Sulfated proteoglycans synthesized by Neuro 2a neuroblastoma cells: Comparison between cells with and without ganglioside-induced neurites

Mouse neuroblastoma Neuro 2a cells are known to extend neurite-like processes in response to gangliosides added to the culture medium. We compared the structural features of proteoglycans (PG) synthesized by conventional Neuro 2a cells with those of neurite-bearing cells. Two different proteoglycans labeled with [³⁵S]sulfate, namely, chondroitin sulfate proteoglycan (CS-PG) and heparan sulfate proteoglycan (HS-PG), were found both in the cell layer and in the culture medium of the conventional cells. CS-PG isolated from the cell layer had a K_av value of 0.38 on Sepharose CL-6B, and had CS side chains with Mr of 27,000. HS-PG in the cell layer was slightly larger (K_av of 0.33) in terms of hydrodynamic size than CS-PG, and the apparent Mr of the heparan sulfate side chains was 10,000. The structural parameters of CS-PG and HS-PG isolated from the medium were almost identical to those of the PGs in the cell layer. In addition to these PGs, single-chain HS, with an average Mr of 2,500, was observed only in the cell layer and this component was the major sulfated component in the cell layers of both control and ganglioside treated cells. The neurite-bearing cells also synthesized both CS-PG and HS-PG which were very similar in hydrodynamic size to those synthesized by the conventional cells, but the size of HS side chains was greater. Radioactivity, as³⁵S, of each sulfated component from the gangliosideteated culture seemed to be slightly less than that of the corresponding component from the control culture. These findings indicate that the marked morphological change in Neuro 2a cells, induced by gangliosides is not accompanied by major changes in the synthesis of PGs.     

Conditioning of native substrates by chondroitin sulfate proteoglycans during cardiac mesenchymal cell migration

It is generally proposed that embryonic mesenchymal cells use sulfated macromolecules during in situ migration. Attempts to resolve the molecular mechanisms for this hypothesis using planar substrates have been met with limited success. In the present study, we provide evidence that the functional significance of certain sulfated macromolecules during mesenchyme migration required the presence of the endogenous migratory template; i.e., native collagen fibrils. Using three-dimensional collagen gel lattices and whole embryo culture procedures to produce metabolically labeled sulfated macromolecules in embryonic chick cardiac tissue, we show that these molecules were primarily proteoglycan (PG) in nature and that their distribution was class specific; i.e., heparan sulfate PG, the minor labeled component (15%), remained pericellular while chondroitin sulfate (CS) PG, the predominately labeled PG (85%), was associated with collagen fibrils as "trails" of 50-60-nm particles when viewed by scanning electron microscopy. Progressive "conditioning" of collagen with CS-PG inhibited the capacity of the template to support subsequent cell migration. Lastly, metabolically labeled, PG-derived CS chains were compared with respect to degree of sulfation in either the C-6 or C-4 position by chromatographic separation of chondroitinase AC digestion products. Results from temporal and regional comparisons of in situ-labeled PGs indicated a positive correlation between the presence of mesenchyme and an enrichment of disaccharide-4S relative to that from regions lacking mesenchyme (i.e., principally myocardial tissue). The suggestion of a mesenchyme-specific CS-PG was substantiated by similarly examining the PGs synthesized solely by cardiac mesenchymal cells migrating within hydrated collagen lattice in culture. These data were incorporated into a model of "substratum conditioning" which provides a molecular mechanism by which secretion of mesenchyme-specific CS-PGs not only provides for directed and sustained cell movement, but ultimately inhibits migration of the cell population as a whole.     

培养的人主动脉平滑肌细胞合成的蛋白聚糖的生化分析

人胎儿主动脉平滑肌细胞(SMC_8)体外培养的第4(T_4)及第10(T_(10))代细胞在含[~(35)S]-硫酸钠的培养液中培养以标记蛋白聚糖(PG)。培养液及细胞层的4mol/L盐酸胍提取液中的PG_8用DEAE-Sephacel离子交换及凝胶过滤柱层析纯化。两代(T_4及T_(10))培养液中均含有一种分子较大及大小类似的硫酸软骨素-PG(CS-PG,在Sepharose CL-4B柱的排阻部位洗脱),其相对含量在T_4为20.8％,T_(10)为12.9％,另外尚均含有一种分子较小及大小类似的硫酸皮肤素-硫酸软骨素-PG(DS-CS-PG,Kd=0.27-0.33,Sepharose CL-4B),其相对含量在T_4为72.7％,T_(10)为81.5％。两代细胞层中除有一种与培养液中大小类似的CS-PG外(其相对含量在T_4为21.7％,T_(10)为10.2％),尚均含有一种分子较小(小于培养液中者)的DS-CS-PG(Kd=0.53,Sepharose CL-4B),其相对含量在T_4为57.4％,T_(10)为75.0％。另外,两代培养液及细胞层中均含有硫酸乙酰肝素-PG(HS-PG),在T_4及T_(10)培养液中分别为6.5％及5.6％;而在细胞层中则分别为20.9％及14.8％。T_(10)的培养液及细胞层中DS-CS-PG的[~(35)S]参入量均高于T_4者;而CS-PG及HS-PG则相反。T_(10)除有PG合成变化外尚有其他衰老迹象(如脂质堆积等),故SMC_8的老化可能与动脉粥样硬化病变形成有关。     

Large disulfide-stabilized proteoglycan complexes are synthesized by the epidermis of axolotl embryos

Proteoglycans (PGs) synthesized by the epidermis during stages crucial to the subepidermal migration of neural crest cells in the trunk of the axolotl (Ambystoma mexicanum, Urodela, Amphibia) embryo were studied. The glycosaminoglycan chains were biosynthetically labeled with [35S]sulfate in vitro during a period corresponding to the onset of migration. After extraction with guanidine HCl, the radiolabeled PGs were separated according to size by molecular-sieve chromatography on Sepharose CL-2B under dissociative conditions. This resulted in the separation of high-molecular-weight PGs, which eluted in the void volume, and low-molecular-weight PGs, eluting in a broad peak with a mean Kav of 0.7. The large PGs were also found to elute in the void volume when chromatographed on a Sephacryl S-1000 column. The low-molecular-weight PGs contained heparan sulfate and chondroitin sulfate (CS) and were not further characterized. The glycosaminoglycan component of the high-molecular-weight PG was completely degraded by chondroitinase ABC, while a large portion was resistant to chondroitinase AC, indicating the presence of dermatan sulfate (DS). These CS/DS chains were of unusually large size (Mr approximately 150,000) as estimated by chromatography on Sepharose CL-4B, relating the elution position to hyaluronan standards. Moreover, the chains were found to have a lower surface charge density than standard CS, and may therefore be undersulfated. After reduction and alkylation the high-molecular-weight PGs were included on both Sepharose CL-2B and Sephacryl S-1000 columns, eluting at Kav 0.2 and 0.4, respectively. Hence, the high-molecular-weight material appears to consist of large PG complexes, stabilized by intermolecular disulfide bonds. A CS/DSPG of similar size as the reduced monomeric form of the high-molecular-weight PG was found in small amounts in the total extract of 35S-labeled material.     

Distinct synthetic and structural characteristics of proteoglycans produced by cultured artery smooth muscle cells of atherosclerosis-susceptible pigeons

Proteoglycan (PG) metabolism by aortic smooth muscle cell cultures derived from atherosclerosis-susceptible White Carneau (WC) and -resistant Show Racer (SR) pigeons was compared using [35S]sodium sulfate and [3H]serine or [3H]glucosamine as labeling precursors. Chondroitin sulfate (CS) PG and dermatan sulfate (DS) PG were the major PG secreted into the medium by both cell types. Total PG production, whether measured by incorporation of radiolabel into either core protein or glycosaminoglycan chains, was consistently lower in WC compared to SR cultures at several time points. This difference was due in part to lower (30-37%) PG synthesis in WC cells, but degradation of newly synthesized PG was an important contributor. A pulse-chase study indicated that of the total radiolabeled PG present at time O, only 47% was present at 24 h in WC cultures compared to 88% in SR cultures. The large CS-PG appeared to be the primary target for degradation in WC cells, and this selective processing resulted in a higher DS-PG:CS-PG ratio in these cultures. Structural studies indicated similar core protein and glycosaminoglycan chain sizes within a PG type for both cell types. PG monomer composition differed, however, by a higher sulfation of WC CS-PG compared to SR CS-PG and by a disaccharide sulfation position favoring 6-sulfation in WC PG and 4-sulfation in SR PG.     

Easy HPLC-based separation and quantitation of chondroitin sulphate and hyaluronan disaccharides after chondroitinase ABC treatment

The sulphation patterns of glycosaminoglycan (GAG) chains are decisive for the biological activity of their proteoglycan (PG) templates for sugar chain polymerization and sulphation. The amounts and positions of sulphate groups are often determined by HPLC analysis of disaccharides resulting from enzymatic degradation of the GAG chains. While heparan sulphate (HS) and heparin are specifically degraded by heparitinases, chondroitinases not only degrade chondroitin sulphate (CS) and dermatan sulphate (DS), but also the protein-free and unsulphated GAG hyaluronan (HA). Thus, disaccharide preparations derived by chondroitinase degradation may be contaminated by HA disaccharides. The latter will often comigrate in HPLC chromatograms with unsulphated disaccharides derived from CS. We have investigated how variation of pH, amount of enzyme, and incubation time affects disaccharide formation from CS and HA GAG chains. This allowed us to establish conditions where chondroitinase degrades CS completely for quantification of all the resulting disaccharides, with negligible degradation of HA, allowing subsequent HA analysis. In addition, we present simple methodology for disaccharide analysis of small amounts of CS attached to a hybrid PG carrying mostly HS after immune isolation. Both methods are applicable to small amounts of GAGs synthesized by polarized epithelial cells cultured on permeable supports.     

Sulfated glycosaminoglycans: their distinct roles in stem cell biology

Sulfated glycosaminoglycan (GAG) chains are a class of long linear polysaccharides that are covalently attached to multiple core proteins to form proteoglycans (PGs). PGs are major pericellular and extracellular matrix components that surround virtually all mammalian cell surfaces, and create conducive microenvironments for a number of essential cellular events, such as cell adhesion, cell proliferation, differentiation, and cell fate decisions. The multifunctional properties of PGs are mostly mediated by their respective GAG moieties, including chondroitin sulfate (CS), heparan sulfate (HS), and keratan sulfate (KS) chains. Structural divergence of GAG chains is enzymatically generated and strictly regulated by the corresponding biosynthetic machineries, and is the major driving force for PG functions. Recent studies have revealed indispensable roles of GAG chains in stem cell biology and technology. In this review, we summarize the current understanding of GAG chain-mediated stem cell niches, focusing primarily on structural characteristics of GAG chains and their distinct regulatory functions in stem cell maintenance and fate decisions.     

Changes in the sulfation extent of membrane-associated proteoglycans produced by sertoli cells in culture

Sertoli cells in culture synthesize two different membrane-associated proteoglycans (MA-PG): a proteoglycan containing heparan sulfate (HS) and chondroitin sulfate (CS) glycosaminoglycan (GAG) chains and a CS-PG containing only CS-GAG chains. The structure of these molecules is regulated by the presence of fetal calf serum (FCS) in the culture medium. Changes in the concentration of FCS resulted in changes in the total ³⁵SO₄ incorporation into MA-PG and a shift in the elution profile of each component subjected to ion-exchange chromatography. Thus, without FCS, the incorporation was low, while in 1% and 10% FCS, the uptake of the precursor was 1.7 and 4.5 times higher, respectively. MA-PG synthesized by Sertoli cells cultured in 10% FCS eluted from DEAE-Sephacel columns at higher salt concentration than the MA-PG synthesized by cells cultured in 0% or 1% FCS. Double-labeled experiments showed that the ³⁵SO_4/3H-glucosamine ratio incorporated into MA-PG produced by Sertoli cells, increased from 17.6 to 23.6 and 50.9 in cells cultured at 0, 1, and 10% FCS, respectively. However, the presence of FCS affected neither the hydrodynamic size nor the chemical nature of GAG chains of MA-PG. These results show that changes in the FCS concentration promote changes in the sulfation extent of MA-PG molecules produced by Sertoli cells.     

培养的人脐静脉内皮细胞合成的蛋白聚糖

以[~(35)S]-Na_2SO_4为示踪物,观察培养的人脐静脉内皮细胞(EC)合成及分泌的蛋白聚糖(PG),经DEAE-Sephacel离子交换及Sepharose6B凝胶滤柱层析分析发现细胞层及培养液均含有三种PG单体,即硫酸乙酰肝素蛋白聚糖(HS-PG)、硫酸软骨素蛋白聚糖(CS-PG)及硫酸皮肤素蛋白聚糖(DS-PG)HS-PG又可分为大小两种,前者(HS-PG_L)位于V_o处,后者(HS-PG_s)Kd=0.53(sepharose6B);CS-PG/DS-PG分为三个峰,峰Ⅰ位于V_0处,峰Ⅱ、峰Ⅲ的Kd值分别为0.26及0.52(sepharose6B)。汇合前后细胞层及培养液中各种PG的含量不同。细胞层PG总量汇合前低于汇合后,无论是细胞层还是培养液汇合前HS-PG_L均低于汇合后,HS-PG_L与HS-PG_s比值亦为汇合前低于汇合后,而CS-PG/DS-PG含量则高于汇合后。汇合前后EC合成及分泌PG的差异与文献报道的EC损伤及正常者类似。     

Cell density-dependent regulation of proteoglycan synthesis by transforming growth factor-beta(1) in cultured bovine aortic endothelial cells

The regulation of vascular endothelial cell behavior during angiogenesis and in disease by transforming growth factor-beta(1) (TGF-beta(1)) is complex, but it clearly involves growth factor-induced changes in extracellular matrix synthesis. Proteoglycans (PGs) synthesized by endothelial cells contribute to the formation of the vascular extracellular matrix and also influence cellular proliferation and migration. Since the effects of TGF-beta(1) on vascular smooth muscle cell growth are dependent on cell density, it is possible that TGF-beta(1) also directs different patterns of PG synthesis in endothelial cells at different cell densities. In the present study, dense and sparse cultures of bovine aortic endothelial cells were metabolically labeled with [(3)H]glucosamine, [(35)S]sulfate, or (35)S-labeled amino acids in the presence of TGF-beta(1). The labeled PGs were characterized by DEAE-Sephacel ion exchange chromatography and Sepharose CL-4B molecular sieve chromatography. The glycosaminoglycan M(r) and composition were analyzed by Sepharose CL-6B chromatography, and the core protein M(r) was analyzed by SDS-polyacrylamide gel electrophoresis, before and after digestion with papain, heparitinase, or chondroitin ABC lyase. These experiments indicate that the effect of TGF-beta(1) on vascular endothelial cell PG synthesis is dependent on cell density. Specifically, TGF-beta(1) induced an accumulation of small chondroitin/dermatan sulfate PGs (CS/DSPGs) with core proteins of approximately 50 kDa in the medium of both dense and sparse cultures, but a cell layer-associated heparan sulfate PG with a core protein size of approximately 400 kDa accumulated only in dense cultures. Moreover, only in the dense cell cultures did TGF-beta(1) cause CS/DSPG hydrodynamic size to increase, which was due to the synthesis of CS/DSPGs with longer glycosaminoglycan chains. The heparan sulfate PG and CS/DSPG core proteins were identified as perlecan and biglycan, respectively, by Western blot analysis. The present data suggest that TGF-beta(1) promotes the synthesis of both perlecan and biglycan when endothelial cell density is high, whereas only biglycan synthesis is stimulated when the cell density is low. Furthermore, glycosaminoglycan chains are elongated only in biglycan synthesized by the cells at a high cell density.     

正常及损伤后修复的内皮细胞合成的蛋白聚糖

用自制尼龙刷将培养至汇合的人脐静脉内皮细胞刮伤后,造成规则的内皮细胞缺失区。继续培养可见,原有的内皮钿胞很快迁移到缺失区,并分裂增殖。约48小时新生的内皮细胞即将缺失区全部修复而形成新的汇合单层。以DEAE-Sephacel离子交换及Sepharose 6B凝胶过滤柱层析分析损伤后修复的内皮细胞合成的蛋白聚糖时发现所合成的蛋白聚糖总量减少;硫酸乙酰肝素蛋白聚糖合成相对减少、而硫酸软骨素及/或硫酸皮肤素蛋白聚糖合成相对增多。说明:伴随着内皮细胞的损伤后修复其蛋白聚糖的合成也有质和量的改变。     

Identification of monoclonal antibodies that recognize novel epitopes in native chondroitin/dermatan sulfate glycosaminoglycan chains: their use in mapping functionally distinct domains of human skin.

Five monoclonal antibodies (MAb), 7D4, 4C3, 6C3, 4D3, and 3C5, were produced in mice immunized with high buoyant density embryonic chick bone marrow proteoglycans (PGs) as antigen. All of these MAb recognized epitopes in native chick bone marrow and cartilage PGs which could be selectively removed by chondroitinase ABC and chondroitinase AC II, indicating that their epitopes were present in chondroitin sulfate glycosaminoglycans (GAGs). These MAb recognized epitopes present in purified cartilage PGs obtained from a wide variety of different vertebrate species. However, none of the new MAb detected epitopes in Swarm rat chondrosarcoma PG. On the basis of these results, we propose that these MAb recognize novel epitopes located in chondroitin sulfate/dermatan sulfate glycosaminoglycan (CS/DS GAG) chains, representing at least four and possibly five different structures. Immunocytochemical studies have shown that the epitopes identified by these new MAb are differentially distributed in tissues. All of these MAb immunocytochemically detected epitopes in embryonic chick cartilage and bone marrow. Three of them (4C3, 7D4, and 6C3) recognized epitopes in adult human skin. All three detected epitopes in the epidermis, one (6C3) strongly detected epitopes in the papillary dermis, and two (4C3, 7D4) detected epitopes in the reticular dermis. Immunostaining patterns in skin using the new MAb directed against native CS/DS structures were distinctly different from those obtained using MAb against the common CS isomers. The distribution of these CS epitopes in functionally distinct domains of different tissues implies that these structures have functional and biological significance.     

In situ ultrastructural characterization of chondroitin sulfate proteoglycans in normal rabbit aorta.

We used a monoclonal antibody recognizing chondroitin sulfate (CS) to investigate by immunocytochemistry the characteristics displayed in situ by aortic proteoglycans (PG) containing CS side chains. The antibody specifically precipitated metabolically labeled PG from aortic extracts. Anti-CS specificity was also tested directly on tissue sections and was confirmed by the virtual abolition of immunolabeling on those previously digested with CS-specific enzymes. The overall CS-PG distribution assessed by light microscopy after embedding in Lowicryl KM4 by silver-enhanced immunogold recapitulated that obtained on frozen sections with immunoperoxidase. Extracellular concentrations of CS-PG were very high in the innermost regions of aorta and decreased in the media. The reaction was weak and diffuse in the adventitia. By electron microscopy, the detailed labeling of CS-PG discriminated patterns of organization at both the regional and the molecular level and enabled morphometric estimations. In relation to other components of the extracellular matrix, we found that CS-PG and elastin mutually excluded each other, while two types of CS-PG were differently associated with collagen within media or adventitia. The use of high-resolution immunodetection for the in situ characterization of aortic CS-PG could add specific information relevant to many biological processes in which these molecules have been implicated.     

In vitro control of neuronal polarity by glycosaminoglycans.

We have studied the effects of proteoglycans (PGs) and glycosaminoglycans (GAGs) on the growth and morphology of neurons in culture. PGs from glial cells or Engelbreth-Holm-Swarm tumor cells (EHS), pure bovine kidney heparan sulfate (HS), shark cartilage type C chondro?tin sulfate (CSc) and bovine mucosa dermatan sulfate (DS) added to embryonic rat neurons strongly enhanced total neurite growth after 48 h in vitro. No trophic effects were seen when PGs treated with a mixture of glycanases were used. PGs, CSc and HS not only enhanced neurite growth but induced the appearance of a majority of neurons with a single long axon whereas, in contrast, DS increased dendrite growth. GAGs bound to the cell surface and were rapidly internalized, a feature that correlated well with the absence of neurotrophicity of GAGs previously immobilized on the culture substratum. Although the mechanisms involved in GAGs neurotrophic effects and in the separate regulation of neuronal polarity by HS and DS were not elucidated, we found that, as opposed to HS, DS was able to enhance neuronal adhesion and spreading and to maintain a high level of expression of microtubule-associated protein 2 (MAP2), a specific dendritic marker. This finding confirms and extends our previous observations on the role of adhesion in the regulation of dendrite growth.     

Biosynthesis of proteoglycans by proliferating and differentiating normal human keratinocytes cultured in serum-free medium

Normal human keratinocytes (NHK) were cultured in serum-free medium, containing low (0.1 mM) or high (2 mM) calcium, to obtain proliferating and differentiating cultures, respectively. Proteoglycan (PG) synthesis of proliferating and differentiating NHK was investigated. Cultures were labeled with 35S-sulfate, and the PGs were extracted from medium and cell layer. The newly synthesized PGs were isolated by ion-exchange chromatography on a column of DEAE-Sephacel. The molecular properties of the PGs and the size and composition of glycosaminoglycans (GAGs) were determined. In general, the PGs are relatively small size (Mr 70,000-120,000). The PGs of proliferating cultures are larger in molecular size than the PGs of differentiating cultures, and this is due to the degradation of the GAG chains. The molecular weight of the GAG chains of proliferating NHK ranged from 4,800 to 22,000, and the range for GAGs from differentiating cultures varied from 2,800 to 9,600. By compositional analysis, these PGs proved to contain heparan sulfate, chondroitin sulfate, and dermatan sulfate as determined by nitrous acid degradation, and chondroitinase ACII and ABC digestion. No significant differences were found in the overall GAG composition of the medium secreted PGs of proliferating and differentiating cultures. In contrast, cell-associated PGs of differentiating cells had higher levels of heparan sulfate than those of proliferating cells.     

CD44, a cell surface chondroitin sulfate proteoglycan, mediates binding of interferon-gamma and some of its biological effects on human vascular smooth muscle cells.

Several cytokines and growth factors act on cells after their association with the glycosaminoglycan (GAG) moiety of cell surface proteoglycans (PGs). Interferon-gamma (IFN-gamma) binds to GAG; however, the relevance of this interaction for the biological activity of IFN-gamma on human cells remains to be established. Human arterial smooth muscle cells (HASMC), the main cells synthesizing PG in the vascular wall, respond markedly to IFN-gamma. We found that treatment of HASMC with chondroitinase ABC, an enzyme that degrades chondroitin sulfate GAG, reduced IFN-gamma binding by more than 50%. This treatment increased the affinity of 125I-IFN-gamma for cells from a Kd value of about 93 nM to a Kd value of about 33 nM. However, the total binding was reduced from 9. 3 +/- 0.77 pmol/microg to 3.0 +/- 0.23 pmol/mg (n = 4). Interestingly, pretreatment with chondroitinase ABC reduced significantly the cellular response toward IFN-gamma. The interaction of IFN-gamma with chondroitin sulfate GAG was confirmed by affinity chromatography of isolated cell-associated 35S-, 3H-labeled PG on a column with immobilized IFN-gamma. The cell-associated PG that binds to IFN-gamma was a chondroitin sulfate PG (CSPG). This CSPG had a core protein of approximately 110 kDa that was recognized by anti-CD44 antibodies on Western blots. High molecular weight complexes between IFN-gamma and chondroitin 6-sulfate were observed in gel exclusion chromatography. Additions of chondroitin 6-sulfate to cultured HASMC antagonized the antiproliferative effect and expression of major histocompatibility complex II antigens induced by IFN-gamma. These results indicate that IFN-gamma binds with low affinity to the chondroitin sulfate GAG moiety of the cell surface CSPG receptor CD44. This interaction may increase the local concentration of IFN-gamma at the cell surface, thus facilitating its binding to high affinity receptors and modulating the ability of IFN-gamma to signal a cellular response.     

Characterization of proteoglycans synthesized by rat thyroid cells in culture and their response to thyroid-stimulating hormone

A Fisher rat thyroid cell line was maintained in culture and the cells were labeled with [3H]glucosamine, [35S]sulfate, and [35S]cysteine to examine the synthesis of proteoglycans. 3H and 35S radioactivity from these precursors were incorporated into both chondroitin sulfate (CS) and heparan sulfate (HS) proteoglycans. CS proteoglycans were almost exclusively secreted into the medium while HS proteoglycans remained mainly associated with the cell layer. Single chain glycosaminoglycans released by papain digestion or alkaline borohydride treatment of either the CS or HS proteoglycans had average molecular weights of approximately 30,000 on Sepharose CL-6B chromatography. Both CS and HS proteoglycans were relatively small and contained only one or two glycosaminoglycans chains. 3H and 35S incorporation into both CS and HS proteoglycans were increased by thyroid-stimulating hormone (TSH) in a dose-dependent manner, which is in part explained by an adenylate cyclase-dependent mechanism as indicated by a similar effect in response to dibutyryl cAMP. TSH enhanced the incorporation of 35S into CS from [35S]cysteine about 1.5-fold and that from [35S]sulfate about 2-fold. This result demonstrated that the increased 35S incorporation from the [35S]sulfate precursor reflects an actual increase in sulfate incorporation and is not simply a result from an apparent increase in specific activity of the phosphoadenosine phosphosulfate donor. Analysis of disaccharides from chondroitinase digests revealed that the proportion of non-sulfated, 4-sulfated, and 6-sulfated disaccharides was not altered appreciably by TSH. These results, together with the disproportionate increase in 3H incorporation into CS from [3H]glucosamine, indicated that TSH increased the specific activity of the 3H label as well. Chase experiments revealed that CS proteoglycans were rapidly (t1/2 = 15 min) secreted into the medium and that the degradation of cell-associated proteoglycans was enhanced by TSH.     

Perlecan domain 1 recombinant proteoglycan augments BMP-2 activity and osteogenesis

ABSTRACT: BACKGROUND: Many growth factors, such as bone morphogenetic protein (BMP)-2, have been shown to interact with polymers of sulfated disacharrides known as heparan sulfate (HS) glycosaminoglycans (GAGs), which are found on matrix and cell-surface proteoglycans throughout the body. HS GAGs, and some more highly sulfated forms of chondroitin sulfate (CS), regulate cell function by serving as co-factors, or co-receptors, in GF interactions with their receptors, and HS or CS GAGs have been shown to be necessary for inducing signaling and GF activity, even in the osteogenic lineage. Unlike recombinant proteins, however, HS and CS GAGs are quite heterogenous due, in large part, to post-translational addition, then removal, of sulfate groups to various positions along the GAG polymer. We have, therefore, investigated whether it would be feasible to deliver a DNA pro-drug to generate a soluble HS/CS proteoglycan in situ that would augment the activity of growth-factors, including BMP-2, in vivo. RESULTS: Utilizing a purified recombinant human perlecan domain 1 (rhPln.D1) expressed from HEK 293 cells with HS and CS GAGs, tight binding and dose-enhancement of rhBMP-2 activity was demonstrated in vitro. In vitro, the expressed rhPln.D1 was characterized by modification with sulfated HS and CS GAGs. Dose-enhancement of rhBMP-2 by a pln.D1 expression plasmid delivered together as a lyophilized single-phase on a particulate tricalcium phosphate scaffold for 6 or more weeks generated up to 9 fold more bone volume de novo on the maxillary ridge in a rat model than in control sites without the pln.D1 plasmid. Using a significantly lower BMP-2 dose, this combination provided more than 5 times as much maxillary ridge augmentation and greater density than rhBMP-2 delivered on a collagen sponge (InFuse[trade mark sign]). CONCLUSIONS: A recombinant HS/CS PG interacted strongly and functionally with BMP-2 in binding and cell-based assays, and, in vivo, the pln.247 expression plasmid significantly improved the dose-effectiveness of BMP-2 osteogenic activity for in vivo de novo bone generation when delivered together on a scaffold as a single-phase. The use of HS/CS PGs may be useful to augment GF therapeutics, and a plasmid-based approach has been shown here to be highly effective.     

Basement membrane and interstitial proteoglycans produced by MDCK cells correspond to those expressed in the kidney cortex

Multiple proteoglycans (PGs) are present in all basement membranes (BM) and may contribute to their structure and function, but their effects on cell behavior are not well understood. Their postulated functions include: a structural role in maintaining tissue histoarchitecture, or aid in selective filtration processes; sequestration of growth factors; and regulation of cellular differentiation. Furthermore, expression PGs has been found to vary in several disease states. In order to elucidate the role of PGs in the BM, a well-characterized model of polarized epithelium, Madin-Darby canine kidney (MDCK) cells has been utilized. Proteoglycans were prepared from conditioned medium by DEAE anion exchange chromatography. The eluted PGs were treated with heparitinase or chondroitinase ABC (cABC), separately or combined, followed by SDS-PAGE. Western blot analysis, using antibodies specific for various PG core proteins or CS stubs generated by cABC treatment, revealed that both basement membrane and interstitial PGs are secreted by MDCK cells. HSPGs expressed by MDCK cells are perlecan, agrin, and collagen XVIII. Various CSPG core proteins are made by MDCK cells and have been identified as biglycan, bamacan, and versican (PG-M). These PGs are also associated with mammalian kidney tubules in vivo.