Archives of Physiology and Biochemistry, 2009; 115(3): 147–154

O r i g i n a l a r T i C l E

Growth factor-mediated hyper-elongation of glycosaminoglycan chains on biglycan requires transcription and translation

Sundy N. Y. Yang 1, Micah L. Burch1,2, Robel Getachew1, Mandy L. Ballinger1, Narin Osman1,2, and Peter J. Little1,2

1BakerIDI Heart and Diabetes Institute, Diabetes and Cell Biology Laboratory, Melbourne, VIC, 3004, Australia, 2Monash University School of Medicine (Alfred Hospital), Faculty of Medicine, Nursing and Health Sciences, Departments of Medicine and Immunology, Prahran VIC, 3004, Australia

Address for Correspondence: Peter J. Little, Head, Diabetes and Cell Biology Laboratory, BakerIDI Heart and Diabetes Institute, St. Kilda Road Central, PO Box 6492, Melbourne, VIC 8008, Australia. Courier Address: 75 Commercial Road, Melbourne, VIC 3004, Australia. Tel: +61 3 8532 1203. Fax: +61 3 8532 1100. E-mail: peter.little@bakeridi.edu.au

(Received 06 May 2009; revised 02 June 2009; accepted 11 June 2009)

Introduction

Proteoglycans are an integral part of the extracellular matrix of all tissues (Hascall et al., 1991; Wight, 1991). There is considerable interest in the biosynthetic pathways determining proteoglycan and particularly glycosaminoglycan (GAG) structure because GAG chains are implicated in many fundamental biologi-cal and pathological processes (Linhardt et al., 2004). A notable example of such a pathological event is the role of GAG chains in binding to low density lipopro-teins (LDL) (Camejo et al., 1985; Oorni et al., 2000). The accumulation of proteoglycans in the wall of injured blood vessels leads to the trapping of apoli-poproteins and is recognized as an initiating factor in atherosclerosis which underlies most cardiovascular

disease (Little et al., 2008b; Nakashima et al., 2007; O’Brien et al., 1998). This concept was first presented by Williams and Tabas as the “response-to-retention hypothesis” (Williams et al., 1995; Williams et al., 1998) and was recently updated to incorporate the continuing role of proteoglycans in atherosclerosis (Tabas et al., 2007). GAG hyper-elongation has been proposed as a target for prevention of atherosclerosis (Ballinger et al., 2004; Little et al., 2007).

Although GAGs have critical biological roles and many of the potential synthetic enzymes have been cloned, the precise mechanism(s) by which chon-droitin sulphate (CS) GAG polymerization occurs is unknown (Kitagawa et al., 2003; Little et al., 2008a; Sato et al., 2003). Biglycan is synthesized as a CS GAG and the action of epimerase on glucuronic acid

ISSN 1381-3455 print/ISSN 1744-4160 online © 2009 Informa UK LtdDOI: 10.1080/13813450903110754

abstractThe mechanism through which growth factors cause glycosaminoglycan (GAG) hyper-elongation is unclear. We have investigated the role of transcription and translation on the GAG hyper-elongation effect of platelet-derived growth factor (PDGF) in human vascular smooth muscle cells (VSMCs). To determine if the response involves specific signalling pathways or the process of GAG hyper-elongation we have also investigated the effects of epidermal growth factor (EGF), transforming growth factor-β (TGF-β) and thrombin. We report that both actinomycin D and cycloheximide completely abolished the ability of PDGF to stimulate radiosulphate incorporation and GAG elongation into secreted proteoglycans, and to increase the size of xyloside GAGs. Blocking de novo protein synthesis completely prevented the action of all growth factors tested to elongate GAG chains. These results lay a foundation for further investigation into the genes and proteins implicated in this response.

Keywords: Vascular smooth muscle cells; biglycan; proteoglycans; actinomycin D; cycloheximide

http://www.informapharmascience.com/arp

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148 S. N. Y. Yang et al.

residues modifies some chains of the residues and therefore, by definition the GAG chains, to derma-tan sulphate (DS) GAGs. Biglycan is the major lipid binding proteoglycan produced by multiply passaged human vascular smooth muscle cells (VSMC). As well as basal or natural GAG synthesis, this process has superimposed upon it the phenomenon of GAG hyper-elongation (Little et al., 2008a; Little et al., 2008b). In GAG hyper-elongation, agonists, such as hormones and growth factors stimulate signalling pathways that lead to further elongation of the GAG chains and thus an increase in the average molecular weight of the intact proteoglycan (Little et al., 2008a). It is the hyper- elongation effect that can increase the pathogenicity of the proteoglycan (Little et al., 2008b).

Treatment of VSMCs with growth factors such as platelet-derived growth factor (PDGF), transforming growth factor (TGF)-, angiotensin II and metabolic agents including oxidized LDL leads to the synthe-sis and secretion of CS/DS proteoglycans which are larger than those from untreated cells (Chang et al., 2000; Little et al., 2002; Schonherr et al., 1993). PDGF, as the prototypical VSMC growth factor, activates cell surface tyrosine kinase receptors and has a number of effects in VSMCs, such as stimulation of prolif-eration, migration and matrix secretion (Ross, 1993). PDGF stimulates hyper-elongation of GAG chains on proteoglycans produced by VSMCs (Schonherr et al., 1993; Schonherr et al., 1991; Schonherr et al., 1997). When cells are provided with exogenous xyloside, a mimetic for xylose which is the first monosaccharide in the tetrasaccharide linkage region, cells synthesize small, free CS GAG chains. Supplementation of cells with xyloside represents an assay of GAG synthesizing activity in the cell independent of core proteins (Fritz et al., 2001; Potter-Perigo et al., 1992). Treatment of cells with TGF- in the presence of xyloside leads to the synthesis of larger xyloside GAG chains, which also show enhanced binding to LDL (Little et al., 2002).

There have been considerable advances in under-standing the signalling pathways that affect GAG hyper-elongation and on the identification and cloning of the enzymes that mediate GAG polym-erization (Little et al., 2008a). However, there is currently very little knowledge of the biochemical processes that link these two phenomena. As the actual mechanism of CS/DS GAG polymerization is unknown, it is not possible to know if the hyper-elongation effect is simply the further activation of the endogenous synthesis or another biochemical response, for example, increased “transit time” in the Golgi apparatus (Campbell et al., 1988). In order to determine the mechanism through which growth factors cause GAG hyper-elongation in VSMCs, we have investigated whether the response requires

transcription of genes and translation of any protein species. We have investigated the role of transcrip-tion and translation on the GAG elongation effect of the protein tyrosine kinase receptor agonist PDGF in human VSMCs. To dissect if the response is related to signalling pathways or the molecular mechanisms of GAG elongation we have investigated the effects of three other agents with distinct signalling pathways, a protein tyrosine kinase agonist, Epidermal Growth Factor (EGF), the serine/threonine kinase receptor agonist, TGF- and the G protein coupled receptor (GPCR) agonist, thrombin. We report that the GAG hyper-elongation effect of all these vasoactive factors is similarly dependent upon transcription and trans-lation which confirms the existence of a rate limiting step in the process of GAG hyper-elongation as a valid target to prevent this pathogenic response.

Materials and methods

Materials

Cycloheximide, actinomycin D, EGF, PDGF, methyl -D-xylopyranoside (xyloside), Whatman 3MM chro-matography paper, dimethylsulfoxide (DMSO) and DEAE Sephacel were obtained from Sigma Aldrich (St Louis, MO). Human recombinant TGF- was obtained from R&D systems (Minneapolis, USA). Foetal bovine serum (FBS) was obtained from CSL (Parkville, Australia). Scintillation fluid, Instagel, was from Packard (Groningen, The Netherlands). Carrier-free (35S)-SO

4 was from ICN Biomedicals (Irvine, CA,

USA). Cetylpyridinium chloride (CPC) was from Uni-lab Chemicals and Pharmaceuticals, India. Cell culture materials were from GIBCO BRL (Grand Island, USA).

Cell Culture

Human VSMCs were isolated using the explant tech-nique from discarded segments of the saphenous veins from a variety of patient donors undergoing sur-gery at the Alfred Hospital (Melbourne, Australia). The acquisition of the vessels was approved by the Alfred Hospital Ethics Committee. Cells were seeded into 24 well plates at 50,000 cells/well in low glucose (5 mM) DMEM with 10% FBS and antibiotics and maintained until confluent. Cells were rendered quiescent by cul-turing in low glucose (5 mM) DMEM with 0.1% FBS for 48 hours prior to experimentation. Experiments were conducted on cells passage 5–22. Human VSMC were treated with actinomycin D (100 ng/ml) and cycloheximide (10 µg/ml) (and in some experiments 0.5 mM xyloside) under basal conditions and in the

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Mechanism of glycosaminoglycan elongation 149

presence of PDGF, EGF, TGF- and thrombin for 24 h with (35S)-SO

4 (50 µCi/mL). Parallel plates without

radiolabel were established to assess cell numbers per well which were then used to normalise the data. The drugs used at the concentrations shown did not affect cell numbers in these experiments.

Measurement of (35S)-SO4 incorporation into proteoglycans

Proteoglycans were extracted from the medium, as previously described (Chang et al., 1983; Wight et al., 1983). Incorporation of (35S)-SO

4 into proteoglycans

was measured by CPC precipitation as previously described (Nigro et al., 2004). Briefly, aliquots (50 µl) of the medium were spotted on 3MM paper and washed five times for 40 min in 1% CPC with 0.05 M NaCl. The amount of precipitate on the dried filter paper was determined by liquid scintillation counting.

Ion-exchange chromatography

Proteoglycans were isolated on DEAE-Trisacryl mini-columns (250 µl resin), which were washed extensively with 8M urea, 2 mM disodium EDTA, 0.5% Triton X-100, 0.25M NaCl, 50 mM Tris/HCl, pH 7.5 to remove glycoproteins and free (35S)-SO

4. Proteoglycans were

eluted with the same buffer containing a higher salt concentration (3M NaCl). The isolated material was further concentrated by ethanol/potassium acetate precipitation.

Gel electrophoresis

SDS-PAGE was performed according to the proce-dure of Laemmli (1970) on 4–20% acrylamide gradi-ent gels with a 3% stacking gel. The labelled prote-oglycans were visualized by exposing dried gels to a phosphor-imaging screen (Fuji Photo Film Co, Japan) for approximately 3 days, and then scanned on a Bio-imaging analyser BAS-1000 MacBas (Fuji Photo Film Co, Japan).

Statistics

Data are presented as mean ± SEM from at least three independent experiments conducted in triplicate. Data from three independent experiments were sta-tistically analysed using a one-way ANOVA for the determination of the least significant difference upon which statistical significance is reported.

Results

We investigated the role of gene transcription in the GAG elongation response to growth factors, spe-cifically PDGF, occurring in human VSMCs (Little et al., 2008a). We utilized the transcription inhibitor, actinomycin D, which binds to DNA at the transcrip-tion initiation complexes and prevents the action of RNA polymerase. Human VSMCs were treated with PDGF (50ng/ml) and labelled with (35S)-SO

4

(50 µCi/ ml) for 24 h in the presence of actinomycin D (30, 100 and 300 ng/ml). The basal rate of (35S)-SO

4

incorporation was 11.4 ± 1.3 cpm/cell over 24 h and this was increased to 21.0 ± 1.7 cpm/cell (p < 0.01) in the presence of PDGF (Figure 1A). This effect of PDGF is consistent with earlier reports (Schonherr et al., 1993; Schonherr et al., 1991). Actinomycin D had a concentration-dependent inhibitory effect on PDGF stimulated (35S)-SO

4 incorporation with partial

inhibition apparent at 30 ng/ml and total inhibition at 300 ng/ml at which concentration the level of prote-oglycan production and secretion was slightly below the basal level of (35S)-SO

4 incorporation (Figure 1A).

Increased (35S)-SO4 incorporation can be due to

increased proteoglycan core protein synthesis (hence more GAG initiation sites), elongation of the GAG chains or increased GAG chain sulfation (Ballinger et al., 2004). Our interest was predominantly in the regulation of GAG elongation. Changes in GAG length can be assessed by the migration of the radio-labelled molecules on SDS-PAGE. This method correlates very closely with GAG size as determined by gel filtration chromatography (Ballinger et al., 2009; Ivey et al., 2008; Little et al., 2002; Nigro et al., 2004). The major proteoglycan of interest is biglycan (Figure 1B). For VSMCs treated with PDGF the migration of biglycan was decreased indicating an increase in the size of biglycan compared to control (Figure 1B, lane 1 versus 5). In the presence of actinomycin D, the transcrip-tion inhibitor caused a concentration-dependent increase in electrophoretic mobility indicating inhi-bition of PDGF mediated GAG elongation (Figure 1B lanes 6, 7 and 8). The alterations in the biglycan size correlated with the changes in total secreted (35S)-SO

4

as shown in the histogram (Figure 1A). This indicates that changes in GAG size make a major contribution to changes in total (35S)-SO

4 incorporation.

In VSMCs treated with actinomycin D (100 ng/ml) in the presence of exogenous xyloside (0.5 mM), the basal level of secreted proteoglycans was reduced and the response to PDGF assessed as ( 35S)-SO

4 incorpora-

tion was almost totally abolished (Figure 1C). The xylo-side GAGs appear as very broad bands at the bottom of the SDS-PAGE gel (Figure 1D). The SDS-PAGE shows that xyloside GAGs secreted from PDGF treated cells

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150 S. N. Y. Yang et al.

are larger than control (lane 1 versus 2) and in the pres-ence of actinomycin D, the ability of PDGF to increase xyloside GAG size is abolished (lanes 3 and 4).

We then evaluated the role of de novo protein syn-thesis in the GAG elongation response to PDGF using the antibiotic, cycloheximide. Human VSMCs were stimulated with PDGF in the presence and absence of cycloheximide (10 µg/ml), metabolically labelled with (35S)-SO

4 and secreted proteoglycans collected

after 24 h. PDGF caused a two fold increase in total (35S)-SO

4 incorporation (Figure 2A). In the presence

of cycloheximide the basal rate of proteoglycan secre-tion was markedly reduced and the response to PDGF was abolished (Figure 2A). SDS-PAGE analysis con-firmed the GAG elongation effect of PDGF (Figure 2B, lanes 1 and 2) but in the presence of cycloheximide the GAG elongation effect was abolished (Figure 2B, lanes 3 and 4). We then examined the effect of block-ing de novo protein synthesis in the presence of exog-enous xyloside. PDGF stimulated an almost two-fold increase in (35S)-SO

4 incorporation in the presence of

xyloside. In the presence of cycloheximide and xylo-side the basal rate of (35S)-SO

4 was markedly reduced

and the stimulatory response to PDGF was abolished (Figure 2C). Evaluation of the size of the xyloside GAGs

by SDS-PAGE demonstrated that the stimulatory effect of PDGF on the size of xyloside GAGs was abol-ished in the presence of cycloheximide (Figure 2D). These data demonstrate that de novo protein synthe-sis is required for the GAG elongation effect of PDGF in VSMCs.

To investigate if the effect of transcription and translation was specific for an individual growth factor signalling pathway we determined the GAG elongation response to three factors which utilize completely different signalling pathways. We used EGF (10 ng/ ml), TGF- (2 ng/ml) and thrombin (10 units/ ml), each of which we have previously dem-onstrated to mediate GAG hyper-elongation (Ballinger et al., 2009; Dadlani et al., 2008; Ivey et al., 2008). Each of the three agonists stimulated total (35S)-SO

4 incor-

poration into secreted proteoglycan with an efficacy TGF- > thrombin > EGF at maximally active concen-trations of the growth factors (Figure 3A). Consistent with the (35S)-SO

4 incorporation, examination of

the size of the secreted proteoglycans by SDS-PAGE showed that each growth factor caused an increase in the size of the proteoglycans with an efficacy TGF- > thrombin > EGF (Figure 3B, lanes 1–4). In the presence of cycloheximide the basal rate of secretion

− PDGF − PDGFkDa

Xyloside-initiated GAG

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PDGF

Actinomycin D(ng/mL)

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2209766

Actinomycin D (ng/ml)

Biglycan

PDGFB

Figure 1. Role of gene transcription in the GAG elongation effect of PDGF on proteoglycans and free GAG chains synthesized in the presence of xyloside. Human VSMC were treated with actinomycin D (100 ng/ml) under basal conditions or in the presence of PDGF (50 ng/ mL) for 24 hours. [35S]-SO

4 was added at 50 µCi/mL. A. [35S]-SO

4 associated with proteoglycans. B. SDS-PAGE (4–13% acrylamide)

analysis. C. [35S]-SO4 associated with proteoglycans and xyloside associated GAG chains. D. Xyloside associated GAG chains were ana-

lysed by SDS-PAGE (4–20% acrylamide). (**p < 0.01; n.s. = not significant vs PDGF in the presence of actinomycin D).

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Mechanism of glycosaminoglycan elongation 151

− PDGF − PDGFkDa

Xyloside

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− PDGF − PDGF

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Xyloside30

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220Biglycan

cyclo

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B

Figure 2. Role of de novo protein synthesis in the GAG elongation effect of PDGF on proteoglycans and free GAG chains synthesized in the presence of xyloside. Human VSMC were treated with cycloheximide (cyclo) (10 µg/ml) under basal conditions or in the presence of PDGF (50 ng/mL) and [35S]-SO

4 (50 µCi/mL ) for 24 h. A. [35S]-SO

4 associated with proteoglycans. B. SDS-PAGE (4–13% acrylamide) analy-

sis. C. [35S]-SO4 associated with proteoglycans and xyloside associated GAG chains. D. Xyloside associated GAG chains were analysed by

SDS-PAGE (4–20% acrylamide). (**p < 0.01; n.s. = not significant compare to PDGF in the presence of cycloheximide).

kDa

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cycloheximidecycloheximideA B

− EGF TGF-β Thr − EGF TGF-β Thr

− EGF TGF-β Thr − EGF TGF-β Thr

Xyloside

Biglycan

cycloheximide

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cycloheximide

C D

− EGF TGF-β Thr − EGF TGF-β Thr

Figure 3. Role of de novo protein synthesis in the GAG elongation effect of three vasocatiove agonists which utilize three distinct cell surface receptors. Experiments were conducted as described in Fig. 2 legend using EGF (10 ng/ml), TGF- (2 ng/ml) or thrombin (10units/ ml) as agonists. A. [35S]-SO

4 associated with proteoglycans. B. SDS-PAGE (4–13% acrylamide) analysis. C. [35S]-SO

4 associated

with proteoglycans and xyloside associated GAG chains. D. Xyloside associated GAG chains were analysed by SDS-PAGE (4–20% acryla-mide). (**p < 0.01; n.s. = not significant compare to EGF or TGF- or thrombin alone in the presence of cycloheximide).

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152 S. N. Y. Yang et al.

of (35S)-SO4 was reduced and the stimulatory response

to the three growth factors was abolished (Figure 3A). The inhibitory effect of blocking de novo protein syn-thesis completely attenuated the GAG elongation effect of the three growth factors (Figure 3B, lanes 6–9). Finally, we examined the GAG elongation effect of the three growth factors on the synthesis of xyloside GAGs. In the presence of xyloside (0.5 mM) all three growth factors stimulated (35S)-SO

4 incorporation into

secreted proteoglycans and xyloside GAGs and the effect was abolished in cells treated with cyclohex-imide (Figure 3C). SDS-PAGE analysis of the size of the xyloside GAGs indicated that all three growth fac-tors stimulated an increase in the size of the xyloside GAGs and that the effect was totally abolished in cells treated with cycloheximide (Figure 3D). These data suggest that the signalling pathways have a point of coalescence which is rate limiting for the polymerisa-tion of GAG chains and this point maybe at the late stage of the signal transduction pathways or at the level of the factors which lead directly to GAG hyper-elongation.

Discussion

We evaluated the role of transcription and transla-tion in the process in which growth factors cause hyper-elongation of GAG chains on the proteoglycan, biglycan, using the transcription inhibitor, actinomy-cin D and the translation inhibitor, cycloheximide. VSMCs were treated separately with actinomycin D and cycloheximide and stimulated with PDGF and GAG synthesis monitored by radio-sulphate incor-poration into secreted proteoglycans over 24 h. Both actinomycin D and cycloheximide completely abol-ished the ability of PDGF to stimulate radio-sulphate incorporation into secreted proteoglycans. Further analysis of proteoglycan size, revealed that the GAG elongation effect was also prevented when transcrip-tion and translation were blocked by actinomycin D and cycloheximide, respectively. The effect of PDGF to increase the size of secreted xyloside GAGs was also abolished by treatment of VSMCs with actinomycin D and cycloheximide.

To investigate if the inhibition was specific for one signalling pathway, we investigated the responses to three growth factors with distinct signalling path-ways. We used the protein tyrosine kinase agonist EGF, protein serine threonine kinase agonist TGF- and the GPCR agonist thrombin. Blocking de novo protein synthesis with cycloheximide completely prevented the action of all three growth factors to elongate the GAG chains on biglycan. Similarly, in the presence of exogenous xyloside, the GAG elongation

effect assessed as xyloside GAG size was completely abolished in the presence of cycloheximide.

We have addressed the question as to whether or not agonist-mediated hyper-elongation involves gene transcription and de novo protein synthesis utilizing the well established pharmacological intervention strategies of actinomycin D and cycloheximide treat-ment, respectively. These experiments show that the synthesis of natural GAGs does not involve de novo protein synthesis, because natural length GAG chains are formed intact or only minimally reduced in size in the presence of actinomycin D and cycloheximide. However, the ability of PDGF to cause GAG hyper-elongation is completely dependent upon transcrip-tion and de novo protein synthesis. The best current explanation for these data is that the integral mem-brane enzymes mediating the synthesis of natural GAGs are only slowly turning over, possibly due to a stable environment in the membranes of the Golgi apparatus. In the time frame of these experiments (24 h), synthesis of GAG occurs normally and does not require transcription and protein translation and uti-lizes the existing pool of proteoglycan core proteins. In contrast, the action of PDGF to hyper-elongate GAGs is clearly dependent upon gene transcription and the synthesis of a new protein associated with the signal-ling pathway or the synthesis of longer GAG chains (see Figures 1B, 2B and 3B). There are at least two distinct possibilities for the role of this newly synthesized pro-tein in mediating GAG elongation – either the agonist is causing the increased expression and synthesis of a factor which is rate limiting in basal GAG synthesis, for example, chondroitin polymerizing factor (ChPF) (Izumikawa et al., 2007) or alternatively, the signalling pathway could involve gene expression and new pro-tein synthesis ultimately mediating the effect on GAG synthesis (Ungefroren et al., 2005). For example, in the pathways that TGF- stimulates biglycan expression in PANC-1 cells, the initial stimulation of Smad phos-phorylation is followed by the expression and syn-thesis of GADD45 which activates p38 MAP kinase (Ungefroren et al., 2005). This is a rapid response and it would be expected that acute actinomycin D and cycloheximide treatment could block this signalling pathway. This data exemplifies the difference between natural GAG synthesis and GAG hyper-elongation and further adds to the validity of the signalling pathways controlling the synthesis and structure of GAGs as a target for the prevention of atherosclerosis (Ballinger et al., 2004; Little et al., 2007).

In the presence of the blockade of transcription and translation the cells initially continue to produce proteoglycans as observed in the electrophoretic analysis (see Figures 1A and 2B). These proteoglycans, specifically biglycan, have the same size GAG chains

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Mechanism of glycosaminoglycan elongation 153

as normally produced on biglycan. Furthermore, the provision of xyloside in the presence of the blockade of transcription and translation still results in the pro-duction and secretion of xyloside GAGs of normal size. These data indicate that the GAG synthesizing machin-ery in the cells continues unaltered for up to 24 h after cessation of transcription and translation. Thus, the cells use the available supply of biglycan core protein on which to synthesize normal length GAG chains and similarly use exogenous xyloside to produce normal xyloside GAGs. In the situation of synthesis in the absence of de novo transcription and transla-tion the total radio sulphate incorporation is reduced under basal condition which indicates the exhaustion of the supply of biglycan core protein. However, the data clearly demonstrates that the effect of growth factors to mediate GAG elongation is inhibited when transcription and translation are blocked. That this occurs for multiple receptor mediated mechanisms of GAG elongation indicates that the critical biochemical target(s) for GAG hyper-elongation is downstream of the variety of cell surface receptors that mediate GAG elongation. The likely ultimate targets include the GAG polymerizing enzymes and those related proteins that act as chaperones and co-polymerization factors and also potentially the carbohydrate transporters in the Golgi apparatus that supply the substrates required for GAG polymerization (Hirschberg et al., 1998; Toma et al., 1996). These remain to be identified.

There have been earlier studies in which the role of transcription and translation in overall proteoglycan synthesis have been investigated. Vijayagopal and colleagues examined the effect of bovine endothe-lial cell (EC) conditioned medium on bovine VSMC proteoglycan production (Vijayagopal et al., 1992). EC conditioned medium stimulated radiolabel incor-poration into secreted proteoglycans due to both an increase in size of the chains and an increase in chain number (Vijayagopal et al., 1992). The effect was attributable particularly to TGF- (20%) and to other unknown factors which we would now know to include endothelin-1 which we have recently shown stimulates proteoglycan synthesis in VSMCs (Ivey et al., 2008). Both actinomycin D and cycloheximide inhibited the ability of EC conditioned medium to stimulate radio-sulphate incorporation into secreted proteoglycans, as found in the current study. The effect was attrib-uted to the supply of new proteoglycan core proteins and chain number rather than the elongation effect of the agonists (Vijayagopal et al., 1992). Berrou and colleagues (Berrou et al., 1991) investigated the effect of porcine EC conditioned medium on the synthesis of proteoglycans by porcine VSMCs. The conditioned medium caused a modest increase in radio-sulphate incorporation into secreted proteoglycans but the

effect was not affected by cycloheximide (Berrou et al., 1991). This is not inconsistent with the current results because Berrou used a very short labelling time (20 min) and thus concluded that the result was due to the utilization of existing core protein pool within the cell and the increased radio-sulphate incorporation was attributed to increased glycosylation of the prote-oglycan core proteins (Berrou et al., 1991).

In conclusion, we have shown that transcription and translation are required for GAG hyper- elongation. Using a group of growth factors with different signal-ling pathways, but all of which mediate GAG hyper-elongation, we have shown that the response occurs downstream of the cell surface receptors. These results now lay a clear foundation for further inves-tigation into the specific genes and proteins that are implicated in this response. Detailed discussion of this area has been published previously (Little et al., 2008b). As multiple growth factors are present in the wall of a diseased blood vessel these data indicate the existence of a common rate limiting step in GAG hyper-elongation and further validate the process as a target for the development of an agent which pre-vents GAG hyper-elongation, reduces lipid binding to proteoglycans and attenuates the development and progression of atherosclerosis, the major underlying cause of most cardiovascular disease (Ballinger et al., 2004; Little et al., 2007).

Acknowledgments

This study was supported by National Health and Medical Research Council project grant (#472611) (PJL) and Fellowship (PJL) and a National Heart Foundation of Australia grant-in-aid (PJL).

Declaration of interest: The authors report no conflict of interest.

References

Ballinger ML, Ivey ME, Osman N, Thomas WG, Little PJ. (2009). Endothelin-1 activates ETA receptors on human vascular smooth muscle cells to yield proteoglycans with increased binding to LDL. Atherosclerosis (In Press).

Ballinger ML, Nigro J, Frontanilla KV, Dart AM, Little PJ. (2004). Regulation of glycosaminoglycan structure and atherogen-esis. Cell Mol Life Sci 61: 1296–306.

Berrou E, Breton M, Deudon E, Picard J. (1991). Stimulation of large proteoglycan synthesis in cultured smooth muscle cells from pig aorta by endothelial cell-conditioned medium. J Cell Physiol 149: 436–43.

Camejo G, Hurt E, Romano M. (1985). Properties of lipoprotein complexes isolated by affinity chromatography from human aorta. Biomed Biochim Acta 44: 389–401.

Arc

hive

s of

Phy

siol

ogy

and

Bio

chem

istr

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

onas

h U

nive

rsity

Fo

r pe

rson

al u

se o

nly.

154 S. N. Y. Yang et al.

Campbell SC, Schwartz NB. (1988). Kinetics of intracellular processing of chondroitin sulphate proteoglycan core pro-tein and other matrix components. J Cell Biol 106: 2191–202.

Chang MY, Potter-Perigo S, Tsoi C, Chait A, Wight TN. (2000). Oxidized low density lipoproteins regulate synthesis of monkey aortic smooth muscle cell proteoglycans that have enhanced native low density lipoprotein binding properties. J Biol Chem 275: 4766–73.

Chang Y, Yanagishita M, Hascall VC, Wight TN. (1983). Proteoglycans synthesized by smooth muscle cells derived from monkey (Macaca nemestrina) aorta. J Biol Chem 258: 5679–88.

Dadlani H, Ballinger ML, Osman N, Getachew R, Little PJ. (2008). Smad and p38 MAP kinase-mediated signalling of prote-oglycan synthesis in vascular smooth muscle. J Biol Chem 283: 7844–52.

Fritz TA, Esko JD. (2001). Xyloside priming of glycosaminoglycan biosynthesis and inhibition of proteoglycan assembly. In: Iozzo RV, editor. Proteoglycan protocols. Totowa, NJ: Humana Press Inc, 317–24.

Hascall VC, Heinegard DK, Wight TN. (1991). Proteoglycans: Metabolism and pathology. In: Hay ED, editor. Cell biology of extracellular matrix. New York: Plenum Press, 149–75.

Hirschberg CB, Robbins PW, Abeijon C. (1998). Transporters of nucleotide sugars, ATP, and nucleotide sulphate in the endoplasmic reticulum and Golgi apparatus. Annu Rev Biochem 67: 49–69.

Ivey ME, Little PJ. (2008). Thrombin regulates vascular smooth muscle cell proteoglycan synthesis via PAR-1 and multiple downstream signalling pathways. Thromb Res 123: 288–297.

Izumikawa T, Uyama T, Okuura Y, Sugahara K, Kitagawa H. (2007). Involvement of chondroitin sulphate synthase-3 (chondroi-tin synthase–2) in chondroitin polymerization through its interaction with chondroitin synthase-1 or chondroitin- polymerizing factor. Biochem J 403: 545–52.

Kitagawa H, Izumikawa T, Uyama T, Sugahara K. (2003). Molecular cloning of a chondroitin polymerizing factor that cooperates with chondroitin synthase for chondroitin polymerization. J Biol Chem 278: 23666–71.

Laemmli UK. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–5.

Linhardt RJ, Toida T. (2004). Role of glycosaminoglycans in cel-lular communication. Acc Chem Res 37: 431–8.

Little PJ, Ballinger ML, Burch ML, Osman N. (2008a). Biosynthesis of natural and hyperelongated chondroitin sulphate gly-cosaminoglycans: new insights into an elusive process. Open Biochem J 2: 135–42.

Little PJ, Ballinger ML, Osman N. (2007). Vascular wall proteogly-can synthesis and structure as a target for the prevention of atherosclerosis. Vasc Health Risk Man 3: 1–8.

Little PJ, Osman N, O’Brien KD. (2008b). Hyperelongated big-lycan: the surreptitious initiator of atherosclerosis. Current Opinion in Lipidology 19: 448–454.

Little PJ, Tannock L, Olin KL, Chait A, Wight TN. (2002). Proteoglycans synthesized by arterial smooth muscle cells in the presence of transforming growth factor-beta1 exhibit increased binding to LDLs. Arterioscler Thromb Vasc Biol 22: 55–60.

Nakashima Y, Fujii H, Sumiyoshi S, Wight TN, Sueishi K. (2007). Early human atherosclerosis: accumulation of lipid and pro-teoglycans in intimal thickenings followed by macrophage infiltration. Arterioscler Thromb Vasc Biol 27: 1159–65.

Nigro J, Ballinger M, Dilley R, Jennings G, Wight T, Little P. (2004). Fenofibrate modifies human vascular smooth muscle proteoglycans and reduces LDL binding. Diabetologia 47: 2105–13.

O’Brien KD, Olin KL, Alpers CE, Chiu W, Ferguson M, Hudkins K, Wight TN, Chait A. (1998). Comparison of apolipoprotein and proteoglycan deposits in human coronary atheroscle-rotic plaques. colocalization of biglycan with apolipopro-teins. Circulation 98: 519–27.

Oorni K, Pentikainen MO, Ala-Korpela M, Kovanen PT. (2000). Aggregation, fusion, and vesicle formation of modified low density lipoprotein particles: molecular mechanisms and effects on matrix interactions. J Lipid Res 41: 1703–14.

Potter-Perigo S, Braun KR, Schonherr E, Wight TN. (1992). Altered proteoglycan synthesis via the false acceptor pathway can be dissociated from beta-D-xyloside inhibition of proliferation. Arch Biochem Biophys 297: 101–9.

Ross R. (1993). The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 362: 801–9.

Sato T, Gotoh M, Kiyohara K, Akashima T, Iwasaki H, Kameyama A, Mochizuki H, Yada T, Inaba N, Togayachi A, Kudo T, Asada M, Watanabe H, Imamura T, Kimata K, Narimatsu H. (2003). Differential roles of two N-acetylgalactosaminyltransferases, CSGalNAcT-1, and a novel enzyme, CSGalNAcT-2. Initiation and elongation in synthesis of chondroitin sulphate. J Biol Chem 278: 3063–71.

Schonherr E, Jarvelainen HT, Kinsella MG, Sandell LJ, Wight TN. (1993). Platelet-derived growth factor and transforming growth factor-beta 1 differentially affect the synthesis of big-lycan and decorin by monkey arterial smooth muscle cells. Arterioscler Thromb 13: 1026–36.

Schonherr E, Jarvelainen HT, Sandell LJ, Wight TN. (1991). Effects of platelet-derived growth factor and transforming growth factor-beta 1 on the synthesis of a large versican-like chon-droitin sulphate proteoglycan by arterial smooth muscle cells. J Biol Chem 266: 17640–7.

Schonherr E, Kinsella MG, Wight TN. (1997). Genistein selec-tively inhibits platelet-derived growth factor-stimulated ver-sican biosynthesis in monkey arterial smooth muscle cells. Arch Biochem Biophys 339: 353–61.

Tabas I, Williams KJ, Boren J. (2007). Subendothelial lipopro-tein retention as the initiating process in atherosclerosis: update and therapeutic implications. Circulation 116: 1832–44.

Toma L, Pinhal MA, Dietrich CP, Nader HB, Hirschberg CB. (1996). Transport of UDP-galactose into the Golgi lumen regulates the biosynthesis of proteoglycans. J Biol Chem 271: 3897–901.

Ungefroren H, Groth S, Ruhnke M, Kalthoff H, Fandrich F. (2005). Transforming growth factor-beta (TGF-beta) type I recep-tor/ALK5-dependent activation of the GADD45beta gene mediates the induction of biglycan expression by TGF-beta. J Biol Chem 280: 2644–52.

Vijayagopal P, Ciolino HP, Berenson GS. (1992). Endothelial cell-conditioned medium modulates the synthesis and structure of proteoglycans in vascular smooth muscle cells. Biochim Biophys Acta 1135: 129–40.

Wight TN (1991). Dynamic iInteraction of proteoglycans. In: Gotlieb AI, editor. Atherosclerosis. New York: Plenum Press, 115–125.

Wight TN, Hascall VC. (1983). Proteoglycans in primate arter-ies. III. Characterization of the proteoglycans synthesized by arterial smooth muscle cells in culture. J Cell Biol 96: 167–76.

Williams KJ, Tabas I. (1995). The response-to-retention hypoth-esis of early atherogenesis. Arterioscler Thromb Vasc Biol 15: 551–61.

Williams KJ, Tabas I. (1998). The response-to-retention hypothesis of atherogenesis reinforced. Curr Opin Lipidol 9: 471–4.

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