Laminin Γ1 Research Articles

Basement Membrane Deposition of Nidogen 1 but Not Nidogen 2 Requires the Nidogen Binding Module of the Laminin γ1 Chain

The nidogen-laminin interaction is proposed to play a key role in basement membrane (BM) assembly. However, though there are similarities, the phenotypes in mice lacking nidogen 1 and 2 (nidogen double null) differ to those of mice lacking the nidogen binding module (γ1III4) of the laminin γ1 chain. This indicates different cell- and tissue-specific functions for nidogens and their interaction with laminin and poses the question of whether the phenotypes in nidogen double null mice are caused by the loss of the laminin-nidogen interaction or rather by other unknown nidogen functions. To investigate this, we analyzed BMs, in particular those in the skin of mice lacking the nidogen binding module. In contrast to nidogen double null mice, all skin BMs in γ1III4-deficient mice appeared normal. Furthermore, although nidogen 1 deposition was strongly reduced, nidogen 2 appeared unchanged. Mice with additional deletion of the laminin γ3 chain, which contains a γ1-like nidogen binding module, showed a further reduction of nidogen 1 in the dermoepidermal BM; however, this again did not affect nidogen 2. This demonstrates that in vivo only nidogen 1 deposition is critically dependent on the nidogen binding modules of the laminin γ1 and γ3 chains, whereas nidogen 2 is independently recruited either by binding to an alternative site on laminin or to other BM proteins.

MiR-124a is frequently down-regulated in glioblastoma and is involved in migration and invasion

Glioblastoma (GBM) represents a formidable clinical challenge for both patients and treating physicians. Due to better local treatments and prolonged patient survival, remote recurrences are increasingly observed, underpinning the importance of targeting tumour migration and attachment. Aberrant expression of microRNA (miRNA) is commonly associated with cancer and loss of miR-124a has previously been implicated to function as a tumour suppressor. The assessment of miR-124a in clinical specimens has been limited and a potential role in migration and invasion has been unexplored until now. We measured the expression levels of mature miR-124a in a retrospective series of 119 cases of histologically confirmed GBM and found its expression was markedly lower in over 80% of the GBM clinical specimens compared to normal brain tissue. The level of reduction in the clinical cohort varied significantly and patients with lower than the average miR-124a expression levels displayed shorter survival times. Endogenous miR-124a expression and the protein expression of three of its targets; IQ motif containing GTPase activating protein 1 (IQGAP1), laminin γ1 (LAMC1) and integrin β1 (ITGB1) were significantly reciprocally associated in the majority of the clinical cases. We confirmed this association in our in vitro model. Functionally, the ectopic expression of mature miR-124a in a GBM cell line resulted in significant inhibition of migration and invasion, demonstrating a role for miR-124a in promoting tumour invasiveness. Our results suggest that miR-124a may play a role in GBM migration, and that targeted delivery of miR-124a may be a novel inhibitor of GBM invasion.

Immunohistochemical evaluation of type IV collagen and laminin-332 γ2 chain expression in well-differentiated oral squamous cell carcinoma and oral verrucous carcinoma: a new recommended cut-off

Invasion and metastasis are two characteristics of malignant tumors, which perform by proteolytic destruction of the components of basement membrane (BM) and cell migration. The aim of this study was to evaluate the immunohistochemical (IHC) assessment of type IV collagen and laminin-332 γ2 (Ln-332 γ2) chain expression in well-differentiated oral squamous cell carcinoma (OSCC) and oral verrucous carcinoma (OVC), because these two lesions have same histopathologic findings whereas they have different biological behaviors. Destruction of BM and cell migration were evaluated by IHC in 15 cases of epithelial hyperplasia with no dysplasia (A group), 15 cases of OVC (B group) and 15 cases of well-differentiated OSCC (C group). There was a significant difference in type IV collagen immunohistochemical staining between three groups, but there were no significant differences between B and C groups. Expression of Ln-332 γ2 chain was not detected in A group. Ln-332 γ2 chain labeling index had significantly difference between B and C groups. The number of Ln-332 γ2 chain immunostaining positive cells was less than 5% in B group and over than 5% in C group which there were significantly differences between these two groups. Isolated immunohistochemical study of type IV collagen does not clearly define that a lesion is invasive or non-invasive and evaluation of Ln-332 γ2 chain expression (cut-off 5%) may be useful as a marker for description of biological differences and diagnosis of OVC from well-differentiated OSCC, especially in doubtful cases.

Intraepidermal erbium:YAG laser resurfacing: Impact on the dermal matrix

Various minimally invasive treatments enhance the skin's appearance. Little is known about the molecular mechanisms whereby treatments working at the epidermal level might alter the dermis. We sought to quantify the molecular changes that result from erbium:yttrium-aluminium-garnet (Er:YAG) laser microablative resurfacing. We performed biochemical analyses after intraepidermal Er:YAG laser resurfacing of 10 patients. Immunohistochemical analysis and polymerase chain reaction technology were utilized to measure key biomarkers. The basement membrane remained intact after intraepidermal microablation, as demonstrated by laminin γ2 immunostaining. Epidermal injury was demonstrated with acute up-regulation of keratin 16. An inflammatory response ensued as indicated by increases in cytokines interleukin 1 beta (IL-1β) and IL-8 as well as a substantial neutrophil infiltrate. Levels of cJun and JunB proteins, components of the transcription factor AP-1 complex, were also elevated. Up-regulation of extracellular matrix degrading proteinases matrix metalloproteinase 1 (MMP-1), MMP-3, and MMP-9 was noted. Atransient increase in keratinocyte proliferation, as indicated by staining for Ki67, was observed. Increased expression of type I and type III procollagen was demonstrated. The data presented are those that resulted from a single treatment session. Although microablation was confined to the uppermost epidermis, marked changes in epidermal and dermal structure and function were demonstrated after Er:YAG laser microablative resurfacing. We demonstrated substantial dermal matrix remodeling, including a degree of collagen production that compares favorably with some more invasive interventions. Dermal remodeling and stimulation of collagen production are associated with wrinkle reduction. Thus these results suggest that the skin's appearance may be enhanced by creating dermal changes through the use of superficially acting treatments.

Laminins, via heparan sulfate proteoglycans, participate in zebrafish myotome morphogenesis by modulating the pattern of Bmp responsiveness

In zebrafish, Hedgehog-induced Engrailed expression defines a muscle fibre population that includes both slow and fast fibre types and exhibits an organisational role on myotome and surrounding tissues, such as motoneurons and lateral line. This Engrailed-positive population is restricted in the myotome to a central domain. To understand how this population is established, we have analysed the phenotype of the sly/lamc1 mutation in the Laminin γ1 chain that was shown to specifically affect Engrailed expression in pioneers. We find that the sly mutation affects Engrailed expression in the entire central domain and that Hedgehog signalling does not mediate this effect. We show that Bmp-responding cells are excluded from the central domain and that this pattern is modulated by laminins, but not by Hedgehog signalling. Knockdown of Bmp signalling rescues Engrailed expression in the sly mutant and ectopically activates Engrailed expression in slow and fast lineages in wild-type embryos. Last, extracellular matrix-associated heparan sulfate proteoglycans are absent in sly and their enzymatic removal mimics the sly phenotype. Our results therefore show that laminins, via heparan sulfate proteoglycans, are instrumental in patterning Bmp responsiveness and that Bmp signalling restricts Engrailed expression to the central domain. This study underlines the importance of extracellular cues for the precise spatial modulation of cell response to morphogens.

Development of a simple enzyme-linked immunosorbent assay for the detection of autoantibodies in anti-p200 pemphigoid

Anti-p200 pemphigoid is a subepidermal blistering skin disease characterized by autoantibodies against a 200-kDa protein (p200) of the dermal-epidermal junction. The laminin γ1 chain has recently been identified as target antigen in this disease and the C-terminus was described as an immunodominant region of laminin γ1. Diagnosis of anti-p200 pemphigoid requires detection of serum IgG at the dermal side of 1 mol L(-1) salt-split skin by indirect immunofluorescence microscopy and labelling of a 200-kDa protein by Western blotting of dermal extract. However, preparation of dermal extract is not widely available, limiting the possibility of diagnosing this disease to a few laboratories. To develop a simple, sensitive and specific diagnostic tool for anti-p200 pemphigoid. Sera from patients with anti-p200 pemphigoid (n = 35), bullous pemphigoid (BP, n = 101), epidermolysis bullosa acquisita (EBA, n = 10), antilaminin 332 mucous membrane pemphigoid (MMP, n = 14), pemphigus vulgaris (PV, n = 51) and healthy volunteers (HV, n = 131) were tested by a novel enzyme-linked immunosorbent assay (ELISA) that employed a recombinant monomeric C-terminal fragment of human laminin γ1 (hLAMC1-cterm) expressed in Escherichia coli. Serum reactivity with hLAMC1-cterm was detected in sera from 24 of 35 (69%) patients with anti-p200 pemphigoid, two of 101 (2%) with BP, 0 of 10 with EBA, two of 14 (14%) with anti-laminin 332 MMP, 0 of 51 with PV, and 0 of 131 HV. This novel ELISA will facilitate the diagnosis of anti-p200 pemphigoid.

Mesenchymal stem cells facilitate axon sorting, myelination, and functional recovery in paralyzed mice deficient in Schwann cell‐derived laminin

Peripheral nerve function depends on a regulated process of axon and Schwann cell development. Schwann cells interact with peripheral neurons to sort and ensheath individual axons. Ablation of laminin γ1 in the peripheral nervous system (PNS) arrests Schwann cell development prior to radial sorting of axons. Peripheral nerves of laminin-deficient animals are disorganized and hypomyelinated. In this study, sciatic nerves of laminin-deficient mice were treated with syngenic murine adipose-derived stem cells (ADSCs). ADSCs expressed laminin in vitro and in vivo following transplant into mutant sciatic nerves. ADSC-treatment of mutant nerves caused endogenous Schwann cells to differentiate past the point of developmental arrest to sort and myelinate axons. This was shown by (1) functional, (2) ultrastructural, and (3) immunohistochemical analysis. Treatment of laminin-deficient nerves with either soluble laminin or the immortalized laminin-expressing cell line 3T3/L1 did not overcome endogenous Schwann cell developmental arrest. In summary, these results indicate that (1) laminin-deficient Schwann cells can be rescued, (2) a cell-based approach is beneficial in comparison with soluble protein treatment, and (3) mesenchymal stem cells modify sciatic nerve function via trophic effects rather than transdifferentiation in this system.

Laminins, via heparan sulfate proteoglycans, participate in zebrafish myotome morphogenesis by modulating the pattern of Bmp responsiveness

In zebrafish, Hedgehog-induced Engrailed expression defines a muscle fibre population that includes both slow and fast fibre types and exhibits an organisational role on myotome and surrounding tissues, such as motoneurons and lateral line. This Engrailed-positive population is restricted in the myotome to a central domain. To understand how this population is established, we have analysed the phenotype of the sly/lamc1 mutation in the Laminin γ1 chain that was shown to specifically affect Engrailed expression in pioneers. We find that the sly mutation affects Engrailed expression in the entire central domain and that Hedgehog signalling does not mediate this effect. We show that Bmp-responding cells are excluded from the central domain and that this pattern is modulated by laminins, but not by Hedgehog signalling. Knockdown of Bmp signalling rescues Engrailed expression in the sly mutant and ectopically activates Engrailed expression in slow and fast lineages in wild-type embryos. Last, extracellular matrix-associated heparan sulfate proteoglycans are absent in sly and their enzymatic removal mimics the sly phenotype. Our results therefore show that laminins, via heparan sulfate proteoglycans, are instrumental in patterning Bmp responsiveness and that Bmp signalling restricts Engrailed expression to the central domain. This study underlines the importance of extracellular cues for the precise spatial modulation of cell response to morphogens.

Matrix metalloproteinase-2 and matrix metalloproteinase-9 codistribute with transcription factors RUNX1/AML1 and ETV5/ERM at the invasive front of endometrial and ovarian carcinoma

Several matrix metalloproteinases (MMPs) are implicated in the degradation of the epithelial basement membrane (BM), invasiveness, and malignancy of endometrial and ovarian carcinomas. We have recently proposed a cooperative role for RUNX1/AML1 and ETV5/ERM in myometrial infiltration during endometrioid endometrial invasiveness. In the present work, we have characterized the occurrence, levels of expression, and codistribution of gelatinases MMP-2 and -9, and the transcription factors RUNX1/AML1 and ETV5/ERM, together with collagen type IV and laminin chains of the epithelial BM in endometrioid endometrial (EEC) and ovarian endometrioid carcinoma (OEC). MMP-2 and -9 expression levels were up-regulated at the invasive front of both carcinomas, and they showed a relatively high degree of volume codistribution with RUNX1/AML1 and ETV5/ERM. EEC tissue microarrays showed similar significant expression and correlation for MMPs and the transcription factors. When the array samples were grouped according to the carcinoma stages, there was significant correlation in the expression levels for both MMP-2 and -9 with ETV5/ERM. Colocalization of MMP-2 and -9 with epithelial basement membrane component collagen type IV showed close spatial association for both MMPs and discontinuation of collagen type IV expression at the invasive front in both EEC and OEC. BM components laminin α1, α2, α3, α5, and γ2 chains, laminin α5 receptor basal cell adhesion molecule (BCAM), and laminin 332 were all detected both in EEC and OEC. Highest expression levels in EEC were for laminin α3 and in OEC for laminin α5 chain. Laminin γ2 chain and laminin 332 showed discontinuous immunoreactivity in the epithelial basement membrane suggestive of proteolytic degradation. These results indicate concurrent mechanisms in expression of MMP-2 and -9, RUNX1/AML1 and ETV5/ERM, and several of the basement membrane components, which are likely to associate with the invasive stage of EEC and OEC.

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.

EGF/TGFβ1 co-stimulation of oral squamous cell carcinoma cells causes an epithelial-mesenchymal transition cell phenotype expressing laminin 332

Epithelial-mesenchymal transition (EMT) is suggested to be crucial for the development of an invasive and metastatic carcinoma cell phenotype. Therefore, the definition of this phenotype is of great clinical interest. We recently evidenced vimentin positive cells in oral squamous cell carcinoma (OSCC) invasive front expressing laminin γ2 chain mRNA implicating an EMT origin of these cells. To further elucidate the nature of these cells, we have investigated the relation between EMT criteria and laminin-332 expression in a cell culture model of transforming growth factor beta-1 (TGFβ1)/epithelial growth factor (EGF) long time co-stimulation. We demonstrate that in contrast to TGFβ1 or EGF alone, co-stimulation induces phenotype transition in OSCC cells which fulfils the criteria of EMT in terms of vimentin up-regulation and E-cadherin down-regulation on protein level as well as cell scattering. Furthermore, cells displayed a strongly enhanced invasiveness and adhesion to type I-IV collagens. Phenotype transition is accompanied by an enhanced expression of laminin-332, especially of its γ2 chain. We further analyse the expression of extracellular matrix related genes by RT-PCR profiling. With respect to strongly enhanced proteins, data confirm the EMT phenotype of co-stimulated OSCC cells and expression of laminin-332. Furthermore, alpha catenin, collagen type 16, the integrin α7 and β1 chains, and MMP11 are suggested as candidates with potential role in EMT in OSCC. In summary we are able to show that EMT in OSCC is mediated by multiple growth factors and is accompanied by laminin γ2 chain up-regulation evidencing the existence of an intermediate Vim(+) /Ln332(+) EMT phenotype as seen in situ.

Expression of laminin γ2 chain monomer enhances invasive growth of human carcinoma cells in vivo

Laminin gamma2 chain is a subunit of the heterotrimeric basement membrane protein laminin-332 (alpha3beta3gamma2). The gamma2 chain is highly expressed by human cancers at the invasion fronts and this expression correlates with poor prognosis of the cancers. Our previous study showed that the gamma2 chain is expressed as a monomer form in invading carcinoma cells. However, the role of the gamma2 protein in tumor invasion remains unknown. Here, we demonstrate that the monomeric gamma2 chain promotes invasive growth of human cancer cells in vivo. First, we analyzed regulatory factors for the gamma2 chain expression using 2 gastric carcinoma cell lines. It was found that tumor necrosis factor-alpha, by itself or in a combination with transforming growth factor-beta1, strongly induced the secretion of the monomeric gamma2 chain. In addition, epidermal growth factor families appeared to function as the gamma2 chain inducers in human cancers. Next, we established T-24 bladder carcinoma cell lines expressing the full-length or the short arm of the laminin gamma2 chain. When these cell lines were i.p. injected into nude mice, they produced larger tumors in the abdominal cavity and showed much stronger invasive growth onto the diaphragms than the control cell line. The gamma2-expressing T-24 cells often produced ascites fluid, but scarcely the control cells. In culture, the gamma2-expressing cells migrated through Matrigel more efficiently than the control cells. These findings imply that the gamma2 monomer is induced in human cancers by inflammatory and stromal cytokines and promotes their invasive growth in vivo.

Pemphigoid without mucosal involvement showing autoantibodies against laminin-332 γ2 subunit

British Journal of DermatologyVolume 163, Issue 5 p. 1120-1122 Pemphigoid without mucosal involvement showing autoantibodies against laminin-332 γ2 subunit Y. Endo, Y. Endo Department of Dermatology, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, JapanSearch for more papers by this authorM. Kato, M. Kato Department of Dermatology, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, JapanSearch for more papers by this authorA. Kitoh, A. Kitoh Department of Dermatology, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, JapanSearch for more papers by this authorS. Kore-eda, S. Kore-eda Department of Dermatology, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, JapanSearch for more papers by this authorS. Fukuda, S. Fukuda Department of Dermatology, School of Medicine, Kurume University, Fukuoka, JapanSearch for more papers by this authorT. Hashimoto, T. Hashimoto Department of Dermatology, School of Medicine, Kurume University, Fukuoka, JapanSearch for more papers by this authorK. Toda, K. Toda Department of Dermatology, Kitano Hospital, Osaka, JapanSearch for more papers by this authorY. Miyachi, Y. Miyachi Department of Dermatology, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, JapanSearch for more papers by this authorA. Utani, A. Utani Department of Dermatology, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan Department of Dermatology, Graduate School of Medicine, Nagasaki University, Nagasaki, JapanE-mail: yendou-tky@umin.ac.jpSearch for more papers by this author Y. Endo, Y. Endo Department of Dermatology, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, JapanSearch for more papers by this authorM. Kato, M. Kato Department of Dermatology, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, JapanSearch for more papers by this authorA. Kitoh, A. Kitoh Department of Dermatology, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, JapanSearch for more papers by this authorS. Kore-eda, S. Kore-eda Department of Dermatology, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, JapanSearch for more papers by this authorS. Fukuda, S. Fukuda Department of Dermatology, School of Medicine, Kurume University, Fukuoka, JapanSearch for more papers by this authorT. Hashimoto, T. Hashimoto Department of Dermatology, School of Medicine, Kurume University, Fukuoka, JapanSearch for more papers by this authorK. Toda, K. Toda Department of Dermatology, Kitano Hospital, Osaka, JapanSearch for more papers by this authorY. Miyachi, Y. Miyachi Department of Dermatology, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, JapanSearch for more papers by this authorA. Utani, A. Utani Department of Dermatology, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan Department of Dermatology, Graduate School of Medicine, Nagasaki University, Nagasaki, JapanE-mail: yendou-tky@umin.ac.jpSearch for more papers by this author First published: 26 October 2010 https://doi.org/10.1111/j.1365-2133.2010.09922.xCitations: 5 Conflicts of interest: none declared. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume163, Issue5November 2010Pages 1120-1122 RelatedInformation

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.

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.

Insulin-like Growth Factor-binding Protein-5-induced Laminin γ1 Transcription Requires Filamin A

Insulin-like growth factor-binding protein-5 (IGFBP-5) has IGF-1-independent intranuclear effects that are poorly defined. Treatment of cells with IGFBP-5 induces migration, prevents apoptosis, and leads to increased laminin subunit transcription. Similarly, filamin A (FLNa), an actin-binding protein that participates in cell attachment, plays important additional roles in signal transduction and modulation of transcriptional responses. In this report, we show that IGFBP-5 leads to dephosphorylation of FLNa with subsequent FLNa cleavage. Following cleavage, there is enhanced recruitment of Smad3/4 to a C-terminal FLNa fragment with nuclear translocation and subsequent binding to the promoter region of the laminin gamma1 (lamc1) gene. FLNa knockdown prevents IGFBP-5-mediated increases in lamc1 transcription. These data indicate that IGFBP-5 induces formation of a FLNa-based nuclear shuttle that recruits transcription factors and regulates transcription of IGFBP-5 target genes. These studies provide new insights into the mechanisms whereby IGFBP-5 and FLNa exert intranuclear effects.

Upregulation proliferation and senescence markers in hyperplasia induced in mouse skin by sulfur mustard exposure

Chemical vesicants such as sulfur mustard (SM) can produce severe tissue damage. A time‐course study using mouse ear skin exposed to a low dose (0.08 mg) of SM resulted in excessive keratinocyte hyperplasia about three days after exposure. The abnormal cell proliferation was reminiscent of that observed in several types of cancer and potential oncogenic hyperproliferative signal molecules were examined to determine if the two systems were comparable. Immunohistochemical, Western blotting and RT‐PCR studies were performed and markers of skin injury and repair were examined for various time‐points. The protein, p16‐INK4a, which triggers either growth arrest or apoptosis was hyperexpressed in the hyperplasia areas of injured skin. The p16‐INK4a co‐expressed with laminin γ2, a polypeptide that promotes epithelial sheet migration over a wound bed. The endoplasmic reticulum stress‐regulated gene, GADD153/CHOP had a similar expression pattern to that of p16‐INK4a. In contrast, both keratin 5, an intermediate filament protein of basal epithelial cells and Ki67, a marker of cells in the growth stage of the cell cycle had a much more restricted distribution. Understanding the basic mechanism of action of vesicant‐induced skin injury and repair should help to identify new targets of potential medical countermeasures against chemical vesicants. This work is supported by ES005022, EY09056, and AR055073.Grant Funding Source: ES005022, EY09056, and AR055073.

Expression and function of laminin and integrins on adhesion/migration of primary culture cells derived from rat oral epithelium

It remains controversial whether or not the junctional epithelium cells that are directly attached to teeth migrate on the enamel surface, as those cells are able to adhere firmly to the enamel. The aim of this study was to investigate the expression patterns of laminin gamma(2), integrin beta(4) and integrin alpha(3), and to examine their potential function in cell migration. Oral epithelium cells obtained from Sprague-Dawley rats were established in primary culture. We employed a wound-healing assay to characterize the direction of cell extension at the start of cell migration, and observed different localizations of laminin and integrins using immunofluorescence. For functional analyses of integrins, we employed a phosphatidylinositol-3-kinase (PI3K) activator to promote integrin beta(4) function and used P1B5 to inhibit integrin alpha(3) function, and we analyzed the percentage of re-epithelialization as the migration function. Marked accumulation of laminin gamma(2) was detected in the peripheral cytoplasm of cells adjacent to the wound area, as shown by the results of the migration assay. Integrin beta(4) was detected in the distal cell processes of actively migrating cells, while integrin alpha(3) was found in cell membranes of cells adjacent to the wound area. In the functional analyses, the percentage of re-epithelialization was significantly lower in the PI3K-activator group and in the P1B5-treated group (2.5% and 7.2%, respectively) than in the control group (39.0%) (p < 0.01). The results suggest that laminin gamma(2) is secreted as a foothold for cell migration, that integrin beta(4) participates in cell adhesion and that integrin alpha(3) is involved in cell migration in the primary culture cells.

Stromal laminin chain distribution in normal, hyperplastic and malignant oral mucosa: relation to myofibroblast occurrence and vessel formation

The contribution of stromal laminin chain expression to malignant potential, tumour stroma reorganization and vessel formation in oral squamous cell carcinoma (OSCC) is not fully understood. Therefore, the expression of the laminin chains alpha2, alpha3, alpha4, alpha5 and gamma2 in the stromal compartment/vascular structures in OSCC was analysed. Frozen tissue of OSCC (9x G1, 24x G2, 8x G3) and normal (2x)/hyperplastic (11x) oral mucosa was subjected to laminin chain and alpha-smooth muscle actin (ASMA) immunohistochemistry. Results were correlated to tumour grade. The relation of laminin chain positive vessels to total vessel number was assessed by immunofluorescence double labelling with CD31. Stromal laminin alpha2 chain significantly decreases and alpha3, alpha4, alpha5 and gamma2 chains and also ASMA significantly increase with rising grade. The amount of stromal alpha3, alpha4 and gamma2 chains significantly increased with rising ASMA positivity. There is a significant decrease in alpha3 chain positive vessels with neoplastic transformation. Mediated by myofibroblasts, OSCC development is associated with a stromal up-regulation of laminin isoforms possibly contributing to a migration promoting microenvironment. A vascular basement membrane reorganization concerning alpha3 and gamma2 chain laminins during tumour angioneogenesis is suggested.

Nerve invasion distance is dependent on laminin γ2 in tumors of pancreatic cancer

The distance of nerve invasion is an important prognostic factor in pancreatic cancer. The extracellular matrix (ECM) of nerve, mainly composed of laminin, collagen IV and anchoring fibrils, might affect nerve invasion. However, this relationship has not been demonstrated. Our study aimed at discovering the promoting factor of nerve invasion within the tumoral ECM. An animal model was established to evaluate the distance of nerve invasion in murine sciatic nerves by intraneural injection of 6 human pancreatic cancer cell lines. mRNA expression of laminins and anchoring fibrils was compared to the distance of nerve invasion for each cancer cell line. A target molecule provided the strong association between mRNA expression and the distance of nerve invasion. To evaluate the role of a target molecule in nerve invasion, protein expression and function were examined using an animal model and surgical cases. Cancer cells with high laminin gamma2 mRNA and protein expression in their basement membranes were associated with long nerve invasion. Knockdown of laminin gamma2 in cancer cells significantly shortened nerve invasion in the animal model. In 75 patients with pancreatic cancer, a large distance of nerve invasion was associated with high expression levels of laminin gamma2 mRNA and basement membranous deposition of laminin gamma2 protein. Our results indicate that laminin gamma2 plays an important role in nerve invasion. The measurement of the nerve invasion distance in our mouse nerve invasion model is useful for evaluating the molecular mechanisms of nerve invasion.