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Cell Growth & Differentiation Vol. 12, 497-504, October 2001
© 2001 American Association for Cancer Research

Characterization of Syndecan-4 Expression in 3T3-F442A Mouse Adipocytes

Link between Syndecan-4 Induction and Cell Proliferation1

Reiko Landry2, Vincent Rioux and André Bensadoun3

Division of Nutritional Sciences, Cornell University, Ithaca, New York 14853


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Heparan sulfate proteoglycans are found on the surface of most cells. Syndecan-4 is a widely expressed transmembrane heparan sulfate proteoglycan. Using quantitative RNase protection assays and immunoblotting, syndecan-4 expression was characterized in 3T3-F442A mouse adipoblasts. These cells exhibit dramatic changes in their biological and morphological characteristics during differentiation to adipocytes. During this process, the levels of syndecan-4 protein and mRNA expression changed dramatically. They peaked at the time when quiescent cells reentered the cell cycle before differentiation. Serum depletion-repletion also replicated the syndecan-4 mRNA induction when the cells were released back into proliferation, and a cycloheximide treatment abolished the peak of induction. In addition, inhibiting syndecan-4 induction with antisense oligonucleotides inhibited the proliferation of 3T3-F442A cells. In the terminally differentiated adipocytes characterized by the loss of proliferation capability, the serum inducibility of syndecan-4 is repressed, emphasizing the link between syndecan-4 induction in 3T3-F442A cells and cell proliferation.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Syndecan-4 is a member of the syndecan family of transmembrane-type HSPGs.4 The syndecan family consists of four HSPGs, syndecan-1, -2, -3, and -4 (1) . Their structure is characterized by hybrid glycosylation with HS or chondroitin sulfate chains (2, 3, 4) , conserved transmembrane and cytoplasmic domains, and a diverse extracellular domain. The length of glycosaminoglycan chains and the degree of sulfation are not uniform, resulting in considerable heterogeneity in molecular weight and charge (5) . Because of both the polyanionic nature of HS chains and the binding potential of the core proteins, syndecans bind many biological molecules, thereby affecting a spectrum of biological activities. These activities include modulation of growth factor activities (6, 7, 8, 9) , anticoagulation , 11, 12, 13, 14) , cell adhesion (15 , 16) , extracellular chaperone activity (17) , and lipoprotein metabolism (18 , 19) . Although they share common structural characteristics, the spatial and temporal expression pattern of each syndecan is markedly different, suggesting specialization of function. Syndecan-4 is the most widely expressed syndecan, although it is expressed in relatively low amounts in quiescent cells. The types of cells that express syndecan-4 include fibroblasts, adipocytes, vascular smooth muscle and endothelial cells (20 , 21) , and lung, liver, and kidney cells (22) . Syndecan-4 is also expressed at the sites of focal adhesion (23) and cutaneous wound repair (24 , 25) . Such wide distribution may indicate the role of syndecan-4 in some basic cellular function common to many cell types. Syndecan-4 is expressed in adipocytes. In this study, we examined the profile of syndecan-4 expression in 3T3-F442A mouse preadipocytes that undergo differentiation into mature adipocytes.

3T3-F442A is a preadipocyte cell line whose adipocyte lineage is committed (26, 27, 28) . 3T3-F442A cells are morphologically similar to the fibroblastic preadipocytes in the stroma of adipose tissue. Preadipocytes go through specific stages of differentiation over the course of 5–14 days to become mature adipocytes: (a) cell-cell contact and growth inhibition; (b) induction; (c) clonal expansion (cell cycle resumption); (d) exit from cell cycle; (e) early differentiation; and (f) late differentiation (29, 30, 31) . Through this differentiation process, they lose their elongated fibroblast-like appearance and acquire the phenotype of mature adipocytes characterized by the presence of triglyceride oil droplet inclusions.

Soon after reaching confluence, cell division of 3T3-F442A preadipocytes ceases. On induction with fetal bovine serum and a high concentration of insulin, cells proceed to the mitotic clonal expansion stage, in which proliferation-arrested cells reenter the cell cycle. After at least one round of cell division, the cells exit the cycle permanently. Adipocyte differentiation is a typical example of terminal differentiation characterized by the loss of proliferation capability (32) .

After hormonal induction, 3T3-F442A preadipocytes dramatically change their biological and morphological properties. We investigated how the levels of syndecan-4 expression change over the course of differentiation in this cell line to correlate syndecan-4 expression with previously defined events of adipocyte differentiation. With the use of antisense oligonucleotides, we tested the hypothesis that syndecan-4 expression is required for cell proliferation.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The Level of Syndecan-4 mRNA in 3T3-F442A Cells Temporarily Surges during Early Adipocyte Differentiation.
Syndecan-4 is expressed in adipose tissue (20) as well as in a number of other tissues. To investigate the possible role of syndecan-4 in the development of adipose tissue, we used the highly adipogenic adipoblast cell line 3T3-F442A derived from 17–19-day-old mouse embryos (28) . First, the mRNA levels of syndecan-4 were measured during the course of differentiation. The day that the differentiating medium was added to cells is designated day 0.

Fig. 1Citation shows that during the differentiation of adipoblast 3T3-F442A cells, syndecan-4 mRNA expression peaked during the initial 24 h after the confluent cells were switched to the differentiation medium. The induction of syndecan-4 mRNA peaked within 8 h after the cells were exposed to the differentiation medium, and it decreased to the basal level after 32 h. This peak period corresponds to the transitional phase of quiescent cells in G0 reentering the cell cycle (G1) due to clonal expansion. The level of syndecan-4 protein expression paralleled that of mRNA as determined by immunoblotting (Fig. 2)Citation . The level of syndecan-4 protein decreases as pre-adipocytes become confluent and stop dividing (Fig. 2ACitation , Lanes 1–3). Twelve h after the addition of the differentiation medium (Fig. 2ACitation , Lane 4), the level of syndecan-4 protein is at its highest concentration. It then decreases to a baseline level by day 2 of differentiation.



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Fig. 1. Syndecan-4 mRNA expression during adipocyte differentiation. A, 3T3-F442A cultures were harvested at each time indicated, and the extracted total RNA was subjected to RNase protection assay to measure syndecan-4 mRNA. Each sample was analyzed in duplicate. Each point represents the mean from the two samples, and the range of values is indicated. The profile was confirmed in independent experiments. B, 10 µg of total RNA from the same samples at each time point were loaded on a RNA gel, transferred to membrane, and probed with a 32P mouse acidic ribosomal phosphoprotein PO.

 


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Fig. 2. Syndecan-4 protein expression during adipocyte differentiation. Dishes of 3T3-F442A cells were harvested at different times during differentiation to adipocyte. Day 0 is the day when differentiation was initiated by the protocol described in "Materials and Methods." Cell lysate proteins (100 µg) digested with chondroitinase ABC and heparin sulfate lyase were loaded in each lane. The proteins were separated by SDS-PAGE, transferred to membrane, and visualized by Western blotting as described in "Materials and Methods." Lanes 1–11 correspond to cells harvested at -4, -2, 0, 0.5, 1, 2, 4, 6, 8, 10, and 12 days after the addition of the differentiating medium.

 
Syndecan-4 Is Induced at G0-G1-phase Transition.
One plausible explanation for the prominent increase in syndecan-4 expression during the clonal expansion is that it is highly induced during the G0-G1-phase transition because the peak corresponds temporally to the G0-G1 transition phase of clonal expansion. At this point, growth-arrested cells resume the cell cycle on hormonal induction. Syndecan-4 induction could be associated with the cell cycle resumption. To see whether this is the case, we synchronized cells in G0 by serum deprivation under normal growth conditions and conducted serum induction experiments to investigate whether we could reproduce the syndecan-4 mRNA induction when the cells were refed serum-containing medium. Serum deprivation experiments using nondifferentiated preadipocytes were performed in the presence and absence of cycloheximide on 50% subconfluent cultures by depriving cells of serum for 24 h. The treatment with 10 µM cycloheximide was started 12 h before serum induction because the complete inhibition of protein synthesis by cycloheximide requires several hours. Within 4 h of re-exposure to serum and, presumably, release from G0, the cells did indeed exhibit high levels of syndecan-4 mRNA (Fig. 3)Citation . Cycloheximide treatment attenuated syndecan-4 induction, suggesting that the induction of syndecan-4 mRNA requires prior synthesis of some proteins. The induction of syndecan-4 was rapid, as evidenced by the fact that the increase started as early as 1 h after serum addition. These results suggest that syndecan-4 is induced during the G0-G1-phase transition in 3T3-F442A cells.



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Fig. 3. Serum induction of syndecan-4 mRNA. 3T3-F442A cells (50% confluent) were deprived of serum for 24 h. A group of cells was treated with 10 µM cycloheximide (CHX). The treatment with cycloheximide was started 12 h before serum induction. Cells were harvested at each time point indicated, and the extracted total RNA was subjected to RNase protection assay. Each point represents the mean from two distinct samples, and the range of values is indicated.

 
Syndecan-4 Antisense Transfection Prevents Cell Proliferation.
To investigate whether syndecan-4 expression was necessary for 3T3-F442A proliferation, we inhibited its expression by an antisense approach. 3T3-F442A cells at about 80% confluence were transfected with 0 nM, 200 nM, 500 nM, and 1 µM syndecan-4 antisense oligonucleotides by a liposome-mediated method (Lipofectin; Life Technologies, Inc.) in serum-free conditions. As a control, we used an oligonucleotide with a similar nt composition, but in a randomized sequence. After 6 h of transfection, the liposome-oligonucleotide complex was removed, and fresh growth medium was added. The cells were harvested at 4 h after serum addition, which, according to the previous experiments, should be the time of highest induction of the syndecan-4 mRNA. Fig. 4Citation shows that the attenuation of syndecan-4 mRNA expression by antisense treatment was dose dependent. At 1 µM, the antisense attenuation of syndecan-4 mRNA is almost complete. Therefore, this concentration was used in the next experiment to verify the effect of antisense treatment on syndecan-4 protein expression (Fig. 4B)Citation .



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Fig. 4. Dose-dependent inhibition of syndecan-4 expression on antisense oligonucleotide transfection. A, 3T3-F442A cells were transfected with various concentrations of syndecan-4 antisense oligonucleotide by using a liposome-mediated method. As a control, an oligonucleotide with a similar nt composition but in a randomized sequence was used. After 6 h of transfection, fresh growth medium was added. After 4 h, the cells were harvested, and the extracted total RNA was subjected to RNase protection assay. Values represent the means from pools of two dishes. B, Western blot for syndecan-4 protein. The sequence of media changes is the same as that described in A. Lane 1, control cells that were not exposed to oligonucleotides. Lane 2, cells exposed to 1 µM antisense oligonucleotides. Lane 3, cells exposed to 1 µM control randomized sequence.

 
Cell proliferation was assessed and compared with the cells transfected with the control oligonucleotides. Cell proliferation was assessed by two methods: (a) the BrdUrd incorporation assay (Amersham); and (b) the tetrazolium bioreduction method (Promega). The cells were transfected with 1 µM antisense or control oligonucleotides in serum-free conditions in a 96-well microtiter plate. After 6 h, the liposome-oligonucleotide complex was removed, fresh growth medium with 10 µM BrdUrd was added, and cells were incubated at 37°C in 10% CO2 for 24 h. Subsequently, the BrdUrd incorporation by the cells during the next 24 h was measured by ELISA with anti-BrdUrd antibody (Cell Proliferation ELISA system; Amersham). Fig. 5Citation shows that BrdUrd incorporation was reduced approximately 70% in the cells treated with syndecan-4 antisense oligonucleotides as compared with the control cells.



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Fig. 5. Effect of inhibition of syndecan-4 expression on cell proliferation. 3T3-F442A cells were transfected with 1 µM antisense or control oligonucleotides as described in the text. After 6 h, 10 µM BrdUrd was added, and after 24 h, BrdUrd incorporation was measured by ELISA with anti-BrdUrd antibody (Cell Proliferation ELISA system; Amersham). Values are the means± SD (n = 15). A representative assay from three separate trials is shown. ***, significant differences with P < 0.001.

 
The increase in cell number was also monitored as a measure of cell division after the antisense oligonucleotide treatment. This was accomplished by monitoring the number of viable cells represented by the amount of formazan produced by bioreduction of the tetrazolium compound. The transfection with antisense or control oligonucleotides was performed as described above. The liposome-oligonucleotide complex was removed, and cells were cultured with the tetrazolium solution (CellTiter 96 Aqueous One Solution Cell Assay System; Promega) for 3 h at 0, 24, and 48 h after transfection. The cell number of both groups right after oligonucleotide treatment did not differ significantly (0 h in Fig. 6Citation ). However, at 24 h, whereas the control cells showed an increase in cell number, the number of antisense-treated cells remained constant. At 48 h, the difference in total cell number in the two groups remains, although by this time point, the antisense-treated cells have also resumed cell division, suggesting that the antisense oligonucleotides had been depleted. These results show that proliferation is inhibited by syndecan-4 antisense treatment and that the effect is maintained for at least 24 h.



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Fig. 6. Effect of inhibition of syndecan-4 expression on cell number. Transfection with antisense or control oligonucleotides was performed in a 96-well microtiter plate as described in the text. The number of living cells was determined by a tetrazolium bioreduction assay. The liposome-oligonucleotide complex was removed, and cells were cultured with the tetrazolium solution (CellTiter 96 Aqueous One Solution Cell Assay System; Promega) for 3 h at 0, 24, and 48 h after transfection. Values are the means± SD (n = 20).

 
Serum Inducibility of Syndecan-4 Is Repressed in Terminally Differentiated 3T3-F442A Adipocytes.
Adipocyte differentiation is a typical example of terminal differentiation characterized by the loss of proliferation potential. If, as the previous data suggest, the syndecan-4 induction is truly associated with the cells’ transition from G0 to G1, then such an induction may be repressed in mature differentiated adipocytes because these cells lack proliferation capability. To investigate this hypothesis, the serum inducibility of syndecan-4 in mature differentiated 3T3-F442A adipocytes was investigated. Serum deprivation using mature adipocytes was performed on differentiated adipocyte cultures on day 15 by depriving cells of serum for 3 h. On day 15, >90% of the cells were differentiated. Control cells were day 1 preadipocytes after hormonal induction. The control cells were maintained in culture for the same length of time as the differentiated cultures. Both groups of cells were deprived of serum for 3 h, and dishes of cells were harvested for syndecan-4 mRNA measurements over 24 h after serum refeeding. Fig. 7Citation shows that the serum induction of syndecan-4 mRNA was indeed severely repressed in the terminally differentiated 3T3-F442A adipocytes, whereas the control preadipocytes demonstrated induction of syndecan-4 mRNA that peaked at 4 h after serum repletion. Syndecan-4 protein expression paralleled the mRNA induction (Fig. 8)Citation . The control preadipocytes showed rapid induction of syndecan-4 protein that reached a maximum at 8 h. On the other hand, such an induction of syndecan-4 protein was not observed with mature adipocytes.



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Fig. 7. Serum induction of syndecan-4 mRNA in preadipocytes and terminally differentiated adipocytes. The serum induction experiment was conducted as described in the text. Cells were harvested at the time points indicated, and the extracted total RNA was subjected to RNase protection assay. Each sample was analyzed in duplicate. Each point represents the mean from two samples, and the range of the values is indicated.

 


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Fig. 8. Serum induction of syndecan-4 protein in day 1 adipocytes and terminally differentiated adipocytes. Serum induction experiments were conducted in day 1 adipocytes (Lanes 1–5) and terminally differentiated adipocytes (Lanes 6–10). Cells were deprived of serum for 3 h and then refed serum-containing medium. Cells were harvested at 0 (Lanes 1 and 6), 1 (Lanes 2 and 7), 4 (Lanes 3 and 8), 8 (Lanes 4 and 9), and 24 h (Lanes 5 and 10), and each cell extract sample containing 100 µg of total protein was digested with 5 milliunits of HT and 80 milliunits of chondroitinase ABC. Time 0 is the time of serum refeeding. The proteins were separated by SDS-PAGE, transferred to membrane, and visualized by Western blotting as described in "Materials and Methods."

 
Ectopic Expression of Syndecan-4 in 3T3-F442A Cells.
Because high syndecan-4 expression coincides with the times when cell division of undifferentiated 3T3-F442A cells occurs, it was of interest to investigate whether constitutive expression of syndecan-4 would extend the clonal expansion and inhibit adipocyte differentiation. Cells stably transfected with pcDNA3-Syn4 expressed approximately 30% more immunoreactive syndecan-4 than control cells stably transfected with the control vector pcDNA3. The time course of triglyceride accumulation in both cell lines was not significantly different. At 2, 3, 5, and 8 days, the triglyceride (µg triglyceride/µg DNA) levels were 4.1, 4.0, 6.3, and 29.9 in adipocytes expressing syndecan-4 constitutively and 2.8, 2.4, 4.9, and 24.3 in control cells, respectively.


    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
3T3-F442A adipoblasts exhibit dramatic changes in their biological and morphological characteristics during adipocyte differentiation. During this process, we noticed that syndecan-4 was highly induced during the initial 24 h after confluent cells were switched to differentiation medium (Fig. 1)Citation . The peak occurred in quiescent cells reentering the cell cycle due to clonal expansion. Consistent with this notion, syndecan-4 was highly induced by serum depletion-repletion. Cycloheximide abolished the syndecan-4 inducibility, indicating that the induction of syndecan-4 mRNA requires prior synthesis of some proteins. The high concentration of syndecan-4 expression observed at the wound edge (24 , 25) may also be explained as a phenomenon that occurs with the sudden burst of proliferation of cells that have been quiescent. Fig. 2Citation shows a significant expression of syndecan-4 before the addition of differentiation medium on days -4 and -2. This may be due to the fact that some cells start differentiating spontaneously or because syndecan-4 is expressed in cycling cells, not just in cells entering the cell cycle from the G0 state.

Our attempt to create a cell line of 3T3-F442A that constitutively produces a high level of antisense syndecan-4 mRNA to abrogate the syndecan-4 induction failed. In retrospect, this may be due to the selective disadvantage of those cells during cell division, and it is consistent with a role for syndecan-4 induction in proliferation. We therefore used synthetic antisense oligonucleotides to decrease syndecan-4 expression. Inhibition of syndecan-4 with antisense treatment prevented proliferation of 3T3-F442A cells. In fact, HSPGs have been shown to be involved in proliferation as well as angiogenesis, differentiation, and morphogenesis (1 , 33 , 34) . A variety of growth factors including FGFs (6) , vascular endothelial growth factor (7 , 8) , and heparin-binding EGF (9) have been shown to bind to heparin-like molecules. The binding of each of these growth factors to its cognate receptor has been shown to require the presence of HS chains. Therefore, the involvement of HSPGs in these biological activities may be largely through growth factor-growth factor receptor signaling. Such biological properties of growth factors can be abolished by HT digestion, demonstrating the importance of the HS chains (6, 7, 8, 9) . Richardson et al. (35) showed that bFGF-dependent migration and proliferation in primary corneal stromal fibroblasts are regulated mainly by the changes in HSPG expression and not by changes in the bFGF receptor number. In particular, the change in syndecan-4 expression correlated well with the bFGF binding and activity of the cells, which result in cell migration and proliferation. Syndecan-4 was also highly expressed in all liver cancer cases examined (36) .

Currently, we do not know whether syndecan-4-dependent proliferation is conferred by the glycosaminoglycan chains or the core protein. In vitro experiments show that all of the HSPG subspecies examined (syndecan-1, -2, and -4 and glypican-1) can promote FGF-FGF receptor interactions and signaling when expressed in K562 hematopoietic cells (37) . On the other hand, recent advances in research suggest that the core protein also plays important roles in signal transduction (38, 39, 40, 41) . In addition, syndecan-4-specific actions such as protein kinase C activation (23 , 42) may be indispensable for the syndecan-4-dependent proliferation. To our knowledge, such an acute pattern of expression has not been reported for any other HSPG (21 , 43) and suggests that the acute expression of syndecan-4 may act as a critical switch in preadipoblast differentiation. As documented by Asundi and Carey (44) , syndecans self-associate to form multimers. This association is concentration dependent, and more association (and hence a higher degree of multimerization) occurs as concentration increases. Importantly, with syndecan-4, self-association of cytoplasmic tails was shown to be necessary for protein kinase C potentiation (23 , 42) . The induced high level of syndecan-4 we observed may be necessary for effective signaling.

In the terminally differentiated adipocytes, the serum inducibility of syndecan-4 is severely repressed (Figs. 7Citation and 8Citation ). Adipocytes are characterized by the permanent cessation of proliferation. This observation suggests the existence of some inhibitory factors or the lack of some activators for syndecan-4 induction in terminally differentiated cells. Repression of syndecan-4 induction could be an important control mechanism to modulate the activity of growth factors.

Constitutive expression of syndecan-4 had no effect on adipocyte differentiation, as measured by triglyceride accumulation. This may be due to the low level of overexpression achieved. Stably transfected 3T3-F442A cells expressed only 40–50% more syndecan-4 than did control cells. We speculate that constitutive expression of syndecan-4 may have resulted in alteration of expression of other protein factors that regulate syndecan-4 turnover. Recent reports indicate that proteases may be intimately linked to fat cell differentiation. Alexander et al. (45) have shown both in vivo and in an adipocyte cell culture model that stromelysin-1 inhibits fat cell differentiation, whereas a plasma kallikrein-dependent plasminogen pathway is required for adipocyte differentiation (46) .

In summary, syndecan-4 induction in 3T3-F442A cells temporally coincides with the time when quiescent cells reenter the cell cycle. Serum depletion-repletion results in syndecan-4 induction. The inhibition of syndecan-4 induction by an antisense approach prevents cell proliferation. In addition, the link between syndecan-4 induction and 3T3-F442A cell proliferation is emphasized by the finding that this serum inducibility of syndecan-4 is severely repressed in the terminally differentiated adipocytes that have lost proliferative capacity.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cell Culture.
The 3T3-F442A mouse preadipocytes were obtained as a generous gift from Dr. H. Green (28) . They were maintained in growth medium (10% calf serum, 2 mM L-glutamine, and 25 mM HEPES in DMEM) at 37°C in 10% CO2. For differentiation, cells were cultured until they reached confluence, and then they were treated with differentiation medium (10% fetal bovine serum, 5 µg/ml insulin, 2 mM L-glutamine, and 25 mM HEPES in DMEM).

Mouse Syndecan-4 cDNA.
The mouse syndecan-4 cDNA was isolated by a combination of PCR and hybridization screening from a mouse adipocyte 3T3-L1 cDNA library in the {lambda}ZAP vector (Stratagene) generously provided by Dr. Daniel Lane. The library was amplified in Escherichia coli BB4, and 50,000 plaque-forming units/dish were plated on ten 150-mm dishes. The lysate from each plate was analyzed by PCR with the following oligonucleotides: 5'-CCTCCCGGACGATGAAGAC-3' and 5'-AGGAAAACGGCGAAGAGGATGC-3'. The sequences of these oligonucleotides were taken from the published rat syndecan-4 sequence (20) . After two rounds of PCR screening, positive clones were isolated by screening of the positive lysates with a random primed {alpha}-32P-labeled probe. The probe consisted of the PCR product generated by the two oligonucleotides listed above. The longest clone isolated extended from nt -107 to 1219, in which the 25th nt is the translation start site. The putative syndecan-4 cDNA sequence was transiently expressed in Cos-7 cells to verify that it expressed a proteoglycan with a core protein of appropriate size. The syndecan-4 cDNA was tagged with a c-myc epitope (EQKLISEEDL) by inserting the corresponding nt sequence after nt 117. The presence of this c-myc epitope was detected by Western blot. A core protein could be identified only after digestion with HT (heparinase III; IBEX Technologies, Inc., Montreal, Canada).

RNA Isolation and RNase Protection Assay.
Total cellular RNA was isolated from cells at each of the respective days of differentiation using Trizol reagent (Life Technologies, Inc.). RNase protection assays were performed with the RNase protection assay kit (Ambion), with some modifications. The probe for syndecan-4 extended from nt 112 to 432. The nt numbering system is that used by Tsuzuki et al. (22) . Briefly, 10 µg of total RNA were hybridized to the 32P-labeled syndecan-4 antisense RNA probe, and the unhybridized probe was digested with RNase. The hybridized probe was precipitated and then filtered through a GF/C glass fiber filter (Whatman). The filter was washed once with 10 ml of 7.5% trichloroacetic acid and once with 10 ml of 95% ethanol, and the total number of cpm bound to the filter was determined by scintillation counting. The lack of nonspecific hybridization was confirmed separately by electrophoresis. The quantity of syndecan-4 mRNA in the sample was obtained by comparing the signal intensity of the sample with that of a standard curve made with syndecan-4 standard mRNA. The standard syndecan-4 mRNA was synthesized using MEGAscript (Ambion) and SP6 RNA polymerase. For the experiment summarized in Fig. 1Citation , aliquots of the same total RNA samples were separated on agarose/formaldehyde gels, and the RNA was transferred to Nytran SuperCharge nylon membrane (Scleicher and Schuell) with the TurboBlotter (Scleicher and Schuell) following the manufacturer’s directions. As a measure of a relatively constant reference mRNA species, the blot was hybridized with a probe for mouse acidic ribosomal phosphoprotein PO (accession number GenBank BC003833). The probe was produced by the random prime labeling procedure using the strip-EZ DNA probe synthesis kit (Ambion). The template for the 583-nt probe was generated by PCR using the following primers: (a) forward primer, 5'-TCTGGAGAAACTGCTGCCTC-3'; and (b) reverse primer, 5'-GAAGGCCTTGACCTTTTCAG-3'. The membrane was prehybridized for 1 h at 68°C in PerfctHyb Plus buffer (Sigma Chemical Co.), and the probe was then added at 1.5 x 106 cpm/ml for 4.5 h at 68°C. The membrane was washed once at room temperature with 2x SSC and 0.1% SDS, washed twice with 0.5x SSC and 0.1% SDS at 68°C, and washed once with 0.1x SSC and 0.1% SDS at 68°C. Each of these washes were conducted for 30 min.

Antibody against Syndecan-4 Extracellular Domain.
The antibody was generated by immunizing a rabbit with recombinant 6-His-tagged syndecan-4 extracellular domain. The syndecan-4 cDNA sequence between nt 118 and 435 was subcloned into pQE30 (Qiagen) and transformed into DH5{alpha} E. coli. After culture and centrifugation of the transformed E. coli, the bacterial pellet was lysed in 100 ml of 6 M guanidine-HCl, 0.2 M NaH2PO4, 0.01 M Tris-HCl, and 0.1 M NaCl (pH 8.0) for each liter of culture. The lysate was sonicated at 100 W, stirred for 1 h at room temperature, and clarified by centrifugation. The recombinant protein was affinity-purified on Ni-NTA resin (Qiagen). The resin was washed with 8 M urea, 0.1 M NaH2PO4, 0.01 M Tris base, and 0.01 M NaCl (pH 8.0). The resin was also washed with 50 mM NaH2PO4, 300 mM NaCl, and 20 mM imidazole (pH 8.0) until the A280 nm of the wash solution was below 0.05. The recombinant protein was eluted with 50 mM NaH2PO4, 300 mM NaCl, and 250 mM imidazole (pH 8.0). The 6-His-tagged protein was further purified by denaturing SDS-PAGE with 6 M urea, followed by electroelution in 40 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid and 60 mM Tris (pH 9.4) using a Bio-Rad Whole Electroeluter set at 200 mA for 30 min. The recombinant protein has 129 amino acids. The sequence includes a 12-amino acid extension on the NH2 terminus (MRGSHHHHHHGS) of the protein and an 11-residue addition at the COOH terminus (SRVDLQPSLIS). The calculated molecular weight is 14,030. Four mg of recombinant syndecan-4 extracellular domain were coupled to Affi-PrepR 15 (Bio-Rad) to generate an affinity column that was used for purification of immunoglobulins. Antiserum was diluted 5-fold in the equilibration buffer [0.2 M Tris base and 0.5 M NaCl (pH 8.0)] and applied to the affinity column. After washing the column with the equilibration buffer, the immunoglobulins were eluted with 0.2 M glycine and 0.5 M NaCl (pH 2.8) into vials containing sufficient 1 M Tris base (pH 9.0) to bring the pH to 7.4.

Immunoblot Analysis.
Cells were extracted with lysis buffer (0.75 M NaCl, 10 mM HEPES, 1 mM EDTA, and 1% Triton X-100) supplemented with protease inhibitors (1 µg/ml leupeptin, 1 µg/ml antipain, 10 µg/ml benzamidine, 10 IU/ml trasylol, 1 µg/ml chymostatin, and 1 µg/ml pepstatin A) and sonicated twice (20 s, 100 W) to obtain a whole cellular lysate. The lysates were dialyzed overnight against 50 mM NaCl, 4 mM CaCl2, and 20 mM Tris-HCl (pH 7.4) and freeze-dried. The samples were resuspended in 250 µl of HT buffer [3 mM calcium acetate, 10 mM EDTA, 0.05% Triton X-100, 10 mM N-ethylmaleimide, and 10 mM HEPES (pH 7.0)] supplemented with the protease inhibitor mixture given above. The protein content was determined, and 50 µl of the sample were digested for 4 h at 37°C with a mixture of 80 milliunits of chondroitin sulfate ABC lyase (chondrotinase ABC, EC 4.2.2.4; Sigma Chemical Co.) and 5 milliunits of heparin sulfate lyase (heparinase III, EC 4.2.28; IBEX Technologies, Inc.).

The digested cell extracts were separated by SDS-PAGE, and the proteins were transferred to Immobilon P membrane (Millipore). The membrane was blocked with 5% nonfat dry milk in PBS containing 0.05% (w/v) Tween 20 for 2 h at room temperature and incubated overnight with 0.1 µg/ml affinity-purified anti-syndecan-4 antibody. The membrane was then incubated with goat antirabbit IgG horseradish peroxidase conjugate (Amersham) for 1 h. The immunoreactive protein was visualized by enhanced chemiluminescence. Molecular weights were estimated using prestained standards (Sigma Chemical Co.) and the BOA protein marker (MoBiTec, Marco Island, FL).

Syndecan-4 Antisense Oligonucleotide Treatment.
The antisense oligonucleotides were obtained from Oligo, etc. Co. The syndecan-4 antisense oligonucleotide was designed to cover the translation start site. The oligophosphothiate nt sequence was: 5'-GCAGGCGCCATGGCTTCAACAG-3'. The control oligonucleotide was designed to have a similar nt composition but in a randomized sequence. The sequence was 5'-GCAGCCGGCAACTTGACGAACT-3'. The same number of cells was plated in 96-well plates and cultured for 24 h before the antisense treatment. The cells were transfected with syndecan-4 antisense oligophosphothioate nts or the control oligophosphothioate nts with a liposome transfection method (Lipofectin; Life Technologies, Inc.). After 6 h, the nt-liposome complex was removed, and the cells were fed fresh growth medium.

Ectopic Expression of Syndecan-4.
The syndecan-4 cDNA in Bluescript was digested with SmaI and XhoI and subcloned into pcDNA3 to obtain pcDNA3-Syn4. 3T3-F442A cells were transfected with this expression vector with LipofectAMINE PLUS (Life Technologies, Inc.) according to the manufacturer’s protocol, and pools of transfected cells were selected in the presence of G418 (400 µg/ml). To study the effect of syndecan-4 ectopic expression on adipocyte differentiation, three 100-mm dishes of 3T3-F442A cells [either control cells (cells transfected with empty pcDNA3) or cells transfected with pcDNA3-Syn4] were collected at each time point and lysed for triglyceride (Triglyceride-int kit; Sigma Chemical Co.) and DNA (47) assays. Cells were lysed with 4 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, 50 mM NH4OH, and 3 units/ml heparin (pH 8.1). Dishes were harvested on days -4, -2, 0, 0.33, 0.42, 1, 2, 3, 5, and 6. Day 0 is the day that differentiation medium was fed to cells.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by NIH Grant HL-14990. Back

2 Present address: Regulatory Biology, The Salk Institute, La Jolla, CA 92186-5000. Back

3 To whom requests for reprints should addressed, at Division of Nutritional Sciences, Savage Hall, Cornell University, Ithaca, NY 14853. Phone: (607) 255-1927; Fax: (607) 255-1033; E-mail: ab55{at}cornell.edu Back

4 The abbreviations used are: HSPG, heparan sulfate proteoglycan; bFGF, basic fibroblast growth factor; FGF, fibroblast growth factor; HS, heparan sulfate; HT, heparitinase; BrdUrd, bromodeoxyuridine; nt, nucleotide. Back

Received for publication 4/ 4/01. Revision received 8/20/01. Accepted for publication 8/29/01.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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