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1.  Laminin121 – Recombinant expression and interactions with integrins 
Laminin-121, previously referred as to laminin-3, was expressed recombinantly in human embryonic kidney (HEK) 293 cells by triple transfection of full-length cDNAs encoding mouse laminin α1, β2 and γ1 chains. The recombinant laminin-121 was purified using Heparin-Sepharose followed by molecular sieve chromatography and shown to be correctly folded by electron microscopy and circular dichroism (CD). The CD spectra of recombinant laminin-121 were very similar to those of laminin-111 isolated from Engelbreth-Holm Swarm tumor (EHS-laminin) but its Tm value was smaller than EHS-laminin and recombinant lamnin-111 suggesting that the replacement of the β chain reduced the stability of the coiled-coil structure of laminin-121. Its binding to integrins was compared with EHS-laminin, laminin-3A32 purified from murine epidermal cell line and recombinantly expressed laminins-111, -211 and -221. Laminin-121 showed the highest affinity to α6β1 and α7β1 integrins and furthermore, laminin-121 most effectively supported neurite outgrowth. Together, this suggests that the β2 laminins have higher affinity for integrins than the β1 laminins.
doi:10.1016/j.matbio.2010.05.004
PMCID: PMC2939186  PMID: 20566382
extracellular matrix; basement membrane; laminin; recombinant expression; integrin; neurite outgrowth
2.  Nidogen-1 regulates laminin-1-dependent mammary-specific gene expression 
Journal of cell science  2000;113(Pt 5):849-858.
Summary
Nidogen-1 (entactin) acts as a bridge between the extracellular matrix molecules laminin-1 and type IV collagen, and thus participates in the assembly of basement membranes. To investigate the role of nidogen-1 in regulating cell-type-specific gene expression in mammary epithelium, we designed a culture microecosystem in which each component, including epithelial cells, mesenchymal cells, lactogenic hormones and extracellular matrix, could be controlled. We found that primary and established mesenchymal and myoepithelial cells synthesized and secreted nidogen-1, whereas expression was absent in primary and established epithelial cells. In an epithelial cell line containing mesenchymal cells, nidogen-1 was produced by the mesenchymal cells but deposited between the epithelial cells. In this mixed culture, mammary epithelial cells express β-casein in the presence of lactogenic hormones. Addition of either laminin-1 plus nidogen-1, or laminin-1 alone, to mammary epithelial cells induced β-casein production. We asked whether recombinant nidogen-1 alone could signal directly for β-casein. Nidogen-1 did not induce β-casein synthesis in epithelial cells, but it augmented the inductive capacity of laminin-1. These data suggest that nidogen-1 can cooperate with laminin-1 to regulate β-casein expression. Addition of full-length nidogen-1 to the mixed cultures had no effect on β-casein gene expression; however, a nidogen-1 fragment containing the laminin-1 binding domain, but lacking the type IV collagen-binding domain, had a dominant negative effect on β-casein expression. These data point to a physiological role for nidogen-1 in the basement membrane-induced gene expression by epithelial cells.
PMCID: PMC2933215  PMID: 10671374
Nidogen-1; Entactin; Basement membrane; Extracellular matrix; Tissue-specific gene expression; Epithelial-mesenchymal interaction; Mammary gland
3.  Expression of ECM proteins fibulin-1 and -2 in acute and chronic liver disease and in cultured rat liver cells 
Cell and Tissue Research  2009;337(3):449-462.
Fibulin-2 has previously been considered as a marker to distinguish rat liver myofibroblasts from hepatic stellate cells. The function of other fibulins in acute or chronic liver damage has not yet been investigated. The aim of this study has been to evaluate the expression of fibulin-1 and -2 in models of rat liver injury and in human liver cirrhosis. Their cellular sources have also been investigated. In normal rat liver, fibulin-1 and -2 were both mainly present in the portal field. Fibulin-1-coding transcripts were detected in total RNA of normal rat liver, whereas fibulin-2 mRNA was only detected by sensitive, real-time quantitative polymerase chain reaction. In acute liver injury, the expression of fibulin-1 was significantly increased (17.23-fold after 48 h), whereas that of fibulin-2 was not modified. The expression of both fibulin-1 and -2 was increased in experimental rat liver cirrhosis (19.16- and 26.47-fold, respectively). At the cellular level, fibulin-1 was detectable in hepatocytes, “activated” hepatic stellate cells, and liver myofibroblasts (2.71-, 122.65-, and 469.48-fold over the expression in normal rat liver), whereas fibulin-2 was restricted to liver myofibroblasts and was regulated by transforming growth factor beta-1 (TGF-β1) in 2-day-old hepatocyte cultures and in liver myofibroblasts. Thus, fibulin-1 and -2 respond differentially to single and repeated damaging noxae, and their expression is differently present in liver cells. Expression of the fibulin-2 gene is regulated by TGF-β1 in liver myofibroblasts.
doi:10.1007/s00441-009-0823-9
PMCID: PMC2728066  PMID: 19609566
Carbon tetrachloride; Extracellular matrix; Hepatic stellate cells; Liver cirrhosis; Liver myofibroblasts; Rat (Wistar); Human
4.  Compositional Differences between Infant and Adult Human Corneal Basement Membranes 
Purpose
Adult human corneal epithelial basement membrane (EBM) and Descemet's membrane (DM) components exhibit heterogeneous distribution. The purpose of the study was to identify changes of these components during postnatal corneal development.
Methods
Thirty healthy adult corneas and 10 corneas from 12-day- to 3-year-old children were studied by immunofluorescence with antibodies against BM components.
Results
Type IV collagen composition of infant corneal central EBM over Bowman's layer changed from α1-α2 to α3-α4 chains after 3 years of life; in the adult, α1-α2 chains were retained only in the limbal BM. Laminin α2 and β2 chains were present in the adult limbal BM where epithelial stem cells are located. By 3 years of age, β2 chain appeared in the limbal BM. In all corneas, limbal BM contained laminin γ3 chain. In the infant DM, type IV collagen α1-α6 chains, perlecan, nidogen-1, nidogen-2, and netrin-4 were found on both faces, but they remained only on the endothelial face of the adult DM. The stromal face of the infant but not the adult DM was positive for tenascin-C, fibrillin-1, SPARC, and laminin-332. Type VIII collagen shifted from the endothelial face of infant DM to its stromal face in the adult. Matrilin-4 largely disappeared after the age of 3 years.
Conclusions
The distribution of laminin γ3 chain, nidogen-2, netrin-4, matrilin-2, and matrilin-4 is described in the cornea for the first time. The observed differences between adult and infant corneal BMs may relate to changes in their mechanical strength, corneal cell adhesion and differentiation in the process of postnatal corneal maturation.
doi:10.1167/iovs.07-0654
PMCID: PMC2151758  PMID: 17962449
5.  Chondrogenic Activity of the Heparan Sulfate Proteoglycan Perlecan Maps to the N-terminal Domain I 
C3H10T1/2 cells differentiate along a chondrogenic pathway when plated onto the extracellular matrix (ECM) protein perlecan (Pln). To identify the region(s) within the large Pln molecule that provides a differentiation signal, recombinant Pln-sequence-based polypeptides representing distinct structural domains were assayed for their ability to promote chondrogenesis in C3H10T1/2 cells. Five distinct domains, along with structural variations, were tested. The N-terminal domain I was tested in two forms (IA and IB) that contain only heparan sulfate (HS) chains or both HS and chondroitin sulfate (CS) chains, respectively. A mutant form of domain I lacking attachment sites for both HS and CS (Pln Imut) was tested also. Other constructs consecutively designated Pln domains II, III(A-C), IV(A,B), and V(A,B) were used to complete the structure-function analysis. Cells plated onto Pln IA or Pln IB but no other domain rapidly assembled into cellular aggregates of 40-120 μm on average. Aggregate formation was dependent on the presence of glycosaminoglycan (GAG) chains, because Pln I-based polypeptides lacking GAG chains either by enzymatic removal or mutation of HS/CS attachment sites were inactive. Aggregates formed on GAG-bearing Pln IA stained with Alcian Blue and were recognized by antibodies to collagen type II and aggrecan but were not recognized by an antibody to collagen type X, a marker of chondrocyte hypertrophy. Collectively, these studies indicate that the GAG-bearing domain I of Pln provides a sufficient signal to trigger C3H10T1/2 cells to enter a chondrogenic differentiation pathway. Thus, this matrix proteoglycan (PG) found at sites of cartilage formation in vivo is likely to enhance early stage differentiation induced by soluble chondrogenic factors.
PMCID: PMC1774590  PMID: 11771669
perlecan; cartilage; chondrogenesis; proteoglycan
6.  Opposing Roles of Integrin α6Aβ1 and Dystroglycan in Laminin-mediated Extracellular Signal-regulated Kinase Activation 
Molecular Biology of the Cell  2003;14(5):2088-2103.
Laminin–integrin interactions can in some settings activate the extracellular signal-regulated kinases (ERKs) but the control mechanisms are poorly understood. Herein, we studied ERK activation in response to two laminins isoforms (-1 and -10/11) in two epithelial cell lines. Both cell lines expressed β1-containing integrins and dystroglycan but lacked integrin α6β4. Antibody perturbation assays showed that both cell lines bound to laminin-10/11 via the α3β1and α6β1 integrins. Although laminin-10/11 was a stronger adhesion complex than laminin-1 for both cell lines, both laminins activated ERK in only one of the two cell lines. The ERK activation was mediated by integrin α6β1 and not by α3β1 or dystroglycan. Instead, we found that dystroglycan-binding domains of both laminin-1 and -10/11 suppressed integrin α6β1-mediated ERK activation. Moreover, the responding cell line expressed the two integrin α6 splice variants, α6A and α6B, whereas the nonresponding cell line expressed only α6B. Furthermore, ERK activation was seen in cells transfected with the integrin α6A subunit, but not in α6B-transfected cells. We conclude that laminin-1 and -10/11 share the ability to induce ERK activation, that this is regulated by integrin α6Aβ1, and suggest a novel role for dystroglycan-binding laminin domains as suppressors of this activation.
doi:10.1091/mbc.E03-01-0852
PMCID: PMC165099  PMID: 12802077
7.  Endostatin reduces vascularization, blood flow, and growth in a rat gliosarcoma. 
Neuro-Oncology  2002;4(1):1-8.
Endostatin, the 20-kDa C-terminal fragment of collagen XVIII, has previously been shown to inhibit growth and induce regression of different experimental tumors in rodents. In this study, we show that recombinant murine and human endostatin, produced in 293 EBNA cells and yeast, respectively, inhibit ectotopic as well as orthotopic growing BT4Cn gliosarcomas in BD-IX rats. In rats in which s.c. gliomas were grown for a total of 29 days, systemic treatment with recombinant murine endostatin induced about 50% reduction of intratumoral blood flow and tumor size after only 10 days of therapy. In contrast, the blood flow to irrelevant organs was unaffected by endostatin, indicating its specificity of action. Tumors were not observed to increase in size or regrow after cessation of therapy. Furthermore, endostatin-treated rats with i.c. tumors had significantly longer survival time than did untreated controls. In the treated rats, endostatin therapy resulted in a reduced tumor blood vessel volume and an increased tumor cell density with an increased apoptotic index within a given tumor volume, as verified by flow cytometry and by staining with deoxynucleotidyltransferase-mediated dUTP nick-end labeling. This work verifies the general anti-angiogenic and antitumor effects of endostatin and indicates that the protein may also be considered as a treatment strategy for malignant brain tumors.
PMCID: PMC1920634  PMID: 11772427
8.  Gene Structure and Functional Analysis of the Mouse Nidogen-2 Gene: Nidogen-2 Is Not Essential for Basement Membrane Formation in Mice 
Molecular and Cellular Biology  2002;22(19):6820-6830.
Nidogens are highly conserved proteins in vertebrates and invertebrates and are found in almost all basement membranes. According to the classical hypothesis of basement membrane organization, nidogens connect the laminin and collagen IV networks, so stabilizing the basement membrane, and integrate other proteins. In mammals two nidogen proteins, nidogen-1 and nidogen-2, have been discovered. Nidogen-2 is typically enriched in endothelial basement membranes, whereas nidogen-1 shows broader localization in most basement membranes. Surprisingly, analysis of nidogen-1 gene knockout mice presented evidence that nidogen-1 is not essential for basement membrane formation and may be compensated for by nidogen-2. In order to assess the structure and in vivo function of the nidogen-2 gene in mice, we cloned the gene and determined its structure and chromosomal location. Next we analyzed mice carrying an insertional mutation in the nidogen-2 gene that was generated by the secretory gene trap approach. Our molecular and biochemical characterization identified the mutation as a phenotypic null allele. Nidogen-2-deficient mice show no overt abnormalities and are fertile, and basement membranes appear normal by ultrastructural analysis and immunostaining. Nidogen-2 deficiency does not lead to hemorrhages in mice as one may have expected. Our results show that nidogen-2 is not essential for basement membrane formation or maintenance.
doi:10.1128/MCB.22.19.6820-6830.2002
PMCID: PMC135501  PMID: 12215539
9.  Perinatal Lethality and Endothelial Cell Abnormalities in Several Vessel Compartments of Fibulin-1-Deficient Mice 
Molecular and Cellular Biology  2001;21(20):7025-7034.
The extracellular matrix protein fibulin-1 is a distinct component of vessel walls and can be associated with other ligands present in basement membranes, microfibrils, and elastic fibers. Its biological role was investigated by the targeted inactivation of the fibulin-1 gene in mice. This led to massive hemorrhages in several tissues starting at midgestation, ultimately resulting in the death of almost all homozygous embryos upon birth. Histological analysis demonstrated dilation and ruptures in the endothelial lining of various small vessels but not in that of larger vessels. Kidneys displayed a distinct malformation of glomeruli and disorganization of podocytes. A delayed development of lung alveoli suggested impairment in lung inflation. Immunohistology demonstrated the absence of fibulin-1 in its typical localizations but no aberrant patterns for several other extracellular matrix proteins. Electron microscopy revealed intact basement membranes but very irregular cytoplasmic processes of capillary endothelial cells in the organs that were most severely affected. Absence of fibulin-1 caused considerable blood loss but did not compromise blood clotting. The data indicate a strong but restricted abnormality in some endothelial compartments which, together with some kidney and lung defects, may be responsible for early death.
doi:10.1128/MCB.21.20.7025-7034.2001
PMCID: PMC99878  PMID: 11564885
10.  Perlecan Maintains the Integrity of Cartilage and Some Basement Membranes 
The Journal of Cell Biology  1999;147(5):1109-1122.
Perlecan is a heparan sulfate proteoglycan that is expressed in all basement membranes (BMs), in cartilage, and several other mesenchymal tissues during development. Perlecan binds growth factors and interacts with various extracellular matrix proteins and cell adhesion molecules. Homozygous mice with a null mutation in the perlecan gene exhibit normal formation of BMs. However, BMs deteriorate in regions with increased mechanical stress such as the contracting myocardium and the expanding brain vesicles showing that perlecan is crucial for maintaining BM integrity. As a consequence, small clefts are formed in the cardiac muscle leading to blood leakage into the pericardial cavity and an arrest of heart function. The defects in the BM separating the brain from the adjacent mesenchyme caused invasion of brain tissue into the overlaying ectoderm leading to abnormal expansion of neuroepithelium, neuronal ectopias, and exencephaly. Finally, homozygotes developed a severe defect in cartilage, a tissue that lacks BMs. The chondrodysplasia is characterized by a reduction of the fibrillar collagen network, shortened collagen fibers, and elevated expression of cartilage extracellular matrix genes, suggesting that perlecan protects cartilage extracellular matrix from degradation.
PMCID: PMC2169352  PMID: 10579729
perlecan; basement membrane; cardiac muscle; exencephaly; chondrodysplasia

Results 1-10 (10)