A novel fibrotic disorder associated with increased dermal fibroblast proliferation and downregulation of genes of the microfibrillar network.

Clinical evaluation of a young woman with subcutaneous fibrotic nodules, progressive distal joint contractures and marfanoid stature revealed a previously unrecognized fibrotic disorder characterized by several unique phenotypic features and some features overlapping with known disorders. Mutational analysis of the FBN1 and FBN2 genes excluded Marfan syndrome and congenital contractural arachnodactyly. MMP2 gene sequence analysis excluded multicentric osteolysis, nodulosis and arthropathy. The lack of mutations within the MAGP2 gene also excluded an MAGP2-associated disorder. In order to establish the mechanistic basis for the severe skin pathology noted in this patient, we performed transcriptional profiling of dermal fibroblasts, and candidate gene expression studies in conjunction with immunocytochemistry and cell-based and functional assays. Data from these experiments have further excluded any previously recognized fibrotic disorder and identified a unique pattern of gene expression in this patient consistent with progressive fibrosis. The pathogenic mechanisms included persistent proliferation of dermal fibroblasts in coexistence with a disarray of the microfibrillar network. Collagen accumulation, moreover, could be linked to extensive crosslinking resulting from increased activities of lysyl oxidases (LOX and LOXL), and lack of remodelling due to deficiencies in collagenolytic matrix metalloproteinases. The disorder may represent a novel syndrome in which transforming growth factor-ß1-independent dermal fibrosis, unlike known microfibrillar disorders caused by single gene deficiencies, associates with a disarray of the microfibrillar network.

Active lysyl oxidase (LOX) correlates with focal adhesion kinase (FAK)/paxillin activation and migration in invasive astrocytes.

The extracellular matrix (ECM) plays a critical role during the development and invasion of primary brain tumours. However, the function of ECM components and signalling between a permissive ECM and invasive astrocytes is not fully understood. We have recently reported the ECM enzyme, lysyl oxidase (LOX), in the central nervous system and observed up-regulation of LOX in anaplastic astrocytoma cells. While the catalytic function of LOX is essential for cross-linking of ECM proteins, we also reported that LOX induced invasive and metastatic properties in breast tumour epithelial cells through hydrogen peroxide-mediated FAK/Src activation. In this study, we tested the hypothesis that active LOX is expressed in anaplastic astrocytes and promotes FAK activation and invasive/migratory behaviour. Results demonstrate that increased expression and activity of LOX positively correlated with invasive phenotype of malignant astrocytoma cell lines. Immunohistochemistry detected increased LOX within tumour cells and ECM in grade I-IV astrocytic neoplasm compared with normal brain and coincidence of increased LOX with the loss of glial fibrillary acidic protein in higher-grade tumours. Increased active LOX in invasive astrocytes was accompanied by phosphorylation of FAK[Tyr576] and paxillin[Tyr118]; furthermore, both FAK and paxillin tyrosine phosphorylation were diminished by beta-aminopropionitrile inhibition of LOX activity and depletion of H(2)O(2) via catalase treatment. Additionally, we provide evidence that in astrocytes, LOX is likely processed by bone morphogenic protein-1 and LOX activity might be further stimulated by the expression of fibronectin in these cells. These results demonstrate an important LOX-mediated mechanism that promotes migratory/invasive behaviour of malignant astrocytes.

Poxvirus infection rapidly activates tyrosine kinase signal transduction.

Viruses have evolved a number of strategies to gain entry and replicate in host target cells that, for human immunodeficiency virus (HIV) and the poxvirus, myxoma virus, involve appropriating chemokine receptors. In this report we demonstrate that activation of multiple intracellular tyrosine phosphorylation events rapidly ensues following virus adsorption to NIH 3T3.CD4.CCR5 cells and affects the ultimate level of myxoma virus replication. UV-inactivated myxoma virus induces the rapid phosphorylation of CCR5 on tyrosine residues, the association of CCR5 with Jaks and p56(lck), and their phosphorylation-activation within minutes of virus adsorption. Additionally, we provide evidence for myxoma virus-inducible signal transducers and activators of transcription (Stat) and insulin receptor substrate (IRS) activation. In contrast to CCR5 activation effected by HIV Env protein, these myxoma virus-inducible phosphorylation events are not sensitive to pertussis toxin treatment. Moreover, in cells that are non-permissive for myxoma virus infection, we provide evidence that myxoma virus fails to invoke this tyrosine phosphorylation cascade. Consistent with the observation that infection of CCR5-expressing cells is blocked by herbimycin A and the Jak 2 inhibitor, tyrophostin AG490, we infer that viral infectivity may be dependent on non-G-protein-coupled signal transduction pathways triggered by the infecting myxoma virus particle. This provides a novel post-binding mechanism by which viruses can co-opt a cellular receptor to permit productive virus infection.

Impaired elastic-fiber assembly by fibroblasts from patients with either Morquio B disease or infantile GM1-gangliosidosis is linked to deficiency in the 67-kD spliced variant of beta-galactosidase.

We have previously shown that intracellular trafficking and extracellular assembly of tropoelastin into elastic fibers is facilitated by the 67-kD elastin-binding protein identical to an enzymatically inactive, alternatively spliced variant of beta-galactosidase (S-Gal). In the present study, we investigated elastic-fiber assembly in cultures of dermal fibroblasts from patients with either Morquio B disease or GM1-gangliosidosis who bore different mutations of the beta-galactosidase gene. We found that fibroblasts taken from patients with an adult form of GM1-gangliosidosis and from patients with an infantile form, carrying a missense mutations in the beta-galactosidase gene-mutations that caused deficiency in lysosomal beta-galactosidase but not in S-Gal-assembled normal elastic fibers. In contrast, fibroblasts from two cases of infantile GM1-gangliosidosis that bear nonsense mutations of the beta-galactosidase gene, as well as fibroblasts from four patients with Morquio B who had mutations causing deficiency in both forms of beta-galactosidase, did not assemble elastic fibers. We also demonstrated that S-Gal-deficient fibroblasts from patients with either GM1-gangliosidosis or Morquio B can acquire the S-Gal protein, produced by coculturing of Chinese hamster ovary cells permanently transected with S-Gal cDNA, resulting in improved deposition of elastic fibers. The present study provides a novel and natural model validating functional roles of S-Gal in elastogenesis and elucidates an association between impaired elastogenesis and the development of connective-tissue disorders in patients with Morquio B disease and in patients with an infantile form of GM1-gangliosidosis.

Characterization of beta-galactosidase mutations Asp332-->Asn and Arg148-->Ser, and a polymorphism, Ser532-->Gly, in a case of GM1 gangliosidosis.

We have identified and characterized three missense mutations in a patient with type 1 G(M1) gangliosidosis, namely a substitution of G for A at nucleotide position 1044 (G1044-->A; in exon 10) on one allele, which converts Asp(332) into asparagine, and both a mutation (C492-->A in exon 4, leading to the amino acid change of Arg(148)-->Ser) and a polymorphism (A1644-->G in exon 15, leading to a change of Ser(532)-->Gly) on the other allele. This patient had less than 1% residual beta-galactosidase activity and minimally detectable levels of immunoreactive beta-galactosidase protein in fibroblasts. To account for the above findings, a series of expression and immunolocalization studies were undertaken to assess the impact of each mutation. Transient overexpression in COS-1 cells of cDNAs encoding Asp(332)Asn, Arg(148)Ser and Ser(532)Gly mutant beta-galactosidases produced abundant amounts of precursor beta-galactosidase, with activities of 0, 84 and 81% compared with the cDNA clone for wild-type beta-galactosidase (GP8). Since the level of vector-driven expression is much less in Chinese hamster ovary (CHO) cells than in COS-1 cells, and we knew that exogenous beta-galactosidase undergoes lysosomal processing when expressed in these cells, transient expression studies were performed of Arg(148)Ser and Ser(532)Gly, which yielded active forms of the enzyme. In this case, the Arg(148)Ser and Ser(532)Gly products gave rise to 11% and 86% of the control activity respectively. These results were not unexpected, since the Arg(148)Ser mutation introduced a major conformational change into the protein, and we anticipated that it would be degraded in the endoplasmic reticulum (ER), whereas the polymorphism was expected to produce near-normal activity. To examine the effect of the Asp(332)Asn mutation on the catalytic activity, we isolated CHO clones permanently transfected with the Asp(332)Asn and Asp(332)Glu constructs, purified the enzymes by substrate-analogue-affinity chromatography, and determined their kinetic parameters. The V(max) values of both mutant recombinant enzymes were markedly reduced (less than 0.9% of the control), and the K(m) values were unchanged compared with the corresponding wild-type enzyme isolated at the same time. Both the Arg(148)Ser beta-galactosidase in CHO cells and Asp(332)Asn beta-galactosidases (in COS-1 and CHO cells) produced abundant immunoreaction in the perinuclear area, consistent with localization in the ER. A low amount was detected in lysosomes. Incubation of patient fibroblasts in the presence of leupeptin, which reduces the rate of degradation of lysosomal beta-galactosidase by thiol proteases, had no effect on residual enzyme activity, and immunostaining was again detected largely in the perinuclear area (localized to the ER) with much lower amounts in the lysosomes. In summary, the Arg(148)Ser mutation has no effect on catalytic activity, whereas the Asp(332)Asn mutation seriously reduces catalytic activity, suggesting that Asp(332) might play a role in the active site. Immunofluorescence studies indicate the expressed mutant proteins with Arg(148)Ser and Asp(332)Asn mutations are held up in the ER, where they are probably degraded, resulting in only minimum amounts of the enzyme becoming localized in the lysosomes. These results are completely consistent with findings in the cultured fibroblasts. Our results imply that most of the missense mutations described in G(M1) gangliosidosis to date have little effect on catalytic activity, but do affect protein conformation such that the resulting protein cannot be transported out of the ER and fails to arrive in the lysosome. This accounts for the minimal amounts of enzyme protein and activity seen in most G(M1) gangliosidosis patient fibroblasts.

Decreased elastin deposition and high proliferation of fibroblasts from Costello syndrome are related to functional deficiency in the 67-kD elastin-binding protein.

Costello syndrome is characterized by mental retardation, loose skin, coarse face, skeletal deformations, cardiomyopathy, and predisposition to numerous malignancies. The genetic origin of Costello syndrome has not yet been defined. Using immunohistochemistry and metabolic labeling with [3H]-valine, we have established that cultured skin fibroblasts obtained from patients with Costello syndrome did not assemble elastic fibers, despite an adequate synthesis of tropoelastin and normal deposition of the microfibrillar scaffold. We found that impaired production of elastic fibers by these fibroblasts is associated with a functional deficiency of the 67-kD elastin-binding protein (EBP), which is normally required to chaperone tropoelastin through the secretory pathways and to its extracellular assembly. Metabolic pulse labeling of the 67-kD EBP with radioactive serine and further chase of this tracer indicated that both normal fibroblasts and fibroblasts from patients with Costello syndrome initially synthesized comparable amounts of this protein; however, the fibroblasts from Costello syndrome patients quickly lost it into the conditioned media. Because the normal association between EBP and tropoelastin can be disrupted on contact with galactosugar-bearing moieties, and the fibroblasts from patients with Costello syndrome revealed an unusual accumulation of chondroitin sulfate-bearing proteoglycans (CD44 and biglycan), we postulate that a chondroitin sulfate may be responsible for shedding EBP from Costello cells and in turn for their impaired elastogenesis. This was further supported by the fact that exposure to chondroitinase ABC, an enzyme capable of chondroitin sulfate degradation, restored normal production of elastic fibers by fibroblasts from patients with Costello syndrome. We also present evidence that loss of EBP from fibroblasts of Costello syndrome patients is associated with an unusually high rate of cellular proliferation.

Impaired elastogenesis in Hurler disease: dermatan sulfate accumulation linked to deficiency in elastin-binding protein and elastic fiber assembly.

Hurler disease resulting from a deficiency in alpha-L-iduronidase, which causes an accumulation of dermatan sulfate and heparan sulfate glycosaminoglycans, is characterized by connective tissue and skeletal deformations, cardiomyopathy, cardiac valve defects, and progressive coronary artery stenosis. In this report, we present evidence that accumulation of dermatan sulfate but not heparan sulfate moieties is linked to impaired elastic fiber assembly that, in turn, contributes substantially to the development of the clinical phenotype in Hurler disease. Our data suggest that dermatan sulfate-bearing moieties bind to and cause functional inactivation of the 67-kd elastin-binding protein, a molecular chaperone for tropoelastin, which normally facilitates its secretion and assembly into elastic fibers. We demonstrate that, in contrast to normal skin fibroblasts and cells from Sanfilippo disease, which accumulate heparan sulfate, Hurler fibroblasts show reduced expression of elastin-binding protein and do not assemble elastic fibers, despite an adequate synthesis of tropoelastin and sufficient production of a microfibrillar scaffold of elastic fibers. Because cultured Hurler fibroblasts proliferate more quickly than their normal counterparts and the addition of exogenous insoluble elastin reduces their proliferation, we suggest that cell contacts with insoluble elastin play an important role in controlling their proliferation.

Bioengineering of elastic cartilage with aggregated porcine and human auricular chondrocytes and hydrogels containing alginate, collagen, and kappa-elastin.

Transplantation of isolated chondrocytes has long been acknowledged as a potential method for rebuilding small defects in damaged or deformed cartilages. Recent advances in tissue engineering permit us to focus on production of larger amounts of cartilaginous tissue, such as might be needed for reconstructive surgery of the entire auricle. In this report we describe modification of the basic techniques that lead to production of a large amount of elastic cartilage originated from porcine and human isolated chondrocytes. Small fragments of auricular cartilage were harvested from children undergoing ear reconstruction for microtia or extirpation of preauricular tags and from ears of juvenile pigs. Enzymatically isolated elastic chondrocytes were then agitated in suspension to form the chondronlike aggregates, which were further embedded in molded hydrogel constructs made of alginate and type I collagen augmented with kappa-elastin. The constructs were then implanted in nude mice and harvested 4 and 12 weeks after heterotransplantation. The resulting neocartilage closely resembled native auricular cartilage at the gross, microscopic, and ultrastructural levels. Immunohistochemistry and electron microscopy additionally confirmed that the newly produced cartilage contained the major components of the elastic cartilage-specific matrix, including collagen type II, proteoglycans, and well-assembled elastic fibers.

Inhibitory mechanisms by which suramin may attenuate neointimal formation after balloon angioplasty.

Restenotic neointimal lesions, a major limitation to coronary angioplasty, develop in response to diverse signals and depend on three properties of activated arterial smooth muscle cells (SMCs): proliferation, migration, and abnormal production of extracellular matrix. Most of the pharmacologic approaches targeting specific pathogenic factors facilitating development of restenosis have failed in clinical trials. Our results indicate that the polysulfonated naphthylurea suramin, a "non-specific drug" that interferes with multiple cellular proteins, inhibits neointimal formation in rabbit iliac arteries after balloon-catheter injury administered throughout the critical period of several weeks after the procedure. In vitro studies aimed at dissecting the mechanism(s) underlying the suramin-dependent effect demonstrated that, in addition to an inhibitory effect on SMC proliferation, suramin inhibited fibronectin and elastin deposition and the migration of SMCs through elastin membranes and into scratch gaps of monolayer cultures. We also demonstrated that suramin causes cell-surface accumulation of the elastin binding protein, a receptor that not only anchors SMCs to the extracellular matrix, but also inhibits SMC response to interleukin-1beta (IL-1beta). We conclude that suramin acts as a multitarget inhibitor of SMC activation and has a therapeutic potential as an agent that may attenuate arterial restenosis after angioplasty.

Cell surface aggregation of elastin receptor molecules caused by suramin amplified signals leading to proliferation of human glioma cells.

We have recently shown that glioma cell lines, as well as cells of human malignant gliomas in situ, synthesize tropoelastin. In addition, glioma cells degrade tropoelastin using metalloproteinase(s), and the resulting peptides, incapable of assembling in the extracellular fibers, interact with the 67-kDa cell surface elastin binding protein (EBP), to transduce signals leading to up-regulation of cell proliferation. In this report, we show that exposure to the polysulfonated bis-naphthylurea suramin causes accumulation of physiologically active EBP molecules on the cell surface of a panel of glioma cell lines (U87, MG, U251 MG, U343 MG-A, U373 MG, SF 126, SF188, SF539), which results in an increase of cellular attachment to elastin-coated dishes and in an efficient binding of radiolabeled tropoelastin. Moreover, 100-200 microM suramin stimulates [3H]-thymidine incorporation by those tropoelastin-producing glioma cell lines, but not by A 2058 melanoma cells, which do not produce elastin. Treatment of all glioma cell lines with 100 microM suramin consistently increased expression of cyclin A and its cyclin-dependent kinase, cdk 2, to levels reached following the exposure to exogenous elastin-degradation products (kappa-elastin). Our data suggest that a suramin-stimulated accumulation of EBP molecules on the cell surface of glioma cells amplifies the elastin-derived signals, leading to their progression through the cell cycle.

UVB irradiation stimulates deposition of new elastic fibers by modified epithelial cells surrounding the hair follicles and sebaceous glands in mice.

UVB irradiation stimulates the synthesis of elastin in the skin of humans and experimental animals. In this study we localized the site and the cells that are responsible for the synthesis of murine dermal elastic fibers. SKH-1 hairless mice were irradiated with UVB and the skin removed for light microscopy, electron microscopy, in situ hybridization, immunohistochemistry, and biochemical studies. In response to chronic low doses of UVB there was an initial moderate increase in tropoelastin mRNA in the papillary dermis. By contrast, there was a continuous marked elevation of collagen alpha1(I) message localizing to sites of inflammatory cell influx throughout the upper and lower dermis. After 25 wk of UV irradiation there was a 2-fold increase in skin elastin, yet total collagen remained unchanged. Serial desmosine analysis from en face sections indicated the increase in elastin content was due to dermal elastic fibers, an increase in the size and number of the dermal cysts, and an increase in subpanniculus elastic fibers. Elastin stains of en face sections suggested that the elastic fibers in the upper dermis were exclusively derived from cells lining the epithelial root sheath and sebaceous glands. In response to UV irradiation, the elastic fibers increased in number and size, wrapping around these structures and aligning in both directions as long fibers parallel to the body axis. Electron micrographs indicated that modified epithelial cells in close proximity to the flattened epithelial cells that encircled the root sheath and sebaceous glands were the source of the elastic fibers.

Astrocytoma cell interaction with elastin substrates: implications for astrocytoma invasive potential.

Elastin has been identified within the meninges and the microvasculature of the normal human brain. However, the role that elastin plays in either facilitating astrocytoma cell attachment to these structures or modulating astrocytoma invasion has not been previously characterized. We have recently shown that astrocytoma cell lines and specimens produce tropoelastin, and express the 67 kDa elastin binding protein (EBP). In the present report, we have established that astrocytoma cells attach to elastin as a substrate in vitro. The U87 MG astrocytoma cell line demonstrated the greatest degree of adhesion. In addition, all astrocytoma cell lines examined were capable of penetrating and migrating through an intact elastin membrane, and of degrading tritiated-elastin, a process that could be prevented by the pre-incubation of astrocytoma cells with EDTA, but not with alpha1-antitrypsin. Astrocytoma cells were also capable of penetrating 1 mm sections of human brain tissue maintained as organotypic cultures. Interestingly, the invasive potential of cultured astrocytoma cells plated on organotypic cultures of human brain was significantly increased after exposure to elastin degradation products (kappa-elastin), which interact with astrocytoma cell surface EBP. Our data show that astrocytoma cells express a functional 67 kDa EBP, enabling them to potentially recognize and attach to elastin as a substrate. These data also suggest that this elastin receptor may be involved in processes which regulate regional astrocytoma invasion.

Antibody raised to AKAAAKAAAKA sequence on tropoelastin recognizes tropoelastin but not mature crosslinked elastin: A new tool in metabolic and structural studies of elastogenesis.

Tropoelastin, which is secreted from the cell in a soluble form, contains specific alanine rich repeat domains that are destined to form covalent desmosine and isodesmosine crosslinks in mature insoluble elastin. We raised a monospecific polyclonal antibody to a AKAAAKAAAKA synthetic peptide (AKA) which represents this alanine rich region of tropoelastin. The antibody was reactive with the original peptide antigen and purified tropoelastin, but not with mature crosslinked elastin isolated from several animal species. Conditioned media from chick aorta smooth muscle cells in culture reacted in an ELISA with the AKA antibody, but only in the presence of BAPN to block the conversion of the epsilon-amino groups to aldehydes. Immunofluorescence demonstrated that the AKA antibody decorated newly deposited tropoelastin assembled in fine fibrils in matrix produced by cultured human skin fibroblasts. EM-immunogold specifically localized this antibody to the immature elastic fibers present in fetal sheep ductus arteriosus. Moreover, immunohistochemistry demonstrated that the antibody recognized nonpolymerized tropoelastin assembled on the periphery of elastic fibers in the aorta of chicks raised on copper deficient and BAPN containing diets. These studies demonstrate that this new anti-tropoelastin antibody can be used as a useful tool to investigate elastin metabolism where it is important to distinguish between tropoelastin and mature crosslinked elastin.

A Pro504 --> Ser substitution in the beta-subunit of beta-hexosaminidase A inhibits alpha-subunit hydrolysis of GM2 ganglioside, resulting in chronic Sandhoff disease.

The GM2 gangliosidoses are caused by mutations in the genes encoding the alpha- (Tay-Sachs) or beta- (Sandhoff) subunits of heterodimeric beta-hexosaminidase A (Hex A), or the GM2 activator protein (AB variant), a substrate-specific co-factor for Hex A. Although the active site associated with the hydrolysis of GM2 ganglioside, as well as part of the binding site for the ganglioside-activator complex, is associated with the alpha-subunit, elements of the beta-subunit are also involved. Missense mutations in these genes normally result in the mutant protein being retained in the endoplasmic reticulum and degraded. The mutations associated with the B1-variant of Tay-Sachs are rare exceptions that directly affect residues in the alpha-active site. We have previously reported two sisters with chronic Sandhoff disease who were heterozygous for the common HEXB deletion allele. Cells from these patients had higher than expected levels of mature beta-protein and residual Hex A activity, approximately 20%. We now identify these patients' second mutant allele as a C1510T transition encoding a beta-Pro504 --> Ser substitution. Biochemical characterization of Hex A from both patient cells and cotransfected CHO cells demonstrated that this substitution (a) decreases the level of heterodimer transport out of the endoplasmic reticulum by approximately 45%, (b) lowers its heat stability, (c) does not affect its Km for neutral or charged artificial substrates, and (d) lowers the ratio of units of ganglioside/units of artificial substrate hydrolyzed by a factor of 3. We concluded that the beta-Pro504 --> Ser mutation directly affects the ability of Hex A to hydrolyze its natural substrate but not its artificial substrates. The effect of the mutation on ganglioside hydrolysis, combined with its effect on intracellular transport, produces chronic Sandhoff disease.

Tropoelastin and elastin degradation products promote proliferation of human astrocytoma cell lines.

Expression of tropoelastin, the precursor of insoluble elastin and a major component of elastic fibers, has not yet been demonstrated in astrocytomas nor has it been linked to their proliferation. Here we report that human astrocytoma cell lines (U87 MG, U251 MG, U343 MG-A, U373 MG, SF 126, SF188, SF 539), as well as surgical specimens of malignant human astrocytomas, express intracellular tropoelastin. The tropoelastin produced by astrocytoma cells is, however, susceptible to proteolytic trimming to the extent that it cannot be assembled into extracellular elastic fibers. Astrocytoma cells also express the cell surface 67-kDa elastin binding protein (EBP), which binds elastin degradation products, leading to the upregulation of cyclin A and cdk2 and increased incorporation of [3H]-thymidine. The elastin-dependent mitogenic response of astrocytoma cells is abolished by lactose and chondroitin sulfate, factors which cause shedding of this 67-kDa elastin receptor from the cell surface and by blocking anti-EBP antibody. We therefore suggest that, in astrocytomas, endogenous tropoelastin degradation products bind to EBP and generate signals leading to cell cycle progression in an autocrine or paracrine manner. This is the first report implicating elastin-derived peptides as possible mitogens in malignant astrocytomas.

The 67-kDa enzymatically inactive alternatively spliced variant of beta-galactosidase is identical to the elastin/laminin-binding protein.

Our previous studies showed immunological and functional similarities, as well as partial sequence homology, between the enzymatically inactive alternatively spliced variant of human beta-galactosidase (S-gal) and the 67-kDa elastin/laminin-binding protein (EBP) from sheep. To define the genetic origin of the EBP further, a full-length human S-gal cDNA clone was constructed and subjected to in vitro transcription/translation. The cDNA was also transfected into COS-1 cells and into the EBP-deficient smooth muscle cells (SMC) from sheep ductus arteriosus (DA). In vitro translation yielded an unglycosylated form of the S-gal protein, which immunoreacted with anti-beta-galactosidase antibodies and bound to elastin and laminin affinity columns. S-gal cDNA transfections into COS-1 and DA SMC increased expression of a 67-kDa protein that immunolocalized intracellularly and to the cell surface and, when extracted from the cells, bound to elastin. The S-gal-transfected cells displayed increased adherence to elastin-covered dishes, consistent with the cell surface distribution of the newly produced S-gal-encoded protein. Transfection of DA SMC additionally corrected their impaired elastic fiber assembly. These results conclusively identify the 67-kDa splice variant of beta-galactosidase as EBP.

Actin filaments participate in the relocalization of phosphatidylinositol3-kinase to glucose transporter-containing compartments and in the stimulation of glucose uptake in 3T3-L1 adipocytes.

Insulin stimulates the rate of glucose uptake into muscle and adipose cells by translocation of glucose transporters from an intracellular storage pool to the plasma membrane. This event requires the prior activation of phosphatidylinositol 3-kinase (PI 3-kinase). Here we report that insulin causes an increase in wortmannin-sensitive PI 3-kinase activity and a gain in the enzyme's regulatory and catalytic subunits p85alpha and p110beta (but not p110alpha) in the intracellular compartments containing glucose transporters. The hormone also caused a marked reorganization of actin filaments, which was prevented by cytochalasin D. Cytochalasin D also decreased significantly the insulin-dependent association of PI 3-kinase activity and the levels of insulin receptor substrate (IRS)-1, p85alpha and p110beta with immunopurified GLUT4-containing compartments. In contrast, the drug did not alter the insulin-induced tyrosine phosphorylation of IRS-1, the association of PI 3-kinase with IRS-1, or the stimulation of PI 3-kinase by insulin in anti-(IRS-1) or anti-p85 immunoprecipitates from whole cell lysates. Cytochalasin D, and the chemically unrelated latrunculin B, which also inhibits actin filament reassembly, prevented the insulin stimulation of glucose transport by approx. 50%. Cytochalasin D decreased by about one-half the insulin-dependent translocation to the plasma membrane of the GLUT1 and GLUT4 glucose transporters. The results suggest that the existence of intact actin filament is correlated with the full recruitment of glucose transporters by insulin. The underlying function of the actin filaments might be to facilitate the insulin-mediated association of the p85-p110 PI 3-kinase with glucose-transporter-containing compartments.

Proteolytic activity of human non-Hodgkin's lymphomas.

This study was conducted to assess the net proteolytic activity of human non-Hodgkin's lymphomas (NHLs). We have compared the extracellular matrix (ECM)-degradative abilities of human NHLs, reactive lymphoid hyperplasias, and established lymphoid cell lines using Matrigel invasion and elastin degradation assays. The inhibition studies allowed identification of the classes of proteinases involved in ECM degradation. Our results indicate that lymphocytes and other leukocytes derived from both human NHLs and reactive lymphoid hyperplasias are capable of Matrigel penetration, but only cells derived from the high-grade human NHLs degrade elastin in vitro. Established lymphoid cell lines (both malignant and Epstein-Barr virus immortalized) do not produce MMP-9, do not penetrate the Matrigel, and do not degrade elastin. Moreover, in human NHLs, elastolytic activity is blocked by metalloproteinase inhibitors, while inhibitors of the other classes of proteolytic enzymes have only minor effects. This study identifies metalloproteinases as the most important class of proteinases involved in ECM degradation by NHLs. The previous studies suggest that, within this class, MMP-9 represents the key enzyme that plays a role in the biological aggressiveness of human NHLs.

Macrophage phagocytosis of polyethylene particulate in vitro.

In this study, an in vitro model has been developed to examine the interactions of macrophages with ultrahigh molecular-weight polyethylene (UHMWPE) and high-density polyethylene (HDPE) particles. Polyethylene particles are the major constituent of the material debris formed as a result of orthopedic implant wear. However, the study of polyethylene particle interactions with cells has been limited. UHMWPE (18-20 microns) and HDPE (4-10 microns) were suspended in soluble collagen type I and subsequently solidified on glass coverslips. The particle chemistry was characterized by Fourier transform infra-red spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS). Mouse cell line macrophages (IC-21) were established on the collagen-particle substrata and maintained for up to 24 h. The response of the cells to the particles was examined by light and transmission electron microscopy (LM and TEM), as well as by scanning electron microscopy (SEM), and compared to cells on control collagen surfaces without particles. Histological analysis of the samples revealed that the macrophages surrounded larger particles (18-20 microns) and the cells appeared to be attached to the surface of the particles, and the smaller particles (4-10 microns) had been phagocytosed within 2 h. Inflammatory cytokines (TNF-alpha, IL-1 alpha, IL-1 beta, and IL-6), lysosomal enzymes (beta-galactosidase and hexosaminidase), and prostaglandin E2 were released into the medium, and IL-1 alpha, IL-1 beta, PGE2, beta-galactosidase, and hexosaminidase levels were significantly increased over collagen control values. The results demonstrate active phagochemotaxis by macrophages for wear particulates and validate this model as a means of studying the specific in vitro interactions of polyethylene with cells.

Elastin receptor and cell-matrix interactions in heart transplant-associated arteriosclerosis.

Vascular cells, as well as monocytes, neutrophils, and lymphocytes which may infiltrate vascular walls and tissues express a multifunctional 67 kD protein which also serves as a subunit of the cell surface "elastin receptor". This protein differs structurally and functionally from other matrix adhesion molecules. Unlike the integrins or cadherins, it is not a transmembrane molecule, but can be immobilized on the cell surface by association with two other membrane-anchored proteins. Once expressed on the cell surface, it may mediate cell-matrix interaction in a calcium-independent manner. Unlike most integrins, which recognize the linear sequence on the matrix ligands (RGD), it recognizes the secondary structure of the matrix macromolecules and binds to several non identical domains on different matrix components, as long as they form the appropriate hydrophobic conformation. Similarly to the transmembrane selectins, the 67 kD protein has lectin-like properties with the galactosugars' binding specificity. However, binding of galactosugar-bearing ligands interrupts its contacts with matrix proteins and displaces the 67 kD protein from the cell surface. Moreover, the 67 kD protein also serves as an intracellular chaperone which facilitates secretion of tropoelastin and assembly of elastic fibers. In this review I will address the role of this 67 kD protein in mechanisms of mutual interaction between vascular smooth muscle cells, infiltrating leukocytes, and several components of extracellular matrix during the development of heart-transplant associated arteriosclerosis.