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1.  Interaction between the ADAMTS-12 metalloprotease and fibulin-2 induces tumor-suppressive effects in breast cancer cells 
Oncotarget  2014;5(5):1253-1264.
Balance between pro-tumor and anti-tumor effects may be affected by molecular interactions within tumor microenvironment. On this basis we searched for molecular partners of ADAMTS-12, a secreted metalloprotease that shows both oncogenic and tumor-suppressive effects. Using its spacer region as a bait in a yeast two-hybrid screen, we identified fibulin-2 as a potential ADAMTS-12-interacting protein. Fibulins are components of basement membranes and elastic matrix fibers in connective tissue. Besides this structural function, fibulins also play crucial roles in different biological events, including tumorigenesis. To examine the functional consequences of the ADAMTS-12/fibulin-2 interaction, we performed different in vitro assays using two breast cancer cell lines: the poorly invasive MCF-7 and the highly invasive MDA-MB-231. Overall our data indicate that this interaction promotes anti-tumor effects in breast cancer cells. To assess the in vivo relevance of this interaction, we induced tumors in nude mice using MCF-7 cells expressing both ADAMTS-12 and fibulin-2 that showed a remarkable growth deficiency. Additionally, we also found that ADAMTS-12 may elicit pro-tumor effects in the absence of fibulin-2. Immunohistochemical staining of breast cancer samples allowed the detection of both ADAMTS-12 and fibulin-2 in the connective tissue surrounding tumor area in less aggressive carcinomas. However, both proteins are hardly detected in more aggressive tumors. These data and survival analysis plots of breast cancer patients suggest that concomitant detection of ADAMTS-12 and fibulin-2 could be a good prognosis marker in breast cancer diagnosis.
PMCID: PMC4012729  PMID: 24457941
extracellular matrix; metalloprotease; ADAMTS; fibulin; breast cancer
2.  Laminin and integrin expression in the ventral ectodermal ridge of the mouse embryo: implications for regulation of BMP signalling 
The ventral ectodermal ridge (VER) is an important signalling centre in the mouse tail-bud following completion of gastrulation. BMP regulation is essential for VER function, but how these signals are transmitted between adjacent tissues is unclear.
We investigated the idea that extracellular matrix components might be involved, using immunohistochemistry and in situ hybridisation to detect all known α, β and γ laminin chains and their mRNAs in the early tail bud. We identified an apparently novel laminin variant, comprising α5, β3 and γ2 chains, as a major component of the VER basement membrane at E9.5. Strikingly, only the mRNAs for these chains were co-expressed in VER cells, suggesting that lamin532 may be the sole basement membrane laminin at this stage. Since α6 integrin was also expressed in VER cells, this raises the possibility of cell-matrix interactions regulating BMP signalling at this site of caudal morphogenesis.
Laminin532 could interact with α6-containing integrin to direct differentiation of the specialised VER cells from surface ectoderm.
PMCID: PMC3629792  PMID: 22911573
mouse; embryo; tail bud; ventral ectodermal ridge; laminin; extracellular matrix; basement membrane; integrin; bone morphogenetic protein
3.  Regional differences in the expression of laminin isoforms during mouse neural tube development 
Many significant human birth defects originate around the time of neural tube closure or early during post-closure nervous system development. For example, failure of the neural tube to close generates anencephaly and spina bifida, faulty cell cycle progression is implicated in primary microcephaly, while defective migration of neuroblasts can lead to neuronal migration disorders such as lissencephaly. At the stage of neural tube closure, basement membranes are becoming organised around the neuroepithelium, and beneath the adjacent non-neural surface ectoderm. While there is circumstantial evidence to implicate basement membrane dynamics in neural tube and surface ectodermal development, we have an incomplete understanding of the molecular composition of basement membranes at this stage. In the present study, we examined the developing basement membranes of the mouse embryo at mid-gestation (embryonic day 9.5), with particular reference to laminin composition. We performed in situ hybridization to detect the mRNAs of all eleven individual laminin chains, and immunohistochemistry to identify which laminin chains are present in the basement membranes. From this information, we inferred the likely laminin variants and their tissues of origin: that is, whether a given basement membrane laminin is contributed by epithelium, mesenchyme, or both. Our findings reveal major differences in basement composition along the body axis, with the rostral neural tube (at mandibular arch and heart levels) exhibiting many distinct laminin variants, while the lumbar level where the neural tube is just closing shows a much simpler laminin profile. Moreover, there appears to be a marked difference in the extent to which the mesenchyme contributes laminin variants to the basement membrane, with potential contribution of several laminins rostrally, but no contribution caudally. This information paves the way towards a mechanistic analysis of basement membrane laminin function during early neural tube development in mammals.
PMCID: PMC3565558  PMID: 21524702
Neural tube closure; laminin; mouse embryo; neural tube defects
4.  Cleavage of Nidogen-1 by Cathepsin S Impairs Its Binding to Basement Membrane Partners 
PLoS ONE  2012;7(8):e43494.
Cathepsin S (catS), which is expressed in normal human keratinocytes and localized close to the dermal-epidermal junction (DEJ) degrades some of major basement membrane (BM) constituents. Among them, catS readily hydrolyzed in a time and dose dependent manner human nidogen-1 (nid-1) and nidogen-2, which are key proteins in the BM structure. CatS preferentially cleaved nid-1 at both acid and neutral pH. Hydrolysis of nid-1 was hampered in murine ctss−/− spleen lysates pretreated with inhibitors of other classes of proteases. Nid-1 was cleaved within its G2 and G3 globular domains that are both involved in interactions with other BM components. Binding assays with soluble and immobilized ligands indicated that catS altered the formation of complexes between nid-1 and other BM components. Assuming that the cleavage of nid-1 impairs its ability to crosslink with BM partners and perturbs the viscoelastic properties of BM matrix, these data indicate that catS may participate in BM proteolysis, in addition to already identified proteases.
PMCID: PMC3429489  PMID: 22952693
5.  Conversion of mechanical force into TGF-β-mediated biochemical signals 
Current biology : CB  2011;21(11):933-941.
Mechanical forces influence homeostasis in virtually every tissue [1–2]. Tendon, constantly exposed to variable mechanical force, is an excellent model in which to study the conversion of mechanical stimuli into a biochemical response [3–5]. Here we show in a mouse model of acute tendon injury and in vitro that physical forces regulate the release of active transforming growth factor (TGF)-β from the extracellular matrix (ECM). The quantity of active TGF-β detected in tissue exposed to various levels of tensile loading correlates directly with the extent of physical forces. At physiological levels, mechanical forces maintain, through TGF-β/Smad2/3-mediated signaling, the expression of Scleraxis (Scx), a transcription factor specific for tenocytes and their progenitors. The gradual and temporary loss of tensile loading causes reversible loss of Scx expression, whereas sudden interruption, such as in transection tendon injury, destabilizes the structural organization of the ECM and leads to excessive release of active TGF-β and massive tenocyte death, which can be prevented by the TGF-β type I receptor inhibitor SD208. Our findings demonstrate a critical role for mechanical force in adult tendon homeostasis. Furthermore, this mechanism could translate physical force into biochemical signals in much broader variety of tissues or systems in the body.
PMCID: PMC3118584  PMID: 21600772
Scleraxis; Tendon; Tensile loading; Mechanical force; TGF-β; Smad2/3
6.  A fibronectin-independent mechanism of collagen fibrillogenesis in adult liver remodeling 
Gastroenterology  2011;140(5):1653-1663.
Fibrosis is an abnormal extension of the wound healing process that follows tissue damage; it is involved in pathogenesis in a variety of chronic diseases. The formation of extracellular matrix is an essential response in wound healing. Although it has been proposed that collagen organization and assembly depend on the fibronectin matrix in culture, the contribution of fibronectin to these processes remains to be defined in vivo.
We generated a conditional, fibronectin-deficient mouse model of liver injury and explored whether fibronectin would be a suitable target for preventing extensive collagen deposits and scar formation that could lead to liver fibrosis.
The lack of fibronectin did not interfere with reconstruction of collagen fibril organization in response to liver injury. Signaling by transforming growth factor (TGF)-β and type V collagen were required for collagen fibrillogenesis during remodeling of adult liver tissue.
TGF-β and type V collagen are targets for regulating the initial fibrogenic response to liver damage.
PMCID: PMC3081910  PMID: 21320502
liver disease; hepatic stellate cells; mouse model of fibrosis; conditional knockout; ECM
7.  Microenvironmental Regulation by Fibrillin-1 
PLoS Genetics  2012;8(1):e1002425.
Fibrillin-1 is a ubiquitous extracellular matrix molecule that sequesters latent growth factor complexes. A role for fibrillin-1 in specifying tissue microenvironments has not been elucidated, even though the concept that fibrillin-1 provides extracellular control of growth factor signaling is currently appreciated. Mutations in FBN1 are mainly responsible for the Marfan syndrome (MFS), recognized by its pleiotropic clinical features including tall stature and arachnodactyly, aortic dilatation and dissection, and ectopia lentis. Each of the many different mutations in FBN1 known to cause MFS must lead to similar clinical features through common mechanisms, proceeding principally through the activation of TGFβ signaling. Here we show that a novel FBN1 mutation in a family with Weill-Marchesani syndrome (WMS) causes thick skin, short stature, and brachydactyly when replicated in mice. WMS mice confirm that this mutation does not cause MFS. The mutation deletes three domains in fibrillin-1, abolishing a binding site utilized by ADAMTSLIKE-2, -3, -6, and papilin. Our results place these ADAMTSLIKE proteins in a molecular pathway involving fibrillin-1 and ADAMTS-10. Investigations of microfibril ultrastructure in WMS humans and mice demonstrate that modulation of the fibrillin microfibril scaffold can influence local tissue microenvironments and link fibrillin-1 function to skin homeostasis and the regulation of dermal collagen production. Hence, pathogenetic mechanisms caused by dysregulated WMS microenvironments diverge from Marfan pathogenetic mechanisms, which lead to broad activation of TGFβ signaling in multiple tissues. We conclude that local tissue-specific microenvironments, affected in WMS, are maintained by a fibrillin-1 microfibril scaffold, modulated by ADAMTSLIKE proteins in concert with ADAMTS enzymes.
Author Summary
The microenvironment is specified by cell-surface molecules, growth factors, and the extracellular matrix. Here we report genetic evidence that implicates fibrillin-1, a ubiquitous extracellular matrix molecule that sequesters latent growth factor complexes, as a key determinant in the local control of musculoskeletal and skin microenvironments. A novel mutation in fibrillin-1 demonstrates that modulation of the fibrillin microfibril scaffold can influence tissue microenvironments and result in the clinical features of Weill-Marchesani syndrome (WMS), including thick skin, short stature, and brachydactyly. Dysregulated WMS microenvironments diverge from Marfan pathogenetic mechanisms, which lead to broad activation of TGFβ signaling in multiple tissues.
PMCID: PMC3252277  PMID: 22242013
8.  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.
PMCID: PMC2939186  PMID: 20566382
extracellular matrix; basement membrane; laminin; recombinant expression; integrin; neurite outgrowth
9.  Perlecan deficiency causes muscle hypertrophy, a decrease in myostatin expression, and changes in muscle fiber composition 
Perlecan is a component of the basement membrane that surrounds skeletal muscle. The aim of the present study is to identify the role of perlecan in skeletal muscle hypertrophy and myostatin signaling, with and without mechanical stress, using a mouse model (Hspg2−/−-Tg) deficient in skeletal muscle perlecan. We found that myosin heavy chain (MHC) type IIb fibers in the tibialis anterior (TA) muscle of Hspg2−/−-Tg mice had a significantly increased fiber cross-sectional area (CSA) compared to control (WT-Tg) mice. Hspg2−/−-Tg mice also had an increased number of type IIx fibers in the TA muscle. Myostatin and its type I receptor (ALK4) expression was substantially decreased in the Hspg2−/−-Tg TA muscle. Myostatin-induced Smad activation was also reduced in a culture of myotubes from the Hspg2−/−-Tg muscle, suggesting that myostatin expression and its signaling were decreased in the Hspg2−/−-Tg muscle. To examine the effects of mechanical overload or unload on fast and slow muscles in Hspg2−/−-Tg mice, we performed tenotomy of the plantaris (fast) muscle and the soleus (slow) muscle. Mechanical overload on the plantaris muscle of Hspg2−/−-Tg mice significantly increased wet weights compared to those of control mice, and unloaded plantaris muscles of Hspg2−/−-Tg mice caused less decrease in wet weights compared to those of control mice. The decrease in myostatin expression was significantly profound in the overloaded plantaris muscle of Hspg2−/−-Tg mice, compared with that of control mice. In contrast, overloading the soleus muscle caused no changes in either type of muscle. These results suggest that perlecan is critical for maintaining fast muscle mass and fiber composition, and for regulating myostatin signaling.
PMCID: PMC2939214  PMID: 20541011
Perlecan; Muscle hypertrophy; Mechanical stress; Myostatin
10.  Crystal Structure of Human Collagen XVIII Trimerization Domain: a Novel Collagen Trimerization Fold 
Journal of molecular biology  2009;392(3):787-802.
Collagens contain a unique triple helical structure with a repeating sequence -G-X-Y-, where proline and hydroxyproline are major constituents in X and Y positions, respectively. Folding of the collagen triple helix requires trimerization domains. Once trimerized, collagen chains are correctly aligned and the folding of the triple helix proceeds in a zipper-like fashion. Here we report the isolation, characterization and crystal structure of the trimerization domain of human type XVIII collagen, a member of the multiplexin family. This domain differs from all other known trimerization domains in other collagens and exhibits a high trimerization potential at picomolar concentrations. Strong chain association and high specificity of binding are needed for multiplexins, which are present at very low levels.
PMCID: PMC3048824  PMID: 19631658
collagen XVIII and XV; crystal structure; trimerization domain; non-collagenous domain; endostatin
11.  Quiescence and Activation of Stem and Precursor Cell Populations in the Subependymal Zone of the Mammalian Brain Are Associated with Distinct Cellular and Extracellular Matrix Signals 
The Journal of Neuroscience  2010;30(29):9771-9781.
The subependymal zone (SEZ) of the lateral ventricles is one of the areas of the adult brain where new neurons are continuously generated from neural stem cells (NSCs), via rapidly dividing precursors. This neurogenic niche is a complex cellular and extracellular microenvironment, highly vascularized compared to non-neurogenic periventricular areas, within which NSCs and precursors exhibit distinct behavior. Here, we investigate the possible mechanisms by which extracellular matrix molecules and their receptors might regulate this differential behavior. We show that NSCs and precursors proceed through mitosis in the same domains within the SEZ of adult male mice—albeit with NSCs nearer ependymal cells—and that distance from the ventricle is a stronger limiting factor for neurogenic activity than distance from blood vessels. Furthermore, we show that NSCs and precursors are embedded in a laminin-rich extracellular matrix, to which they can both contribute. Importantly, they express differential levels of extracellular matrix receptors, with NSCs expressing low levels of α6β1 integrin, syndecan-1, and lutheran, and in vivo blocking of β1 integrin selectively induced the proliferation and ectopic migration of precursors. Finally, when NSCs are activated to reconstitute the niche after depletion of precursors, expression of laminin receptors is upregulated. These results indicate that the distinct behavior of adult NSCs and precursors is not necessarily regulated via exposure to differential extracellular signals, but rather via intrinsic regulation of their interaction with their microenvironment.
PMCID: PMC3842479  PMID: 20660259
12.  Fibulin-2 and fibulin-5 cooperatively function to form the internal elastic lamina and protect from vascular injury 
Recent findings on the role of fibulin-5 (Fbln5) have provided substantial progress in understanding the molecular mechanism of elastic fiber assembly in vitro. However, little is known about differential roles of fibulins in the elastogenesis of blood vessels. Here, we generated double knockout mice for Fbln5 and Fbln2 (termed DKO) and examined the role of fibulins-2 and -5 in development and injury response of the blood vessel wall.
Methods and Results
Fibulin-2 is distinctly located in the subendothelial matrix, whereas fibulin-5 is observed throughout the vessel wall. All of the elastic laminae, including the internal elastic lamina (IEL), were severely disorganized in DKO mice, which was not observed in single knockout mice for Fbln2 or Fbln5. Furthermore, DKO vessels displayed upregulation of vascular adhesion molecules, tissue factor expression and thrombus formation with marked dilation and thinning of the vessel wall after carotid artery ligation-injury.
Fibulin-2 and fibulin-5 cooperatively function to form the IEL during postnatal development by directing the assembly of elastic fibers, and are responsible for maintenance of the adult vessel wall after injury. The DKO mouse will serve as a unique animal model to test the effect of vessel integrity during various pathological insults.
PMCID: PMC2800831  PMID: 19893004
internal elastic lamina; vascular remodeling; development; injury
13.  ADAMTS metalloproteases generate active versican fragments that regulate interdigital web regression 
Developmental cell  2009;17(5):687-698.
We show that combinatorial mouse alleles for the secreted metalloproteases Adamts5, Adamts20 (bt), and Adamts9 result in fully penetrant soft-tissue syndactyly. Interdigital webs in Adamts5−/−; bt/bt mice had reduced apoptosis and decreased cleavage of the proteoglycan versican; however, the BMP-FGF axis, which regulates interdigital apoptosis was unaffected. BMP4 induced apoptosis, but without concomitant versican proteolysis. Haploinsufficiency of either Vcan or Fbln1, a co-factor for versican processing by ADAMTS5, led to highly penetrant syndactyly in bt mice, suggesting that cleaved versican was essential for web regression. The local application of an amino-terminal versican fragment corresponding to ADAMTS-processed versican, induced cell death in Adamts5−/−; bt/bt webs. Thus, ADAMTS proteases cooperatively maintain versican proteolysis above a required threshold to create a permissive environment for apoptosis. The data highlight the developmental significance of proteolytic action on the ECM, not only as a clearance mechanism, but also as a means to generate bioactive versican fragments.
PMCID: PMC2780442  PMID: 19922873
Limb development; Interdigital web; Morphogenesis; Extracellular matrix; Proteoglycan; ADAMTS; Syndactyly; Apoptosis; Versican; Fibulin
14.  Alterations of Extracellular Matrix Components and Proteinases in Human Corneal Buttons With INTACS for Post–Laser In Situ Keratomileusis Keratectasia and Keratoconus 
Cornea  2008;27(5):565-573.
To perform an immunohistochemical evaluation of corneas with INTACS for post–laser in situ keratomileusis (LASIK) keratectasia and keratoconus, obtained after corneal transplantation.
Corneas from 1 patient with INTACS for post-LASIK keratectasia and 2 patients with INTACS for keratoconus were obtained within 3 hours after penetrating keratoplasty, and cryostat sections were analyzed by immunostaining for 35 extracellular matrix (ECM) components and proteinases.
In the stroma of all corneas next to an INTACS implant, ECM components typically associated with fibrosis were observed. These included tenascin-C, fibrillin-1, and types III, IV (α1/α2 chains), and XIV collagen. Also, significant deposition of perlecan, nidogen-2, and cellular fibronectin was revealed in the same locations. The keratoconus cases displayed typical Bowman layer breaks and subepithelial fibrosis with deposition of various ECM components. In all cases, some keratocytes around INTACS were positive for specific proteinases associated with stromal remodeling, including cathepsins F and H, matrix metalloproteinase (MMP)-1, MMP-3, and MMP-10. Staining for MMP-7 was variable; MMP-2 and MMP-9 were mostly negative. Patterns of type IV collagen α3, α4, and α6 chains; types VI and VIII collagen; laminin-332,α4, α5,β1, β2, and γ1 laminin chains; vitronectin; thrombospondin-1; urokinase; EMMPRIN; and cathepsins B and L were unchanged around INTACS in all 3 cases compared with normal.
Abnormal accumulation of fibrotic ECM components and proteinases near INTACS suggests ongoing lysis and remodeling of corneal stroma. Specific changes observed in each case may be related to underlying pathology.
PMCID: PMC2746565  PMID: 18520507
INTACS; keratoconus; laser in situ keratomileusis; cornea; extracellular matrix; fibrosis; matrix metalloproteinase; tenascin-C; cathepsin; nidogen
15.  Compositional Differences between Infant and Adult Human Corneal Basement Membranes 
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.
Thirty healthy adult corneas and 10 corneas from 12-day- to 3-year-old children were studied by immunofluorescence with antibodies against BM components.
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.
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.
PMCID: PMC2151758  PMID: 17962449
16.  Proteinase and Growth Factor Alterations Revealed by Gene Microarray Analysis of Human Diabetic Corneas 
PURPOSE. To identify proteinases and growth factors abnormally expressed in human corneas of donors with diabetic retinopathy (DR), additional to previously described matrix metalloproteinase (MMP)-10 and -3 and insulin-like growth factor (IGF)-I.
METHODS. RNA was isolated from 35 normal, diabetic, and DR autopsy human corneas ex vivo or after organ culture. Amplified cRNA was analyzed using 22,000-gene microarrays (Agi-lent Technologies, Palo Alto, CA). Gene expression in each diabetic corneal cRNA was assessed against pooled cRNA from 7 to 9 normal corneas. Select differentially expressed genes were validated by quantitative real-time RT-PCR (QPCR) and immunohistochemistry. Organ cultures were treated with a cathepsin inhibitor, cystatin C, or MMP-10.
RESULTS. More than 100 genes were upregulated and 2200 were downregulated in DR corneas. Expression of cathepsin F and hepatocyte growth factor (HGF) genes was increased in ex vivo and organ-cultured DR corneas compared with normal corneas. HGF receptor c-met, fibroblast growth factor (FGF)-3, its receptor FGFR3, tissue inhibitor of metalloproteinase (TIMP)-4, laminin α4 chain, and thymosin β4 genes were down-regulated. The data were corroborated by QPCR and immuno-histochemistry analyses; main changes of these components occurred in corneal epithelium. In organ-cultured DR corneas, cystatin C increased laminin-10 and integrin α3β1, whereas in normal corneas MMP-10 decreased laminin-10 and integrin α3β1 expression.
CONCLUSIONS. Elevated cathepsin F and the ability of its inhibitor to produce a more normal phenotype in diabetic corneas suggest increased proteolysis in these corneas. Proteinase changes may result from abnormalities of growth factors, such as HGF and FGF-3, in DR corneas. Specific modulation of proteinases and growth factors could reduce diabetic corneal epitheliopathy.
PMCID: PMC1459105  PMID: 16186340
17.  Nidogen 1 and 2 gene promoters are aberrantly methylated in human gastrointestinal cancer 
Molecular Cancer  2007;6:17.
Nidogens are highly conserved proteins of basement membranes. Two nidogen proteins, nidogen 1 and nidogen 2, are known in mammals.
We show that CpG islands of both NID1 and NID2 genes are aberrantly methylated in human cancer samples and cancer cell lines. For both genes, methylation was correlated with loss of gene transcription in human cell lines. Furthermore, demethylation of the NID1 and NID2 promoters restored gene transcription, demonstrating that methylation was responsible for silencing nidogen genes. In primary tumors, we detected NID1 promoter methylation in 67% of colon cancer samples and in 90% of gastric cancers. NID2 promoter was methylated in 29% of colon and 95% of gastric cancers. Immuno-staining for nidogen-2 confirmed the correlation between aberrant methylation and loss of nidogen expression also in primary tumors, implying that aberrant methylation was a mechanism for inhibiting nidogens expression in human gastrointestinal tumors.
These results suggest that loss of nidogens expression has a potential pathogenetic role in colon and stomach tumorigenesis. Nidogens are believed to connect laminin and collagen IV networks, hence stabilizing the basement membrane structure. Nidogens are also important for cell adhesion, as they establish contacts with various cellular integrins. Loss of nidogen expression may favor invasion and metastasis of cancer cells by loosening cell interaction with basal membrane and by weakening the strength of the basement membrane itself, first barrier from the connective vascularized matrix.
PMCID: PMC1831485  PMID: 17328794
18.  Overexpression of β1-chain-containing laminins in capillary basement membranes of human breast cancer and its metastases 
Breast Cancer Research  2005;7(4):R411-R421.
Laminins are the major components of vascular and parenchymal basement membranes. We previously documented a switch in the expression of vascular laminins containing the α4 chain from predominantly laminin-9 (α4β2γ1) to predominantly laminin-8 (α4β1γ1) during progression of human brain gliomas to high-grade glioblastoma multiforme. Here, differential expression of laminins was studied in blood vessels and ductal epithelium of the breast.
In the present study the expressions of laminin isoforms α1–α5, β1–β3, γ1, and γ2 were examined during progression of breast cancer. Forty-five clinical samples of breast tissues including normal breast, ductal carcinomas in situ, invasive ductal carcinomas, and their metastases to the brain were compared using Western blot analysis and immunohistochemistry for various chains of laminin, in particular laminin-8 and laminin-9.
Laminin α4 chain was observed in vascular basement membranes of most studied tissues, with the highest expression in metastases. At the same time, the expression of laminin β2 chain (a constituent of laminin-9) was mostly seen in normal breast and carcinomas in situ but not in invasive carcinomas or metastases. In contrast, laminin β1 chain (a constituent of laminin-8) was typically found in vessel walls of carcinomas and their metastases but not in those of normal breast. The expression of laminin-8 increased in a progression-dependent manner. A similar change was observed from laminin-11 (α5β2γ1) to laminin-10 (α5β1γ1) during breast tumor progression. Additionally, laminin-2 (α2β1γ1) appeared in vascular basement membranes of invasive carcinomas and metastases. Chains of laminin-5 (α3β3γ2) were expressed in the ductal epithelium basement membranes of the breast and diminished with tumor progression.
These results suggest that laminin-2, laminin-8, and laminin-10 are important components of tumor microvessels and may associate with breast tumor progression. Angiogenic switch from laminin-9 and laminin-11 to laminin-8 and laminin-10 first occurs in carcinomas in situ and becomes more pronounced with progression of carcinomas to the invasive stage. Similar to high-grade brain gliomas, the expression of laminin-8 (and laminin-10) in breast cancer tissue may be a predictive factor for tumor neovascularization and invasion.
PMCID: PMC1175051  PMID: 15987446
19.  Activities of the Matrix Metalloproteinase Stromelysin-2 (MMP-10) in Matrix Degradation and Keratinocyte Organization in Wounded Skin 
Molecular Biology of the Cell  2004;15(12):5242-5254.
The matrix metalloproteinase stromelysin-2 is expressed in keratinocytes of the epithelial tongue of skin wounds, suggesting a role in keratinocyte migration. Here, we show that stromelysin-2 enhances migration of cultured keratinocytes. To gain insight into the in vivo activities of stromelysin-2 in epithelial repair, we generated transgenic mice expressing a constitutively active stromelysin-2 mutant in keratinocytes. These animals had no alterations in skin architecture, and the healing rate of skin wounds was normal. Histologically, however, we found abnormalities in the organization of the wound epithelium. Keratinocytes at the migrating epidermal tip were scattered in most sections of mice with high expression level, and there was a reduced deposition of new matrix. In particular, the staining pattern of laminin-5 at the wound site was altered. This may be due to proteolytic processing of laminin-5 by stromelysin-2, because degradation of laminin-5 by this enzyme was observed in vitro. The inappropriate matrix contact of keratinocytes was accompanied by aberrant localization of β1-integrins and phosphorylated focal adhesion kinase, as well as by increased apoptosis of wound keratinocytes. These results suggest that a tightly regulated expression level of stromelysin-2 is required for limited matrix degradation at the wound site, thereby controlling keratinocyte migration.
PMCID: PMC532007  PMID: 15371548
20.  Laminin 
The Journal of Cell Biology  2004;164(7):959-963.
Laminin-1 is emerging as the key molecule in early embryonic basement membrane assembly. Here we review recent insights into its functions gained from the synergistic application of genetic and structural methods.
PMCID: PMC2172061  PMID: 15037599
extracellular matrix; embryo development; mutagenesis; structure determination
21.  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
22.  Characteristics of a Novel Type of Bovine Cryptosporidium andersoni 
We isolated oocysts that resemble Cryptosporidium andersoni from cattle grazing on a farm in Japan. The partial sequences of genes from the isolate were coincident with published sequences of genes of C. andersoni. Since the isolate was able to infect SCID mice, the isolate appears to be a novel type of C. andersoni.
PMCID: PMC152376  PMID: 12514063

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