Fibrin induces IL-8 expression from human oral squamous cell carcinoma cells.

In recent studies, we have demonstrated that fibrin is present in association with tumor cells in oral squamous cell carcinoma (OSCC) in vivo. We hypothesized that this fibrin can directly induce the expression of known angiogenic factors from oral tumor cells. Since IL-8 is known to be the major inducer of angiogenesis caused by these cells, we examined the ability of fibrin to stimulate IL-8 expression from OSCC cells in vitro. A physiologically relevant concentration of fibrin was found to cause a dose and time-dependent stimulation of IL-8 expression from oral and pharyngeal tumor cells but not from a non-tumorigenic oral cell line. Fibrinogen, thrombin and collagen were all unable to induce significant IL-8 expression, establishing the specificity of fibrin in causing this response. Gel filtration chromatography confirmed the molecular identity of the IL-8 antigen detected in the ELISA system used. These results suggest that fibrin may promote angiogenesis in oral tumors in vivo by directly upregulating the expression of IL-8 from tumor cells.

Aggrecan domains expected to traffic through the exocytic pathway are misdirected to the nucleus.

In this article, we report the misdirected targeting of expressed aggrecan domains. Aggrecan, the chondroitin sulfate (CS) proteoglycan of cartilage, normally progresses through the exocytic pathway. Proteins expressed from constructs containing the putative aggrecan signal sequence (i.e., the first 23 N-terminal amino acids), specified globular (G) domains G1 and/or G3, and a segment of the CS domain were detected in the endoplasmic reticulum (ER) and Golgi complex. Although proteins expressed from constructs containing the putative signal and G3, but lacking G1, were detected to a limited extent in the secretory pathway, they primarily accumulated in nuclei. Discrete nuclear inclusions were seen when G3 was expressed. Immunoelectron microscopic characterization of the inclusions suggested the association of nuclear G3 with other proteins. When signal-free G3 constructs and those with G3 immediately following the N-terminal signal were expressed, abundant dispersed accumulations filled the nucleoplasm. The data suggest first, that signal-free and signal-containing G3 proteins enter the nucleus from the cytosol, and second, that the entry of signal-containing G3 proteins into the ER lumen is inefficient. Hsp25, Hsp70, and ubiquitin were colocalized with nuclear G3, indicating the involvement of chaperones and the degradative machinery in the formation and/or attempted disposal of the abnormal nuclear inclusions. Overall, the results focus attention on (1) intracellular protein trafficking at the ER membrane and the nuclear envelope and (2) chaperone interactions and mechanisms leading to abnormal protein deposition in the nucleus.

Aggrecan synthesis and secretion. A paradigm for molecular and cellular coordination of multiglobular protein folding and intracellular trafficking.

Each globular domain of exported multiglobular proteins putatively undergoes chaperone surveillance in the endoplasmic reticulum lumen. It is difficult to visualize how surveillance of multiple globular domains might be orchestrated and regulated. Aggrecan core protein has been used as a prototype for this problem by examining transfection of informative constructs into Chinese hamster ovary cells. The salient results are as follows: 1) aggrecan's N-terminal G1 domain is minimally secreted, and its flanking Golgi reporter sites are not decorated with glycsoaminoglycan chains; in contrast, its C-terminal G3 domain is readily secreted with flanking GAG chains, and G3 also facilitates G1 secretion; 2) G3 but not G1 can be intracellularly cross-linked to chaperone Hsp25; 3) G3 and Hsp25 remain noncovalently bound and are secreted together when G3 is situated N-terminal to its normal location; 4) exon 15, which encodes the center of G3's C-lectin subdomain, is necessary and sufficient for G3 secretion. A model is proposed in which Hsp25 piggybacks onto nascent G3 in the cytosol during a translocational pause and enters the ER lumen with G3, and once G3 properly folds, Hsp25 releases G3 and recycles to the nucleus while G3 continues to the Golgi stacks, providing passage for the entire core protein.

Calreticulin-integrin bidirectional signaling complex.

Calreticulin has multiple functions, diverse cellular locations, and putative isoforms. It likely maintains integrin avidity by binding alpha integrin cytoplasmic tails and is a surface lectin which triggers cell spreading. In the present study, we have immunocaptured a cell surface complex from B16 mouse melanoma cells which contains alpha 6 beta 1 integrin, two molecular forms of calreticulin, and KDEL docking protein (KDEL-R). One of the calreticulins, "endocalreticulin", a 52 kDa protein, does not become surface biotinylated, and is probably bound to alpha integrin cytoplasmic tails; it disappears when B16 cells adhere to laminin, and two ubiquitinated calreticulins appear. One ubiquitinated species, a 125 kDa protein, is restricted to focal contacts whereas a second species, a 75 kDa protein, is in focal contacts and surrounding plasma membrane; it also arises when cells bind non-specific surfaces. The other calreticulin, "ectocalreticulin", a 62 kDa protein, becomes surface biotinylated, is probably anchored to surface KDEL-R, and cooperates with alpha 6 beta 1 integrin, triggering cell spreading. The present results suggest a model in which calreticulin-integrin surface complex functions as a symbiotic unit, transmitting information in both directions across the plasma membrane.

Divergent secretory behavior of the opposite ends of aggrecan.

The proteoglycan, aggrecan has a globular domain, G1, at the N terminus and a different globular domain, G3, at the C terminus. Aggrecan produced by mutant nanomelic chickens is truncated due to a premature stop codon and consequently lacks G3 and a minor portion of its chondroitin sulfate domain (Li, H., Schwartz, N. B., and Vertel, B. M.(1993) J. Biol. Chem. 268, 23504-23511). The mutant protein is retained in the endoplasmic reticulum and fails to enter the Golgi stacks (Vertel, B. M., Walters, L. M., Grier, B., Maine, N. , and Goetinck, P. F.(1993) J. Cell Sci. 104, 939-948). The homozygous mutant is lethal because of failure of chondrogenesis and osteogenesis, while the heterozygous mutant is dwarfed. To further elucidate the pathogenetic mechanisms underlying nanomelia and to determine if G1 and G3 are themselves secreted, we expressed them in transfected host cells. Expression was performed in wild type Chinese hamster ovary (CHO) cells and in mutant CHO cells which are unable to link glycosaminoglycan (GAG) chains to core proteins. We compared: (a) secretion of expressed G1 and G3 constructs containing contiguous GAG chain consensus sites and (b) GAG chain modification of the secreted proteins. We find that: 1) G3 is 24-100 times more rapidly secreted than G1; 2) secreted G3 contains contiguous chondroitin sulfate GAG chains, while secreted G1 lacks contiguous GAG chains; 3) G3 secretion is not coupled to xylosylation of contiguous GAG chain consensus sites. These results imply that G1 and G3 intrinsically differ in passage through the cell secretory route.

Calreticulin binding affinity for glycosylated laminin.

Several lines of evidence indicate that calreticulin has lectin-like properties. As a molecular chaperone, calreticulin binds preferentially to nascent glycoproteins via their immature carbohydrates; this property closely resembles that seen for calnexin, a chaperone with extensive molecular identity to calreticulin. A cell surface form of calreticulin also exhibits lectin-like properties, binding specific oligomannosides including those covalently linked to laminin. In the present study we examined the interaction between calreticulin and laminin by means of surface plasmon resonance. The results show that calreticulin specifically binds to glycosylated laminin but fails to specifically bind tunicamycin-derived unglycosylated laminin or bovine serum albumin. Calreticulin binding to glycosylated laminin requires calcium and is abolished in the presence of EDTA. Scatchard analysis of binding yields an apparent association constant, Ka, of 2.1 +/- 0.9 x 10(6) m-1 while kinetic analysis yields an estimate of the association on rate, (Kassoc), as 2 x 10(5) m-1 s-1. The composite results support calreticulin's lectin-like properties as well as its proposed role in laminin recognition, both in the cell interior and on the cell surface.

Temporal and spatial appearance of alpha-dystroglycan in differentiated mouse myoblasts in culture.

The dystrophin-glycoprotein complex plays an important role in muscle function. One of the components of the complex, a 156-kDa cell surface glycoprotein (alpha-dystroglycan) binds to laminin, thereby connecting the basal lamina and muscle cells. We have examined the progressive appearance of alpha-dystroglycan and laminin in muscle cells that differentiate in culture. We find that nondifferentiated cultures of C2C12 myoblasts express low amounts of dystroglycan mRNA and, in contrast, this gene is prominently expressed in differentiated myotubes. Immunofluorescence analysis with a monoclonal antibody against alpha-dystroglycan shows its progressive appearance during myoblast differentiation into myotubes. Immunostaining with a monoclonal antibody against laminin shows that it is not present on the surface of undifferentiated myoblasts. Subsequently, laminin becomes apparent on the surface of differentiated myotubes where it codistributes with immunostained alpha-dystroglycan. Immunoblotting of isolated surface membrane proteins of differentiated myotubes with antibodies against alpha-dystroglycan identifies a broad band of about 140-160 kDa, resembling alpha-dystroglycan from rabbit muscle. The composite results indicate that alpha-dystroglycan and laminin appear and become co-distributed on the surface of cultured C2C12 during the progression of differentiation.

Cell surface calreticulin is a putative mannoside lectin which triggers mouse melanoma cell spreading.

B16 mouse melanoma cells adhere to and spread on laminin. We have previously shown that cell spreading is uncoupled from adhesion when unglycosylated laminin is used as a substratum; spreading was restored by a Pronase digest of laminin which became inactive when it was specifically depleted of its mannoside peptides; spreading was also specifically restored by mannosides such as mannan, Man9, and Man6, but not Man3. The effector mannosides bind to a cell surface receptor, previously shown by direct and indirect methods. We have now identified the receptor as cell surface calreticulin by isolating it via mannan affinity chromatography and showing its sequence identity with mouse calreticulin. Anti-calreticulin antibodies confirm this identity, decorate the B16 cell surface, and block cell spreading. Purified B16 cell calreticulin from whole cell lysates successfully competes with cell surface calreticulin and prevents cell spreading. The composite data implicate cell surface calreticulin as a putative lectin that must be occupied to initiate spreading of laminin-adherent B16 cells.

Rodent myoblast interactions with laminin require cell surface glycoconjugates but not laminin glycosyl groups.

Laminin glycosyl groups are necessary for the spreading of murine melanoma cells which become attached to this glycoprotein. Laminin has been implicated in myogenesis but the potential role of its glycosyl groups in this process has not been examined. In this study we report the effects of the carbohydrate moieties of laminin on myoblast adhesion, spreading, and differentiation. Unglycosylated laminin from tunicamycin-treated cultures of a mouse cell line, M1536 B3, was used in the experiments. Glycosylated laminin from a murine tumor and from cultures of M1563 B3 cells served as controls. Cell binding experiments with C2C12 mouse myoblasts showed that the cells preferred a laminin-coated surface, compared to the uncoated plastic surface (nontissue culture wells). Myoblasts did not distinguish between glycosylated and unglycosylated laminin substrates. Both glycosylated and unglycosylated forms of laminin promoted myoblast growth and differentiation. In contrast, cells on uncoated plastic surfaces grew very slowly and did not further differentiate. The L6 rat myoblast response to glycosylated and unglycosylated laminin was the same. These results indicate that although rodent myoblasts in culture require a laminin substratum for spreading, growth, and differentiation on a proprietary plastic surface, laminin carbohydrates are not implicated in those cellular responses. In contrast, parallel studies using the lectin, Con A, indicate that cell surface glycoconjugates of myoblasts are implicated in the response of these cells to a laminin substratum.

Oligomannosides initiate cell spreading of laminin-adherent murine melanoma cells.

Murine melanoma cells readily bind and spread on murine laminin. Uncoupling of spreading from adhesion occurs when unglycosylated laminin is used as the cellular substratum; spreading is restored by soluble glycosylated laminin or soluble glycopeptides of laminin. In this study, using kifunensine, we produced and characterized an oligomannoside-rich glycoform of laminin. When used as a substratum for cell attachment and spreading, this laminin was as effective as mature glycosylated laminin. When added in solution to unglycosylated laminin-adherent cells, kifunensine-laminin was more effective in promoting cell spreading than mature glycosylated laminin. Reconstitution with soluble polysaccharides showed that cell spreading was initiated rapidly by microgram amounts of mannan but not other polysaccharides and approached a maximum within 1 h; titration with mannan yielded an adsorption isotherm profile. Mannose was an antagonist, preventing mannan from restoring cell spreading, but it was not an agonist. A Pronase digest of mature glycosylated laminin, depleted of its oligomannoside-peptides, was unable to restore cell spreading, whereas a control digest was fully active. Melanoma cells were unable to bind to three different neoglycoprotein surfaces, but when soluble unglycosylated laminin was present in the medium the cells adhered to and spread only upon mannosylated bovine serum albumin. Of the various cell lines known to interact with glycosylated laminin only murine melanoma cells showed oligomannoside-dependent spreading on an unglycosylated laminin substratum. The composite results indicate that oligomannosides are necessary to initiate murine melanoma cell spreading on laminin but are not sufficient for cell adhesion.

Characterization of oligomannoside binding to the surface of murine melanoma cells. Potential relationship to oligomannoside-initiated cell spreading.

The spreading of murine melanoma cells on unglycosylated laminin is initiated by soluble glycosylated laminins containing oligomannosides, or by mannan, and is inhibited by mannose (Chandrasekaran, S., Tanzer, M. L., and Giniger, M. S. (1994) J. Biol. Chem. 269, 3356-3366). In the present study, melanoma cell recognition of oligomannosides was explored by several different methods. Comparison of cell spreading, initiated by related branched oligomannosides, showed that micromolar concentrations of Man6 and Man9 were able to restore cell spreading maximally, whereas Man3 was ineffective. Man9 bound to the melanoma cells in a bimodal manner at micromolar concentrations, reaching saturation, whereas Man3 showed linear binding in the same and higher ranges. Comparison of Man9 binding with Man9-initiated cell spreading demonstrated that maximal spreading occurred at approximately half-saturation. Competition experiments showed that mannose or mannan effectively impaired Man9 binding to the cells, whereas galactose or fucose was ineffective. Mannan-conjugated fluorescent beads bound in a diffuse pattern to the surface of melanoma cells, whereas underivatized beads did not bind. The distribution of laminin-binding beta 1 integrin on the cell surface was compared with surface mannan binding. The beta 1 integrin staining pattern did not match the mannan staining pattern either in attached, nonspread cells or in attached, spread cells. The composite results support a model in which occupancy of both an integrin and a cell surface lectin is required for murine melanoma cells to spread on glycosylated laminin.

Molecular cloning and analysis of the protein modules of aggrecans.

The large aggregating chondroitin sulfate proteoglycan of cartilage, aggrecan, has served as a prototype of proteoglycan structure. Molecular cloning has elucidated its primary structure and revealed both known and unknown domains. To date the complete structures of chicken, rat and human aggrecans have been deduced, while partial sequences have been reported for bovine aggrecan. A related proteoglycan, human versican, has also been cloned and sequenced. Both aggrecan and versican have two lectin domains, one at the amino-terminus which binds hyaluronic acid and one at the carboxyl-terminus whose physiological ligand is unknown. Both lectins have homologous counterparts in other types of proteins. Within the aggrecans the keratan sulfate domain may be variably present and also has a prominent repeat in some species. The chondroitin sulfate domain has three distinct regions which vary in their prominence in different species. The complex molecular structure of aggrecans is consistent with the concept of exon shuffling and aggrecans serve as suitable prototypes for comprehending the evolution of multi-domain proteins.

Extracellular matrix. 3: Evolution of the extracellular matrix in invertebrates.

Invertebrates comprise about 95% of animal species, yet most studies of extracellular matrices have centered on vertebrates. Comparative studies of invertebrates will enhance comprehension of evolutionary processes and appreciation of the diversity of extracellular matrices. Moreover, new functions and new structures will be revealed over a wide range of organismic needs. Another important perspective is that several invertebrate species have provided insight into developmental processes, and those processes often have direct relevance to vertebrate development. Thus, studies of fruit flies, nematodes, and sea urchins have revealed common features of cell biology, embryonic development, and matrix properties that pertain throughout the animal kingdom. The advantages of invertebrates are their rapid rates of embryonic development, their amenability to genetic manipulation, availability of innumerable mutants, and their ease of study in the laboratory. Extracellular matrices themselves are readily compared. Invertebrates display a wide diversity of such matrices, at the levels of both tissue architecture and molecular anatomy. Knowledge of that diversity leads to an appreciation of evolutionary variety and eventually to comprehension of the organization of extracellular matrices and of the properties of their constituent macromolecules. The expanding knowledge of unique matrix molecules from invertebrates also has economic potential and is beginning to provide new materials for biotechnology.

Molecular cloning and analysis of the protein modules of aggrecans.

The large aggregating chondroitin sulfate proteoglycan of cartilage, aggrecan, has served as a prototype of proteoglycan structure. Molecular cloning has elucidated its primary structure and revealed both known and unknown domains. To date the complete structures of chicken, rat and human aggrecans have been deduced, while partial sequences have been reported for bovine aggrecan. A related proteoglycan, human versican, has also been cloned and sequenced. Both aggrecan and versican have two lectin domains, one at the amino-terminus which binds hyaluronic acid and one at the carboxyl-terminus whose physiological ligand is unknown. Both lectins have homologous counterparts in other types of proteins. Within the aggrecans the keratan sulfate domain may be variably present and also has a prominent repeat in some species. The chondroitin sulfate domain has three distinct regions which vary in their prominence in different species. The complex molecular structure of aggrecans is consistent with the concept of exon shuffling and aggrecans serve as suitable prototypes for comprehending the evolution of multi-domain proteins.

Laminin oligosaccharides play a pivotal role in cell spreading.

The basement membrane glycoprotein laminin promotes cell adhesion, spreading and neurite outgrowth. We can uncouple cell adhesion and spreading (or neurite outgrowth) when unglycosylated laminin is used as a substratum. Mouse melanoma cells, B16F1 line, readily attach to unglycosylated laminin but fail to spread once adherent. Spreading can be restored by titration with glycosylated laminin or with laminin glycopeptides. When the laminin substratum is absent in the test chambers, the cells do not adhere when either intact laminin or its glycopeptides are then added. Analyses show that these added substances are recoverable from the culture medium and do not bind to the chamber surfaces. Use of selective inhibitors which interfere with carbohydrate processing yields several glycoforms of laminin which we have isolated and examined for their ability to support cell adhesion and spreading. Laminin which is enriched in high mannose oligosaccharides is much more effective in promoting cell spreading than laminin which is enriched in hybrid oligosaccharides. These results are consistent with earlier studies which showed that ConA, which primarily recognizes mannose residues, could also uncouple cell adhesion and spreading. Although mono- and disaccharides failed to restore cell spreading, we have found that addition of various mannose oligosaccharides to adherent cells effectively reestablishes their spreading behavior. The extent of cell spreading which is achieved by the added saccharides is related to their amount, their duration of addition, and their molecular structures.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of a type IX collagen-related mRNA in an invertebrate, the marine annelid Nereis virens.

Fibrous and non-fibrous collagens have been described in both vertebrate and invertebrate animals. However, there has been limited characterization of non-fibrous collagens and their corresponding genes in invertebrate animals. In the present study we have used as a probe an avian cDNA clone which encompasses the COL3, NC3 and part of the COL2 domain of the collagen alpha 3(IX) subunit. This probe hybridized to mRNA obtained from the cuticle and body of the marine annelid, Nereis virens. Northern blot hybridization exhibited an mRNA of ca. 7.5-8 kilobases which in situ hybridization shows to be most abundant over cuticle-associated cells. Dot-blot hybridization, comparing cuticle mRNA and body mRNA, indicates that this collagen mRNA is five times more abundant in the cuticle. The composite data suggest evolutionary conservation, in both vertebrate and invertebrate animals, of a non-fibrillar collagen.

Role of laminin carbohydrates on cellular interactions.

Laminins, a family of large multidomain glycoproteins of the basal lamina, have been implicated in the development and maintenance of cellular and tissue organization. Considerable interest has arisen concerning the ways in which laminin carries out its biological functions. Previously these biologic responses have been primarily attributed to the peptide sequences of laminin, however, newer studies suggest that laminin carbohydrates may also participate in such cellular activities. Recently, a subpopulation of laminin molecules purified from EHS sarcoma by lectin affinity chromatography has been shown to contain about 25 to 30% carbohydrate. Most of the carbohydrates present are complex-type asparagine-linked oligosaccharides encompassing many different structures, some of which are unique to laminin. To date, the biological function of the carbohydrates of laminin remains somewhat unclear. They do not appear to be needed for heparin binding or to enhance proteinase stability, however, current evidence suggests they are important in cellular spreading and neurite outgrowth. It is our hypothesis that in the covalently-linked carbohydrate moieties of laminin will ultimately prove to be involved in information transfer to responsive cells. It is the purpose of this review to delineate current concepts of the structure and function of this unique glycoprotein's sugar chains.