LAMB3 Research Articles

Expression of Laminin β1 and Integrin α2 in the Anterior Temporal Neocortex Tissue of Patients With Intractable Epilepsy

ABSTRACTWe investigated the expression of laminin β1 and integrin α2 in the anterior temporal neocortex tissue of patients with intractable epilepsy and explored the role of these molecules in the pathogenesis of this disease. Immunohistochemistry and immunofluorescence were used to test the expression of laminin β1 and integrin α2 in samples (from the brain bank of our department, n = 32) of surgically removed anterior temporal neocortex tissues from intractable epilepsy patients, and the results were compared with those of controls (n = 10). We found that laminin β1 and integrin α2 protein expression was significantly increased in the anterior temporal neocortex as compared with controls (immunohistochemistry optical density: laminin β1 = 0.36 ± 0.01 vs. 0.10 ± 0.03 for control; integrin α2 = 0.42 ± 0.02 vs. 0.04 ± 0.01 for control; p < .05). Immunofluorescence staining indicated that laminin β1 and integrin α2 accumulated in the plasma membrane and cytoplasm, with strong fluorescence intensity in the anterior temporal neocortex tissue of patients with intractable epilepsy. Thus, our work demonstrates that laminin β1 and integrin α2 expression is elevated in the anterior temporal neocortex tissue from patients with intractable epilepsy.

Aberrant up-regulation of LAMB3 and LAMC2 by promoter demethylation in gastric cancer

The LAMB3 and LAMC2 genes encode the laminin-5 β3 and γ2 chains, respectively, which are parts of laminin-5, one of the major components of the basement membrane zone. Here, we report the frequent up-regulation of LAMB3 and LAMC2 by promoter demethylation in gastric cancer. Gene expression data analysis showed that LAMB3 and LAMC2 were up-regulated in various tumor tissues. Combined analyses of DNA methylation and gene expression of both genes in gastric cancer cell lines and tissues showed that DNA hypomethylation was associated with the up-regulation of both genes. Treatment with a methylation inhibitor induced LAMB3 and LAMC2 expression in gastric cancer cell lines in which both genes were silenced. By chromatin immunoprecipitation assay, we showed the activation histone mark H3K4me3 was associated with the expression of both genes. The expression level of LAMB3 affected multiple malignant phenotypes in gastric cancer cell lines. These results suggest that epigenetic activation of LAMB3 and LAMC2 may play an important role in gastric carcinogenesis.

Tendon Is Covered by a Basement Membrane Epithelium That Is Required for Cell Retention and the Prevention of Adhesion Formation

The ability of tendons to glide smoothly during muscle contraction is impaired after injury by fibrous adhesions that form between the damaged tendon surface and surrounding tissues. To understand how adhesions form we incubated excised tendons in fibrin gels (to mimic the homeostatic environment at the injury site) and assessed cell migration. We noticed cells exiting the tendon from only the cut ends. Furthermore, treatment of the tendon with trypsin resulted in cell extravagation from the shaft of the tendons. Electron microscopy and immunolocalisation studies showed that the tendons are covered by a novel cell layer in which a collagen type IV/laminin basement membrane (BM) overlies a keratinised epithelium. PCR and western blot analyses confirmed the expression of laminin β1 in surface cells, only. To evaluate the cell retentive properties of the BM in vivo we examined the tendons of the Col4a1+/Svc mouse that is heterozygous for a G-to-A transition in the Col4a1 gene that produces a G1064D substitution in the α1(IV) chain of collagen IV. The flexor tendons had a discontinuous BM, developed fibrous adhesions with overlying tissues, and were acellular at sites of adhesion formation. In further experiments, tenotomy of wild-type mice resulted in expression of laminin throughout the adhesion. In conclusion, we show the existence of a novel tendon BM-epithelium that is required to prevent adhesion formation. The Col4a1+/Svc mouse is an effective animal model for studying adhesion formation because of the presence of a structurally-defective collagen type IV-containing BM.

Genetic Deletion of Laminin Isoforms β2 and γ3 Induces a Reduction in Kir4.1 and Aquaporin-4 Expression and Function in the Retina

BackgroundGlial cells such as retinal Müller glial cells are involved in potassium ion and water homeostasis of the neural tissue. In these cells, inwardly rectifying potassium (Kir) channels and aquaporin-4 water channels play an important role in the process of spatial potassium buffering and water drainage. Moreover, Kir4.1 channels are involved in the maintenance of the negative Müller cell membrane potential. The subcellular distribution of Kir4.1 and aquaporin-4 channels appears to be maintained by interactions with extracellular and intracellular molecules. Laminins in the extracellular matrix, dystroglycan in the membrane, and dystrophins in the cytomatrix form a complex mediating the polarized expression of Kir4.1 and aquaporin-4 in Müller cells.Methodology/Principal FindingsThe aim of the present study was to test the function of the β2 and γ3 containing laminins in murine Müller cells. We used knockout mice with genetic deletion of both β2 and γ3 laminin genes to assay the effects on Kir4.1 and aquaporin-4. We studied protein and mRNA expression by immunohistochemistry, Western Blot, and quantitative RT-PCR, respectively, and membrane currents of isolated cells by patch-clamp experiments. We found a down-regulation of mRNA and protein of Kir4.1 as well as of aquaporin-4 protein in laminin knockout mice. Moreover, Müller cells from laminin β2 and γ3 knockout mice had reduced Kir-mediated inward currents and their membrane potentials were more positive than those in age-matched wild-type mice.ConclusionThese findings demonstrate a strong impact of laminin β2 and γ3 subunits on the expression and function of both aquaporin-4 and Kir4.1, two important membrane proteins in Müller cells.

Calcium Channels Link the Muscle-Derived Synapse Organizer Laminin β2 to Bassoon and CAST/Erc2 to Organize Presynaptic Active Zones

Synapse formation requires the organization of presynaptic active zones, the synaptic vesicle release sites, in precise apposition to postsynaptic neurotransmitter receptor clusters; however, the molecular mechanisms responsible for these processes remain unclear. Here, we show that P/Q-type and N-type voltage-dependent calcium channels (VDCCs) play essential roles as scaffolding proteins in the organization of presynaptic active zones. The neuromuscular junction of double knock-out mice for P/Q- and N-type VDCCs displayed a normal size but had significantly reduced numbers of active zones and docked vesicles and featured an attenuation of the active-zone proteins Bassoon, Piccolo, and CAST/Erc2. Consistent with this phenotype, direct interactions of the VDCC β1b or β4 subunits and the active zone-specific proteins Bassoon or CAST/Erc2 were confirmed by immunoprecipitation. A decrease in the number of active zones caused by a loss of presynaptic VDCCs resembled the pathological conditions observed in the autoimmune neuromuscular disorder Lambert-Eaton myasthenic syndrome. At the synaptic cleft of double knock-out mice, we also observed a decrease of the synaptic organizer laminin β2 protein, an extracellular ligand of P/Q- and N-type VDCCs. However, the transcription level of laminin β2 did not decrease in double knock-out mice, suggesting that the synaptic accumulation of laminin β2 protein required its interaction with presynaptic VDCCs. These results suggest that presynaptic VDCCs link the target-derived synapse organizer laminin β2 to active-zone proteins and function as scaffolding proteins to anchor active-zone proteins to the presynaptic membrane.

Novel mutations in steroid-resistant nephrotic syndrome diagnosed in Tunisian children

Steroid-resistant nephrotic syndrome (NS) remains one of the most intractable causes of end-stage renal disease in the first two decades of life. Several genes have been involved including NPHS1, NPHS2, WT1, PLCE1, and LAMB2. Our aim was to identify causative mutations in these genes, in 24 children belonging to 13 families with NS manifesting with various ages of onset. We performed haplotype analysis and direct exon sequencing of NPHS1, NPHS2, PLCE1, LAMB2, and the relevant exons 8 and 9 of WT1. Ten different pathogenic mutations were detected in seven families concerning four genes (NPHS1 (3/7), LAMB2 (2/7), NPHS2 (1/7), and WT1 (1/7)). Five of the detected mutations were novel; IVS9+2 T>C and p.D616G in NPHS1; p.E371fsX16 in NPHS2, and p.E705X and p.D1151fsX23 in LAMB2. Nine of 24 patients failed to be categorized by mutational analysis. Our study extends the spectrum of abnormalities underlying NS, by reporting novel mutations in the NPHS1 and NPHS2 genes and the first cases of LAMB2 mutations in Tunisia. Congenital and infantile NS can be explained by mutations in NPHS1, NPHS2, WT1, or LAMB2 genes. The identification of additional genes mutated in NS can be anticipated.

Integrin Alpha 7 Interacts with High Temperature Requirement A2 (HtrA2) to Induce Prostate Cancer Cell Death

Integrins are a family of receptors for extracellular matrix proteins that have critical roles in human tissue development. Previous studies identified down-regulation and/or mutations of integrin alpha7 (ITGA7) in prostate cancer, liver cancer, soft tissue leiomyosarcoma, and glioblastoma multiforme. Here we report that expression of ITGA7 induced apoptosis in the human prostate cancer cell lines PC3 and DU145. Yeast two-hybrid analysis revealed that the C-terminus of ITGA7 interacts with high temperature requirement A2 (HtrA2), a serine protease with a critical role in apoptosis. Expression of ITGA7 increases the protease activity of HtrA2 both in vitro and in vivo. Deletion of the HtrA2 interaction domain abrogates the cell death activity of ITGA7, whereas down-regulation of HtrA2 dramatically reduced cell death mediated by ITGA7. In addition, site-directed protease-null mutant HtrA2S306A expression blocked apoptosis induced by ITGA7. Interestingly, interaction between ITGA7 and its ligand laminin 2 appears to protect against cell death, since depleting laminin beta2 with a small-interfering RNA significantly exacerbated apoptosis induced by ITGA7 expression. This report provides a novel insight into the mechanism by which ITGA7 acts as a tumor suppressor.

A Novel Disulfide Pattern in Laminin-Type Epidermal Growth Factor-like (LE) Modules of Laminin β1 and γ1 Chains

In-depth mass spectrometric analysis of disulfide bond patterns in recombinant mouse laminin β1 and γ1 chain N-terminal fragments comprising the laminin N-terminal (LN) domain and the first four laminin epidermal growth factor-like (LE) domains revealed a novel disulfide pattern for LE domains. This showed a (2-3, 4-5, 6-7, 8-1) connectivity with the last cysteine of one LE domain being connected to the first cysteine of the following LE domain. The same pattern was also found in E4, the N-terminal β1 chain fragment derived by elastase digestion of mouse EHS tumor laminin-111, showing that this pattern occurs in native laminin. The strictly linear pattern with an interdomain disulfide has not been described previously for EGF domains. The N-terminal portions of laminin short arms, consisting of the LN domain and LE domains 1-4, are essential for laminin-laminin self-interactions, whereas the internal LE domains 7-9 in the laminin γ1 chain harbor the nidogen binding site and have a conventional disulfide pattern. This suggests that LE domains differing in function also differ in their disulfide patterns.

Temporal expression pattern of adhesion genes in human oral mucosal keratinocytes on type IV collagen-coated titanium

The cell adhesion extracellular matrix (ECM), and the cell adhesive system mediate various adhesion molecules. Cell adhesion regulates proliferation and differentiation. However, the cell adhesive systems between titanium and epithelial cells are still not clearly understood. In this study, we cultured human epithelial cells on type IV collagen-coated titanium (TiCol-4) and non-coated titanium (Ti). We used titanium disks of 30 mm diameter and 1.0 mm thickness. The titanium disks were first abraded using #800, #1200, #2400, and #4000 diamond pads and 0.3 mum alumina, and then washed with acetone, ethanol, and ultra-pure water in an ultrasonic cleaner for 10 min each. Human oral keratinocytes (hOMK) were seeded with 5.0 x 10(4) cells on TiCol-4 and Ti and placed in 6-well culture dishes. The cell adhesion examination was conducted with a Cell Coulter Counter after 1, 3, and 5 h. hOMK were observed using scanning electron microscopy (SEM). Real-time PCR was performed with four primers: gene laminin beta3, integrin beta4, integrin alpha1, and integrin alpha3. The results suggest that TiCol-4 could be used as a means for obtaining better hOMK than Ti. Type IV collagen could provide an excellent substratum for hOMK attachment on titanium surfaces.

Distribution of laminin β2, collagen type IV, fibronectin and MMP-9 in ovaries of the teleost fish

Extracellular matrix in the ovarian follicle has been characterised for several mammalian species but there are no reports that describe the immunolocalisation of the extracellular matrix elements, matrix metalloproteinases, and its relation to plasma 17β estradiol levels and follicular apoptosis during the teleost's reproductive cycle. The present study used immunohistochemistry to characterise the distribution of laminin β2, collagen type IV, fibronectin and matrix metalloproteinases-9 (MMP-9). The TUNEL in situ technique was used to quantify apoptosis and indirect immunofluorimetric to determine plasma 17β estradiol levels. The TUNEL-positive reaction associated with morphological features exhibited follicular apoptosis. During postovulatory follicle involution, the drop in plasma 17β estradiol levels after spawning contributed to the intense apoptosis observed. By immunohistochemical analysis, laminin β2 and collagen type IV were identified as the major constituents of the basement membrane. The loss of integrity of the basement membrane occurred due to lyses of the major constituents, and coincides with increased follicular apoptosis. The integrity of the basement membrane is important for the survival of follicular cells. Furthermore, the MMP-9 results suggest that this enzyme is involved in final oocyte maturation and regression of postovulatory follicles. Fibronectin was observed on the surface of follicular cells of the postovulatory follicle in P. argenteus, this being important for maintaining normal cell adhesion to extracellular matrix. In conclusion, our results suggest that the structure and composition of the extracellular matrix, and plasma 17β estradiol levels related to apoptosis, play an important role during the follicular development and post-spawning involution in teleost fishes.

Acute hyperglycemia rapidly stimulates VEGF mRNA translation in the kidney. Role of angiotensin type 2 receptor (AT2)

Angiotensin II (Ang II) and vascular endothelial growth factor (VEGF) are important mediators of kidney injury in diabetes. Acute hyperglycemia increased synthesis of intrarenal Ang I and Ang II and resulted in activation of both Ang II receptors, AT1 and AT2, in the kidney. Losartan (specific AT1 antagonist) or PD123319 (specific AT2 antagonist) did not affect hyperglycemia but prevented activation of renal AT1 and AT2, respectively. In murine renal cortex, acute hyperglycemia increased VEGF protein but not mRNA content after 24 h, which suggested translational regulation. Blockade of AT2, but not AT1, prevented increase in VEGF synthesis by inhibiting translation of VEGF mRNA in renal cortex. Acute hyperglycemia increased VEGF expression in wild type but not in AT2 knockout mice. Binding of heterogeneous nuclear ribonucleoprotein K to VEGF mRNA, which stimulates its translation, was prevented by blockade of AT2, but not AT1. The Akt-mTOR-p70 S6K signaling pathway, involved in the activation of mRNA translation, was activated in hyperglycemic kidneys and was blocked by the AT2 antagonist. Elongation phase is an important step of mRNA translation that is controlled by elongation factor 1A (eEF1A) and 2 (eEF2). Expression of eEF1A and activity of eEF2 was higher in kidney cortex from hyperglycemic mice and only the AT2 antagonist prevented these changes. To assess selectivity of translational control of VEGF expression, we measured expression of fibronectin (FN) and laminin β1 (lamβ1): acute hyperglycemia increased FN expression at both protein and mRNA levels, indicating transcriptional control, and did not affect the expression of lamβ1. To confirm results obtained with PD123319, we induced hyperglycemia in AT2 knockout mice and found that in the absence of AT2, translational control of VEGF expression by hyperglycemia was abolished. Our data show that acute hyperglycemia stimulates Ang II synthesis in murine kidney cortex, this leads to AT2 activation and stimulation of VEGF mRNA translation, via the Akt-mTOR-p70 S6K signaling pathway. Our data show that exclusive translational control of protein expression in the kidney by acute hyperglycemia is not a general phenomenon, but do not prove that it is restricted to VEGF.

Expanding the clinical spectrum of congenital nephrotic syndrome caused by NPHS1 mutations

Nephrotic syndrome (NS) is a life-threatening condition when starting within the 3 months after birth [1–3]. Descriptively, NS is classified as congenital (CNS) when manifesting in utero or during the first three months of life, infantile (INS) with onset later during the first year (4–12 months) and childhood when it develops thereafter [1–3]. CNS may be also described as syndromic or isolated, depending on the presence or not of extrarenal malformations including dysmorphic facial features (e.g. microcephaly) [1–3]. The identification over the last decade of disease-causing mutations in various podocyte-expressed proteins has significantly improved early diagnosis, clinical management and genetic counselling of hereditary CNS [1–3]. Mutations in the gene NPHS1 encoding nephrin were found as the most common cause of CNS [4]. Originally known as the Finnish type of CNS (CNF) [5], this autosomal recessive condition is nowadays preferentially called nephrotic syndrome type 1 (NPHS1) is CNS, due to about 100 distinct mutations found in Finnish as well as non-Finnish cohorts [1–3]. The second most frequent gene causing CNS is the podocin (NPHS2) gene [6], first implicated in sporadic cases of childhood steroid-resistant NS with focal glomerulosclerosis [7]. In a recent large cohort of patients with an isolated NS occurring within the first year of life, two-thirds of the cases were due to mutations in either the NPHS1 or the NPHS2 genes [4]. In the remaining cases, the responsible gene was found to be the transcription factor WT1 (WT1) gene, the laminin-beta 2 (LAMB2) gene or another, non-identified gene [4]. Interestingly, mutations in WT1 and LAMB2 genes have been both associated not only to isolated CNS [4,8] but also to more complex entities including developmental disorders [9] and distinct ocular anomalies (Pierson syndrome) [10]. Lastly, mutations in two additional genes were identified in some cases of nonsyndromic CNS: PLCE1 gene involved in the RAS/MAPK signalling pathway and COQ2 gene involved in the coenzyme Q10 biosynthetic [11,12]. This paper is an Editorial Comment that reports on a cohort of 67 patients (from 62 different families belonging to 21 different ethnicities) with CNS [13]. In 36 families, the two disease-causing changes were identified in the NPHS1 gene [13], confirming a former study from the same group showing that one-half of CNS among a large multi-ethnic cohort of patients is caused by recessive mutations in this gene [4]. In nine other affected children, a single mutation in the NPHS1 gene was identified [13]. Interestingly, despite the wide variety of ethnic background, 26 families showed homozygous disease-causing mutation (26 of 36, 72%), mostly explained by a high degree of consanguinity as observed in previous studies on NPHS1 mutational spectrum [14,15]. Overall 37 different NPHS1 mutations, 19 of which are novel, were identified in 44 unrelated patients [13]. Most novel mutations found in this cohort were missense (11 of 19, 58%) but also included some splice-site [4] and nonsense mutations [3] and one small deletion. It is worthy to note that two novel homozygous recessive NPHS1 mutations (p.L587R and p.S910P) were identified in two siblings and two unrelated children [13]. All four patients had an unusually late onset of nephrotic syndrome ranging from the age of 90 days to the age of 2 years [13]. Nephrin is a 1241-residue transmembrane adhesion protein consisting of eight extracellular Ig-like domains, one fibronectin type III motif and a cytosolic C-terminal tail [16]. Nephrin directly participates in the structural basis of the slit diaphragm by its ability to homoor heterodimerize [17]. Available in vitro studies have showed that most NPHS1 missense mutations lead to abnormal retention of nephrin in the endoplasmic reticulum, and therefore it fails to traffic out to the cell surface [18]. These functional data suggested then that the majority of NPHS1 mutations (i.e truncated and missense) result in a loss of function of nephrin which might then explain severe and early-onset phenotype. Strikingly, mutations in the NPHS1 gene were recent-

Amino Acid Sequence Requirements of Laminin β1 Chain Peptide B133 (DISTKYFQMSLE) for Amyloid-like Fibril Formation, Syndecan Binding, and Neurite Outgrowth Promotion

Peptide B133 (DSITKYFQMSLE), derived from mouse laminin beta1 chain (residues 1298-1309), promotes cell attachment, neurite outgrowth, and amyloid-like fibril formation. Previously, we showed that the N-terminal Asp-deleted peptide B133a (SITKYFQMSLE) promotes integrin alpha2beta1-mediated cell attachment and spreading but does not form amyloid-like fibrils, and that the C-terminal Glu-deleted peptide B133g (DSITKYFQMSL) attaches cells without cell spreading and forms amyloid-like fibrils. In this study, we further investigated the amino acid sequence requirements of B133 for biological function using a set of truncated and Ala-substituted peptides. Attachment of cells to B133g was inhibited by only heparin, and Congo Red analysis indicated that the amyloid-like fibril formation activity of B133g was stronger than that of B133. Alanine scan analysis for the B133g peptide indicated that Asp and Ile residues are essential for cell attachment. Additionally, the N-terminal Asp residue was required for neurite outgrowth. Further, amyloid-like fibril formation required Asp and Ile residues. These data suggest that the amyloid-like fibril formation of B133g is required for cell attachment activity. We also evaluated the attachment of cells to the peptides using syndecan- and glypican-overexpressing cells. B133g attached to syndecan-overexpressing cells but not to glypican-overexpressing cells, suggesting that the amyloidogenic peptides promote syndecan-mediated cell attachment. These findings were useful for clarifying the mechanism of amyloid-like fibril formation and biological functions. The B133 peptide promotes amyloid-like fibril formation, syndecan-mediated cell attachment, and neurite outgrowth and has the potential for use as a biomaterial for tissue engineering.

Mutations in the human laminin beta2 (LAMB2) gene and the associated phenotypic spectrum.

Mutations of LAMB2 typically cause autosomal recessive Pierson syndrome, a disorder characterized by congenital nephrotic syndrome, ocular and neurologic abnormalities, but may occasionally be associated with milder or oligosymptomatic disease variants. LAMB2 encodes the basement membrane protein laminin beta2, which is incorporated in specific heterotrimeric laminin isoforms and has an expression pattern corresponding to the pattern of organ manifestations in Pierson syndrome. Herein we review all previously reported and several novel LAMB2 mutations in relation to the associated phenotype in patients from 39 unrelated families. The majority of disease-causing LAMB2 mutations are truncating, consistent with the hypothesis that loss of laminin beta2 function is the molecular basis of Pierson syndrome. Although truncating mutations are distributed across the entire gene, missense mutations are clearly clustered in the N-terminal LN domain, which is important for intermolecular interactions. There is an association of missense mutations and small in frame deletions with a higher mean age at onset of renal disease and with absence of neurologic abnormalities, thus suggesting that at least some of these may represent hypomorphic alleles. Nevertheless, genotype alone does not appear to explain the full range of clinical variability, and therefore hitherto unidentified modifiers are likely to exist.

Laminin-121—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.

B133 (DSITKYFQMSLE), a laminin β1-derived peptide, contains distinct core sequences for both integrin α2β1-mediated cell adhesion and amyloid-like fibril formation

The B133 peptide (DSITKYFQMSLE, mouse laminin β1 chain 1319–1330) promotes cell attachment, and forms amyloid-like fibrils. Here, we evaluated the active core sequences using B133 deletion peptides. B133a, lacking the N-terminal Asp residue, promoted cell spreading via integrin α2β1, whereas B133g, lacking the C-terminal Glu residue, lost the activity. Congo red analysis using the truncated peptides determined that B133g forms amyloid-like fibrils but B133a did not. These results suggest that the N- and C-terminal amino acids contribute to integrin α2β1 binding and to fibril formation, respectively. Further analyses using the truncated peptides showed that the C-terminal eight residues (B133d: KYFQMSLE) are a minimum active sequence for integrin α2β1-mediated cell attachment and the N-terminal nine residues (B133i: DSITKYFQM) are critical for amyloid-like fibril formation. These results suggest that peptide B133 is multifunctional with two different active core sequences: integrin α2β1-mediated cell attachment and amyloid-like fibril formation. Moreover, alanine substitution analysis of B133a indicated that six amino acids, Ile, Thr, Tyr, Phe, Met, and Glu, are important for cell attachment activity. When the Ser residue at the 9th position of B133a was replaced with Ala, the cell attachment activity was enhanced. Further mutation analysis at the 9th position of B133a using various amino acids suggests that hydrophobic amino acids are effective for the integrin α2β1-mediated cell attachment activity. These findings define multifunctional and overlapping sites on the B133 peptide and are useful for designing multifunctional synthetic molecules.

Molecular interactions in the retinal basement membrane system: A proteomic approach

Basement membranes (BMs) are physiologically insoluble extracellular matrix sheets present in all multicellular organisms. They play an important role in providing mechanical strength to tissues and regulating cell behavior. Proteomic analysis of BM proteins is challenged by their high molecular weights and extensive post-translational modifications. Here, we describe the direct analysis of an in vivo BM system using a mass spectrometry (MS) based proteomics approach. Retinal BMs were isolated from embryonic chick eyes. The BM macromolecules were deglycosylated and separated by low percentage gradient SDS PAGE, in-gel digested and analyzed by LC-MS/MS. This identified over 27 extracellular matrix proteins in the retinal BM. A semi-quantitative measure of protein abundance distinguished, nidogens-1 and -2, laminin subunits α1, α5, β2, and γ1, agrin, collagen XVIII, perlecan, FRAS1 and FREM2 as the most abundant BM protein components. Laminin subunits α3, β1, γ2, γ3 and collagen IV subunits α5 and α6 were minor constituents. To examine binding interactions that contribute to the stability of the retinal BM, we applied the LC-MS/MS based approach to detect potential BM complexes from the vitreous. Affinity-captured nidogen- and heparin-binding proteins from the vitreous contained >10 and >200 proteins respectively. Comparison of these protein lists with the retinal BM proteome reveals that glycosaminoglycan and nidogen binding interactions play a central role in the internal structure and formation of the retinal BM. In addition, we studied the biomechanical qualities of the retinal BM before and after deglycosylation using atomic force microscopy. These results show that the glycosaminoglycan side chains of the proteoglycans play a dominant role in regulating the thickness and elasticity of the BMs by binding water to the extracellular matrix. To our knowledge, this is the first large-scale investigation of an in vivo BM system using MS-based proteomics.

Tissue distribution of the laminin β1 and β2 chain during embryonic and fetal human development

Laminins are the major glycoproteins present in all basement membranes. Previously, we showed that perlecan is present during human development. Although an overview of mRNA-expression of the laminin β1 and β2 chains in various developing fetal organs is already available, a systematic localization of the laminin β1 and β2 chains on the protein level during embryonic and fetal human development is missing. Therefore, we studied the immunohistochemical expression and tissue distribution of the laminin β1 and β2 chains in various developing embryonic and fetal human organs between gestational weeks 8 and 12. The laminin β1 chain was ubiquitously expressed in the basement membrane zones of the brain, ganglia, blood vessels, liver, kidney, skin, pancreas, intestine, heart and skeletal system. Furthermore, the laminin β2 chain was present in the basement membrane zones of the brain, ganglia, skin, heart and skeletal system. The findings of this study support and expand upon the theory that these two laminin chains are important during human development.

Integrin alpha 7 interacts with HtrA2 to induce prostate cancer cell death

Integrins are a family of receptors for extracellular matrix proteins that have critical roles in human tissue development. Previous studies identified down‐regulation and/or mutations of integrin alpha 7 (ITGA7) in prostate cancer (PC), liver cancer, soft tissue leiomyosarcoma (STL) and glioblastoma multiforme. Here we report that expression of ITGA7 induced apoptosis in the human prostate cancer cell lines PC3 and DU145. Yeast two‐hybrid analysis revealed that the C‐terminus of ITGA7 interacts with HtrA2, a serine protease with a critical role in apoptosis. Expression of ITGA7 increases the protease activity of HtrA2 both in vitro and in vivo. Deletion of the HtrA2 interaction domain abrogates the cell death activity of ITGA7, while down regulation of HtrA2 dramatically reduced cell death mediated by ITGA7. In addition, site‐directed protease‐null mutant HtrA2S306A expression blocked apoptosis induced by ITGA7. Interestingly, interaction between ITGA7 and its ligand laminin 2 appears protecting against cell death, since depleting laminin beta 2 with a small interfering RNA (siRNA) significantly exacerbated apoptosis induced by ITGA7 expression. This report provides a novel insight into the mechanism by which ITGA7 acts as a tumor suppressor.

Differential expression of focimatrix and steroidogenic enzymes before size deviation during waves of follicular development in bovine ovarian follicles

During the growth of bovine follicles, one emerges from a wave as the largest and dominant follicle. What regulates dominance is not known but candidates include oestradiol, transforming growth factor beta β1 (TGFB1), and recently CYP11AI (cholesterol side-chain cleavage) and focal intra-epithelial matrix (focimatrix). To examine this, pairs of bovine ovaries with 2 or more follicles of equal size (>5 mm) and hence in a wave before deviation, were collected at an abattoir (6.7 ± SEM 0.1 mm diameter; n = 14 animals, 35 follicles in total). These follicles were dissected and follicular fluid collected to measure progesterone and oestradiol concentrations. A portion of the follicle wall was processed for histological classification of health or atresia and granulosa cells were harvested for quantitative RT-PCR of focimatrix components [ COL4A1 (collagen type IV α1), LAMB2 (laminin β2) and HSPG2 (perlecan)], steroidogenic enzymes [ CYP11A1 and CYP19A1] and TGFB1. For statistical analyses follicles within each animal were grouped into either the highest (oestradiol, CYP11A1) or lowest ( TGFB1) expression ( n = 14) for comparison with the remaining follicles ( n = 21). When grouped on oestradiol no other parameters differed significantly, and when grouped on TGFB1 some parameters were different however the levels were also lower, and not higher as expected. When grouped on CYP11A1 other parameters were significantly elevated in the high CYP11A1 group ( COL4A1 P < 0.05; LAMB2 P < 0.01; HSPG2 P < 0.01 and CYP19A1 P < 0.001). This suggests that steroidogenesis and focimatrix might be important in a follicle attaining dominance.