Collagenase-3 Induction in Rat Lung Fibroblasts Requires the Combined Effects of Tumor Necrosis Factor-α and 12-Lipoxygenase Metabolites: A Model of Macrophage-induced, Fibroblast-driven Extracellular Matrix Remodeling during Inflammatory Lung Injury

The mechanisms responsible for the induction of matrix-degrading proteases during lung injury are ill defined. Macrophage-derived mediators are believed to play a role in regulating synthesis and turnover of extracellular matrix at sites of inflammation. We find a localized increase in the expression of the rat interstitial collagenase (MMP-13; collagenase-3) gene from fibroblastic cells directly adjacent to macrophages within silicotic rat lung granulomas. Conditioned medium from macrophages isolated from silicotic rat lungs was found to induce rat lung fibroblast interstitial collagenase gene expression. Conditioned medium from primary rat lung macrophages or J774 monocytic cells activated by particulates in vitro also induced interstitial collagenase gene expression. Tumor necrosis factor-α (TNF-α) alone did not induce interstitial collagenase expression in rat lung fibroblasts but did in rat skin fibroblasts, revealing tissue specificity in the regulation of this gene. The activity of the conditioned medium was found to be dependent on the combined effects of TNF-α and 12-lipoxygenase-derived arachidonic acid metabolites. The fibroblast response to this conditioned medium was dependent on de novo protein synthesis and involved the induction of nuclear activator protein-1 activity. These data reveal a novel requirement for macrophage-derived 12-lipoxygenase metabolites in lung fibroblast MMP induction and provide a mechanism for the induction of resident cell MMP gene expression during inflammatory lung processes.

Overexpression of the Matrix Metalloproteinase Matrilysin Results in Premature Mammary Gland Differentiation and Male Infertility

To examine the role of matrilysin (MAT), an epithelial cell-specific matrix metalloproteinase, in the normal development and function of reproductive tissues, we generated transgenic animals that overexpress MAT in several reproductive organs. Three distinct forms of human MAT (wild-type, active, and inactive) were placed under the control of the murine mammary tumor virus promoter/enhancer. Although wild-type, active, and inactive forms of the human MAT protein could be produced in an in vitro culture system, mutations of the MAT cDNA significantly decreased the efficiency with which the MAT protein was produced in vivo. Therefore, animals carrying the wild-type MAT transgene that expressed high levels of human MAT in vivo were further examined. Mammary glands from female transgenic animals were morphologically normal throughout mammary development, but displayed an increased ability to produce β-casein protein in virgin animals. In addition, beginning at approximately 8 mo of age, the testes of male transgenic animals became disorganized with apparent disintegration of interstitial tissue that normally surrounds the seminiferous tubules. The disruption of testis morphology was concurrent with the onset of infertility. These results suggest that overexpression of the matrix-degrading enzyme MAT alters the integrity of the extracellular matrix and thereby induces cellular differentiation and cellular destruction in a tissue-specific manner.

Integrin regulation of c-Abl tyrosine kinase activity and cytoplasmic–nuclear transport

The product of the c-abl protooncogene is a nonreceptor tyrosine kinase found in both the cytoplasm and the nucleus. We report herein that cell adhesion regulates the kinase activity and subcellular localization of c-Abl. When fibroblastic cells are detached from the extracellular matrix, kinase activity of both cytoplasmic and nuclear c-Abl decreases, but there is no detectable alteration in the subcellular distribution. Upon adhesion to the extracellular matrix protein fibronectin, a transient recruitment of a subset of c-Abl to early focal contacts is observed coincident with the export of c-Abl from the nucleus to the cytoplasm. The cytoplasmic pool of c-Abl is reactivated within 5 min of adhesion, but the nuclear c-Abl is reactivated after 30 min, correlating closely with its return to the nucleus and suggesting that the active nuclear c-Abl originates in the cytoplasm. In quiescent cells where nuclear c-Abl activity is low, the cytoplasmic c-Abl is similarly regulated by adhesion but the nuclear c-Abl is not activated upon cell attachment. These results show that c-Abl activation requires cell adhesion and that this tyrosine kinase can transmit integrin signals to the nucleus where it may function to integrate adhesion and cell cycle signals.

NF-κB activation and interleukin 6 production in fibroblasts by estrogen receptor-negative breast cancer cell-derived interleukin 1α

Several angiogenic factors and extracellular matrix-degrading enzymes that promote invasion and metastasis of cancer are produced by stromal fibroblasts that surround cancer cells. The expression of genes that code for some of these proteins is regulated by the transcription factor NF-κB. In this report, we demonstrate that conditioned medium (CM) from estrogen receptor (ER)-negative but not ER-positive breast cancer cells induces NF-κB in fibroblasts. In contrast, CM from both ER-positive and ER-negative breast cancer cells induces NF-κB in macrophages and endothelial cells. NF-κB activation in fibroblasts was accompanied by induction of interleukin 6 (IL-6) and urokinase plasminogen activator (uPA), both of which promote angiogenesis and metastasis. A survey of cytokines known for their ability to induce NF-κB identified IL-1α as the factor responsible for NF-κB activation in fibroblasts. Analysis of primary breast carcinomas revealed the presence of IL-1α transcripts in majority of lymph node-positive breast cancers. These results along with the known role of IL-1α and IL-6 in osteoclast formation provide insight into the mechanism of metastasis and hypercalcemia in advanced breast cancers.

Polygalacturonase Gene Expression in Ripe Melon Fruit Supports a Role for Polygalacturonase in Ripening-Associated Pectin Disassembly

Ripening-associated pectin disassembly in melon is characterized by a decrease in molecular mass and an increase in the solubilization of polyuronide, modifications that in other fruit have been attributed to the activity of polygalacturonase (PG). Although it has been reported that PG activity is absent during melon fruit ripening, a mechanism for PG-independent pectin disassembly has not been positively identified. Here we provide evidence that pectin disassembly in melon (Cucumis melo) may be PG mediated. Three melon cDNA clones with significant homology to other cloned PGs were isolated from the rapidly ripening cultivar Charentais (C. melo cv Reticulatus F1 Alpha) and were expressed at high levels during fruit ripening. The expression pattern correlated temporally with an increase in pectin-degrading activity and a decrease in the molecular mass of cell wall pectins, suggesting that these genes encode functional PGs. MPG1 and MPG2 were closely related to peach fruit and tomato abscission zone PGs, and MPG3 was closely related to tomato fruit PG. MPG1, the most abundant melon PG mRNA, was expressed in Aspergillus oryzae. The culture filtrate exponentially decreased the viscosity of a pectin solution and catalyzed the linear release of reducing groups, suggesting that MPG1 encodes an endo-PG with the potential to depolymerize melon fruit cell wall pectin. Because MPG1 belongs to a group of PGs divergent from the well-characterized tomato fruit PG, this supports the involvement of a second class of PGs in fruit ripening-associated pectin disassembly.

Activation of a 15-kDa endonuclease in hypoxia/reoxygenation injury without morphologic features of apoptosis.

Hypoxia/reoxygenation is an important cause of tissue injury in a variety of organs and is classically considered to be a necrotic form of cell death. We examined the role of endonuclease activation, considered a characteristic feature of apoptosis, in hypoxia/reoxygenation injury. We demonstrate that subjecting rat renal proximal tubules to hypoxia/reoxygenation results in DNA strand breaks and DNA fragmentation (both by an in situ technique and by agarose gel electrophoresis), which precedes cell death. Hypoxia/reoxygenation resulted in an increase in DNA-degrading activity with an apparent molecular mass of 15 kDa on a substrate gel. This DNA-degrading activity was entirely calcium dependent and was blocked by the endonuclease inhibitor aurintricarboxylic acid. The protein extract from tubules subjected to hypoxia/reoxygenation cleaved intact nuclear DNA obtained from normal proximal tubules into small fragments, which further supports the presence of endonuclease activity. Despite unequivocal evidence of endonuclease activation, the morphologic features of apoptosis, including chromatin condensation, were not observed by light and electron microscopy. Endonuclease inhibitors, aurintricarboxylic acid and Evans blue, provided complete protection against DNA damage induced by hypoxia/reoxygenation but only partial protection against cell death. Taken together, our data provide strong evidence for a role of endonuclease activation as an early event, which is entirely responsible for the DNA damage and partially responsible for the cell death that occurs during hypoxia/reoxygenation injury. Our data also indicate that in hypoxia/reoxygenation injury endonuclease activation and DNA fragmentation occur without the morphological features of apoptosis.

Regulation of matrix metalloproteinase-9 and inhibition of tumor invasion by the membrane-anchored glycoprotein RECK

A human fibroblast cDNA expression library was screened for cDNA clones giving rise to flat colonies when transfected into v-Ki-ras-transformed NIH 3T3 cells. One such gene, RECK, encodes a membrane-anchored glycoprotein of about 110 kDa with multiple epidermal growth factor-like repeats and serine-protease inhibitor-like domains. While RECK mRNA is expressed in various human tissues and untransformed cells, it is undetectable in tumor-derived cell lines and oncogenically transformed cells. Restored expression of RECK in malignant cells resulted in suppression of invasive activity with concomitant decrease in the secretion of matrix metalloproteinase-9 (MMP-9), a key enzyme involved in tumor invasion and metastasis. Moreover, purified RECK protein was found to bind to, and inhibit the proteolytic activity of, MMP-9. Thus, RECK may link oncogenic signals to tumor invasion and metastasis.

Attachment to the nuclear matrix mediates specific alterations in chromatin structure

The DNA in eukaryotic chromosomes is organized into a series of loops that are permanently attached at their bases to the nuclear scaffold or matrix at sequences known as scaffold-attachment or matrix-attachment regions. At present, it is not clear what effect affixation to the nuclear matrix has on chromatin architecture in important regulatory regions such as origins of replication or the promoter regions of genes. In the present study, we have investigated cell-cycle-dependent changes in the chromatin structure of a well characterized replication initiation zone in the amplified dihydrofolate reductase domain of the methotrexate-resistant Chinese hamster ovary cell line CHOC 400. Replication can initiate at any of multiple potential sites scattered throughout the 55-kilobase intergenic region in this domain, with two subregions (termed ori-β and ori-γ) being somewhat preferred. We show here that the chromatin in the ori-β and ori-γ regions undergoes dramatic alterations in micrococcal nuclease hypersensitivity as cells cross the G1/S boundary, but only in those copies of the amplicon that are affixed to the nuclear matrix. In contrast, the fine structure of chromatin in the promoter of the dihydrofolate reductase gene does not change detectably as a function of matrix attachment or cell-cycle position. We suggest that attachment of DNA to the nuclear matrix plays an important role in modulating chromatin architecture, and this could facilitate the activity of origins of replication.

Carbohydrate binding and resistance to proteolysis control insecticidal activity of Griffonia simplicifolia lectin II

Griffonia simplicifolia leaf lectin II (GSII), a plant defense protein against certain insects, consists of an N-acetylglucosamine (GlcNAc)-binding large subunit with a small subunit having sequence homology to class III chitinases. Much of the insecticidal activity of GSII is attributable to the large lectin subunit, because bacterially expressed recombinant large subunit (rGSII) inhibited growth and development of the cowpea bruchid, Callosobruchus maculatus (F). Site-specific mutations were introduced into rGSII to generate proteins with altered GlcNAc binding, and the different rGSII proteins were evaluated for insecticidal activity when added to the diet of the cowpea bruchid. At pH 5.5, close to the physiological pH of the cowpea bruchid midgut lumen, rGSII recombinant proteins were categorized as having high (rGSII, rGSII-Y134F, and rGSII-N196D mutant proteins), low (rGSII-N136D), or no (rGSII-D88N, rGSII-Y134G, rGSII-Y134D, and rGSII-N136Q) GlcNAc-binding activity. Insecticidal activity of the recombinant proteins correlated with their GlcNAc-binding activity. Furthermore, insecticidal activity correlated with the resistance to proteolytic degradation by cowpea bruchid midgut extracts and with GlcNAc-specific binding to the insect digestive tract. Together, these results establish that insecticidal activity of GSII is functionally linked to carbohydrate binding, presumably to the midgut epithelium or the peritrophic matrix, and to biochemical stability of the protein to digestive proteolysis.

α1 and α2 Integrins Mediate Invasive Activity of Mouse Mammary Carcinoma Cells through Regulation of Stromelysin-1 Expression

Tumor cell invasion relies on cell migration and extracellular matrix proteolysis. We investigated the contribution of different integrins to the invasive activity of mouse mammary carcinoma cells. Antibodies against integrin subunits α6 and β1, but not against α1 and α2, inhibited cell locomotion on a reconstituted basement membrane in two-dimensional cell migration assays, whereas antibodies against β1, but not against α6 or α2, interfered with cell adhesion to basement membrane constituents. Blocking antibodies against α1 integrins impaired only cell adhesion to type IV collagen. Antibodies against α1, α2, α6, and β1, but not α5, integrin subunits reduced invasion of a reconstituted basement membrane. Integrins α1 and α2, which contributed only marginally to motility and adhesion, regulated proteinase production. Antibodies against α1 and α2, but not α6 and β1, integrin subunits inhibited both transcription and protein expression of the matrix metalloproteinase stromelysin-1. Inhibition of tumor cell invasion by antibodies against α1 and α2 was reversed by addition of recombinant stromelysin-1. In contrast, stromelysin-1 could not rescue invasion inhibited by anti-α6 antibodies. Our data indicate that α1 and α2 integrins confer invasive behavior by regulating stromelysin-1 expression, whereas α6 integrins regulate cell motility. These results provide new insights into the specific functions of integrins during tumor cell invasion.

Characterization of ATM Expression, Localization, and Associated DNA-dependent Protein Kinase Activity

Ataxia telangiectasia–mutated gene (ATM) is a 350-kDa protein whose function is defective in the autosomal recessive disorder ataxia telangiectasia (AT). Affinity-purified polyclonal antibodies were used to characterize ATM. Steady-state levels of ATM protein varied from undetectable in most AT cell lines to highly expressed in HeLa, U2OS, and normal human fibroblasts. Subcellular fractionation showed that ATM is predominantly a nuclear protein associated with the chromatin and nuclear matrix. ATM protein levels remained constant throughout the cell cycle and did not change in response to serum stimulation. Ionizing radiation had no significant effect on either the expression or distribution of ATM. ATM immunoprecipitates from HeLa cells and the human DNA-dependent protein kinase null cell line MO59J, but not from AT cells, phosphorylated the 34-kDa subunit of replication protein A (RPA) complex in a single-stranded and linear double-stranded DNA–dependent manner. Phosphorylation of p34 RPA occurred on threonine and serine residues. Phosphopeptide analysis demonstrates that the ATM-associated protein kinase phosphorylates p34 RPA on similar residues observed in vivo. The DNA-dependent protein kinase activity observed for ATM immunocomplexes, along with the association of ATM with chromatin, suggests that DNA damage can induce ATM or a stably associated protein kinase to phosphorylate proteins in the DNA damage response pathway.

α5β1 Integrin Controls Cyclin D1 Expression by Sustaining Mitogen-activated Protein Kinase Activity in Growth Factor-treated Cells

Cyclin D1 expression is jointly regulated by growth factors and cell adhesion to the extracellular matrix in many cell types. Growth factors are thought to regulate cyclin D1 expression because they stimulate sustained extracellular signal-regulated kinase (ERK) activity. However, we show here that growth factors induce transient ERK activity when added to suspended fibroblasts and sustained ERK activity only when added to adherent fibroblasts. Cell attachment to fibronectin or anti-α5β1 integrin is sufficient to sustain the ERK signal and to induce cyclin D1 in growth factor-treated cells. Moreover, when we force the sustained activation of ERK, by conditional expression of a constitutively active MAP kinase/ERK kinase, we overcome the adhesion requirement for expression of cyclin D1. Thus, at least in part, fibroblasts are mitogen and anchorage dependent, because integrin action allows for a sustained ERK signal and the expression of cyclin D1 in growth factor-treated cells.

Cell-Matrix Adhesions Differentially Regulate Fascin Phosphorylation

Cell adhesion to individual macromolecules of the extracellular matrix has dramatic effects on the subcellular localization of the actin-bundling protein fascin and on the ability of cells to form stable fascin microspikes. The actin-binding activity of fascin is down-regulated by phosphorylation, and we used two differentiated cell types, C2C12 skeletal myoblasts and LLC-PK1 kidney epithelial cells, to examine the hypothesis that cell adhesion to the matrix components fibronectin, laminin-1, and thrombospondin-1 differentially regulates fascin phosphorylation. In both cell types, treatment with the PKC activator 12-tetradecanoyl phorbol 13-acetate (TPA) or adhesion to fibronectin led to a diffuse distribution of fascin after 1 h. C2C12 cells contain the PKC family members α, γ, and λ, and PKCα localization was altered upon cell adhesion to fibronectin. Two-dimensional isoelectric focusing/SDS-polyacrylamide gels were used to determine that fascin became phosphorylated in cells adherent to fibronectin and was inhibited by the PKC inhibitors calphostin C and chelerythrine chloride. Phosphorylation of fascin was not detected in cells adherent to thrombospondin-1 or to laminin-1. LLC-PK1 cells expressing green fluorescent protein (GFP)-fascin also displayed similar regulation of fascin phosphorylation. LLC-PK1 cells expressing GFP-fascin S39A, a nonphosphorylatable mutant, did not undergo spreading and focal contact organization on fibronectin, whereas cells expressing a GFP-fascin S39D mutant with constitutive negative charge spread more extensively than wild-type cells. In contrast, C2C12 cells coexpressing S39A fascin with endogenous fascin remained competent to form microspikes on thrombospondin-1, and cells that expressed fascin S39D attached to thrombospondin-1 but did not form microspikes. Blockade of PKCα activity by TPA-induced down-regulation led to actin association of wild-type fascin in fibronectin-adherent C2C12 and LLC-PK1 cells but did not alter the distribution of S39A or S39D fascins. The association of fascin with actin in fibronectin-adherent cells was also evident in the presence of an inhibitory antibody to integrin α5 subunit. These novel results establish matrix-initiated PKC-dependent regulation of fascin phosphorylation at serine 39 as a mechanism whereby matrix adhesion is coupled to the organization of cytoskeletal structure.

Identification of a nuclear domain with deacetylase activity

Here, we describe the identification and characterization of a nuclear body (matrix-associated deacetylase body) whose formation and integrity depend on deacetylase activity. Typically, there are 20–40 0.5-μM bodies per nucleus, although the size and number can vary substantially. The structure appears to contain both class I and the recently described class II histone deacetylases (HDAC)5 and 7 along with the nuclear receptor corepressors SMRT (silencing mediator for retinoid and thyroid receptor) and N-CoR (nuclear receptor corepressor). Addition of the deacetylase inhibitors trichostatin A and sodium butyrate completely disrupt these nuclear bodies, providing a demonstration that the integrity of a nuclear body is enzyme dependent. We demonstrate that HDAC5 and 7 can associate with at least 12 distinct proteins, including several members of the NuRD and Sin3A repression complexes, and appear to define a new but related complex.

Interaction of voltage-gated sodium channels with the extracellular matrix molecules tenascin-C and tenascin-R

The type IIA rat brain sodium channel is composed of three subunits: a large pore-forming α subunit and two smaller auxiliary subunits, β1 and β2. The β subunits are single membrane-spanning glycoproteins with one Ig-like motif in their extracellular domains. The Ig motif of the β2 subunit has close structural similarity to one of the six Ig motifs in the extracellular domain of the cell adhesion molecule contactin (also called F3 or F11), which binds to the extracellular matrix molecules tenascin-C and tenascin-R. We investigated the binding of the purified sodium channel and the extracellular domain of the β2 subunit to tenascin-C and tenascin-R in vitro. Incubation of purified sodium channels on microtiter plates coated with tenascin-C revealed saturable and specific binding with an apparent Kd of ≈15 nM. Glutathione S-transferase-tagged fusion proteins containing various segments of tenascin-C and tenascin-R were purified, digested with thrombin to remove the epitope tag, immobilized on microtiter dishes, and tested for their ability to bind purified sodium channel or the epitope-tagged extracellular domain of β2 subunits. Both purified sodium channels and the extracellular domain of the β2 subunit bound specifically to fibronectin type III repeats 1–2, A, B, and 6–8 of tenascin-C and fibronectin type III repeats 1–2 and 6–8 of tenascin-R but not to the epidermal growth factor-like domain or the fibrinogen-like domain of these molecules. The binding of neuronal sodium channels to extracellular matrix molecules such as tenascin-C and tenascin-R may play a crucial role in localizing sodium channels in high density at axon initial segments and nodes of Ranvier or in regulating the activity of immobilized sodium channels in these locations.