– mice.
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To determine whether the increase in triglyceride, containing an altered composition of fatty acids, found in subcutaneous tissues from TNX–/– mice is regulated at the transcriptional level, we compared mRNA levels for various genes involved in lipogensis by semiquantitative RT-PCR analysis. The lipogenic enzymes include not only genes for fatty acid biosynthesis, such as fatty acid synthase, acetyl-CoA carboxylase and stearoyl-CoA desaturase, but also glycerol-3-phosphate acyltransferase for triglyceride synthesis and malic enzyme that provides the NADPH for reductive biosynthesis. Acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase are lipogenic enzymes that play a central role in the production of major long-chain monounsaturated fatty acids in mammals by conversion of acetyl-CoA to palmitoleic acid and oleic acid. Sterol regulatory element-binding protein-1 (SREBP-1) is an important transcription factor that regulates the syntheses of enzymes for fatty acid and triglyceride biosynthesis such as acetyl-CoA carboxylase, fatty acid synthase and stearoyl-CoA desaturase as well as glycerol-3-phosphate acyltransferase. GM3 synthase is a key enzyme for the synthesis of ganglioside GM3. As expected, the mRNA levels for fatty acid synthase, acetyl-CoA carboxylase, stearoyl-CoA desaturase, and glycerol-3-phosphate acyltransferase were markedly elevated in TNX–/– mice (Fig. 4). Interestingly, the mRNA level for SREBP-1, which controls the mRNA expressions for these lipogenic genes, was also significantly increased in TNX–/– mice. In contrast, TNX–/– mice showed only a slight increase in malic enzyme mRNA compared with that in wild-type mice and almost the same amount of GM3 synthase mRNA as that in wild-type mice. These results indicated that the absence of TNX causes significant increases in mRNA levels of lipogenic genes as well as the SREBP-1 gene in TNX–/– mice.
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To further confirm that TNX regulates the level of triglyceride and the composition of triglyceride-associated fatty acids, TNX was transiently over-expressed in primary TNX-null fibroblasts (Fig. 5A), and then the amount of triglyceride was investigated by HPTLC analyses (Fig. 5B). We speculated that over-expression of TNX would result in a reduction in the amount of triglyceride in the cells. At first we confirmed the increased level of triglyceride in TNX-null fibroblasts compared with that in wild-type fibroblasts as observed in the skin of TNX–/– mice (Fig. 5B). Over-expression of TNX by transfection of the pSecFTNX-2 vector in TNX-null fibroblasts resulted in a decrease in the triglyceride amount compared with that in parental TNX null-fibroblasts and in cells transfected with the empty pSecTag2/Hygro B vector (Fig. 5B). Likewise, we also examined the level of triglyceride in the cell lines that stably over-express TNX protein. In the stable cell lines, we expected the steady results irrespective of the transfection efficiency. Initially, we attempted to establish stable TNX-expressing cell lines in TNX-null fibroblasts. Unfortunately, we could not obtain the cell lines in TNX-null fibroblasts. Instead, we were able to establish two cell lines FTNX-12 and FTNX-2 that stably over-express TNX protein in wild-type fibroblasts. Since endogenous TNX protein at approximately 450 kDa could not be detected in the fibroblast cell lines (Fig. 5A, lanes 3–5), we speculated that the effect of endogenous TNX on triglyceride synthesis is lower than that of exogenous TNX in the TNX-over-expressed FTNX-12 and FTNX-2 cells. As a control, we used an empty vector-transfected cell line (FTNX-9). In Fig. 5A, a prominent exogenous TNX protein at approximately 350 kDa was detected in FTNX-12 and FTNX-2 cells. Over-expression of TNX resulted in a significant decrease in the amount of triglyceride in FTNX-12 and FTNX-2 cells compared with that in FTNX-9 cells (Fig. 5B). Actually, histological analysis with Oil Red O staining revealed that many cells with lipid droplets was observed in FTNX-9 cells compared to FTNX-12 cells (Fig. 5C). We also investigated the composition of triglyceride-associated fatty acids in FTNX-9 and FTNX-12 cells (Fig. 6A,B). The relative proportion of each fatty acid with respect to the total of the six major fatty acids in control FTNX-9 and TNX-over-expressed FTNX-12 cells was compared. In FTNX-12 cells, the distribution of the saturated fatty acid stearic acid (C18 : 0) was significantly increased and those of the unsaturated fatty acids palmitoleic acid (C16 : 1), oleic acid (C18 : 1), and linoleic acid (C18 : 2) were significantly decreased compared with those in FTNX-9 cells (P < 0.05) (Fig. 6C). These results suggested that over-expression of TNX in the cells leads to an increase in the composition of saturated fatty acid and decrease in the composition of unsaturated fatty acids.
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Discussion |
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We found that the thicknesses of subcutaneous adipose tissues from TNX–/– mice were greater than those from wild-type mice. Two possible reasons for this, i.e. adipocyte hypertrophy due to the increased lipid accumulation in adipocytes or adipocyte hyperplasia due to increased preadipocyte proliferation, were considered. Based on the results showing increased amounts of triglyceride in TNX–/– mice, it seems more likely that adipocyte hypertrophy contributes to the increased thickness of subcutaneous adipose tissues. Furthermore, the results of preliminary quantitative analysis of proliferating adipocytes detected by in vivo incorporation of bromodeoxyuridine (BrdU) showed that the labelling index of adipocytes of TNX–/– mice was almost the same as that of wild-type mice (data not shown), indicating that the increased thickness of adipose tissues observed in TNX–/– mice is due to adipocyte hypertrophy, not to hyperplasia. Thus increased thicknesses of adipose tissues in the skin from TNX–/– mice may be due to the larger adipocytes.
We previously demonstrated that TNX–/– mice have enhanced activities of MMPs, especially MMP-2 and MMP-9, due to the induction of expression of MMPs at the transcriptional level, resulting in the promotion of tumour invasion and metastasis (Matsumoto et al. 2001). It has been shown that treatment with MMP inhibitors of a preadipocyte cell line decreases adipocyte differentiation, indicating involvement of MMPs in adipose differentiation (Bouloumiéet al. 2001; Croissandeau et al. 2002). It has also been reported that the extracellular matrix is directly involved in the accumulation of triglyceride (Nakajima et al. 2002). Inhibition of the synthesis of collagen V and VI by a specific inhibitor impaired triglyceride accumulation in a dose-dependent manner. Therefore, it is possible that the increase in the activities of MMPs caused by TNX deficiency leads to the increase in the amount of triglyceride and to the alteration in composition of fatty acids. Actually, FTNX-12 cells in which TNX was over-expressed showed not only suppression of triglyceride synthesis followed by alteration in fatty acid composition but also a decrease in the level of MMP-2 activity (Matsumoto et al. 2004). Thus, the regulation of lipogenesis and the regulation of MMP activity by TNX might occur at the same time. It is necessary to determine whether triglyceride accumulation is suppressed when increased MMP activity is restrained by MMP inhibitors in TNX–/– mice. If triglyceride accumulation is not suppressed by the administration of MMP inhibitors, TNX would be involved in lipogenesis without the participation of MMP activity.
A deficiency of TNX has been reported to be associated with Ehlers-Danlos syndrome (Burch et al. 1997; Schalkwijk et al. 2001). Ehlers-Danlos syndrome is characterized by hyperextensibility of the skin, hypermobility of joints, and tissue fragility. Ultrastructural examinations of the skin in patients with Ehlers-Danlos syndrome have revealed abnormalities of collagen fibrils, indicating that the syndrome is a disorder of collagen fibril deposition. Besides TNX gene mutations, specific mutations in the genes encoding collagen types I, III, and V as well as collagen-modifying enzymes have been found in patients with this syndrome (Mao & Bristow 2001). Mao et al. (2002) reported that independently derived TNX–/– mice exhibit progressive skin hyperextensibility similar to that in patients with Ehlers-Danlos syndrome due to an alteration in the organization of collagen fibrils. The TNX–/– mice generated by us also show almost the same phenotype as progressive skin hyperextensibility and reduced tensile strength owing to an abnormality of collagen fibrils (our unpublished observations). Thus, the main reason for the skin hyperextensibility in TNX–/– mice would be alteration in the organization of collagen fibrils. However, the composition of fatty acids in the skin might also be involved in the phenotype such as the skin hyperextensibility in TNX–/– mice, because the melting points of saturated fatty acids are much higher than those of unsaturated fatty acids. For example, the melting points of the saturated fatty acids myristic acid (C14 : 0), palmitic acid (C16 : 0) and stearic acid (C18 : 0) are 53.9 °C, 63.1 °C and 69.6 °C, respectively. On the other hand, those of the unsaturated fatty acids palmitoleic acid (C16 : 1), oleic acid (C18 : 1) and linoleic acid (C18 : 2) are 0.5 °C, 11 °C, and –5 °C, respectively. As shown in Fig. 3C, in the skin of TNX–/– mice, the composition of saturated fatty acid palmitoic acid was decreased, whereas the compositions of unsaturated fatty acids palmitoleic acid and oleic acid were increased, implying that the fluidity of physical properties of the TNX-deficient skin is promoted more than that of wild-type skin. It would therefore be interesting to examine whether the fatty acid composition is altered in the skin of patients with Ehlers-Danlos syndrome caused by TNX deficiency.
At present, the molecular mechanism by which TNX deficiency leads to considerable induction of mRNAs of fatty acid synthetic genes such as fatty acid synthase at the transcriptional level is not clear. It is necessary to examine whether TNX suppresses the promoter activities of the fatty acid synthetic genes and further important to investigate a TNX-mediated intracellular signalling pathway that regulates the expression of fatty acid synthetic genes. In TNX–/– mice, the amount of ganglioside GM3 was slightly increased. However, the mRNA levels of GM3 synthase in wild-type and TNX–/– mice were almost the same. Thus, it is possible that the stability and/or translation efficiency of GM3 synthase mRNA was increased by TNX deficiency. It is interesting that GM3 is able to regulate the adhesion and migration of cells through the modulation of MMP-9 activities (Wang et al. 2003). Wang et al. (2003) demonstrated that GM3 over-expression resulted in decreases in the expression and activity levels of MMP-9. Thus, the increased level of GM3 might be related, at least in part, to the increased activity levels of MMPs detected in TNX–/– mice. Furthermore, gangliosides are known to act as modulators of transmembrane signalling by regulating various receptor functions. As for insulin signalling, Tagami et al. (2002) reported that insulin resistance induced by tumour necrosis factor- (TNF-) is mediated by an increased level of GM3 followed by suppression of tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and glucose uptake. Exogenous GM3 itself mimicked the effects of TNF - on insulin signalling. In TNX–/– mice the mRNA level of TNF- might also increase, leading to the insulin resistance. It is known that insulin resistance often accompanies obesity (Hotamisligil et al. 1993). Accordingly, an increase in the level of GM3 might result in an increase in the amount of triglyceride by inhibiting the insulin signalling pathway.
In conclusion, we have presented evidence for the first time that TNX regulates triglyceride synthesis and the composition of triglyceride-associated fatty acids. Since over-expression of TNX in cells resulted in a decrease in triglyceride synthesis, it is possible that TNX represents a new, interesting therapeutical target for the inhibition of triglyceride synthesis leading to obesity.
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Experimental procedures |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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The generation of TNX–/– mice by homologous recombination using embryonic stem (ES) cells has been previously described (Matsumoto et al. 2001). The genetic background of wild-type (TNX+/+) and TNX–/– mice is a mixture of C57BL/6, CBA and ICR strains. The mice were housed in laminar flow cabinets under specific pathogen-free conditions. All animal experiments were carried out in accordance with the NIH Guide for Care and Use of Laboratory Animals and approved by the Animal Care and Use Committee of Hokkaido University.
Cells and transfection
Wild-type or TNX–/– mice were dissected on postnatal day 1 with scissors in phosphate-buffered saline (PBS)/2.5% trypsin (Becton Dickinson, Tokyo, Japan) supplemented with penicillin (200 units/mL) and streptomycin (200 µg/mL). A clump of cells was stirred for 30 min at 37 °C to disperse the cells. The cells were then centrifuged, collected, and cultured in Dulbecco's modified Eagle's medium (DMEM) (Nissui, Tokyo, Japan) supplemented with 10% foetal bovine serum (FBS) (JRH Biosciences, USA). During the two or three subcultures of these cells, most of the cells that attached to the dish were fibroblasts.
TNX-null fibroblasts were trypsinized and resuspended at a concentration of 4 x 107/mL in HBS buffer (25 mM HEPES, pH 7.05, 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 6 mM glucose). The cell suspension was electroporated with 50 µg of a plasmid DNA (pSecFTNX-2) encoding mouse TNX in a cuvette using a Bio-Rad (Tokyo, Japan) Gene Pulser 250 V, 960 µF. pSecFTNX-2 encodes a short alternatively spliced form lacking M3 and M15-M22 FNIII repeats (Ikuta et al. 1998, 2000). The estimated molecular size is approximately 350 kDa. As a negative control, empty vector pSecTag2/Hygro B was transfected into the TNX-null fibroblasts as well. The electroporated cells were allowed to stand for 10 min, plated at 5 x 106 cells per 10-cm dish, and then cultured. Twelve hours later, the cells were recultured in fresh DMEM/10% FBS for further 48 h. Then cells were collected.
To obtain immortal fibroblasts, subculture of the fibroblasts was repeated 90 times. To generate stably transfected fibroblasts with high levels of expression of TNX, wild-type fibroblasts were transfected by the calcium phosphate co-precipitation method (Minamitani et al. 2000) in the presence of 10 µg of pSecFTNX-2 plasmid, selected in 400 µg/mL hygromycin-B (Wako, Osaka, Japan), and cloned by limiting dilution. Two clones, FTNX-12 and FTNX-2, in which TNX is highly expressed was obtained. Empty vector (pSecTag2/Hygro B)-transfected cell, FTNX-9 cell, was used as controls.
Immunohistochemistry
Freshly obtained caudal dorsal skins from three male TNX–/– mice and three weight-matched wild-type mice at 2 months of age were embedded in Tissue-Tek OCT compound (Sakura Finetechnical, Tokyo, Japan) and frozen in liquid nitrogen. Tissue sections of 8 µm in thickness were cut on a cryostat and mounted on slides. The slides were processed for immunohistochemical staining as previously described (Matsumoto et al. 2002a). Briefly, the sections were fixed in 4% paraformaldehyde, washed in PBS, and incubated with rabbit polyclonal anti-mouse TNX antibodies (pAbM18). After extensive washing with PBS, the slides were treated with a peroxidase polymer-conjugated secondary antibody, Envision (DAKO, Carpinteria, USA) and counterstained with haematoxylin.
Oil Red O staining
To determine lipid deposition, freshly obtained skins of caudal dorsal regions were embedded in Tissue-Tek OCT compound and frozen in liquid nitrogen. Tissue sections of 8 µm in thickness were cut using a cryostat. The sections were fixed in 10% formalin for 1 h, rinsed in distilled water, and stained with 0.5% Oil Red O solution (Chroma Gesellschaft Schmidt, Germany) for 15 min at room temperature. Sections were rinsed with 60% isopropanol and counterstained with haematoxylin for 1 min. All tissues were observed and photographed using an Axiophot 2 microscope. Serial sections were stained with haematoxylin and eosin. Quantification of the thicknesses of adipose tissues was then performed using NIH Image software. The Mann-Whitney U-test was used for statistical analysis. The staining of FTNX-9 and FTNX-12 cells with 0.29% Oil Red O solution was performed for 30 min according to the same procedure as described for the mouse skin specimens.
Lipid analysis
Skin was removed from the back of each 2-month-old weight-matched male wild-type and TNX–/– mouse, and the hypodermis was scratched off and was massed (approximately 200 mg). For transient TNX-over-expressed cells, the cell lines FTNX-9 and FTNX-12, more than 5 x 107 cells were harvested. Total lipids were extracted from the cells with chloroform/methanol (1 : 1 and 1 : 2), successively. Following final extraction, residual precipitates that include proteins was dissolved in 0.5 M NaOH solution, and then protein concentration was determined using a BCA assay kit (Pierce, IL, USA). For separation of simple lipids such as triglycerides, the total lipids were evaporated to dryness, and the lipids with equal amounts adjusted by protein amounts (approximately 0.25 mg protein) were separated by silica gel high-performance thin-layer chromatography (HPTLC) (Merck, Darmstadt, Germany) with hexane/diethyl ether/acetic acid (80 : 30 : 1). For phospholipid analysis, the lipids were separated with chloroform/methanol/aqueous 12 mM MgCl2 (65 : 25 : 4). The lipids were visualized by heating with cupric phosphoric acid. For HPTLC analysis, lipid extracts of equal amounts were loaded on plates. The quantity of lipid in each band was measured with a dual-wavelength flying spot scanner (CS9300-PC, Shimadzu, Kyoto, Japan) in the reflectance mode at 500 nm with integrated areas. For glycolipid analysis, total lipids were applied to a DEAE-Sephadex A-25 column (Amersham Biosciences, Tokyo, Japan) (acetate form, 2.4-mL bed volume). Neutral lipids were eluted with five volumes of chloroform/methanol/water (30 : 60 : 8). Acidic lipids such as gangliosides were eluted with five volumes of chloroform/methanol/aqueous 1 M sodium acetate (30 : 60 : 8). The neutral and acidic lipid fractions were evaporated to dryness, and contaminating esters were methanolysed with methanolic 0.1 M NaOH for 2 h at 40 °C. The solution was neutralized with 1 M HCl in methanol, diluted with an equal volume of 50 mM aqueous NaCl, and applied to a Sep–Pak C18 reverse-phase cartridge (Water Associates, MA, USA). The cartridge was washed with water, and then lipids were eluted with methanol and chloroform/methanol (1 : 2), successively. The eluate was evaporated to dryness, and the lipids were analysed by HPTLC. The plates were then developed with chloroform/methanol/aqueous 12 mM MgCl2 (65 : 25 : 4) for neutral lipids or with chloroform/methanol/aqueous 0.9% CaCl2 (50 : 50 : 9) for acidic lipids. Glycolipids were visualized by spraying orcinol-sulphuric acid. For acidic glycolipids such as gangliosides, another extraction was also done using a previously described method (Ladisch & Gillard 1985). Briefly, total lipids were dispersed in an appropriate volume of a mixture of diisopropyl ether (DIPE) and butanol (3 : 2) by vortexing and sonication. Fifty mM of aqueous NaCl, one-half the volume of the organic solvent mixture, was added, and the sample was again mixed by vortexing and sonication for several minutes. After centrifugation of the sample, the ganglioside-containing lower aqueous phase was collected, diluted with a 1.67-fold volume of 50 mM aqueous NaCl, and applied to a Sep–Pak C18 reverse-phase cartridge. The gangliosides were eluted as described above. Purified standard lipids used for HPTLC analysis were purchased from Matreya Inc. (Pleasant Cap, PA, USA).
Fatty acid composition
For analysis of triglyceride-associated fatty acids, the plates were soaked with isopropanol/aqueous 0.2% CaCl2/methanol (40 : 20 : 7) for 20 s after TLC. Then the triglyceride samples on the plates were transferred to PVDF membranes (Millipore, MA, USA) at 180 °C for 50 s. The samples were extracted with chloroform/methanol (1 : 1) from the PVDF membranes, and the extracts were dried with N2 at room temperature. Then 5% hydrogen chloride methanol solution (Wako) was added to the dry residue, and transesterification was performed at 95 °C for 2 h. Following the transesterification step, H2O was added and the fatty acid methyl esters were extracted with hexane (Merck). The hexane extracts were dried under a stream of N2 at room temperature, redissolved in 10-µL hexane, and subjected to gas chromatography-mass spectrometry (GC-MS). The conditions of the GC-MS analysis of fatty acids were as follows. The GC-MS analysis was carried out on a HP 6890 series gas chromatogragh (Hewlett-Packard Co., Palo Alto, CA, USA) equipped with a 30-m DB-23 (i.d. = 0.25 mm, film thickness of 0.25 µm) (J & W Scientific, Folsom, CA, USA) and a mass spectrometer JMS-700TZ (JEOL Ltd, Tokyo, Japan). The sample injection volume was 0.6 µL with a split ratio (1 : 10). The GC oven temperature was set at 40 °C for the first 1 min. The temperature of the column was increased by 30 °C/min up to 130 °C, by 6 °C/min up to 170 °C, and then by 3 °C/min up to 215 °C, and then the column was then kept at that temperature for 10 min. The temperature of the column was then increased by 1 °C/min up to 230 °C. The ionization temperature, energy and current were 230 °C, 70 eV and 300 µA, respectively. Ultrahigh-purity-grade helium was used as a carrier gas at a flow rate of 1.5 mL/min. The temperatures of the injection port, interface and ion source were 250 °C, 250 °C and 230 °C, respectively. Electron ionization was used. The ionization energy and current were 70 eV and 300 µA, respectively. Spectra were recorded from 40 to 500 m/z.
RNA purification and semiquantitative reverse transcription-PCR (RT-PCR)
Dorsal skin from adult (2-months-old) wild-type and TNX–/– mice was dissected, frozen, and powdered in liquid nitrogen. Total RNA was isolated, and poly(A)-rich RNA was selected using an mRNA purification kit (Amersham Biosciences, Tokyo, Japan). RT-PCR analysis was performed to identify the mRNAs from the genes that participate in lipogenesis. RT-PCR conditions were the same as those reported previously (Matsumoto et al. 2002b). To obtain the following quantitative information on each mRNA expression, PCR conditions that showed linear kinetics of amplification were used. Then the optimum condition was used as follows. Initial denaturation at 94 °C for 1 min was followed by 25 PCR cycles at 98 °C for 20 s and at 68 °C for 5 min. After a final extension at 72 °C for 10 min, PCR products were separated on 2% agarose gel. The following PCR primers were used for detection of fatty acid synthase, acetyl-CoA carboxylase, stearoyl-CoA desaturase, glycerol-3-phosphate acyltransferase, malic enzyme, SREBP-1, and GM3 synthase mRNAs. For the detection of mouse fatty acid synthase mRNA (GENBANK accession number: KM_126624), forward fmFatty primer 5'-ATGAGGCTGTGAAGCCGTTGGGAGTGA-3' and reverse rmFatty primer 5'-TTCGTACCTCCTTGGCAAACACACCTT-3' were used. A 459-bp band was expected. For the detection of mouse acetyl-CoA carboxylase (DDBJ accession number: BC022940), forward fmAcetyl primer 5'-CGAGCAGCCCATTCTCATCTATATC-3' and reverse rmAcetyl primer 5'-ACTGTGTGTGCTCGTGGTTCAGCTC-3' were used. A 515-bp band was expected. For the detection of mouse fatty acid synthase mRNA (GENBANK accession number: KM_126624), forward fmFatty primer 5'-ATGAGGCTGTGAAGCCGTTGGGAGTGA-3' and reverse rmFatty primer 5'-TTCGTACCTCCTTGGCAAACACACCTT-3' were used. A 459-bp band was expected. For the detection of mouse stearoyl-CoA desaturase mRNA (DDBJ accession no. BC040384), forward fmStearo primer 5'-TGAGGCTCTTCCTCATCATTGCCAAC-3' and reverse rmStearo primer 5'-AGGGGAAGGCGTGGTGGTAGTTGTGG-3' were used. A 516-bp band was expected. For the detection of mouse glycerol-3-phosphate acyltransferase mRNA (DDBJ accession no. M77003), forward fmGlycerol primer 5'-ACCTAGGCTTCTCCGGGAATTCAGA-3' and reverse rmGlycerol primer 5'-AGATAGCAATCTCGCTGCTCCTCACC-3' were used. A 550-bp band was expected. For the detection of mouse malic enzyme mRNA (GENBANK accession no. XM_122579 [GenBank] ), forward fmMalic primer 5'-CTGCCTTCAACGAGCGGCCCATCAT-3' and reverse rmMalic primer 5'-ACTTCTGCAGGCCACGGATAACAAT-3' were used. A 505-bp band was expected. For the detection of mouse SREBP-1 mRNA (DDBJ accession no. BC006051), forward fmSREBP primer 5'-TGGTGGGCCTCACTGACAGCTGTGGT-3' and reverse rmSREBP primer 5'-CAGCCGAGCTGTGGCCTCATGTAGG-3' were used. A 510-bp band was expected. As a control, ß-actin mRNA was used. For the detection of mouse ß-actin mRNA (DDBJ accession no. X03765), forward fmAct primer 5'-TACCACGGGCATTGTGATGG-3' and reverse rmAct primer 5'-GATCTTGATCTTCATGGTGC-3' were used. A 546-bp band was expected.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: kematsum{at}pharm.hokudai.ac.jp
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Received: 12 January 2004
Accepted: 10 May 2004
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