Regulation of angiogenesis by extracellular matrix

During angiogenesis, endothelial cell growth, migration, and tube formation are regulated by pro- and anti-angiogenic factors, matrix-degrading proteases, and cell-extracellular matrix interactions. Temporal and spatial regulation of extracellular matrix remodeling events allows for local changes in net matrix deposition or degradation, which in turn contributes to control of cell growth, migration, and differentiation during different stages of angiogenesis. Remodeling of the extracellular matrix can have either pro- or anti-angiogenic effects. Extracellular matrix remodeling by proteases promotes cell migration, a critical event in the formation of new vessels. Matrix-bound growth factors released by proteases and/or by angiogenic factors promote angiogenesis by enhancing endothelial migration and growth. Extracellular matrix molecules, such as thrombospondin-1 and -2, and proteolytic fragments of matrix molecules, such as endostatin, can exert anti-angiogenic effects by inhibiting endothelial cell proliferation, migration and tube formation. In contrast, other matrix molecules promote endothelial cell growth and morphogenesis, and/or stabilize nascent blood vessels. Hence, extracellular matrix molecules and extracellular matrix remodelling events play a key role in regulating angiogenesis.     

Structure of the major concanavalin A reactive oligosaccharides of the extracellular matrix component laminin

Laminin, a high molecular weight (1,000,000) glycoprotein component of basement membranes, was isolated from the EHS murine tumor as a noncovalent complex with entactin by lectin affinity chromatography using the alpha-D-galactosyl binding lectin Griffonia simplicifolia I (GS I). Entactin was removed from this complex by passage over Sephacryl S-1000 in the presence of SDS. Compositional analysis showed that the affinity-purified laminin contained 25-30% carbohydrate by weight. Methylation analysis revealed that the oligosaccharides of laminin contained bi- and triantennary chains, the blood group I structure, and repeating sequences of 3Gal beta 1,4GlcNAc beta 1 units. Free oligosaccharides were derived from the asparagine-linked glycans of affinity-purified laminin by hydrazinolysis, re-N-acetylation, and reduction with NaB3H4. When fractionated by affinity chromatography on concanavalin A (Con A)-Sepharose, 80% of the oligosaccharides passed through the column unretarded and a single peak corresponding to 20% of the oligosaccharides was adsorbed and specifically eluted with a linear gradient of 0-30 mM methyl alpha-D-glucopyranoside. Further fractionation of the Con A reactive oligosaccharides on GS I-Sepharose demonstrated that 70% of these oligosaccharides possess at least one terminal nonreducing alpha-D-galactopyranosyl unit. The Con A reactive oligosaccharides were subjected to sequential digestion with endo- and exoglycosidases, and the reaction products were analyzed by gel filtration chromatography on a column of Bio-Gel P4. We thereby obtained evidence for a variety of structures not previously reported to exist on murine laminin including hybrid biantennary complex and biantennary complex structures containing poly(lactosaminyl) repeating units. The poly(lactosaminyl) units occur either on one or on both branches of the biantennary chains, as well as in more highly branched blood group I poly(lactosamine) structures. All sialic acid is present as N-acetylneuraminic acid linked alpha 2,3 to galactose.     

The role of microvilli in the agglutination of cells by concanavalin A

Morphological correlates of lectin agglutinability were examined in eight cell lines of varying sensitivity to agglutination by concanavalin A (ConA). The number of microvilli on the surface of cells growing in monolayers was positively correlated with agglutinability. However, when cells were brought into suspension, they all developed numerous microvilli which persisted when the cells were treated with ConA regardless of whether or not they were agglutinated by the lectin. Treatment of cells with dibutyryl cyclic AMP (db-cAMP) and theophylline caused a parallel decrease in agglutinability and numbers of microvilli in monolayer cultures, but suspended cells from control and treated cultures were identical in appearance in the absence or presence of ConA. The surface morphology of cells agglutinated by ConA was very similar to that of cells that spontaneously agglutinated in the absence of the lectin, and surface bound ConA was rapidly withdrawn from microvilli on all cell types. Neither the morphology of cells nor the surface distribution of ConA can explain observed differences in agglutinability.     

Regulation of plasminogen activation by components of the extracellular matrix

The kinetics of activation of Glu-plasminogen (Glu-Pg) and Lys77-Pg by two-chain recombinant tissue plasminogen activator (t-PA) were determined in the presence of isolated protein components of the extracellular matrix (ECM) and compared to activation in the presence of fibrinogen and fibrinogen fragments and in the absence of added protein. Several ECM protein components were as effective as fibrinogen fragments at stimulating Pg activation. Stimulation of Glu-Pg activation resulted from both a decrease in Km and an increase in Vmax, whereas stimulation of Lys77-Pg was due primarily to increases in Vmax. The most effective stimulators of activation were basement membrane type IV collagen and gelatin which resulted in a 21- and 55-fold increase, respectively, in the kcat/Km of Glu-Pg (relative to a 10-fold increase observed with fibrinogen fragments). Amidolytic activity of t-PA was also enhanced up to 12-fold by ECM proteins. However, plasmin amidolytic activity was unaffected by the presence of added proteins. These data suggest that several ECM-associated proteins can enhanced the activation of Pg in the absence of fibrin.     

Regulation of cancer invasiveness by the physical extracellular matrix environment

Long-term clinical outcomes are dependent on whether carcinoma cells leave the primary tumor site and invade through adjacent tissue. Recent evidence links tissue rigidity to alterations in cancer cell phenotype and tumor progression. We found that rigid extracellular matrix (ECM) substrates promote invasiveness of tumor cells via increased activity of invadopodia, subcellular protrusions with associated ECM-degrading proteinases. Although the subcellular mechanism by which substrate rigidity promotes invadopodia function remains to be determined, force sensing does appear to occur through myosin-based contractility and the mechanosensing proteins FAK and p130^Cas. In addition to rigidity, a number of ECM characteristics may regulate the ability of cells to invade through tissues, including matrix density and crosslinking. 3-D biological hydrogels based on type I collagen and reconstituted basement membrane are commonly used to study invasive behavior; however, these models lack some of the tissue-specific properties found in vivo. Thus, new in vitro organotypic and synthetic polymer ECM substrate models will be useful to either mimic the properties of specific ECM microenvironments encountered by invading cancer cells or to manipulate ECM substrate properties and independently test the role of rigidity, integrin ligands, pore size and proteolytic activity in cancer invasion of various tissues.Key words: cancer, invasion, invadopodia, rigidity, mechanotransduction, microenvironmentIn multicellular organisms, cells must sense and respond to multiple cues for proper functioning within tissues. Although most experimental research has focused on the regulation of cellular processes by external chemical signals, there is increasing recognition that mechanical forces also regulate critical cellular functions. Indeed, rigidity of the extracellular environment has been shown to regulate such diverse processes as muscle cell differentiation, stem cell lineage fate, breast epithelial signaling and phenotype, and fibroblast motility.^1–5In breast cancer, accumulating evidence suggests a role for tissue rigidity in promoting both the formation and invasiveness of tumors. Mammographic density of breast tissue has been correlated with increased cancer risk and included in models to predict the likelihood of in situ and invasive breast cancers.⁶ Histologically, dense breast tissue has increased stromal collagen content and in vitro analyses have shown that cancerous breast tissue is much stiffer than normal tissue (as represented by values for the elastic or Young''s moduli).^3,7 In addition, experimentally increased expression of collagen fibrils in a mouse mammary model of spontaneous breast cancer was recently shown to promote tumor formation, invasion and metastasis.⁸ Therefore, both clinical and animal data suggest a correlation between tissue density and cancer aggressiveness, and mechanical factors appear likely to play a role in this process.⁹A well-established mechanism by which extracellular matrix (ECM) rigidity signals can drive phenotypic transformations is through mechano-signal transduction (mechanotransduction) pathways in which external forces are transmitted via integrin receptors at linear focal adhesion structures to cytoskeletal and signaling proteins inside the cell. Actomyosin contractility leads to stretching and activation of proteins such as talin, p130^Cas and potentially focal adhesion kinase (FAK).^10–12 For example, stem cell lineage was found to be dependent on formation of cellular focal adhesions and actomyosin contractility in response to substrate tensile properties.² Mammary epithelial cells grown on compliant matrices will differentiate and polarize to form lactating 3-D structures that resemble in vivo acini but fail to do so on stiff matrices due to increased cytoskeletal contractility.³ Activation of mechanotransduction molecules, such as FAK, Rho and ROCK, are required for the rigidity-induced phenotype changes.^3,5 Using polyacrylamide (PA) gel systems, Yu-li Wang''s group found that rigid substrates induce fibroblast and epithelial cells to migrate away from each other instead of aggregating to form tissue-like structures.¹³ This transformation in phenotype is characteristic of the epithelial to mesenchymal transition and thought to be crucial for tumor cell migration.¹⁴A critical feature of tumor aggressiveness is the ability to invade across tissue boundaries, through degradation of ECM. The subcellular structures responsible for this invasive activity are thought to be invadopodia: actin-rich, finger-like cellular protrusions that proteolytically degrade local ECM. These structures are characteristic of invasive cells and have been implicated in tumor cell metastasis due to their association with ECM degradation.¹⁵ Similar structures, podosomes, are formed in src-transformed cells, as well as normal cells such as osteoclasts and dendritic cells that need to degrade matrix and/or cross tissue boundaries.¹⁶ In addition to mediating ECM degradation, podosomes have been postulated to function as adhesion structures, since well-characterized adhesion proteins localize to podosomes and many podosome-expressing cells no longer express focal adhesions.¹⁷ Furthermore, podosomes have been shown to be essential for chemotactic motility and transendothelial migration, although not for chemokinetic motility.^18,19We recently found that ECM rigidity increases both the number and activity of invadopodia, and this effect was dependent on the cellular contractile machinery (Fig. 1A).²⁰ Consistent with a role for mechanotransduction in this process, we found localization of the active, phosphorylated forms of the mechanosensing proteins FAK and p130^Cas in actively degrading invadopodia and an increase in invadopodia-associated degradation in breast cancer cells overexpressing FAK and p130^Cas. These results suggest that in breast cancer, increases in tissue rigidity may directly lead to increased cellular invasiveness and tumor progression.Open in a separate windowFigure 1Potential rigidity sensing mechanisms by invadopodia. (A) Invadopodia are typically identified by colocalization of fluorescent antibodies for actin and cortactin at puncta that correspond to areas of ECM degradation visualized as dark regions in FITC-labeled fibronectin (Fn) overlaying gelatin. In this case, ECM was layered on top of either soft (storage modulus = 360 Pa) or hard (storage modulus = 3,300 Pa) polyacrylamide gels (PA) to determine if invadopodia activity was regulated by differences in mechanical properties. On hard PA, invasive MCF10ACA1d breast carcinoma cells produced more invadopodia and degraded more ECM than on soft PA. Yellow arrows indicate examples of invadopodia. (B) The localization of rings of the contractile protein myosin IIA (myoIIA) surrounding invadopodia (actin puncta) suggests a role for these structures in mechanosensing by potentially linking invadopodia with the contractile apparatus to detect differences in substrate rigidity. An example ring structure is indicated with a yellow arrow and shown in the zoomed portion of the myosin IIA image, and an example of no or weak localization of myosin IIA with an invadopodium is indicated with the red arrow. (C) Activated forms of FAK and p130^Cas localize to invadopodia and depend on cytoskeletal contractility.²⁰ Rings of myosin IIA also frequently surround invadopodia. These results suggest that invadopodia may act as mechanosensing organelles, either directly through localized mechanoresponsiveness at the invadopodia or through longer-range connections to neighboring or even distant focal adhesions. In either case, traction forces may be generated as a result of changes in cytoskeletal tension in response to ECM properties. Alternatively, invadopodia function could be regulated in the absence of local traction forces, secondary to distant intracellular signaling that leads to alterations in whole cell phenotypic changes. (A and B) are reprinted from Current Biology, Volume 18, Nelson R. Alexander, Kevin M. Branch, Aron Parekh, Emily S. Clark, Izuchukwu C. Iwueke, Scott A. Guelcher and Alissa M. Weaver, Extracellular Matrix Rigidity Promotes Invadopodia Activity, pp. 1295–9, 2008; with permission from Elsevier.The localization of phosphorylated FAK and p130^Cas at invadopodia and the requirement for actomyosin contractility in our study suggests that invadopodia have the potential to act as mechanosensing organelles. This concept is supported by our finding that ∼40% of breast cancer cells cultured on rigid substrates had rings of myosin IIA surrounding invadopodia (Fig. 1B)²⁰ and the recent finding that similar podosome structures can exert local traction forces.²¹ In addition, a few studies have implicated integrin activity in invadopodia function as well as localized β1 and β3 integrins to invadopodia.^22–25 However, whether invadopodia can serve as tension-generating adhesion structures is controversial, in part because of the presence of both focal adhesions and invadopodia in many cancer cells (Fig. 1C).Regulation of invadopodia and podosome function is also not straightforward. Although our data,²⁰ along with results from Collin et al.,²¹ suggests that mechanical tension promotes invadopodia and podosome activity, in some systems podosome formation is promoted by a loss rather than a gain of cytoskeletal tension. That is, local cytoskeletal relaxation has been shown to promote podosome formation coincident with focal adhesion dissolution in both vascular smooth muscle cells treated with phorbol ester²⁶ and neuroblastoma cells.²⁷ A yin-yang activity between focal adhesions and podosomes has been known for many years, whereby activation of src kinase leads to both disassembly of focal adhesions²⁸ and formation of podosomes.²⁹ However, the role of tension in this process is unclear, particularly since activation of src kinase occurs downstream of mechanical stimuli³⁰ and should promote podosome/invadopodia activity, yet loss of tension apparently induces biological activities dependent on src kinase (focal adhesion disassembly and podosome formation).^26,27 For invadopodia, the role of tension is even less clear. Basic characterization studies need to be performed to establish molecular and structural differences between invadopodia and focal adhesions and to measure force profiles at the two structures. Since invadopodia have much smaller diameters compared to podosomes (50–100 nm vs ∼1 µm, respectively),^15,16 the latter task of determining traction forces may be difficult due to resolution limitations in measuring potentially tiny substrate displacements. The standard identification of invadopodia, by association of actin-rich puncta with sites of degradation of fluorescent ECM, adds another technical limitation since the thickness and fluorescence of the ECM matrix used to identify proteolytic activity may hinder visualization of embedded fluorescent beads in the underlying PA gel (displacement of beads is typically used to calculate traction forces).³¹ Thus, an important future direction should be the development of new in vitro experimental systems that have manipulable substrate properties and allow simultaneous identification of subcellular forces and proteolytic activity.The cellular response to rigidity is often characterized using PA gels with tunable stiffness in the range spanning that of normal and cancerous breast tissue (elastic moduli = 100–10,000 Pa).^3,7 PA gels will likely continue to be invaluable tools for understanding cellular responses to rigidity. However, this system is inherently simple and cannot fully replicate cellular events occurring in a complex in vivo ECM microenvironment. Given that invading breast cancer cells are likely to experience different microenvironments as they cross through the basement membrane (BM) and into neighboring collagenous stromal tissue (Fig. 2), biological hydrogels such as reconstituted basement membrane (Matrigel) and type I collagen gels are often utilized to mimic these ECM substrates. However, both of these models lack many of the chemical, physical, and mechanical characteristics of tissues found in vivo and have been recently questioned as suitable models for studying cancer cell invasion.³² Type I collagen gels have a fibrillar architecture but a low density and high porosity³³ and frequently lack crosslinking sites.³⁴ Although Matrigel contains many of the biochemical components of the BM, it is tumor-derived³⁵ and the major component is laminin-1, which is only abundant in fetal tissues.³⁶ By contrast, the major component of normal BM is type IV collagen. In addition, Matrigel is a solubilized preparation that lacks crosslinks³⁷ and a fibrillar component.³⁸ Both sparse collagen gels and Matrigel are quite compliant with Young''s moduli of ∼1,000 and ∼200 Pa, respectively;³ therefore, without further manipulation these substrates lack the rigidity required to mimic tumor-associated ECM.Open in a separate windowFigure 2Navigation of basement membranes and stromal collagen by invading cancer cells. Invasive cancer cells are thought to navigate different tissue microenvironments in the process of invasion. In order for invasion to occur, tumor cells must first breach the basement membrane, a thin and highly crosslinked specialized ECM that requires proteolytic degradation for subsequent transmigration. Once past this barrier, cells must proceed through the neighboring stroma composed of collagenous connective tissue. The meshwork in the stroma is looser and may facilitate diverse migration modes dependent on local microenvironmental conditions and cellular cohesiveness. These modes of migration include a single cell, proteinase-independent amoeboidal phenotype (left) and single cell (middle) and collective (right) proteinase-dependent mesenchymal phenotypes that locally degrade matrix at enzymatically active invadopodia. Note the absence of collagen stroma surrounding and along the migration track of proteolytically active cells. New physiologically relevant models that mimic these interactions in vitro will be useful to elucidate mechanisms of cancer cell migration and invasion in various tissues.In order to invade neighboring stromal tissue, carcinoma cells must first breach the BM, a complex, interwoven meshwork composed of type IV collagen, laminin, nidogen/entactin, and various proteoglycans and glycoproteins.³² The highly ordered and crosslinked type IV collagen network is regarded as the limiting barrier to cancer cell invasion since it forms pores on the order of 100 nm that are too small for passage of cells without proteolytic degradation of the BM.³² In addition to degradation, decreased BM synthesis may contribute to the initial steps of cancer invasion by altering the balance between BM formation and remodeling.³⁹ Once cancer cells cross the BM, they encounter stromal collagen tissue. In tumors, this desmoplastic stroma is frequently fibrotic due to increased ECM deposition and crosslinking by carcinoma-associated fibroblasts.⁹ Although controversial, cancer cells are thought to use a nonproteolytic, amoeboid mode to traverse this connective tissue;⁴⁰ therefore, different modes of migration may be necessary to traverse BM or stromal collagenous matrices (Fig. 2). However, the amoeboid phenotype has been described using either sparse collagen gels without crosslinks⁴¹ or Matrigel.⁴² In vivo, the process of invading through tumor-associated stromal collagen is likely to depend on the pore size, the crosslinking status, and whether cells are migrating collectively or individually.^34,43In light of these concerns and many others, there has been a push for more physiologically relevant in vitro models that represent closer approximations of BM or stromal collagen tissue. Successful models, whether natural or synthetic, must be able to mimic the composition, architecture and mechanical properties of the in vivo environment as well as support cell culture in ex vivo conditions. Natural substrates can be produced by cultured cells, such as the epithelial basement membranes synthesized by MDCK cells.³⁷ Alternatively, organotypic models derived from biological specimens have recently been utilized to study invasion. These materials can be based on processed biological tissue, such as detergent-extracted mouse embryo sections,⁴⁴ homogenized involution matrix,³⁸ and decellularized human dermis,⁴⁵ or on native tissue such as chick chorioallantoic membrane⁴⁶ and explanted peritoneal or mammary tissue.^34,37 In addition, the field of tissue engineering has already provided novel hybrid scaffolds and advanced tissue culturing methods that can be utilized for cancer research.⁴⁷ Biological materials developed for clinical use in tissue reconstruction and regeneration, such as small intestinal submucosa and urinary bladder matrix, are attractive candidates as new in vitro models since they maintain their tissue-like properties and have been extensively characterized.^48,49 These tissue-derived scaffolds are composed of well-defined structural and functional proteins, originally produced by cells in vivo, and maintain their complex 3-D architecture. Thus, such materials can provide an environment that recapitulates the chemical, physical and mechanical properties found in vivo.⁴⁸ In addition, synthetic materials, such as poly(ethylene glycol)-based hydrogels, will likely play a large role in cancer research since they can be designed with defined chemistries to obtain appropriate physical and mechanical properties as well as specific spatial arrangements of biologically relevant moieties on relevant length scales.^33,50 Similarly, engineered adhesive microenvironments created with microfabrication techniques can also be utilized to probe molecular and cellular phenomena.⁵¹ Due to this flexibility in fabrication, these materials are good candidates for novel in vitro models to probe the effects of specific mechanical, topographical and chemical factors on cellular migration and invasion.In summary, the physical microenvironment is increasingly recognized as a major influence on cellular phenotype. Recent data emphasizes the importance of mechanical factors in tumor progression, including cellular invasiveness. Exciting future directions include understanding how stromal and BM environments affect cellular invasiveness at multiple scales, including subcellular and molecular regulation of ECM degradation in response to ECM rigidity and the role of proteinases in crossing diverse tissue barriers. The development of novel model systems with appropriate biological and physical properties will facilitate all of these goals.     

Regulation of macrophage ApoE expression and processing by extracellular matrix

Macrophage-derived apoE in the vessel wall has important effects on atherogenesis in vivo, making it important to understand factors that regulate its expression. Vessel wall macrophages are embedded in an extracellular matrix produced largely by arterial smooth muscle cells and endothelial cells. In this series of studies, we evaluated the influence of extracellular matrix on macrophage apoE expression. Subendothelial matrix, fibronectin, or collagen I stimulated macrophage apoE gene expression and apoE synthesis. Adhesion of macrophages to a polylysine substrate had no effect. An increase in apoE synthesis after plating on fibronectin could be observed by 2 h and was inhibited by blocking antibodies to the alpha(5)beta(1) integrin receptor for fibronectin. Fibronectin also regulated the post-translational processing of newly synthesized macrophage apoE by inhibiting its degradation. The increment in apoE resulting from suppressed degradation was retained in the cell-fibronectin monolayer in a pool that was resistant to release by exogenous high density lipoprotein subfraction 3. These observations establish a new pathway for the regulation of macrophage apoE expression in the vessel wall. The composition of the extracellular matrix changes after vessel wall injury and in response to locally produced cytokines and growth factors. The evolving composition of this matrix will, therefore, be important for regulating apoE expression and processing by vessel wall macrophages.     

Regulation of autophagy by extracellular matrix glycoproteins in HeLa cells

Macroautophagy is a major lysosomal degradation pathway for cellular components in eukaryotic cells. Baseline macroautophagy is important for quality control of the cytoplasm in order to avoid the accumulation of cytotoxic products. Its stimulation by various stressful situations, including nutrient starvation, is important in maintaining cell survival. Here we demonstrate that macroautophagy is regulated differently depending on whether HeLa cells adhere to collagen I or collagen IV, proteins typical of connective tissue and basal membrane, respectively. We observed that the basal levels of macroautophagy were higher in cells plated on collagen IV than in cells plated on collagen I or on uncoated substrate. However, the stimulation of macroautophagy by nutrient starvation, as reflected by the buildup of autophagosomes and the increase in the autophagic flux, was higher in cells plated on collagen I than in cells plated on collagen IV. These contrasting results were not due to differences in the starvation-dependent inhibition of mTOR complex 1 signaling. Interestingly, cells plated on collagen IV formed numerous focal adhesions (FAs), whereas fewer FAs were observed in cells plated on the other substrates. This implies that focal adhesion kinase (FAK) was more robustly activated by collagen IV. Silencing the expression of FAK by siRNA in cells plated on collagen IV shifted the autophagic phenotype of these cells to an "uncoated substrate autophagic phenotype" under both basal and starvation-induced conditions. Moreover, cells plated on collagen IV were less dependent on autophagy to survive in the absence of nutrients. We conclude that extracellular matrix components can modulate macroautophagy and mitigate its role in cell survival.     

Regulation of rat mammary gene expression by extracellular matrix components

In the mammary gland the induction and maintenance of differentiation are dependent on both lactogenic hormones and the extracellular matrix (ECM). Since mammary epithelial cells differentiate on a basement membrane in vivo we have examined the effects of basement membrane components on the expression of milk protein genes in primary rat mammary cultures. We examined the effects of a basement membrane gel derived from the Englebreth-Holm-Swarm tumor as well as its major component, laminin, on the expression of a group of milk protein genes. We demonstrate that the basement membrane gel induces alpha-casein and alpha-lactalbumin (alpha-LA) accumulation up to 160- and 70-fold, respectively, of that on tissue culture plastic. Laminin, a major component of the basement membrane, also caused significant induction of these same proteins. In order to determine whether these ECM effects occurred at a translational or post-translational level, pulse-chase experiments were performed. These experiments demonstrated that a laminin substratum selectively effects milk protein turnover and secretion. In order to demonstrate whether ECM effects occurred at the level of steady state accumulation of mRNA we performed dot blot and Northern analyses using cloned cDNA probes for alpha-, beta-, and gamma-caseins and alpha-LA. These studies demonstrated that ECM components induced alpha- and beta-caseins up to 10-fold, and alpha-LA up to 3-fold, with no significant effect on gamma-casein. These results demonstrate that milk protein genes are not coordinately regulated by ECM components. Furthermore, since the amount of induction of milk proteins exceeds the amount of induction of mRNAs for these proteins, we conclude that in our system a major effect of ECM components is at the translational and/or post-translational levels. Based on these findings we propose a model in which basement membrane components effect mammary gene expression at multiple levels.     

Effect of metabolic state on agglutination of human erythrocytes by concanavalin A.

Intact freshly drawn or stored human erythrocytes, which show little agglutination by concanavalin A, become agglutinable by this lectin in the presence of adenosine. alpha-Methylglucose (10 mM) completely inhibits this agglutination. The concanavalin A agglutination shows no sensitivity to vinblastine or cytochalasin B. Resealed membranes preparaed with ATP in lysing and resealing medium give modest agglutinability, while the presence of adenosine in both the lysing and the resealing medium results in a substantial agglutinability of the resealed membranes. Mild trypsin treatment of the erythrocytes causes an enhanced sensitivity to adenosine activation of the concanavalin A agglutination, while extensive trypsin treatment produced highly agglutinable erythrocytes that shown no response to the presence of adenosine in the lectin solution. The extensively treated erythrocytes also show concanavalin A agglutination at temperatures below 37 degrees C, under conditions in which intact or moderately treated erythrocytes do not agglutinate, with or without adenosine present. Results suggest that the adenosine activation of concanavalin A agglutination of intact human erythrocytes is mediated through a metabolic conversion of adenosine to a rapidly turned over metabolite which participates directly in the activation of agglutination. The agglutinability does not appear to depend on whole cell ATP levels, but may involve a particular pool of ATP. The effect of variation of cellular metabolic state and the response of particular systems involved in lectin-mediated agglutinability to cellular metabolism seem to be worth consideration in explaining the frequently large differences in agglutinability of und in cells in different biological states, such as those encountered in normal and transformed cells.     

Regulation of cell activity by the extracellular matrix: the concept of matrikines

The activity of connective tissue cells is modulated by a number of factors present in their environment. In addition to the soluble factors such as hormones, cytokines or growth factors, cells also receive signals from the surrounding extracellular matrix (ECM) macromolecules. Moreover, they may degrade the ECM proteins and liberate peptides which may by themselves constitute new signals for the surrounding cells. Therefore, an actual regulation loop exists in connective tissue, constituted by peptides generated by ECM degradation and connective tissue cells. The term of "matrikine" has been proposed to designate such ECM-derived peptides able to regulate cell activity. In this review, we summarize some data obtained in our laboratory with two different matrikines: the tripeptide glycyl-histidyl-lysine (GHK) and the heptapeptide cysteinyl-asparaginyl-tyrosyl-tyrosyl-seryl-asparaginyl-serine (CNYYSNS). GHK is a potent activator of ECM synthesis and remodeling, whereas CNYYSNS is able to inhibit polymorphonuclear leukocytes activation and decrease the invasive capacities of cancer cells.     

Regulation of the extrinsic apoptotic pathway by the extracellular matrix glycoprotein EMILIN2

Elastin microfibril interface-located proteins (EMILINs) constitute a family of extracellular matrix (ECM) glycoproteins characterized by the presence of an EMI domain at the N terminus and a gC1q domain at the C terminus. EMILIN1, the archetype molecule of the family, is involved in elastogenesis and hypertension etiology, whereas the function of EMILIN2 has not been resolved. Here, we provide evidence that the expression of EMILIN2 triggers the apoptosis of different cell lines. Cell death depends on the activation of the extrinsic apoptotic pathway following EMILIN2 binding to the TRAIL receptors DR4 and, to a lesser extent, DR5. Binding is followed by receptor clustering, colocalization with lipid rafts, death-inducing signaling complex assembly, and caspase activation. The direct activation of death receptors by an ECM molecule that mimics the activity of the known death receptor ligands is novel. The knockdown of EMILIN2 increases transformed cell survival, and overexpression impairs clonogenicity in soft agar and three-dimensional growth in natural matrices due to massive apoptosis. These data demonstrate an unexpected direct and functional interaction of an ECM constituent with death receptors and discloses an additional mechanism by which ECM cues can negatively affect cell survival.     

Effect of adenosine on concanavalin A agglutination of human erythrocytes

We have attempted to correlate the functional activity of protein 3 with its activity as a receptor for concanavalin A. The concanavalin A agglutination of human erythrocytes is enhanced by adenosine. It varies with time of storage of the blood and is dependent on the concentration of adenosine in the medium. Adenine and/or inosine, which increase cellular ATP, do not substitute for adenosine in enhancing agglutination, and adenosine enhances agglutination of fresh erythrocytes with normal levels of ATP. Thus, it appears that cellular ATP levels are not directly involved in modulation of concanavalin A agglutination by adenosine. Trypsin, which hydrolyzes most of the exposed proteins of the cell surface but does not alter protein 3, enhances concanavalin A agglutination without altering the relative response of the cell to adenosine.Glucose, as well as the glucose transport inhibitors maltose and cellobiose, inhibits agglutination. High concentrations of adenosine reverse the inhibition by glucose and enhance agglutination in the presence of maltose and cellobiose.Treatment of erythrocytes with 4,4′-diisothiocyanostilbene-2,2-disulfonic acid disodium salt, which selectively inhibits the anion transport function of protein 3, substantially inhibits adenosine-supported concanavalin A agglutination.Treatment of erythrocytes with iodoacetate under conditions in which it selectively reacts with glyceraldehyde-3-phosphate dehydrogenase inhibits agglutination. Adenosine protects this dehydrogenase in erythrocytes from inactivation by iodoacetate, over the same concentration range in which it enhances agglutination.