Laminin B1 Chain Research Articles

Early interstitial accumulation of collagen type I discriminates chronic rejection from chronic cyclosporine nephrotoxicity.

Little is known regarding the composition of the interstitial extracellular matrix of kidney allografts with deteriorating function. Collagen I, III, and IV, the collagen IV alpha3 chain, and the laminin beta2 chain were investigated in biopsies of allografted kidneys with chronic cyclosporine A nephrotoxicity (CsAT) (n = 17), chronic rejection (CR) (n = 12), or chronic allograft nephropathy (CAN) (n = 19). alpha-Smooth muscle actin expression was also examined. Normal native kidneys were used as control samples (n = 11). Biopsy samples were studied with routine light microscopy and immunostaining. The mean interstitial fibrosis scores were significantly higher for the CR and CAN groups, compared with the chronic CsAT group. The cortical tubulointerstitial areas of the CR and CAN groups, but not the chronic CsAT group, contained more collagen I than did normal control samples. Differences were noted even in biopsies with mild fibrosis. Accumulation of collagen III, IV, and IV alpha3 was increased in all patient groups. Collagen III accumulation was greater in the CR and CAN groups than in the chronic CsAT group. Receiver-operating characteristic curve analysis demonstrated that collagen I staining had the best discriminatory value in differentiating CR from chronic CsAT, with a sensitivity of 63% and a specificity of 94% at a cutoff value of 19%. Laminin beta2 staining did not differentiate CR from CsAT. Increased alpha-smooth muscle actin staining did not differ among the three groups. It was concluded that, during chronic CsAT, collagen III and IV were preferentially accumulated in the tubulointerstitium. Early increases in the deposition of collagen I, with collagen III and IV, were more specific for CR. CR seems to elicit a more pronounced fibrotic response than does chronic CsAT.

Up-regulating effect of hepatitis C virus core protein on laminin B1 chain gene promoter

Up-regulating effect of hepatitis C virus core protein on laminin B1 chain gene promoter

Identification of redundant angiogenic sites in laminin α1 and γ1 chains

The degradation of the extracellular matrix is one of the first steps involved in angiogenesis, the formation of new vessels from preexisting ones. Laminin, a large extracellular matrix protein, has many biological activities, including the promotion of angiogenesis. Screening of the laminin-1 chains identified 20 angiogenic peptides, of which, A13 and C16, from the α1 and γ1 chains, respectively, were the most active. We recently identified the receptors for C16 as the integrins α5β1 and αvβ3. Here, we show unexpectedly that A13 is a redundant active site to C16 present in the N-terminal globular domain of the α1 chain. The peptides are located in homologous sites present in the last globular domains of their respective chains, and their amino acids are 66% conserved, as compared to the inactive homologous site in the β1 chain, B19 to B20, which is only 18%–23% conserved. Cell attachment studies demonstrated that both A13 and C16 reciprocally inhibited their adhesion activity, whereas the corresponding laminin β1 chain peptides were inactive. Chorioallantoic membrane assays showed that the in vivo angiogenic activity of A13 is blocked by a C16 antagonist, C16S, which also binds to the same integrin receptors. A13 affinity chromatography and immunoprecipitation analysis showed that the αvβ3 and α5β1 integrin receptors bind to this sequence. We have therefore identified redundant activity on two laminin chains. These highly conserved functional sites are likely important mediators of the biological responses of laminins because either one or both of these chains (active sites) are present in almost all laminin isoforms identified to date.

Focusing on the Glomerular Slit Diaphragm: Podocin Enters the Picture

The nature of the glomerular filtration barrier is currently one of the most intensely studied and exciting problems in nephrology research, primarily because of the relatively recent discoveries that two novel and two previously known genes are intimately involved in glomerular filtration. The two novel genes, NPHS1 and NPHS2 (encoding nephrin and podocin, respectively) were identified by positional cloning. NPHS1 is mutated in congenital nephrotic syndrome of the Finnish type, 1 and NPHS2 is mutated in steroid resistant nephrotic syndrome. 2 The other two genes, previously characterized but not known to be involved in filtration, are ACTN4, encoding α-actinin-4, and Cd2ap, encoding CD2-associated protein (CD2AP). ACTN4 was found to be mutated in several families exhibiting inherited focal segmental glomerulosclerosis, 3 and knockout mice lacking CD2AP exhibit congenital nephrotic syndrome. 4 Several recent reviews have addressed the potential roles of these molecules in glomerular filtration. 5-10

Regulation of laminin beta2 chain gene expression in human cancer cell lines.

The laminin beta2 chain is a basement membrane component expressed in a tissue- and developmental stage-specific manner. In this report we have examined the transcriptional and post-transcriptional regulation of the human laminin beta2 chain in human tumor cell lines. Both the A204 rhabdomyosarcoma and clone A colon carcinoma cells express the laminin beta2 chain mRNA, but only the A204 cells secrete laminin heterotrimers containing the beta2 chain. Segments of the beta2 chain gene promoter region were cloned into luciferase reporter vectors, and their ability to stimulate transcription was tested by transient transfection. Sequences downstream of the transcription start site between nucleotides +91 and +120 were found to be essential for luciferase activity in the two cell lines. Additional positive regulatory regions were present further upstream, between nucleotides -164 to -667 and between nucleotides -667 to -1724. Genomic DNA at the 3' end of the gene also appeared to have enhancer activity, as a 1.1-kb fragment located downstream of the last exon stimulated the luciferase activity of the nucleotides -667/+297 promoter segment approximately threefold. Alternative splicing of the first intron of the human laminin beta2 chain gene generates two isoforms of the 5' untranslated region of the beta2 chain mRNA. The translational efficiencies of the two laminin beta2 chain leaders did not differ significantly, when assayed by polysome profile analysis of endogenous clone A cell beta2 chain mRNA, transient transfection of chimeric beta2 chain leader/luciferase expression plasmids in clone A cells, and translation of in vitro synthesized RNAs in rabbit reticulocyte lysates.

Cell Adhesive Sequences in Mouse Laminin β1 Chain

Laminin-1, a major component of the basement membrane, consists of three different chains, α1, β1, and γ1. We sought to identify cell adhesive sequences from the mouse laminin β1 chain by testing HT-1080 fibrosarcoma and B16-F10 melanoma cells for binding to 187 overlapping synthetic peptides which covered the entire chain. Fourteen peptides showed cell adhesive activities with either peptide-conjugated Sepharose beads or peptide-coated plates or both. Additional cells, including neuronal, endothelial, and salivary gland cells, showed biological responses in a cell type-specific manner. B-7, B-133, and B-160 showed the most potent cell attachment. Cell binding on three peptides (B-34, B-133, and B-160) was inhibited by EDTA. Cell adhesion to 11 of the 12 active peptides was inhibited to varying degrees by heparin. Of the 17 active peptides identified in the laminin β1 chain in this and other studies, 8 are clustered on the amino terminal globular domain, suggesting a possible important role in cell binding for this domain that may be multifunctional. These data demonstrate that the laminin β1 chain has multiple active sites for cell adhesion, some of which are cell-type specific.

Glycosaminoglycan-Binding Properties and Secondary Structure of the C-Terminus of Netrin-1

Netrins are soluble neurite-outgrowth-promoting proteins related to the laminin B2 chain. Since these proteins and their receptor DCC (the “deleted in colorectal carcinoma” gene product) bind heparin, glycosaminoglycans may modulate their biological actions in a similar fashion as described for several other ligand-receptor systems. Here we show that a polypeptide encompassing the C-terminal cluster of basic amino acids of netrin-1 (i) adopts an α-helical conformation in water-trifluoroethanol mixtures according to circular dichroism experiments and (ii) binds electrostatically to heparin with high affinity under physiological ionic conditions (KD = 15 nM for the binding to immobilized heparin according to surface plasmon resonance, KD = 50 nM in solution as determined with isothermal titration calorimetry). These data indicate that the cluster of basic amino acids at the C-terminus of netrin-1 forms an α-helical structural element which can contribute to the glycosaminoglycan-binding activity of this neurotrophic guidance molecule.

Distinct Activities of p52/NF-κB Required for Proper Secondary Lymphoid Organ Microarchitecture: Functions Enhanced by Bcl-3

AbstractMice rendered deficient in p52, a subunit of NF-κB, or in Bcl-3, an IκB-related regulator that associates with p52 homodimers, share defects in the microarchitecture of secondary lymphoid organs. The mutant mice are impaired in formation of B cell follicles and are unable to form proper follicular dendritic cell (FDC) networks upon antigenic challenge. The defects in formation of B cell follicles may be attributed, at least in part, to impaired production of the B lymphocyte chemoattractant (BLC) chemokine, possibly a result of defective FDCs. The p52- and Bcl-3-deficient mice exhibit additional defects within the splenic marginal zone, including reduced numbers of metallophilic macrophages, reduced deposition of the laminin-β2 chain and impaired expression of a mucosal addressin marker on sinus-lining cells. Whereas p52-deficient mice are severely defective in all of these aspects, Bcl-3-deficient mice are only partially defective. We determined that FDCs or other non-hemopoietic cells that underlie FDCs are intrinsically impaired in p52-deficient mice. Adoptive transfers of wild-type bone marrow into p52-deficient mice failed to restore FDC networks or follicles. The transfers did restore metallophilic macrophages to the marginal zone, however. Together, the results suggest that p52 carries out functions essential for a proper splenic microarchitecture in both hemopoietic and non-hemopoietic cells and that Bcl-3 is important in enhancing these essential activities of p52.

Bronchial smooth muscle hypoplasia in mouse embryonic lungs exposed to a laminin β1 chain antisense oligonucleotide

We used an antisense oligonucleotide (ODN) to inhibit laminin (LM) β1 chain synthesis in mouse embryonic lung explants and cell cultures. The ODN spanned 17 bases located 13 bases downstream the initiation codon and contained phosphorothioate and C-5 propynyl pyrimidine modifications. Penetration of the ODN into the lung explants was confirmed by fluorescein isothiocyanate (FITC) tagging. 50 μM of antisense ODN decreased LM β1 chain synthesis by 82±6.9% with no significant changes in the synthesis of other LM chains. The same antisense probe but without C-5 propynyl pyrimidine modification, another 17-mer ODN complementary to the LM β1 initiation codon, and a 17-mer ODN complementary to the LM α1 initiation codon had no antisense activity. Lung explants exposed to the active LM β1 antisense ODN showed decreased LM-1 and collagen type IV deposition at the epithelial–mesenchymal interface and an arrest in bronchial smooth muscle (SM) development. Histological examination and cell motility assays suggested that this arrest was due to impaired spreading and migration of SM cell precursors over the defective basement membrane (BM). Our studies indicate that β1-chain containing LMs play a role in bronchial myogenesis.

Disruption of Laminin β2 Chain Production Causes Alterations in Morphology and Function in the CNS

From the elegant studies of Ramon y Cajal (1909) to the current advances in molecular cloning (e.g., Farber and Danciger, 1997), the retina has served as an ideal model for the entire CNS. We have taken advantage of the well described anatomy, physiology, and molecular biology of the retina to begin to examine the role of the laminins, one component of the extracellular matrix, on the processes of neuronal differentiation and synapse formation in the CNS. We have examined the effect of the deletion of one laminin chain, the beta2 chain, on retinal development. The gross development of retinas from laminin beta2 chain-deficient animals appears normal, and photoreceptors are formed. However, these retinas exhibit several pathologies: laminin beta2 chain-deficient mice display abnormal outer segment elongation, abnormal electroretinograms, and abnormal rod photoreceptor synapses. Morphologically, the outer segments are reduced by 50% in length; the outer plexiform layer of mutant animals is disrupted specifically, because only 7% of observed rod invaginating synapses appear normal, whereas the inner plexiform layer is undisturbed; finally, the rate of apoptosis in the mutant photoreceptor layer is twice that of control mice. Physiologically, the electroretinogram is altered; the amplitude of the b-wave and the slope of the b-wave intensity-response function are both decreased, consistent with synaptic disruption in the outer retina. Together, these results emphasize the prominence of the extracellular matrix and, in particular, the laminins in the development and maintenance of synaptic function and morphogenesis in the CNS.

Characterization of the human laminin β2 chain locus (LAMB2): linkage to a gene containing a nonprocessed, transcribed LAMB2-like pseudogene (LAMB2L) and to the gene encoding glutaminyl tRNA synthetase (QARS)

The laminin β2 chain is an important constituent of certain kidney and muscle basement membranes. We have generated a detailed physical map of a 110-kb genomic DNA segment surrounding the human laminin β2 chain gene (LAMB2) on chromosome 3p21.3→p21.2, a region paralogous with the chromosome 7q22→q31 region that contains the laminin β1 chain gene locus (LAMB1). Several CpG islands and a novel polymorphic microsatellite marker (D3S4594) were identified. The 3′ end of LAMB2 lies 16 kb from the 5′ end of the glutaminyl tRNA synthetase gene (QARS). About 20 kb upstream of LAMB2 we found a gene encoding a transcribed, non-processed LAMB2-like pseudogene (LAMB2L). The sequence of 1.75 kb of genomic DNA at the 3′ end of LAMB2L was similar to exons 8–12 of the laminin β2 chain gene. The LAMB2L–LAMB2–QARS cluster lies telomeric to the gene encoding the laminin-binding protein dystroglycan (DAG1).

Experimental AA amyloidogenesis is associated with differential expression of extracellular matrix genes

An abnormality in basement membrane metabolism has been postulated to play an important role in the pathogenesis of experimental murine AA amyloidosis. The potential contribution of the structural basement membrane proteins laminin, type IV collagen and entactin to amyloidogenesis in this model was investigated with a kinetic analysis of the expression of the corresponding genes during amyloid formation. Splenic AA amyloid deposition was stimulated by the concomitant administration of subcutaneous silver nitrate, as an inflammatory stimulus, and intravenous amyloid enhancing factor. Using a reverse transcription-poly-merase chain reaction assay, a differential pattern of expression of these genes was observed at the mRNA level. Whereas laminin Bl mRNA levels did not change at any time during amyloidogenesis, a 2.2 to 3 fold induction of laminin B2, entactin and al-type IVcollagen mRNAs coincided with the initial detection of splenic amyloid deposits at 48 hours post-stimulation, as detected by immunohis-tochemistry. Temporal and spatial codeposition of laminin and type IV collagen with amyloid was demonstrated by immunohistochemistry. A 1.4, 2.3 and 2.2-fold increase in laminin B2, entactin and al-type IV'collagen mRNA levels, respectively, was detected at 24 hours post-stimulation, a point at which amyloid deposits could not be detected. Neither inflammation nor amyloid enhancing factor alone influenced laminin, entactin or type IV collagen expression at the protein or mRNA level. These observations suggest that the laminin B2 chain and al-type IV collagen chain account, at least in part, for the observed laminin and collagen IV immunoreactivity inAA amyloid deposits and that entactin may also be a component of the amyloid deposit. The onset of the induction of laminin B2, entactin and al-type IV collagen gene expression prior to the appearance of amyloid deposits, and our previous data with the heparan sulfate proteoglycan, perlecan, suggests these basement membrane proteins may play a role in the initial stages of AA fibrillogenesis.

Laminin alpha2 chain-deficient congenital muscular dystrophy: variable epitope expression in severe and mild cases.

To characterize the expression of distinct fragments of laminin alpha2 chain in patients with partial laminin alpha2 chain deficiency and variable clinical severity. Deficiency of laminin alpha2 chain caused by mutations of the LAMA2 gene on chromosome 6q2 account for approximately 50% of cases of congenital muscular dystrophy (CMD) in white patients. The complete absence of laminin alpha2 is usually associated with a severe phenotype affecting skeletal muscle and the peripheral and central nervous systems. Quantitative assessment of immunofluorescence to study the expression of C- and N-terminal portions of laminin alpha2 chain in five patients with partial laminin alpha2 chain deficiency and variable phenotype. All five patients showed abnormal T2 signal on brain MRI. Immunohistochemistry of muscle specimens showed preserved or minimally reduced expression of the C-terminal region of the laminin alpha2 chain (67 to 74%), but a marked reduction of the N-terminal region in four patients (13 to 19%). One patient with a mild phenotype had a partial reduction (45%) of the C-terminal and the N-terminal (51%) portions of the laminin alpha2 chain. Two patients were unable to walk or sit, although the C-terminal portion of the laminin alpha2 chain was expressed at significant levels (67 to 74%). In contrast, two patients with a similar expression of the C-terminus (67 to 70%) had a milder phenotype and became ambulatory. It was impossible to predict the phenotypes in these four patients with a strong expression of the C-terminus and with low levels of the N-terminus based on the amount of protein expressed. In addition, the laminin beta2 chain was moderately reduced (54 to 75%) in all patients with laminin alpha2 chain deficiency. A strong correlation between the amount of the C-terminus but not for the N-terminus and laminin beta2 reduction could be observed. N-terminal antibodies to the laminin alpha2 chain provide a more precise immunohistochemical detection of partially laminin alpha2 chain-deficient CMD. The secondary reduction of laminin beta2 chain may better define laminin alpha2 chain-deficient CMD. More data are needed to predict which portions of C-terminus and midrod region of the laminin alpha2 chain result in a semifunctional protein and a milder phenotype.

Regulation by thyroid hormone of the expression of basement membrane components in rat prepubertal Sertoli cells.

The present study reports the modulation of basement membrane (BM) components, laminin, entactin, and type IV collagen, expression in prepubertal rat Sertoli cell by the thyroid hormone T3. Immunocytochemical studies of permeabilized Sertoli cells in culture showed that T3 treatment (10[-7] M for 24 h) increased the number of cells staining positive for laminin and/or entactin (from 58 +/- 5.3% to 86.4 +/- 6.5%, P < 0.01). In contrast, a strong inhibition of type IV collagen immunopositivity was observed. Western blot analysis of Sertoli cell-conditioned media indicated that T3 treatment significantly (P < 0.01) increased the level of secreted entactin by 60-65% without affecting the levels of laminin A and B1/B2 chains. Moreover, thyroid hormone treatment of Sertoli cells significantly reduced type IV collagen secretion by 62% (P < 0.05). Slot blot analysis of poly-A RNA demonstrated a significant (P < 0.01) increase in the level of entactin messenger RNA (mRNA) by 140% (P < 0.01) and a 50% reduction of type IV collagen alpha1 chain mRNA after thyroid hormone treatment. No effect of the hormone was observed on the accumulation of the laminin B1 and B2 chain mRNAs in Sertoli cell cultures. These effects cannot be ascribed to changes in the degradation of BM components, because no effect of thyroid hormone was observed on plasminogen activators or metalloproteinase secretion by Sertoli cells. These observations indicate the Sertoli cell as a source of entactin within the testis, demonstrate the ability of T3 to differentially regulate the expression of BM components, and can be regarded as a part of the integrated mechanism by which thyroid hormone affects testicular development and differentiation.

Ontogeny of Extracellular Matrix Gene Expression by Rat Lung Cells at Late Fetal Gestation

Lung epithelial and mesenchymal cells are separated by a basement membrane. At late fetal gestation, this basement membrane in fenestrated, allowing epithelial cytoplasmic extensions to reach in close proximity of the interstitial fibroblast. The enzymes responsible for this focal basement membrane remodelling, and their cellular origin, remains to be defined. Basement membrane remodelling generally involves a special class of matrix-degrading enzymes, called metalloproteinases. Herein, we report that fetal lung cells originating from both tissue layers, mesoderm and endoderm, express the metalloproteinase genes, MMP-1 or interstitial collagenase, and MMP-3 or stromelysin. The inhibitor of metalloproteinases, TIMP-1, is mainly expressed in fetal lung fibroblasts. During late fetal development, MMP-1 mRNA expression in both cell types increases close to term (day 21, term = 22 days), while that of stromelysin and TIMP-1 remain constant. Both fibroblasts and epithelial cells express fibronectin (FN) mRNA. The expression of the FN gene in epithelial cells decreases slightly at the canalicular stage of lung development (days 19–20), whereas FN expression in fibroblasts is not changed with advancing gestation. Procollagen α₁(I) mRNA is predominantly detected in fibroblasts whereas message for laminin B1 chain is primarily found in epithelial cells. Expression of procollagen α₁(I) mRNA decreases in fibroblasts during the canalicular stage of fetal lung development compared to the pseudoglandular stage (day 18) but increases thereafter at the saccular stage (day 21) of development. Laminin B1 expression in epithelial cells declines with advancing gestation. These data are consistent with a process of basement membrane thinning during the canalicular stage, followed by metalloproteinase-mediated penetration. Further, a progressive reduction in laminin expression is consistent with progressive epithelial differentiation.

Sequences 5' of the basement membrane laminin β1 chain gene ( LAMB1) direct the expression of β-galactosidase during development of the mouse testis and ovary

The murine LAMB1 gene encoding laminin β1 is expressed in the developing male and female gonads and mesonephros. To identify the cis-acting elements regulating the expression of LAMB1, murine transgenic lines were generated by fusing regions of the LAMB1 gene to the Eschericia coli lacZ gene. The p3.9LAMβgal construct contained approximately 4 kb of 5′ flanking sequence and directed β-galactosidase expression in many different organs including the kidney, mammary gland, and the male and female genital systems, the focus of this report. In male embryos, between gestational ages E 14.5 and birth β-galactosidase was transiently expressed in the prospermatogonia cells of the testis and in the differentiating epithelial cells in the ductus deferens, ductus epididymis, and seminal vesicles. In female embryos, β-galactosidase was not detected in the ovary until about 1 week after birth; at this time, β-galactosidase was expressed by oocytes of primary and secondary follicles. In contrast, transgenic mice carrying the first 0.7 kb of LAMB1 fused to the lacZ gene expressed β-galactosidase only in the prospermatogonia cells of the testis. Thus, the cis-acting element(s) necessary for the expression of the LAMB1 gene in prospermatogonia cells are located in the first 0.7 kb of LAMB1 5′ flanking sequence; element(s) required for expression of the LAMB1 gene in oocytes and epithelial cells of the mesonephric ducts, mesonephric tubules, the ductus deferens, ductus epididymis, and seminal vesicles are located within 4 kb 5′ of the transcription initiation site.

Laminin chain-specific gene expression during mouse oocyte maturation.

Expression patterns of laminin chain mRNAs (A, B1, and B2) during mouse oocyte maturation were examined using the competitive reverse transcription-polymerase chain reaction (RT-PCR) method. Total and poly (A)-rich mRNAs isolated from various stages of maturing oocytes in vitro were subjected to RT-PCR and the precise amount of laminin chain-specific mRNA transcripts was estimated by adding externally known amounts of in vitro transcribed mutant cRNA transcripts as an internal control. The estimated copy numbers for A, B1, and B2 chain mRNAs in a single germinal vesicle-stage oocyte were 1.34 +/- 0.19 x 10(5), 6.95 +/- 0.32 x 10(6), and 2.0 +/- 0.56 x 10(5), respectively. Although notable changes of all laminin chain mRNA levels were not observed at any stage of meiotic maturation in total mRNA preparation, chain- and meiotic stage-dependent alterations of poly (A)-tailed mRNA quantities were observed in poly (A)-rich mRNA preparation. A potent RNA synthesis blocker, alpha-amanitin did not influence the changes of mRNA levels, implying the presence of posttranscriptional regulation mechanism in the expression of laminin chains during mouse oocyte maturation. Discrete and time-dependent deadenylation of A and B1 chain, but not B2 chain mRNA, was observed during oocyte maturation by a rapid amplification of cDNA ends (RACE)-PCR. In germinal vesicle (GV)-stage oocytes, only B1 chain was found to be present in a highly polyadenylated state and subsequent deadenylation was observed as meiosis progressed. The poly (A) tail modification was dependent on the initiation of meiotic resumption. Although all laminin chain mRNAs were found in fully grown and meiotically competent mouse oocytes, Western blot analysis detected the B1 chain polypeptide only in GV- and polar body (PB)-stage eggs. These results suggest that the expression of laminin B1 chain in mouse oocytes may be due to its large amount of mRNA transcripts and/or high level of polyadenylation state that is efficient for translational activation.

Changes of laminin β2 chain expression in congenital muscular dystrophy

We studied the distribution of laminin β2 chain in the skeletal muscle basement membrane of 16 patients with congenital muscular dystrophy (CMD) by immunohistochemistry. A dramatic reduction in the laminin β2 staining was observed in four patients with classical merosin-negative CMD. A moderate reduction of laminin β2 labelling was observed in four patients with partial merosin deficiency and two patients with merosin-positive CMD. Two patients with merosin-positive CMD had no apparent changes in the expression of laminin β2. In three patients and one fetus diagnosed as Walker-Warburg syndrome (WWS) the laminin β2 pattern was similar to normal controls. We conclude that a primary deficiency in the laminin α2 chain may lead to a vast or moderate reduction in the laminin β2 chain in the skeletal muscle membrane.

Regulatory sequences for the transcription of the laminin B2 gene in astrocytes

Astrocytes synthesize only the B2 chain of laminin and that this chain is sufficient to stimulate neurite outgrowth. In this study, we have examined laminin B1 and B2 promoter constructs in various cell types in order to understand the transcriptional regulation of laminin B2 gene in astrocytes. Comparison of nuclear factor binding by Southwestern analysis with the highly active B2 promoter fragment revealed different patterns of nuclear factor binding. In HepG2 cells, two proteins of 105 and 98 kDa were identified while, in primary astrocytes, human U251 and rat C6 glioma cells, a greater number of nuclear proteins ranging from 43 to 212 kDa were detected. The laminin B1 promoter construct was inactive in transient transfection experiments in astrocytes yet active in the HepG2 hepatoma cells which synthesize both the B1 and B2 chains. In contrast, the laminin B2 promoter construct was active in both astrocytes and HepG2 cells. These results are consistent with the lack of laminin B1 mRNA expression in astrocytes and suggest that the differential regulation of the laminin B1 and B2 gene is controlled at the transcriptional level. Delineation of the 5′-flanking regions responsible for basal levels of B2 laminin promoter activity revealed a silencer-like segment between −830 and −224 which reduced promoter activity. Deletion analysis further revealed that B2 laminin promoter possesses a highly active short promoter (−94 to +106) and basal transcriptional activity resides within −61 to +106. DNase 1 footprinting, gel-shift competition assays and site-directed mutagenesis of a highly active short promoter revealed that this region contained binding sites for cell-type nuclear factors. The shortest construct containing only residues −21 to +106 was inactive in HepG2 and U251 glioma cells. In primary astrocytes, however, this construct showed a high level of transcriptional activity. Deletion of 47 bp (+59 to +106) in 5′-UTR completely blocked promoter activity in astrocytes confirming that this downstream region is important for transcriptional activity in primary astrocytes. Together, these results suggest that astrocytes may utilize mutually exclusive transcription factors and regulatory sequences, in addition to common factors in the control of the laminin B2 promoter.

Tight Junctional Damage in Experimental Mumps‐Associated Hydrocephalus

AbstractTight junctions in the central nervous system (CNS) are a major component of brain barriers including the blood‐brain barrier (BBB) and blood‐CSF barrier, which regulate solute entry and protect against invasion by microorganisms. In this study, we examined the breach of tight junctions in mumps virus‐induced hydrocephalic brain in hamsters using antibodies to Laminin B1 chain and zonula occludentes (ZO‐1) immuno‐histochemistry, and evaluated the role of tight junctions in the pathogenesis of hydrocephalus after mumps virus infection.In suckling hamsters intracerebrally inoculated with mumps virus, severe periventricular edema, ependymal cell loss, and ventricular dilation were observed. ZO‐1‐immunoreactive tight junctions in the hydrocephalic brains were severely damaged in the choroid plexus and ependyma, and in white matter capillaries as early as 3 days after inoculation. These changes were suspected to increase the permeability in blood‐brain and blood‐CSF barriers, leading to periventricular edema. We concluded that the pathologic features of mumps virus‐induced hydrocephalus are intimately related to the breach of tight junctions.