Major Glycoprotein Component Research Articles

Dysfunction of 67-kDa Laminin Receptor Disrupts BBB Integrity via Impaired Dystrophin/AQP4 Complex and p38 MAPK/VEGF Activation Following Status Epilepticus.

Status epilepticus (SE, a prolonged seizure activity) impairs brain-blood barrier (BBB) integrity, which results in secondary complications following SE. The non-integrin 67-kDa laminin receptor (67-kDa LR) plays a role in cell adherence to laminin (a major glycoprotein component in basement membrane), and participates laminin-mediated signaling pathways including p38 mitogen-activated protein kinase (p38 MAPK). Thus, we investigated the role of 67-kDa LR in SE-induced vasogenic edema formation in the rat piriform cortex (PC). SE diminished 67-kDa LR expression, but increased laminin expression, in endothelial cells accompanied by the reduced SMI-71 (a rat BBB barrier marker) expression. Astroglial 67-kDa LR expression was also reduced in the PC due to massive astroglial loss. 67-kDa LR neutralization led to serum extravasation in the PC concomitant with the reduced SMI-71 expression. 67-kDa LR neutralization also decreased expressions of dystrophin and aquaporin-4 (AQP4). In addition, it increased p38 MAPK phosphorylation and expressions of vascular endothelial growth factor (VEGF), laminin and endothelial nitric oxide synthase (eNOS), which were abrogated by SB202190, a p38 MAPK inhibitor. Therefore, our findings indicate that 67-kDa LR dysfunction may disrupt dystrophin-AQP4 complex, which would evoke vasogenic edema formation and subsequent laminin over-expression via activating p38 MAPK/VEGF axis.

Effect of mucus layer on the transcellular absorption of lipophilic drugs in rat small intestine

PurposeThe mucus layer, which is adjacent to the intestinal wall, is considered to be a physiological barrier to intestinal drug absorption, since a drug must diffuse through this lipophilic layer prior to permeation across the intestinal epithelial cells. Thus, mucus layer permeation is suggested to be a rate‐limiting step in the intestinal absorption of lipophilic drugs. The major glycoprotein components of the mucus layer are mucins, which presumably play a pharmaceutical role in intestinal drug absorption, however, there have been no reports that describe the contribution of mucins to intestinal drug absorption at the molecular level. In this study, we examined the involvement of the mucus layer in the intestinal permeability of various lipophilic drugs in rat small intestine. Furthermore, we evaluated the regional distributions of mucins (MUCs) in rat gastrointestinal tract and explored the relationship between the effect of the mucus layer on passive transcellular transport and the expression pattern of mucins.MethodsDrug permeation experiments were conducted using the in vitro sac method. The amount of mucus glycoproteins liberated from the intestinal wall were determined by the alcian blue method. The mRNA and protein expression levels of mucins were evaluated by quantitative real‐time RT‐PCR (qPCR) and Western blotting, respectively.Results and DiscussionThe membrane permeability of the lipophilic drugs in the rat gastrointestinal tract was increased by pretreatment with dithiothreitol (DTT), which is commonly used as a mucus remover, and the alternation was remarkable in the upper part of the small intestine. Furthermore, the enhancing effects of DTT tended to be pronounced on the permeability of drugs with higher log D values in the case of neutral drugs with non‐dissociable groups. These results indicated that the effect of the mucus layer on intestinal permeability depends on the log D values and the physiological charge of the compound. On the other hand, the mRNA expression analysis of MUC genes revealed that the intestinal expression of MUC5AC is considerably higher in the duodenum, and those of MUC1, MUC2, and MUC3A are gradually increased toward the lower intestine in rats. Furthermore, Western blotting showed abundant MUC5AC protein in the mucus solution liberated from jejunum, but not from ileum and colon by the treatment with DTT. Therefore, these results suggest that rMUC5AC is involved in the absorption of lipophilic drugs in the upper part of rat small intestine.ConclusionsMUC5AC could be involved in the barrier function to limit the transcellular permeation of hydrophobic drugs in the upper part of the gastrointestinal tract.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of trans Protein QTL for Secreted Airway Mucins in Mice and a Causal Role for Bpifb1.

Mucus hyper-secretion is a hallmark feature of asthma and other muco-obstructive airway diseases. The mucin proteins MUC5AC and MUC5B are the major glycoprotein components of mucus and have critical roles in airway defense. Despite the biomedical importance of these two proteins, the loci that regulate them in the context of natural genetic variation have not been studied. To identify genes that underlie variation in airway mucin levels, we performed genetic analyses in founder strains and incipient lines of the Collaborative Cross (CC) in a house dust mite mouse model of asthma. CC founder strains exhibited significant differences in MUC5AC and MUC5B, providing evidence of heritability. Analysis of gene and protein expression of Muc5ac and Muc5b in incipient CC lines (n = 154) suggested that post-transcriptional events were important regulators of mucin protein content in the airways. Quantitative trait locus (QTL) mapping identified distinct, trans protein QTL for MUC5AC (chromosome 13) and MUC5B (chromosome 2). These two QTL explained 18 and 20% of phenotypic variance, respectively. Examination of the MUC5B QTL allele effects and subsequent phylogenetic analysis allowed us to narrow the MUC5B QTL and identify Bpifb1 as a candidate gene. Bpifb1 mRNA and protein expression were upregulated in parallel to MUC5B after allergen challenge, and Bpifb1 knockout mice exhibited higher MUC5B expression. Thus, BPIFB1 is a novel regulator of MUC5B.

Expression, purification, and characterization of recombinant human and murine milk fat globule-epidermal growth factor-factor 8

Milk fat globule-epidermal growth factor-factor 8 (MFG-E8), as its name suggests, is a major glycoprotein component of milk fat globules secreted by the mammary epithelium. Although its role in milk fat production is unclear, MFG-E8 has been shown to act as a bridge linking apoptotic cells to phagocytes for removal of these dying cells. MFG-E8 is capable of bridging these two very different cell types via interactions through both its epidermal growth factor (EGF)-like domain(s) and its lectin-type C domains. The EGF-like domain interacts with αVβ3 and αVβ5 integrins on the surface of phagocytes, whereas the C domains bind phosphatidylserine found on the surface of apoptotic cells. In an attempt to purify full-length, recombinant MFG-E8 expressed in either insect cells or CHO cells, we find that it is highly aggregated. Systematic truncation of the domain architecture of MFG-E8 indicates that the C domains are mainly responsible for the aggregation propensity. Addition of Triton X-100 to the conditioned cell culture media allowed partial recovery of non-aggregated, full-length MFG-E8. A more comprehensive detergent screen identified CHAPS as a stabilizer of MFG-E8 and allowed purification of a significant portion of non-aggregated, full-length protein. The CHAPS-stabilized recombinant MFG-E8 retained its natural ability to bind both αVβ3 and αVβ5 integrins and phosphatidylserine suggesting that it is properly folded and active. Herein we describe an efficient purification method for production of non-aggregated, full-length MFG-E8.

Exploring the role and diversity of mucins in health and disease with special insight into non-communicable diseases.

Mucins are major glycoprotein components of the mucus that coats the surfaces of cells lining the respiratory, digestive, gastrointestinal and urogenital tracts. They function to protect epithelial cells from infection, dehydration and physical or chemical injury, as well as to aid the passage of materials through a tract i.e., lubrication. They are also implicated in the pathogenesis of benign and malignant diseases of secretory epithelial cells. In Human there are two types of mucins, membrane-bound and secreted that are originated from mucous producing goblet cells localized in the epithelial cell layer or in mucous producing glands and encoded by MUC gene. Mucins belong to a heterogeneous family of high molecular weight proteins composed of a long peptidic chain with a large number of tandem repeats that form the so-called mucin domain. The molecular weight is generally high, ranging between 0.2 and 10 million Dalton and all mucins contain one or more domains which are highly glycosylated. The size and number of repeats vary between mucins and the genetic polymorphism represents number of repeats (VNTR polymorphisms), which means the size of individual mucins can differ substantially between individuals which can be used as markers. In human it is only MUC1 and MUC7 that have mucin domains with less than 40% serine and threonine which in turn could reduce number of PTS domains. Mucins can be considered as powerful two-edged sword, as its normal function protects from unwanted substances and organisms at an arm's length while, malfunction of mucus may be an important factor in human diseases. In this review we have unearthed the current status of different mucin proteins in understanding its role and function in various non-communicable diseases in human with special reference to its organ specific locations. The findings described in this review may be of direct relevance to the major research area in biomedicine with reference to mucin and mucin associated diseases.

Cell receptors for influenza a viruses and the innate immune response.

The interaction of the hemagglutinin (HA) of the influenza A viruses (IAV) with the cell surface is a key factor for entry of the virus and productive infection of the cell. This glycoprotein has affinity for sialic acids (SA), and different strains present specificity for SA bound through α2,3 or α2,6 linkages to the underlying sugar chain, which is usually related with host and cell tropism. Nucleic acid recognizing receptors (mainly RIG-I and Toll-like receptors) are the most extensively studied pattern recognition receptors for IAV. However, due to the ability of the HA of avian, swine, or human influenza viruses to bind differently linked SA and also to the high levels and variability of glycosylations of their major virion glycoprotein components, HA and NA, IAV interacting proteins on the cell surface could also play an important role in initiating different signaling pathways to elicit the immune response in infected cells. But, at present, these processes are not well understood. In this mini-review we discuss how the interactions of IAV with cell surface receptors on immune cells might be important for the induction of specific innate immune responses and as a result, for pathogenicity in humans.

Proteomic identification of proteinase inhibitors in the porcine enamel matrix derivative, EMD®

The porcine enamel matrix derivative, EMD(®), which is the active component of Emdogain(®), is used widely in periodontics because of its ability to promote the regeneration of soft and hard tissues and to reduce inflammation. Previous studies have used indirect methods to explain its angiogenic and proliferative effects on cells associated with wound healing. In this study we used proteomic techniques to identify proteins in EMD other than amelogenins. Proteins in EMD were separated by two-dimensional gel electrophoresis and were identified using mass spectrometry. Proteomic results were validated by western blot analysis of Emdogain. Fourteen proteins of porcine origin were identified and included the serine and cysteine proteinase inhibitors alpha1-antichymotrypsin and fetuin A, respectively. Alpha1-antichymotrypsin is an acute-phase factor that has been reported to indirectly down-regulate the expression of the gelatinase MMP-9. Fetuin A, a major glycoprotein component of bone and teeth, is a potent inhibitor of ectopic calcification of vascular and soft tissues and has been implicated in both osteogenesis and bone resorption. It also facilitates plasma membrane repair in damaged fibroblasts. EMD contains a number of high-molecular-weight compounds which include the proteinase inhibitors, fetuin A and alpha1-antichymotrypsin.

A single heterozygous nucleotide substitution displays two different altered mechanisms in the FBN1 gene of five Italian marfan patients

The Fibrillin-1 gene ( FBN1; chromosome 15q21.1) encodes a major glycoprotein component of the extracellular matrix. Mutations in FBN1, TGFBR1, TGFBR2 are known to cause Marfan syndrome (MIM 154700), a pleiotropic disorder. In the present study, we describe five novel missense FBN1 mutations in five Marfan patients that have the peculiarity to activate two contemporary mutational mechanisms: a missense mutation and exon skipping.

Extracellular Interactions between GluR2 and N-Cadherin in Spine Regulation

Via its extracellular N-terminal domain (NTD), the AMPA receptor subunit GluR2 promotes the formation and growth of dendritic spines in cultured hippocampal neurons. Here we show that the first N-terminal 92 amino acids of the extracellular domain are necessary and sufficient for GluR2's spine-promoting activity. Moreover, overexpression of this extracellular domain increases the frequency of miniature excitatory postsynaptic currents (mEPSCs). Biochemically, the NTD of GluR2 can interact directly with the cell adhesion molecule N-cadherin, in cis or in trans. N-cadherin-coated beads recruit GluR2 on the surface of hippocampal neurons, and N-cadherin immobilization decreases GluR2 lateral diffusion on the neuronal surface. RNAi knockdown of N-cadherin prevents the enhancing effect of GluR2 on spine morphogenesis and mEPSC frequency. Our data indicate that in hippocampal neurons N-cadherin and GluR2 form a synaptic complex that stimulates presynaptic development and function as well as promoting dendritic spine formation.

Correlation between laminin and cathespin D expressions in breast carcinoma

ObjectiveLaminin is a major glycoprotein component of basement membrance which is an important barrier to tumor cells which must be breeched before metastatic spread can occur. Proteolytic enzymes play an important role in mediating the passage of cancer cells through the basement membrane (BM) and extracellular matrix. We compared the patterns of laminin and cathepsin D (CD) expressions in a range of benign and malignant breast lesions to better understand the process of tumor progression.MethodsOne hundred and sixty-two cases of breast samples comprising 18 fibroadeomas, 22 cases of fibrocystic disease, 96 cases of invasive ductal carcinoma and 26 carcinomas with intraductal components were evaluated for laminin and cathepsin D expressions by immunohistochemical staining.ResultsThe prevalence of CD positivity in both neoplastic and stromal cell components were significantly higher in higher histological grade tumors compared to lower grades (P<0.001). Various severity of BM disruption correlated with histological grade of the carcinomas (P<0.001). There was a negative correlation between the laminin expression and CD presence.ConclusionIn the process of cancer cell invasion and metastasis, the basement membrane is disrupted by proteinase secreted by cancer cells, especially by stroma cells of cancer.

New monoclonal antibodies to non-glycosylated domains of the secreted mucins MUC5B and MUC7.

The separation and characterization of salivary mucins is not straightforward because of their large size, heterogeneity, and molecular interactions. The MUC5B and MUC7 mucins are major glycoprotein components of saliva that are thought to play a vital role in maintaining oral health. MUC5B is also a major component of respiratory mucus and is produced by the tracheal and bronchial glands, while MUC7 has a more limited pattern of expression in the bronchial tree. MUC5B is a gel-forming mucin and thus confers viscosity, whereas MUC7 is much smaller. MUC7 has anti-fungal activity, and both mucins interact with bacteria. The aim of this work was to produce new monoclonal antibodies that can be used to quantify and characterize these mucins by standard laboratory procedures. Peptide sequences in non-conserved and non-glycosylated regions were selected and monoclonal antibodies produced by an efficient immunization and cloning strategy, and screening against purified mucins. Three new antibodies-EU-MUC5Ba and EU-MUC5Bb (against MUC5B) and EU-MUC7a (against MUC7)-were isolated that do not show cross-reactivity with other gel-forming mucins. All work on immunohistochemistry can be used for semi-quantitative immunoblotting after agarose gel electrophoresis. These reagents are valuable tools to study changes in these mucins in oral and respiratory disease, and unlike other monoclonal antibodies to these mucins they recognize epitopes that are not affected by glycosylation.

Cell Adhesion to Fibrillin-1 Molecules and Microfibrils Is Mediated by α5β1 and αvβ3 Integrins

Fibrillins are the major glycoprotein components of microfibrils that form a template for tropoelastin during elastic fibrillogenesis. We have examined cell adhesion to assembled purified microfibrils, and its molecular basis. Human dermal fibroblasts exhibited Arg-Gly-Asp and cation-dependent adhesion to microfibrils and recombinant fibrillin-1 protein fragments. Strong integrin alpha 5 beta 1 interactions with fibrillin ligands were identified, but integrin alpha v beta 3 also contributed to cell adhesion. Fluorescence-activated cell sorting analysis confirmed the presence of abundant alpha 5 beta 1 and some alpha v beta 3 receptors on these cells. Adhesion to microfibrils and to Arg-Gly-Asp containing fibrillin-1 protein fragments induced signaling events that led to cell spreading, altered cytoskeletal organization, and enhanced extracellular fibrillin-1 deposition. Differences in cell shape when plated on fibrillin or fibronectin implied substrate-specific alpha 5 beta 1-mediated cellular responses. An Arg-Gly-Asp-independent cell adhesion sequence was also identified within fibrillin-1. Adhesion and spreading of smooth muscle cells on fibrillin ligands was enhanced by antibody-induced beta1 integrin activation. A375-SM melanoma cells bound Arg-Gly-Asp-containing fibrillin-1 protein fragments mainly through alpha v beta 3, whereas HT1080 cells used mainly alpha 5 beta 1. This study has shown that fibrillin microfibrils mediate cell adhesion, that alpha 5 beta 1 and alpha v beta 3 are both important but cell-specific fibrillin-1 receptors, and that cellular interactions with fibrillin-1 influence cell behavior.

Laminin-8/9 is synthesized by rat glomerular mesangial cells and is required for PDGF-induced mesangial cell migration

Laminin (LM), the major glycoprotein component of basement membranes is expressed as multiple isoforms in a developmentally regulated and tissue-specific manner. LM alpha4 has a limited tissue distribution and is highly expressed in the developing glomerulus. In the present study, we investigate the in vivo and in vitro expression and function of LM alpha4 in the glomerulus. LM alpha4 expression was examined by Northern blot, reverse transcription polymerase chain reaction (RT-PCR), Western blot, and immunofluorescence microscopy. Mesangial cells (MC) were plated on purified LM-1, LM-2, and LM-8/9. Immunofluorescence microscopy was performed to examine the cellular phenotypes induced by LM-1 and LM-8/9. A modified Boyden chamber method was used to assess laminin participation in platelet-derived growth factor (PDGF)-stimulated migration. mRNA for LMalpha4 is expressed in cultured rat MC, and isolated rat and mouse glomeruli, but not in cultured rat glomerular epithelial cells or glomerular endothelial cells. Using antibodies specific for LM alpha4, a 240 kD band was detected in MC extract and a slightly smaller band was identified in extracted rat glomeruli. Purified LM-8/9 had MC adhesive activity comparable to LM-1 and LM-2. MC attached to LM-8/9 exhibited a unique phenotype. In contrast to LM-1, attachment of MC to LM-8/9 produced a highly arborized cell morphology with significantly reduced formation of focal contacts or stress fibers. LM alpha4 is utilized by MC during PDGF-stimulated migration. LM alpha4 is synthesized by MC and persists in the mature glomerulus. LM-8/9 stimulates a unique cellular morphology, and they are utilized in PDGF-induced migration. These factors suggest that LM alpha4 plays an important role in MC differentiation and in the maintenance of MC phenotype.

Correlation between laminin and cathepsin D expressions in breast carcinoma.

Laminin is a major glycoprotein component of basement membrane which is an important barrier to tumor cells which must be breached before metastatic spread can occur. Proteolytic enzymes play an important role in mediating the passage of cancer cells through the basement membrane (BM) and extracellular matrix. We have compared the patterns of laminin and cathepsin D (CD) expressions in a range of benign and malignant breast lesions to better understand the process of tumor progression. One hundred and sixty-two cases of breast samples comprising 18 fibroadenomas, 22 cases of fibrocystic disease, 96 cases of invasive ductal carcinoma and 26 carcinomas with intraductal components were evaluated for laminin and cathepsin D expressions by immunohistochemical staining. The prevalence of CD positivity in both neoplastic and stromal cell components were significantly higher in higher histological grade tumors compared to lower grades (P <0.01). Various severity of BM disruption correlated with histological grade of the carcinomas (P <0.001). There was a negative correlation between the laminin expression and CD presence. It was confirmed that in a process of cancer cell invasion and metastasis, it could be necessary with the basement membrane disruption by proteinase secreted by cancer cells and especially by stroma cells of cancer.

Group A Streptococci Bind to Mucin and Human Pharyngeal Cells through Sialic Acid-Containing Receptors

The first step in the colonization of group A streptococci (Streptococcus pyogenes) is adherence to pharyngeal epithelial cells. Prior to adherence to their target tissue, the first barrier that the streptococci encounter is the mucous layer of the respiratory tract. The present study was undertaken to characterize the interaction between mucin, the major glycoprotein component of mucus, and streptococci. We report here that S. pyogenes is able to bind to bovine submaxillary mucin in solid-phase microtiter plate assays. Western blots probed with (125)I-labeled mucin and a panel of monoclonal antibodies revealed that the streptococcal M protein is one of two cell wall-associated proteins responsible for this binding. The binding was further localized to the N-terminal portion of the M molecule. Further analysis revealed that the M protein binds to the sialic acid moieties on mucin, and this interaction seems to be based on M-protein conformation rather than specific amino acid sequences. We found that sialic acid also plays a critical role in the adherence of an M6 streptococcal strain to the Detroit 562 human pharyngeal cell line and have identified alpha2-6-linked sialic acid as an important sialylated linkage for M-protein recognition. Western blot analysis of extracted pharyngeal cell membrane proteins identified three potential sialic acid-containing receptors for the M protein. The results are the first to show that sialic acid not only is involved in the binding of the streptococci to mucin but also plays an important role in adherence of group A streptococci to the pharyngeal cell surface.

Morphological and Biochemical Changes of Isolated Chicken Egg-Envelope During Sperm Penetration: Degradation of the 97-Kilodalton Glycoprotein Is Involved in Sperm-Driven Hole Formation on the Egg-Envelope1

The chicken egg-envelope is made of two major glycoprotein components, which are designated as gp97 and gp42 (after their molecular masses). To elucidate how these two components are involved in macromolecular organization of the chicken egg-envelope, the isolated egg-envelope was characterized by immunochemical and biochemical methods. The gp97 was suggested to be a homologue of mouse ZPB based on the similarities of N-terminal and internal sequences. Immunoblotting using anti-gp97 monoclonal antibodies and two-dimensional gel electrophoresis with or without mercaptoethanol treatment revealed that gp97 formed a homodimer through disulfide bonds, whereas gp42 did not. Under indirect immunofluorescence microscopy, the anti-gp97 antibody visualized indistinct, small spots on the egg-envelope, whereas the anti-gp42 antibody showed a meshwork of blurry, fibrous structures. The hole formation on the egg-envelope by in vitro sperm penetration was completely inhibited by two anti-gp97 monoclonal antibodies. Interestingly, the anti-gp97 monoclonal antibodies blocked the proteolysis not only of gp97 but also of gp42 during incubation of the egg-envelope with either sperm or the crude chicken acrosin. Taken together, these results indicate that gp97 may play pivotal roles not only in constitution of the macromolecular organization of the egg-envelope but also in triggering hydrolysis of the egg-envelope during sperm penetration.

A comparison of the patterns of laminin expression in fibroadenoma, fibrocystic diseases, pre-invasive and invasive ductal breast carcinoma

The basement membrane (BM), of which laminin is a major glycoprotein component, is an important barrier to tumour cells which must be breeched before metastatic spread can occur. We have compared the pattern of laminin expression in a range of benign and malignant breast lesions to better understand the process of tumour progression. A total of 162 cases of breast samples, comprising 18 fibroadenomas, 22 cases of fibrocystic disease, 96 cases of invasive ductal carcinoma and 26 carcinomas with intraductal components, were evaluated for laminin expression by a standard immunoperoxidase method on formalin-fixed, paraffin-embedded histological sections, using a commercial antibody against human laminin. The pattern of laminin expression was charted as follows: Type I, > 70% of BM complete/continuous; Type II, > 70% of BM moderately disrupted; Type III, > 70% of BM completely disrupted. The Type I pattern was observed in all cases of fibroadenoma and fibrocystic diseases, and in 77% of intraductal carcinoma components. Various patterns of BM disruption were observed in invasive ductal carcinoma. Severity of BM disruption correlated with histological grade of the carcinomas (P < 0.001). Small-sized tumours, those without lymphatic invasion and lymph node-negative tumours showed more complete patterns of laminin expression. The current study suggests that tumour cells with high histological grade possess an enhanced capacity to disrupt the basement membrane, an important step in the metastatic process. The detection of BM disruption by immunohistochemical staining for laminin is technically easy and may be usefully applied for the differentiation of in situ and microinvasive carcinoma.

Fibronectin fragments induce MMP activity in mouse mammary epithelial cells: evidence for a role in mammary tissue remodeling.

Mammary gland form and function are regulated by interactions between epithelium and extracellular matrix. Major glycoprotein components of extracellular matrix have been identified that give survival, proliferation and differentiation signals to mammary epithelial cells. We provide evidence that proteolytic fragments of the extracellular matrix glycoprotein, fibronectin, suppress growth and can promote apoptosis of mouse mammary epithelial cells. During mammary gland involution, total fibronectin and fibronectin fragment levels are increased. The peak levels of fibronectin protein and fragments are observed 4-6 days post-weaning, coincident with the peak in epithelial cell death. Using a model for hormone withdrawal-induced death of mammary epithelium, elevated levels of fibronectin proteolytic fragments were associated with apoptosis in TM-6 cells, a tumorigenic mouse mammary epithelial cell line. Treatment of TM-6 cells with exogenous fibronectin fragments (FN120) reduced cell number, and induced apoptosis and matrix degrading protease activity. Inhibition of matrix protease activity rescued TM-6 cell viability, indicating that FN120-induced cell loss is mediated through matrix protease activity. In a three-dimensional model for mammary gland development, FN120 reduced alveolar-like and promoted ductal-like development by a matrix protease-dependent mechanism. These data suggest that during post-lactational involution, fibronectin fragments may contribute to epithelial cell loss and dissolution of mammary alveoli by inducing matrix degrading proteinases.

Endothelial Cell Integrin Laminin Receptor Expression in Multiple Sclerosis Lesions

Laminin, a major glycoprotein component of vessel basement membranes, is recognized by beta1- and beta3-integrins expressed on endothelial cells. To determine how endothelial cell integrins might function in multiple sclerosis (MS) lesions, integrin laminin receptors and laminin were analyzed in central nervous system samples from MS patients and controls by immunohistochemistry. In active MS lesions, endothelial cell VLA-6 and beta1 subunits were decreased compared to controls whereas alpha(v) subunit and VLA-1 were increased. In chronic inactive lesions beta1, VLA-6 and alpha(v) were the same as controls but VLA-1 remained increased. Alpha3 subunit was constant in all samples. By immunoelectron microscopy VLA-1, VLA-6, beta1, and laminin were distributed throughout endothelial cells; alpha(v) was adjacent to and on luminal surfaces; alpha(v) and VLA-1 were on intercellular junctions. These results indicate distinct regulation and functions of these integrins in different lesion stages. In active lesions decreased endothelial cell beta1/VLA-6 could result in their detachment from laminin thereby facilitating leukocyte transvascular migration and blood-brain barrier breakdown. Alpha(v) and VLA-1 on intercellular junctions may participate in re-establishing vessel integrity after leukocyte migration. Luminal surface alpha(v) also likely binds intraluminal ligands and cells. In chronic inactive plaques persistently elevated endothelial cell VLA-1 correlates with long-standing endothelial cell and blood-brain barrier dysfunction.

Expression and localization of laminin-5 subunits during mouse tooth development.

Tooth morphogenesis is regulated by epithelial-mesenchymal interactions mediated by the basement membrane (BM). Laminins are major glycoprotein components of the BMs, which are involved in several cellular activities. The expression and localization of the alpha3, beta3, and gamma2 laminin-5 subunits have been analyzed by in situ hybridization and immunohistochemistry during mouse molar development. Initially (E12), mRNAs of all subunits were detected in the entire dental epithelium and the corresponding proteins were located in the BM. During cap formation (E13-14), transcripts for the alpha3 and gamma2 subunits were localized in the outer dental epithelium (ODE), whereas the beta3 subunit mRNA was present in the inner dental epithelium (IDE). During the early bell stage (E16), immunoreactivity for all subunits disappeared from the BM along the IDE, although intense signals for beta3 mRNA were detectable in cells of the IDE. Subsequently, when the dentinal matrix was secreted by odontoblasts (E18-19.5), mRNAs of all three subunits were re-expressed by ameloblasts, and the corresponding proteins were detected in ameloblasts and in the enamel matrix. Tissue recombination experiments demonstrated that when E16 IDE or ODE was associated with E18 dental papilla mesenchyme, immunostaining for all laminin-5 subunits disappeared from the BM, whereas when cultured with non-dental limb bud mesenchyme, they remained positive after 48 hr of culture. These results suggest that the temporospatial expression of laminin-5 subunits in tooth development, which appears to be differentially controlled by the dental mesenchyme, might be related to the enamel organ histo-morphogenesis and the ameloblast differentiation.