Pancreatic cancer cells respond to type I collagen by inducing snail expression to promote membrane type 1 matrix metalloproteinase-dependent collagen invasion

Pancreatic ductal adenocarcinoma (PDAC) is characterized by pronounced fibrotic reaction composed primarily of type I collagen. Although type I collagen functions as a barrier to invasion, pancreatic cancer cells have been shown to respond to type I collagen by becoming more motile and invasive. Because epithelial-mesenchymal transition is also associated with cancer invasion, we examined the extent to which collagen modulated the expression of Snail, a well known regulator of epithelial-mesenchymal transition. Relative to cells grown on tissue culture plastic, PDAC cells grown in three-dimensional collagen gels induced Snail. Inhibiting the activity or expression of the TGF-β type I receptor abrogated collagen-induced Snail. Downstream of the receptor, we showed that Smad3 and Smad4 were critical for the induction of Snail by collagen. In contrast, Smad2 or ERK1/2 was not involved in collagen-mediated Snail expression. Overexpression of Snail in PDAC cells resulted in a robust membrane type 1-matrix metalloproteinase (MT1-MMP, MMP-14)-dependent invasion through collagen-coated transwell chambers. Snail-expressing PDAC cells also demonstrated MT1-MMP-dependent scattering in three-dimensional collagen gels. Mechanistically, Snail increased the expression of MT1-MMP through activation of ERK-MAPK signaling, and inhibiting ERK signaling in Snail-expressing cells blocked two-dimensional collagen invasion and attenuated scattering in three-dimensional collagen. To provide in vivo support for our findings that Snail can regulate MT1-MMP, we examined the expression of Snail and MT1-MMP in human PDAC tumors and found a statistically significant positive correlation between MT1-MMP and Snail in these tumors. Overall, our data demonstrate that pancreatic cancer cells increase Snail on encountering collagen-rich milieu and suggest that the desmoplastic reaction actively contributes to PDAC progression.     

Interaction of the NG2 chondroitin sulfate proteoglycan with type VI collagen

The NG2 chondroitin sulfate proteoglycan is a membrane-associated molecule of approximately 500 kD with a core glycoprotein of 300 kD. Both the complete proteoglycan and a smaller quantity of the 300-kD core are immunoprecipitable with polyclonal and monoclonal antibodies against purified NG2. From some cell lines, the antibodies coprecipitate NG2 and type VI collagen, the latter appearing on SDS- PAGE as components of 140 and 250 kD under reducing conditions. The immunoprecipitation of type VI collagen does not seem to be due to recognition of the collagen by the antibodies, but rather to binding of the collagen to NG2. Studies on the NG2-type VI collagen complex suggest that binding between the two molecules is mediated by protein- protein interactions rather than by ionic interactions involving the glycosaminoglycans. Immunofluorescence double labeling in frozen sections of embryonic rat shows that NG2 and type VI collagen are colocalized in structures such as the intervertebral discs and arteries of the spinal column. In vitro the two molecules are highly colocalized on the surface of several cell lines. Treatment of these cells resulting in a change in the distribution of NG2 on the cell surface also causes a parallel change in type VI collagen distribution. Our results suggest that cell surface NG2 may mediate cellular interactions with the extracellular matrix by binding to type VI collagen.     

CD44/chondroitin sulfate proteoglycan and alpha 2 beta 1 integrin mediate human melanoma cell migration on type IV collagen and invasion of basement membranes.

Tumor cell invasion of basement membranes (BM) represents one of the critical steps in the metastatic process. Tumor cell recognition of individual BM matrix components may involve individual cell adhesion receptors, such as integrins or cell surface proteoglycans, or may involve a coordinate action of both types of receptors. In this study, we have focused on the identification of a cell surface CD44/chondroitin sulfate proteoglycan (CSPG) and alpha 2 beta 1 integrin on human melanoma cells that are both directly involved in the in vitro invasion of reconstituted BM via a type IV collagen-dependent mechanism. Interfering with cell surface expression of human melanoma CSPG with either p-nitro-phenyl-beta-D-xylopyranoside treatment or anti-CD44 monoclonal antibody (mAb) preincubation (mAb) preincubation inhibits melanoma cell invasion through reconstituted BM. These treatments also strongly inhibit melanoma cell migration on type IV collagen, however, they are ineffective at inhibiting cell adhesion to type IV collagen. Purified melanoma cell surface CD44/CSPG, or purified chondroitin sulfate, bind to type IV collagen affinity columns, consistent with a role for CD44/CSPG-type IV collagen interactions in mediating tumor cell invasion. In contrast, melanoma cell migration on laminin (LM) does not involve CD44/CSPG, nor does CD44/CSPG bind to LM, suggesting that CD44/CSPG-type IV collagen interactions are specific in nature. Additionally, anti-alpha 2 and anti-beta 1 integrin mAbs are capable of blocking melanoma cell invasion of reconstituted BM. Both of these anti-integrin mAbs inhibit melanoma cell adhesion and migration on type IV collagen, whereas only anti-beta 1 mAb inhibits cell adhesion to LM. Collectively, these results indicate that melanoma cell adhesion to type IV collagen is an important consideration in invasion of reconstituted BM in vitro, and suggest that CD44/CSPG and alpha 2 beta 1 integrin may collaborate to promote human melanoma cell adhesion, migration, and invasion in vivo.     

A cell surface chondroitin sulfate proteoglycan, immunologically related to CD44, is involved in type I collagen-mediated melanoma cell motility and invasion

The metastatic spread of tumor cells occurs through a complex series of events, one of which involves the adhesion of tumor cells to extracellular matrix (ECM) components. Multiple interactions between cell surface receptors of an adherent tumor cell and the surrounding ECM contribute to cell motility and invasion. The current studies evaluate the role of a cell surface chondroitin sulfate proteoglycan (CSPG) in the adhesion, motility, and invasive behavior of a highly metastatic mouse melanoma cell line (K1735 M4) on type I collagen matrices. By blocking mouse melanoma cell production of CSPG with p-nitrophenyl beta-D-xylopyranoside (beta-D-xyloside), a compound that uncouples chondroitin sulfate from CSPG core protein synthesis, we observed a corresponding decrease in melanoma cell motility on type I collagen and invasive behavior into type I collagen gels. Melanoma cell motility on type I collagen could also be inhibited by removing cell surface chondroitin sulfate with chondroitinase. In contrast, type I collagen-mediated melanoma cell adhesion and spreading were not affected by either beta-D-xyloside or chondroitinase treatments. These results suggest that mouse melanoma CSPG is not a primary cell adhesion receptor, but may play a role in melanoma cell motility and invasion at the level of cellular translocation. Furthermore, purified mouse melanoma cell surface CSPG was shown, by affinity chromatography and in solid phase binding assays, to bind to type I collagen and this interaction was shown to be mediated, at least in part, by chondroitin sulfate. Additionally we have determined that mouse melanoma CSPG is composed of a 110-kD core protein that is recognized by anti-CD44 antibodies on Western blots. Collectively, our data suggests that interactions between a cell surface CD44-related CSPG and type I collagen in the ECM may play an important role in mouse melanoma cell motility and invasion, and that the chondroitin sulfate portion of the proteoglycan seems to be a critical component in mediating this effect.     

Interactions between chondroitin sulfate proteoglycan, fibronectin, and collagen

A proteoglycan isolated from a rat yolk sac tumor and characterized as a chondroitin sulfate proteoglycan with a smaller amount of dermatan sulfate was studied with respect to complex formation with collagen and fibronectin. The proteoglycan co-precipitated with native collagen from neutral salt solutions at 6 degrees C and 37 degrees C. Addition of fibronectin in such precipitation mixtures resulted in incorporation of fibronectin to the precipitate. Treatment of the proteoglycan with alkali to separate the glycosaminoglycan chains from the protein part and digestion of the protein part with papain greatly reduced the capacity of the proteoglycan to precipitate collagen and fibronectin. A defined extracellular matrix as represented by the complexes of collagen, proteoglycan, and fibronectin constructed here may be useful for studies on the biological effects of extracellular matrices. The multiple interactions of matrix macromolecules exemplified by these results may play a role in the formation of extracellular matrices and in the maintenance of their integrity.     

Basement membrane zone type XV collagen is a disulfide-bonded chondroitin sulfate proteoglycan in human tissues and cultured cells

Type XV collagen has a widespread distribution in human tissues, but a nearly restricted localization in basement membrane zones. The alpha1(XV) chain contains a highly interrupted collagenous region of 577 residues, and noncollagenous amino- and carboxyl-terminal domains of 530 and 256 residues, respectively. Cysteines are present in each domain and consensus sequences for O-linked glycosaminoglycans are situated in the amino terminus and in two large, noncollagenous interruptions. We now report that type XV collagen is a chondroitin sulfate proteoglycan in human tissues and cultured cells, and that the alpha chains are covalently linked by interchain disulfide bonds only between the two cysteines in the collagenous region. Western blotting of tissue extracts revealed a diffuse smear with a mean size >/=400 kDa, which after chondroitinase digestion resolved into a 250-kDa band in umbilical cord, and 250- and 225-kDa bands in placenta, lung, colon, and skeletal muscle. The latter two bands were also directly visualized by alcian blue/silver staining of a purified placenta extract. In a human rhabdomyosarcoma cell line, almost all of the newly synthesized type XV collagen was secreted into the medium and upon chondroitinase digestion just the 250-kDa alpha chain was generated. Chondroitinase plus collagenase digestion of tissue and medium proteins followed by Western blotting using domain-specific antibodies revealed a 135-kDa amino-terminal fragment containing glycosaminoglycan chains and a 27-kDa fragment representing the intact carboxyl terminus. However, a truncated carboxyl peptide of approximately 8-kDa was also evident in tissue extracts containing the 225-kDa form. Our data suggest that the 225-kDa form arises from differential carboxyl cleavage of the 250-kDa form, and could explain the approximately 19-kDa endostatin-related fragments (John, H., Preissner, K. T., Forssmann, W.-G., and St?ndker, L. (1999) Biochemistry 38, 10217-10224), which may be liberated from the alpha1(XV) chain.     

Melanoma chondroitin sulfate proteoglycan enhances FAK and ERK activation by distinct mechanisms

Melanoma chondroitin sulfate proteoglycan (MCSP) is an early cell surface melanoma progression marker implicated in stimulating tumor cell proliferation, migration, and invasion. Focal adhesion kinase (FAK) plays a pivotal role in integrating growth factor and adhesion-related signaling pathways, facilitating cell spreading and migration. Extracellular signal-regulated kinase (ERK) 1 and 2, implicated in tumor growth and survival, has also been linked to clinical melanoma progression. We have cloned the MCSP core protein and expressed it in the MCSP-negative melanoma cell line WM1552C. Expression of MCSP enhances integrin-mediated cell spreading, FAK phosphorylation, and activation of ERK1/2. MCSP transfectants exhibit extensive MCSP-rich microspikes on adherent cells, where it also colocalizes with alpha4 integrin. Enhanced activation of FAK and ERK1/2 by MCSP appears to involve independent mechanisms because inhibition of FAK activation had no effect on ERK1/2 phosphorylation. These results indicate that MCSP may facilitate primary melanoma progression by enhancing the activation of key signaling pathways important for tumor invasion and growth.     

Immunofluorescence studies on cartilage matrix synthesis : The synthesis of link protein, chondroitin sulfate proteoglycan monomer and type II collagen

A comparison of the synthesis and deposition of fibrous type II collagen and the constituents of chondroitin sulfate proteoglycan (CSPG) aggregates, CSPG monomer and link protein, was made for chicken sternal chondrocytes in culture, using simultaneous double immunofluorescence and lectin localization. Chondrocytes deposited only CSPG constituents--and not type II collagen--into the extracellular matrix (ECM). Intracellular precursors of CSPG monomer were localized primarily in perinuclear regions, but were observed in other cytoplasmic vesicles as well. Link protein antibodies stained the same intracellular structures, but stained the perinuclear cytoplasm less intensely. In contrast, type II procollagen was distributed in vesicles throughout the cytoplasm and was clearly absent from the distinctive, CSPG precursor-containing vesicles. Fluorescence-labelled lectins were used to further identify intracellular membrane compartments. Wheat germ agglutinin (WGA) and Ricinus lectins (which recognize carbohydrates added in the Golgi) stained the perinuclear cytoplasm, while concanavalin A (conA) (which recognizes mannose-rich oligosaccharides added co-translationally) stained vesicles throughout the rest of the cytoplasm and not the perinuclear cytoplasm. The distinctive CSPG-containing vesicles were not stained with WGA or Ricinus agglutinins. Data presented elsewhere demonstrate that the vesicles do not react with monoclonal antibodies which recognize chondroitin sulfate (CS) or keratan sulfate (KS) determinants. Thus, we conclude that the vesicles accumulate CSPG precursors which have not been modified by Golgi-mediated processes. The data indicate that matrix molecules may be segregated selectively prior to transit through the Golgi complex. The co-distribution of link protein and CSPG monomer precursors in vesicles prior to further, Golgi-mediated modification may reflect an as yet undetermined function of these vesicles in the processing or assembly of CSPG.     

Oversulfated chondroitin sulfate proteoglycan in cultured human peritoneal macrophages

Human peritoneal macrophages were cultured in vitro and labeled with [35S]-sulfate. Both on day 1 and day 6 in culture the cells were found to synthesize exclusively chondroitin sulfate proteoglycan, the main part (70%) being associated with the medium after a 20 hour pulse. The glycosaminoglycan chains were found to be oversulfated both after 1 and 6 days in culture, due to the presence of disulfated disaccharide units.     

Cell density-dependent expression of chondroitin sulfate proteoglycan in cultured human monocytes

Monocytes were isolated and established in vitro at different cell densities. The incorporation of [35S]sulfate into macromolecules in monocytes (day 1 in culture) and monocyte-derived macrophages (day 5 in culture) was found to increase with decreasing cell density in approximately the same way in both day 1 and day 5 cell cultures. [35S]Sulfate was found to be incorporated almost exclusively into chondroitin sulfate proteoglycan (CSPG) in both high and low density monocyte and monocyte-derived macrophage cultures. The molecular size of the [35S]CSPGs produced by the high and low cell density cultures were not found to differ as judged by gel chromatography elution patterns. The molecular size and the structure of the glycosaminoglycan chains were found to be almost similar in high and low density day 1 and day 5 cultures. Only a small degree of proteoglycan degradation could be observed in both high and low density cultures. Furthermore, cell density-dependent differences in CSPG biosynthesis could be observed already 2 h after the establishment of the cultures, indicating that a process of "down-regulation" in high density cultures was already in operation. The glycosaminoglycan synthesis in high cell density day 1 cultures could be increased slightly following exposure to 0.5 mM benzyl-beta-D-xyloside, but not to the same level as that observed in untreated low cell density cultures. By contrast, the expression of 35S-macromolecules by cells cultured at high cell density for 5 days could be increased by xyloside treatment almost to the same level as that observed in the low density cultures.     

DACS, novel matrix structure composed of chondroitin sulfate proteoglycan in the brain

Chondroitin sulfate proteoglycans (CSPGs) are major components of the extracellular matrix (ECM) in the brain. In the adult cerebral cortex, there are special CSPG-containing structures known as perineuronal nets (PNNs), which are highly condensed ECM structures. Here, we report a novel CSPG-containing structure distinct from PNNs in the adult mouse cerebral cortex. An anti-chondroitin sulfate antibody CS56 delineated a structure with a unique morphology like a dandelion clock. Accordingly, we named it DAndelion Clock-like Structure (DACS). Immunohistochemical evidence showed that DACSs surrounded a group of NeuN-positive/GABA-negative neurons. At ultrastructural level, CS56-immunoreactivities were localized in the cytoplasm and on the membrane of astrocytes. As the postnatal cerebral cortex matured, DACSs became visible around the end of the critical period. This is the first report demonstrating the presence of an ECM structure DACS composed of CSPGs around a group of cortical neurons in the adult cerebral cortex.     

Presence of a laminin-binding chondroitin sulfate proteoglycan at the cell surface of a human melanoma cell Mel-85

Working with Mel-85 (a human melanoma cell line), we have been able to detect a laminin-binding molecule with an apparent molecular mass of 100/110 kDa (Mel-85-LBM). Reduction with -mercaptoethanol decreases its molecular mass but does not affect its ability to bind laminin. This laminin interaction seems to be very specific since Mel-85-LBM binds laminin, but not fibronectin, vitronectin or type I collagen in affinity chromatography experiments. The molecule has a negative net charge at physiological pH and binds laminin in a divalent cation dependent way. Mel-85-LBM was metabolically radiolabeled with sodium [³⁵S]-sulfate and chemical -elimination of purified Mel-85-LBM releases chondroitin sulfate chains. Mel-85-LBM is also sensitive to chondroitinase ABC digestion. These findings show that this molecule is a chondroitin sulfate proteoglycan. The location of this proteoglycan at the cell surface is evidenced by experiments using a polyclonal antiserum raised against purified Mel-85LBM, that specifically reacts with just one molecule by western blotting among Mel-85 total cell extract as well as produces a positive signal by flow cytometry and a fluorescence profile of Mel-85 cells adhered on laminin.     

Kinetics of intracellular processing of chondroitin sulfate proteoglycan core protein and other matrix components

Pulse-chase labeling techniques are used in conjunction with subcellular fractionation and quantitative immunoprecipitation to define the kinetics of intracellular translocation and secretion of proteoglycan core protein, along with link protein and type II collagen. In embryonic chick chondrocytes the core protein is processed very rapidly, exhibiting a t 1/2 in both the rough endoplasmic reticulum and golgi region of less than 10 min. Link protein appears to be processed as rapidly as the core protein, but the kinetics of type II collagen secretion is 3-4 times slower. These results are consistent with possible segregation and coordinate intracellular processing of link protein and core protein, macromolecules which are known to associate extracellularly. In contrast, rat chondrosarcoma chondrocytes translocated and secreted the core protein much more slowly (t 1/2 = 40 min) than the chick cells, perhaps due to the significantly reduced levels of galactosyltransferase I observed in the transformed chondrocytes.     

Modulation of the expression of chondroitin sulfate proteoglycan in stimulated human monocytes

Proteoglycan biosynthesis was studied in human monocytes and monocyte-derived macrophages (MDM) after exposure to typical activators of the monocyte/macrophage system: interferon-gamma (IFN-gamma), lipopolysaccharide (LPS), and phorbol 12-myristate 13-acetate (PMA). By morphological examination, both monocytes and MDM were stimulated by these activators. Treatment with IFN-gamma resulted in a slight decrease in the expression of [35S]chondroitin sulfate proteoglycan (CSPG) in both monocytes and MDM, whereas LPS treatment increased the [35S]CSPG expression 1.8 and 2.2 times, respectively. PMA, in contrast, decreased the CSPG expression 0.4 times in monocytes, whereas MDM were stimulated to increase the biosynthesis 1.9 times. An increase in the sulfate density of the chondroitin sulfate chains was evident following differentiation of monocytes into MDM due to the expression of disulfated disaccharide units of the chondroitin sulfate E type (CS-E). However, monocytes exposed to PMA did also express disaccharides of the chondroitin sulfate E type. Furthermore, the expression of CS-E in MDM was increased 2 times following PMA treatment. An inactive phorbol ester, phorbol 12,13-diacetate, did not affect the expression of CS-E in either monocytes or MDM when compared with control cultures, suggesting that protein kinase C-dependent signal pathways may be involved in the regulation of sulfation of CSPG. Exposure to LPS or IFN-gamma did not lead to any changes in the sulfation of the chondroitin sulfate chains.     

Intracellular features of type II procollagen and chondroitin sulfate proteoglycan synthesis in chondrocytes

The intracellular compartments of chondrocytes involved in the synthesis and processing of type II procollagen and chondroitin sulfate proteoglycan (CSPG) monomer were investigated using simultaneous double immunofluorescence and lectin localization reactions. Type II procollagen was distributed in vesicles throughout the cytoplasm, whereas intracellular precursors of CSPG monomer were accumulated in the perinuclear cytoplasm. In this study, cytoplasmic vesicles that stained intensely with antibodies directed against CSPG monomer but did not react with type II collagen antibodies, also were observed. A monoclonal antibody, 5-D-4, that recognizes keratan sulfate determinants was used to identify the Golgi complex (the site of keratan sulfate chain elongation). Staining with 5-D-4 was restricted to the perinuclear cytoplasm. The vesicles outside the perinuclear cytoplasm that stained intensely with antibodies to CSPG monomer did not react with 5-D-4. Fluorescent lectins were used to characterize further subcellular compartments. Concanavalin A, which reacts with mannose-rich oligosaccharides, did not stain the perinuclear region, but it did stain vesicles throughout the rest of the cytoplasm. Because mannose oligosaccharides are added cotranslationally, the stained vesicles throughout the cytoplasm presumably correspond to the rough endoplasmic reticulum. Wheat germ agglutinin, which recognizes N-acetyl-D-glucosamine and sialic acid (carbohydrates added in the Golgi), stained exclusively the perinuclear cytoplasm. By several criteria (staining with the monoclonal antibody 5-D-4 and with wheat germ agglutinin), the perinuclear cytoplasm seems to correspond to the Golgi complex. The cytoplasmic vesicles that react with anti-CSPG monomer and not with anti-type II collagen contain precursors of CSPG monomer not yet modified by Golgi-mediated oligosaccharide additions (because they are not stained with wheat germ agglutinin or with the anti-keratan sulfate antibody); these vesicles may have a unique function in the processing of CSPG.     

Melanoma chondroitin sulphate proteoglycan regulates cell spreading through Cdc42, Ack-1 and p130cas

Melanoma chondroitin sulphate proteoglycan (MCSP) is a cell-surface antigen that has been implicated in the growth and invasion of melanoma tumours. Although this antigen is expressed early in melanoma progression, its biological function is unknown. MCSP can stimulate the integrin-alpha4 beta1-mediated adhesion and spreading of melanoma cells. Here we show that stimulated MCSP recruits tyrosine-phosphorylated p130 cas, an adaptor protein important in tumour cell motility and invasion. MCSP stimulation also results in a pronounced activation and recruitment of the Rho-family GTPase Cdc42. MCSP-induced spreading of melanoma cells is dependent upon active Cdc42, a Cdc42-associated tyrosine kinase (Ack-1) and tyrosine phosphorylation of p130cas. Furthermore, vectors inhibiting Ack-1 or Cdc42 expression and/or function abrogate MCSP-induced tyrosine phosphorylation and recruitment of p130cas. Our findings indicate that MCSP may modify tumour growth or invasion by a unique signal-transduction pathway that links Cdc42 activation to downstream tyrosine phosphorylation and subsequent cytoskeletal reorganization.     

Cell shape regulates collagen type I expression in human tendon fibroblasts

Understanding the relationship between cell shape and cellular function is important for study of cell biology in general and for regulation of cell phenotype in tissue engineering in particular. In this study, microcontact printing technique was used to create cell-adhesive rectangular and circular islands. The rectangular islands had three aspect ratios: 19.6, 4.9, and 2.2, respectively, whereas circular islands had a diameter of 50 microm. Both rectangular and circular islands had the same area of 1960 microm(2). In culture, we found that human tendon fibroblasts (HTFs) assumed the shapes of these islands. Quantitative immunofluorescence measurement showed that more elongated cells expressed higher collagen type I than did less stretched cells even though cell spreading area was the same. This suggests that HTFs, which assume an elongated shape in vivo, have optimal morphology in terms of expression of collagen type I, which is a major component of normal tendons. Using immunohistochemistry along with cell traction force microscopy (CTFM), we further found that these HTFs with different shapes exhibited variations in actin cytoskeletal structure, spatial arrangement of focal adhesions, and spatial distribution and magnitude of cell traction forces. The changes in the actin cytoskeletal structure, focal adhesion distributions, and traction forces in cells with different shapes may be responsible for altered collagen expression, as they are known to be involved in cellular mechanotransduction.     

Immunocytochemical investigation on the distribution of small chondroitin sulfate-dermatan sulfate proteoglycan in the human

Polyclonal antibodies against the core protein of the small chondroitin sulfate-dermatan sulfate proteoglycan from human skin fibroblast secretions were used, after affinity-purification, as a probe to study localization of crossreactive material in several human tissues by indirect immunocytochemistry. In contrast to skin, kidney, and the adventitial layer of aorta, positive staining of brain, liver, cartilage, and intimal and medial layers of aorta required pre-treatment of tissue sections with chondroitin ABC lyase. In all tissues investigated, antigenic material was present in the interstitial space. Filamentous structures were perpendicularly oriented towards basement membranes. In liver, specific staining was seen along the sinusoidal walls. Reticular fibers with or without focal condensations were seen in cerebral cortex and cerebellum. The results suggest a role of small chondroitin sulfate-dermatan sulfate proteoglycan in cell-matrix interactions.     

Uterine stromal cell chondroitin sulfate proteoglycans bind to collagen type I and inhibit embryo outgrowth in vitro.

Chondroitin sulfate proteoglycans (CSPGs) are the major class of proteoglycans synthesized by mouse uterine stroma in vitro (Jacobs, A. L., and Carson, D. D. (1991). J. Biol. Chem. 266, 15,464-15,473). In the present study, stromal CSPGs were isolated and examined with regard to their ability to bind to specific extracellular matrix (ECM) components. Of a variety of ECM components tested, only collagen type I formed stable complexes with stromal CSPGs in both solid phase and solution binding assays. Proteolytic digestion of the CSPGs did not affect binding and suggested that the protein cores did not participate directly in binding. Furthermore, free chondroitin sulfate polysaccharides do not compete effectively in the binding assays. Therefore, interactions with multiple CS chains and/or the higher charge density afforded by intact CSPGs appear to be required for retention by collagen type I. Intact CSPGs were examined for their ability to modulate embryo attachment and outgrowth in vitro on fibronectin- or collagen type I-coated surfaces. In both cases, intact CSPGs, but not their constituent protein cores or polysaccharides, inhibited both the rate and the extent of outgrowth formation. In addition, embryo outgrowth on stromal ECM was enhanced by predigestion with chondroitinase. Addition of exogenous CSPG markedly retarded embryo outgrowth on stromal matrix. Collectively, these data indicate that stromal cell-derived CSPGs are retained by collagen type I in the stromal interstitial ECM where these molecules may attenuate trophoblast invasive behavior.     

Role of melanoma chondroitin sulphate proteoglycan in patterning stem cells in human interfollicular epidermis

Human interfollicular epidermis is renewed by stem cells that are clustered in the basal layer in a patterned, non-random distribution. Stem cells can be distinguished from other keratinocytes by high expression of beta1 integrins and lack of expression of terminal differentiation markers; they divide infrequently in vivo but form actively growing colonies in culture. In a search for additional stem cell markers, we observed heterogeneous epidermal expression of melanoma chondroitin sulphate proteoglycan (MCSP). MCSP was expressed by those keratinocytes with the highest beta1 integrin levels. In interfollicular epidermis, expression was confined to non-cycling cells and, in culture, to self-renewing clones. However, fluorescence-activated cell sorting on the basis of MCSP and beta1 integrin expression gave no more enrichment for clonogenic keratinocytes than sorting for beta1 integrins alone. To interfere with endogenous MCSP, we retrovirally infected keratinocytes with a chimera of the CD8 extracellular domain and the MCSP cytoplasmic domain. CD8/MCSP did not affect keratinocyte proliferation or differentiation but the cohesiveness of keratinocytes in isolated clones or reconstituted epidermal sheets was greatly reduced. CD8/MCSP caused stem cell progeny to scatter without differentiating. CD8/MCSP did not alter keratinocyte motility but disturbed cadherin-mediated cell-cell adhesion and the cortical actin cytoskeleton, effects that could be mimicked by inhibiting Rho. We conclude that MCSP is a novel marker for epidermal stem cells that contributes to their patterned distribution by promoting stem cell clustering.