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Role of laminin receptor in tumor cell migration.  


Polyclonal antisera were made against biochemically purified laminin receptor protein as well as against synthetic peptides deduced from a complementary DNA clone corresponding to the COOH-terminal end of the laminin receptor (U.M. Wewer et al., Proc. Natl. Acad. Sci. USA, 83: 7137-7141, 1986). These antisera were used to study the potential role of laminin receptor in laminin-mediated attachment and haptotactic migration of human A2058 melanoma cells. The anti-laminin receptor antisera reacted with the surface of suspended, nonpermeabilized melanoma and carcinoma cells. The anti-laminin receptor antisera blocked the surface interaction of A2058 cells with endogenous laminin, resulting in the inhibition of laminin-mediated cell attachment. The A2058 melanoma cells migrated toward a gradient of solid phase laminin or fibronectin (haptotaxis). Anti-laminin antiserum abolished haptotaxis on laminin but not on fibronectin. Synthetic peptide GRGDS corresponding to the fibronectin cell-binding domain inhibited haptotaxis on fibronectin but not on laminin. Both types of anti-laminin receptor antisera inhibited haptotaxis on laminin but not on fibronectin. Using immunohistochemistry, invading human carcinoma cells in vivo exhibited a marked cytoplasmic immunoreactivity for the receptor antigen. Together these findings indicate a specific role for the laminin receptor in laminin-mediated migration and that the ligand binding of the laminin receptor is encompassed in the COOH-terminal end of the protein. PMID:2959357

Wewer, U M; Taraboletti, G; Sobel, M E; Albrechtsen, R; Liotta, L A



Presence of Laminin Receptors in Staphylococcus aureus  

NASA Astrophysics Data System (ADS)

A characteristic feature of infection by Staphylococcus aureus is bloodstream invasion and widespread metastatic abscess formation. The ability to extravasate, which entails crossing the vascular basement membrane, appears to be critical for the organism's pathogenicity. Extravasation by normal and neoplastic mammalian cells has been correlated with the presence of specific cell surface receptors for the basement membrane glycoprotein laminin. Similar laminin receptors were found in Staphylococcus aureus but not in Staphylococcus epidermidis, a noninvasive pathogen. There were about 100 binding sites per cell, with an apparent binding affinity of 2.9 nanomolar. The molecular weight of the receptor was 50,000 and pI was 4.2. Eukaryotic laminin receptors were visualized by means of the binding of S. aureus in the presence of laminin. Prokaryotic and eukaryotic invasive cells might utilize similar, if not identical, mechanisms for invasion.

Lopes, J. D.; Dos Reis, M.; Brentani, R. R.



Schwann cell myelination requires integration of laminin activities  

PubMed Central

Summary Laminins promote early stages of peripheral nerve myelination by assembling basement membranes (BMs) on Schwann cell surfaces, leading to activation of ?1 integrins and other receptors. The BM composition, structural bonds and ligands needed to mediate this process, however, are not well understood. Mice hypomorphic for laminin ?1-subunit expression that assembled endoneurial BMs with reduced component density exhibited an axonal sorting defect with amyelination but normal Schwann cell proliferation, the latter unlike the null. To identify the basis for this, and to dissect participating laminin interactions, LAMC1 gene-inactivated dorsal root ganglia were treated with recombinant laminin-211 and -111 lacking different architecture-forming and receptor-binding activities, to induce myelination. Myelin-wrapping of axons by Schwann cells was found to require higher laminin concentrations than either proliferation or axonal ensheathment. Laminins that were unable to polymerize through deletions that removed critical N-terminal (LN) domains, or that lacked cell-adhesive globular (LG) domains, caused reduced BMs and almost no myelination. Laminins engineered to bind weakly to ?6?1 and/or ?7?1 integrins through their LG domains, even though they could effectively assemble BMs, decreased myelination. Proliferation depended upon both integrin binding to LG domains and polymerization. Collectively these findings reveal that laminins integrate scaffold-forming and cell-adhesion activities to assemble an endoneurial BM, with myelination and proliferation requiring additional ?6?1/?7?1-laminin LG domain interactions, and that a high BM ligand/structural density is needed for efficient myelination.

McKee, Karen K.; Yang, Dong-Hua; Patel, Rajesh; Chen, Zu-Lin; Strickland, Sidney; Takagi, Junichi; Sekiguchi, Kiyotoshi; Yurchenco, Peter D.



Laminin Receptor Involvement in the Anti-angiogenic Activity of Pigment Epithelium-derived Factor*S??  

PubMed Central

Pigment epithelium-derived factor (PEDF) is a multifunctional protein with neurotrophic, anti-oxidative, and anti-inflammatory properties. It is also one of the most potent endogenous inhibitors of angiogenesis, playing an important role in restricting tumor growth, invasion, and metastasis. Studies show that PEDF binds to cell surface proteins, but little is known about how it exerts its effects. Recently, research identified phospholipase A2/nutrin/patatin-like phospholipase domain-containing 2 as one PEDF receptor. To identify other receptors, we performed yeast two-hybrid screening using PEDF as bait and discovered that the non-integrin 37/67-kDa laminin receptor (LR) is another PEDF receptor. Co-immunoprecipitation, His tag pulldown, and surface plasmon resonance assays confirmed the interaction between PEDF and LR. Using the yeast two-hybrid method, we further restricted the LR-interacting domain on PEDF to a 34-amino acid (aa) peptide (aa 44–77) and the PEDF-interacting domain on LR to a 91-aa fragment (aa 120–210). A 25-mer peptide named P46 (aa 46–70), derived from 34-mer, interacts with LR in surface plasmon resonance assays and binds to endothelial cell (EC) membranes. This peptide induces EC apoptosis and inhibits EC migration, tube-like network formation in vitro, and retinal angiogenesis ex vivo, like PEDF. Our results suggest that LR is a real PEDF receptor that mediates PEDF angiogenesis inhibition.

Bernard, Adrien; Gao-Li, Jacqueline; Franco, Claudio-Areias; Bouceba, Tahar; Huet, Alexis; Li, Zhenlin



The Human Laminin Receptor is a Member of the Integrin Family of Cell Adhesion Receptors  

NASA Astrophysics Data System (ADS)

A receptor for the adhesive basement membrane protein, laminin, was isolated from human glioblastoma cells by affinity chromatography on laminin. This receptor has a heterodimeric structure similar to that of receptors for other extracellular matrix proteins such as fibronectin and vitronectin. Incorporation of the laminin receptor into liposomal membranes makes it possible for liposomes to attach to surfaces coated with laminin. The receptor liposomes also attached to some extent to surfaces coated with fibronectin, but not with other matrix proteins. These properties identify the laminin receptor as a member of the integrin family of cell adhesion receptors.

Gehlsen, Kurt R.; Dillner, Lena; Engvall, Eva; Ruoslahti, Erkki



Expression and distribution of laminin receptor precursor/laminin receptor in rabbit tissues.  


The 37/67-kDa laminin receptor precursor (LRP)/laminin receptor (LR) is a cell surface receptor for cellular prion proteins and misfolded pathological prions. Previous research has shown that blocking or decreasing LRP/LP levels by anti-LRP/LR antibodies or small interfering RNAs (siRNAs) can prolong the incubation phase of experimental prion infection. This study aimed to investigate potential mechanisms contributing to prion resistance/susceptibility by using the rabbit, a species unsusceptible to prion infection, as a model. We investigated the expression level and distribution of LRP/LR in rabbit tissues by real-time polymerase chain reaction and by immunochemical analysis with a monoclonal anti-67 kDa LR antibody. Our results showed LRP/LR mRNA expression in all the tissues examined. Very low LRP/LR expression levels were observed in central nervous system (CNS) tissues, whereas high expression levels were observed in reproductive and digestive tissues, which differed from the expression patterns that have been reported for prion-susceptible animals. The immunochemical staining results were generally consistent with the mRNA findings, although no LR protein was detected in CNS tissues. Our findings provide a basis for further studies on prion resistance in rabbits and other animal species. PMID:23715696

Wang, Huinuan; Yang, Lifeng; Kouadir, Mohammed; Tan, Rongrong; Wu, Wenyu; Zou, Huarong; Wang, Jin; Khan, Sher Hayat; Li, Dongfeng; Zhou, Xiangmei; Yin, Xiaomin; Wang, Yunsheng; Zhao, Deming



Crystal Structure of the Human Laminin Receptor Precursor  

SciTech Connect

The human laminin receptor (LamR) interacts with many ligands, including laminin, prions, Sindbis virus, and the polyphenol (-)-epigallocatechin-3-gallate (EGCG), and has been implicated in a number of diseases. LamR is overexpressed on tumor cells, and targeting LamR elicits anti-cancer effects. Here, we report the crystal structure of human LamR, which provides insights into its function and should facilitate the design of novel therapeutics targeting LamR.

Jamieson,K.; Wu, J.; Hubbard, S.; Meruelo, D.



Laminin receptor complementary DNA-deduced synthetic peptide inhibits cancer cell attachment to endothelium.  


Stable attachment of cancer cells to the endothelium is a key step in the formation of metastasis. In this study, we have investigated the possibility that interaction between laminin and its Mr 67,000 high-affinity receptor (67 LR) could play a major role in this process. Scatchard analysis of laminin-binding studies showed that bovine aortic endothelial cells exhibit 46,000 high-affinity receptors that mediate, at least in part, the attachment of highly invasive melanoma cells. This endothelial cell-melanoma cell interaction was significantly inhibited by soluble laminin and by anti-laminin antibodies. Peptide G, an eicosapeptide derived from the complementary DNA sequence of the 67 LR precursor (IPCNNKGAHSVGLMWWMLAR) that specifically binds to laminin and presumably contains the active ligand-binding site of the receptor, specifically prevented attachment of the melanoma cells to both the bovine aortic endothelial cell monolayer and human umbilical vein endothelium. Thus, peptide G may selectively interfere with the metastatic cascade by inhibiting tumor cell attachment to endothelium via the laminin-67 LR pathway and is a potential new antimetastatic agent. PMID:1833053

Castronovo, V; Taraboletti, G; Sobel, M E



Modulation of laminin receptor expression by estrogen and progestins in human breast cancer cell lines.  


The effects of estradiol and two synthetic progestins (ORG2058 and R5020) on the expression of the high-affinity, metastasis-associated laminin receptor in two human breast carcinoma cell lines were examined. The T47D cell line contains estrogen and progesterone receptors, but the MDA-MB 231 cell line lacks both receptors. Treatment of T47D cells with 10(-9) M estradiol alone results in a three-fold increase (P less than or equal to .05) in the steady-state level of laminin receptor mRNA determined by RNA blot analysis as well as in cell-surface, laminin receptor expression that is evaluated by immunofluorescence. No effects of estradiol on the receptor-negative MDA-MB 231 cells were observed. Untreated and steroid-treated MDA-MB 231 cells had higher levels of laminin receptor mRNA than did untreated or estradiol-treated T47D cells. A more dramatic increase (five-fold; P less than or equal to .005) of mRNA and cell-surface expression in T47D cells was observed after treatment with estradiol plus 10(-8) M progestin or with progestin alone. Estradiol treatment also increased chemotaxis and haptotaxis of T47D cells but not of MDA-MB 231 cells to laminin; it had no effect on the attachment of these latter cells to laminin. Interestingly, treatment with estradiol plus progestin or progestin alone significantly increased the attachment of T47D cells to laminin but did not have an effect on either haptotaxis or chemotaxis to laminin. These results suggest that the various cell-laminin interactions are mediated by different mechanisms. The augmentation of laminin receptor mRNA by estrogen and progesterone treatment in hormone receptor-positive cells, but not in cells that lack these receptors, may relate functionally to the difference in the clinical aggressiveness between classes of breast cancers. PMID:2523976

Castronovo, V; Taraboletti, G; Liotta, L A; Sobel, M E



Functional domains of the 67-kDa laminin receptor precursor.  


We report the characterization of two functional domains of the metastasis-associated 67-kDa laminin receptor (67-LR). Using synthetic peptides deduced from the cDNA sequence of the 37-kDa precursor of the laminin receptor (37-LRP) as well as their corresponding affinity-purified polyclonal antibodies, we identified a unique laminin binding site as well as a membrane-associated domain of the receptor. In laminin dot blot and solid phase radioligand assays, a 20 amino acid synthetic peptide (IPCNNKGAHSVGLMWWMLAR, amino acid residues 161-180, designated peptide G) specifically bound to laminin with high affinity (Kd = 5 x 10(-8) M). Peptide G also specifically eluted the 67-LR from a laminin affinity column. Peptide G and laminin reacted with a 1:1 stoichiometry, suggesting that there is one recognition site on laminin for the peptide G domain. Immunofluorescence studies, performed on permeabilized and nonpermeabilized human A2058 melanoma cells using 10 different affinity-purified antibodies to distinct regions of the 37-LRP, identified an unusually short membrane-associated domain that was consistent with a computer predicted transmembrane domain (residues 86-101). Our data demonstrate for the first time that the 37-LRP has two functional domains consistent with the characteristics of the mature 67-LR. Furthermore, we propose peptide G as a potential inhibitor of tumor cell interactions with laminin. PMID:1834645

Castronovo, V; Taraboletti, G; Sobel, M E



Comprehensive Proteomic Analysis of Non-Integrin Laminin Receptor Interacting Proteins  

PubMed Central

Human non-integrin laminin receptor is a multifunctional protein acting as an integral component of the ribosome and a cell surface receptor for laminin-1. Laminin receptor is overexpressed in several human cancers and is also the cell surface receptor for several viruses and pathogenic prion protein, making it a pathologically significant protein. This study focused on the proteomic characterization of laminin receptor interacting proteins from mus musculus. The use of affinity chromatography with immobilized recombinant laminin receptor coupled with mass spectrometry analysis identified 45 proteins with high confidence. Following validation through co-immunoprecipitation, the proteins were classified based on predicted function into ribosomal, RNA processing, signal transduction/ metabolism, protein processing, cytoskeleton/ cell anchorage, DNA/ chromatin and unknown functions. A significant portion of the identified proteins is related to functions or localizations previously described for laminin receptor. This work represents a comprehensive proteomic approach to studying laminin receptor, and provides an essential stepping-stone to a better mechanistic understanding of this protein’s diverse functions.

Venticinque, Lisa; Meruelo, Daniel



Schwannoma cell-derived inhibitor of the neurite-promoting activity of laminin  

Microsoft Academic Search

During the purification of laminin-proteo- glycan complexes from rat RN22 Schwannoma cell- conditioned medium, a laminin-rich fraction was ob- tained which lacked neurite-promoting activity. Since laminin from several sources is known to have potent neurite-promoting activity, this result suggested that ei- ther this laminin was inactive or its activity was some- how masked by associated molecule(s). The latter pos- sibility

David Muir; Eva Engvall; Sflvio Varon; Marston Manthorpe



Multiple cell surface receptors for the short arms of laminin: alpha 1 beta 1 integrin and RGD-dependent proteins mediate cell attachment only to domains III in murine tumor laminin.  


Cell surface molecules that interact with the cross formed by the three short arms of murine tumor laminin were studied using thermal perturbation, antibody and peptide blocking, and affinity chromatography. Several potential receptors for the laminin short arms were revealed that differed from those mediating cell attachment to the E8 (long arm) fragment. Two cell lines, Rugli and L8 attached well to E1-X (short arm) fragments of laminin. This attachment was blocked by antibodies against alpha 1 integrin chains. Other cells were unable to attach strongly to E1-X, but attached to P1. This attachment was unaffected by anti-beta 1 integrin antibodies, but specifically blocked by the peptide GRGDS. By contrast, binding of Rugli cells was RGD independent and blocked by anti-beta 1 integrin antibodies. G7 and C2C12 myoblasts were very sensitive to GRGDS (ID50 approximately 2 for attachment to P1 which implied that a non-beta 1 series integrin, possibly alpha v beta 3, was involved. On heat denaturation of P1(3) attachment remained sensitive to RGDS and ID50 was unchanged. On heat denaturation of E1-X, attachment remained sensitive to RGDS but the ID50 increased to approximately 200 Cellular beta 1 integrins were retained on laminin affinity columns. A beta 1 integrin with an approximately 190 kD alpha-chain could be isolated from Rugli cells whose attachment could be blocked by anti-alpha 1 antibodies and not from cells blocked by RGDS peptides. Anti-alpha 1 antibodies blocked Rugli attachment to native laminin, but only when the E8 cell binding sites on laminin were also blocked. Thus, a receptor related to alpha 1 beta 1 integrin can function simultaneously with a receptor for E8. Anti-alpha 1 also blocked attachment to heated laminin, suggesting that the heat-stable attachment activity in laminin involved the E1-X binding site. Thus, at least two putative receptors mediate attachment to the short arms of laminin. One, related to alpha 1 beta 1 integrin, recognizes RGDS-independent sites in E1-X defined by P1 (within domains III, IIIa, IIIb), and one is an RGD-dependent molecule recognizing sites in P1, and is not a beta 1 integrin. PMID:1827447

Goodman, S L; Aumailley, M; von der Mark, H



Laminin-1 redistributes postsynaptic proteins and requires rapsyn, tyrosine phosphorylation, and Src and Fyn to stably cluster acetylcholine receptors  

PubMed Central

Clustering of acetylcholine receptors (AChRs) is a critical step in neuromuscular synaptogenesis, and is induced by agrin and laminin which are thought to act through different signaling mechanisms. We addressed whether laminin redistributes postsynaptic proteins and requires key elements of the agrin signaling pathway to cause AChR aggregation. In myotubes, laminin-1 rearranged dystroglycans and syntrophins into a laminin-like network, whereas inducing AChR-containing clusters of dystrobrevin, utrophin, and, to a marginal degree, MuSK. Laminin-1 also caused extensive coclustering of rapsyn and phosphotyrosine with AChRs, but none of these clusters were observed in rapsyn ?/? myotubes. In parallel with clustering, laminin-1 induced tyrosine phosphorylation of AChR ? and ? subunits. Staurosporine and herbimycin, inhibitors of tyrosine kinases, prevented laminin-induced AChR phosphorylation and AChR and phosphotyrosine clustering, and caused rapid dispersal of clusters previously induced by laminin-1. Finally, laminin-1 caused normal aggregation of AChRs and phosphotyrosine in myotubes lacking both Src and Fyn kinases, but these clusters dispersed rapidly after laminin withdrawal. Thus, laminin-1 redistributes postsynaptic proteins and, like agrin, requires tyrosine kinases for AChR phosphorylation and clustering, and rapsyn for AChR cluster formation, whereas cluster stabilization depends on Src and Fyn. Therefore, the laminin and agrin signaling pathways overlap intracellularly, which may be important for neuromuscular synapse formation.

Marangi, P. Angelo; Wieland, Simon T.; Fuhrer, Christian



Laminin-1-induced migration of multiple myeloma cells involves the high-affinity 67 kD laminin receptor  

PubMed Central

The 67?kD laminin receptor (67LR) binds laminin-1 (LN), major component of the basement membrane, with high affinity. In this study, we demonstrated that human multiple myeloma cell lines (HMCL) and murine 5T2MM cells express 67LR. CD38bright+ plasma cells in fresh multiple myeloma (MM) bone marrow (BM) samples showed weaker 67LR expression, but expression increased after direct exposure to a BM endothelial cell line (4LHBMEC). LN stimulated the in vitro migration of 3 HMCL (MM5.1, U266 and MMS.1), primary MM cells and the murine 5T2MM cells. 67LR has been shown to mediate the actions of LN through binding to CDPGYIGSR, a 9 amino acid sequence from the B1 chain of LN. MM cell migration was partially blocked by peptide 11, a synthetic nonapeptide derived from this amino sequence and also by a blocking antiserum against 67LR. Co-injection of peptide 11 with 5T2MM cells in a murine in vivo model of MM resulted in a decreased homing of 5T2MM cells to the BM compartment. In conclusion, LN acts as a chemoattractant for MM cells by interaction with 67LR. This interaction might be important during extravasation of circulating MM cells. © 2001 Cancer Research Campaign

Vande Broek, I; Vanderkerken, K; De Greef, C; Asosingh, K; Straetmans, N; Van Camp, B; Van Riet, I



The Potential Roles of a Laminin Receptor in Adhesion and Apoptosis of Cells of the Marine Bivalve Meretrix meretrix  

PubMed Central

Background The laminin receptors (LRs) play important roles in cell adhesion to the extracellular matrix, certain cell-cell adhesions, and the activation of many intracellular signaling pathways. Studies of LRs have primarily focused on mammals, while few studies of LRs in marine invertebrates have been reported. The functions of LRs in marine bivalve species are still unclear. Methodology/Principal Findings In this study, we cloned and sequenced an LR gene, MmeLR, from the clam Meretrix meretrix. The MmeLR mRNA and protein detected by realtime PCR and western blots were primarily distributed in muscle tissues. Far-western analysis showed a specific interaction between recombinant MmeLR and the LR ligand laminin. The results of the binding assay suggested a role of LR in cell adhesion and apoptosis in cultured primary cells of mantle tissues from M. meretrix. The Bcl-2 mRNA expression level in primary cells cultured in matrigel (mainly laminin) coated plates was significantly higher than in cells cultured in non-coated plates at 48 h of culture, while the p53 mRNA expression pattern was inversely related to that of bcl-2, suggesting that MmeLR is involved in p53-dependent apoptosis, and the binding between MmeLR and laminin inhibits apoptosis during primary cell culture. Conclusions Our results suggest that MmeLR may be involved in cell adhesion and apoptosis. This study may increase the understanding of the role of laminin receptor in cell adhesion and apoptosis and help to improve the culture of primary cells of marine invertebrates.

You, Yanan; Huan, Pin; Wang, Xiaomei; Liu, Baozhong



Human microvascular endothelial cells use beta 1 and beta 3 integrin receptor complexes to attach to laminin  

PubMed Central

Microvascular endothelial cells (MEC) use a set of surface receptors to adhere not only to the vascular basement membrane but, during angiogenic stimulation, to the interstitium. We examined how cultured human MEC interact with laminin-rich basement membranes. By using a panel of monoclonal antibodies, we found that MEC cells express a number of integrin-related receptor complexes, including alpha 1 beta 1, alpha 2 beta 1, alpha 3 beta 1, alpha 5 beta 1, alpha 6 beta 1, alpha V beta 3. Attachment to laminin, a major adhesive protein in basement membranes, was studied in detail. Blocking monoclonal antibodies specific to different integrin receptor complexes showed that the alpha 6 beta 1 complex was important for MEC adhesion to laminin. In addition, blocking antibody also implicated the vitronectin receptor (alpha V beta 3) in laminin adhesion. We used ligand affinity chromatography of detergent-solubilized receptor complexes to further define receptor specificity. On laminin-Sepharose columns, we identified several integrin receptor complexes whose affinity for the ligand was dependent on the type of divalent cation present. Several beta 1 complexes, including alpha 1 beta 1, alpha 2 beta 1, and alpha 6 beta 1 bound strongly to laminin. In agreement with the antibody blocking experiments, alpha V beta 3 was found to bind well to laminin. However, unlike binding to its other ligands (e.g., vitronectin, fibrinogen, von Willebrand factor), alpha V beta 3 interaction with laminin did not appear to be Arg-Gly-Asp (RGD) sensitive. Finally, immunofluorescent staining demonstrated both beta 1 and beta 3 complexes in vinculin-positive focal adhesion plaques on the basal surface of MEC adhering to laminin-coated substrates. The results indicate that both these subfamilies of integrin heterodimers are involved in promoting MEC adhesion to laminin and the vascular basement membrane.



Green Tea Polyphenols Precondition against Cell Death Induced by Oxygen-Glucose Deprivation via Stimulation of Laminin Receptor, Generation of Reactive Oxygen Species, and Activation of Protein Kinase C?  

PubMed Central

As the development of synthetic drugs for the prevention of stroke has proven challenging, utilization of natural products capable of preconditioning neuronal cells against ischemia-induced cell death would be a highly useful complementary approach. In this study using an oxygen-glucose deprivation and reoxygenation (OGD/R) model in PC12 cells, we show that 2-day pretreatment with green tea polyphenols (GTPP) and their active ingredient, epigallocatechin-3-gallate (EGCG), protects cells from subsequent OGD/R-induced cell death. A synergistic interaction was observed between GTPP constituents, with unfractionated GTPP more potently preconditioning cells than EGCG. GTPP-induced preconditioning required the 67-kDa laminin receptor (67LR), to which EGCG binds with high affinity. 67LR also mediated the generation of reactive oxygen species (ROS) via activation of NADPH oxidase. An exogenous ROS-generating system bypassed 67LR to induce preconditioning, suggesting that sublethal levels of ROS are indeed an important mediator in GTPP-induced preconditioning. This role for ROS was further supported by the fact that antioxidants blocked GTPP-induced preconditioning. Additionally, ROS induced an activation and translocation of protein kinase C (PKC), particularly PKC? from the cytosol to the membrane/mitochondria, which was also blocked by antioxidants. The crucial role of PKC in GTPP-induced preconditioning was supported by use of its specific inhibitors. Preconditioning was increased by conditional overexpression of PKC? and decreased by its knock-out with siRNA. Collectively, these results suggest that GTPP stimulates 67LR and thereby induces NADPH oxidase-dependent generation of ROS, which in turn induces activation of PKC, particularly prosurvival isoenzyme PKC?, resulting in preconditioning against cell death induced by OGD/R.

Gundimeda, Usha; McNeill, Thomas H.; Elhiani, Albert A.; Schiffman, Jason E.; Hinton, David R.; Gopalakrishna, Rayudu



Correlation between Laminin5 ?2 Chain Expression and Epidermal Growth Factor Receptor Expression and Its Clinicopathological Significance in Squamous Cell Carcinoma of the Tongue  

Microsoft Academic Search

Recent investigations have revealed that growth factors may influence the invasive activity of tumor cells. Expression of laminin-5 ?2 chain (LN-5 ?2) and epidermal growth factor receptor (EGFR) in squamous cell carcinomas of the tongue in 104 patients with stage II, III, and IVA, B (excluding the cases with distant metastasis) was examined immunohistochemically to determine the correlation between the

Kazuhide Katoh; Yukihiro Nakanishi; Shingo Akimoto; Kimio Yoshimura; Minoru Takagi; Michiie Sakamoto; Setsuo Hirohashi



Laminin receptor 37/67LR regulates adhesion and proliferation of normal human intestinal epithelial cells.  


Interactions between the cell basal membrane domain and the basement membrane are involved in several cell functions including proliferation, migration and differentiation. Intestinal epithelial cells can interact with laminin, a major intestinal basement membrane glycoprotein, via several cell-surface laminin-binding proteins including integrin and non-integrin receptors. The 37/67kDa laminin receptor (37/67LR) is one of these but its role in normal epithelial cells is still unknown. The aim of this study was to characterise the expression pattern and determine the main function of 37/67LR in the normal human small intestinal epithelium. Immunolocalization studies revealed that 37/67LR was predominantly present in the undifferentiated/proliferative region of the human intestinal crypt in both the immature and adult intestine. Using a human intestinal epithelial crypt (HIEC) cell line as experimental model, we determined that 37/67LR was expressed in proliferative cells in both the cytoplasmic and membrane compartments. Small-interfering RNA-mediated reduction of 37/67LR expression led to HIEC cell-cycle reduction and loss of the ability to adhere to laminin-related peptides under conditions not altering ribosomal function. Taken together, these findings indicate that 37/67LR regulates proliferation and adhesion in normal intestinal epithelial cells independently of its known association with ribosomal function. PMID:23991217

Khalfaoui, Taoufik; Groulx, Jean-François; Sabra, Georges; GuezGuez, Amel; Basora, Nuria; Vermette, Patrick; Beaulieu, Jean-François



Alterations in integrin receptor expression on chemically transformed human cells: specific enhancement of laminin and collagen receptor complexes  

PubMed Central

The abilities of malignant tumor cells to bind and migrate through basement membranes are important steps in invasion and metastasis. Malignant tumor cells would therefore be expected to express receptors on their surfaces for basement membrane and stromal components, such as collagens, laminin, and fibronectin, although the pattern of expression of these receptors on the malignant cells may be different from that on their normal progenitors. We report here that chemically transformed tumorigenic human cells express an altered pattern of integrin receptors on their cell surfaces as compared with their untransformed nontumorigenic counterparts. Specifically, N-methyl-N'-nitro-N- nitrosoguanidine transformation of HOS cells into highly tumorigenic cells results in a significant specific increase in the expression of (in descending order of level of cell surface expression) the integrins alpha 6/beta 1, alpha 2/beta 1, and alpha 1/beta 1, which are receptors for laminin, collagens, and collagen type IV and laminin, respectively. The level of expression of two fibronectin receptor integrins, alpha 5/beta 1 and alpha 3/beta 1, are, however, unaltered, whereas the level of expression of vitronectin receptor integrin, alpha v/beta 3, is drastically reduced on the transformed cells. Consistent with the increased expression of laminin and collagen receptors and the decreased expression of vitronectin receptors on the transformed cells, these cells attached three- to fivefold more strongly to laminin and collagen but attached very poorly to vitronectin. The MNNG-HOS cells were also found to have a greater potential for invasion through reconstituted basement membrane, matrigel, the major components of which are laminin and type IV collagen. The invasion of both the HOS and MNNG-HOS cells was inhibited 45-50% by a polyclonal anti- fibronectin receptor antibody. However, although the invasion of HOS cells could be inhibited up to 75% by an anti-alpha 6 monoclonal antibody, a similar concentration of this antibody had no effect on the alpha 6-overproducing MNNG-HOS cells. A fivefold higher concentration of this antibody did result in partial inhibition of MNNG-HOS invasion. These data indicate a critical role for the alpha 6/beta 1 laminin receptor in the invasion of these cells through basement membranes and demonstrate that chemical transformation of nontumorigenic human cells to highly tumorigenic cells is associated with an altered pattern of integrin expression which may play a direct role in the increased capacity of these cells to bind and invade through basement membranes.



Laminin-6 is activated by proteolytic processing and regulates cellular adhesion and migration differently from laminin-5.  


Laminin-6 (LN6) and laminin-5 (LN5), which share the common integrin-binding domain in the laminin alpha3 chain, are thought to cooperatively regulate cellular functions, but the former has poorly been characterized. Human fibrosarcoma HT1080 cells expressing an exogenous alpha3 chain were found to secrete LN6 with the full-length alpha3 chain and a smaller amount of its processed form lacking the carboxyl-terminal G4-5 domain, besides mature LN5 without G4-5 (mat-LN5). We prepared the unprocessed LN6 and mat-LN5, as well as LN6 mutants without G4-5 (LN6DeltaG4-5) or G5 (LN6DeltaG5). These laminins supported attachment of HT1080 cells and human keratinocytes (HaCaT) through integrins alpha(3)beta(1) and/or alpha(6)beta(1). LN6DeltaG4-5, LN6DeltaG5, and mat-LN5 promoted rapid cell spreading, whereas LN6 did hardly. A purified G4-5 fragment of the laminin alpha3 chain supported cell attachment through interaction with heparan sulfate proteoglycans and promoted cell spreading in combination with mat-LN5 or LN6DeltaG4-5. These results imply that the G4-5 domain within the LN6 molecule suppresses cell adhesion, while the released G4-5 promotes it. The presence of G5 rather than the heparin-binding domain G4 was responsible for the impaired cell spreading activity of LN6. However, the unprocessed LN6 promoted cell spreading in the presence of mat-LN5. Unlike mat-LN5, both LN6DeltaG4-5 and LN6 did weakly or did not stimulate cell motility. These findings demonstrate that LN6 and LN5 have distinct biological activities, but they may cooperatively support cell adhesion. The proteolytic processing of the alpha3 chain seems to regulate the physiological functions of LN6. PMID:12379663

Hirosaki, Tomomi; Tsubota, Yoshiaki; Kariya, Yoshinobu; Moriyama, Kayano; Mizushima, Hiroto; Miyazaki, Kaoru



Expression of Laminin Isoforms, Receptors and Binding Proteins Unique to Nucleus Pulposus Cells of Immature Intervertebral Disc  

PubMed Central

Intervertebral disc (IVD) disorders are believed to be related to aging-related cell loss and phenotypic changes, as well as biochemical and structural changes in the extracellular matrix of the nucleus pulposus (NP) region. Previously, we found that the laminin ?1 chain was more highly expressed in immature NP porcine tissues, in parallel with the expression pattern for a laminin receptor, integrin ?6 subunit, as compared to adjacent anulus fibrosus region; suggesting that cell-matrix interactions may be unique to the immature NP. However, the identity of laminin isoforms specific to immature or mature NP tissues, their associated receptors and functional significance are still poorly understood. In this study, we evaluated the zonal-specific expression of the laminin chains, receptors (i.e. integrins) and other binding proteins in immature tissue and isolated cells of rat, porcine and human intervertebral disc, towards the goal of revealing features of cellular environment and cell-matrix interactions in the immature NP. Results from both immuno-histochemical staining and flow cytometry analysis found that NP cells expressed higher levels of the laminin ?5 chain, laminin receptors (integrin ?3, ?6, ?4 subunit and CD239) and related binding proteins (CD151), as compared to cells from adjacent anulus fibrosus. These differences suggest that laminin interactions with NP cells are distinct from that of the anulus fibrosus, and that laminins may be important contributors to region-specific IVD biology. The revealed laminin isoforms, their receptors and related binding proteins may be used as distinguishing features of these immature NP cells in the intervertebral disc.

Chen, Jun; Jing, Liufang; Gilchrist, Christopher L; Richardson, William J; Fitch, Robert D; Setton, Lori A



Expression of the 67 kDa laminin receptor and the ?6 integrin subunit in serous ovarian carcinoma  

Microsoft Academic Search

The aim of this study was to analyze the expression of two laminin receptors, the 67kDa laminin receptor (LBP) precursor and\\u000a the ?6 integrin subunit, in effusions and solid tumors of patients diagnosed with serous ovarian carcinoma and to evaluate\\u000a their predictive role. Eighty-eight effusions and one hundred sixteen primary (= forty-one) and metastatic (= seventy-five)\\u000a ovarian carcinomas were evaluated

Vered Givant-Horwitz; Ben Davidson; Gregg van de Putte; Hiep Phuc Dong; Iris Goldberg; Sivan Amir; Gunnar B. Kristensen; Reuven Reich



Laminin Receptor Complementary DNA-deduced Synthetic Peptide Inhibits Cancer Cell Attachment to Endothelium  

Microsoft Academic Search

Stable attachment of cancer cells to the endothelium is a key step in the formation of metastasis. In this study, we have investigated the possibility that interaction between laminili and its M, 67,000 high- affinity receptor (67 LR) could play a major role in this process. Scat- chard analysis of laminin-binding studies showed that bovine aortic endothelial cells exhibit 46,000

Vincent Castronovo; Giulia Taraboletti; Mark E. Sobel



Green Tea Polyphenol EGCG Sensing Motif on the 67-kDa Laminin Receptor  

PubMed Central

Background We previously identified the 67-kDa laminin receptor (67LR) as the cell-surface receptor conferring the major green tea polyphenol (–)-epigallocatechin-3-O-gallate (EGCG) responsiveness to cancer cells. However, the underlying mechanism for interaction between EGCG and 67LR remains unclear. In this study, we investigated the possible role of EGCG-67LR interaction responsible for its bioactivities. Methodology/Principal Findings We synthesized various peptides deduced from the extracellular domain corresponding to the 102-295 region of human 67LR encoding a 295-amino acid. The neutralizing activity of these peptides toward EGCG cell-surface binding and inhibition of cancer cell growth were assayed. Both activities were inhibited by a peptide containing the 10-amino acid residues, IPCNNKGAHS, corresponding to residues 161-170. Furthermore, mass spectrometric analysis revealed the formation of a EGCG-LR161-170 peptide complex. A study of the amino acid deletion/replacement of the peptide LR161-170 indicated that the 10-amino acid length and two basic amino acids, K166 and H169, have a critical role in neutralizing EGCG’s activities. Moreover, neutralizing activity against the anti-proliferation action of EGCG was observed in a recombinant protein of the extracellular domain of 67LR, and this effect was abrogated by a deletion of residues 161-170. These findings support that the 10 amino-acid sequence, IPCNNKGAHS, might be the functional domain responsible for the anti-cancer activity of EGCG. Conclusions/Significance Overall, our results highlight the nature of the EGCG-67LR interaction and provide novel structural insights into the understanding of 67LR-mediated functions of EGCG, and could aid in the development of potential anti-cancer compounds for chemopreventive or therapeutic uses that can mimic EGCG-67LR interactions.

Sugihara, Kaori; Tsukamoto, Shuntaro; Yamada, Koji; Tachibana, Hirofumi



Distinct Roles for Laminin Globular Domains in Laminin alpha1 Chain Mediated Rescue of Murine Laminin alpha2 Chain Deficiency  

Microsoft Academic Search

BackgroundLaminin ?2 chain mutations cause congenital muscular dystrophy with dysmyelination neuropathy (MDC1A). Previously, we demonstrated that laminin ?1 chain ameliorates the disease in mice. Dystroglycan and integrins are major laminin receptors. Unlike laminin ?2 chain, ?1 chain binds the receptors by separate domains; laminin globular (LG) domains 4 and LG1-3, respectively. Thus, the laminin ?1 chain is an excellent tool

Kinga I. Gawlik; Mikael Åkerlund; Virginie Carmignac; Harri Elamaa; Madeleine Durbeej; Antoni L. Andreu



Opposing Roles of Integrin ?6A?1 and Dystroglycan in Laminin-mediated Extracellular Signal-regulated Kinase Activation  

PubMed Central

Laminin–integrin interactions can in some settings activate the extracellular signal-regulated kinases (ERKs) but the control mechanisms are poorly understood. Herein, we studied ERK activation in response to two laminins isoforms (-1 and -10/11) in two epithelial cell lines. Both cell lines expressed ?1-containing integrins and dystroglycan but lacked integrin ?6?4. Antibody perturbation assays showed that both cell lines bound to laminin-10/11 via the ?3?1and ?6?1 integrins. Although laminin-10/11 was a stronger adhesion complex than laminin-1 for both cell lines, both laminins activated ERK in only one of the two cell lines. The ERK activation was mediated by integrin ?6?1 and not by ?3?1 or dystroglycan. Instead, we found that dystroglycan-binding domains of both laminin-1 and -10/11 suppressed integrin ?6?1-mediated ERK activation. Moreover, the responding cell line expressed the two integrin ?6 splice variants, ?6A and ?6B, whereas the nonresponding cell line expressed only ?6B. Furthermore, ERK activation was seen in cells transfected with the integrin ?6A subunit, but not in ?6B-transfected cells. We conclude that laminin-1 and -10/11 share the ability to induce ERK activation, that this is regulated by integrin ?6A?1, and suggest a novel role for dystroglycan-binding laminin domains as suppressors of this activation.

Ferletta, Maria; Kikkawa, Yamato; Yu, Hao; Talts, Jan F.; Durbeej, Madeleine; Sonnenberg, Arnoud; Timpl, Rupert; Campbell, Kevin P.; Ekblom, Peter; Genersch, Elke



Laminin Receptor 1 Precursor Protein (37LRP) epitope delineated by an Hepatocellular carcinoma specific antibody  

US Patent & Trademark Office Database

This invention relates to the diagnosis and treatment of cancerous diseases, particularly to such diagnosis and treatment which revolves around the ability of the 5LAC-23 monoclonal antibody (or antigenic binding fragments derived therefrom) to bind with the Laminin Receptor 1 Precursor Protein 37LRP; and most particularly to diagnosis and treatment of Hepatocellular Carcinoma by various means which rely upon direct binding of 5LAC-23 with the particular antigenic moiety specifically recognized thereby and generally overexpressed in Hepatocellular carcinoma cells. The invention additionally relates to the treatment of such cells with conjugated moieties effective to aid in differentiation, treatment and diagnostic imaging thereof.



Shrimp laminin receptor binds with capsid proteins of two additional shrimp RNA viruses YHV and IMNV.  


Laminin receptor (Lamr) in shrimp was previously proposed to be a potential receptor protein for Taura syndrome virus (TSV) based on yeast two-hybrid assays. Since shrimp Lamr bound to the VP1 capsid protein of TSV, we were interested to know whether capsid/envelope proteins from other shrimp viruses would also bind to Lamr. Thus, capsid/envelope encoding genes from 5 additional shrimp viruses were examined. These were Penaeus stylirostris densovirus (PstDNV), white spot syndrome virus (WSSV), infectious myonecrosis virus (IMNV), Macrobrachium rosenbergii nodavirus (MrNV), and yellow head virus (YHV). Protein interaction analysis using yeast two-hybrid assay revealed that Lamr specifically interacted with capsid/envelope proteins of RNA viruses IMNV and YHV but not MrNV and not with the capsid/envelope proteins of DNA viruses PstDNV and WSSV. In vitro pull-down assay also confirmed the interaction between Lamr and YHV gp116 envelope protein, and injection of recombinant Lamr (rLamr) protein produced in yeast cells protected shrimp against YHV in laboratory challenge tests. PMID:21414409

Busayarat, Nattaphon; Senapin, Saengchan; Tonganunt, Moltira; Phiwsaiya, Kornsunee; Meemetta, Watcharachai; Unajak, Sasimanas; Jitrapakdee, Sarawut; Lo, Chu-Fang; Phongdara, Amornrat



Laminin on Toxoplasma gondii mediates parasite binding to the beta 1 integrin receptor alpha 6 beta 1 on human foreskin fibroblasts and Chinese hamster ovary cells.  


We investigated the role of parasite-bound laminin and the host cell beta 1 integrin receptors for this extracellular matrix protein in Toxoplasma gondii binding to fibroblasts. Laminin but not fibronectin was detected on extracellular tachyzoites by immunofluorescence and immunoblotting. Binding of parasites to CHO cells was inhibited by polyclonal antibodies to laminin and by a monoclonal antibody directed against the globular carboxyl-terminal portion of the long arm of laminin (at or near the suggested ligand-binding sites for alpha 3 beta 1 and alpha 6 beta 1), but not by a monoclonal antibody directed against the lateral short arms of laminin near the cross region of the molecule. Antibodies to the alpha 6 but not the alpha 2, alpha 3, or alpha 5 chains of the beta 1 family of integrins blocked parasite attachment to human foreskin fibroblasts and CHO cells. Attachment of T. gondii to cells via laminin on the parasite surface and laminin receptors on the mammalian cell is consistent with the capacity of the parasite to invade almost all nucleated cells. PMID:1399003

Furtado, G C; Cao, Y; Joiner, K A



Retinal Pigment Epithelial Cells Synthesize Laminins, Including Laminin 5, and Adhere to Them through ?3- and ?6-Containing Integrins  

PubMed Central

Purpose Retinal diseases are often accompanied by changes in the structure of the multilayered extracellular matrix underlying the retina, Bruch's membrane (BrM). These structural revisions potentially lead to alterations in retinal pigment epithelium (RPE) adhesion, likely via modification of interactions with extracellular matrix (ECM) proteins including laminins in BrM. The purpose of this study was to identify specific laminins in BrM and their receptors in RPE cells. Methods The laminin composition of BrM was determined using biochemical, molecular biological, and immunohistochemical techniques of rat, bovine, and human tissue and cell lines. An adhesion assay was used to test RPE attachment to laminins and the receptors used for this attachment. Results BrM contained laminin chains that could form laminin heterotrimers including laminins 1, 5, 10, and 11. RPE cells synthesized these laminin chains in vitro. Therefore, RPE cells may synthesize BrM laminins. The RPE cells preferentially adhered to potential BrM laminins. Although the cells adhered to the BrM component collagen IV, these cells preferentially adhered to laminins. Of the laminins tested, the RPE cells adhered preferentially to laminin 5. The cells interacted with these laminins via specific integrins and attained a different morphology on each laminin. In particular, the RPE cells rapidly attached and flattened on laminin 5. Conclusions BrM contains specific laminins, and RPE cells express integrin receptors for those laminins. The interaction of these specific laminins and integrins most likely leads to differential behavior of RPE cells.

Aisenbrey, Sabine; Zhang, Minlei; Bacher, Daniel; Yee, Jason; Brunken, William J.; Hunter, Dale D.



Two distinct cell-binding domains in laminin can independently promote nonneuronal cell adhesion and spreading.  


Two subfragments of laminin, E8, a major part of the long arm, and E1-4, the three short arms, promote cell adhesion and spreading. Three distinct types of adhesive behavior are seen in short term (1 h) assays, typified by secondary murine fibroblasts, adherent only on fibronectin; secondary murine myoblasts, adherent on fibronectin, laminin, and the E8 fragment; and Rugli human glioblastoma cells, adherent on fibronectin, laminin, E8, and E1-4. E8-specific polyclonal antibodies block myoblast adhesion to E8 and to laminin with identical concentration dependence; Rugli binding to E8 but not to laminin is also totally blocked by these antibodies. Heating of E8 and laminin to approximately 60 degrees C abolishes cell attachment-promoting activity for myoblasts. Adhesion of Rugli cells to E8 is also lost, but on laminin the attachment-promoting activity remains constant. This is due to an increase in the activity of E1-4 fragment as it is heated. Thus, major sites for initial cell adhesion to and spreading on laminin lie within the E8 and E1-4 fragments, but not all cells binding to laminin will bind to both fragments. These data may tentatively be explained by the existence of more than one type of receptor for laminin at the cell surface; one is needed for each fragment. PMID:3038930

Goodman, S L; Deutzmann, R; von der Mark, K




PubMed Central

Laminins are major constituents of basement membranes. At least 16 isoforms have now been described, each with distinct spatio-temporal expression patterns and functions. The laminin-511 heterotrimer (?5?1?1) is one of the more recent isoforms to be identified and a potent adhesive and pro-migratory substrate for a variety of normal and tumor cell lines in vitro. As our understanding of its precise function in normal tissues and in pathologies is rapidly unraveling, current evidence suggests an important regulatory role in cancer. This review describes published data on laminin-511 expression in several malignancies and experimental evidence from both in vitro and in vivo studies supporting its functional role during tumor progression. A particular emphasis is put on more recent studies from our laboratory and that of others indicating that laminin-511 contributes to tumor dissemination and metastasis in advanced breast carcinomas and other tumor types. Collectively, the experimental evidence suggests that high expression of laminin-511 has prognostic significance and that targeting tumor-laminin-511 interactions may have therapeutic potential in advanced cancer patients.

Pouliot, Normand; Kusuma, Nicole



Activated Notch1 maintains the phenotype of radial glial cells and promotes their adhesion to laminin by upregulating nidogen  

PubMed Central

Radial glia are neural stem cells that exist only transiently during CNS development where they serve as scaffolds for neuronal migration. Their instability makes them difficult to study and therefore we have isolated stabilized radial glial clones from E14.5 cortical progenitors (e.g. L2.3) after expression of v-myc. Activated Notch1 intracellular region (actNotch1) promotes radial glia in the embryonic mouse forebrain (Gaiano et al. 2000) and when it was introduced into E14.5 cortical progenitors or radial glial clone L2.3, the cells exhibited enhanced radial morphology and increased expression of the radial glial marker BLBP. A representative clone of L2.3 cells expressing actNotch1 called NL2.3–4 migrated more extensively than L2.3 cells in culture and in white matter of adult rat spinal cord. Microarray and RT-PCR comparisons of mRNAs expressed in these closely related clones showed extensive similarities but differed significantly for certain mRNAs including several cell adhesion molecules. Cell adhesion assays demonstrated significantly enhanced adhesion to laminin of NL2.3–4 by comparison to L2.3 cells. The laminin binding protein nidogen was the most highly induced adhesion molecule in NL2.3–4, and immunological analyses indicated that radial glia synthesize and secrete nidogen. Adhesion of NL2.3-4 cells to laminin was inhibited by anti-nidogen antibodies and required the nidogen binding region in laminin, indicating that nidogen promotes cell adhesion to laminin. The combined results indicate that persistent expression of activated Notch1 maintains the phenotype of radial glial cells, inhibits their differentiation, and promotes their adhesion and migration on a laminin/nidogen complex.

Li, Hedong; Chang, Yu-Wen; Mohan, Kriti; Su, Hui-Wen; Ricupero, Christopher L.; Baridi, Ajoeb; Hart, Ronald P.; Grumet, Martin



Expression of High-Affinity Laminin Receptor mRNA Correlates with Cell Proliferation Rather Than Invasion in Human Papillomavirus-associated Cervical Neoplasms1  

Microsoft Academic Search

Induction of the expression of the M, 67,000 high-affinity laminin receptor gene has been postulated as playing a role in the progression of human tumors to invasive cancers. We tested this hypothesis by exam ining histopathological sections of a large number of epithelial lesions of the genital tract associated with human papillomaviruses. In situ hybrid ization was performed with a

Lisa M. Demeter; Mark H. Stoler; Mark E. Sobel; Thomas R. Broker; Louise T. Chow


Laminin-10 and Its Receptors in Breast Carcinoma: Cooperation of Alpha6Beta4 and Alpha3Beta1 Integrin Laminin Receptors in Breast Carcinoma.  

National Technical Information Service (NTIS)

Insulin receptor substrate 1 and 2 are downstream signaling molecules for growth factors and adhesion receptors that are implicated in breast cancer. In this report, the author dissects the individual roles of IRS-1 and IRS-2 in the promotion and progress...

R. A. Awwad



Green tea polyphenol epigallocatechin-3-gallate inhibits TLR4 signaling through the 67-kDa laminin receptor on lipopolysaccharide-stimulated dendritic cells.  


Epigallocatechin-3-gallate (EGCG), a major active polyphenol of green tea, has been shown to down-regulate inflammatory responses in dendritic cells (DCs); however, the underlying mechanism has not been understood. Recently, we identified the 67-kDa laminin receptor (67LR) as a cell-surface EGCG receptor. In this study, we showed the molecular basis for the down-regulation of toll-like receptor 4 (TLR4) signal transduction by EGCG in DCs. The expressions of CD80, CD86, and MHC class I and II, which are molecules essential for antigen presentation by DCs, were inhibited by EGCG via 67LR. In addition, EGCG-treated DCs inhibited lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines (tumor necrosis factor [TNF]-?, interleukin [IL]-1?, and IL-6) and activation of mitogen-activated protein kinases (MAPKs), e.g., extracellular signal-regulated kinase 1/2 (ERK1/2), p38, c-Jun N-terminal kinase (JNK), and nuclear factor ?B (NF-?B) p65 translocation through 67LR. Interestingly, we also found that EGCG markedly elevated the expression of the Tollip protein, a negative regulator of TLR signaling, through 67LR. These novel findings provide new insight into the understanding of negative regulatory mechanisms of the TLR4 signaling pathway and consequent inflammatory responses that are implicated in the development and progression of many chronic diseases. PMID:22960171

Byun, Eui-Baek; Choi, Han-Gyu; Sung, Nak-Yun; Byun, Eui-Hong



Adhesomes: specific granules containing receptors for laminin, C3bi/fibrinogen, fibronectin, and vitronectin in human polymorphonuclear leukocytes and monocytes  

PubMed Central

We have localized several major extracellular matrix protein receptors in the specific granules of human polymorphonuclear (PMN) and monocytic leukocytes using double label immunoelectron microscopy (IEM) with ultrathin frozen sections and colloidal-gold conjugates. Rabbit antibodies to 67-kD human laminin receptor (LNR) were located on the inner surface of the specific granule membrane and within its internal matrix. LNR antigens co-distributed with lactoferrin, a marker of specific granules, but did not co-localize with elastase in azurophilic granules of PMNs. Further, CD11b/CD18 (leukocyte receptor for C3bi, fibrinogen, endothelial cells, and endotoxin), mammalian fibronectin receptor (FNR), and vitronectin receptor (VNR) antigens were also co- localized with LNR in PMN specific granules. A similar type of granule was found in monocytes which stained for LNR, FNR, VNR, CD18, and lysozyme. Activation of PMNs with either PMA, f-met-leu-phe (fMLP), tumor necrosis factor (TNF), or monocytic leukocytes with lipopolysaccharide (LPS), induced fusion of specific granules with the cell membrane and expression of both LNR and CD18 antigens on the outer cell surface. Further, stimulation led to augmented PMN adhesion on LN substrata, and six- to eightfold increases in specific binding of soluble LN that was inhibited by LNR antibody. These results indicate that four types of extracellular matrix receptors are located in leukocyte specific granules, and suggest that up-regulation of these receptors during inflammation may mediate leukocyte adhesion and extravasation. We have thus termed leukocyte specific granules adhesomes.



Modulation of the Metastatic Activity of Melanoma Cells by Laminin and Fibronectin  

NASA Astrophysics Data System (ADS)

Metastatic mouse melanoma cells have a high affinity for the basement membrane and the ability to degrade it; these properties may allow tumor cells to invade the membrane and disseminate. In this study it was found that the metastatic potential of mouse melanoma cells varied when the cells were exposed in culture to fibronectin or laminin. After removal of fibronectin or exposure to laminin, the cells had an increased affinity for basement membrane collagen, were more invasive of basement membranes in vitro, and produced more lung colonies in vivo. These changes are correlated with and may be due to an increase in the laminin-binding capacity of the tumor cell surface.

Terranova, Victor P.; Williams, Jeannette E.; Liotta, Lance A.; Martin, George R.



Integrin alphavbeta3 binding to human alpha5-laminins facilitates FGF-2- and VEGF-induced proliferation of human ECV304 carcinoma cells.  


Human ECV304 cells respond reproducibly by tube formation to complex basement membrane matrices. Laminins are major glycoproteins of basement membranes. We therefore studied the ability of ECV304 cells to attach to defined laminin isoforms and to fibronectin, and identified the involved laminin receptors. The cells bound poorly to fibronectin, to some extent to laminin-1, whereas laminin-2/4 and -10/11 were strong adhesive substrates. Antibody perturbation assays showed that adhesion to laminin-1 was mediated by integrin alpha6beta1, and adhesion to laminin-2/4 by cooperative activity of integrins alpha3beta1 and alpha6beta1. Adhesion of ECV 304 cells to laminin-10/11 was mainly mediated by integrins alpha3beta1, with minor involvement of alpha6beta1/4 and alphavbeta3. Solid-phase binding assays confirmed that integrin alphavbeta3 binds human laminin-10/11 and -10, in an RGD-dependent fashion. Although integrin alphavbeta3 played a very minor role in cell adhesion to laminin-10/11, this interaction facilitated growth factor-induced proliferation of ECV304 cells. In response to FGF-2 or VEGF, the cells proliferated better when attached on laminin-10/11 than on laminin-1, -2/4, or gelatin. The proliferation induced by the joint application of laminin-10/11 and either one of the growth factors could be blocked by antibodies against integrin alphavbeta3. Fragments of several other basement membrane components are known to interact with alphavbeta3. The current data show that that integrin alphavbeta3 can bind intact alpha5-containing laminin trimers. Since the laminin alpha5 chain is broadly expressed in adult basement membranes, this interaction could be physiologically important. Our data suggest that this interaction is involved in the regulation of cellular responses to growth factors known to be involved in epithelial and endothelial development. PMID:12691260

Genersch, Elke; Ferletta, Maria; Virtanen, Ismo; Haller, Hermann; Ekblom, Peter



Activity-dependent retrograde laminin A signaling regulates synapse growth at Drosophila neuromuscular junctions.  


Retrograde signals induced by synaptic activities are derived from postsynaptic cells to potentiate presynaptic properties, such as cytoskeletal dynamics, gene expression, and synaptic growth. However, it is not known whether activity-dependent retrograde signals can also depotentiate synaptic properties. Here we report that laminin A (LanA) functions as a retrograde signal to suppress synapse growth at Drosophila neuromuscular junctions (NMJs). The presynaptic integrin pathway consists of the integrin subunit ?? and focal adhesion kinase 56 (Fak56), both of which are required to suppress crawling activity-dependent NMJ growth. LanA protein is localized in the synaptic cleft and only muscle-derived LanA is functional in modulating NMJ growth. The LanA level at NMJs is inversely correlated with NMJ size and regulated by larval crawling activity, synapse excitability, postsynaptic response, and anterograde Wnt/Wingless signaling, all of which modulate NMJ growth through LanA and ??. Our data indicate that synaptic activities down-regulate levels of the retrograde signal LanA to promote NMJ growth. PMID:23054837

Tsai, Pei-I; Wang, Manyu; Kao, Hsiu-Hua; Cheng, Ying-Ju; Lin, Yu-Jing; Chen, Ruey-Hwa; Chien, Cheng-Ting



Dystroglycan receptor is involved in integrin activation in intestinal epithelia  

PubMed Central

The dystroglycans (?-DG and ?-DG), which play important roles in the formation of basement membranes, have been well studied in skeletal muscle and nerve, but their expression and localization in intestinal epithelial cells has not been previously investigated. Here, we demonstrated that the DG complex, composed of ?-DG, ?-DG, and utrophin, is specifically expressed in the basolateral membrane of the Caco-2-BBE monolayer. The DG complex coprecipitated with ?1-integrin, suggesting a possible interaction among these proteins. In addition, we observed that activation of DG receptors by laminin-1 enhanced the interaction between ?1-integrin and laminin-1, whereas activation of DG receptors by laminin-2 reduced the interaction between ?1-integrin and laminin-2. Finally, we demonstrated that the intracellular COOH-terminal tail of ?-DG and its binding to the DG binding domain of utrophin are crucial for the interactions between laminin-1/-2 and ?1-integrin. Collectively, these novel results indicate that dystroglycans play important roles in the regulation of interactions between intestinal epithelial cells and the extracellular matrix.

Driss, Adel; Charrier, Laetitia; Yan, Yutao; Nduati, Vivienne; Sitaraman, Shanthi; Merlin, Didier



Laminin 332 in squamous-cell carcinoma  

Microsoft Academic Search

Basement membranes can be a barrier to tumour growth, but basement membrane molecules, including laminins, are also important autocrine factors produced by cancers to promote tumorigenesis. Many studies have shown the importance of laminin 332 (previously known as laminin 5) in this process, especially in squamous cell carcinoma. Through interactions with several cell-surface receptors (including ?6?4 and ?3?1 integrins, epidermal

M. Peter Marinkovich



Identification and immunolocalization of laminin in cartilage.  


In a previous study on the role of integrins in the interaction of human chondrocytes with extracellular collagen and fibronectin (Dürr et al., (1993) Exp. Cell Res. 207, 235) we showed that chondrocytes adhere to laminin-1 (LN-1) in a beta 1-integrin-dependent manner. FACS analysis with various integrin antibodies including the monoclonal antibody GOH3 indicated the presence of the alpha 6 beta 1-laminin receptor on the chondrocyte surface. Anti-alpha 6 antibodies inhibited adhesion to the LN-1/E8 fragment, but not to whole laminin or heat-denatured Laminin-1, indicating that chondrocytes utilize at least two beta 1-integrins for laminin adhesion, one of which is alpha 6 beta 1 recognizing the LN-1/E8 fragment. The presence of alpha 6 beta 1-integrin on the chondrocyte surface also suggested the existence of laminin-like molecules in cartilage. Here we provide immunological and biochemical evidence in support of this possibility. Several polyclonal antibodies raised against laminin-1 or the LN-1/E8 fragment revealed a strong pericellular reaction in sections of human fetal epiphyseal cartilage and adult articular cartilage. In the fetal epiphysis laminin staining was most prominent in mature, large chondrocytes appearing in the secondary ossification zone, in particular, in the vicinity of invading capillary sprouts. Chondrocytes in the proliferating and hypertrophic zone of the growth plate and perichondrium cells were negative. All chondrocytes that stained for alpha 6-integrin also stained for laminin-1. A laminin-1-like molecule was extracted from hyaline cartilage with two bands migrating slightly faster than the alpha 1 and beta 1/gamma 1 subunits of laminin on SDS-gel electrophoresis. The two bands stained with anti-laminin-1 antibodies and could be immunoprecipitated with the same antibodies from metabolically labeled chondrocyte cultures. These findings suggests a role for laminin in developing cartilage and thus additional roles for laminins outside basement membranes. PMID:8549667

Dürr, J; Lammi, P; Goodman, S L; Aigner, T; von der Mark, K



Expression of the 67-kD laminin receptor, galectin-1, and galectin-3 in advanced human uterine adenocarcinoma  

Microsoft Academic Search

Alterations of tumor cell interactions with laminin, a basement membrane glycoprotein, are consistent features of the invasive and metastatic phenotype. Qualitative and quantitative changes in the expression of cell surface laminin-binding proteins have been correlated with the ability of cancer cells to cross basement membranes during the metastatic cascade. Such phenotypic modifications are usually associated with poor prognosis. In this

Frédéric A Van Den Brûle; Crina Buicu; Andy Berchuck; Robert C Bast; Manuel Deprez; Fu-tong Liu; Douglas N. W Cooper; Claudette Pieters; Mark E Sobel; Vincent Castronovo



Laminins in basement membrane assembly.  


The heterotrimeric laminins are a defining component of all basement membranes and self-assemble into a cell-associated network. The three short arms of the cross-shaped laminin molecule form the network nodes, with a strict requirement for one ?, one ? and one ? arm. The globular domain at the end of the long arm binds to cellular receptors, including integrins, ?-dystroglycan, heparan sulfates and sulfated glycolipids. Collateral anchorage of the laminin network is provided by the proteoglycans perlecan and agrin. A second network is then formed by type IV collagen, which interacts with the laminin network through the heparan sulfate chains of perlecan and agrin and additional linkage by nidogen. This maturation of basement membranes becomes essential at later stages of embryo development. PMID:23076216

Hohenester, Erhard; Yurchenco, Peter D



Laminin-induced activation of Rac1 and JNKp46 is initiated by Src family kinases and mimics the effects of skeletal muscle contraction1  

PubMed Central

Laminin-binding to dystroglycan in the dystrophin glycoprotein complex2 causes signaling through dystroglycan-syntrophin-grb2-SOS1-Rac1-PAK1-JNK. Laminin-binding also causes syntrophin tyrosine phosphorylation to initiate signaling. The kinase responsible was investigated here. PP2 and SU6656, specific inhibitors of Src family kinases, decreased the amount of phosphotyrosine-syntrophin and decreased active Rac1 in laminin-treated myoblasts, myotubes or skeletal muscle microsomes. c-Src and c-Fyn both phosphorylate syntrophin and inhibition of either with specific siRNAs diminish syntrophin phosphorylation. When the rat gastrocnemius was contracted, Rac1 activation increased compared to the relaxed control muscle and Rac1 co-localized with ?-dystroglycan. Similar results were obtained when the muscle was stretched. Contracted muscle also contained more activated c-Jun N-terminal Kinase, JNKp46. E3, an expressed protein containing only laminin domains LG4–5, increased proliferation of myoblasts and PP2 prevented cell proliferation. In addition, Src family kinases co-localized with activated Rac1 and with laminin-Sepharose in solid-phase binding assays. Thus, contraction, stretching, or laminin-binding cause Src-family kinase recruitment to the dystrophin glycoprotein complex, activating Rac1 and inducing downstream signaling. The DGC likely represents a mechanoreceptor in skeletal muscle regulating muscle growth in response to muscle activity. Src-family kinases play an initiating and critical role.

Zhou, YanWen; Jiang, Daifeng; Thomason, Donald B.; Jarrett, Harry W.



Receptor functions for the integrin VLA3: fibronectin, collagen, and laminin binding are differentially influenced by Arg-Gly-Asp peptide and by divalent cations  

Microsoft Academic Search

The capability of the integrin VLA-3 to function as a receptor for collagen (Coil), laminin (Lm), and fibronectin (Fn) was addressed using both whole cell adhesion assays and Iigand affinity columns. Analysis of VLA-3-mediated cell adhesion was facilitated by the use of a small cell lung carci- noma line (NCI-H69), which expresses VLA-3 but few other integrins. While VLA-3 interaction

M. J. Elices; Lisa A. Urry; Martin E. Hemler



Control pathways of the 67 kDa laminin binding protein: surface expression and activity of a new ligand binding domain.  


A number of papers have been published on the clinical correlation of the expression of the 67 kDa laminin binding protein (LBP) with the metastatic potential of solid tumors. Both mRNA and protein expression levels have been reported, but both the relationship between them and the molecular nature of the 67 kDa surface product remain unclear. We have utilized a homotypic overexpression system to investigate the cell surface presentation of the 67 kDa LBP and the contribution of this protein to the invasive phenotype of cultured cell lines. We report here that the cellular mRNA levels do not directly reflect the levels of the 67 kDa LBP observed on the cell surface in this overexpression system. Methotrexate amplification of transfected plasmids expressing the 67 kDa LBP leads to an initial elevation of both the LBP mRNA and surface protein levels. This is accompanied by an altered, more flattened, cell morphology. Later, apparent adaptation of the cells to methotrexate is accompanied by a down-regulation of the surface expression of the protein. mRNA levels, however, remain elevated. A nine amino acid sequence, CDPGYIGSR (peptide 11), within the beta chain of laminin 1 has been identified as a probable binding domain for the 67 kDa LBP. Previous studies have identified a region of the 67 kDa LBP which may be involved in laminin interaction, although not necessarily via the peptide 11 domain. We have identified a second site within the amino acid coding sequence of the 67 kDa LBP which also shows biological activity both in vitro and in vivo. A peptide with this sequence, LBP residues 205-229, binds laminin-1 in a peptide 11 inhibitable manner. The receptor-derived peptide modulates invasion of basement membrane matrix in vitro and inhibits experimental lung colony formation when injected along with B16BL6 mouse melanoma cells. However, pretreatment of the melanoma cells with the peptide enhances lung colony formation. Thus, the interaction of the 67 kDa LBP with basement membrane matrix appears to involve a complex series of events including multiple adhesive sites and tight regulation of cell surface expression. PMID:7641420

Landowski, T H; Uthayakumar, S; Starkey, J R



INS-1 cell glucose-stimulated insulin secretion is reduced by the downregulation of the 67?kDa laminin receptor.  


Understanding ? cell-extracellular matrix (ECM) interactions can advance our knowledge of the mechanisms that control glucose homeostasis and improve culture methods used in islet transplantation for the treatment of diabetes. Laminin is the main constituent of the basement membrane and is involved in pancreatic ? cell survival and function, even enhancing glucose-stimulated insulin secretion. Most of the studies on cell responses towards laminin have focused on integrin-mediated interactions, while much less attention has been paid on non-integrin receptors, such as the 67?kDa laminin receptor (67LR). The specificity of the receptor-ligand interaction through the adhesion of INS-1 cells (a rat insulinoma cell line) to CDPGYIGSR-, GRGDSPC- or CDPGYIGSR?+?GRGDSPC-covered surfaces was evaluated. Also, the effects of the 67LR knocking down over glucose-stimulated insulin secretion were investigated. Culture of the INS-1 cells on the bioactive surfaces was improved compared to the low-fouling carboxymethyl dextran (CMD) surfaces, while downregulation of the 67LR resulted in reduced cell adhesion to surfaces bearing the CDPGYIGSR peptide. Glucose-stimulated insulin secretion was hindered by downregulation of the 67LR, regardless of the biological motif available on the biomimetic surfaces on which the cells were cultured. This finding illustrates the importance of the 67LR in glucose-stimulated insulin secretion and points to a possible role of the 67LR in the mechanisms of insulin secretion. Copyright © 2013 John Wiley & Sons, Ltd. PMID:23362185

Sabra, Georges; Dubiel, Evan A; Kuehn, Carina; Khalfaoui, Taoufik; Beaulieu, Jean-François; Vermette, Patrick



Green tea (-)-epigallocatechin gallate inhibits insulin stimulation of 3T3-L1 preadipocyte mitogenesis via the 67-kDa laminin receptor pathway.  


Insulin and (-)-epigallocatechin gallate (EGCG) have been reported to regulate fat cell mitogenesis and adipogenesis, respectively. This study investigated the pathways involved in EGCG modulation of insulin-stimulated mitogenesis in 3T3-L1 preadipocytes. EGCG inhibited insulin stimulation of preadipocyte proliferation in a dose- and time-dependent manner. EGCG also suppressed insulin-stimulated phosphorylation of the insulin receptor-beta, insulin receptor (IR) substrates 1 and 2 (IRS1 and IRS2), and mitogen-activated protein kinase pathway proteins, RAF1, MEK1/2, and ERK1/2, but not JNK. Furthermore, EGCG inhibited the association of IR with the IRS1 and IRS2 proteins, but not with the IRS4 protein. These data suggest that EGCG selectively affects particular types of IRS and MAPK family members. Generally, EGCG was more effective than epicatechin, epicatechin gallate, and epigallocatechin in modulating insulin-stimulated mitogenic signaling. We identified the EGCG receptor [also known as the 67-kDa laminin receptor (67LR)] in fat cells and found that its expression was sensitive to growth phase, tissue type, and differentiation state. Pretreatment of preadipocytes with 67LR antiserum prevented the effects of EGCG on insulin-stimulated phosphorylation of IRS2, RAF1, and ERK1/2 and insulin-stimulated preadipocyte proliferation (cell number and bromodeoxyuridine incorporation). Moreover, EGCG tended to increase insulin-stimulated associations between the 67LR and IR, IRS1, IRS2, and IRS4 proteins. These data suggest that EGCG mediates anti-insulin signaling in preadipocyte mitogenesis via the 67LR pathway. PMID:19176763

Ku, Hui-Chen; Chang, Hsin-Huei; Liu, Hsien-Chun; Hsiao, Chiao-Hsin; Lee, Meng-Jung; Hu, Yu-Jung; Hung, Pei-Fang; Liu, Chi-Wei; Kao, Yung-Hsi



Dystrophin Glycoprotein Complex-associated G?? Subunits Activate Phosphatidyl Inositol-3-Kinase/Akt signaling in Skeletal Muscle in a Laminin-dependent Manner  

PubMed Central

Previously, we showed that laminin-binding to the dystrophin glycoprotein complex (DGC) of skeletal muscle causes a heterotrimeric G-protein, (G???) to bind, changing the activation state of the Gs? subunit. Others have shown that laminin-binding to the DGC also leads to Akt activation. G??, released when Gs? is activated, is known to bind phosphatidylinositol 3-kinase (PI3K), which activates Akt in other cells. Here, we investigate whether muscle Akt activation results from G??, using immunoprecipitation and immunoblotting, and purified G??. In the presence of laminin, PI3K-binding to the DGC increases and Akt becomes phosphorylated and activated (pAkt), and glycogen synthase kinase is phosphorylated. Antibodies, which specifically block laminin-binding to ?-dystroglycan, prevent PI3K-binding to the DGC. Purified bovine brain G?? also caused PI3K and Akt activation. These results show that DGC-G?? is binding PI3K and activating pAkt in a laminin-dependent manner. Mdx mice, which have greatly diminished amounts of DGC proteins, display elevated pAkt signaling and increased expression of integrin ?1 compared to normal muscle. This integrin binds laminin, G??, and PI3K. Collectively, these suggest that PI3K is an important target for the G??, which normally binds to DGC syntrophin, and activates PI3K/Akt signaling. Disruption of the DGC in mdx mouse is causing dis-regulation of the laminin-DGC-G??-PI3K-Akt signaling and is likely to be important to the pathogenesis of muscular dystrophy. Up-regulating integrin ?1 expression and activating the PI3K/Akt pathway in muscular dystrophy may partially compensate for the loss of the DGC. The results suggest new therapeutic approaches to muscle disease.

Xiong, Yongmin; Zhou, Yanwen; Jarrett, Harry W.



Photodynamic treatment of epithelial tissue derived from patients with endometrial cancer: a contribution to the role of laminin and epidermal growth factor receptor in photodynamic therapy  

NASA Astrophysics Data System (ADS)

Photodynamic therapy (PDT) was used to treat endometrial G1 cancer tissue derived from patients who had undergone a total hysterectomy and bilateral salpingo-oophorectomy. After surgical treatment the cancerous tissue was kept in a medium containing Dulbecco solution, fetal calf serum, and antibiotics. The tissue was then exposed to hematoporphyrin derivative (0.1 mg/l) and 24 h later exposed to light (total light dose--18 J/sq cm). Necrosis depth was evaluated 24 h later using a light microscope. In order to assess the possible role of the basal membrane component laminin, as well as epidermal growth factor receptor susceptibility to PDT, immunohistochemical studies were carried out. Additionally, nucleolar organizer regions evaluation was performed. Our experiment confirmed that PDT results in the necrosis in the treated endometrial cancer, while not affecting the laminin in the cancerous tissue. In contrast, PDT strongly affects the epidermal growth factor receptor and nucleolar organizer regions in cancer cells. We suggest that laminin may contribute to the prevention of cancer dissemination in the cases where PDT has to be repeated, and that after PDT the cells become less susceptible to a mitogen, like, e.g., epidermal growth factor.

Ziolkowski, Piotr P.; Symonowicz, Krzysztof; Osiecka, Beata J.; Rabczynski, Jerzy; Gerber, Jerzy



Induction of cytotoxic T-cell responses against the oncofetal antigen-immature laminin receptor for the treatment of hematologic malignancies.  


The oncofetal antigen immature laminin receptor protein (OFA-iLRP) is a highly conserved protein that is preferentially expressed in fetal tissues and in many types of cancer, including hematopoietic malignancies, whereas OFA-iLRP is not detectable on healthy differentiated adult cells. To investigate whether OFA-iLRP-specific cytotoxic T lymphocytes (CTLs) are capable of killing OFA-iLRP-expressing hematologic targets, CTLs were generated from healthy HLA-A*0201-positive volunteers by incubating T cells with autologous dendritic cells (DCs) transfected with OFA-iLRP RNA. OFA-iLRP-specific CTLs lysed HLA-A2+ OFA-iLRP+ tumor cells, including several lymphoma and leukemia cell lines, as well as fresh leukemic targets from patients with acute myeloid leukemia (AML) and chronic lymphatic leukemia (CLL), indicating that OFA-iLRP-derived peptides are naturally processed and presented by hematologic tumors. Healthy OFA-iLRP-negative target cells (CD14+ monocytes, activated B cells, DCs, bone marrow cells) were not attacked by OFA-iLRP-specific CTLs. Furthermore, in an established murine B-cell lymphoma model (A20), treatment with syngeneic DCs transfected with OFA-iLRP-coding RNA resulted in powerful antitumor effects in a significant portion of mice. For the first time, these data show that OFA-iLRP can be used as a target for T-cell-based immunotherapeutic strategies against hematologic malignancies. PMID:12869512

Siegel, Sandra; Wagner, Andreas; Kabelitz, Dieter; Marget, Matthias; Coggin, Joseph; Barsoum, Adel; Rohrer, James; Schmitz, Norbert; Zeis, Matthias



Proinflammatory Signals and the Loss of Lymphatic Vessel Hyaluronan Receptor-1 (LYVE-1) in the Early Pathogenesis of Laminin Alpha2-deficient Skeletal Muscle  

PubMed Central

Congenital muscular dystrophy type 1A, a severe neuromuscular disease characterized by early-onset muscle weakness and degeneration, is caused by insufficient levels of laminin ?2 (LAMA2) in the basal lamina surrounding muscle fibers and other cells. A better understanding of the molecular mechanisms leading to muscle loss is needed to develop therapeutic interventions for this disease. Here, the authors show that inflammation is an early feature of pathogenesis in Lama2-deficient mouse muscle, indicated by elevated expression of tenascin C in the endomysium around muscle fibers, infiltration of macrophages, and induction of the inflammatory cytokines tumor necrosis factor ? (TNF?) and IL-1?. In addition, the expression of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), a specific marker for lymphatic vessel endothelial cells, is dramatically reduced early in Lama2-deficient muscle pathogenesis. LYVE-1 expression, which is inhibited by TNF?, is also decreased in muscles undergoing degeneration due to dystrophin deficiency and cardiotoxin damage. LYVE-1 expression thus provides a useful biomarker to monitor the onset of muscle pathogenesis, likely serving as an indicator of inflammatory signals present in muscles. Together, the data show that inflammatory pathways are activated in the earliest stages of Lama2-deficient disease progression and could play a role in early muscle degeneration.

Wardrop, Katherine E.; Dominov, Janice A.



Expression of laminins and their integrin receptors in different conditions of synovial membrane and synovial membrane-like interface tissue  

PubMed Central

OBJECTIVE—To demonstrate the expression of laminins (Lns) and their integrin (Int) receptors in different synovial samples and synovial membrane-like interface tissues from well fixed and aseptically loosened total hip replacement (THR), and the potential role of Ln-Int interaction in the production of collagenases and cytokines.?METHODS—Immunohistochemical staining was done to detect the distribution of EHS Ln, Ln ?2, ?3, ?5, ?1, ?2 chains and Int ?1, ?2, ?3, ?6, ?1, ?4 subunits in different samples. Double immunofluorescence labelling was used to find colocalisation of Int ?6 subunit and collagenase-1/collagenase-3/TNF?/IL6.?RESULTS—General Ln immunoreactivity was detected in all specimens. Ln ?5, ?1 and ?2, but not ?2 and ?3 chains were seen in the synovial lining and the basement membrane of blood vessels with the intensity/extent of labelling in the following rank order: rheumatoid arthritis (RA) loosened prostheses, osteoarthritis, well fixed prostheses, traumatic knees. Among Int subunits, staining for ?1 was usually the strongest, followed by staining for Int ?6, ?1, ?3, and ?2 subunits, with the same rank order for overall expression of Lns. Int ?4 subunit was not detectable in most of the specimens. Double labelling focused on Int ?6 subunit disclosed its frequent colocalisation with collagenases 1 and 3 and with tumour necrosis factor ? and interleukin 6 in synovial lining.?CONCLUSION—Synovial lining contains Ln-10, Ln-11, and Int ?6?1 and ?1?1 receptors. In aseptic loosening of THR, interface tissue has a similar Ln subtype and Int receptor composition as RA synovium, which confirms its "lining-like" phenotype. Synovial lining does not contain Ln-5 (?3?3?2) or Int ?6?4, which are components of epithelial hemidesmosomes. The expression of Lns and their Int receptors is upregulated in inflammation. The close spatial relation between Ln and its Int receptors in synovial lining cells containing proteinases and cytokines suggests a potential role in joint destruction and prosthetic loosening.??

Konttinen, Y.; Li, T. F.; Xu, J. W.; Tagaki, M.; Pirila, L.; Silvennoinen, T.; Santavirta, S.; Virtanen, I.




PubMed Central

The basement membrane is a highly intricate and organized portion of the extracellular matrix that interfaces with a variety of cell types including epithelial, endothelial, muscle, nerve, and fat cells. The laminin family of glycoproteins is a major constituent of the basement membrane. The sixteen known laminin isoforms are formed from combinations of ?, ?, and ? chains, with each chain containing specific domains capable of interacting with cellular receptors such as integrins and other extracellular ligands. In addition to its role in the assembly and architectural integrity of the basement membrane, laminins interact with cells to influence proliferation, differentiation, adhesion, and migration, processes activated in normal and pathologic states. In vitro these functions are regulated by the posttranslational modifications of the individual laminin chains. In vivo laminin knock-out mouse studies have been particularly instructive in defining the function of specific laminins in mammalian development and have also highlighted its role as a key component of the basement membrane. In this review, we will define how laminin structure complements function and explore its role in both normal and pathologic processes.

Tzu, Julia; Marinkovich, M. Peter



Humoral immune responses against the immature laminin receptor protein show prognostic significance in patients with chronic lymphocytic leukemia.  


Chronic lymphocytic leukemia (CLL) is characterized by a highly variable clinical course. The role of an autologous tumor-specific immune control contributing to the variable length of survival in CLL is poorly understood. We investigated whether humoral immunity specific for the CLL-associated Ag oncofetal Ag/immature laminin receptor (OFA/iLR) has a prognostic value in CLL. Among sera of 67 untreated patients with CLL, 23 (34.3%) had detectable OFA/iLR Abs that were reactive for at least one specific OFA/iLR epitope. Patients with humoral responses compared with patients with nonreactive sera had a longer progression-free survival (p = 0.029). IgG subclass analyses showed a predominant IgG1 and IgG3 response. OFA/iLR Abs were capable of recognizing and selectively killing OFA/iLR-expressing CLL cells in complement-mediated and Ab-dependent cellular cytotoxicity assays. In the analysis of 11 CLL patients after allogeneic hematopoietic stem cell transplantation, 8 showed high values for OFA/iLR Abs that specifically recognized the extracellular domain of the protein, suggesting a potential role of anti-OFA/iLR-directed immune responses to the graft-vs-leukemia effect in CLL. Our data suggest that spontaneous tumor-specific humoral immune responses against OFA/iLR exist in a significant proportion of CLL patients and that superior progression-free survival in those patients could reflect autologous immune control. PMID:18424761

Friedrichs, Birte; Siegel, Sandra; Kloess, Marita; Barsoum, Adel; Coggin, Joseph; Rohrer, James; Jakob, Ilja; Tiemann, Markus; Heidorn, Klaus; Schulte, Christoph; Kabelitz, Dieter; Steinmann, Jörg; Schmitz, Norbert; Zeis, Matthias



Multiple Functions of the 37/67-kd Laminin Receptor Make It a Suitable Target for Novel Cancer Gene Therapy  

PubMed Central

The 37/67-kd laminin receptor, LAMR, is a multifunctional protein that associates with the 40S ribosomal subunit and also localizes to the cell membrane to interact with the extracellular matrix. LAMR is overexpressed in many types of cancer, playing important roles in tumor-cell migration and invasion. Here, we show that LAMR is also vital for tumor-cell proliferation, survival, and protein translation. Small-interfering RNA (siRNA)–mediated reduction in expression of LAMR leads to G1 phase cell-cycle arrest in vitro by altering cyclins A2/B1, cyclin-dependent kinases (CDKs) 1/2, Survivin, and p21 expression levels. In vivo, reduction in LAMR expression results in inhibition of HT1080 cells to develop tumors. We also found that LAMR's ribosomal functions are critical for translation as reduction in LAMR expression leads to a dramatic decrease in newly synthesized proteins. Further, cells with lower expression of LAMR have fewer 40S subunits and 80S monosomes, causing an increase in free 60S ribosomal subunits. These results indicate that LAMR is able to regulate tumor development in many ways; further enhancing its potential as a target for gene therapy. To test this, we developed a novel Sindbis/Lenti pseudotype vector carrying short-hairpin RNA (shRNA) designed against lamr. This pseudotype vector effectively reduces LAMR expression and specifically targets tumors in vivo. Treatment of tumor-bearing severe combine immunodeficient (SCID) mice with this pseudotype vector significantly inhibits tumor growth. Thus, we show that LAMR can be used as a target in novel therapy for tumor reduction and elimination.

Scheiman, Jonathan; Tseng, Jen-Chieh; Zheng, Yun; Meruelo, Daniel



Structure and function of an early divergent form of laminin in hydra: a structurally conserved ECM component that is essential for epithelial morphogenesis.  


As a major component of the extracellular matrix (ECM), laminin has been found in many vertebrate and invertebrate organisms. Its molecular structure is very similar across species lines and its biological function in the ECM has been extensively studied. In an effort to study ECM structure and function in hydra, we have cloned a partial hydra laminin alpha chain and the full-length hydra laminin beta chain using ECM-enriched cDNA libraries. Analysis of deduced amino acid sequences indicated that both polypeptides have high sequence similarity to a number of invertebrate and vertebrate laminin alpha and beta subunits. Rotary shadow analysis of isolated hydra laminin indicates it has a heterotrimeric organization that is characteristic of vertebrate laminins. A putative integrin-class protein was also identified using a cell-binding peptide sequence from the laminin beta chain as an affinity probe, indicating that integrins are possible cell surface receptors in hydra. In agreement with previous results for the hydra laminin beta chain, in situ hybridization experiments revealed that hydra laminin alpha chain mRNA is restricted to endodermal cells. As with a number of other hydra ECM components, higher levels of laminin alpha chain mRNA are localized to regions where cell migration and differentiation are actively undertaken such as the base of tentacles, the peduncle region, buds, regenerating tentacles, and at the head end during regeneration. The role of laminin in morphogenesis was studied using an antisense approach and the results indicated that translation of the laminin alpha chain is required for head regeneration. PMID:12012231

Zhang, Xiaoming; Fei, Kaiyin; Agbas, Abdulbaki; Yan, Li; Zhang, Jinsong; O'Reilly, Brooke; Deutzmann, Rainer; Sarras, Michael P



Polymorphonuclear leukocytes present laminin peptides in endocytic compartments.  


Rat and Human neutrophils presented cytoplasmic vacuoles immunoreactive for laminin at the electron microscopy level. Colocalization of the anti-laminin labeling with albumin-gold complexes and alkaline phosphatase activity in rat PMN suggest an endocytic nature for this compartment. Immunoblot analysis of human normal peripheral blood neutrophils revealed the presence of two laminin peptides around 100 kDa. PMID:8630048

Vannier-Santos, M A; de Souza, S J; Brentani, R R; de Souza, W



The laminin family.  


Laminins are large molecular weight glycoproteins constituted by the assembly of three disulfide-linked polypeptides, the ?, ? and ? chains. The human genome encodes 11 genetically distinct laminin chains. Structurally, laminin chains differ by the number, size and organization of a few constitutive domains, endowing the various members of the laminin family with common and unique important functions. In particular, laminins are indispensable building blocks for cellular networks physically bridging the intracellular and extracellular compartments and relaying signals critical for cellular behavior, and for extracellular polymers determining the architecture and the physiology of basement membranes. PMID:23263632

Aumailley, Monique



Laminin isoforms in development and disease  

Microsoft Academic Search

The members of the laminin family of heterotrimers are major constituents of all basement membranes, sheet-like extracellular\\u000a structures, present in almost all organs. The laminins bind to cell surface receptors and thereby tightly connect the basement\\u000a membrane to the adjacent cell layer. This provides for the specific basement membrane functions to stabilize cellular structures,\\u000a to serve as effective physical barriers,

Susanne Schéele; Alexander Nyström; Madeleine Durbeej; Jan F. Talts; Marja Ekblom; Peter Ekblom



Expression of the 67 kD laminin receptor in human ovarian carcinomas as defined by a monoclonal antibody, MLuC5.  


Previous immunohistochemical data from our laboratory have demonstrated that expression of the 67 kD laminin receptor (67LR), a cancer-associated, high-affinity laminin-binding protein, is upregulated in ovarian carcinoma cells compared with normal serosal cells, and that this increased expression in cancer cells could be related to patient outcome. The aim of this study was to validate MLuC5, a monoclonal antibody that recognises the 67LR, as a tool to perform future immunohistochemical studies on larger populations of ovarian carcinoma patients. Expression of the 67LR was determined in 51 primary human ovarian carcinoma samples using immunohistochemistry and MLuC5. The 67LR was detected in ovarian carcinoma cell clusters of variable extent. Analysis of the data determined that 67LR expression was significantly increased in the samples from patients with disease progression, compared with those with no evidence of disease after completion of primary therapy, and in pooled grade 2 and 3 tumours compared to borderline and grade 1 tumours (P < 0.05, chi-squared test). No other significant correlation between 67LR expression and other clinicopathological parameters could be established. These data suggest that the 67LR is correlated to ovarian tumour progression. Detection of the 67LR using this monoclonal antibody could constitute an interesting parameter in prognosis determination of ovarian cancer. PMID:8911124

van den Brûle, F A; Castronovo, V; Ménard, S; Giavazzi, R; Marzola, M; Belotti, D; Taraboletti, G



Cell surface galactosyltransferase mediates the initiation of neurite outgrowth from PC12 cells on laminin.  


Neurite outgrowth from PC12 pheochromocytoma cells, as well as from peripheral and central nervous system neurons in vitro, is mediated by the extracellular matrix molecule, laminin. We have recently shown that mesenchymal cell spreading and migration on laminin is mediated, in part, by the cell surface enzyme, beta 1,4 galactosyltransferase (GalTase). GalTase is localized on lamellipodia of migrating cells where it functions as a laminin receptor by binding to specific N-linked oligosaccharides in laminin (Runyan et al., 1988; Eckstein and Shur, 1989). In the present study, we examined whether GalTase functions similarly during neutrite outgrowth on laminin using biochemical and immunological analyses. PC12 neurite outgrowth was inhibited by reagents that perturb cell surface GalTase activity, including anti-GalTase IgG and Fab fragments, as well as the GalTase modifier protein alpha-lactalbumin. Control reagents had no effect on neurite outgrowth. Furthermore, blocking GalTase substrates on laminin matrices by earlier galactosyltion or enzymatic removal of GalTase substrates also inhibited neurite outgrowth. Conversely, neurite outgrowth was enhanced by the addition of UDP-galactose, which completes the GalTase enzymatic reaction, while inappropriate sugar nucleotides had no effect. The effects of all these treatments were dose and/or time dependent. Surface GalTase was shown to function during both neurite initiation and elongation, although the effects of GalTase perturbation were most striking during the initiation stages of neurite formation. Consistent with this, surface GalTase was localized by indirect immunofluorescence to the growth cone and developing neurite. Collectively, these results demonstrate that GalTase mediates the initiation of neurite outgrowth on laminin, and to a lesser extent, neurite elongation. Furthermore, this study demonstrates that process extension from both mesenchymal cells and neuronal cells is partly dependent upon specific oligosaccharide residues in laminin. PMID:2105324

Begovac, P C; Shur, B D



Cellular prion protein/laminin receptor: distribution in adult central nervous system and characterization of an isoform associated with a subtype of cortical neurons.  


The 67-kDa LR protein was originally discovered as a non-integrin laminin receptor. Several more recent in vitro studies demonstrated the function of 67-kDa LR and its related 'precursor' form 37-kDa LRP as receptors of cellular prion protein and their implication in abnormal prion protein propagation in vitro. In addition, expression of both proteins was shown to increase considerably in the brain of scrapie-infected mice and hamsters. While LRP/LR are thus likely to play important roles in neuronal cell adhesion, survival and homeostasis and during pathological disorders, little is known so far about their fine cellular distribution in adult central nervous system. Using immunocytochemistry and western blotting, we show here that the 67-kDa LR is the major receptor form in adult rat brain and spinal cord, expressed within the cytoplasm and at the plasma membrane of most neurons and in a subset of glial cells. The overall distribution of LR correlates well with that reported for laminin-1 but also with brain regions classically associated with prion-related neurodegeneration. In contrast to LR, the 37-kDa LRP form is much less abundant in adult than in postnatal central nervous system. Characterization of a novel antibody allowed us to study the distribution across tissues of cell membrane-associated LRP. Interestingly, this form is almost exclusively found on a subclass of parvalbumin-immunoreactive cortical interneurons known to degenerate during the early stages of Creutzfeldt-Jakob disease. Our demonstration of local differences in the expression of particular LRP/LR isoforms may be a first step towards unraveling their specific molecular interactions. PMID:15548204

Baloui, Hasna; von Boxberg, Ysander; Vinh, Joëlle; Weiss, Stefan; Rossier, Jean; Nothias, Fatiha; Stettler, Olivier



Akt\\/PKB regulates laminin and collagen IV isotypes of the basement membrane  

Microsoft Academic Search

Basement membranes are important for epithelial differentiation, cell survival, and normal and metastatic cell migration. Much is known about their breakdown and remodeling, yet their positive regulation is poorly understood. Our previous analysis of a fibroblast growth factor (FGF) receptor mutation raised the possibility that protein kinase B (Akt\\/PKB) activated by FGF is connected to the expression of certain laminin

Xiaofeng Li; Ulrika Talts; Jan F. Talts; Esther Arman; Peter Ekblom; Peter Lonai



High expression of the immature laminin receptor protein correlates with mutated IGVH status and predicts a favorable prognosis in chronic lymphocytic leukemia.  


The immature laminin receptor (iLR) is a tumor-associated antigen. We analyzed the expression of iLR on malignant B cells of 134 unselected patient samples with CLL and hypothesized that iLR expression would have prognostic significance due to a differential expression pattern. High ILR expression (cut-off value 30%) was correlated with mutated IGVH status (p<0.0001). Patients with high iLR-expression had a significantly longer time to progression (p=0.039). Combination of CD38, ZAP-70, and iLR by flow cytometry can be used to construct a diagnostic score identifying patients with a median progression free survival of 80 months, if no adverse marker is present. PMID:21055809

Friedrichs, Birte; Siegel, Sandra; Reimer, Rudolph; Barsoum, Adel; Coggin, Joseph; Kabelitz, Dieter; Heidorn, Klaus; Schulte, Christoph; Schmitz, Norbert; Zeis, Matthias



Processing of Laminin5 and Its Functional Consequences: Role of Plasmin and Tissue-type Plasminogen Activator  

Microsoft Academic Search

The laminin-5 component of the extracellu- lar matrices of certain cultured cells such as the rat epi- thelial cell line 804G and the human breast epithelial cell MCF-10A is capable of nucleating assembly of cell- matrix adhesive devices called hemidesmosomes when other cells are plated upon them. These matrices also impede cell motility. In contrast, cells plated onto the laminin-5-rich

Lawrence E. Goldfinger; M. Sharon Stack; Jonathan C. R. Jones



Laminin enhances ?2-adrenergic receptor stimulation of L-type Ca2+ current via cytosolic phospholipase A2 signalling in cat atrial myocytes  

PubMed Central

We previously reported that attachment of atrial myocytes to the extracellular matrix protein laminin (LMN), decreases adenylate cyclase (AC)/cAMP and increases ?2-adrenergic receptor (AR) stimulation of L-type Ca2+ current (ICa,L). This study therefore sought to determine whether LMN enhances ?2-AR signalling via a cAMP-independent mechanism, i.e. cytosolic phospholipase A2 (cPLA2) signalling. Studies were performed on acutely isolated atrial myocytes plated on uncoated coverslips (?LMN) or coverslips coated with LMN (+LMN). As previously reported, 0.1 ?m zinterol (zint-?2-AR) stimulation of ICa,L was larger in +LMN than ?LMN myocytes. In +LMN myocytes, zint-?2-AR stimulation of ICa,L was inhibited by inhibition of cPLA2 by arachidonyltrifluoromethyl ketone (AACOCF3; 10 ?m), inhibition of Gi by pertussis toxin and chelation of intracellular Ca2+ by 10 ?m BAPTA-AM. In contrast to zinterol, stimulation of ICa,L by fenoterol (fen-?2-AR), a ?2-AR agonist that acts exclusively via Gs signalling, was smaller in +LMN than ?LMN myocytes. Arachidonic acid (AA; 5 ?m) stimulated ICa,L to a similar extent in ?LMN and +LMN myocytes. Inhibition of cAMP-dependent protein kinase A (cAMP/PKA) by either 5 ?m H?89 or 1 ?m KT5720 in ?LMN myocytes mimicked the effects of +LMN myocytes to enhance zint-?2-AR stimulation of ICa,L, which was blocked by 10 ?m AACOCF3. In contrast, H?89 inhibited fen-?2-AR stimulation of ICa,L, which was unchanged by AACOCF3. Inhibition of ERK1/2 by 1 ?m U0126 inhibited zint-?2-AR stimulation of ICa,L in +LMN myocytes and ?LMN myocytes in which cAMP/PKA was inhibited by KT5720. In ?LMN myocytes, cytochalasin D prevented inhibition of cAMP/PKA from enhancing zint-?2-AR stimulation of ICa,L. We conclude that LMN enhances zint-?2-AR stimulation of ICa,L via Gi/ERK1/2/cPLA2/AA signalling which is activated by concomitant inhibition of cAMP/PKA signalling and dependent on the actin cytoskeleton. These findings provide new insight into the cellular mechanisms by which the extracellular matrix can remodel ?2-AR signalling in atrial muscle.

Pabbidi, M R; Ji, X; Samarel, A M; Lipsius, S L



Enhanced adhesion to laminin by apoptotic eosinophils.  


Apoptotic cells are regarded as inert bodies that turn off intracellular processes and functional capabilities. The objective was to study adhesion by eosinophils in relation to the apoptotic process. Eosinophils were cultured for up to 72 h. The living cells were separated from the apoptotic cells, and their adhesion to transfected cell lines expressing vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), E-selectin and laminin was measured. To relate the functional studies with cell structure, the surface receptor expression of beta1- and beta2-integrins was investigated by flow cytometry. Apoptotic eosinophils evidenced an increased expression of the alpha-chain of the laminin receptor and CD49f and an increased ability to adhere to a laminin-coated surface. Adhesion to the endothelial cell adhesion receptors E-selectin, VCAM-1 and ICAM-1 was absent in apoptotic eosinophils and was paralleled by a low expression of CD11b, CD29, CD49d and CD66b. The specifically increased adhesion to laminin and expression of the laminin receptor alpha-chain is a unique feature of apoptotic eosinophils. When an eosinophil goes into apoptosis, it still possesses the ability to interact with its environment. Our results point to new ideas as to how the apoptotic eosinophil behaves in apoptosis. PMID:14507306

Seton, K; Håkansson, L; Venge, P



Thyroid Hormone Regulates the Extracellular Organization of Laminin on Astrocytes  

Microsoft Academic Search

Astrocytes produce laminin, a key extracellular matrix guidance molecule in the developing brain. Laminin is bound to transmem- brane receptors on the surface of astrocytes known as integrins, which are, in turn, bound to the microfilament meshwork inside the astro- cyte. Previous studies have shown that T4 regulates the pattern of integrin distribution in astrocytes by modulating the organization of




Identification of Two Laminin-Binding Fimbriae, the Type 1 Fimbria of Salmonella enterica Serovar Typhimurium and the G Fimbria of Escherichia coli, as Plasminogen Receptors  

PubMed Central

Escherichia coli strains carrying recombinant plasmids encoding either the type 1 fimbria of Salmonella enterica serovar Typhimurium or the G fimbria of E. coli exhibited binding of human 125I-Glu-plasminogen and enhanced the tissue-type plasminogen activator-catalyzed conversion of plasminogen to plasmin. Purified type 1 or G fimbriae similarly bound plasminogen and enhanced its activation. The binding of plasminogen did not involve the characteristic carbohydrate-binding property of the fimbriae but was inhibited at low concentrations by the lysine analog ?-aminocaproic acid. Because these fimbrial types bind to laminin of basement membranes (M. Kukkonen et al., Mol. Microbiol. 7:229–237, 1993; S. Saarela et al., Infect. Immun. 64:2857–2860, 1996), the results demonstrate a structural unity in the creation and targeting of bacterium-bound proteolytic plasmin activity to basement membranes.

Kukkonen, Maini; Saarela, Sirkku; Lahteenmaki, Kaarina; Hynonen, Ulla; Westerlund-Wikstrom, Benita; Rhen, Mikael; Korhonen, Timo K.



Akt/PKB regulates laminin and collagen IV isotypes of the basement membrane  

PubMed Central

Basement membranes are important for epithelial differentiation, cell survival, and normal and metastatic cell migration. Much is known about their breakdown and remodeling, yet their positive regulation is poorly understood. Our previous analysis of a fibroblast growth factor (FGF) receptor mutation raised the possibility that protein kinase B (Akt/PKB) activated by FGF is connected to the expression of certain laminin and type IV collagen isotypes. Here we test this hypothesis and demonstrate that constitutively active Akt/PKB, an important downstream element of phosphoinositide 3?-kinase signaling, induces the synthesis of laminin-1 and collagen IV isotypes and causes their translocation to the basement membrane. By using promoter–reporter constructs, we show that constitutively active phosphoinositide 3?-kinase-p110 or Akt/PKB activates, whereas dominant negative Akt/PKB inhibits, transcription of laminin ?1 and collagen IV ?1 in differentiating C2 myoblast- and insulin-induced Chinese hamster ovary–T cell cultures. These results suggest that Akt/PKB activated by receptor tyrosine kinases is involved in the positive regulation of basement membrane formation. The possible role of Akt/PKB-induced laminin and collagen IV synthesis in cell survival and differentiation will be discussed.

Li, Xiaofeng; Talts, Ulrika; Talts, Jan F.; Arman, Esther; Ekblom, Peter; Lonai, Peter



Extraribosomal Functions Associated with the C Terminus of the 37/67 kDa Laminin Receptor are Required for Maintaining Cell Viability  

SciTech Connect

The 37/67 kDa laminin receptor (LAMR) is a multifunctional protein, acting as an extracellular receptor, localizing to the nucleus, and playing roles in rRNA processing and ribosome assembly. LAMR is important for cell viability; however, it is unclear which of its functions are essential. We developed a silent mutant LAMR construct, resistant to siRNA, to rescue the phenotypic effects of knocking down endogenous LAMR, which include inhibition of protein synthesis, cell cycle arrest, and apoptosis. In addition, we generated a C-terminal-truncated silent mutant LAMR construct structurally homologous to the Archaeoglobus fulgidus S2 ribosomal protein and missing the C-terminal 75 residues of LAMR, which displays more sequence divergence. We found that HT1080 cells stably expressing either silent mutant LAMR construct still undergo arrest in the G{sub 1} phase of the cell cycle when treated with siRNA. However, the expression of full-length silent mutant LAMR rescues cell viability, whereas the expression of the C-terminal-truncated LAMR does not. Interestingly, we also found that both silent mutant constructs restore protein translation and localize to the nucleus. Our findings indicate that the ability of LAMR to regulate viability is associated with its C-terminal 75 residues. Furthermore, this function is distinct from its role in cell proliferation, independent of its ribosomal functions, and may be regulated by a nonnuclear localization.

J Scheiman; K Jamieson; J Ziello; J Tseng; D Meruelo



Extraribosomal functions associated with the C terminus of the 37/67 kDa laminin receptor are required for maintaining cell viability  

PubMed Central

The 37/67?kDa laminin receptor (LAMR) is a multifunctional protein, acting as an extracellular receptor, localizing to the nucleus, and playing roles in rRNA processing and ribosome assembly. LAMR is important for cell viability; however, it is unclear which of its functions are essential. We developed a silent mutant LAMR construct, resistant to siRNA, to rescue the phenotypic effects of knocking down endogenous LAMR, which include inhibition of protein synthesis, cell cycle arrest, and apoptosis. In addition, we generated a C-terminal-truncated silent mutant LAMR construct structurally homologous to the Archaeoglobus fulgidus S2 ribosomal protein and missing the C-terminal 75 residues of LAMR, which displays more sequence divergence. We found that HT1080 cells stably expressing either silent mutant LAMR construct still undergo arrest in the G1 phase of the cell cycle when treated with siRNA. However, the expression of full-length silent mutant LAMR rescues cell viability, whereas the expression of the C-terminal-truncated LAMR does not. Interestingly, we also found that both silent mutant constructs restore protein translation and localize to the nucleus. Our findings indicate that the ability of LAMR to regulate viability is associated with its C-terminal 75 residues. Furthermore, this function is distinct from its role in cell proliferation, independent of its ribosomal functions, and may be regulated by a nonnuclear localization.

Scheiman, J; Jamieson, K V; Ziello, J; Tseng, J-C; Meruelo, D



The ?1 Cytoplasmic Domain Regulates the Laminin-binding Specificity of the ?7X1 Integrin  

PubMed Central

During muscle development, the laminin-specific ?7 integrin is alternatively spliced in the putative ligand-binding domain to yield either the ?7X1 or the ?7X2 variant. The relative level of ?7X1 and ?7X2 is developmentally regulated. Similarly, the partner ?1 integrin cytoplasmic domain is converted from the ?1A to the ?1D splice variant. To determine whether ?1D modulates the activity of the ?7 receptor, cells were transfected with ?7X1 and ?1D cDNA. ?7X1 coupled with ?1A failed to adhere to laminin-1, whereas cotransfectants expressing ?7X1 and ?1D showed strong adhesion. Interestingly, ?7X1 complexed with ?1A and ?1D displayed the same level of poor adhesion to laminin-2/4 or strong adhesion to laminin-10/11. These findings indicate that ?7 function is regulated not only by X1/X2 in its extracellular domain but also by ?1 cytoplasmic splice variants. It is likely that expression of ?1D alters ?7X1 binding to laminin isoforms by a process related to ligand affinity modulation. Functional regulation of ?7?1 by developmentally regulated splicing events may be important during myogenic differentiation and repair because the integrin mediates adhesion, motility, and cell survival.

Yeh, Ming-Guang; Ziober, Barry L.; Liu, Baomei; Lipkina, Galina; Vizirianakis, Ioannis S.; Kramer, Randall H.



Laminin Interactions with Head and Neck Cancer Cells under Low Fluid Shear Conditions Lead to Integrin Activation and Binding*  

PubMed Central

Lymphatic metastasis of cancer cells involves movement from the primary tumor site to the lymph node, where the cells must be able to productively lodge and grow. It is there that tumor cells encounter cellular and non-cellular constituent elements that make up the lymph node parenchyma. Our work shows that head and neck squamous cell carcinoma (HNSCC) cell lines are able to bind to laminin, fibronectin, vitronectin, and hyaluronic acid, which are extracellular matrix elements within the lymph node parenchyma. HNSCC cell lines bound to laminin under lymphodynamic low shear stress (0.07 dynes/cm2), consistent with lymph flow via ?1 integrins, including ?2?1, ?3?1, and ?6?1. Binding occurred in the presence of shear stress and not in the absence of flow. Additionally, tumor cell binding to laminin under flow did result in calcium signaling. Our data indicate a novel role for ?1 integrin-mediated binding of HNSCC cells to laminin under conditions of lymphodynamic flow that results in intracellular calcium signaling within the cancer cell.

Fennewald, Susan M.; Kantara, Carla; Sastry, Sarita K.; Resto, Vicente A.



Macrophage interactions with laminin: PMA selectively induces the adherence and spreading of mouse macrophages on a laminin substratum  

Microsoft Academic Search

The ability of thioglycollate (TG)-elicited mouse peritoneal macrophages to adhere to a laminin substratum has been studied. These cells do not ad- here to laminin-coated (20 ~tg\\/ml) surfaces, but the ad- dition of phorbol myristate acetate (PMA; 50 ng\\/ml) results in their rapid adherence and spreading on this substratum. TG-elicited and PMA-activated macro- phages, however, can bind soluble laminin. Macro-

Arthur M. Mercurio; Leslie M. Shaw



Identification of HLA-A*0201-presented T cell epitopes derived from the oncofetal antigen-immature laminin receptor protein in patients with hematological malignancies.  


The oncofetal Ag immature laminin receptor (OFA-iLR) is a potential target molecule for immunotherapeutic studies in several tumor entities, including hematological malignancies. In the present study, we characterize two HLA-A*0201-presented epitopes eliciting strong OFA-iLR peptide-specific human cytotoxic T cell (CTLs) responses in vitro. Both allogeneic HLA-A*0201-matched and autologous CTLs recognized and killed endogenously OFA-iLR-expressing tumor cell lines and primary malignant cells from patients with hemopoietic malignancies in an MHC-restricted fashion but spared nonmalignant hemopoietic cells. Spontaneous OFA-iLR peptide-specific T cell reactivity was detectable in a significant proportion of leukemia patients. Interestingly, in patients with chronic lymphocytic leukemia and multiple myeloma but not in those with acute myeloid leukemia, significant frequencies of OFA peptide-specific CTLs could be detected in an early stage of disease but disappeared in patients with progressive disease. The identification of OFA-iLR-derived peptide epitopes provides a basis for tumor immunological studies and therapeutic vaccination strategies in patients with OFA-iLR-expressing malignancies. PMID:16709854

Siegel, Sandra; Wagner, Andreas; Friedrichs, Birte; Wendeler, Anneke; Wendel, Lena; Kabelitz, Dieter; Steinmann, Jörg; Barsoum, Adel; Coggin, Joseph; Rohrer, James; Dreger, Peter; Schmitz, Norbert; Zeis, Matthias



Laminin-332-Integrin Interaction: A Target For Cancer Therapy?  

PubMed Central

For many years, extracellular matrix (ECM) was considered to function as a tissue support and filler. However, we now know that ECM proteins control many cellular events through their interaction with cell-surface receptors and cytoplasmic signaling pathways. For example, they regulate cell proliferation, cell division, cell adhesion, cell migration, and apoptosis. We focus in this review on a laminin isoform, laminin-332 (formerly termed laminin-5), a major component of the basement membrane (BM) of skin and other epithelial tissues. It is composed of 3 subunits (?3, ?3, and ?2) and interacts with at least two integrin receptors expressed by epithelial cells (?3?1 and ?6?4 integrin). Mutations in either laminin-332 or integrin ?6?4 result in junctional epidermolysis bullosa, a blistering skin disease, while targeting of laminin-332 by autoantibodies in cicatricial pemphigoid leads to dysadhesion of epithelial cells from their underlying connective tissue. Abnormal expression of laminin-332 and its integrin receptors is also a hallmark of certain tumor types and is believed to promote invasion of colon, breast and skin cancer cells. Moreover, there is emerging evidence that laminin-332 and its protease degradation products are not only found at the leading front of several tumors but also likely induce and/or promote tumor cell migration. Thus, in this review, we focus specifically on the role of laminin-332 and its integrin receptors in adhesion, proliferation, and migration/invasion of cancer cells. Finally, we discuss strategies for the development of laminin-332-based antagonists for the treatment of malignant tumors.

Tsuruta, Daisuke; Kobayashi, Hiromi; Imanishi, Hisayoshi; Sugawara, Koji; Ishii, Masamitsu; Jones, Jonathan C.R.



Antibodies against the VRT101 laminin epitope correlate with human SLE disease activity and can be removed by extracorporeal immunoadsorption  

Microsoft Academic Search

Objective. We have previously shown that murine pathogenic lupus autoantibodies bind to VRT101, a 21-mer peptide located at the globular part of the laminin-? chain. In this study, we evaluated whether VRT101 also serves as a target for human lupus antibodies, upholding the hypothesis that VRT101 may serve as a potential target in the therapy of lupus. Methods. Anti-VRT101 and

H. Amital; M. Heilweil-Harel; R. Ulmansky; M. Harlev; E. Toubi; A. Hershko; Y. Naparstek



YIGSR, a Synthetic Laminin Pentapeptide, Inhibits Experimental Metastasis Formation  

Microsoft Academic Search

The invasion of tumor cells through basement membranes is a critical step in the formation of metastases. The binding of the malignant cells to laminin in the basement membranes allows their attachment and activates their invasiveness. Recently a synthetic nonapeptide from the B1 chain sequence of laminin was identified as a major site for cell binding. A pentapeptide within the

Yukihide Iwamoto; Frank A. Robey; Jeannette Graf; Makoto Sasaki; Hynda K. Kleinman; Yoshihiko Yamada; George R. Martin



Therapeutic applications of laminin and laminin-derived protein fragments  

US Patent & Trademark Office Database

Laminin and specific laminin-derived protein fragments are disclosed as potent inhibitors of Alzheimer's disease type amyloidoses. A specific region is identified within laminin which interacts with the Alzheimer's disease beta-amyloid protein and contributes to the observed inhibitory and therapeutic effects. A prominent .about.130 kilodalton band in laminin was found in human serum and cerebrospinal fluid which primarily interacted with A.beta. as determined by ligand blotting methodology. This .about.130 kilodalton laminin fragment is known as the E8 fragment and is also believed to consist of the globular domains of the laminin A chain. The interaction of specific laminin fragments such as the newly discovered .about.130 kDa protein is believed to bind A.beta. in biological fluids and keep it in a soluble state.



Pancreatic carcinomas deposit laminin-5, preferably adhere to laminin-5, and migrate on the newly deposited basement membrane.  

PubMed Central

We studied the adhesion mechanism of pancreatic carcinoma using in vitro adhesion and migration assays of stable cell lines and tumors grown from these cell lines in nude mice. We also compared the results with the expression profiles of laminins and their receptors in pancreatic carcinomas to evaluate the relevance of these mechanisms in vivo. All of the cell lines preferably adhered to laminin-5, irrespective of their capability to synthesize laminin-5. Cell migration was studied in the presence of hepatocyte growth factor, as it increased the speed of migration manyfold. Herbimycin A treatment and antibodies against the beta 1 and alpha 3 integrin subunits and laminin alpha 3 chain almost entirely blocked cell migration of the BxPC-3 cell line, whereas migration was nearly unaffected by RGD peptide and only moderately inhibited by antibody against the alpha 6 integrin subunit. Indirect immunofluorescence microscopy of wounded BxPC-3 cells suggested a rapid endocytosis of alpha 3 integrin subunit in the cells at the margin of the wound and a rapid, polarized rearrangement of the alpha 6 beta 4 integrin. Especially HGF-treated cultures showed a prominent cytoplasmic reaction for laminin-5 at the margin of the wound. Xenografted cells formed tumors that produced and deposited the same laminin chains as the in vitro cultures. Frozen sections of human pancreatic carcinomas showed reactivity for laminin chains suggestive for expression of laminin-1 and laminin-5. Both xenografted tumors and human pancreatic carcinomas also showed stromal reactivity for laminin-5. Electron microscopy of the human tumors suggested that this was due to an abundant reduplication the basement-membrane-like material around the nests of malignant cells. Our results suggest that pancreatic carcinomas synthesize and deposit laminin-5 in the basement membrane in an abnormal manner. Invading cells adhere to this newly produced basement membrane and migrate on it by using the alpha 3 beta 1 integrin receptor recognizing laminin-5. Images Figure 1 Figure 2 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10

Tani, T.; Lumme, A.; Linnala, A.; Kivilaakso, E.; Kiviluoto, T.; Burgeson, R. E.; Kangas, L.; Leivo, I.; Virtanen, I.



Connectin: Cell Surface Protein That Binds Both Laminin and Actin  

NASA Astrophysics Data System (ADS)

A purified cell surface receptor protein for laminin (Mr = 70,000) isolated from mouse fibrosarcoma cells binds to actin with specificity and high affinity. This binding was demonstrated both by cosedimentation of the receptor with actin and binding of the receptor to actin immobilized on nitrocellulose filters. Specificity was demonstrated by displacement of 35S-labeled receptor by unlabeled receptor. Scatchard analysis of receptor binding to actin yielded a Kd of 6 × 10-7 M. The receptor was observed to reduce the viscosity of actin filaments. It also caused the formation of bundles of parallel filaments. This observation and the stoichiometry of binding suggest that the receptor binds along the sides of actin filaments. Based on the ability of this receptor to bind both extracellular laminin and intracellular actin, we have named this protein ``connectin.'' Connectin may be an example of a transmembrane protein that is capable of mediating the interaction of a cell with its extracellular matrix.

Brown, Susan S.; Malinoff, Herbert L.; Wicha, Max S.



Adenosine receptor activation and nociception  

Microsoft Academic Search

Adenosine and ATP exert multiple influences on pain transmission at peripheral and spinal sites. At peripheral nerve terminals in rodents, adenosine A1 receptor activation produces antinociception by decreasing, while adenosine A2 receptor activation produces pronociceptive or pain enhancing properties by increasing, cyclic AMP levels in the sensory nerve terminal. Adenosine A3 receptor activation produces pain behaviours due to the release

Jana Sawynok



Laminins promote postsynaptic maturation by an autocrine mechanism at the neuromuscular junction.  


A prominent feature of synaptic maturation at the neuromuscular junction (NMJ) is the topological transformation of the acetylcholine receptor (AChR)-rich postsynaptic membrane from an ovoid plaque into a complex array of branches. We show here that laminins play an autocrine role in promoting this transformation. Laminins containing the alpha4, alpha5, and beta2 subunits are synthesized by muscle fibers and concentrated in the small portion of the basal lamina that passes through the synaptic cleft at the NMJ. Topological maturation of AChR clusters was delayed in targeted mutant mice lacking laminin alpha5 and arrested in mutants lacking both alpha4 and alpha5. Analysis of chimeric laminins in vivo and of mutant myotubes cultured aneurally demonstrated that the laminins act directly on muscle cells to promote postsynaptic maturation. Immunohistochemical studies in vivo and in vitro along with analysis of targeted mutants provide evidence that laminin-dependent aggregation of dystroglycan in the postsynaptic membrane is a key step in synaptic maturation. Another synaptically concentrated laminin receptor, Bcam, is dispensable. Together with previous studies implicating laminins as organizers of presynaptic differentiation, these results show that laminins coordinate post- with presynaptic maturation. PMID:18794334

Nishimune, Hiroshi; Valdez, Gregorio; Jarad, George; Moulson, Casey L; Müller, Ulrich; Miner, Jeffrey H; Sanes, Joshua R



Identification of laminin binding proteins in cell membranes of a human colon adenocarcinoma cell line  

Microsoft Academic Search

The invasion of malignant cells through the basement membrane is a critical step in local infiltration and metastasis. Adhesion and invasion of malignant cells may be modulated by their receptor mediated binding to the basement membrane glycoprotein laminin. We studied the specific adhesion of human colon adenocarcinoma derived HT 29 cells to laminin and its proteolytic fragments. The major cell

A Stallmach; D Schuppan; J Dax; C Hanski; E O Riecken



Biological function of laminin-5 and pathogenic impact of its deficiency  

Microsoft Academic Search

The basement membrane glycoprotein laminin-5 is a key component of the anchoring complex connecting keratinocytes to the underlying dermis. It is secreted by keratinocytes as a cross-shaped heterotrimer of ?3, ?3 and ?2 chains and serves as a ligand of various transmembrane receptors, thereby regulating keratinocyte adhesion, motility and proliferation. In intact skin, laminin-5 provides essential links to both the

Holm Schneider; Christiane Mühle; Frederic Pacho



Mixed peptide-chitosan membranes to mimic the biological activities of a multifunctional laminin alpha1 chain LG4 module.  


Laminin alpha1 chain LG4 module is multifunctional and interacts with syndecans and integrin alpha2beta1 via AG73 (RKRLQVQLSIRT) and EF-I (DYATLQLQEGRLHFMFDLG) sites, respectively. Here, we conjugated the AG73 and EF1zz (ATLQLQEGRLHFXFDLGKGR, X: Nle) peptides on a chitosan membrane in various ratios to develop an LG4 mimic biomaterial. The AG73-chitosan membrane promoted strong cell attachment with membrane ruffling and the EF1zz-chitosan membrane promoted integrin-mediated cell adhesion with well-organized actin stress fibers. When AG73 and EF1zz were conjugated on a chitosan membrane with 1:9 molar ratio, the mixed peptide-chitosan membrane promoted the strong cell attachment and neurite outgrowth similar to that on the recombinant LG4 protein. Well-organized actin stress fibers and vinculin accumulated focal contacts were observed in the cells attached on the AG73:EF1zz (molar ratio=1:9)-chitosan membrane. These results suggest that the mixed peptide-chitosan membrane interacts with both syndecans and integrin alpha2beta1 and mimics the cell adhesion of a multifunctional LG4 protein. The mixed peptide-chitosan approach has potential as a multifunctional biomaterial for cell and tissue engineering. PMID:19124151

Hozumi, Kentaro; Yamagata, Natsumi; Otagiri, Dai; Fujimori, Chikara; Kikkawa, Yamato; Kadoya, Yuichi; Nomizu, Motoyoshi



Molecular analysis of spontaneous nephrotropic anti-laminin antibodies in an autoimmune MRL-lpr/lpr mouse.  


To explore the genetic relationship between anti-laminin and anti-DNA autoantibodies (autoAb), VH gene and gene family expression were determined among autoAb derived from an individual 6-mo-old MRL-lpr/lpr mouse. Whereas 85% of the anti-DNA Ig were identified by one of two VH family probes, 7183 and VHJ558, none of the anti-laminin antibodies (Ab) examined were recognized by these probes. Subsequent V region sequence analysis of three of the anti-laminin Ab revealed that they in fact utilized a J558 VH gene (VH50). Furthermore, FR2 and CDR2 oligonucleotide probes complementary to VH50 recognized multiple anti-laminin Ab by Northern blot analysis; the FR2 probe recognized two control anti-DNA Ab, but neither probe recognized anti-DNA Ab from the same mouse. Polymerase chain reaction amplification of MRL-lpr/lpr genomic liver DNA using primers generated from VH50 and Vk50 sequences indicated that all three anti-laminin Ig have a single replacement mutation in both their VH and Vk genes. Search of the nucleic acid databases revealed that both germline VH and Vk genes are expressed unmutated by murine lupus anti-dsDNA autoAb, previously sequenced in other laboratories. Sequence comparisons suggest that differences in anti-DNA and anti-laminin reactivity may be dependent upon somatically generated differences in the CDR3 regions of the H and L chains. The results indicate that lupus anti-laminin Ab can arise from distinct B cell populations but express the same unmutated germline V region genes as lupus anti-dsDNA autoAb. They further raise the possibility that these distinct B cell populations may be activated and expanded either: independently, by distinct Ig receptor ligands such as the Ag, laminin and DNA; or simultaneously, by a common ligand such as an anti-Id recognizing a common V region epitope. PMID:8335911

Foster, M H; Sabbaga, J; Line, S R; Thompson, K S; Barrett, K J; Madaio, M P



Binding of laminin to oral and endocarditis strains of viridans streptococci.  

PubMed Central

Attachment of bacteria to the host tissue is regarded as a crucial step in the development of many types of infections. Recent studies by us and others have shown that matrix proteins which serve as adhesion proteins for eucaryotic cells may also be recognized by some bacteria. In the present communication, we report that several strains of viridans streptococci are able to bind to laminin. Most strains isolated from blood and heart valves of patients with endocarditis expressed laminin receptors, whereas only a few of the strains isolated from the oral cavity recognized this protein. This observation indicates that laminin binding might be an important factor in the pathogenesis of viridans endocarditis. Laminin binding to two strains (Streptococcus mitis UAB594 and UAB597) isolated from patients with endocarditis was characterized further. The bacterial cells expressed a limited number of laminin receptors (4 X 10(2) to 1 X 10(3) per cell) which bound the protein in a high-affinity interaction (Kd, 40 to 80 nM). This receptor of S. mitis UAB594 was heat labile and could be solubilized from bacteria by brief digestion with trypsin. Solubilized receptors which competed with cell-bound receptors for 125I-laminin could be adsorbed on laminin-Sepharose but not on Sepharose substituted with fibrinogen or fibronectin. Comparison of laminin receptors from S. mitis with those previously described for Streptococcus pyogenes suggest that different sites in the laminin molecule are recognized by the two bacteria and hence that the corresponding receptor molecules are not identical.

Switalski, L M; Murchison, H; Timpl, R; Curtiss, R; Hook, M



Sialic acid-dependent recognition of laminin and fibrinogen by Aspergillus fumigatus conidia.  

PubMed Central

In an attempt to define the molecular basis of the adherence of Aspergillus fumigatus conidia to the host tissues, a step which might be mediated by the recognition of basement membrane laminin or fibrinogen, we analyzed the binding of these glycoproteins by flow cytometry and a microtiter plate adherence assay. Flow cytometry revealed that the binding of fluorescein isothiocyanate-labeled laminin to conidia was saturable and specific. Moreover, the ability of conidia to bind laminin increased with their maturation. Competition experiments showed a cross-reactivity between laminin and fibrinogen binding and a lack of interactions with glycosaminoglycans. In addition, the binding of laminin was not inhibited by the different adhesive synthetic peptides tested. Furthermore, the microtiter plate assay of adherence to chymotrypsin degradation products of laminin or fibrinogen purified by gel filtration suggested a unique binding site common to sequential degradation fragments or the presence of multiple binding sites on the two ligands. Therefore, the role of carbohydrates in the recognition process was investigated. Among the carbohydrates tested, constitutive of the conidial wall or of the oligosaccharide side chains of laminin and fibrinogen, only N-acetylneuraminic acid and sialyllactose inhibited the binding of these glycoproteins to conidia. In conclusion, these results strengthen the idea that the laminin and fibrinogen receptors in A. fumigatus are identical and suggest an interaction mediated by a sialic acid-specific lectin of the conidial wall.

Bouchara, J P; Sanchez, M; Chevailler, A; Marot-Leblond, A; Lissitzky, J C; Tronchin, G; Chabasse, D



Laminin Isoforms and Laminin-Producing Cells in Rat Anterior Pituitary  

PubMed Central

Laminin is a key component of the basement membrane and is involved in the structural scaffold and in cell proliferation and differentiation. Research has identified 19 laminin isoforms, which are assemblies of ?, ?, and ? chains (eg, the ?1, ?1, and ?1 chains form the laminin 111 isoform). Although laminin is known to be present in the anterior pituitary, the specific laminin isoforms have not been identified. This study used molecular biological and histochemical techniques—namely, RT-PCR, immunohistochemistry, and in situ hybridization—to identify the laminin isoforms and laminin-producing cells in rat anterior pituitary. RT-PCR showed that laminin ?1, ?3, and ?4 genes were expressed in anterior pituitary. Immunohistochemistry revealed laminin ?1 in gonadotrophs and laminin ?4 in almost all vascular endothelial cells. Laminin ?3 was seen in a subset of vascular endothelial cells. We then performed in situ hybridization to localize ? and ? chains in these cells and found that laminin ?1, ?2, and ?1 were expressed in gonadotrophs and that laminin ?1 and ?1 were expressed in endothelial cells. In conclusion, we identified gonadotroph-type (laminin 111 and 121) and vascular-type (laminin 411 and 311) laminin isoforms in rat anterior pituitary.

Ramadhani, Dini; Tsukada, Takehiro; Fujiwara, Ken; Horiguchi, Kotaro; Kikuchi, Motoshi; Yashiro, Takashi



Sulfatide-binding domain of the laminin A chain.  


A sulfatide-binding site on the globular end region of the long arm of laminin has been identified. Following proteolytic digestion with thermolysin, an intact fragment of the laminin A chain carboxyl-terminal domain exhibiting sulfatide-binding activity was isolated using gel filtration and heparin affinity chromatography. This fragment is composed of two peptides that are covalently linked by at least one disulfide bond and encompass the carboxyl-terminal 394 amino acids of the A chain. The clusters of charged residues in the primary structure of these fragments are sufficient for heparin-binding activity but not sulfatide binding since reduction and alkylation of the fragments abolished sulfatide binding under conditions in which heparin binding was retained. Thus, sulfatide binding requires an intact three-dimensional structure. The iodinated fragment bound to A2058 melanoma and T47D breast carcinoma cells and could be displaced by the unlabeled fragment. Based on incorporation of [35S] sulfate, both cell lines synthesize sulfated glycolipids that bind to laminin. In agreement with previous data that indicate a synergistic interaction of the sulfatide-binding domain with other laminin-binding sites on melanoma cells during attachment, the isolated sulfatide-binding fragment significantly inhibited interaction of labeled intact laminin with melanoma and breast carcinoma cells in direct binding assays. PMID:2373692

Taraboletti, G; Rao, C N; Krutzsch, H C; Liotta, L A; Roberts, D D



Laminin in the cutaneous basement membrane as a potential target in lewisite vesication.  


The epidermal-dermal junction has a complex molecular architecture, with numerous components playing key roles in adhesion of the epidermis to the dermis. The purpose of this study was to examine structural components of the epidermal-dermal junction as potential targets for toxicity by lewisite (dichloro(2-chlorovinyl)arsine). This was accomplished by (1) immunocytochemical mapping of laminin, type IV collagen, and bullous pemphigoid antigen (BPA) in lewisite-treated isolated perfused porcine skin flaps (IPPSF), (2) evaluation of protease activity in IPPSF blister fluid against laminin substrate from murine EHS tumor and human keratinocytes, and (3) examination of human keratinocyte laminin for direct chemical modification by lewisite. Lewisite-induced epidermal-dermal separation was localized to the lamina lucida. Localization of the separation suggested that laminin, a cysteine-rich and highly protease-sensitive adhesive glycoprotein, is a potential target for lewisite action. It was hypothesized that chemical modification of laminin directly (via chemical alkylation of laminin thiols by the arsenical) or indirectly (due to lewisite-induced cytotoxic release of proteases) could result in blister formation. Employing sensitive methodology, no evidence of proteolytic activity against EHS tumor laminin or human keratinocyte laminin was identified in the blister fluid. In addition, no evidence for direct chemical modification of laminin by lewisite was demonstrated. However, up to 36% of the thiol groups in human keratinocyte laminin immunoprecipitates was potentially available for reaction with alkylating agents. While these studies did not demonstrate a lewisite-induced chemical modification of laminin, they do not rule out the possibility that other adhesive molecules of the basement membrane are targets for lewisite action. Further evaluation of the molecular role that these binding modalities play in vesicant-induced separation may provide new insights into therapeutic and prophylactic strategies against the toxicity of such compounds and contribute to a better understanding of basement membrane biochemistry. PMID:8184425

King, J R; Peters, B P; Monteiro-Riviere, N A



Responses of cultured neural retinal cells to substratum-bound laminin and other extracellular matrix molecules.  


The responses of cultured chick embryo retinal neurons to several extracellular matrix molecules are described. Retinal cell suspensions in serum-free medium containing the "N1" supplement (J. E. Bottenstein, S. D. Skaper, S. Varon, and J. Sato, 1980, Exp. Cell Res. 125, 183-190) were seeded on tissue culture plastic surfaces pretreated with polyornithine (PORN) and with one of the factors to be tested. Substantial cell survival could be observed after 72 hr in vitro on PORN pretreated with serum or laminin, whereas most cells appeared to be degenerating on untreated PORN, PORN-fibronectin, and PORN-chondronectin. Cell attachment, although quantitatively similar for all these substrata, was temperature-dependent on serum and laminin but not on fibronectin or untreated PORN. In a short-term bioassay, neurite development was abundant on laminin, scarce on serum and fibronectin, and absent on PORN. No positive correlation between cell spreading and neurite production could be seen: cell spreading was more extensive on PORN and fibronectin than on laminin or serum, while on laminin-treated dishes, spreading was similar for neurite-bearing and non-neurite-bearing cells. Laminin effects on retinal neurons were clearly substratum dependent. When bound to tissue culture plastic, laminin showed a dose-dependent inhibitory effect on cell attachment and did not stimulate neurite development. PORN-bound laminin, on the other hand, did not affect cell attachment but caused marked stimulation of neurite development, suggesting that laminin conformation and/or the spatial distribution of active sites play an important role in the neurite-promoting function of this extracellular matrix molecule. Investigation of the embryonic retina with ELISA and immunocytochemical methods showed that laminin is present in this organ during development. Therefore, in vivo and in vitro observations are consistent with the possibility that laminin might influence neuronal development in the retina. PMID:3902534

Adler, R; Jerdan, J; Hewitt, A T



Laminin ?5 guides tissue patterning and organogenesis  

PubMed Central

Laminins (LM) are extracellular matrix molecules that contribute to and are required for the formation of basement membranes. They participate in the modulation of epithelial/mesenchymal interactions and are implicated in organogenesis and maintenance of organ homeostasis. Among the LM molecules, the LM ?5 chain (LM?5) is one of the most widely distributed LM in the developing and mature organism. Its presence in some basement membranes during embryogenesis is absolutely required for maintenance of basement membrane integrity and thus for proper organogenesis. LM?5 also regulates the expression of genes important for major biological processes, in part by repressing or activating signaling pathways, depending upon the physiological context.

Spenle, Caroline; Simon-Assmann, Patricia; Orend, Gertraud; Miner, Jeffrey H.



Laminin alters cell shape and stimulates motility and proliferation of murine skeletal myoblasts.  


Proliferating skeletal myoblasts show multiple specific responses to laminin, one of the major glycoprotein components of basement membranes. Using MM14Dy myoblasts, a myogenic cell strain derived from a normal adult mouse skeletal muscle, we show in this study that substrate-bound laminin but not other matrix proteins such as collagens or fibronectin specifically and rapidly induces the outgrowth of cell processes, resulting in bipolar, spindle-shaped cells. This effect is independent from the presence of collagens or serum, and was also observed in primary cultures of fetal mouse skeletal myoblasts. The outgrowth of cell processes on laminin is associated with a dramatic stimulation of cell motility: MM14 myoblasts migrate about five times faster on laminin than on fibronectin. In another series of experiments the effect of laminin and fibronectin on thymidine uptake and proliferation of myoblasts was tested. On top of a type I collagen substrate which was provided to ensure complete adhesion even at low doses of laminin or fibronectin, laminin stimulated myoblast proliferation and incorporation of [3H]thymidine in a dose-dependent manner. The stimulation is two- to threefold higher than on dishes coated with equivalent amounts of fibronectin and is observed both in the presence and in the absence of serum. These results suggest that laminin, a major component of the muscle basal lamina, may be actively involved in the development and regeneration of skeletal muscle. PMID:3334715

Ocalan, M; Goodman, S L; Kühl, U; Hauschka, S D; von der Mark, K



Design and activity of multifunctional fibrils using receptor-specific small peptides.  


We have designed multifunctional peptide fibrils using bioactive laminin-derived peptides and evaluated their potential as a biomedical material for tissue engineering. The Leu-Arg-Gly-Asp-Asn (LRGDN) peptide derived from laminin-111, which contains an RGD sequence bound to integrin alphavbeta3, was added to the N-terminus of the four amyloidogenic cell-adhesive laminin-derived peptides (A119: LSNIDYILIKAS, AG97: SAKVDAIGLEIV, B133: DISTKYFQMSLE, and B160: VILQQSAADIAR). The RGD-conjugated peptides were stained with Congo red and exhibited amyloid-like fibril formation in the electron microscopic. The RGD-conjugated peptides promoted human dermal fibroblasts spreading with well-organized actin stress fibers and focal contacts. Human dermal fibroblast attachment to the RGD-conjugated peptides was inhibited by anti-alphav integrin antibody. Further, cell attachment to B133 was inhibited by anti-alpha2 and anti-beta1 integrin antibodies, whereas attachment to RGD-B133 was inhibited by anti-alphav and anti-beta1 integrin antibodies. These results suggest that the RGD-conjugated peptides interact with integrin alphavbeta3 and that RGD-B133 interacts with both integrin alphavbeta3 and integrin beta1. The RGD-conjugated peptide fibrils promoted neurite outgrowth in a peptide-dependent manner. These results support that biologically active sequence-conjugated peptide fibrils interact in a receptor-specific manner with cells and promote multifunctional activities. These fibrils may have use as biological supports for cell-specific tissue engineering. PMID:19765823

Ohga, Yukiko; Katagiri, Fumihiko; Takeyama, Kazuki; Hozumi, Kentaro; Kikkawa, Yamato; Nishi, Norio; Nomizu, Motoyoshi



Degradation of laminin by human tumor cathepsin B  

Microsoft Academic Search

Cathepsin B activity, including that of a plasma membrane-associated cathepsin B, has been linked to tumor malignancy. As cathepsin B at the tumor cell surface has been hypothesized to play a role in the focal degradation of basement membrane during the metastatic cascade, we have examined the ability of human tumor cathepsin B to degrade laminin, an adhesive glycoprotein found

Tamara T. Lah; Michael R. Buck; Kenneth V. Honn; John D. Crissman; Nagesawa C. Rao; Lance A. Liotta; Bonnie F. Sloane



The disintegrin kistrin inhibits neurite extension from spiral ganglion explants cultured on laminin.  


The influence of laminin-1 (LN) and tenascin-C (TN), extracellular matrix molecules expressed spatially and temporally along the neural growth route from spiral ganglion (SG) neurons to the cochlear sensory cells, was evaluated in cultured SG explants from postnatal day 4 rats. Increasing concentrations of LN resulted in a strong, dose-dependent increase in the length of neurites and in a higher number of neural processes, while varying TN concentrations had relatively minor effects on both parameters. The results suggest differential receptor activation by LN and TN. When explants grown on LN were treated with Kistrin, an inhibitor of the alphavbeta3 integrin, the LN-induced increase in neurite length was reduced in a dose-dependent manner. However, the number of extending neurites was not affected, indicating that different receptors mediate this response, perhaps by increasing neuronal survival. PMID:11385179

Aletsee, C; Mullen, L; Kim, D; Pak, K; Brors, D; Dazert, S; Ryan, A F


Alpha 6 beta 4 integrin and newly deposited laminin-1 and laminin-5 form the adhesion mechanism of gastric carcinoma. Continuous expression of laminins but not that of collagen VII is preserved in invasive parts of the carcinomas: implications for acquisition of the invading phenotype.  

PubMed Central

We studied the expression and distribution of different laminin chains, the alpha 6 beta 4 integrin and type VII collagen, i.e., components of the epithelial adhesion complex, in gastric carcinomas and in suggested preneoplastic stages of this malignancy. Intestinal-type gastric carcinomas showed strong reactivity for laminin alpha 1, alpha 3, beta 1, and beta 3 chains, the components of laminin-1 and -5, at the interface between malignant cells and tumor stroma. The reactivities were continuous throughout the carcinomas, even in structures invading through the smooth muscle layers of the gastric wall. The expression of different laminin chains was accompanied by strong polarized reactivity for the alpha 6 beta 4 integrin, which is a receptor for both laminin-1 and laminin-5. Collagen type VII was only occasionally present at sites showing reactivity for laminin-5 and was totally absent from the cell islands invading through the gastric wall. Intestinalized gastric epithelium showed a similar expression pattern of laminins and the alpha 6 beta 4 integrin as the gastric carcinomas. Our results suggest that gastric carcinomas use the alpha 6 beta 4 integrin and newly deposited laminin-1 and -5, accompanied by the disappearance of type VII collagen, as their mechanism of adhesion during the invasion through surrounding tissues. Unlike in previous studies, the reactivity for the laminin-5 protein was not restricted to the invading cells but surrounded the malignant glandular structures throughout the tumor. Our results also show that both intestinal-type gastric carcinoma, and intestinal metaplasia mimic the gastric surface epithelium in the expression pattern of laminins and the beta 4 integrin subunit. This supports previous studies proposing a pathogenetic sequence from intestinal metaplasia to gastric carcinoma. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5

Tani, T.; Karttunen, T.; Kiviluoto, T.; Kivilaakso, E.; Burgeson, R. E.; Sipponen, P.; Virtanen, I.



Improvement of Sciatic Nerve Regeneration Using Laminin-Binding Human NGF-?  

PubMed Central

Background Sciatic nerve injuries often cause partial or total loss of motor, sensory and autonomic functions due to the axon discontinuity, degeneration, and eventual death which finally result in substantial functional loss and decreased quality of life. Nerve growth factor (NGF) plays a critical role in peripheral nerve regeneration. However, the lack of efficient NGF delivery approach limits its clinical applications. We reported here by fusing with the N-terminal domain of agrin (NtA), NGF-? could target to nerve cells and improve nerve regeneration. Methods Laminin-binding assay and sustained release assay of NGF-? fused with NtA (LBD-NGF) from laminin in vitro were carried out. The bioactivity of LBD-NGF on laminin in vitro was also measured. Using the rat sciatic nerve crush injury model, the nerve repair and functional restoration by utilizing LBD-NGF were tested. Findings LBD-NGF could specifically bind to laminin and maintain NGF activity both in vitro and in vivo. In the rat sciatic nerve crush injury model, we found that LBD-NGF could be retained and concentrated at the nerve injury sites to promote nerve repair and enhance functional restoration following nerve damages. Conclusion Fused with NtA, NGF-? could bind to laminin specifically. Since laminin is the major component of nerve extracellular matrix, laminin binding NGF could target to nerve cells and improve the repair of peripheral nerve injuries.

Sun, Wenjie; Sun, Changkai; Zhao, Hui; Lin, Hang; Han, Qianqian; Wang, Jingyu; Ma, Hui; Chen, Bing; Xiao, Zhifeng; Dai, Jianwu



The E8 subfragment of laminin promotes locomotion of myoblasts over extracellular matrix.  


The locomotion of murine myoblasts over the extracellular matrix components laminin and fibronectin was analyzed using quantitative videomicroscopy, and the organization of the cytoskeleton was observed in parallel immunofluorescence studies. Cells plated on the laminin-nidogen complex locomoted twice as fast as on laminin alone. The main form of translocation on laminin was a jerky cycle of prolonged lamellipod extension followed by rapid (approximately 200- less than 500 microh h-1) movement of the cell body into the extended lamellipod. The locomotion-stimulating activity of laminin resides in the elastase digestion fragment E8, part of the laminin long arm, while the E1-4 fragment containing the three short arms is inactive. Myoblasts moved poorly over fibronectin irrespective of whether high, intermediate, or low coating concentrations were used (approximately 5,000- approximately 10 fmol cm-2). In contrast, the locomotory responses both to laminin and to E8 peaked sharply at coating concentrations approximately 20-50 fmol cm-2 and decreased at higher concentrations. This response corresponds to that expected for a haptotactic stimulant. When cells locomoted over a mixed substrate of laminin and fibronectin, the fibronectin effects appeared to predominate. The cytoskeleton has been implicated in many cellular motile processes. Within 6 h on fibronectin many cells expressed vinculin-containing focal contacts, elaborated stress fibers and had periodically organized alpha actinin, whereas on laminin, most cells showed diffuse vinculin and alpha actinin and a fine meshlike actin cytoskeleton. We conclude that the poor locomotion of cells over fibronectin is because of the cytoskeletal stabilization it induces. PMID:2503526

Goodman, S L; Risse, G; von der Mark, K



The E8 subfragment of laminin promotes locomotion of myoblasts over extracellular matrix  

PubMed Central

The locomotion of murine myoblasts over the extracellular matrix components laminin and fibronectin was analyzed using quantitative videomicroscopy, and the organization of the cytoskeleton was observed in parallel immunofluorescence studies. Cells plated on the laminin- nidogen complex locomoted twice as fast as on laminin alone. The main form of translocation on laminin was a jerky cycle of prolonged lamellipod extension followed by rapid (approximately 200- less than 500 microh h-1) movement of the cell body into the extended lamellipod. The locomotion-stimulating activity of laminin resides in the elastase digestion fragment E8, part of the laminin long arm, while the E1-4 fragment containing the three short arms is inactive. Myoblasts moved poorly over fibronectin irrespective of whether high, intermediate, or low coating concentrations were used (approximately 5,000- approximately 10 fmol cm-2). In contrast, the locomotory responses both to laminin and to E8 peaked sharply at coating concentrations approximately 20-50 fmol cm-2 and decreased at higher concentrations. This response corresponds to that expected for a haptotactic stimulant. When cells locomoted over a mixed substrate of laminin and fibronectin, the fibronectin effects appeared to predominate. The cytoskeleton has been implicated in many cellular motile processes. Within 6 h on fibronectin many cells expressed vinculin-containing focal contacts, elaborated stress fibers and had periodically organized alpha actinin, whereas on laminin, most cells showed diffuse vinculin and alpha actinin and a fine meshlike actin cytoskeleton. We conclude that the poor locomotion of cells over fibronectin is because of the cytoskeletal stabilization it induces.



Human Anti-Laminin 5 Autoantibodies Induce Subepidermal Blisters in an Experimental Human Skin Graft Model  

Microsoft Academic Search

Patients with one form of cicatricial pemphigoid have IgG antibasement membrane autoantibodies against laminin 5 (?3?3?2). Although passive transfer of rabbit anti-laminin 5 IgG to neonatal mice has been shown to induce subepidermal blisters that mimic those in patients, it has not been possible to directly assess the pathogenic activity of human autoantibodies in this animal model because the latter

Zelmira Lazarova; Roger Hsu; Carole Yee; Kim B. Yancey



Laminin121 - Recombinant expression and interactions with integrins  

PubMed Central

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 ?1, ?2 and ?1 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 ? 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 ?6?1 and ?7?1 integrins and furthermore, laminin-121 most effectively supported neurite outgrowth. Together, this suggests that the ?2 laminins have higher affinity for integrins than the ?1 laminins.

Sasaki, Takako; Takagi, Junichi; Giudici, Camilla; Yamada, Yoshihiko; Arikawa-Hirasawa, Eri; Deutzmann, Rainer; Timpl, Rupert; Sonnenberg, Arnoud; Bachinger, Hans Peter; Tonge, David



Structural requirements for the stimulation of neurite outgrowth by two variants of laminin and their inhibition by antibodies  

PubMed Central

Laminin derived from the Engelbreth-Holm-Swarm (EHS) tumor and a lamininlike molecule synthesized by RN22 Schwannoma cells both stimulate rapid neurite outgrowth, consistent with a common neurite- promoting site. However, antilaminin antisera can only inhibit the activity of the EHS laminin. The blocking antibodies in such sera are directed against the terminal heparin-binding domain of the laminin long arm (Edgar, D., R. Timpl, and H. Thoenen. 1984. EMBO [Eur. Mol. Biol. Organ.] J. 3: 1463-1468). These epitopes are demonstrated by immunoblotting to be part of the A chain and to be absent in RN22 laminin, showing (through metabolic labeling) that the cells synthesized little if any 440-kD A chain. This indicates that the antibody inhibition was probably due to steric hindrance, a common neurite-promoting site, apparently not being antigenic in native molecules. Antibodies raised against a 25-kD proteolytic fragment derived from the long arm of laminin were then used as probes to identify other potential neurite-promoting structures. Although these antibodies do not cross-react with native laminin, they recognized the B chains of denatured EHS and RN22 molecules on immunoblots. The antibodies also bound to the large proteolytic fragment, derived from the long arm of laminin that contains the neurite-promoting site, thus inhibiting its activity. Taken together, these results point to the localization of normally nonantigenic, defined, B chain sequences within or close to the neurite-promoting site of laminin.



Protease-Activated Receptor-2 Activation  

PubMed Central

We have investigated the specific contribution of protease-activated receptor-2 (PAR2) to host defense during Porphyromonas gingivalis infection. Culture supernatants from P. gingivalis strains 33277 and W50 provoked Ca2+ mobilization in cells transfected with PAR2 (PAR2-KNRK) and desensitized the subsequent responses to PAR2-selective agonist. In addition, culture supernatants of P. gingivalis E8 (RgpA/RgpB double knockout) did not cause calcium response in PAR2-KNRK cells, evidencing the involvement of the arginine-specific cysteine proteases RgpA and RgpB in PAR2 activation by P. gingivalis. Injection of P. gingivalis into mouse subcutaneous chambers provoked an increased proteolytic activity, which was inhibited by serine protease inhibitors. Fluids collected from chambers of P. gingivalis-injected mice were able to activate PAR2 and this activation was inhibited by serine protease inhibitors. P. gingivalis inoculation into subcutaneous chambers of wild-type mice induced an inflammatory response that was inhibited by a serine protease inhibitor and was significantly reduced in PAR2-deficient mice. Finally, mice orally challenged with P. gingivalis developed alveolar bone loss, which was significantly reduced in PAR2-deficient mice at 42 and 60 days after P. gingivalis infection. We conclude that PAR2 is activated on P. gingivalis infection, in which it plays an important role in the host inflammatory response.

Holzhausen, Marinella; Spolidorio, Luis Carlos; Ellen, Richard P.; Jobin, Marie-Claude; Steinhoff, Martin; Andrade-Gordon, Patricia; Vergnolle, Nathalie



Pathology is alleviated by doxycycline in a laminin-?2-null model of congenital muscular dystrophy  

PubMed Central

Objective Congenital muscular dystrophy type 1A (MDC1A) is an autosomal recessive disease that is caused by loss-of-function mutations in the laminin-?2 gene and results in motor nerve and skeletal muscle dysfunction. In a previous study, we used genetic modifications to show that inappropriate induction of apoptosis was a significant contributor to pathogenesis in a laminin-?2-deficient mouse model of MDC1A. To identify a possible pharmacological therapy for laminin-?2-deficency, this study was designed to determine if treatment with minocycline or doxycycline, which are tetracycline derivatives reported to have anti-apoptotic effects in mammals, would significantly increase lifespan and improve neuromuscular function in laminin-?2-deficient mice. Methods Mice that were homozygous for a targeted, inactivating mutation of the laminin-?2-gene were placed into control, minocycline-treated, or doxycycline-treated groups. Drug treatment began within two weeks of birth and the progression of disease was followed over time using behavioral, growth, histological, and molecular assays. Results We found that treatment with either minocycline or doxycycline increased the median lifespan of laminin-?2-null mice from ?32 days to ?70 days. Furthermore, doxycycline improved postnatal growth rate and delayed the onset of hindlimb paralysis. Doxycycline-treated laminin-?2-deficient muscles had increased Akt phosphorylation, decreased inflammation, and decreased levels of Bax protein, TUNEL-positive myonuclei, and activated caspase-3. Interpretation Doxycycline or other drugs with similar functional profiles may be a possible route to improving neuromuscular dysfunction due to laminin-?2-deficiency.

Girgenrath, Mahasweta; Beermann, Mary Lou; Vishnudas, Vivek K.; Homma, Sachiko; Miller, Jeffrey Boone



Protease activated receptors: theme and variations  

Microsoft Academic Search

The four PAR family members are G protein coupled receptors that are normally activated by proteolytic exposure of an occult tethered ligand. Three of the family members are thrombin receptors. The fourth (PAR2) is not activated by thrombin, but can be activated by other proteases, including trypsin, tryptase and Factor Xa. This review focuses on recent information about the manner

Peter J O'Brien; Marina Molino; Mark Kahn; Lawrence F Brass



Calcium sensing receptor activators: calcimimetics.  


The calcium sensing receptor (CaR) is a G protein-coupled receptor (GPCR) that plays a fundamental role in serum calcium homeostasis. The CaR is expressed on the chief cells of the parathyroid gland and is responsible for controlling the secretion of parathyroid hormone (PTH). PTH acts on several organs including the bone, kidney, and intestine to tightly regulate the concentration of serum calcium. Substances other than calcium that activate the CaR are referred to as calcimimetics. Calcimimetics that bind to the CaR as agonists are referred to as type I. Type II calcimimetics bind to a site that is distinct from the physiological ligand and function as positive allosteric modulators of the CaR. Type II calcimimetics amplify the sensitivity of the CaR to serum calcium and are thus able to lower the concentration of serum PTH. Calcimimetics are being pursued as therapeutics for the treatment of disorders that are characterized by elevated levels of PTH such as primary and secondary hyperparathyroidism (primary HPT and secondary HPT). In this review, we provide an overview of key results in the discovery of cinacalcet HCl (Sensipar in the US, Mimpara in Europe). In addition, other recently disclosed type II calcimimetics are discussed. PMID:18220738

Harrington, Paul E; Fotsch, Christopher



Purification and Characterization of Mammalian Integrins Expressed by a Rat Neuronal Cell Line (PC12): Evidence That They Function as t\\/13 Heterodimeric Receptors for Laminin and Type IV Collagen  

Microsoft Academic Search

Cells of the rat neuronal line, PC12, adhere well to substrates coated with laminin and type IV col- lagen, but attach poorly to fibronectin. Adhesion and neurite extension in response to these extracellular ma- trix proteins are inhibited by Fab fragments of an an- tiserum (anti-ECMR) that recognizes PC12 cell sur- face integrin subunits of M~ 120,000, 140,000, and 180,000

Kevin J. Tomaselli; Caroline H. Damsky; Louis E Reichardt


Turnover of fibronectin and laminin by alveolar epithelial cells.  


Type II pulmonary epithelial cells in primary culture synthesize and assemble a multicomponent extracellular matrix which exhibits biological activity in vitro. Simultaneously, the pneumocytes degrade components of the underlying matrix, such that matrix composition may be determined by the balance of synthesis and turnover. The present work defines turnover of the specific matrix glycoproteins, fibronectin and laminin, both in the type II cell and in its extracellular matrix. Pulse-chase experiments demonstrate that both fibronectin and laminin, identified by immunoprecipitation, turn over rapidly in the cell and extracellular matrix compartments, with half-lives < 10 h. In the cell compartment, initial rates of laminin turnover are more rapid than those of fibronectin on culture day 2, but these rates are similar on day 6. Matrix fibronectin also turns over rapidly, with similar rates on day 2 and day 6. During the chase interval, small but increasing amounts of immunoprecipitable fibronectin are detected in the medium, suggesting that a portion of the glycoprotein may be released to the extracellular compartment, rather than degraded. Alternatively, release of immunoreactive glycoprotein may involve ongoing processing and secretion of residual radiolabeled fibronectin by the cells. The results suggest that matrix composition may be determined by turnover, as well as synthesis, of its components. PMID:8572238

Dunsmore, S E; Martinez-Williams, C; Goodman, R A; Rannels, D E



The activation of the CGRP receptor.  


The CGRP (calcitonin gene-related peptide) receptor is a family B GPCR (G-protein-coupled receptor). It consists of a GPCR, CLR (calcitonin receptor-like receptor) and an accessory protein, RAMP1 (receptor activity modifying protein 1). RAMP1 is needed for CGRP binding and also cell-surface expression of CLR. CLR is an example of a family B GPCR. Unlike family A GPCRs, little is known about how these receptors are activated by their endogenous ligands. This review considers what is known about the activation of family B GPCRs and then considers how this might be applied to CLR, particularly in light of new knowledge of the crystal structures of family A GPCRs. PMID:23356280

Barwell, James; Wheatley, Mark; Conner, Alex C; Taddese, Bruck; Vohra, Shabana; Reynolds, Christopher A; Poyner, David R



Activation of the rat kidney mineralocorticoid receptor  

SciTech Connect

Activation of the rat kidney mineralocorticoid receptor was investigated using DEAE-cellulose, DNA-cellulose and gel permeation chromatography. Specific labeling of the mineralocorticoid receptor was achieved by labeling with (/sup 3/H)aldosterone in the presence of the pure glucocorticoid RU28362. The specificity of labeling was confirmed by the lack of immunoreactivity of (/sup 3/H)aldosterone-labeled material with the monoclonal antiglucocorticoid receptor antibody BUGR-1. The unactivated aldosterone-mineralocorticoid receptor complex did not bind to DNA-cellulose, was eluted from DEAE-cellulose at relatively high salt (190 mM KCl) concentration and had an apparent Stokes radius when chromatographed on Sephacryl S300 of 6.3 nm. After activation (at 25/sup 0/C for 20 min), the aldosterone-mineralocorticoid receptor complex had increased affinity for DNA-cellulose, decreased affinity for DEAE-cellulose and appeared as a smaller complex when chromatographed on Sephacryl S300. These changes were blocked by sodium molybdate. The authors results indicate that activation of the rat kidney mineralocorticoid receptor is analogous to activation of the glucocorticoid receptor and suggest that activation of the mineralocorticoid receptor involves dissociation of a multimeric receptor form.

Eisen, L.P.; Harmon, J.M.



Deflazacort increases laminin expression and myogenic repair, and induces early persistent functional gain in mdx mouse muscular dystrophy.  


Deflazacort slows the progress of Duchenne muscular dystrophy (DMD) with fewer side effects than prednisone. In mdx mice, deflazacort treatment augments repair and growth of new muscle fibers. We tested the hypothesis that deflazacort improves muscle function and promotes repair by increasing myogenic cell proliferation and fiber differentiation. mdx mice (3.5 weeks old) were treated with deflazacort (1.2 mg/kg) or vehicle for 4 weeks. Forelimb grip strength was measured. After 4 weeks, the right tibialis anterior muscle (TA) was crush injured to induce synchronous regeneration. DNA was labeled using different markers 24 and 2 h before collecting tissues 4 days after injury. The expression of creatine kinase (CK) isoforms, laminin-2 (merosin) mRNA and protein, and proliferation by myogenic cells were measured and satellite cells were identified by immunolocalization of c-met receptor. Peak grip strength increased 15% within 10 days of treatment, and was maintained up to 6 weeks after the end of treatment in a second experiment. Expression of CK MM in the regenerating TA rose from 46% to 55% of total CK activity after deflazacort treatment. Satellite cells were more numerous and appeared earlier on new fibers, in concert with a threefold increase in proliferation by myogenin+ (but not MyoD+) myoblasts. alpha2-Laminin mRNA expression and protein increased 1.3-5.5-fold relative to MM CK in regenerating and dystrophic TA, respectively. These studies support the hypothesis that deflazacort promotes functional gains, myogenic differentiation, myoblast fusion, and laminin expression in regenerating dystrophic muscle. The potential to augment precursor specification, strength, and possible membrane stability may be useful in directing long-term benefits for DMD patients and short-term amplification of precursors prior to myoblast transfer. PMID:11038071

Anderson, J E; Weber, M; Vargas, C


Reductions in laminin ?2 mRNA translation are responsible for impaired IGFBP-5-mediated mesangial cell migration in the presence of high glucose  

PubMed Central

Insulin-like growth factor binding protein-5 (IGFBP-5) mediates mesangial cell migration through activation of cdc42, and laminin421 binding to ?6?1-integrin (Berfield AK, Hansen KM, Abrass CK. Am J Physiol Cell Physiol 291: C589–C599, 2006). Because glomerular expression of laminin ?2 is reduced in diabetic rats (Abrass CK, Spicer D, Berfield AK, St. John PL, Abrahamson DR. Am J Pathol 151: 1131–1140, 1997), we directly examined the effect of hyperglycemia on mesangial cell migration and laminin ?2 expression. Migration mediated by IGFBP-5 is impaired in the presence of 25 mM glucose. This reduction in migration was found to result from a loss in mesangial cell synthesis of laminin421, and IGFBP-5-induced migration could be restored by replacing laminin421. Additional studies showed that there was selective reduction in mRNA translation of laminin ?2 in the presence of high glucose. Preserved synthesis of laminin ?1 indicates that not all proteins are reduced by high glucose and confirms prior data showing that laminin411 cannot substitute for laminin421 in IGFBP-5-mediated migration. Given the importance of mesangial migration in the reparative response to diabetes-associated mesangiolysis, these findings provide new insights into abnormalities associated with diabetic nephropathy and the potential importance of differential control of protein translation in determination of alterations of protein expression.

Schaeffer, Valerie; Hansen, Kim M.; Morris, David R.



The C-terminal Region of Laminin ? Chains Modulates the Integrin Binding Affinities of Laminins*S?  

PubMed Central

Laminins are major cell-adhesive proteins in basement membranes that are capable of binding to integrins. Laminins consist of three chains (?, ?, and ?), in which three laminin globular modules in the ? chain and the Glu residue in the C-terminal tail of the ? chain have been shown to be prerequisites for binding to integrins. However, it remains unknown whether any part of the ? chain is involved in laminin-integrin interactions. We compared the binding affinities of pairs of laminin isoforms containing the ?1 or ?2 chain toward a panel of laminin-binding integrins, and we found that ?2 chain-containing laminins (?2-laminins) bound more avidly to ?3?1 and ?7X2?1 integrins than ?1 chain-containing laminins (?1-laminins), whereas ?6?1, ?6?4, and ?7X1?1 integrins did not show any preference toward ?2-laminins. Because ?3?1 contains the “X2-type” variable region in the ?3 subunit and ?6?1 and ?6?4 contain the “X1-type” region in the ?6 subunit, we hypothesized that only integrins containing the X2-type region were capable of discriminating between ?1-laminins and ?2-laminins. In support of this possibility, a putative X2-type variant of ?6?1 was produced and found to bind preferentially to ?2-laminins. Production of a series of swap mutants between the ?1 and ?2 chains revealed that the C-terminal 20 amino acids in the coiled-coil domain were responsible for the enhanced integrin binding by ?2-laminins. Taken together, the results provide evidence that the C-terminal region of ? chains is involved in laminin recognition by integrins and modulates the binding affinities of laminins toward X2-type integrins.

Taniguchi, Yukimasa; Ido, Hiroyuki; Sanzen, Noriko; Hayashi, Maria; Sato-Nishiuchi, Ryoko; Futaki, Sugiko; Sekiguchi, Kiyotoshi



Proteinase-Activated Receptors and Arthritis  

Microsoft Academic Search

\\u000a The novel family of proteinase-activated receptors (PARs) is activated through proteolytic cleavage by serine proteinases.\\u000a This family of G protein-coupled receptors and their activating enzymes are found widely throughout the body. It has been\\u000a known for some time that during arthritic conditions, high levels of serine proteinases are released from joint tissue and\\u000a contribute to joint degradation. Expression of PARs

Fiona A. Russell; Jason J. McDougall


Alignment and composition of laminin-polycaprolactone nanofiber blends enhance peripheral nerve regeneration.  


Peripheral nerve transection occurs commonly in traumatic injury, causing deficits distal to the injury site. Conduits for repair currently on the market are hollow tubes; however, they often fail due to slow regeneration over long gaps. To facilitate increased regeneration speed and functional recovery, the ideal conduit should provide biochemically relevant signals and physical guidance cues, thus playing an active role in regeneration. To that end, laminin and laminin-polycaprolactone (PCL) blend nanofibers were fabricated to mimic peripheral nerve basement membrane. In vitro assays established 10% (wt) laminin content is sufficient to retain neurite-promoting effects of laminin. In addition, modified collector plate design to introduce an insulating gap enabled the fabrication of aligned nanofibers. The effects of laminin content and fiber orientation were evaluated in rat tibial nerve defect model. The lumens of conduits were filled with nanofiber meshes of varying laminin content and alignment to assess changes in motor and sensory recovery. Retrograde nerve conduction speed at 6 weeks was significantly faster in animals receiving aligned nanofiber conduits than in those receiving random nanofiber conduits. Animals receiving nanofiber-filled conduits showed some conduction in both anterograde and retrograde directions, whereas in animals receiving hollow conduits, no impulse conduction was detected. Aligned PCL nanofibers significantly improved motor function; aligned laminin blend nanofibers yielded the best sensory function recovery. In both cases, nanofiber-filled conduits resulted in better functional recovery than hollow conduits. These studies provide a firm foundation for the use of natural-synthetic blend electrospun nanofibers to enhance existing hollow nerve guidance conduits. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2011. PMID:22106069

Neal, Rebekah A; Tholpady, Sunil S; Foley, Patricia L; Swami, Nathan; Ogle, Roy C; Botchwey, Edward A



Factors Modulating Estrogen Receptor Activity.  

National Technical Information Service (NTIS)

Our goal to elucidate the molecular mechanisms of transcriptional regulation by the estrogen receptor alpha (ER) in breast cancer. ER is a hormone-dependent transcription factor involved in the regulation of both normal and malignant breast cell growth by...

M. Garabedian



Antibodies to laminin in Chagas' disease  

PubMed Central

We have found that sera from humans with Chagas' disease and Rhesus monkeys infected with Trypanosoma cruzi contain IgM and IgG antibodies, which react with structures in a variety of connective tissues. These antibodies react with laminin but not with various other purified connective tissue components like collagen types I, III, IV, and V, fibronectin, heparan sulfate (BM-1) proteoglycan, or chondronectin. The tissue-reacting antibodies were isolated by absorption to a laminin- Sepharose column. The bound fraction contained all the tissue-reacting antibodies. These antibodies strongly stained trypomastigotes and amastigotes, but weakly stained epimastigotes. These studies show that sera from T. cruzi-infected primates contain antilaminin antibodies, which may be produced by those host in response to a laminin-like molecule present in the parasite.



Changes of laminin ?2 chain expression in congenital muscular dystrophy  

Microsoft Academic Search

We studied the distribution of laminin ?2 chain in the skeletal muscle basement membrane of 16 patients with congenital muscular dystrophy (CMD) by immunohistochemistry. A dramatic reduction in the laminin ?2 staining was observed in four patients with classical merosin-negative CMD. A moderate reduction of laminin ?2 labelling was observed in four patients with partial merosin deficiency and two patients

Ronald D. Cohn; Ralf Herrmann; Ulla M. Wewer; Thomas Voit



Laminin is produced by early rat astrocytes in primary culture  

Microsoft Academic Search

The production of laminin by early rat astrocytes in primary culture was investigated by double immunoffuorescence staining for laminin and the gliat fibrillary acidic protein (GFAP), a defined astrocyte marker. In early cultures (3 d in vitro; 3 DIV) cytoplasmic laminin was detected in all the GFAP-positive cells which formed the major population (80%) of the nonneuronal cells present in

P. Liesi; D. DAHL; A. VAHERI



Amyloid ? peptide oligomers directly activate NMDA receptors.  


Amyloid beta (A?) oligomers accumulate in the brain tissue of Alzheimer disease patients and are related to disease pathogenesis. The precise mechanisms by which A? oligomers cause neurotoxicity remain unknown. We recently reported that A? oligomers cause intracellular Ca(2+) overload and neuronal death that can be prevented by NMDA receptor antagonists. This study investigated whether A? oligomers directly activated NMDA receptors (NMDARs) using NR1/NR2A and NR1/NR2B receptors that were heterologously expressed in Xenopus laevis oocytes. Indeed, A? oligomers induced inward non-desensitizing currents that were blocked in the presence of the NMDA receptor antagonists memantine, APV, and MK-801. Intriguingly, the amplitude of the responses to A? oligomers was greater for NR1/NR2A heteromers than for NR1/NR2B heteromers expressed in oocytes. Consistent with these findings, we observed that the increase in the cytosolic concentration of Ca(2+) induced by A? oligomers in cortical neurons is prevented by AP5, a broad spectrum NMDA receptor antagonist, but slightly attenuated by ifenprodil which blocks receptors with the NR2B subunit. Together, these results indicate that A? oligomers directly activate NMDA receptors, particularly those with the NR2A subunit, and further suggest that drugs that attenuate the activity of such receptors may prevent A? damage to neurons in Alzheimer?s disease. PMID:21349580

Texidó, Laura; Martín-Satué, Mireia; Alberdi, Elena; Solsona, Carles; Matute, Carlos



Laminin database: a tool to retrieve high-throughput and curated data for studies on laminins.  


The Laminin(LM)-database, hosted at, is the first database focusing a non-collagenous extracellular matrix protein family, the LMs. Part of the knowledge available in this website is automatically retrieved, whereas a significant amount of information is curated and annotated, thus placing LM-database beyond a simple repository of data. In its home page, an overview of the rationale for the database is seen and readers can access a tutorial to facilitate navigation in the website, which in turn is presented with tabs subdivided into LMs, receptors, extracellular binding and other related proteins. Each tab opens into a given LM or LM-related molecule, where the reader finds a series of further tabs for 'protein', 'gene structure', 'gene expression' and 'tissue distribution' and 'therapy'. Data are separated as a function of species, comprising Homo sapiens, Mus musculus and Rattus novergicus. Furthermore, there is specific tab displaying the LM nomenclatures. In another tab, a direct link to PubMed, which can be then consulted in a specific way, in terms of the biological functions of each molecule, knockout animals and genetic diseases, immune response and lymphomas/leukemias. LM-database will hopefully be a relevant tool for retrieving information concerning LMs in health and disease, particularly regarding the hemopoietic system. PMID:21087995

Golbert, Daiane C F; Linhares-Lacerda, Leandra; Almeida, Luiz G; Correa-de-Santana, Eliane; de Oliveira, Alice R; Mundstein, Alex S; Savino, Wilson; de Vasconcelos, Ana T R



Laminins, derivatives, and compositions including same and methods for their therapeutic use  

US Patent & Trademark Office Database

In various embodiments, the present disclosure provides a method of treating a subject using laminin or a composition that includes laminin. In one embodiment, the method is used to enhance muscle regeneration, maintenance, or repair in a subject. In another embodiment, the method is used to promote wound healing. The method, in yet another embodiment, is used to prevent or reduce muscle damage or injury. In specific implementations of these methods, the laminin or composition that includes laminin is administered in a therapeutically effective amount. In some implementations, the laminin is a complete laminin protein. In other implementations, the laminin is a laminin fragment, a laminin derivative, or a laminin analogue.

Burkin; Dean J. (Sparks, NV); Rooney; Jachinta E. (Reno, NV)



The regulation of lymphocyte activation by inhibitory receptors  

Microsoft Academic Search

The ability of activating immune recognition receptors on lymphocytes to regulate cellular activation and function can be profoundly altered by co-stimulation with inhibitory receptors. Inhibitory receptors, such as the MHC-recognizing inhibitory receptors expressed on NK cells and subpopulations of activated T cells, can fully block the generation of any cytotoxic function by targeting proximal signals. Inhibitory Fc receptors on B

Paul J Leibson



Activation of caspases by death receptors.  


Caspases form a family of cysteine proteases which are implicated in multiple forms of apoptosis. Their role has been intensively studied during apoptosis triggered by death receptors, but their exact biological functions and mechanisms of activation remain unclear. Here, we summarize the knowledge about the signal transduction pathways of death receptors leading to the activation of caspases as the key effector components of apoptotic cell death. PMID:9889414

Bantel, H; Brüning, T; Schulze-Osthoff, K



Central mineralocorticoid receptors, sympathetic activity, and hypertension  

Microsoft Academic Search

Recent evidence highlighting the presence of corticosteroid receptors in the central nervous system has prompted further investigation\\u000a regarding their role in the pathogenesis of hypertension. The sympathetic nervous system, an important factor in the pathogenesis\\u000a of hypertension, is influenced by sodium and volume status. Activation of central nervous system mineralocorticoid receptors\\u000a is known to affect sympathetic activity, although the processes

Frances McManus; Scott M. MacKenzie; E. Marie Freel



Renal collecting system growth and function depend upon embryonic ?1 laminin expression  

PubMed Central

In order to understand the functions of laminins in the renal collecting system, the Lamc1 gene was inactivated in the developing mouse ureteric bud (UB). Embryos bearing null alleles exhibited laminin deficiency prior to mesenchymal tubular induction and either failed to develop a UB with involution of the mesenchyme, or developed small kidneys with decreased proliferation and branching, delayed renal vesicle formation and postnatal emergence of a water transport deficit. Embryonic day 12.5 kidneys revealed an almost complete absence of basement membrane proteins and reduced levels of ?6 integrin and FGF2. mRNA levels for fibroblast growth factor 2 (FGF2) and mediators of the GDNF/RET and WNT11 signaling pathway were also decreased. Furthermore, collecting duct cells derived from laminin-deficient kidneys and grown in collagen gels were found to proliferate and branch slowly. The laminin-deficient cells exhibited decreased activation of growth factor- and integrin-dependent pathways, whereas heparin lyase-treated and ?1 integrin-null cells exhibited more selective decreases. Collectively, these data support a requirement of ?1 laminins for assembly of the collecting duct system basement membrane, in which immobilized ligands act as solid-phase agonists to promote branching morphogenesis, growth and water transport functions.

Yang, Dong-Hua; McKee, Karen K.; Chen, Zu-Lin; Mernaugh, Glenda; Strickland, Sidney; Zent, Roy; Yurchenco, Peter D.



Amyloid-? Peptides Activate ?1-Adrenergic Cardiovascular Receptors.  


Alzheimer disease features amyloid-? (A?) peptide deposition in brain and blood vessels and is associated with hypertension. A? peptide can cause vasoconstriction and endothelial dysfunction. We observed that A? peptides exert a chronotropic effect in neonatal cardiomyocytes, similar to ?1-adrenergic receptor autoantibodies that we described earlier. Recently, it was shown that ?1-adrenergic receptor could impair blood-brain flow. We hypothesized that A? peptides might elicit a signal transduction pathway in vascular cells, induced by ?1-adrenergic receptor activation. A? (25-35) and A? (10-35) induced a positive chronotropic effect in the cardiac contraction assay (28.75±1.15 and 29.40±0.98 bpm), which was attenuated by ?1-adrenergic receptor blockers (urapidil, 1.53±1.17 bpm; prazosin, 0.30±0.96 bpm). Both A? peptides induced an intracellular calcium release in vascular smooth muscle cells. Chronotropic activity and calcium response elicited by A? (25-35) were blocked with peptides corresponding to the first extracellular loop of the ?1-adrenergic receptor. We observed an induction of extracellular-regulated kinase 1/2 phosphorylation by A? (25-35) in Chinese hamster ovary cells overexpressing ?1-adrenergic receptor, vascular smooth muscle cells, and cardiomyocytes. We generated an activation-state-sensitive ?1-adrenergic receptor antibody and visualized activation of the ?1-adrenergic receptor by A? peptide. A? (25-35) induced vasoconstriction of mouse aortic rings and in coronary arteries in Langendorff-perfused rat hearts that resulted in decreased coronary flow. Both effects could be reversed by ?1-adrenergic receptor blockade. Our data are relevant to the association between Alzheimer disease and hypertension. They may explain impairment of vascular responses by A? and could have therapeutic implications. PMID:24001898

Haase, Nadine; Herse, Florian; Spallek, Bastian; Haase, Hannelore; Morano, Ingo; Qadri, Fatimunnisa; Szijártó, István A; Rohm, Ilonka; Yilmaz, Atilla; Warrington, Junie P; Ryan, Michael J; Gollasch, Maik; Müller, Dominik N; Dechend, Ralf; Wallukat, Gerd



Beta1 integrin and alpha-dystroglycan binding sites are localized to different laminin-G-domain-like (LG) modules within the laminin alpha5 chain G domain.  

PubMed Central

Laminins are a group of extracellular-matrix proteins important in development and disease. They are heterotrimers, and specific domains in the different chains have specialized functions. The G domain of the alpha5 chain has now been produced in transfected mammalian cells as single modules and two tandem arrays, alpha5LG1-3 and alpha5LG4-5 (LG is laminin G domain-like). Using these fragments we produced specific polyclonal antibodies functional in immunoblotting and immunofluorescence studies and in solid-phase assays. Both alpha5LG tandem arrays had physiologically relevant affinities for sulphated ligands such as heparin and sulphatides. Cells adhered to these fragments and acquired a spread morphology when plated on alpha5LG1-3. Binding of integrins alpha3beta1 and alpha6beta1 was localized to the alpha5LG1-3 modules, and alpha-dystroglycan binding was localized to the alpha5LG4-5 modules, thus locating these activities to different LG modules within the laminin alpha5 G domain. However, both these activities were of relatively low affinity, indicating that integrin-mediated cell adhesion to the laminin 10/11 alpha5G domain depends on contributions from the other chains of the heterotrimer and that high-affinity alpha-dystroglycan binding could be dependent on specific Ca(2+)-ion-co-ordinating amino acids absent from alpha5LG4-5.

Yu, Hao; Talts, Jan F



Laminin5 in Epithelial Tumour Invasion  

Microsoft Academic Search

Laminin-5 (LN-5), consisting of 3-, 3-, and 2-chains, is a component of the cell adhesion complex containing hemidesmosomes and anchoring fibrils. This protein is a major constituent of the extracellular matrix and has recently proved to be an invasion marker for epithelial cells in many immunohistochemical surveys, indicating that it is frequently expressed in the invading edges of epithelial tumour

Masahiko Katayama; Kiyotoshi Sekiguchi



Modulating Estrogen Receptor-related Receptor-? Activity Inhibits Cell Proliferation*  

PubMed Central

High expression of the estrogen receptor-related receptor (ERR)-? in human tumors is correlated to a poor prognosis, suggesting an involvement of the receptor in cell proliferation. In this study, we show that a synthetic compound (XCT790) that modulates the activity of ERR? reduces the proliferation of various cell lines and blocks the G1/S transition of the cell cycle in an ERR?-dependent manner. XCT790 induces, in a p53-independent manner, the expression of the cell cycle inhibitor p21waf/cip1 at the protein, mRNA, and promoter level, leading to an accumulation of hypophosphorylated Rb. Finally, XCT790 reduces cell tumorigenicity in Nude mice.

Bianco, Stephanie; Lanvin, Olivia; Tribollet, Violaine; Macari, Claire; North, Sophie; Vanacker, Jean-Marc



Interdomain movements in metabotropic glutamate receptor activation.  


Many cell surface receptors are multimeric proteins, composed of several structural domains, some involved in ligand recognition, whereas others are responsible for signal transduction. In most cases, the mechanism of how ligand interaction in the extracellular domains leads to the activation of effector domains remains largely unknown. Here we examined how the extracellular ligand binding to the venus flytrap (VFT) domains of the dimeric metabotropic glutamate receptors activate the seven transmembrane (7TM) domains responsible for G protein activation. These two domains are interconnected by a cysteine-rich domain (CRD). We show that any of the four disulfide bridges of the CRD are required for the allosteric coupling between the VFT and the 7TM domains. More importantly, we show that a specific association of the two CRDs corresponds to the active state of the receptor. Indeed, a specific crosslinking of the CRDs with intersubunit disulfide bridges leads to fully constitutively active receptors, no longer activated by agonists nor by allosteric modulators. These data demonstrate that intersubunit movement at the level of the CRDs represents a key step in metabotropic glutamate receptor activation. PMID:21896740

Huang, Siluo; Cao, Jianhua; Jiang, Ming; Labesse, Gilles; Liu, Jianfeng; Pin, Jean-Philippe; Rondard, Philippe



Interdomain movements in metabotropic glutamate receptor activation  

PubMed Central

Many cell surface receptors are multimeric proteins, composed of several structural domains, some involved in ligand recognition, whereas others are responsible for signal transduction. In most cases, the mechanism of how ligand interaction in the extracellular domains leads to the activation of effector domains remains largely unknown. Here we examined how the extracellular ligand binding to the venus flytrap (VFT) domains of the dimeric metabotropic glutamate receptors activate the seven transmembrane (7TM) domains responsible for G protein activation. These two domains are interconnected by a cysteine-rich domain (CRD). We show that any of the four disulfide bridges of the CRD are required for the allosteric coupling between the VFT and the 7TM domains. More importantly, we show that a specific association of the two CRDs corresponds to the active state of the receptor. Indeed, a specific crosslinking of the CRDs with intersubunit disulfide bridges leads to fully constitutively active receptors, no longer activated by agonists nor by allosteric modulators. These data demonstrate that intersubunit movement at the level of the CRDs represents a key step in metabotropic glutamate receptor activation.

Huang, Siluo; Cao, Jianhua; Jiang, Ming; Labesse, Gilles; Liu, Jianfeng; Pin, Jean-Philippe; Rondard, Philippe



Keratinocyte motility induced by TGF-beta1 is accompanied by dramatic changes in cellular interactions with laminin 5.  


Transforming growth factor-beta1 (TGF-beta1) has the ability to induce epithelial cell migration while stopping proliferation. In this study, we show that, concomitant to promoting migration of normal human keratinocytes in vitro, TGF-beta1 induced a marked decrease in their adhesion capacity to processed alpha3-containing laminin 5-coated surfaces. Indeed, the expression levels of alpha3 and alpha6 integrin subunit mRNA and protein, as well as the cell surface alpha3beta1 and alpha6beta4 integrins, were down-regulated. Recent studies showed that keratinocytes over express and deposit laminin 5 during migration and we have shown that laminin 5 found in the matrix of TGF-beta1 induced migrating keratinocytes is present in its unprocessed form [Décline and Rousselle, 2001: J. Cell Sci. 114:811-823]. We show here that TGF-beta1 treatment of the cells promoted a significant increase in their adhesion to the alpha3 chain carboxy-terminal LG4/5 subdomain and that this interaction is likely to be mediated by a heparan sulfate proteoglycan type of receptor. Our results indicate that alpha6beta4 and alpha3beta1 integrin interactions with laminin 5 are diminished during migration while a specific interaction occurs between an additional cellular receptor and the alpha3 LG4/5 module present on unprocessed laminin 5. PMID:12451596

Décline, Françoise; Okamoto, Osamu; Mallein-Gerin, Frédéric; Helbert, Bruno; Bernaud, Janine; Rigal, Dominique; Rousselle, Patricia



Co-activation of epidermal growth factor receptor and c-MET defines a distinct subset of lung adenocarcinomas.  


Epidermal growth factor receptor (EGFR) and MET are molecular targets for lung cancer treatment. The relationships between expression, activation, and gene abnormalities of these two targets are currently unclear. Here, we demonstrate that a panel of 40 lung cancer cell lines could be classified into two groups. Group I was characterized by (1) high phosphorylations of MET and EGFR, (2) frequent mutation or amplification of EGFR, MET, and human epidermal growth factor receptor-2 (HER2), (3) high expressions of bronchial epithelial markers (thyroid transcription factor-1 (TTF-1), MUC1, and Cytokeratin 7 (CK7)); and (4) high expressions of MET, human epidermal growth factor receptor-3, E-cadherin, cyclooxygenase-2, and laminin gamma2. In contrast, Group II exhibited little or no phosphorylation of MET and EGFR; no mutation or amplification of EGFR, MET, and HER2; were triple-negative for TTF-1, MUC1, and CK7; and showed high expressions of vimentin, fibroblast growth factor receptor-1, and transcription factor 8. Importantly, Group I was more sensitive to gefitinib and more resistant to cisplatin and paclitaxel than Group II. The clinical relevance was confirmed in publicly available data on 442 primary lung adenocarcinoma patients; survival benefits by postoperative chemotherapy were seen in only patients with tumors corresponding to Group II. Overall, co-activation of EGFR and MET defines a distinct subgroup of lung carcinoma with characteristic genetic abnormalities, gene expression pattern, and response to chemotherapeutic reagents. PMID:20934974

Matsubara, Daisuke; Ishikawa, Shumpei; Sachiko, Oguni; Aburatani, Hiroyuki; Fukayama, Masashi; Niki, Toshiro



Fatty acid activation of peroxisome proliferator-activated receptor (PPAR)  

Microsoft Academic Search

Peroxisome proliferators such as clofibric acid, nafenopin, and WY-14,643 have been shown to activate peroxisome proliferator-activated receptor (PPAR), a member of the steroid nuclear receptor superfamily. We have cloned the cDNA from rat that is homologous to that from mouse, which encodes a 97% similar protein. To search for physiologically occurring activators, we established a transcriptional transaction assay by stably

Carlos Bocos; Martin Göttlicher; Katy Gearing; Carol Banner; Eva Enmark; Michèle Teboul; Anja Crickmore; Jan-Åke Gustaffson



Keratinocyte-Targeted Expression of Human Laminin ?2 Rescues Skin Blistering and Early Lethality of Laminin ?2 Deficient Mice  

PubMed Central

Laminin-332 is a heterotrimeric basement membrane component comprised of the ?3, ß3, and ?2 laminin chains. Laminin-332 modulates epithelial cell processes, such as adhesion, migration, and differentiation and is prominent in many embryonic and adult tissues. In skin, laminin-332 is secreted by keratinocytes and is a key component of hemidesmosomes connecting the keratinocytes to the underlying dermis. In mice, lack of expression of any of the three Laminin-332 chains result in impaired anchorage and detachment of the epidermis, similar to that seen in human junctional epidermolysis bullosa, and death occurs within a few days after birth. To bypass the early lethality of laminin-332 deficiency caused by the knockout of the mouse laminin ?2 chain, we expressed a dox-controllable human laminin ?2 transgene under a keratinocyte-specific promoter on the laminin ?2 (Lamc2) knockout background. These mice appear similar to their wild-type littermates, do not develop skin blisters, are fertile, and survive >1.5 years. Immunofluorescence analyses of the skin showed that human laminin ?2 colocalized with mouse laminin ?3 and ß3 in the basement membrane zone underlying the epidermis. Furthermore, the presence of “humanized” laminin-332 in the epidermal basement membrane zone rescued the alterations in the deposition of hemidesmosomal components, such as plectin, collagen type XVII/BP180, and integrin ?6 and ß4 chains, seen in conventional Lamc2 knockout mice, leading to restored formation of hemidesmosomes. These mice will be a valuable tool for studies of organs deficient in laminin-332 and the role of laminin-332 in skin, including wound healing.

Adair-Kirk, Tracy L.; Griffin, Gail L.; Meyer, Michelle J.; Kelley, Diane G.; Miner, Jeffrey H.; Keene, Douglas R.; Marinkovich, M. Peter; Ruppert, J. Michael; Uitto, Jouni; Senior, Robert M.



EGF receptor activities in mammalian development.  


The receptor for epidermal growth factor (EGF) and its analog transforming growth factor alpha (TGF alpha) is ubiquitous, implying quite general roles for EGF/TGF alpha in cell viability and tissue maintenance in adult tissues. There is also evidence that the EGF receptor is active in promoting wound healing and tissue regeneration in adult organs, such as skin, liver, and intestinal epithelium. It is likely that EGF receptors have more specific roles during the gestation period. For example, we have detected EGF receptors on the 3.5-day blastocyst (trophectoderm) surface and since TGF alpha-like mRNA sequences and peptides have been detected at this time (Rappolee et al., Science 241:1823, 1988), there is a strong implication for autocrine stimulation in pre- and peri-implantation stage embryos. Paracrine stimulation between the embryo and maternal tissues is also likely since both receptors and TGF alpha are present in decidual cells. Therefore EGF receptors may take part in growth regulation of the early embryo and in the process of implantation. Other examples where EGF receptors may play specific roles during embryonic development are discussed. PMID:2271181

Adamson, E D



Mouse Acetylcholinesterase Enhances Neurite Outgrowth of Rat R28 Cells Through Interaction With Laminin-1  

PubMed Central

The enzyme acetylcholinesterase (AChE) terminates synaptic transmission at cholinergic synapses by hydrolyzing the neurotransmitter acetylcholine, but can also exert ‘non-classical’, morpho-regulatory effects on developing neurons such as stimulation of neurite outgrowth. Here, we investigated the role of AChE binding to laminin-1 on the regulation of neurite outgrowth by using cell culture, immunocytochemistry, and molecular biological approaches. To explore the role of AChE, we examined fiber growth of cells overexpressing different forms of AChE, and/or during their growth on laminin-1. A significant increase of neuritic growth as compared with controls was observed for neurons over-expressing AChE. Accordingly, addition of globular AChE to the medium increased total length of neurites. Co-transfection with PRIMA, a membrane anchor of AChE, led to an increase in fiber length similar to AChE overexpressing cells. Transfection with an AChE mutant that leads to the retention of AChE within cells had no stimulatory effect on neurite length. Noticeably, the longest neurites were produced by neurons overexpressing AChE and growing on laminin-1, suggesting that the AChE/laminin interaction is involved in regulating neurite outgrowth. Our findings demonstrate that binding of AChE to laminin-1 alters AChE activity and leads to increased neurite growth in culture. A possible mechanism of the AChE effect on neurite outgrowth is proposed due to the interaction of AChE with laminin-1.

Sperling, Laura E.; Klaczinski, Janine; Schutz, Corina; Rudolph, Lydia; Layer, Paul G.



NMDA Receptor Activity in Neuropsychiatric Disorders  

PubMed Central

N-Methyl-d-aspartate (NMDA) receptors play a variety of physiologic roles and their proper signaling is essential for cellular homeostasis. Any disruption in this pathway, leading to either enhanced or decreased activity, may result in the manifestation of neuropsychiatric pathologies such as schizophrenia, mood disorders, substance induced psychosis, Huntington’s disease, Alzheimer’s disease, and neuropsychiatric systemic lupus erythematosus. Here, we explore the notion that the overlap in activity of at least one biochemical pathway, the NMDA receptor pathway, may be the link to understanding the overlap in psychotic symptoms between diseases. This review intends to present a broad overview of those neuropsychiatric disorders for which alternations in NMDA receptor activity is prominent thus suggesting that continued direction of pharmaceutical intervention to this pathway may present a viable option for managing symptoms.

Lakhan, Shaheen E.; Caro, Mario; Hadzimichalis, Norell



Like-acetylglucosaminyltransferase (LARGE)-dependent modification of dystroglycan at Thr-317/319 is required for laminin binding and arenavirus infection  

PubMed Central

?-dystroglycan is a highly O-glycosylated extracellular matrix receptor that is required for anchoring of the basement membrane to the cell surface and for the entry of Old World arenaviruses into cells. Like-acetylglucosaminyltransferase (LARGE) is a key molecule that binds to the N-terminal domain of ?-dystroglycan and attaches ligand-binding moieties to phosphorylated O-mannose on ?-dystroglycan. Here we show that the LARGE modification required for laminin- and virus-binding occurs on specific Thr residues located at the extreme N terminus of the mucin-like domain of ?-dystroglycan. Deletion and mutation analyses demonstrate that the ligand-binding activity of ?-dystroglycan is conferred primarily by LARGE modification at Thr-317 and -319, within the highly conserved first 18 amino acids of the mucin-like domain. The importance of these paired residues in laminin-binding and clustering activity on myoblasts and in arenavirus cell entry is confirmed by mutational analysis with full-length dystroglycan. We further demonstrate that a sequence of five amino acids, Thr317ProThr319ProVal, contains phosphorylated O-glycosylation and, when modified by LARGE is sufficient for laminin-binding. Because the N-terminal region adjacent to the paired Thr residues is removed during posttranslational maturation of dystroglycan, our results demonstrate that the ligand-binding activity resides at the extreme N terminus of mature ?-dystroglycan and is crucial for ?-dystroglycan to coordinate the assembly of extracellular matrix proteins and to bind arenaviruses on the cell surface.

Hara, Yuji; Kanagawa, Motoi; Kunz, Stefan; Yoshida-Moriguchi, Takako; Satz, Jakob S.; Kobayashi, Yvonne M.; Zhu, Zihan; Burden, Steven J.; Oldstone, Michael B. A.; Campbell, Kevin P.



Thrombin receptors activate potassium and chloride channels.  


We used DAMI human megakaryocytic leukemia cells to study transmembrane ion currents activated through the G-protein-coupled thrombin receptor pathway. When the cells were stimulated by thrombin receptor-activating peptide, an increase in cytosolic Ca2+ ([Ca2+]i) developed as predicted by the known effect that thrombin exerts in the platelet. We then monitored the membrane potentials of individual DAMI cells during this response and observed complex, triphasic changes that could not be accounted for by Ca2+ fluxes alone. These consisted of rapid hyperpolarization, followed by depolarization to values more positive than the resting potential and then by slow repolarization. For the purpose of this study, we focused on the hyperpolarizing current that developed immediately after thrombin receptor activation. This proved to be composed of (1) a Ca(2+)-independent, outwardly rectifying Cl- current and (2) a strongly hyperpolarizing, inwardly rectifying, Ba(2+)-sensitive K+ current that required an increase of [Ca2+]i for activation. By analogy with their functions in other cell systems, it is logical to conclude that these prominent K+ and Cl- conductances may serve to regulate the complex volume changes that accompany thrombin receptor activation and/or to increase the electromotive drive that supports Ca2+ influx under these conditions through hyperpolarization of the cell membrane. PMID:8555487

Sullivan, R; Kunze, D L; Kroll, M H



Phorbol esters enhance attachment of NIH/3T3 cells to laminin and type IV collagen substrates  

SciTech Connect

The effect of phorbol esters on the adhesive properties of NIH/3T3 mouse fibroblasts was investigated using plastic substrates precoated with the extracellular matrix proteins fibronectin, collagen, and laminin. Treatment with phorbol 12-myristate 13-acetate (PMA) enhanced NIH/3T3 cell attachment to laminin and type IV collagen substrates but had little or no effect on attachment to fibronectin and type I collagen substrates. The effect of PMA in enhancing cell attachment to laminin and type IV collagen substrates was dose dependent between 10{sup {minus}9} and 10{sup {minus}7} M. PMA was effective as early as 30 min; the effect reached a maximum at 2 h and decreased gradually. Phorbol 12, 13-dibenzoate and phorbol 12, 13-diacetate were effective but to a lesser extent and phorbol 12-myristate and phorbol 13-acetate showed little or no effect. These results suggest that PMA may enhance NIH/3T3 cell adhesion through effects on laminin and type IV collagen receptors. Retinoic acid, which itself requires at least 6 h to show an effect on attachment, did not have any effect on cell attachment in 2 h and, if anything, slightly inhibited PMA-enhanced cell attachment to laminin and type IV collagen substrates.

Kato, Shigemi; Ben, T.L.; De Luca, L.M. (National Institutes of Health, Bethesda, MD (USA))



Presence of multiple laminin- and fibronectin-binding proteins in cell wall extract of Candida albicans: influence of dialysis.  


Candida albicans has been reported to express only one to three proteins that bind extracellular matrix proteins, such as laminin and fibrinogen. In those reports, cell wall extracts were subjected to various processing steps, such as dialysis and lyophilization, prior to Western blot analysis. Here, we demonstrate that dialysis for only 2 h of cell wall protein extracts results in a substantial loss (40-60%) of protein. With overnight dialysis, the loss was increased further. After 2 h of dialysis, wall extracts contained fewer laminin- and fibronectin-reactive proteins. In addition, the number of wall proteins in the extracts detected by a polyclonal anti-human fibronectin receptor antiserum decreased after dialysis. These results demonstrate that the C. albicans yeast cell wall contains multiple proteins capable of binding laminin and fibronectin and many of these proteins are not functionally detectable following dialysis. PMID:8786472

Glee, P M; Masuoka, J; Ozier, W T; Hazen, K C


A conformational intermediate in glutamate receptor activation.  


Ionotropic glutamate receptors (iGluRs) transduce the chemical signal of neurotransmitter release into membrane depolarization at excitatory synapses in the brain. The opening of the transmembrane ion channel of these ligand-gated receptors is driven by conformational transitions that are induced by the association of glutamate molecules to the ligand-binding domains (LBDs). Here, we describe the crystal structure of a GluA2 LBD tetramer in a configuration that involves an ?30° rotation of the LBD dimers relative to the crystal structure of the full-length receptor. The configuration is stabilized by an engineered disulfide crosslink. Biochemical and electrophysiological studies on full-length receptors incorporating either this crosslink or an engineered metal bridge show that this LBD configuration corresponds to an intermediate state of receptor activation. GluA2 activation therefore involves a combination of both intra-LBD (cleft closure) and inter-LBD dimer conformational transitions. Overall, these results provide a comprehensive structural characterization of an iGluR intermediate state. PMID:23931998

Lau, Albert Y; Salazar, Héctor; Blachowicz, Lydia; Ghisi, Valentina; Plested, Andrew J R; Roux, Benoît



Adhesion, growth, and matrix production by fibroblasts on laminin substrates  

Microsoft Academic Search

Human embryonic skin fibroblasts have been shown to attach and spread on laminin substrates in the absence of protein synthesis and presence of fibronectin-depleted serum and anti-fibronectin antibodies. Rates of attachment and the type of spreading are virtually identical on fibronectin and laminin-coated substrates with the development of microfilament bundles and focal adhesions. Antibodies to laminin, but not fibronectin, will




Epigenetic regulation of pregnane X receptor activity.  


Pregnane X receptor (PXR, NR1I2) is a ligand-dependent nuclear receptor (NR) that functions as a xenobiotic sensor and effector in coordinately regulating expression of genes of the xenobiotic detoxification network. PXR exerts its transcriptional regulatory functions by dimerization with retinoic X receptor RXR, and PXR-RXR complex binds to specific DNA sequences for regulating gene expression. PXR functions are regulated at the epigenetic level by chromatin modifications, DNA methylation and noncoding RNA. Chromatin modifications are carried out, in part, through interaction with coregulator complexes, including steroid coactivators (SRCs), corepressors (NcoR/SMRT), hepatocyte nuclear factor 4 alpha, proliferator activated receptor ? coactivator 1 alpha and protein arginine methyltransferase 1. PXR can be modified by acetylation, phosphorylation and sumoylation, and the promoter of PXR can be methylated at the "CpG" island. These factors collectively determine the ways in which PXR activity can be regulated, thereby affecting the magnitude and duration of the PXR-regulated drug metabolic responses. Most studies of PXR focus on its role as a transcription factor, which is responsible for the generation of messenger RNA. Recent emerging evidence suggests that PXR regulates gene expression at both transcriptional and translational levels. This review highlights recent research on the epigenetic mechanisms that are found to be important for the gene-regulatory activity of PXR and discusses their implications in xenobiotic metabolism and adverse drug responses. PMID:23600685

Tian, Yanan



Fatty Acids Activate a Chimera of the Clofibric Acid-Activated Receptor and the Glucocorticoid Receptor  

Microsoft Academic Search

Peroxisome proliferators such as clofibric acid, nafenopin, and WY-14,643 have been shown to activate PPAR (peroxisome proliferator-activated receptor), a member of the steroid nuclear receptor superfamily. We have cloned the cDNA from the rat that is homologous to that from the mouse [Issemann, I. & Green, S. (1990) Nature (London) 347, 645-650], which encodes a 97% similar protein with a

Martin Gottlicher; Eva Widmark; Qiao Li; Jan-Ake Gustafsson



Change of laminin density stimulates axon branching via growth cone myosin II-mediated adhesion.  


Axon branching enables neurons to contact with multiple targets and respond to their microenvironment. Owing to its importance in neuronal network formation, axon branching has been studied extensively during the past decades. The chemical properties of extracellular matrices have been proposed to regulate axonal development, but the effects of their density changes on axon branching are not well understood. Here, we demonstrate that both the sharp broadening of substrate geometry and the sharp change of laminin density stimulate axon branching by using microcontact printing (?CP) and microfluidic printing (?FP) techniques. We also found that the change of axon branching stimulated by laminin density depends on myosin II activity. The change of laminin density induces asymmetric extensions of filopodia on the growth cone, which is the precondition for axon branching. These previously unknown mechanisms of change of laminin density-stimulated axon branching may explain how the extracellular matrices regulate axon branching in vivo and facilitate the establishment of neuronal networks in vitro. PMID:23959160

Liu, Wenwen; Xing, Shige; Yuan, Bo; Zheng, Wenfu; Jiang, Xingyu



Clinical and immunological heterogeneity of canine subepidermal blistering dermatoses with anti-laminin-332 (laminin-5) auto-antibodies.  


Laminin-332 (laminin-5) is a basement membrane heterotrimeric protein composed of alpha-3, beta-3 and gamma-2 laminin chains. Laminin-332 polypeptides are targeted by auto-antibodies in human patients with mucous membrane (cicatricial) pemphigoid or, more rarely, subepidermal vesicular diseases that resemble epidermolysis bullosa acquisita (EBA) or bullous pemphigoid (BP). The objectives of this report were to characterize the clinical, histopathological and immunological characteristics of nine dogs with auto-antibodies targeting laminin-332. Immunological investigations consisted of direct immunofluorescence (IF), indirect IF with intact and salt-split canine gingival, and salt-split normal or laminin-332-deficient human skin, immunoblotting with purified human laminin-332 and immunoblotting with recombinant NC1 domain of human collagen VII. All dogs exhibited varying degrees of skin blistering and ulceration associated with microscopic subepidermal vesiculation with or without inflammatory cells. Indirect IF established that circulating IgG auto-antibodies bound the dermal side of salt-split canine lip and human skin. In five dogs, IgG variably recognized the basement membrane of laminin-332-deficient human skin (three dogs negative, two dogs positive). In all nine dogs, IgG auto-antibodies detected purified human laminin-332 by immunoblotting. In two dogs, additional targeting of collagen VII-NC1 was present. These observations establish laminin-332 as a novel basement membrane antigen in dogs with autoimmune blistering diseases with variable clinical phenotypes. The names 'acquired junctional epidermolysis bullosa', 'anti-laminin-332 mucous membrane pemphigoid (MMP)' and 'mixed auto-immune subepidermal blistering dermatosis' are proposed for dogs with clinical signs reminiscent of EBA, MMP or BP respectively. PMID:20456722

Olivry, Thierry; Bizikova, Petra; Dunston, Stanley M; Bond, Ross; Halliwell, Richard; Loeffler, Anette; Pucheu-Haston, Cherie M; Chen, Mei; Marinkovich, M Peter



Adipogenic effect of calcium sensing receptor activation.  


We established that human adipose cells and the human adipose cell line LS14 express the calcium-sensing receptor (CaSR) and that its activation induces inflammatory cytokine production. Also, its expression is enhanced upon exposure to obesity-associated proinflammatory cytokines. We have thus proposed that CaSR activation may be associated with adipose dysfunction. Here, we evaluated a possible effect on adipogenesis. We induced adipose differentiation of primary and LS14 human preadipocytes with or without the simultaneous activation of CaSR, by the exposure to the calcimimetic cinacalcet. Activation of the receptor for 24 h decreased by 40 % the early differentiation marker CCAAT/enhancer-binding protein ?. However, upon longer-term (10 day) exposure to the adipogenic cocktail, cinacalcet exerted the opposite effect, causing a dose-response increase in the expression of the mature adipose markers adipocyte protein 2, adiponectin, peroxisome proliferator-activated receptor ?, fatty acid synthase, and glycerol-3-phosphate dehydrogenase. To assess whether there was a time-sensitive effect of CaSR activation on adipogenesis, we evaluated the 10 day effect of cinacalcet exposure for the first 6, 24, 48 h, 6, and 10 days. Our observations suggest that regardless of the period of exposure, 10 day adipogenesis is elevated by cinacalcet. CaSR activation may interfere with the initial stages of adipocyte differentiation; however, these events do not seem to preclude adipogenesis from continuing. Even though adipogenesis (particularly in subcutaneous depots) is associated with insulin sensitivity and adequate adipose function, the implications of our findings in visceral adipocytes, especially in the context of inflamed AT and overnutrition, remain to be established. PMID:24005534

Villarroel, Pia; Reyes, Marcela; Fuentes, Cecilia; Segovia, María Pia; Tobar, Nicolás; Villalobos, Elisa; Martínez, Jorge; Hugo, Eric; Ben-Jonathan, Nira; Cifuentes, Mariana



Activation of Adenosine Receptor on Gingival Fibroblasts  

PubMed Central

CD73 (ecto-5?-nucleotidase) on human gingival fibroblasts plays a role in the regulation of intracellular cAMP levels through the generation of adenosine, which subsequently activates adenosine receptors. In this study, we examined the involvement of ecto-adenosine deaminase, which can be anchored to CD26 on human gingival fibroblasts, in metabolizing adenosine generated by CD73, and thus attenuating adenosine receptor activation. Ecto-adenosine deaminase expression on fibroblasts could be increased by pre-treatment with a lysate of Jurkat cells, a cell line rich in cytoplasmic adenosine deaminase. Interestingly, the cAMP response to adenosine generated from 5?-AMP via CD73 and the ability of 5?-AMP to induce hyaluronan synthase 1 mRNA were significantly decreased by the pre-treatment of fibroblasts with Jurkat cell lysate. This inhibitory effect was reversed by the specific adenosine deaminase inhibitor. These results suggest that ecto-adenosine deaminase metabolizes CD73-generated adenosine and regulates adenosine receptor activation.

Hashikawa, T.; Takedachi, M.; Terakura, M.; Yamada, S.; Thompson, L.F.; Shimabukuro, Y.; Murakami, S.



Interaction of acetylcholinesterase with the G4 domain of the laminin alpha1-chain.  


Although the primary function of AChE (acetylcholinesterase) is the synaptic hydrolysis of acetylcholine, it appears that the protein is also able to promote various non-cholinergic activities, including cell adhesion, neurite outgrowth and amyloidosis. We have observed previously that AChE is able to bind to mouse laminin-111 in vitro by an electrostatic mechanism. We have also observed that certain mAbs (monoclonal antibodies) recognizing AChE's PAS (peripheral anionic site) inhibit both laminin binding and cell adhesion in neuroblastoma cells. Here, we investigated the interaction sites of the two molecules, using docking, synthetic peptides, ELISAs and conformational interaction site mapping. Mouse AChE was observed on docking to bind to a discontinuous, largely basic, structure, Val(2718)-Arg-Lys-Arg-Leu(2722), Tyr(2738)-Tyr(2739), Tyr(2789)-Ile-Lys-Arg-Lys(2793) and Val(2817)-Glu-Arg-Lys(2820), on the mouse laminin alpha1 G4 domain. ELISAs using synthetic peptides confirmed the involvement of the AG-73 site (2719-2729). This site overlaps extensively with laminin's heparin-binding site, and AChE was observed to compete with heparan sulfate for laminin binding. Docking showed the major component of the interaction site on AChE to be the acidic sequence Arg(90)-Glu-Leu-Ser-Glu-Asp(95) on the omega loop, and also the involvement of Pro(40)-Pro-Val(42), Arg(46) (linked to Glu(94) by a salt bridge) and the hexapeptide Asp(61)-Ala-Thr-Thr-Phe-Gln(66). Epitope analysis, using CLiPS technology, of seven adhesion-inhibiting mAbs (three anti-human AChE, one anti-Torpedo AChE and three anti-human anti-anti-idiotypic antibodies) showed their major recognition site to be the sequence Pro(40)-Pro-Met-Gly-Pro-Arg-Arg-Phe(48) (AChE human sequence). The antibodies, however, also reacted with the proline-containing sequences Pro(78)-Gly-Phe-Glu-Gly-Thr-Glu(84) and Pro(88)-Asn-Arg-Glu-Leu-Ser-Glu-Asp(95). Antibodies that recognized other features of the PAS area but not the Arg(90)-Gly-Leu-Ser-Glu-Asp(95) motif interfered neither with laminin binding nor with cell adhesion. These results define sites for the interaction of AChE and laminin and suggest that the interaction plays a role in cell adhesion. They also suggest the strong probability of functional redundancy between AChE and other molecules in early development, particularly heparan sulfate proteoglycans, which may explain the survival of the AChE-knockout mouse. PMID:18215127

Johnson, Glynis; Swart, Chrisna; Moore, Samuel W



Active Nuclear Receptors Exhibit Highly Correlated AF2 Domain Motions  

Microsoft Academic Search

Nuclear receptor ligand binding domains (LBDs) convert ligand binding events into changes in gene expression by recruiting transcriptional coregulators to a conserved activation function-2 (AF-2) surface. While most nuclear receptor LBDs form homo- or heterodimers, the human nuclear receptor pregnane X receptor (PXR) forms a unique and essential homodimer and is proposed to assemble into a functional heterotetramer with the

Denise G. Teotico; Monica L. Frazier; Feng Ding; Nikolay V. Dokholyan; Brenda R. S. Temple; Matthew R. Redinbo



Modulation of Neuritogenesis by Astrocyte Muscarinic Receptors*  

PubMed Central

Astrocytes have been shown to release factors that have promoting or inhibiting effects on neuronal development. However, mechanisms controlling the release of such factors from astrocytes are not well established. Astrocytes express muscarinic receptors whose activation stimulates a robust intracellular signaling, although the role of these receptors in glial cells is not well understood. Acetylcholine and acetylcholine receptors are present in the brain before synaptogenesis occurs and are believed to be involved in neuronal maturation. The present study was undertaken to investigate whether stimulation of muscarinic receptors in astrocytes would modulate neurite outgrowth in hippocampal neurons. Rat hippocampal neurons, co-cultured with rat cortical astrocytes previously exposed to the cholinergic agonist carbachol, displayed longer neurites. The effect of carbachol in astrocytes was due to the activation of M3 muscarinic receptors. Exposure of astrocytes to carbachol increased the expression of the extracellular matrix proteins fibronectin and laminin-1 in these cells. This effect was mediated in part by an increase in laminin-1 and fibronectin mRNA levels and in part by the up-regulation of the production and release of plasminogen activator inhibitor-1, an inhibitor of the proteolytic degradation of the extracellular matrix. The inhibition of fibronectin activity strongly reduced the effect of carbachol on the elongation of all the neurites, whereas inhibition of laminin-1 activity reduced the elongation of minor neurites only. Plasminogen activator inhibitor-1 also induced neurite elongation through a direct effect on neurons. Taken together, these results demonstrate that cholinergic muscarinic stimulation of astrocytes induces the release of permissive factors that accelerate neuronal development.

Guizzetti, Marina; Moore, Nadia H.; Giordano, Gennaro; Costa, Lucio G.



Mycobacterium smegmatis laminin-binding glycoprotein shares epitopes with Mycobacterium tuberculosis heparin-binding haemagglutinin.  


Mycobacterium tuberculosis, the causative agent of tuberculosis, produces a heparin-binding haemagglutinin adhesin (HBHA), which is involved in its epithelial adherence. To ascertain whether HBHA is also present in fast-growing mycobacteria, Mycobacterium smegmatis was studied using anti-HBHA monoclonal antibodies (mAbs). A cross-reactive protein was detected by immunoblotting of M. smegmatis whole-cell lysates. However, the M. tuberculosis HBHA-encoding gene failed to hybridize with M. smegmatis chromosomal DNA in Southern blot analyses. The M. smegmatis protein recognized by the anti-HBHA mAbs was purified by heparin-Sepharose chromatography, and its amino-terminal sequence was found to be identical to that of the previously described histone-like protein, indicating that M. smegmatis does not produce HBHA. Biochemical analysis of the M. smegmatis histone-like protein shows that it is glycosylated like HBHA. Immunoelectron microscopy demonstrated that the M. smegmatis protein is present on the mycobacterial surface, a cellular localization inconsistent with a histone-like function, but compatible with an adhesin activity. In vitro protein interaction assays showed that this glycoprotein binds to laminin, a major component of basement membranes. Therefore, the protein was called M. smegmatis laminin-binding protein (MS-LBP). MS-LBP does not appear to be involved in adherence in the absence of laminin but is responsible for the laminin-mediated mycobacterial adherence to human pneumocytes and macrophages. Homologous laminin-binding adhesins are also produced by virulent mycobacteria such as M. tuberculosis and Mycobacterium leprae, suggesting that this adherence mechanism may contribute to the pathogenesis of mycobacterial diseases. PMID:11123691

Pethe, K; Puech, V; Daffé, M; Josenhans, C; Drobecq, H; Locht, C; Menozzi, F D



Prothymosin Alpha Selectively Enhances Estrogen Receptor Transcriptional Activity by Interacting with a Repressor of Estrogen Receptor Activity  

Microsoft Academic Search

We find that prothymosin alpha (PTa) selectively enhances transcriptional activation by the estrogen receptor (ER) but not transcriptional activity of other nuclear hormone receptors. This selectivity for ER is explained by PTa interaction not with ER, but with a 37-kDa protein denoted REA, for repressor of estrogen receptor activity, a protein that we have previously shown binds to ER, blocking




BeWo choriocarcinoma cells produce laminin 10.  

PubMed Central

BeWo is a choriocarcinoma cell line that generates an extracellular matrix (ECM) rich in laminin and is a useful model for human trophoblast. Immunofluorescence with monoclonal antibodies demonstrates that BeWo ECM contains laminin subunits beta1 and gamma1. Immunoprecipitation from conditioned medium shows that the cells secrete two distinct laminin trimers both containing beta1 and gamma1 but with alpha subunits of approx. 400 and 450 kDa. The culture medium also contains a species thought to be beta1 gamma1 dimer. Immunoprecipitation with monoclonal antibody 4C7, previously thought to recognize the alpha1 subunit, isolates complexes containing only the smaller alpha subunit. A second complex containing the larger alpha subunit along with beta1, gamma1 and a 150 kDa polypeptide is precipitated from 4C7-depleted medium with an anti-(laminin 1) polyclonal antibody. Peptide sequencing demonstrates that the 4C7-reactive species is alpha5, which is present as two similarly sized polypeptides. mRNA species encoding laminin subunits alpha1, alpha5, beta1, beta2 and gamma1 are all present in the cells. These results demonstrate the secretion of a novel laminin isoform, laminin 10, the subunit composition of which is alpha5 beta1 gamma1. Laminin 1 is also produced. No evidence for the secretion of beta2-containing laminin isoforms could be derived despite the presence of beta2 mRNA. Analysis with reverse transcriptase-mediated PCR also showed the presence of laminin alpha5 in first-trimester placenta and decidua.

Church, H J; Aplin, J D



Origin of basal activity in mammalian olfactory receptor neurons  

PubMed Central

Mammalian odorant receptors form a large, diverse group of G protein–coupled receptors that determine the sensitivity and response profile of olfactory receptor neurons. But little is known if odorant receptors control basal and also stimulus-induced cellular properties of olfactory receptor neurons other than ligand specificity. This study demonstrates that different odorant receptors have varying degrees of basal activity, which drives concomitant receptor current fluctuations and basal action potential firing. This basal activity can be suppressed by odorants functioning as inverse agonists. Furthermore, odorant-stimulated olfactory receptor neurons expressing different odorant receptors can have strikingly different response patterns in the later phases of prolonged stimulation. Thus, the influence of odorant receptor choice on response characteristics is much more complex than previously thought, which has important consequences on odor coding and odor information transfer to the brain.



Monoclonal antibodies to the human insulin receptor that activate glucose transport but not insulin receptor kinase activity  

SciTech Connect

Three mouse monoclonal antibodies were produced that reacted with the ..cap alpha.. subunit of the human insulin receptor. All three both immunoprecipitated /sup 125/I-labeled insulin receptors from IM-9 lymphocytes and competitively inhibited /sup 125/I-labeled insulin binding to its receptor. Unlike insulin, the antibodies failed to stimulate receptor autophosphorylation in both intact IM-9 lymphocytes and purified human placental insulin receptors. Moreover, unlike insulin, the antibodies failed to stimulate receptor-mediated phosphorylation of exogenous substrates. However, like insulin, two of the three antibodies stimulated glucose transport in isolated human adipocytes. One antibody, on a molar basis, was as potent as insulin. These studies indicate, therefore, that monoclonal antibodies to the insulin receptor can mimic a major function of insulin without activating receptor kinase activity. They also raise the possibility that certain actions of insulin such as stimulation of glucose transport may not require the activation of receptor kinase activity.

Forsayeth, J.R.; Caro, J.F.; Sinha, M.K.; Maddux, B.A.; Goldfine, I.D.



Monoclonal Antibodies to the Human Insulin Receptor that Activate Glucose Transport but not Insulin Receptor Kinase Activity  

NASA Astrophysics Data System (ADS)

Three mouse monoclonal antibodies were produced that reacted with the ? subunit of the human insulin receptor. All three both immunoprecipitated 125I-labeled insulin receptors from IM-9 lymphocytes and competitively inhibited 125I-labeled insulin binding to its receptor. Unlike insulin, the antibodies failed to stimulate receptor autophosphorylation in both intact IM-9 lymphocytes and purified human placental insulin receptors. Moreover, unlike insulin, the antibodies failed to stimulate receptor-mediated phosphorylation of exogenous substrates. However, like insulin, two of the three antibodies stimulated glucose transport in isolated human adipocytes. One antibody, on a molar basis, was as potent as insulin. These studies indicate, therefore, that monoclonal antibodies to the insulin receptor can mimic a major function of insulin without activating receptor kinase activity. They also raise the possibility that certain actions of insulin such as stimulation of glucose transport may not require the activation of receptor kinase activity.

Forsayeth, John R.; Caro, Jose F.; Sinha, Madhur K.; Maddux, Betty A.; Goldfine, Ira D.



Activating and Inactivating Hormone Receptor Mutations  

Microsoft Academic Search

The unravelling of gene structures of hormones, their receptors and the various components of their signal transduction apparatus has enabled diagnosis of the aetiology of hormone resistance at the molecular level. Inactivating mutations can be found in hormone receptor genes or those encoding components of the post-receptor signal transduction cascade. Another category of receptor mutation is that causing constitutive receptor

Ilpo Huhtaniemi



The Effect of Laminin-1-Doped Nanoroughened Implant Surfaces: Gene Expression and Morphological Evaluation  

PubMed Central

Aim. This study aimed to observe the morphological and molecular effect of laminin-1 doping to nanostructured implant surfaces in a rabbit model. Materials and Methods. Nanostructured implants were coated with laminin-1 (test; dilution, 100??g/mL) and inserted into the rabbit tibiae. Noncoated implants were used as controls. After 2 weeks of healing, the implants were removed and subjected to morphological analysis using scanning electron microscopy (SEM) and gene expression analysis using the real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Results. SEM revealed bony tissue attachment for both control and test implants. Real-time RT-PCR analysis showed that the expression of osteoblast markers RUNX-2, osteocalcin, alkaline phosphatase, and collagen I was higher (1.62-fold, 1.53-fold, 1.97-fold, and 1.04-fold, resp.) for the implants modified by laminin-1 relative to the control. All osteoclast markers investigated in the study presented higher expression on the test implants than controls as follows: tartrate-resistant acid phosphatase (1.67-fold), calcitonin receptor (1.35-fold), and ATPase (1.25-fold). The test implants demonstrated higher expression of inflammatory markers interleukin-10 (1.53-fold) and tumour necrosis factor-? (1.61-fold) relative to controls. Conclusion. The protein-doped surface showed higher gene expression of typical genes involved in the osseointegration cascade than the control surface.

Schwartz-Filho, Humberto Osvaldo; Bougas, Kostas; Coelho, Paulo G.; Xue, Ying; Hayashi, Mariko; Faeda, Rafael Silveira; Marcantonio, Rosemary Adriana Chierici; Ono, Daisuke; Kobayashi, Fumio; Mustafa, Kamal; Wennerberg, Ann; Jimbo, Ryo



Laminin peptide YIGSR induces collagen synthesis in Hs27 human dermal fibroblasts.  


The dermal ECM is synthesized from fibroblasts and is primarily compromised of fibrillar collagen and elastic fibers, which support the mechanical strength and resiliency of skin, respectively. Laminin, a major glycoprotein located in the basement membrane, promotes cell adhesion, cell growth, differentiation, and migration. The laminin tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide, corresponding to the 929-933 sequence of the ?1 chain, is known to be a functional motif with effects on the inhibition of tumor metastasis, the regulation of sensory axonal response and the inhibition of angiogenesis through high affinity to the 67kDa laminin receptor. In this study, we identified a novel function of the YIGSR peptide to enhance collagen synthesis in human dermal fibroblasts. To elucidate this novel function regarding collagen synthesis, we treated human dermal fibroblasts with YIGSR peptide in both a time- and dose-dependent manner. According to subsequent experiments, we found that the YIGSR peptide strongly enhanced collagen type 1 synthesis without changing cell proliferation or cellular MMP-1 level. This YIGSR peptide-mediated collagen type 1 synthesis was modulated by FAK inhibitor and MEK inhibitor. This study clearly reveals that YIGSR peptide plays a novel function on the collagen type 1 synthesis of dermal fibroblasts and also suggests that YIGSR is a strong candidate peptide for the treatment of skin aging and wrinkles. PMID:23111328

Yoon, Jong Hyuk; Kim, Jaeyoon; Lee, Hyeongjoo; Kim, So Young; Jang, Hwan-Hee; Ryu, Sung Ho; Kim, Beom Joon; Lee, Taehoon G



The Discoidin Domain Receptor Tyrosine Kinases Are Activated by Collagen  

Microsoft Academic Search

Two mammalian receptor tyrosine kinases (DDR1 and DDR2) have extracellular domains closely related to a D. discoideum lectin, discoidin, required for cell aggregation. Here, we show that the mammalian DDR receptors bind and are activated by specific types of collagen. Stimulation of DDR receptor tyrosine kinase activity requires the native triple-helical structure of collagen and occurs over an extended period

Wolfgang Vogel; Gerald D Gish; Frauke Alves; Tony Pawson



laminin alpha 1 gene is essential for normal lens development in zebrafish  

Microsoft Academic Search

BACKGROUND: Laminins represent major components of basement membranes and play various roles in embryonic and adult tissues. The functional laminin molecule consists of three chains, alpha, beta and gamma, encoded by separate genes. There are twelve different laminin genes identified in mammals to date that are highly homologous in their sequence but different in their tissue distribution. The laminin alpha

Natalya S Zinkevich; Dmitry V Bosenko; Brian A Link; Elena V Semina



Composition in situ and in vitro of vascular smooth muscle laminin in the rat  

Microsoft Academic Search

Vascular smooth muscle cells are surrounded by a basal lamina containing an array of macromolecules: included among these are the laminins, a family of oligomeric glycoproteins composed of subunits encoded by different genes. In this study, we have used monoclonal antibodies to several of these subunits, including S-laminin, laminin B2, and laminin B1, to study these proteins in tail artery,

H. M. Walker-Caprioglio; D. D. Hunter; P. G. McGuire; S. A. Little; L. J. McGuffee



Skeletal muscle denervation activates acetylcholine receptor genes  

PubMed Central

Transcriptional activity of acetylcholine receptor subunit genes was investigated in innervated and denervated chick skeletal muscle. The sciatic nerve of 3-d-old White Leghorn chicks was sectioned unilaterally; after various intervals, nuclei were isolated from operated and sham-operated animals, and run-on assays performed. Nuclei were incubated with 32P-UTP, and total RNA was extracted and hybridized onto filters containing an excess of subunit-specific DNA. Specific transcripts were detected by autoradiography and quantitated densitometrically. A sharp increase in transcriptional activity was observed to begin approximately 1/2 d after the operation and peak 1 d later when transcriptional rates reached approximately seven-, six-, and fivefold control levels for the alpha-, delta-, and gamma-subunit genes, respectively. The specificity of the effect was ascertained by normalization to total RNA synthesis and by the demonstration that several nonreceptor genes respond differently to denervation. These results suggest that a denervation signal reaches the genome to induce receptor expression. In addition, since the increase in mRNA levels significantly exceeds what can be accounted for by increased gene activity, posttranscriptional effects are suggested.



CB? cannabinoid receptors promote maximal FAK catalytic activity by stimulating cooperative signaling between receptor tyrosine kinases and integrins in neuronal cells.  


Tyrosine phosphorylation (Tyr-P) of focal adhesion kinase (FAK) regulates FAK activation. Phosphorylated FAK Tyr 397 binds Src family kinases (Src), which in turn directly phosphorylate FAK Tyr 576/577 to produce maximal FAK enzymatic activity. CB? cannabinoid receptors (CB?) are abundantly expressed in the nervous system and influence FAK activation by presently unknown mechanisms. The current investigation determined that CB?-stimulated maximal FAK catalytic activity is mediated by Gi/o proteins in N18TG2 neuronal cells, and that G12/13 regulation of Rac1 and RhoA occurs concomitantly. Immunoblotting analyses using antibodies against FAK phospho-Tyr 397 and phospho-Tyr 576/577 demonstrated that the time-course of CB?-stimulated FAK 576/577 Tyr-P occurred in three phases: Phase I (0-2 min) maximal Tyr-P, Phase II (5-20 min) rapid decline in Tyr-P, and Phase III (>20 min) plateau in Tyr-P at submaximal levels. In contrast, FAK 397 Tyr-P was monophasic and significantly lower in magnitude. FAK 397 Tyr-P and Phase I FAK 576/577 Tyr-P involved protein tyrosine phosphatase (PTP1B and Shp1/Shp2)-mediated Src activation, Protein Kinase A (PKA) inhibition, and integrin activation. Phase I maximal FAK 576/577 Tyr-P also required cooperative signaling between receptor tyrosine kinases (RTKs) and integrins. The integrin antagonist RGDS peptide, Flk-1 vascular endothelial growth factor receptor (VEGFR) antagonist SU5416, and epidermal growth factor receptor (EGFR) antagonist AG 1478 blocked Phase I FAK 576/577 Tyr-P. CB? agonists failed to stimulate FAK Tyr-P in the absence of integrin activation upon suspension in serum-free culture media. In contrast, cells grown on the integrin ligands fibronectin and laminin displayed increased FAK 576/577 Tyr-P that was augmented by CB? agonists and blocked by the Src inhibitor PP2 and Flk-1 VEGFR antagonist SU5416. Taken together, these studies have identified a complex integrative pathway utilized by CB? to stimulate maximal FAK 576/577 Tyr-P in neuronal cells. PMID:23571270

Dalton, George D; Peterson, Lynda J; Howlett, Allyn C



Targeting proteinase-activated receptors: therapeutic potential and challenges  

Microsoft Academic Search

Proteinase-activated receptors (PARs), a family of four seven-transmembrane G protein-coupled receptors, act as targets for signalling by various proteolytic enzymes. PARs are characterized by a unique activation mechanism involving the proteolytic unmasking of a tethered ligand that stimulates the receptor. Given the emerging roles of these receptors in cancer as well as in disorders of the cardiovascular, musculoskeletal, gastrointestinal, respiratory

Farshid Noorbakhsh; Kathryn DeFea; Rithwik Ramachandran; Morley D. Hollenberg



Developmentally regulated interactions of human thymocytes with different laminin isoforms  

PubMed Central

The gene family of heterotrimeric laminin molecules consists of at least 15 naturally occurring isoforms which are formed by five different ?, three ? and three ? subunits. The expression pattern of the individual laminin chains in the human thymus was comprehensively analysed in the present study. Whereas laminin isoforms containing the laminin ?1 chain (e.g. LN-1) were not present in the human thymus, laminin isoforms containing the ?2 chain (LN-2/4) or the ?5 chain (LN-10/11) were expressed in the subcapsular epithelium and in thymic blood vessels. Expression of the laminin ?4 chain seemed to be restricted to endothelial cells of the thymus, whereas the LN-5 isoform containing the ?3 chain could be detected on medullary thymic epithelial cells and weakly in the subcapsular epithelium. As revealed by cell attachment assays, early CD4? CD8? thymocytes which are localized in the thymus beneath the subcapsular epithelium adhered strongly to LN-10/11, but not to LN-1, LN-2/4 or LN-5. Adhesion of these thymocytes to LN-10/11 was mediated by the integrin ?6?1. During further development, the cortically localized CD4+ CD8+ thymocytes have lost the capacity to adhere to laminin-10/11. Neither do these cells adhere to any other laminin isoform tested. However, the more differentiated single positive CD8+ thymocytes which were mainly found in the medulla were able to bind to LN-5 which is expressed by medullary epithelial cells. Interactions of CD8+ thymocytes with LN-5 were integrin ?6?4-dependent. These results show that interactions of developing human thymocytes with different laminin isoforms are spatially and developmentally regulated.

Kutlesa, Snjezana; Siler, Ulrich; Speiser, Angelika; Wessels, Johannes T; Virtanen, Ismo; Rousselle, Patricia; Sorokin, Lydia M; Muller, Claudia A; Klein, Gerd



Laminin Synthesis and the Adhesion Characteristics of Immortalized Human Corneal Epithelial Cells to Laminin Isoforms  

Microsoft Academic Search

We have studied the synthesis of laminins (Ln) and determined the specific integrins mediating the adhesion of immortalized human corneal epithelial cells to mouse Ln-1, and human Lns-5 and -10. Immunofluorescence microscopy of the cells demonstrated integrin ?2, ?3, ?6, ?1and ?4subunits, integrins ?6and ?4being found in a typical ‘leopard-skin’ like manner. Immunoprecipitation studies showed that the cells produced ?3,

Sissi Filenius; Marketta Hormia; Jan Rissanen; Robert E. Burgeson; Yashihiko Yamada; Kaoru Araki-Sasaki; Masatsugu Nakamura; Ismo Virtanen; Timo Tervo



Laminin-5 and type I collagen promote adhesion and osteogenic differentiation of animal serum-free expanded human mesenchymal stromal cells.  


Mesenchymal stromal cells (MSC) are differentiation competent cells and may generate, among others, mature osteoblasts or chondrocytes in vitro and in vivo. Laminin-5 and type I collagen are important components of the extracellular matrix. They are involved in a variety of cellular and extracellular activities including cell attachment and osteogenic differentiation of MSC. MSC were isolated and expanded using media conforming good medical practice (GMP)-regulations for medical products. Cells were characterized according to the defined minimal criteria for multipotent MSC. MTT- and BrdU-assays were performed to evaluate protein-dependent (laminin-5, laminin-1, type I collagen) metabolic activity and proliferation of MSC. MSC-attachment assays were performed using protein-coated culture plates. Osteogenic differentiation of MSC was measured by protein-dependant mineralization and expression of osteogenic marker genes (osteopontin, alkaline phophatase, Runx2) after three, seven and 28 days of differentiation. Marker genes were identified using quantitative reverse-transcription polymerase chain reaction. Expansion of MSC in GMP-conforming media yielded vital cells meeting all minimal criteria for MSC. Attachment assay revealed a favorable binding of MSC to laminin-5 and type I collagen at a protein concentration of 1-5 fmol/µL. Compared to plastic, osteogenic differentiation was significantly increased by laminin-5 after 28 days of culture (P<0.04). No significant differences in gene expression patterns were observed. We conclude that laminin-5 and type I collagen promote attachment, but laminin-1 and laminin-5 promote osteogenic differentiation of MSC. This may influence future clinical applications. PMID:23589764

Mittag, Falk; Falkenberg, Eva-Maria; Janczyk, Alexandra; Götze, Marco; Felka, Tino; Aicher, Wilhelm K; Kluba, Torsten



C3d fragment of complement interacts with laminin and binds to basement membranes of glomerulus and trophoblast  

PubMed Central

Two mouse monoclonal antibodies generated against human placental homogenate were found to react specifically with human complement component C3. In immunofluorescence of human tissues, these antibodies gave a bright linear staining outlining the glomerular basement membrane of the adult kidney and the trophoblast basement membrane of placenta. An identical staining pattern was observed with a rabbit C3d antiserum which also prevented binding of the monoclonal antibodies to tissue sections. Only negligible basement membrane staining was observed in the same tissues with antisera to human C3c, C5, IgG, IgA, or IgM. When interactions of C3 with basement membrane proteins were tested in enzyme immunoassays and column chromatography, C3(H2O) was found to bind efficiently to solid-phase laminin. Native C3 from fresh plasma did not bind to laminin but C3 from plasma treated with methylamine bound efficiently. When C3 was cleaved with trypsin, C3b and C3d but not C3c bound to laminin-Sepharose. The interaction of C3 and laminin was inhibited by soluble laminin and by high ionic strength. The results indicate that C3d, a biologically active breakdown product of C3, can be found in glomerular and placental basement membranes in the absence of signs for ongoing local complement activation or immune complex deposition. It is possible that binding affinities between C3 and basement membrane molecules, especially laminin, are involved in the retention of C3d at these sites. Such interactions between C3 and components of the glomerular basement membrane could play important roles in complement-related pathological processes of the glomerulus.



Insulin receptor regulates photoreceptor CNG channel activity.  


Photoreceptor cyclic nucleotide gated (CNG) channels are critical elements in phototransduction and light adaptation. Here we report that insulin receptor (IR), an integral membrane protein, directly phosphorylates the CNGA1 subunit of CNG channels that in turn affects the function of these channels negatively. The IR phosphorylates Tyr(498) and Tyr(503) residues on CNGA1 that are situated at the membrane-cytoplasmic interface. The IR tyrosine kinase activity is essential for the inhibition of CNG channel. To maintain the channels in an off state, it is necessary not only to have a precise balance of the cGMP levels but also to have a control on the cGMP sensitivity of the CNG channels itself. In this study, we observed that the channel opens at a lower concentration of cGMP in IR(-/-) mice. These studies suggest that IR regulates the modulation of CNG channel activity in vivo. PMID:23032687

Gupta, Vivek K; Rajala, Ammaji; Rajala, Raju V S



PDGF enhances IRES-mediated translation of Laminin B1 by cytoplasmic accumulation of La during epithelial to mesenchymal transition  

PubMed Central

The extracellular matrix protein Laminin B1 (LamB1) regulates tumor cell migration and invasion. Carcinoma cells acquire invasive properties by epithelial to mesenchymal transition (EMT), which is a fundamental step in dissemination of metastatic cells from the primary tumor. Recently, we showed that enhanced translation of LamB1 upon EMT of malignant hepatocytes is mediated by an internal ribosome entry site (IRES). We demonstrated that the IRES transacting factor La binds the minimal IRES motif and positively modulates IRES activity of LamB1. Here, we show that platelet-derived growth factor (PDGF) enhances IRES activity of LamB1 by the increasing cytoplasmic localization of La during EMT. Accordingly, cells expressing dominant negative PDGF receptor display reduced cytoplasmic accumulation of La and show no elevation of IRES activity or endogenous LamB1 levels after stimulation with PDGF. Furthermore, La-mediated regulation of LamB1 IRES activity predominantly depends on MAPK/ERK signaling downstream of PDGF. Notably, LamB1 expression is not significantly downregulated by the impairment of the translation initiation factor eIF4E. In vivo, knockdown of La associated with decreased LamB1 expression and reduced tumor growth. Together, these data suggest that PDGF is required for the cytoplasmic accumulation of La that triggers IRES-dependent translation of LamB1 during EMT.

Petz, Michaela; Them, Nicole C. C.; Huber, Heidemarie; Mikulits, Wolfgang



Sustained activation of STAT5 is essential for chromatin remodeling and maintenance of mammary-specific function  

SciTech Connect

Epithelial cells, once dissociated and placed in two-dimensional (2D) cultures, rapidly lose tissue-specific functions. We showed previously that in addition to prolactin, signaling by laminin-111 was necessary to restore functional differentiation of mammary epithelia. Here, we elucidate two additional aspects of laminin-111 action. We show that in 2D cultures, the prolactin receptor is basolaterally localized and physically segregated from its apically placed ligand. Detachment of the cells exposes the receptor to ligation by prolactin leading to signal transducers and activators of transcription protein 5 (STAT5) activation, but only transiently and not sufficiently for induction of milk protein expression. We show that laminin-111 reorganizes mammary cells into polarized acini, allowing both the exposure of the prolactin receptor and sustained activation of STAT5. The use of constitutively active STAT5 constructs showed that the latter is necessary and sufficient for chromatin reorganization and {beta}-casein transcription. These results underscore the crucial role of continuous laminin signaling and polarized tissue architecture in maintenance of transcription factor activation, chromatin organization, and tissue-specific gene expression.

Xu, Ren; Nelson, Celeste M.; Muschler, John L.; Veiseh, Mandana; Vonderhaar, Barbara K.; Bissell, Mina J.



Receptor-activating autoantibodies and disease: preeclampsia and beyond  

PubMed Central

The research reviewed in this article provides examples of autoantibody-mediated receptor activation that likely contributes to disease. The classic example is Graves’ hyperthyroidism, in which autoantibodies activate the thyroid-stimulating hormone receptor resulting in overproduction of thyroid hormones. Other compelling examples come from the cardiovascular literature and include agonistic autoantibodies targeting the cardiac ?1-adrenergic receptor, which are associated with dilated cardiomyopathy. Autoantibodies capable of activating ?1-adrenergic receptors are associated with refractory hypertension and cardiomyopathy. A prominent example is preeclampsia, a hypertensive disease of pregnancy, characterized by the presence of autoantibodies that activate the major angiotensin receptor, AT1. AT1 receptor-activating autoantibodies are also observed in kidney transplant recipients suffering from severe vascular rejection and malignant hypertension. AT1 receptor-activating autoantibodies and antibodies that activate the endothelin-1 receptor, ETA, are prevalent in individuals diagnosed with systemic sclerosis. Thus, the presence of agonistic autoantibodies directed to G protein-coupled receptors has been observed in numerous cardiovascular disease states. Rapidly emerging evidence indicates that receptor-activating autoantibodies contribute to disease, and that efforts to detect and remove these pathogenic autoantibodies or block their actions will provide promising therapeutic possibilities.

Xia, Yang; Kellems, Rodney E



Peroxisome Proliferator Activated Receptors and Lipoprotein Metabolism  

PubMed Central

Plasma lipoproteins are responsible for carrying triglycerides and cholesterol in the blood and ensuring their delivery to target organs. Regulation of lipoprotein metabolism takes place at numerous levels including via changes in gene transcription. An important group of transcription factors that mediates the effect of dietary fatty acids and certain drugs on plasma lipoproteins are the peroxisome proliferator activated receptors (PPARs). Three PPAR isotypes can be distinguished, all of which have a major role in regulating lipoprotein metabolism. PPAR? is the molecular target for the fibrate class of drugs. Activation of PPAR? in mice and humans markedly reduces hepatic triglyceride production and promotes plasma triglyceride clearance, leading to a clinically significant reduction in plasma triglyceride levels. In addition, plasma high-density lipoprotein (HDL)-cholesterol levels are increased upon PPAR? activation in humans. PPAR? is the molecular target for the thiazolidinedione class of drugs. Activation of PPAR? in mice and human is generally associated with a modest increase in plasma HDL-cholesterol and a decrease in plasma triglycerides. The latter effect is caused by an increase in lipoprotein lipase-dependent plasma triglyceride clearance. Analogous to PPAR?, activation of PPAR?/? leads to increased plasma HDL-cholesterol and decreased plasma triglyceride levels. In this paper, a fresh perspective on the relation between PPARs and lipoprotein metabolism is presented. The emphasis is on the physiological role of PPARs and the mechanisms underlying the effect of synthetic PPAR agonists on plasma lipoprotein levels.

Kersten, Sander



Steroid Receptor RNA Activator – A nuclear receptor coregulator with multiple partners: Insights and challenges  

Microsoft Academic Search

Steroid Receptor RNA Activator (SRA) occupies a unique and enigmatic position within the nuclear receptor (NR) field and more broadly in transcriptional regulation. This is as a result of its transcripts having both coding and non-coding coactivator activities along with its protein product SRAP performing mixed coactivator\\/repressor functions. Recent publications have provided greater understanding of SRA gene product activities and

Shane M. Colley; Peter J. Leedman



Differential Activation of Nuclear Receptors by Perfluorinated Fatty Acid Analogs and Natural Fatty Acids: A Comparison of Human, Mouse, and Rat Peroxisome Proliferator-Activated Receptor , - , and - , Liver X Receptor , and Retinoid X Receptor  

Microsoft Academic Search

Administration of ammonium salts of perfluorooctanoate (PFOA) to rats results in peroxisome proliferation and benign liver tumors, events associated with activation of the nuclear receptor (NR) peroxisome proliferator-activated receptor-a (PPARa). Due to its fatty acid structure, PFOA may activate other NRs, such as PPARb ,P PARg, liver X receptor (LXR), or retinoid X receptor (RXR). In this study, the activation

John P. Vanden Heuvel; Jerry T. Thompson; Steven R. Frame; Peter J. Gillies



Modulation of Chemokine Receptor Activity through Dimerization and Crosstalk  

PubMed Central

Chemokines are small, secreted proteins that bind to the chemokine receptor subfamily of class A G-protein coupled receptors. Collectively, these receptor-ligand pairs are responsible for diverse physiological responses including immune cell trafficking, development and mitogenic signaling, both in the context of homeostasis and disease. However, chemokines and their receptors are not isolated entities, but instead function in complex networks involving homo- and heterodimer formation as well as crosstalk with other signaling complexes. Herein, the functional consequences of chemokine receptor activity, from the perspective of both direct physical associations with other receptors and indirect crosstalk with orthogonal signaling pathways, will be reviewed. Modulation of chemokine receptor activity through these mechanisms has significant implications in physiological and pathological processes, as well as drug discovery and drug efficacy. The integration of signals downstream of chemokine and other receptors will be key to understanding how cells fine-tune their response to a variety of stimuli, including therapeutics.

Salanga, Catherina L.; O'Hayre, Morgan; Handel, Tracy



The molecular mechanism of transcriptional activation by the peroxisome proliferator activated-receptor alpha  

Microsoft Academic Search

The peroxisome proliferator-activated receptor (PPAR?) is a member of the nuclear receptor superfamily, a growing class of transcriptional activators. Many of these proteins, including PPAR?, function as intracellular receptors for hormones and upon the binding of agonist, activate the transcription of target genes. PPAR? is an integral regulator of fatty acid metabolism and as such is activated by fatty acids

Kenji Sean Miyata



Mechanism of activation of the TGF-beta receptor  

Microsoft Academic Search

Transforming growth factor-beta (TGF-beta) signals by contacting two distantly related transmem-brane serine\\/threonine kinases called receptors I and II. The role of these molecules in signalling has now been determined. TGF-beta binds directly to receptor II, which is a constitutively active kinase. Bound TGF-beta is then recognized by receptor I which is recruited into the complex and becomes phosphorylated by receptor

Jeffrey L. Wrana; Liliana Attisano; Rotraud Wieser; Francesc Ventura; Joan Massagué



Pronociceptive response elicited by TRPA1 receptor activation in mice  

Microsoft Academic Search

Ankyrin-repeat transient receptor potential 1 (TRPA1) is a member of the transient receptor potential (TRP) channel family and it is found in sensory neurons. In the present study, we found that TRPA1 receptor activation with allyl isothiocyanate or cinnamaldehyde caused dose-dependent spontaneous nociception when injected into the mouse hind paw. Very similar results were obtained when stimulating transient receptor potential

E. L. Andrade; A. P. Luiz; J. Ferreira; J. B. Calixto



Cranin: A Laminin-Binding Protein of Cell Membranes  

Microsoft Academic Search

We report that a 120-kDa glycoprotein is the predominant laminin-binding protein detected within plasma membranes of rodent NG108-15 neural hybrid cells, embryonic chicken brain, and mouse 3T3 fibroblasts. This protein was detected when membrane extracts were separated by PAGE, transferred to nitrocellulose, and incubated with laminin at concentrations as low as 2.8 × 10-11 M, under conditions of physiological ionic

Neil R. Smalheiser; Nancy B. Schwartz



A peptide derived from laminin-?3 reversibly impairs spermatogenesis in rats  

PubMed Central

Cellular events that occur across the seminiferous epithelium of the mammalian testis during spermatogenesis are tightly coordinated by biologically active peptides released from laminin chains. Laminin-?3 domain IV (Lam ?3 DIV) is released at the apical ectoplasmic specialization (ES) during spermiation and mediates restructuring of the blood-testis barrier (BTB), which facilitates the transit of preleptotene spermatocytes. Here we determine the biologically active domain in Lam ?3 DIV, which we designate F5-peptide, and show that overexpression of this domain, or the use of a synthetic F5-peptide, in Sertoli cells with an established functional BTB reversibly perturbs BTB integrity in vitro and in rat testis in vivo. This effect is mediated via changes in protein distribution at the Sertoli and Sertoli-germ cell-cell interface and by phosphorylation of focal adhesion kinase at Tyr407. The consequences are perturbed organization of actin filaments in Sertoli cells, disruption of the BTB and spermatid loss. The impairment of spermatogenesis suggests that this laminin peptide fragment may serve as a contraceptive in male rats.

Su, Linlin; Mruk, Dolores D.; Lie, Pearl P.Y.; Silvestrini, Bruno; Cheng, C. Yan



Receptor Tyrosine Phosphatase ? (RPTP?) Activity and Signaling Are Attenuated by Glycosylation and Subsequent Cell Surface Galectin-1 Binding*  

PubMed Central

O-Mannosyl-linked glycosylation is abundant within the central nervous system, yet very few glycoproteins with this glycan modification have been identified. Congenital diseases with significant neurological defects arise from inactivating mutations found within the glycosyltransferases that act early in the O-mannosyl glycosylation pathway. The N-acetylglucosaminyltransferase known as GnT-Vb or -IX is highly expressed in brain and branches O-mannosyl-linked glycans. Our results using SH-SY5Y neuroblastoma cells indicate that GnT-Vb activity promotes the addition of the O-mannosyl-linked HNK-1 modification found on the developmentally regulated and neuron-specific receptor protein-tyrosine phosphatase ? (RPTP?). These changes in glycosylation accompany decreased cell-cell adhesion and increased rates of migration on laminin. In addition, we show that expression of GnT-Vb promotes its dimerization and inhibits RPTP? intrinsic phosphatase activity, resulting in higher levels of phosphorylated ?-catenin, suggesting a mechanism by which GnT-Vb glycosylation couples to changes in cell adhesion. GnT-Vb-mediated glycosylation of RPTP? promotes galectin-1 binding and RPTP? levels of retention on the cell surface. N-Acetyllactosamine, but not sucrose, treatment of cells results in decreased RPTP retention, showing that galectin-1 binding contributes to the increased retention after GnT-Vb expression. These results place GnT-Vb as a regulator of RPTP? signaling that influences cell-cell and cell-matrix interactions in the developing nervous system.

Abbott, Karen L.; Matthews, Russell T.; Pierce, Michael



Transient expression of laminin {alpha}1 chain in regenerating murine liver: Restricted localization of laminin chains and nidogen-1  

SciTech Connect

Most interstitia between epithelial and endothelial cells contain basal laminae (BLs), as defined by electron microscopy. However, in liver, the sinusoidal interstitium (called space of Disse) between hepatocytes and sinusoidal endothelial cells (SECs) lacks BLs. Because laminins are major components of BLs throughout the body, whether laminins exist in sinusoids has been a controversial issue. Despite recent advances, the distribution and expression of laminin chains have not been well defined in mammalian liver. Here, using a panel of antibodies, we examined laminins in normal and regenerating mouse livers. Of {alpha} chains, {alpha}5 was widely observed in all BLs except for sinusoids, while the other {alpha} chains were variously expressed in Glisson's sheath and central veins. Laminin {gamma}1 was also distributed to all BLs except for sinusoids. Although the {beta}2 chain was observed in all BLs and sinusoids, the expression of {beta}1 chain was restricted to Glisson's sheath. Detailed analysis of regenerating liver revealed that {alpha}1 and {gamma}1 chains appeared in sinusoids and were produced by stellate cells. The staining of {alpha}1 and {gamma}1 chains reached its maximum intensity at 6 days after two-thirds partial hepatectomy (PHx). Moreover, in vitro studies showed that {alpha}1-containing laminin promoted spreading of sinusoidal endothelial cells (SECs) isolated from normal liver, but not other hepatic cells. In addition, SECs isolated from regenerating liver elongated pseudopodia on {alpha}1-containing laminin more so than did cells from normal liver. The transient expression of laminin {alpha}1 may promote formation of sinusoids after PHx.

Kikkawa, Yamato [Department of Pathophysiology, Cancer Research Institute, Sapporo Medical University School of Medicine, South 1, West 17, Chuo-ku, Sapporo 060-8556 (Japan)]. E-mail:; Mochizuki, Yoichi [Department of Pathophysiology, Cancer Research Institute, Sapporo Medical University School of Medicine, South 1, West 17, Chuo-ku, Sapporo 060-8556 (Japan); Miner, Jeffrey H. [Renal Division, Department of Internal Medicine and Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110 (United States); Mitaka, Toshihiro [Department of Pathophysiology, Cancer Research Institute, Sapporo Medical University School of Medicine, South 1, West 17, Chuo-ku, Sapporo 060-8556 (Japan)



Recognition of the laminin E8 cell-binding site by an integrin possessing the alpha 6 subunit is essential for epithelial polarization in developing kidney tubules  

PubMed Central

It has been previously shown that A-chain and domain(E8)-specific antibodies to laminin that inhibit cell adhesion also interfere with the establishment of epithelial cell polarity during kidney tubule development (Klein, G., M. Langegger, R. Timpl, and P. Ekblom. 1988. Cell. 55:331-341). A monoclonal antibody specific for the integrin alpha 6 subunit, which selectively blocks cell binding to E8, was used to study the receptors involved. Immunofluorescence staining of embryonic kidneys and of organ cultures of metanephric mesenchyme demonstrated coappearance of the integrin alpha 6 subunit and the laminin A-chain in regions where nonpolarized mesenchymal cells convert into polarized epithelial cells. Both epitopes showed marked colocalization in basal areas of tubules, while an exclusive immunostaining for alpha 6 was observed in lateral and apical cell surfaces of the tubular epithelial cells. Organ culture studies demonstrated a consistent inhibition of kidney epithelium development by antibodies against the alpha 6 subunit. The data suggest that the recognition of E8 cell-binding site of laminin by a specific integrin is crucial for the formation of kidney tubule epithelium from undifferentiated mesenchymal stem cells. In some other cell types (endothelium, some ureter cells) an exclusive expression of alpha 6 with no apparent colocalization of laminin A-chain in the corresponding basement membrane was seen. Thus, in these cells, integrins possessing the alpha 6 subunit may bind to laminin isoforms that differ from those synthesized by developing tubules.



Interaction of two translational components, lysyl-tRNA synthetase and p40/37LRP, in plasma membrane promotes laminin-dependent cell migration.  


Although human lysyl-tRNA synthetase (KRS), an enzyme for protein synthesis, is often highly expressed in various cancer cells, its pathophysiological implications have not been understood. Here we found that KRS induces cancer cell migration through interaction with the 67-kDa laminin receptor (67LR) that is converted from ribosomal subunit p40. On laminin signal, KRS was phosphorylated at the T52 residue by p38MAPK and dissociated from the cytosolic multi-tRNA synthetase complex for membrane translocation. The importance of T52 phosphorylation for membrane translocation of KRS was confirmed by site-directed mutagenesis. In the membrane, turnover of 67LR was controlled by Nedd4-mediated ubiquitination, and KRS inhibited ubiquitin-dependent degradation of 67LR, thereby enhancing laminin-induced cell migration. This work thus unveiled a unique function of KRS in the control of cell migration and its pathological implication in metastasis. PMID:22751010

Kim, Dae Gyu; Choi, Jin Woo; Lee, Jin Young; Kim, Hyerim; Oh, Young Sun; Lee, Jung Weon; Tak, Yu Kyung; Song, Joon Myong; Razin, Ehud; Yun, Seok-Hyun; Kim, Sunghoon



Constitutive receptor activation and pharmacological modeling: the adenosine A2b receptor as a prototype  

Microsoft Academic Search

In this thesis a combined approach is described to investigate the constitutive activity of G protein protein-coupled receptors (GPCRs) using human adenosine A2B receptors and to evaluate disease-related constitutive GPCR activity as a target for treatment. To this end a yeast expression system together with pharmacological and theoretical receptor models have been applied.\\u000aIn Chapter 2 the advantages of yeasts

Qilan Li



YY1 repressing peroxisome proliferator-activated receptor delta promoter  

Microsoft Academic Search

Peroxisome proliferator-activated receptors delta (PPAR?) is a nuclear hormone receptor belonging to the steroid receptor\\u000a superfamily and is molecular targets for drugs to treat hypertriglyceridemia and type 2 diabetes. Yin Yang 1 (YY1) is a transcription\\u000a factor that can repress or activate transcription of the genes with which it interacts. In this report, we show that YY1 specifically\\u000a interacts with

Cheng-Qiang He; Nai-Zheng Ding; Wei Fan



Rotational motion during three-dimensional morphogenesis of mammary epithelial acini relates to laminin matrix assembly  

PubMed Central

Our understanding of the mechanisms by which ducts and lobules develop is derived from model organisms and three-dimensional (3D) cell culture models wherein mammalian epithelial cells undergo morphogenesis to form multicellular spheres with a hollow central lumen. However, the mechanophysical properties associated with epithelial morphogenesis are poorly understood. We performed multidimensional live-cell imaging analysis to track the morphogenetic process starting from a single cell to the development of a multicellular, spherical structure composed of polarized epithelial cells surrounding a hollow lumen. We report that in addition to actively maintaining apicobasal polarity, the structures underwent rotational motions at rates of 15–20 ?m/h and the structures rotated 360° every 4 h during the early phase of morphogenesis. Rotational motion was independent of the cell cycle, but was blocked by loss of the epithelial polarity proteins Scribble or Pard3, or by inhibition of dynein-based microtubule motors. Interestingly, none of the structures derived from human cancer underwent rotational motion. We found a direct relationship between rotational motion and assembly of endogenous basement membrane matrix around the 3D structures, and that structures that failed to rotate were defective in weaving exogenous laminin matrix. Dissolution of basement membrane around mature, nonrotating acini restored rotational movement and the ability to assemble exogenous laminin. Thus, coordinated rotational movement is a unique mechanophysical process observed during normal 3D morphogenesis that regulates laminin matrix assembly and is lost in cancer-derived epithelial cells.

Wang, Hui; Lacoche, Sam; Huang, Ling; Xue, Bin; Muthuswamy, Senthil K.



Rotational motion during three-dimensional morphogenesis of mammary epithelial acini relates to laminin matrix assembly.  


Our understanding of the mechanisms by which ducts and lobules develop is derived from model organisms and three-dimensional (3D) cell culture models wherein mammalian epithelial cells undergo morphogenesis to form multicellular spheres with a hollow central lumen. However, the mechanophysical properties associated with epithelial morphogenesis are poorly understood. We performed multidimensional live-cell imaging analysis to track the morphogenetic process starting from a single cell to the development of a multicellular, spherical structure composed of polarized epithelial cells surrounding a hollow lumen. We report that in addition to actively maintaining apicobasal polarity, the structures underwent rotational motions at rates of 15-20 ?m/h and the structures rotated 360° every 4 h during the early phase of morphogenesis. Rotational motion was independent of the cell cycle, but was blocked by loss of the epithelial polarity proteins Scribble or Pard3, or by inhibition of dynein-based microtubule motors. Interestingly, none of the structures derived from human cancer underwent rotational motion. We found a direct relationship between rotational motion and assembly of endogenous basement membrane matrix around the 3D structures, and that structures that failed to rotate were defective in weaving exogenous laminin matrix. Dissolution of basement membrane around mature, nonrotating acini restored rotational movement and the ability to assemble exogenous laminin. Thus, coordinated rotational movement is a unique mechanophysical process observed during normal 3D morphogenesis that regulates laminin matrix assembly and is lost in cancer-derived epithelial cells. PMID:23248267

Wang, Hui; Lacoche, Sam; Huang, Ling; Xue, Bin; Muthuswamy, Senthil K



Transcription Activation by the Human Estrogen Receptor Subtype   (ER ) Studied with ER  and ER  Receptor Chimeras  

Microsoft Academic Search

We have studied the two estrogen receptor (ER) subtypes, ERa and ERb, and chimeric constructs with ERa and ERb to examine the bioactivities of these receptors and their responses to estrogen and antiestrogen ligands. Transcriptional activity of ERb is highly de- pendent on cell\\/promoter context and on the nature of the ligand. ERb activated significant levels of transcription in response




Calcitonin receptor-like receptor (CLR) influences posttranslational events of receptor activity-modifying proteins (RAMPs).  


Adrenomedullins (AM) form a multifunctional subfamily of the calcitonin gene-related peptide (CGRP) superfamily, the members of which exert their physiological roles through a 1:1 combination of calcitonin receptor-like receptors (CLRs) and receptor activity-modifying proteins (RAMPs). It has been shown that RAMPs can modify the biochemical properties of CLRs; for example, RAMP escorts CLR to the plasma membrane, affects glycosylation state of CLR, and transforms the ligand selectivity of CLR, but on the other hand the effects of CLRs on the biochemical and functional properties of the partner RAMPs are not well established. In this study, using pufferfish (mefugu, mf) homolog, we revealed that mfCLR1 could affect the post-translational modification and trafficking pathway of mfRAMP1. In addition, mfCLRs boosted mfRAMP1, mfRAMP2b, and mfRAMP3 translocation to cell surface. We further revealed that mfRAMPs, except mfRAMP1 and mfRAMP3, could be expressed as multimers on the plasma membrane. However, only monomeric form of mfRAMP2a, mfRAMP4, and mfRAMP5 could heteromerize with mfCLR1 but not with mfCLR2 or mfCLR3, which was consistent with their abilities to induce cAMP response. Collectively our results indicate that the glycosylation, subcellular trafficking, and pharmacological properties of the components of RAMP-CLR receptor complexes are regulated in an interdependent manner. PMID:22321396

Nag, Kakon; Sultana, Naznin; Hirose, Shigehisa



Sigma receptors suppress multiple aspects of microglial activation.  


During brain injury, microglia become activated and migrate to areas of degenerating neurons. These microglia release proinflammatory cytokines and reactive oxygen species causing additional neuronal death. Microglia express high levels of sigma receptors, however, the function of these receptors in microglia and how they may affect the activation of these cells remain poorly understood. Using primary rat microglial cultures, it was found that sigma receptor activation suppresses the ability of microglia to rearrange their actin cytoskeleton, migrate, and release cytokines in response to the activators adenosine triphosphate (ATP), monocyte chemoattractant protein 1 (MCP-1), and lipopolysaccharide (LPS). Next, the role of sigma receptors in the regulation of calcium signaling during microglial activation was explored. Calcium fluorometry experiments in vitro show that stimulation of sigma receptors suppressed both transient and sustained intracellular calcium elevations associated with the microglial response to these activators. Further experiments showed that sigma receptors suppress microglial activation by interfering with increases in intracellular calcium. In addition, sigma receptor activation also prevented membrane ruffling in a calcium-independent manner, indicating that sigma receptors regulate the function of microglia via multiple mechanisms. PMID:19031439

Hall, Aaron A; Herrera, Yelenis; Ajmo, Craig T; Cuevas, Javier; Pennypacker, Keith R



Vitamin D receptor activation and cardiovascular disease.  


Vitamin D has been recently associated with several renal, cardiovascular and inflammatory diseases, beyond mineral metabolism and bone health. This is due in part to widespread expression of vitamin D receptor (VDR) on tissues and cells such as heart, kidney, immune cells, brain and muscle. In chronic kidney disease (CKD) and other chronic disorders, vitamin D deficiency [serum 25(OH)D <20 ng/mL] is very common and is associated with adverse outcomes. Paricalcitol, a selective activator of VDR, has demonstrated in several experimental and clinical studies of diabetic and non-diabetic CKD a favourable profile compared to other VDR activators, alone or as add-on to standard therapy. These beneficial effects are mediated by different actions such as reduction of oxidative stress, inflammation, downregulation of cardiac and renal renin expression, downregulation of calcifying genes and direct vascular protective effects. Furthermore, paricalcitol beneficial effects may be independent of baseline serum parathyroid hormone (PTH), calcium and phosphate levels. These benefits should be confirmed in large and well-designed ongoing clinical trials. PMID:23258805

Gonzalez-Parra, Emilio; Rojas-Rivera, Jorge; Tuñón, Jose; Praga, Manuel; Ortiz, Alberto; Egido, Jesus



Adenosine receptor activation ameliorates type 1 diabetes  

Microsoft Academic Search

Growing evidence indicates that adeno- sine receptors could be promising therapeutic targets in autoimmune diseases. Here we studied the role of adenosine receptors in controlling the course of type 1 diabetes. Diabetes in CD-1 mice was induced by multi- ple-low-dose-streptozotocin (MLDS) treatment and in nonobese diabetic (NOD) mice by cyclophosphamide injection. The nonselective adenosine receptor agonist 5-N-ethylcarboxamidoadenosine (NECA) prevented diabetes

Zoltan H. Nemeth; David Bleich; Balazs Csoka; Pal Pacher; Jon G. Mabley; Leonora Himer; E. Sylvester Vizi; Edwin A. Deitch; Csaba Szabo; Bruce N. Cronstein; Gyorgy Hasko



Specificity and function of activating Ly-49 receptors.  


Inhibitory Ly-49 receptors allow murine natural killer (NK) cells to kill cells with aberrant class I MHC expression while sparing normal cells. This is accomplished by their recognition of specific class I MHC products and prevention of NK-cell lysis of cells that present a normal repertoire of class I MHC ligands--"the missing self hypothesis". However, Ly-49 receptors that lack the cytoplasmic immunoreceptor tyrosine-based inhibitory motif, which is required for inhibition of killing, have also been described. These receptors were found to stimulate NK killing and are therefore referred to as activating Ly-49 receptors. Interestingly, the activating receptors have class I MHC-binding domains that are nearly indistinguishable from those of the inhibiting receptors, and binding to class I MHC has now been demonstrated for three activating receptors. Presently, there is no defined physiological role for activating Ly-49 receptors. Here we present an overview of current knowledge regarding the diversity, structure and function of activating Ly-49 receptors with a focus on class I MHC specificity, and we discuss their potential role(s) in natural resistance. PMID:11513132

Kane, K P; Silver, E T; Hazes, B



Granzyme K Activates Protease-Activated Receptor-1  

PubMed Central

Granzyme K (GrK) is a trypsin-like serine protease that is elevated in patients with sepsis and acute lung inflammation. While GrK was originally believed to function exclusively as a pro-apoptotic protease, recent studies now suggest that GrK may possess other non-cytotoxic functions. In the context of acute lung inflammation, we hypothesized that GrK induces pro-inflammatory cytokine release through the activation of protease-activated receptors. The direct effect of extracellular GrK on PAR activation, intracellular signaling and cytokine was assessed using cultured human lung fibroblasts. Extracellular GrK induced secretion of IL-6, IL-8 and MCP-1 in a dose- and time-dependent manner in lung fibroblasts. Heat-inactivated GrK did not induce cytokine release indicating that protease activity is required. Furthermore, GrK induced activation of both the ERK1/2 and p38 MAP kinase signaling pathways, and significantly increased fibroblast proliferation. Inhibition of ERK1/2 abrogated the GrK-mediated cytokine release. Through the use of PAR-1 and PAR-2 neutralizing antibodies, it was determined that PAR-1 is essential for GrK-induced IL-6, IL-8 and MCP-1 release. In summary, extracellular GrK is capable of activating PAR-1 and inducing fibroblast cytokine secretion and proliferation.

Cooper, Dawn M.; Pechkovsky, Dmitri V.; Hackett, Tillie L.; Knight, Darryl A.; Granville, David J.



Subunit dissociation as a possible mechanism of glucocorticoid receptor activation.  


For the elucidation of the mechanism of steroid hormone receptor activation, the hydrodynamic properties of the unactivated and activated forms of the nonproteolyzed glucocorticoid receptor from the mouse AtT-20 pituitary tumor cell line were determined. The unactivated, molybdate-stabilized receptor has the following properties: sedimentation coefficient = 9 S; Rs = 8.3 nm; Mr = 317 000; f/f0 = 1.70; axial ratio (prolate ellipsoid) = 14. The activated monomeric receptor has a sedimentation coefficient of 3.2 S, a Stokes radius of 6 nm, a molecular weight of 81 000, a frictional ratio of 1.93, and an axial ratio (prolate ellipsoid) of 18. A receptor species of intermediate size was detected when the analysis was performed in buffer containing both 0.3 M KCl and 20mM Na2MoO4. Its characteristics are as follows: sedimentation coefficient = 5 S; Rs = 8.3 nm; Mr = 176 000; f/f0 = 2.06; axial ratio (prolate ellipsoid) = 22. A preliminary study seemed to indicate that this is an activated form of the receptor. On the basis of the molecular weights, it is likely that the unactivated receptor is a tetramer of identical hormone-binding subunits (Mr = 81 000) while the intermediate form is a homodimer. Alternatively, non-hormone-binding components (receptor-binding factors) may be involved in forming the multimeric, nonactivated receptor complex. In either case, the dissociation of a multimeric, nonactivated receptor into subunits appears to be a possible mechanism of receptor activation. Finally, the addition of high concentrations of 1-thioglycerol promoted activation. Thus, sulfhydryl groups may be involved in receptor subunit interaction. PMID:6849900

Vedeckis, W V



Melatonin blocks the activation of estrogen receptor for DNA binding.  


The present study shows that melatonin prevents, within the first cell cycle, the estradiol-induced growth of synchronized MCF7 breast cancer cells. By using nuclear extracts of these cells, we first examined the binding of estradiol-estrogen receptor complexes to estrogen-responsive elements and found that the addition of estradiol to whole cells activates the binding of the estrogen receptor to DNA whereas melatonin blocks this interaction. By contrast, melatonin neither affects the binding of estradiol to its receptor nor the receptor nuclear localization. Moreover, we also show that addition of estradiol to nuclear extracts stimulates the binding of estrogen receptor to DNA, but this activation is also prevented by melatonin. The inhibitory effect caused by melatonin is saturable at nanomolar concentrations and does not appear to be mediated by RZR nuclear receptors. The effect is also specific, since indol derivatives do not cause significant inhibition. Furthermore, we provide evidence that melatonin does not interact with the estrogen receptor in the absence of estradiol. Together, these results demonstrate that melatonin interferes with the activation of estrogen receptor by estradiol. The effect of melatonin suggests the presence of a receptor that, upon melatonin addition, destabilizes the binding of the estradiol-estrogen receptor complex to the estrogen responsive element. PMID:10224229

Rato, A G; Pedrero, J G; Martinez, M A; del Rio, B; Lazo, P S; Ramos, S



Thrombopoietin receptor activation: transmembrane helix dimerization, rotation, and allosteric modulation  

PubMed Central

We report how rotational variations in transmembrane (TM) helix interactions participate in the activity states of the thrombopoietin receptor (TpoR), a type 1 cytokine receptor that controls the production of blood platelets. We also explore the mechanism of small-molecule agonists that do not mimic the natural ligand. We show, by a combination of cysteine cross-linking, alanine-scanning mutagenesis, and computational simulations, that the TpoR TM dimerizes strongly and can adopt 3 different stable, rotationally related conformations, which may correspond to specific states of the full-length receptor (active, inactive, and partially active). Thus, our data suggest that signaling and inactive states of the receptor are related by receptor subunit rotations, rather than a simple monomer-dimer transition. Moreover, results from experiments with and without agonists in vitro and in cells allow us to propose a novel allosteric mechanism of action for a class of small molecules, in which they activate TpoR by binding to the TM region and by exploiting the rotational states of the dimeric receptor. Overall, our results support the emerging view of the participation of mutual rotations of the TM domains in cytokine receptor activation.—Matthews, E. E., Thévenin, D., Rogers, J. M., Gotow, L., Lira, P. D., Reiter, L. A., Brissette, W. H., Engelman, D. M. Thrombopoietin receptor activation: transmembrane helix dimerization, rotation, and allosteric modulation.

Matthews, Erin E.; Thevenin, Damien; Rogers, Julia M.; Gotow, Lisa; Lira, Paul D.; Reiter, Lawrence A.; Brissette, William H.; Engelman, Donald M.



Molecular mechanism for plant steroid receptor activation by somatic embryogenesis co-receptor kinases.  


Brassinosteroids, which control plant growth and development, are sensed by the leucine-rich repeat (LRR) domain of the membrane receptor kinase BRASSINOSTEROID INSENSITIVE 1 (BRI1), but it is unknown how steroid binding at the cell surface activates the cytoplasmic kinase domain of the receptor. A family of somatic embryogenesis receptor kinases (SERKs) has been genetically implicated in mediating early brassinosteroid signaling events. We found a direct and steroid-dependent interaction between the BRI1 and SERK1 LRR domains by analysis of their complex crystal structure at 3.3 angstrom resolution. We show that the SERK1 LRR domain is involved in steroid sensing and, through receptor-co-receptor heteromerization, in the activation of the BRI1 signaling pathway. Our work reveals how known missense mutations in BRI1 and in SERKs modulate brassinosteroid signaling and the targeting mechanism of BRI1 receptor antagonists. PMID:23929946

Santiago, Julia; Henzler, Christine; Hothorn, Michael



Modulation of Steroid Hormone Receptor Activity  

Microsoft Academic Search

Classical steroid hormones (SHs) – estrogens, androgens, progestins, glucocorticoids and mineralocorticoids – play critical roles in the regulation of reproduction, metabolism and cancer. SHs act via their cognate steroid hormone receptors (SHRs) in multiple target tissues throughout the body, exerting their physiological effects through nuclear receptor (NR)-mediated gene transcription. Since SHRs are the mediators of steroid hormone signalling in cells,

Vladimir Staniši?; David M. Lonard; Bert W. O’Malley



Qualitative alterations in laminin expression in experimental lupus nephritis.  

PubMed Central

Previous studies have revealed quantitative alterations in laminin-1 expression at the mRNA and protein levels during the development of glomerulonephritis and glomerulosclerosis in chronic graft-versus-host disease in mice, a model for lupus nephritis. We have now studied the qualitative alterations in laminin expression with two monoclonal antibodies that recognize epitopes on either the E8 or the P1 fragment of laminin-1. Both of these fragments are involved in cell-matrix and matrix-matrix interactions. In normal glomeruli these laminin epitopes are present only in the mesangial matrix; during embryogenesis, however, they are also present in the glomerular basement membrane. The distribution of laminin epitopes was first studied by using immunofluorescence in kidneys of mice with graft-versus-host disease at different points in time after disease induction. Reflection contrast and immunoelectron microscopy were performed after in vivo injection of the horseradish peroxidase-coupled monoclonal antibodies. In glomeruli of mice 8 weeks after disease induction, both injected antibodies bound specifically in electron-dense immune deposits in the mesangium and subepithelially along the glomerular basement membrane as well as in the expanded mesangial matrix. At 11 and 12 weeks after disease induction, when focal and segmental glomerulosclerosis had developed, the antibodies additionally bound in the matrix subendothelially along the glomerular basement membrane and at the periphery of end-stage sclerotic lesions. To study changes in the distribution of laminin epitopes over time, mice were injected with either monoclonal antibody before induction of graft-versus-host disease. The antibodies were detected 8 and 12 weeks later in the mesangial matrix of mice with lupus nephritis. Once segmental glomerulosclerosis had developed, the antibodies were additionally detected within the thickened glomerular capillary wall. The specific binding of anti-laminin monoclonal antibodies in electron-dense immune deposits further substantiates the hypothesis that anti-laminin autoantibodies participate in glomerular immune complex formation in this model, as suggested by earlier studies. Furthermore, our results show that the distribution of glomerular laminin epitopes in the matrix is altered during the development of glomerular disease. These changes in the structure of the glomerular basement membrane may contribute to the abnormal cell-matrix and matrix-matrix interactions during the development of glomerular disease in this model for lupus nephritis. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 7 Figure 8

Kootstra, C. J.; Bergijk, E. C.; Veninga, A.; Prins, F. A.; de Heer, E.; Abrahamson, D. R.; Bruijn, J. A.



Metal binding is critical for the folding and function of laminin binding protein, Lmb of Streptococcus agalactiae.  


Lmb is a 34 kDa laminin binding surface adhesin of Streptococcus agalactiae. The structure of Lmb reported by us recently has shown that it consists of a metal binding crevice, in which a zinc ion is coordinated to three highly conserved histidines. To elucidate the structural and functional significance of the metal ion in Lmb, these histidines have been mutated to alanine and single, double and triple mutants were generated. These mutations resulted in insolubility of the protein and revealed altered secondary and tertiary structures, as evidenced by circular dichroism and fluorescence spectroscopy studies. The mutations also significantly decreased the binding affinity of Lmb to laminin, implicating the role played by the metal binding residues in maintaining the correct conformation of the protein for its binding to laminin. A highly disordered loop, proposed to be crucial for metal acquisition in homologous structures, was deleted in Lmb by mutation (?Lmb) and its crystal structure was solved at 2.6 Å. The ?Lmb structure was identical to the native Lmb structure with a bound zinc ion and exhibited laminin binding activity similar to wild type protein, suggesting that the loop might not have an important role in metal acquisition or adhesion in Lmb. Targeted mutations of histidine residues confirmed the importance of the zinc binding crevice for the structure and function of the Lmb adhesin. PMID:23826314

Ragunathan, Preethi; Sridaran, Divya; Weigel, Anja; Shabayek, Sarah; Spellerberg, Barbara; Ponnuraj, Karthe



Maintenance of glomerular filtration barrier integrity requires laminin alpha5.  


Mutation of the mouse laminin alpha5 gene results in a variety of developmental defects, including defects in kidney structure and function. Whereas the total absence of laminin alpha5 results in breakdown of the glomerular basement membrane (GBM) and failed glomerular vascularization, a hypomorphic Lama5 mutation (the Lama5(neo) allele) results in proteinuria, hematuria, polycystic kidney disease (PKD), and death 3 to 4 weeks after birth. Here, we examined the role of podocyte-derived laminin alpha5 via podocyte-specific inactivation of Lama5 and podocyte-specific rescue of the Lama5(neo) mutation. Podocyte-specific inactivation of Lama5 resulted in varying degrees of proteinuria and rates of progression to nephrotic syndrome. The GBM of proteinuric mice appeared thickened and "moth-eaten," and podocyte foot processes became effaced. Podocyte-specific restoration of laminin alpha5 production using two distinct strategies in Lama5(neo/neo) mice resulted in the resolution of proteinuria, hematuria, and PKD. These results suggest that the development of normal GBM structure and function requires podocyte-derived laminin alpha5 during and after glomerulogenesis and present a unique mechanism for the pathogenesis of PKD in these mice. PMID:20150535

Goldberg, Seth; Adair-Kirk, Tracy L; Senior, Robert M; Miner, Jeffrey H



Influence of laminin-2 on Schwann cell-axon interactions.  


The dy/dy mouse suffers from a form of muscular dystrophy caused by a substantial reduction in laminin alpha2-chain protein, a major component of both muscle and Schwann cell basal laminae. This article examines the effect of laminin alpha2 deficiency on Schwann cell-axon interactions both in vivo at varying intervals after nerve crush, and in vitro, in cocultures of neurons and Schwann cells. The morphological spectrum of aberrant Schwann cell-axon associations seen in uncrushed dy/dy sciatic nerves was recapitulated during regeneration: myelination of regenerating axons was delayed compared with the process in unaffected mice and the relatively few myelin sheaths which were formed in dy/dy distal nerve stumps were often uncompacted. In vitro, Schwann cells dissociated from adult dy/dy sciatic nerves predictably failed to express detectable laminin alpha2-chain and displayed an unusual multipolar morphology. Branching of neurites, in terms both of numbers of terminal branches and of complexity of branching, from dorsal root ganglia neurons grown on dy/dy Schwann cells, was significantly less extensive than that seen when neurons were cocultured with Schwann cells from unaffected littermates, but this effect was reversed by exogenous laminin-2. Our results lend strong support to the view that laminin-2 is essential for establishing and/or maintaining Schwann cell-axon interactions, in normal and in regenerating nerves. PMID:11008211

Uziyel, Y; Hall, S; Cohen, J



Adenosine receptor activation ameliorates type 1 diabetes  

PubMed Central

Growing evidence indicates that adenosine receptors could be promising therapeutic targets in autoimmune diseases. Here we studied the role of adenosine receptors in controlling the course of type 1 diabetes. Diabetes in CD-1 mice was induced by multiple-low-dose-streptozotocin (MLDS) treatment and in nonobese diabetic (NOD) mice by cyclophosphamide injection. The nonselective adenosine receptor agonist 5?-N-ethylcarboxamidoadenosine (NECA) prevented diabetes development in both MLDS-challenged mice and in cyclophosphamide-treated NOD mice. The effect of NECA was reversed by the selective A2B receptor antagonist N-(4-cyanophenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]acetamide (MRS 1754). The selective A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA) and A3 receptor agonist N6-(3-iodobenzyl)-adenosine-5?-N-methyluronamide (IB-MECA) were less efficacious in ameliorating the course of diabetes. NECA inhibited diabetes in A2A receptor KO mice and the selective A2A receptor agonist 2-p-(2-carboxyethyl)phenethyl-amino-5?-N-ethyl-carboxamidoadenosine (CGS21680) had no effect in normal mice, indicating a lack of role of A2A receptors. NECA failed to prevent cytokine-induced ?-cell death in vitro, but NECA strongly suppressed expression of the proinflammatory cytokines TNF-?, MIP-1?, IL-12, and IFN-? in pancreata, endotoxin, or anti-CD3-stimulated splenic cells, and T helper 1 lymphocytes, indicating that the beneficial effect of NECA was due to immunomodulation. These results demonstrate that adenosine receptor ligands are potential candidates for the treatment of type 1 diabetes.

Nemeth, Zoltan H.; Bleich, David; Csoka, Balazs; Pacher, Pal; Mabley, Jon G.; Himer, Leonora; Vizi, E. Sylvester; Deitch, Edwin A.; Szabo, Csaba; Cronstein, Bruce N.; Hasko, Gyorgy



Dynamic correlation networks in human peroxisome proliferator-activated receptor-? nuclear receptor protein  

Microsoft Academic Search

Peroxisome proliferator-activated receptor-? nuclear receptor (PPAR-?) belongs to the superfamily of nuclear receptor proteins\\u000a that function as ligand-dependent transcription factors and plays a specific physiological role as a regulator of lipid metabolism.\\u000a A number of experimental studies have suggested that allostery plays an important role in the functioning of PPAR-?. Here\\u000a we use normal-mode analysis of PPAR-? to characterize a

Jeremy Fidelak; Silvia Ferrer; Michael Oberlin; Dino Moras; Annick Dejaegere; Roland H. Stote



Bosentan, a dual endothelin receptor antagonist, activates the pregnane X nuclear receptor  

Microsoft Academic Search

Recent clinical studies have shown that bosentan, a dual endothelin receptor antagonist, decreases the exposure to various substrates of cytochrome P450 (CYP) isoenzymes 2C9 and 3A4. The aim of the study was to investigate the effect of bosentan, its metabolites and glibenclamide on the activity of the pregnane X receptor, a nuclear receptor that regulates the transcription of CYP3A4. CV-1

Paul L. M van Giersbergen; Carmela Gnerre; Alexander Treiber; Jasper Dingemanse; Urs A Meyer



A Potential Role of Progestin-Induced Laminin-5/?6-Integrin Signaling in the Formation of Side Branches in the Mammary Gland  

PubMed Central

Mammary organoids from adult mice produce tubules, analogous to mammary ducts in vivo, in response to hepatocyte growth factor (HGF) when cultured in collagen gels. The combination of HGF plus progestin (R5020) causes reduced tubule number and length. We hypothesized that the inhibitory effect on tubulogenesis was due to progestin-mediated alteration of HGF/c-Met signaling. Using molecular inhibitors and short hairpin RNA, it was determined that HGF activation of Ras-related C3 botulinum toxin substrate (Rac1) was required for the formation of cytoplasmic extensions, the first step of tubulogenesis, and that Rac1 activity was Src kinase (Src) and focal adhesion kinase (FAK) dependent. The highly novel finding was that R5020 reduced tubulogenesis by up-regulating and increasing extracellular laminin and ?6-integrin ligation to reduce activation of the Src, focal adhesion kinase, and Rac1 pathway. Receptor activator of nuclear factor-?B ligand, another progesterone-induced paracrine factor, did not replicate this effect of R5020. The inhibitory effect of R5020 on tubulogenesis was likely mediated through progesterone receptor (PR) isoform A (PRA), because PRA is the predominant PR isoform expressed in the organoids, and the progestin-induced effect was prevented by the PR antagonist RU486. These results provide a plausible mechanism that explains progestin/PRA-mediated blunting of HGF-induced tubulogenesis in vitro and is proposed to be relevant to progesterone/PRA-induced side-branching in vivo during pregnancy.

Meyer, Gabriele; Leipprandt, Jeffrey; Xie, Jianwei; Aupperlee, Mark D.



The Orphan Nuclear Receptor TR4 Is a Vitamin A-activated Nuclear Receptor  

SciTech Connect

Testicular receptors 2 and 4 (TR2/4) constitute a subgroup of orphan nuclear receptors that play important roles in spermatogenesis, lipid and lipoprotein regulation, and the development of the central nervous system. Currently, little is known about the structural features and the ligand regulation of these receptors. Here we report the crystal structure of the ligand-free TR4 ligand binding domain, which reveals an autorepressed conformation. The ligand binding pocket of TR4 is filled by the C-terminal half of helix 10, and the cofactor binding site is occupied by the AF-2 helix, thus preventing ligand-independent activation of the receptor. However, TR4 exhibits constitutive transcriptional activity on multiple promoters, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, or ligand binding substantially reduce the transcriptional activity of this receptor. Importantly, both retinol and retinoic acid are able to promote TR4 to recruit coactivators and to activate a TR4-regulated reporter. These findings demonstrate that TR4 is a ligand-regulated nuclear receptor and suggest that retinoids might have a much wider regulatory role via activation of orphan receptors such as TR4.

Zhou, X. Edward; Suino-Powell, Kelly M.; Xu, Yong; Chan, Cee-Wah; Tanabe, Osamu; Kruse, Schoen W.; Reynolds, Ross; Engel, James Douglas; Xu, H. Eric (Van Andel); (Michigan-Med)



Plant cysteine proteases that evoke itch activate protease-activated receptors  

PubMed Central

Background Bromelain, ficin and papain are cysteine proteases from plants that produce itch upon injection into skin. Their mechanism of action has not been considered previously. Objectives To determine the mechanism by which these proteases function. Methods The ability of these proteases to activate protease-activated receptors was determined by ratiometric calcium imaging. Results We show here that bromelain, ficin and papain activate protease-activated receptors 2 and 4. Conclusions Bromelain, ficin and papain function as signalling molecules and activate protease-activated receptors. Activation of these receptors is the likely mechanism by which these proteases evoke itch.

Reddy, V.B.; Lerner, E.A.



Phospholipase C-Coupled Receptors and Activation of TRPC Channels  

Microsoft Academic Search

The canonical transient receptor potential (TRPC) cation channels are mammalian homologs of the photoreceptor channel TRP inDrosophila melanogaster. All seven TRPCs (TRPC1 through TRPC7) can be activated through Gq\\/11 receptors or receptor tyrosine kinase (RTK) by mechanisms downstream of phospholipase C. The last decade saw a rapidly growing interest in understanding the role of TRPC channels in calcium entry pathways

M. Trebak; L. Lemonnier; J. Smyth; G. Vazquez; J. Putney


Activation of estrogen receptors with E2 downregulates peroxisome proliferator-activated receptor ? in hepatocellular carcinoma.  


Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality and occurs more often in men than in women; however, little is known about its underlying molecular mechanisms. The present study investigated the effect of estrogen receptor (ER)? and ER? on peroxisome proliferator-activated receptor ? (PPAR?) expression in Hep3B cells. We examined PPAR?, ER? and ER? mRNA and protein expression by RT-PCR and western blotting. In order to determine whether PPAR? plays a central role in HCC, we screened for PPAR? expression in liver cancer patient tissues and differentially differentiated HCC cell lines (HA22T, Huh-7, Hep3B and HepG2). We found that PPAR? expression was highly expressed in liver cancer tissues and in Hep3B cells. Furthermore, overexpression of ER? and ER? was found to decrease PPAR? expression at the transcriptional as well as at the translational level in a ligand-dependent manner. In summary, the present study demonstrated that both ER? and ? were sufficient to inhibit PPAR? and provide a valuable therapeutic option for the treatment of HCC patients. PMID:24126791

Lin, Yueh-Min; Velmurugan, Bharath Kumar; Yeh, Yu-Lan; Tu, Chuan Chou; Ho, Tsung-Jung; Lai, Tung Yuan; Tsai, Chih-Hao; Tsai, Fuu Jen; Tsai, Chang-Hai; Huang, Chih-Yang



Receptor interactions in modulating ventilatory activity.  


The ventilatory control system utilizes a variety of sensory receptor groups, including chemoreceptors and mechanoreceptors, to provide feedback concerning the status of controlled variables. Most ventilatory responses to altered receptor inputs generally involve a complex interaction between several receptor groups, central integrative mechanisms, and other modulatory inputs (e.g., "state," hormonal, or neurotransmitter status). Because the control system is complex, nonlinear, and dynamic, the ultimate ventilatory response elicited by a given stimulus is not easy to predict based on the reflex effects of individual receptor groups studied in isolation. A full understanding of the role that sensory receptors play in ventilatory control requires information concerning interactions among receptor groups and with other elements of the control system. The complexity of the problem and the lack of a uniform definition of the term "interaction" has hindered research in this area. An interaction is defined as a nonadditive relationship between independent inputs to the system. Within this definition, five domains of interaction are described. 1) Algebraic interactions occur in ventilation and/or its components because of their multiplicative and nonlinear relationship. 2) Closed-loop interactions occur because of the prevalence of feedback loops within the respiratory control system. 3) Neural interactions reflect central nervous system integration of simultaneous receptor inputs and are demonstrated when feedback loops are opened. Three subdomains of neural interactions are defined: modulatory, dynamic, and range-specific neural interactions. 4) Mechanical interactions result from nonlinear transformations of motoneuron output into mechanical actions. 5) Adaptive interactions occur when paired receptor or modulatory inputs alter future responses. To understand the role of any sensory receptor group in ventilatory control, it is necessary to define its interactions with other control system elements in each of these domains. Understanding the mechanisms of these interactions requires detailed information about the physical system subserving ventilatory control (mechanics and gas exchange) and the relevant properties of the neural network coordinating their actions. PMID:2240275

Mitchell, G S; Douse, M A; Foley, K T



P2 receptors activated by uracil nucleotides--an update.  


Pyrimidine nucleotides, including UTP, UDP and UDP-glucose, are important signaling molecules which activate G protein-coupled membrane receptors (GPCRs) of the P2Y family. Four distinct pyrimidine nucleotide-sensitive P2Y receptor subtypes have been cloned, P2Y2, P2Y4, P2Y6 and P2Y14. P2Y2 and P2Y4 receptors are activated by UTP (the P2Y2, and the rat but not the human P2Y4 receptor are also activated by ATP), the P2Y6 receptor is activated by UDP, and the P2Y14 receptor by UDP-glucose. Furthermore, non-P2Y GPCRs, the cysteinylleukotriene receptors (CysLT1R and CysLT2R) have been described to be activated by UDP in addition to activation by cysteinylleukotrienes. While P2Y2, P2Y4, and P2Y6 receptor activation results in stimulation of phospholipase C, the P2Y14 receptor is coupled to inhibition of adenylate cyclase. Derivatives and analogs of the physiological nucleotides UTP, UDP and ATP have been synthesized and evaluated in order to obtain enzymatically stable, subtype-selective agonists. The P2Y2 receptor agonists diuridine tetraphosphate (diquafosol) and the uracil-cytosine dinucleotide denufosol are currently undergoing clinical trials for dry eye disease, retinal detachment disease, upper respiratory tract symptoms, and cystic fibrosis, respectively. The first antagonists for P2Y2 and P2Y6 receptors that appear to be selective versus other P2Y receptor subtypes have recently been described. Selective antagonists for P2Y4 and P2Y14 receptors are still lacking. Uracil nucleotide-sensitive P2Y receptor subtypes may constitute future targets for the treatment of certain cancer types, vascular diseases, inflammatory diseases, and immunomodulatory intervention. They have also been proposed to play a role in neurodegenerative diseases. This article is an updated version of "P2-Pyrimidinergic Receptors and Their Ligands" by C. E. Müller published in Curr. Pharm. Des. 2002, 8, 2353-2369. PMID:16475938

Brunschweiger, Andreas; Müller, Christa E



Peroxisome proliferator-activated receptor ? in malignant diseases  

Microsoft Academic Search

Peroxisome proliferator-activated receptor ? (PPAR-?) belongs to the family of nuclear hormone receptors (NHRs) and is a ligand-activated transcription factor. There are four mRNAs, PPAR-?1, PPAR-?2, PPAR-?3 and PPAR-?4, which encode two proteins, PPAR-?1and PPAR-?2. PPAR-? consists of five or six structural regions (A–F) in four functional domains. The NH2-terminal A\\/B domain harbors a ligand-independent transcriptional activation function (AF-1), the

Tingting Wang; Jian Xu; Xiaofei Yu; Renchi Yang; Zhong Chao Han



The role of LamininB2 ( LanB2 ) during mesoderm differentiation in Drosophila  

Microsoft Academic Search

In Drosophila, four genes encode for laminin subunits and the formation of two laminin heterotrimers has been postulated. We report the\\u000a identification of mutations in the Drosophila LamininB2 (LanB2) gene that encodes for the only laminin ? subunit and is found in both heterotrimers. We describe their effects on embryogenesis,\\u000a in particular the differentiation of visceral tissues with respect to

Georg Wolfstetter; Anne Holz


Capture of an activated receptor complex from the surface of live cells by affinity receptor chromatography.  


Cell surface receptors and their associated signaling pathways on the plasma membrane are key targets in understanding cellular responses. However, the isolation and identification of receptor complexes has been elusive. The Fc receptor was captured from the surface of live cells using microbeads coated with the receptor's cognate ligand, gamma globulin (IgG), and analyzed by liquid chromatography and tandem mass spectrometry (LC-MS/MS) alongside several controls. Live-cell affinity receptor chromatography (LARC) resulted in a partially nonredundant list of 288 proteins that were specific to the Fc receptor complex. The proteins identified were in close agreement with previously determined factors in the Fc receptor complex as demonstrated by genetic and biochemical methods and revealed novel complex members. Confocal microscopy was used to confirm recruitment of SRC, SYK, PLC, PKC, PI3K, SHIP, TEC, CDC42, RAP, PAK, GAP, GEF, GRP, and CRK to the receptor complex upon activation by the same ligand microbeads. The expression of mutants and silencing RNA against specific isoforms were used to demonstrate a functional role for novel members of the Fc receptor complex, including RHOG (RAS homologue member G), p115 RhoGEF (protein of 115-kDa RAS homologue guanine exchange factor), and CRKL (CRK-like). The recruitment of AKT pleckstrin homology (PH) domain green fluorescent protein (GFP) was used to quantify the production of phosphorylated inositol at the activated receptor complex. We conclude that it is feasible to capture an activated receptor complex from the surface of live cells using ligand-coated microbeads for identification of members of a receptor complex or pathway by LC-MS/MS. PMID:18601892

Jankowski, Andy; Zhu, Peihong; Marshall, John G



Forced expression of laminin ?1 in podocytes prevents nephrotic syndrome in mice lacking laminin ?2, a model for Pierson syndrome  

PubMed Central

Pierson syndrome is a congenital nephrotic syndrome with ocular and neurological defects caused by mutations in LAMB2, the gene encoding the basement membrane protein laminin ?2 (Lam?2). It is the kidney glomerular basement membrane (GBM) that is defective in Pierson syndrome, as Lam?2 is a component of laminin-521 (LM-521; ?5?2?1), the major laminin in the mature GBM. In both Pierson syndrome and the Lamb2?/? mouse model for this disease, laminin ?1 (Lam?1), a structurally similar homolog of Lam?2, is marginally increased in the GBM, but it fails to fully compensate for the loss of Lam?2, leading to the filtration barrier defects and nephrotic syndrome. Here we generated several lines of Lam?1 transgenic mice and used them to show that podocyte-specific Lam?1 expression in Lamb2?/? mice abrogates the development of nephrotic syndrome, correlating with a greatly extended lifespan. In addition, the more Lam?1 was expressed, the less urinary albumin was excreted. Transgenic Lam?1 expression increased the level of Lam?5 in the GBM of rescued mice, consistent with the desired increased deposition of laminin-511 (?5?1?1) trimers. Ultrastructural analysis revealed occasional knob-like subepithelial GBM thickening but intact podocyte foot processes in aged rescued mice. These results suggest the possibility that up-regulation of LAMB1 in podocytes, should it become achievable, would likely lessen the severity of nephrotic syndrome in patients carrying LAMB2 mutations.

Suh, Jung Hee; Jarad, George; VanDeVoorde, Rene G.; Miner, Jeffrey H.



Inhibiting Proteasomal Proteolysis Sustains Estrogen Receptor  Activation  

Microsoft Academic Search

Estrogen receptor- (ER) is a ligand-dependent transcription factor that mediates physiological re- sponses to 17-estradiol (E2). Ligand binding rap- idly down-regulates ER levels through proteaso- mal proteolysis, but the functional impact of receptor degradation on cellular responses to E2 has not been fully established. In this study, we investigated the effect of blocking the ubiquitin- proteasome pathway on ER-mediated transcrip-




Targeting a Tumor-Specific Laminin Domain Critical for Human Carcinogenesis  

Microsoft Academic Search

Laminin-332 is critical for squamous cell carcinoma (SCC) tumorigenesis, but targeting it for cancer therapy has been unachievable due to key role of laminin-332 in promoting tissue integrity. Here, we show that a portion of laminin-332, termed G45, which is proteolytically removed and absent in normal tissues, is prominently expressed in most human SCC tumors and plays an important role

Mark Tran; Patricia Rousselle; Pasi Nokelainen; Sruthi Tallapragada; Ngon T. Nguyen; Edgar F. Fincher



Effect of laminin 332 on motility and invasion in bladder cancer.  


We examined the correlation between laminin 332 and malignancy in bladder cancer patients, and, using a strain of invasive bladder cancer cells, determined whether laminin 332 causes bladder cancer motility and invasion. To investigate the correlation between laminin 332 g2 distribution and patient outcome, we performed a semiquantitative immunohistochemical analysis of 35 paraffin-embedded samples using the antibody D4B5, which is specific for the laminin 5 ?2 chain. To evaluate the role of laminin 332 in NBT-II cell motility and invasion, we used a scratch assay and the Boyden chamber chemoinvasion system. Tumor stage and grade were significantly correlated with a loss of laminin 332 ?2 chain from the basement membrane (p = 0.001) and its retention in the cytoplasm (p = 0.001) (Kruskal-Wallis test). Kaplan-Meier survival curves revealed an association between the risk of progression and cytoplasmic retention of the laminin 332 ?2 chain. In addition, an in vitro scratch assay showed an increase in the migration of cells treated with laminin 332 from their cluster. The Boyden chamber assay showed that laminin 332 potentiated NBT-II cell invasion. Immunohistochemistry results showed that bladder cancer patients with a higher malignancy expressed more laminin 332. The in vitro scratch and invasion assay showed that laminin 332 stimulated the motility and invasion of bladder cancer cells. The invasion assay explains the correlation between laminin 332 expression and bladder cancer malignancy. PMID:23906232

Kang, Sung-Gu; Ha, Young-Ran; Ko, Young-Hwii; Kang, Seok-Ho; Joo, Kwan-Joong; Cho, Hyun-Yee; Park, Hong-Seok; Kim, Chul-Hwan; Kwon, Soon-Young; Kim, Je-Jong; Cheon, Jun; Lee, Jeong-Gu



Distribution and Function of Laminins in the Neuromuscular System of Developing, Adult, and Mutant Mice  

Microsoft Academic Search

Laminins, heterotrimers of a , b , and g chains, are prominent constituents of basal laminae (BLs) throughout the body. Previous studies have shown that laminins affect both myogenesis and synap- togenesis in skeletal muscle. Here we have studied the distribution of the 10 known laminin chains in muscle and peripheral nerve, and assayed the ability of several heterotrimers to

Bruce L. Patton; Jeffrey H. Miner; Arlene Y. Chiu; Joshua R. Sanes



Characterization of peroxisome proliferator-activiated receptor alpha (PPARalpha)-independent effects of PPARalpha activators in the rodent liver: Di(2-ethylehexyl) phthalate activates the constitutive activated receptor  

EPA Science Inventory

Peroxisome proliferator chemicals (PPC) are thought to mediate their effects in rodents on hepatocyte growth and liver cancer through the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha). Recent studies indicate that the plasticizer di-2-ethylhexyl ph...


Evidence that a laminin-like insect protein mediates early events in the interaction of a Phytoparasite with its vector's salivary gland.  


Phytomonas species are plant parasites of the family Trypanosomatidae, which are transmitted by phytophagous insects. Some Phytomonas species cause major agricultural damages. The hemipteran Oncopeltus fasciatus is natural and experimental host for several species of trypanosomatids, including Phytomonas spp. The invasion of the insect vectors' salivary glands is one of the most important events for the life cycle of Phytomonas species. In the present study, we show the binding of Phytomonas serpens at the external face of O. fasciatus salivary glands by means of scanning electron microscopy and the in vitro interaction of living parasites with total proteins from the salivary glands in ligand blotting assays. This binding occurs primarily through an interaction with a 130 kDa salivary gland protein. The mass spectrometry of the trypsin-digest of this protein matched 23% of human laminin-5 ?3 chain precursor sequence by 16 digested peptides. A protein sequence search through the transcriptome of O. fasciatus embryo showed a partial sequence with 51% similarity to human laminin ?3 subunit. Anti-human laminin-5 ?3 chain polyclonal antibodies recognized the 130 kDa protein by immunoblotting. The association of parasites with the salivary glands was strongly inhibited by human laminin-5, by the purified 130 kDa insect protein, and by polyclonal antibodies raised against the human laminin-5 ?3 chain. This is the first report demonstrating that a laminin-like molecule from the salivary gland of O. fasciatus acts as a receptor for Phytomonas binding. The results presented in this investigation are important findings that will support further studies that aim at developing new approaches to prevent the transmission of Phytomonas species from insects to plants and vice-versa. PMID:23118944

de Almeida Dias, Felipe; Souza dos Santos, Andre Luis; Santos Lery, Letícia Miranda; Alves e Silva, Thiago Luiz; Oliveira, Mauricio Martins; Bisch, Paulo Mascarello; Saraiva, Elvira Maria; Souto-Padrón, Thaïs Cristina; Lopes, Angela Hampshire



Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by inhibition of epidermal growth factor receptor signaling.  


Phenobarbital is a central nervous system depressant that also indirectly activates nuclear receptor constitutive active androstane receptor (CAR), which promotes drug and energy metabolism, as well as cell growth (and death), in the liver. We found that phenobarbital activated CAR by inhibiting epidermal growth factor receptor (EGFR) signaling. Phenobarbital bound to EGFR and potently inhibited the binding of EGF, which prevented the activation of EGFR. This abrogation of EGFR signaling induced the dephosphorylation of receptor for activated C kinase 1 (RACK1) at Tyr(52), which then promoted the dephosphorylation of CAR at Thr(38) by the catalytic core subunit of protein phosphatase 2A. The findings demonstrated that the phenobarbital-induced mechanism of CAR dephosphorylation and activation is mediated through its direct interaction with and inhibition of EGFR. PMID:23652203

Mutoh, Shingo; Sobhany, Mack; Moore, Rick; Perera, Lalith; Pedersen, Lee; Sueyoshi, Tatsuya; Negishi, Masahiko



Synaptic NMDA receptor activity boosts intrinsic antioxidant defenses  

Microsoft Academic Search

Intrinsic antioxidant defenses are important for neuronal longevity. We found that in rat neurons, synaptic activity, acting via NMDA receptor (NMDAR) signaling, boosted antioxidant defenses by making changes to the thioredoxin-peroxiredoxin (Prx) system. Synaptic activity enhanced thioredoxin activity, facilitated the reduction of overoxidized Prxs and promoted resistance to oxidative stress. Resistance was mediated by coordinated transcriptional changes; synaptic NMDAR activity

Sofia Papadia; Francesc X Soriano; Frédéric Léveillé; Marc-Andre Martel; Kelly A Dakin; Henrik H Hansen; Angela Kaindl; Marco Sifringer; Jill Fowler; Vanya Stefovska; Grahame Mckenzie; Marie Craigon; Roderick Corriveau; Peter Ghazal; Karen Horsburgh; Bruce A Yankner; David J A Wyllie; Chrysanthy Ikonomidou; Giles E Hardingham



Visualization of Chemokine Receptor Activation in Transgenic Mice Reveals Peripheral Activation of CCR2 Receptors in States of Neuropathic Pain  

PubMed Central

CCR2 chemokine receptor signaling has been implicated in the generation of diverse types of neuropathology, including neuropathic pain. For example, ccr2 knock-out mice are resistant to the establishment of neuropathic pain, and mice overexpressing its ligand, monocyte chemoattractant protein-1 (MCP1; also known as CCL2), show enhanced pain sensitivity. However, whether CCR2 receptor activation occurs in the central or peripheral nervous system in states of neuropathic pain has not been clear. We developed a novel method for visualizing CCR2 receptor activation in vivo by generating bitransgenic reporter mice in which the chemokine receptor CCR2 and its ligand MCP1 were labeled by the fluorescent proteins enhanced green fluorescent protein and monomeric red fluorescent protein-1, respectively. CCR2 receptor activation under conditions such as acute inflammation and experimental autoimmune encephalomyelitis could be faithfully visualized by using these mice. We examined the status of CCR2 receptor activation in a demyelination injury model of neuropathic pain and found that MCP1-induced CCR2 receptor activation mainly occurred in the peripheral nervous system, including the injured peripheral nerve and dorsal root ganglia. These data explain the rapid antinociceptive effects of peripherally administered CCR2 antagonists under these circumstances, suggesting that CCR2 antagonists may ameliorate pain by inhibiting CCR2 receptor activation in the periphery. The method developed here for visualizing CCR2 receptor activation in vivo may be extended to G-protein-coupled receptors (GPCRs) in general and will be valuable for studying intercellular GPCR-mediated communication in vivo.

Jung, Hosung; Bhangoo, Sonia; Banisadr, Ghazal; Freitag, Caroline; Ren, Dongjun; White, Fletcher A.; Miller, Richard J.



Activation of Peroxisome Proliferator-Activated Receptor ?/? Induces Lung Cancer Growth via Peroxisome Proliferator-Activated Receptor Coactivator ?-1?  

PubMed Central

We previously demonstrated that a selective agonist of peroxisome proliferator–activated receptor ?/? (PPAR?/?), GW501516, stimulated human non–small cell lung carcinoma (NSCLC) growth, partly through inhibition of phosphatase and tensin homolog deleted on chromosome 10 expression. Here, we show that GW501516 also decreases the phosphorylation of AMP-activated protein kinase ? (AMPK?), a major regulator of energy metabolism. This was mediated through specific activation of PPAR?/?, as a PPAR?/? small interfering RNA inhibited the effect. However, AMPK? did not mediate the growth-promoting effects of GW501516, as silencing of AMPK? did not inhibit GW501516-induced cell proliferation. Instead, we found that GW501516 stimulated peroxisome proliferator–activated receptor coactivator ? (PGC)-1?, which activated the phosphatidylinositol 3 kinase (PI3-K)/Akt mitogenic pathway. An inhibitor of PI3-K, LY294002, had no effect on PGC-1?, consistent with PGC-1? being upstream of PI3-K/Akt. Of note, an activator of AMPK?, 5-amino-4-imidazole carboxamide riboside, inhibited the growth-promoting effects of GW501516, suggesting that although AMPK? is not responsible for the mitogenic effects of GW501516, its activation can oppose these events. This study unveils a novel mechanism by which GW501516 and activation of PPAR?/? stimulate human lung carcinoma cell proliferation, and suggests that activation of AMPK? may oppose this effect.

Han, ShouWei; Ritzenthaler, Jeffrey D.; Sun, XiaoJuan; Zheng, Ying; Roman, Jesse



Dimerization with Cannabinoid Receptors Allosterically Modulates Delta Opioid Receptor Activity during Neuropathic Pain  

PubMed Central

The diversity of receptor signaling is increased by receptor heteromerization leading to dynamic regulation of receptor function. While a number of studies have demonstrated that family A G-protein-coupled receptors are capable of forming heteromers in vitro, the role of these heteromers in normal physiology and disease has been poorly explored. In this study, direct interactions between CB1 cannabinoid and delta opioid receptors in the brain were examined. Additionally, regulation of heteromer levels and signaling in a rodent model of neuropathic pain was explored. First we examined changes in the expression, function and interaction of these receptors in the cerebral cortex of rats with a peripheral nerve lesion that resulted in neuropathic pain. We found that, following the peripheral nerve lesion, the expression of both cannabinoid type 1 receptor (CB1R) and the delta opioid receptor (DOR) are increased in select brain regions. Concomitantly, an increase in CB1R activity and decrease in DOR activity was observed. We hypothesize that this decrease in DOR activity could be due to heteromeric interactions between these two receptors. Using a CB1R-DOR heteromer-specific antibody, we found increased levels of CB1R-DOR heteromer protein in the cortex of neuropathic animals. We subsequently examined the functionality of these heteromers by testing whether low, non-signaling doses of CB1R ligands influenced DOR signaling in the cortex. We found that, in cortical membranes from animals that experienced neuropathic pain, non-signaling doses of CB1R ligands significantly enhanced DOR activity. Moreover, this activity is selectively blocked by a heteromer-specific antibody. Together, these results demonstrate an important role for CB1R-DOR heteromers in altered cortical function of DOR during neuropathic pain. Moreover, they suggest the possibility that a novel heteromer-directed therapeutic strategy for enhancing DOR activity, could potentially be employed to reduce anxiety associated with chronic pain.

Stockton, Steven D.; Miller, Lydia K.; Devi, Lakshmi A.



Peroxisome proliferator-activated receptors (PPARs): Nuclear receptors at the crossroads between lipid metabolism and inflammation  

Microsoft Academic Search

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear receptor family. PPARs function as regulators of lipid and lipoprotein metabolism and glucose homeostasis and influence cellular proliferation, differentiation and apoptosis. PPAR! is highly expressed in tissues such as liver, muscle, kidney and heart, where it stimulates the #-oxidative degradation of fatty acids. PPAR% is predominantly expressed in

G. Chinetti; J.-C. Fruchart; B. Staels



Physiologically active compounds interacting with serotonin (5-hydroxytryptamine) receptors  

NASA Astrophysics Data System (ADS)

The data on the structures of organic compounds active with respect to serotonin (5-hydroxytryptamine) receptors are systematised. Various aspects of their design are considered. The bibliography includes 296 references.

Zefirova, Ol'ga N.; Zefirov, Nikolai S.



Constitutively active ALK2 receptor mutants require type II receptor cooperation.  


Constitutively activating mutations in receptor kinases recruit downstream effector pathways independently of upstream signaling, with consequences ranging from developmental syndromes to cancer. Classic fibrodysplasia ossificans progressiva (FOP) is a congenital syndrome resulting from highly conserved activating mutations of the glycine-serine-rich (GS) regulatory domain of ACVR1, encoding bone morphogenetic protein (BMP) type I receptor ALK2, which lead to inappropriate signaling and heterotopic ossification of soft tissues. It is unclear if constitutively active mutant ALK2 receptors (caALK2) can function independently of signaling complexes with type II receptors and ligands. We found that ablation of BmpRII and ActRIIa abrogated BMP ligand-mediated and caALK2-mediated signaling and transcription in cells and disrupted caALK2-induced heterotopic ossification in mice. Signaling via GS domain ALK2 mutants could be restored by the expression of either BMP type II receptor. The contribution of BMP type II receptors was independent of their ligand-binding or kinase function but was dependent upon an intact cytoplasmic domain. These data demonstrate that GS domain ALK2 mutants act independently of upstream signaling but may require a nonenzymatic scaffolding function provided by type II receptors to form functional, apparently ligand-independent signaling complexes. These findings define the minimal requirements for signaling of GS domain ALK2 mutants, with implications for the therapeutic targeting of their activity in disease. PMID:23572558

Bagarova, Jana; Vonner, Ashley J; Armstrong, Kelli A; Börgermann, Jan; Lai, Carol S C; Deng, Donna Y; Beppu, Hideyuki; Alfano, Ivan; Filippakopoulos, Panagis; Morrell, Nicholas W; Bullock, Alex N; Knaus, Petra; Mishina, Yuji; Yu, Paul B



Heterotrimeric G protein activation by G-protein-coupled receptors  

Microsoft Academic Search

Heterotrimeric G proteins have a crucial role as molecular switches in signal transduction pathways mediated by G-protein-coupled receptors. Extracellular stimuli activate these receptors, which then catalyse GTP–GDP exchange on the G protein ?-subunit. The complex series of interactions and conformational changes that connect agonist binding to G protein activation raise various interesting questions about the structure, biomechanics, kinetics and specificity

William M. Oldham; Heidi E. Hamm



Ah receptor agonist activity in frequently consumed food items  

Microsoft Academic Search

The aryl hydrocarbon receptor (AhR) receives much attention for its role in the toxicity of dioxins and dioxin-like polychlorinated biphenyls. However, many other compounds have also been reported to bind and activate AhR, of which natural food components are of special interest from a human health perspective. Using the dioxin receptor–chemical-activated luciferase gene expression (DR CALUX®) bioassay, extracts from many

W. J. De Waard; J. M. M. J. G. Aarts; A. A. C. M. Peijnenburg; T. M. C. M. De Kok; F.-J. Van Schooten; L. A. P. Hoogenboom



Activation of the Mineralocorticoid Receptor Increases Striatin Levels  

Microsoft Academic Search

BackgroundAldosterone (ALDO), a critical regulator of sodium homeostasis, mediates its effects via activation of the mineralocorticoid receptor (MR) through mechanisms that are not entirely clear. Striatin, a membrane associated protein, interacts with estrogen receptors in endothelial cells.MethodsWe studied the effects of MR activation in vitro and in vivo on striatin levels in vascular tissue.ResultsWe observed that dietary sodium restriction was

Luminita H. Pojoga; Patricia Coutinho; Alicia Rivera; Tham M. Yao; Enrique R. Maldonado; Rodeler Youte; Gail K. Adler; Jonathan Williams; Alexander Turchin; Gordon H. Williams; Jose R. Romero



Activators of the nuclear receptor PPAR? enhance colon polyp formation  

Microsoft Academic Search

A high-fat diet increases the risk of colon, breast and prostate cancer. The molecular mechanism by which dietary lipids promote tumorigenesis is unknown. Their effects may be mediated at least in part by the peroxisome proliferator-activated receptors (PPARs). These ligand-activated nuclear receptors modulate gene expression in response to fatty acids, lipid-derived metabolites and antidiabetic drugs. To explore the role of

Enrique Saez; Peter Tontonoz; Michael C. Nelson; Jacqueline G. A. Alvarez; Tze Ming U; Stephen M. Baird; Vilmos A. Thomazy; Ronald M. Evans



A recombinant catalytic domain of matriptase induces detachment and apoptosis of small-intestinal epithelial IEC-6 cells cultured on laminin-coated surface.  


Matriptase is a type-II transmembrane serine protease that is expressed strongly in the epithelial elements of various organs. In the small intestine, it is expressed prominently at the villus tip where aged epithelial cells undergo shedding and/or apoptosis. This observation, together with the ability of matriptase to cleave laminin (a basement membrane component critical for epithelial cell attachment), prompted us to hypothesize that it plays an important part in the removal of aged epithelial cells in the small intestine. We tested this hypothesis by determining whether a recombinant catalytic domain of rat matriptase (His(6)t-S-CD) causes detachment and/or apoptosis of small-intestinal epithelial IEC-6 cells. His(6)t-S-CD caused detachment of cells attached to laminin-coated plates but did not detach cells attached to fibronectin- or type-IV collagen-coated plates. Pre-treatment of laminin-coated plates with His(6)t-S-CD decreased the attachment of cells, suggesting that the recombinant matriptase caused detachment through a mechanism involving a direct effect on laminin. His(6)t-S-CD was also found to induce apoptosis in the cells cultured on laminin-coated plates, as assessed by annexin-V staining, DNA fragmentation and caspase-3 activity assays. These findings support our hypothesis regarding the role of matriptase in the small intestine. PMID:20855298

Mochida, Seiya; Tsuzuki, Satoshi; Inouye, Kuniyo; Fushiki, Tohru



Airway receptor activity in the developing opossum.  


Characteristics of airway receptor discharge were evaluated in pentobarbital-anesthetized, gallamine-paralyzed, artificially ventilated, open-chest suckling and weanling opossums (Didelphis marsupialis). Animals were tested with single-unit vagal recordings at 20, 30, 55, and 90 days of age; results were compared with previous data from adults. Rapidly adapting airway receptors ( RARs ) comprised a smaller percentage of the sampled population at 20 and 30 days of age than in older groups. The static firing properties of slowly adapting airway receptors (SARs) were examined at 0-20 cmH2O transpulmonary pressure (Ptp). At the higher Ptp levels, average receptor discharge rates increased with increasing age; these results are similar to findings in placental mammals. At 5 cmH2O, however, only the 20-day-old animals exhibited reduced rates of SAR discharge. The dynamic response of SARs in 20- and 30-day-old animals was reduced compared with adult values, as measured by the adaptation of their discharge frequencies; however, the reduction was proportional to the lower receptor firing rates. About 80% of tested SARs were inhibited by CO2 at 20 and 55 days; similar results were obtained in adults. Other results have shown that morphologic development of the vagi in opossums at 50 days of age compares with placental mammals similarly studied at birth. In contrast, the static SAR discharge in the 50-day-old opossum at low Ptp (i.e., 5 cmH2O) is similar to that observed in adults, whereas the newborn placental mammal has a greatly reduced SAR firing rate. This suggests that early utilization of the lungs can contribute to functional maturation of airway receptors. PMID:6426327

Farber, J P; Fisher, J T; Sant'Ambrogio, G



Laminin-332-beta1 integrin interactions negatively regulate invadopodia.  


Adhesion of epithelial cells to basement membranes (BM) occurs through two major structures: actin-associated focal contacts and keratin-associated hemidesmosomes, both of which form on laminin-332 (Ln-332). In epithelial-derived cancer cells, additional actin-linked structures with putative adhesive properties, invadopodia, are frequently present and mediate BM degradation. A recent study proposed that BM invasion requires a proper combination of focal contacts and invadopodia for invading cells to gain traction through degraded BM, and suggested that these structures may compete for common molecular components such as Src kinase. In this study, we tested the role of the Ln-332 in regulating invadopodia in 804G rat bladder carcinoma cells, a cell line that secretes Ln-332 and forms all three types of adhesions. Expression of shRNA to Ln-332 gamma2 chain (gamma2-kd) led to increased numbers of invadopodia and enhanced extracellular matrix degradation. Replating gamma2-kd cells on Ln-332 or collagen-I fully recovered cell spreading and inhibition of invadopodia. Inhibition of alpha3 or beta1, but not alpha6 or beta4, phenocopied the effect of gamma2-kd, suggesting that alpha3beta1-mediated focal contacts, rather than alpha6beta4-mediated hemidesmosome pathways, intersect with invadopodia regulation. gamma2-kd cells exhibited alterations in focal contact-type structures and in activation of focal adhesion kinase (FAK) and Src kinase. Inhibition of FAK also increased invadopodia number, which was reversible with Src inhibition. These data are consistent with a model whereby actin-based adhesions can limit the availability of active Src that is capable of invadopodia initiation and identifies Ln-332-beta1 interactions as a potent upstream regulator that limits cell invasion. PMID:20039268

Liu, Shanshan; Yamashita, Hironobu; Weidow, Brandy; Weaver, Alissa M; Quaranta, Vito



Identification of the B1 and B2 subunits of human placental laminin and rat parietal-yolk-sac laminin using antisera specific for murine laminin-beta-galactosidase fusion proteins.  

PubMed Central

Antisera raised against fusion proteins consisting of murine laminin B1 and B2 subunit sequences fused to the C-terminus of Escherichia coli beta-galactosidase were tested for their subunit specificity on Western blots of deglycosylated murine Engelbreth-Holm-Swarm (EHS) laminin. The antisera raised against B2 subunit sequences (anti-XLB2.1 and anti-XLB2.2) bound only to the EHS laminin B2 subunit. One of the antisera raised against B1 subunit sequences (anti-XLB1.2) was specific for the B1 subunit, whereas two others (anti-XLB1.1 and anti-XLB1.3) cross-reacted with the EHS laminin B2 subunit. Gold-labelled heparin-albumin was shown to bind specifically to the A subunit of deglycosylated EHS laminin on Western blots. These reagents were used to identify the homologous subunits in rat parietal-yolk-sac laminin and human placental laminin. The anti-(fusion protein) antisera identified the B1 and B2 subunits of the rat laminin, and these were similar in size to the murine EHS B subunits. Human placental laminin gave bands of 400, 340, 230, 190 and 180 kDa on reducing SDS/PAGE. The anti-(fusion protein) antisera identified the 230 and 190 kDa bands as the B1 and B2 subunits respectively. Gold-labelled heparin-albumin bound to the 400, 340 and 190 kDa bands of human placental laminin and so did not unambiguously identify a single A subunit. The human placental laminin may contain a mixture of isoforms, with alternative subunits substituting for the A subunit. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6.

Brown, J C; Spragg, J H; Wheeler, G N; Taylor, P W



Mineralocorticoid receptor activation in obesity hypertension  

Microsoft Academic Search

Obesity hypertension and metabolic syndrome have become major public health concerns. Nowadays, aldosterone is recognized as an important mediator of cardiovascular and renal damage. In the kidney, aldosterone injures glomerular visceral epithelial cells (podocytes), the final filtration barrier to plasma macromolecules, leading to proteinuria and glomerulosclerosis. Mineralocorticoid receptor (MR) antagonists effectively ameliorate proteinuria in patients or in animal models of

Miki Nagase; Toshiro Fujita



2Adrenergic receptor activation delays wound healing  

Microsoft Academic Search

Keratinocytes migrate directionally into the wound bed to initiate re-epithelialization, necessary for wound closure and restoration of barrier function. They solely express the 2-adrenergic receptor (2-AR) subtype of -ARs and can also synthesize -AR agonists generating a hormonal mediator network in the skin. Emerging studies from our laboratory demonstrate that -AR agonists decrease keratinocyte migration via a protein phosphatase (PP)

Christine E. Pullar; Jennifer C. Grahn; Wei Liu; R. Rivkah Isseroff



Low temperature and muscarinic receptor activities.  


Lowering temperature from 37 degrees C to 22, 18, and 14 degrees C triggered automaticity of smooth longitudinal muscle of guinea pig isolated ileum. The amplitude of the hypothermia-induced automaticity was dependent on the degree of temperature drop: the greater the temperature drop, the greater the amplitude. However, when the preparation was initially prepared and maintained at 14 degrees C and then the temperature was raised at a similar rate to 18, 22, and 37 degrees C, the automaticity was not observed. This series of observations suggests that cooling rate may be the trigger and/or part of the triggering mechanism for the observed automaticity. Mepenzolate (1.0 x 10(-6) M), a specific muscarinic receptor antagonist, blocked the automaticity suggesting the involvement of muscarinic receptors in the pathogenesis and/or the manifestation of the automaticity. Verapamil (1.0 x 10(-7) M), a calcium channel blocker which inhibits the transmembrane Ca2+ influx into smooth muscle cells during excitation, blocked the automaticity suggesting that transmembrane Ca2+ influx plays a significant role in the pathogenesis and/or manifestation of the automaticity. A specific cytoplasmic calcium channel blocker, 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (1.0 x 10(-6) M) blocked the automaticity, suggesting that cytoplasmic calcium also plays a significant role in the pathogenesis and/or manifestation of the automaticity. In order to characterize the temperature-induced changes in the muscarinic receptors, an attempt was made to use the classic method of Furchgott and Burstyn to determine the dissociation constants of acetylcholine at muscarinic receptors at different temperatures. However, the alkylation of muscarinic receptors with phenoxybenzamine at lower temperatures was erratic and the recovery from the occlusion was too rapid to apply the method of Furchgott and Burstyn. We concluded that the lack of reversibility of the effects of phenoxybenzamine at 37 degrees C is due to the predominance of covalent bonding of phenoxybenzamine with the receptors, whereas at lower temperatures like 24 degrees C, the blockade of the muscarinic receptors by phenoxybenzamine is mainly due to simple occlusion. PMID:2791613

Tsai, C S; Ochillo, R F



Differential trafficking of AMPA receptors following activation of NMDA receptors and mGluRs  

PubMed Central

The removal of AMPA receptors from synapses is a major component of long-term depression (LTD). How this occurs, however, is still only partially understood. To investigate the trafficking of AMPA receptors in real-time we previously tagged the GluA2 subunit of AMPA receptors with ecliptic pHluorin and studied the effects of NMDA receptor activation. In the present study we have compared the effect of NMDA receptor and group I mGluR activation, using GluA2 tagged with super ecliptic pHluorin (SEP-GluA2) expressed in cultured hippocampal neurons. Surprisingly, agonists of the two receptors, which are both able to induce chemical forms of LTD, had clearly distinct effects on AMPA receptor trafficking. In agreement with our previous work we found that transient NMDA receptor activation results in an initial decrease in surface GluA2 from extrasynaptic sites followed by a delayed reduction in GluA2 from puncta (putative synapses). In contrast, transient activation of group I mGluRs, using DHPG, led to a pronounced but more delayed decrease in GluA2 from the dendritic shafts. Surprisingly, there was no average change in the fluorescence of the puncta. Examination of fluorescence at individual puncta, however, indicated that alterations did take place, with some puncta showing an increase and others a decrease in fluorescence. The effects of DHPG were, like DHPG-induced LTD, prevented by treatment with a protein tyrosine phosphatase (PTP) inhibitor. The electrophysiological correlate of the effects of DHPG in the SEP-GluA2 infected cultures was a reduction in mEPSC frequency with no change in amplitude. The implications of these findings for the initial mechanisms of expression of both NMDA receptor- and mGluR-induced LTD are discussed.



Laminin111: A Potential Therapeutic Agent for Duchenne Muscular Dystrophy  

Microsoft Academic Search

Duchenne muscular dystrophy (DMD) still needs effective treatments, and myoblast transplantation (MT) is considered as an approach to repair damaged skeletal muscles. DMD is due to the complete loss of dystrophin from muscles. The lack of link between the contracting apparatus and the extracellular matrix leads to frequent damage to the sarcolemma triggering muscle fiber necrosis. Laminins are major proteins

Sébastien Goudenege; Yann Lamarre; Nicolas Dumont; Joël Rousseau; Jérôme Frenette; Daniel Skuk; Jacques P Tremblay



Expression of laminin subunits in congenital muscular dystrophy  

Microsoft Academic Search

The expression of laminin subunits M, A, B1 and B2 was studied immunocytochemically in 25 cases of classical congenital muscular dystrophy (CMD), 11 hypotonic infants, 20 cases of a variety of inherited and acquired neuromuscular disorders, and 11 controls. Merosin, as indicated by labelling for the M chain, was deficient in 12 (48%) of the cases of classical CMD. Seven

C. A. Sewry; J. Philpot; D. Mahony; L. A. Wilson; F. Muntoni; V. Dubowitz



Polymerized Laminin-332 Matrix Supports Rapid and Tight Adhesion of Keratinocytes, Suppressing Cell Migration  

PubMed Central

Laminin-332 (?3ß3?2) (Lm332) supports the stable anchoring of basal keratinocytes to the epidermal basement membrane, while it functions as a motility factor for wound healing and cancer invasion. To understand these contrasting activities of Lm332, we investigated Lm332 matrices deposited by normal human keratinocytes and other Lm332-expressing cell lines. All types of the cells efficiently deposited Lm332 on the culture plates in specific patterns. On the contrary, laminins containing laminin ß1 and/or ?1 chains, such as Lm511 and Lm311, were not deposited on the culture plates even if secreted into culture medium. The Lm332 deposition was not inhibited by function-blocking antibodies to the ?3 and ?6 integrins but was inhibited by sodium selenate, suggesting that sulfated glycosaminoglycans on cell surface, e.g. heparan sulfate proteoglycans, might be involved in the process. HEK293 cells overexpressing exogenous Lm332 (Lm332-HEK) almost exclusively deposited Lm332 on the plates. The deposited Lm332 matrix showed a mesh-like network structure as analyzed by electron microscopy, suggesting that Lm332 was highly polymerized. When biological activity was analyzed, the Lm332 matrix rather suppressed the migration of keratinocytes as compared with purified Lm332, which highly promoted the cell migration. The Lm332 matrix supported adhesion of keratinocytes much more strongly and stably than purified Lm332. Integrin ?3ß1 bound to the Lm332 matrix at a three times higher level than purified Lm332. Normal keratinocytes prominently showed integrin ?6ß4-containing, hemidesmosome-like structures on the Lm332 matrix but not on the purified one. These results indicate that the polymerized Lm332 matrix supports stable cell adhesion by interacting with both integrin ?6ß4 and ?3ß1, whereas unassembled soluble Lm332 supports cell migration.

Kariya, Yoshinobu; Sato, Hiroki; Katou, Naoko; Kariya, Yukiko; Miyazaki, Kaoru



Monocyte attachment to laminin in diabetes mellitus: The role of ATP.  


Monocyte-extracellular matrix interactions have been implicated in atherosclerosis pathophysiology. Laminin, the main basement membrane protein contains cell binding domains that can be cryptic, presented only after protein modification. In the present study we evaluated monocyte attachment to laminin-1 in the presence of ATP. Monocytes were derived from either healthy volunteers or patients with diabetes mellitus type II. For the estimation of monocyte attachment to laminin the myeloperoxidase assay was used. Monocytes derived from diabetic patients, showed an increased ability to attach to laminin (p = 0.0055). The presence of ATP increased the attachment of control monocytes to laminin (p = 0.0022). On the contrary, the presence of ATP did not affect the attachment of monocytes derived from diabetic patients to laminin. Our results indicate a modified interaction between monocytes and laminin-1 in diabetes mellitus. PMID:19287210

Kostidou, Elena; Trachana, Varvara; Topouridou, Konstantina; Paletas, Konstantinos; Tsapas, Apostolos; Kaloyianni, Martha; Koliakos, George



Distinct changes in the laminin composition of basement membranes in human seminiferous tubules during development and degeneration.  

PubMed Central

We studied the distribution of laminin (Ln) chains and their integrin (Int) receptors in normal developing and adult and in atrophied human testes by using immunohistochemistry. Immunostaining for EHS Ln and type IV collagen was used to identify basement membranes (BMs). In the BM of seminiferous epithelium of fetal testis, a panel of monoclonal antibodies showed immunoreactivity for Ln alpha 1-, alpha 2-, beta 1-, beta 2- and gamma 1-chains, suggestive of the presence of Lns 1 to 3. In BM of adult seminiferous epithelium with active spermatogenesis, immunoreactivity for Ln beta 2- and gamma 1-chains was found but not for Ln alpha-chains, suggesting a complex of Ln chains not compatible with any known trimers. Instead, with polyclonal Ln antiserum and monoclonal antibody to type IV collagen, a distinct BM-like reactivity was seen. In atrophied testes, prominent immunoreactivities for Ln chains, compatible with Lns 1 to 3, were seen in the thickened BM of seminiferous tubules, hence suggestive of reappearance of fetal Lns. Among the subunits of Ln-binding Int receptors in fetal seminiferous tubules, a strong immunoreactivity for Int beta 1- and Int alpha 6-subunits was seen throughout the seminiferous epithelium, other Int subunits being found in interstitial cells. In the adult and atrophied testes, immunoreactivities for Int beta 1- and Int alpha 6-subunits were seen to be confined to the basal aspect of the seminiferous epithelium whereas immunoreactivities for Int alpha 1-, alpha 2-, alpha 3- and beta 4-subunits were seen in the myoid cells. The results show that both maturation and degenerative changes of human testes are accompanied by distinct changes in the Ln expression of BM of seminiferous epithelium, which appears to accompany epithelial differentiation of the Sertoli cells. Furthermore, they suggest the presence of a novel Ln trimer in BM of adult human seminiferous tubules. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6

Virtanen, I.; Lohi, J.; Tani, T.; Korhonen, M.; Burgeson, R. E.; Lehto, V. P.; Leivo, I.



A dual mechanism for impairment of GABAA receptor activity by NMDA receptor activation in rat cerebellum granule cells.  


The function of the GABAA receptor has been studied using the whole cell voltage clamp recording technique in rat cerebellum granule cells in culture. Activation of NMDA-type glutamate receptors causes a reduction in the effect of GABA. Full GABAA receptor activity was recovered after washing out NMDA and NMDA action was prevented in a Mg+2 containing medium. The NMDA effect was also absent when extracellular Ca+2 was replaced by Ba+2 and when 10 mM Bapta was present in the intracellular solution. Charge accumulations via voltage activated Ca+2 channels greater than the ones via NMDA receptors do not cause any reduction in GABAA receptor function, suggesting that Ca+2 influx through NMDA receptor channels is critical for the effect. The NMDA effect was reduced by including adenosine-5'-O-3-thiophosphate (ATP-gamma-S) in the internal solution and there was a reduction in the NMDA effect caused by deltamethrin, a calcineurin inhibitor. Part of the NMDA induced GABAA receptor impairment was prevented by prior treatment with L-arginine. Analogously, part of the NMDA effect was prevented by blockage of NO-synthase activity by N omega-nitro-L-arginine. A combination of NO-synthase and calcineurin inhibitors completely eliminated the NMDA action. An analogous result was obtained by combining the NO-synthase inhibitor with the addition of ATP-gamma-S to the pipette medium. The additivity of the prevention of the NMDA impairment of GABAA receptor by blocking the L-arginine/NO pathway and inhibiting calcineurin activity suggests an independent involvement of these two pathways in the interaction between NMDA and the GABAA receptor. On the one hand Ca+2 influx across NMDA channels activates calcineurin and dephosphorylates the GABAA receptor complex directly or dephosphorylates proteins critical for the function of the receptor. On the other hand, Ca+2 influx activates NO-synthase and induces nitric oxide production, which regulates such receptors via protein kinase G activity. PMID:9037753

Robello, M; Amico, C; Cupello, A



Mycobacterium tuberculosis Activates Human Macrophage Peroxisome Proliferator-Activated Receptor ? Linking Mannose Receptor Recognition to Regulation of Immune Responses  

PubMed Central

Mycobacterium tuberculosis enhances its survival in macrophages by suppressing immune responses in part through its complex cell wall structures. Peroxisome proliferator-activated receptor ? (PPAR?), a nuclear receptor superfamily member, is a transcriptional factor that regulates inflammation and has high expression in alternatively activated alveolar macrophages and macrophage-derived foam cells, both cell types relevant to tuberculosis pathogenesis. In this study, we show that virulent M. tuberculosis and its cell wall mannose-capped lipoarabinomannan induce PPAR? expression through a macrophage mannose receptor-dependent pathway. When activated, PPAR? promotes IL-8 and cyclooxygenase 2 expression, a process modulated by a PPAR? agonist or antagonist. Upstream, MAPK-p38 mediates cytosolic phospholipase A2 activation, which is required for PPAR? ligand production. The induced IL-8 response mediated by mannose-capped lipoarabinomannan and the mannose receptor is independent of TLR2 and NF-?B activation. In contrast, the attenuated Mycobacterium bovis bacillus Calmette-Guérin induces less PPAR? and preferentially uses the NF-?B–mediated pathway to induce IL-8 production. Finally, PPAR? knockdown in human macrophages enhances TNF production and controls the intracellular growth of M. tuberculosis. These data identify a new molecular pathway that links engagement of the mannose receptor, an important pattern recognition receptor for M. tuberculosis, with PPAR? activation, which regulates the macrophage inflammatory response, thereby playing a role in tuberculosis pathogenesis.

Rajaram, Murugesan V. S.; Brooks, Michelle N.; Morris, Jessica D.; Torrelles, Jordi B.; Azad, Abul K.; Schlesinger, Larry S.



Peroxisome Proliferator-Activated Receptors and Progression of Colorectal Cancer  

PubMed Central

The peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily. These receptors are also ligand-dependent transcription factors responsible for the regulation of cellular events that range from glucose and lipid homeostases to cell differentiation and apoptosis. The importance of these receptors in lipid homeostasis and energy balance is well established. In addition to these metabolic and anti-inflammatory properties, emerging evidence indicates that PPARs can function as either tumor suppressors or accelerators, suggesting that these receptors are potential candidates as drug targets for cancer prevention and treatment. However, conflicting results have emerged regarding the role of PPARs on colon carcinogenesis. Therefore, further investigation is warranted prior to considering modulation of PPARs as an efficacious therapy for colorectal cancer chemoprevention and treatment.

Wang, Dingzhi; DuBois, Raymond N.



Functional activity of the human prolactin receptor and its ligands.  


Prolactin receptors (PRLR) have been identified in a number of human tissues and cell lines, although little is known about the human receptor protein. The cloning of the human PRLR cDNA has enabled further characterization of the receptor protein in transfected cells. Since the human cDNA is expressed at lower levels than the rat cDNA, we have constructed a hybrid cDNA (pECE r5'hPRLR) containing nucleotides of the 5' untranslated region and signal peptide of the rat PRLR and the protein coding and 3' untranslated portion of the human receptor. Expression of the hybrid receptor was increased more than two-fold compared to the human receptor as detected by specific binding of 125I-human growth hormone (GH) to transfected COS-7 cells. The relative molecular mass of the receptor was 93,000 Da, as determined by chemical cross-linking studies. Transcriptional assays were used to show the human PRLR was able to activate two milk protein genes; ovine beta-lactoglobulin and rat beta-casein. Transfected cells expressing the human PRLR receptor, treated with human GH or prolactin (PRL), induced a dose-dependent increase in transcriptional activation of the beta-casein/luciferase fusion gene. Glycosylated, and non-glycosylated human PRL, and ovine PRL were equally effective in activating the beta-casein promoter. Human placental lactogen and bovine PRL could also induce a greater than 10-fold induction, whereas insulin did not significantly stimulate the beta-casein promoter. The results show that the human PRLR can activate both beta-lactoglobulin and beta-casein milk gene promoters and that these reporter genes can be used to evaluate the functional activity of agonists and antagonists of the human PRLR. PMID:8674856

Lochnan, H A; Buteau, H; Richards, S; Edery, M; Kelly, P A



The Laminin 511/521 Binding Site on the Lutheran Blood Group Glycoprotein is Located at theFlexible Junction of Ig Domains 2 and 3  

SciTech Connect

The Lutheran blood group glycoprotein, first discovered on erythrocytes, is widely expressed in human tissues. It is a ligand for the {alpha}5 subunit of Laminin 511/521, an extracellular matrix protein. This interaction may contribute to vasocclusive events that are an important cause of morbidity in sickle cell disease. Using X-ray crystallography, small angle X-ray scattering and site directed mutagenesis we show that the extracellular region of Lutheran forms an extended structure with a distinctive bend between the second and third immunoglobulin-like domains. The linker between domains 2 and 3 appears to be flexible and is a critical determinant in maintaining an overall conformation for Lutheran that is capable of binding to Laminin. Mutagenesis studies indicate that Asp312 of Lutheran and the surrounding cluster of negatively charged residues in this linker region form the Laminin binding site. Unusually, receptor binding is therefore not a function of the domains expected to be furthermost from the plasma membrane. These studies imply that structural flexibility of Lutheran may be essential for its interaction with Laminin and present a novel opportunity for the development of therapeutics for sickle cell disease.

Mankelow, Tosti J.; Burton, Nicholas; Stedansdottir, Fanney O.; Spring, Frances A.; Parsons, Stephen F.; Pesersen, Jan S.; Oliveira, Cristiano L.P.; Lammie, Donna; Wess, Timothy; Mohandas, Narla; Chasis, Joel A.; Brady, R. Leo; Anstee, David J.



The Laminin 511/521-binding site on the Lutheran blood group glycoprotein is located at the flexible junction of Ig domains 2 and 3  

PubMed Central

The Lutheran blood group glycoprotein, first discovered on erythrocytes, is widely expressed in human tissues. It is a ligand for the ?5 subunit of Laminin 511/521, an extracellular matrix protein. This interaction may contribute to vaso-occlusive events that are an important cause of morbidity in sickle cell disease. Using x-ray crystallography, small-angle x-ray scattering, and site-directed mutagenesis, we show that the extracellular region of Lutheran forms an extended structure with a distinctive bend between the second and third immunoglobulin-like domains. The linker between domains 2 and 3 appears to be flexible and is a critical determinant in maintaining an overall conformation for Lutheran that is capable of binding to Laminin. Mutagenesis studies indicate that Asp312 of Lutheran and the surrounding cluster of negatively charged residues in this linker region form the Laminin-binding site. Unusually, receptor binding is therefore not a function of the domains expected to be furthermost from the plasma membrane. These studies imply that structural flexibility of Lutheran may be essential for its interaction with Laminin and present a novel opportunity for the development of therapeutics for sickle cell disease.

Burton, Nicholas; Stefansdottir, Fanney O.; Spring, Frances A.; Parsons, Stephen F.; Pedersen, Jan S.; Oliveira, Cristiano L. P.; Lammie, Donna; Wess, Timothy; Mohandas, Narla; Chasis, Joel Anne; Anstee, David J.



Activation of muscarinic receptors modulates NMDA receptor-mediated responses in auditory cortex  

Microsoft Academic Search

The present study examines the ability of muscarinic receptor activation to modulate glutamatergic responses in the in vitro\\u000a rat auditory cortex. Whole-cell patch-clamp recordings were obtained from layer II-III pyramidal neurons and responses elicited\\u000a by either stimulation of deep gray matter or iontophoretic application of glutamate receptor agonists. Iontophoresis of the\\u000a muscarinic agonist acetyl-?-methylcholine (MCh) produced an atropine-sensitive reduction in

V. Bessie Aramakis; Anita E. Bandrowski; J. H. Ashe



Targets of activated steroid hormone receptors: basal transcription factors and receptor interacting proteins  

Microsoft Academic Search

Steroid hormone receptors constitute a family of inducible transcription factors that mediate the multifold effects of steroids\\u000a on development, reproduction, proliferation, and cellular homeostasis. Activation through the binding of the cognate hormone\\u000a enables the receptors to bind with high affinity to specific response elements in the promoters of target genes, resulting\\u000a in stimulation or repression of transcription. While protein-protein interactions

L. Klein-Hitpass; C. Schwerk; S. Kahmann; L. Vaßen



Flavonoids as dietary regulators of nuclear receptor activity  

PubMed Central

Metabolic diseases such as obesity, type II diabetes, and dyslipidemia are a rising cause of mortality worldwide. The progression of many metabolic diseases is fundamentally regulated on the transcriptional level by a family of ligand-activated transcription factors, called nuclear receptors, which detect and respond to metabolic changes. Their role in maintaining metabolic homeostasis makes nuclear receptors an important pharmaceutical and dietary target. This review will present the growing evidence that flavonoids, natural secondary plant metabolites, are important regulators of nuclear receptor activity. Structural similarities between flavonoids and cholesterol derivatives combined with the promiscuous nature of most nuclear receptors provide a wealth of possibilities for pharmaceutical and dietary modulation of metabolism. While the challenges of bringing flavonoid-derived therapeutics to the market are significant, we consider this rapidly growing field to be an essential aspect of the functional food initiative and an important mine for pharmaceutical compounds.

Avior, Yishai; Bomze, David; Ramon, Ory



A peptoid "antibody surrogate" that antagonizes VEGF receptor 2 activity.  


We report a two-color, cell-based screen to identify specific receptor-binding compounds in a combinatorial library of peptoids displayed on beads. We apply this strategy to the isolation of vascular endothelial growth factor receptor 2 (VEGFR2)-binding peptoids. A dimeric derivative of one of these lead compounds is shown to be an antagonist of VEGFR2 activity both in vitro and in vivo. This methodology provides a potentially general route to synthetic molecules that bind integral membrane receptors with affinities and specificities similar to those of antibodies, but which are far smaller and easier to make and manipulate. PMID:18386897

Udugamasooriya, D Gomika; Dineen, Sean P; Brekken, Rolf A; Kodadek, Thomas



Laminin-?1 Impairs Spatial Learning through Inhibition of ERK/MAPK and SGK1 Signaling  

PubMed Central

Laminin is a major structural element of the basal lamina consisting of an ?-chain, a ?-chain, and a ?-chain arranged in a cross-like structure, with their C-terminal inter-coiled. Laminin is abundantly expressed in the hippocampus of mature brain and is implicated in several psychiatric disorders, but its possible role involved in learning and memory function is not known. This issue was examined here. Our results revealed that water maze training significantly decreased laminin-?1 (LB1) expression in the rat hippocampal CA1 area. Transfection of LB1 WT plasmid to hippocampal CA1 neurons impaired water maze performance in rats. Meanwhile, it decreased the phosphorylation level of ERK/MAPK and protein kinase serum- and glucocorticoid-inducible kinase-1 (SGK1). By contrast, knockdown of endogenous LB1 expression using RNA interference (LB1 siRNA) enhanced water maze performance. Meanwhile, it increased the phosphorylation level of ERK/MAPK and SGK1. The enhancing effect of LB1 siRNA on spatial learning and on the phosphorylation of ERK/MAPK and SGK1 was blocked by co-treatment with the MEK inhibitor U0126 at a concentration that did not apparently affect spatial learning and ERK/MAPK phosphorylation alone. Further, the enhancing effect of LB1 siRNA on spatial learning and SGK1 phosphorylation was similarly blocked by co-transfection with SGK1 siRNA at a concentration that did not markedly affect spatial learning and SGK1 expression alone. These results together indicate that LB1 negatively regulates spatial learning in rats. In addition, LB1 impairs spatial learning through decreased activation of the ERK/MAPK–SGK1 signaling pathway in the rat hippocampus.

Yang, Ying C; Ma, Yun L; Liu, Wen T; Lee, Eminy HY



A Peptoid “Antibody Surrogate” That Antagonizes VEGF Receptor 2 Activity  

Microsoft Academic Search

We report a two-color, cell-based screen to identify specific receptor-binding compounds in a combinatorial library of peptoids displayed on beads. We apply this strategy to the isolation of vascular endothelial growth factor receptor 2 (VEGFR2)-binding peptoids. A dimeric derivative of one of these lead compounds is shown to be an antagonist of VEGFR2 activity both in vitro and in vivo.

D. Gomika Udugamasooriya; Sean P. Dineen; Rolf A. Brekken; Thomas Kodadek



Peroxisome proliferator-activated receptor ?—insights from human genetics  

Microsoft Academic Search

Peroxisome proliferator-activated receptor ? (PPAR?), mediates adipocyte differentiation and is the target for the thiazolidinedione (TZD) group of insulin-sensitising antidiabetic agents. We screened the PPAR? gene in 85 subjects with severe insulin resistance and identified heterozygous, missense mutations in several individuals from different families. Functional studies indicate that the receptor mutants are transcriptionally impaired and inhibit wild-type PPAR? action in

V. K. K Chatterjee



Activating Mineralocorticoid Receptor Mutation in Hypertension Exacerbated by Pregnancy  

Microsoft Academic Search

Hypertension and pregnancy-related hypertension are major public health problems of largely unknown causes. We describe a mutation in the mineralocorticoid receptor (MR), S810L, that causes early-onset hypertension that is markedly exacerbated in pregnancy. This mutation results in constitutive MR activity and alters receptor specificity, with progesterone and other steroids lacking 21-hydroxyl groups, normally MR antagonists, becoming potent agonists. Structural and

David S. Geller; Anita Farhi; Nikki Pinkerton; Michael Fradley; Michael Moritz; Adrian Spitzer; Gretchen Meinke; Francis T. F. Tsai; Paul B. Sigler; Richard P. Lifton



The peroxisome proliferator-activated receptor , an integrator of transcriptional repression and nuclear receptor signaling  

Microsoft Academic Search

The three PPAR (peroxisome proliferator-activated receptor) isoforms are critical regulators of lipid homeostasis by controlling the balance between the burning and storage of long chain fatty acids. Whereas PPAR and PPAR have been studied extensively, the function of PPAR remains the most elusive. Intriguingly, in cotransfection experiments, PPAR is a potent inhibitor of ligand-induced transcriptional activity of PPAR and PPAR.

Yanhong Shi; Michelle Hon; Ronald M. Evans



Identification of COUP-TFII Orphan Nuclear Receptor as a Retinoic Acid?Activated Receptor  

SciTech Connect

The chicken ovalbumin upstream promoter-transcription factors (COUP-TFI and II) make up the most conserved subfamily of nuclear receptors that play key roles in angiogenesis, neuronal development, organogenesis, cell fate determination, and metabolic homeostasis. Although the biological functions of COUP-TFs have been studied extensively, little is known of their structural features or aspects of ligand regulation. Here we report the ligand-free 1.48 {angstrom} crystal structure of the human COUP-TFII ligand-binding domain. The structure reveals an autorepressed conformation of the receptor, where helix {alpha}10 is bent into the ligand-binding pocket and the activation function-2 helix is folded into the cofactor binding site, thus preventing the recruitment of coactivators. In contrast, in multiple cell lines, COUP-TFII exhibits constitutive transcriptional activity, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, and ligand binding, substantially reduce the COUP-TFII transcriptional activity. Importantly, retinoid acids are able to promote COUP-TFII to recruit coactivators and activate a COUP-TF reporter construct. Although the concentration needed is higher than the physiological levels of retinoic acids, these findings demonstrate that COUP-TFII is a ligand-regulated nuclear receptor, in which ligands activate the receptor by releasing it from the autorepressed conformation.

Kruse, Schoen W.; Suino-Powell, Kelly; Zhou, X. Edward; Kretschman, Jennifer E.; Reynolds, Ross; Vonrhein, Clemens; Xu, Yong; Wang, Liliang; Tsai, Sophia Y.; Tsai, Ming-Jer; Xu, H. Eric (Baylor); (Van Andel); (Globel Phasing); (Grand Valley)



Activity-dependent phosphorylation of GABAA receptors regulates receptor insertion and tonic current  

PubMed Central

The expression of GABAA receptors and the efficacy of GABAergic neurotransmission are subject to adaptive compensatory regulation as a result of changes in neuronal activity. Here, we show that activation of L-type voltage-gated Ca2+ channels (VGCCs) leads to Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of S383 within the ?3 subunit of the GABAA receptor. Consequently, this results in rapid insertion of GABAA receptors at the cell surface and enhanced tonic current. Furthermore, we demonstrate that acute changes in neuronal activity leads to the rapid modulation of cell surface numbers of GABAA receptors and tonic current, which are critically dependent on Ca2+ influx through L-type VGCCs and CaMKII phosphorylation of ?3S383. These data provide a mechanistic link between activity-dependent changes in Ca2+ influx through L-type channels and the rapid modulation of GABAA receptor cell surface numbers and tonic current, suggesting a homeostatic pathway involved in regulating neuronal intrinsic excitability in response to changes in activity.

Saliba, Richard S; Kretschmannova, Karla; Moss, Stephen J



Kinetic deuterium isotope effects in glucocorticoid receptor activation  

SciTech Connect

Activation and deactivation of the chick thymus glucocorticoid receptor protein was studied in ordinary and heavy water by DNA-cellulose binding of the tritiated triamcinolone acetonide-receptor complex. Activation was significantly slower in heavy water if it was promoted by incubation at elevated temperature in buffers of low ionic strength. In the presence of 300 mM KC1 or after separation from the low molecular weight cytosol constituents, the complex was activated at the same rate in both solvents. Deactivation (time dependent loss of DNA-binding capacity) was much faster in ordinary than in heavy water regardless of gel filtration or the presence of KC1. A model of receptor activation-deactivation was constructed on the basis of these data that accounts for the observed kinetic deuterium isotope effects and reveals some submolecular details of the process.

Aranyi, P.



Glycine Receptor ?2 Subunit Activation Promotes Cortical Interneuron Migration  

PubMed Central

Summary Glycine receptors (GlyRs) are detected in the developing CNS before synaptogenesis, but their function remains elusive. This study demonstrates that functional GlyRs are expressed by embryonic cortical interneurons in vivo. Furthermore, genetic disruption of these receptors leads to interneuron migration defects. We discovered that extrasynaptic activation of GlyRs containing the ?2 subunit in cortical interneurons by endogenous glycine activates voltage-gated calcium channels and promotes calcium influx, which further modulates actomyosin contractility to fine-tune nuclear translocation during migration. Taken together, our data highlight the molecular events triggered by GlyR ?2 activation that control cortical tangential migration during embryogenesis.

Avila, Ariel; Vidal, Pia M.; Dear, T. Neil; Harvey, Robert J.; Rigo, Jean-Michel; Nguyen, Laurent



Decreased GABAA and GABAB receptor functional activity in cannabinoid CB1 receptor knockout mice.  


The interaction between brain GABAergic and endocannabinoid systems was evaluated by examining the quantitative and functional status of GABAergic receptors in cannabinoid CB(1) receptor knockout (CB(1)(-/-)) mice. To this aim, GABA(A) ([(3)H]-Muscimol binding assay), GABA(B) (baclofen-stimulated [(35)S]-GTP?S binding assay), GABA(A)?(1), GABA(A)?(2) and GABA(A)?(2) receptors gene expression (real-time reverse transcriptase polymerase chain reaction [PCR]) were carried out in CB(1)(-/-) and wild-type mice (CB(1)(+/+)). [(3)H]-Muscimol binding assays revealed significant reduction in the density of GABA(A) receptors in CA2 (30%) and DG (28%) of the hippocampus, thalamus (40%), cingulate (28%) and motor cortex (35%) of CB(1)(-/-) mice. Functional activity of metabotropic GABA(B) receptors was measured by evaluating the ability of GABA(B) agonist baclofen to stimulate [(35)S]-GTP?S binding. The results showed significant reduced [(35)S]-GTP?S binding in CA1 (61%), CA3 (51%) and DG (60%) of CB(1)(-/-) mice compared with CB(1)(+/+) mice. Real-time reverse transcriptase PCR was carried out for evaluating gene expression of ?(1), ?(2) and ?(2) subunits of GABA(A) receptor in the amygdala. The results showed significant reduced GABA(A)?(1) (50%) and GABA(A)?(2) (40%) receptor subunits gene expression in the amygdala of CB(1)(-/-) mice. No difference was observed in GABA(A)?(2) receptor subunit gene expression. This study provides strong evidence of the involvement of CB(1) receptors in the control of GABAergic responses mediated by GABA(A) and GABA(B) receptors, and suggests a possible role of the endocannabinoid system in the regulation of anxiety-related disorders. PMID:20142297

Urigüen, Leyre; García-Gutiérrez, María S; Manzanares, Jorge



Ethanol impairs muscarinic receptor-induced neuritogenesis in rat hippocampal slices: role of astrocytes and extracellular matrix proteins  

PubMed Central

In an in vitro co-culture system of astrocytes and neurons, stimulation of cholinergic muscarinic receptors in astrocytes had been shown to cause neuritogenesis in hippocampal neurons, and this effect was inhibited by ethanol. The present study sought to confirm these earlier findings in a more complex system, in vitro rat hippocampal slices in culture. Exposure of hippocampal slices to the cholinergic agonist carbachol (1 mM for 24 h) induced neurite outgrowth in hippocampal pyramidal neurons, which was mediated by activation of muscarinic M3 receptors. Specifically, carbachol induced a >4-fold increase in the length of the longest neurite, and a 4-fold increase in the length of minor neurites and in the number of branches. Co-incubation of carbachol with ethanol (50 mM) resulted in significant inhibition of the effects induced by carbachol on all parameters measured. Neurite outgrowth in CNS neurons is dependent on various permissive factors that are produced and released by glial cells. In hippocampal slices carbachol increased the levels of two extracellular matrix protein, fibronectin and laminin-1, by 1.6-fold, as measured by Western blot. Co-incubation of carbachol with ethanol significantly inhibited these increases. Carbachol-induced increases in levels of extracellular matrix proteins were antagonized by a M3 muscarinic receptor antagonist. Furthermore, function-blocking fibronectin or laminin-1 antibodies antagonized the effect of carbachol on neurite outgrowth. These results indicate that in hippocampal slices stimulation of muscarinic M3 receptors induces neurite outgrowth, which is mediated by fibronectin and laminin-1, two extracellular matrix proteins released by astrocytes. By decreasing fibronectin and laminin levels ethanol prevents carbachol-induced neuritogenesis. These findings highlight the importance of glial-neuronal interactions as important targets in the developmental neurotoxicity of alcohol.

Giordano, Gennaro; Guizzetti, Marina; Dao, Khoi; Mattison, Hayley A.; Costa, Lucio G.



Activation of group I metabotropic glutamate receptors potentiates heteromeric kainate receptors.  


Kainate receptors (KARs), a family of ionotropic glutamate receptors, are widely expressed in the central nervous system and are critically involved in synaptic transmission. KAR activation is influenced by metabotropic glutamate receptor (mGlu) signaling, but the underlying mechanisms are not understood. We undertook studies to examine how mGlu modulation affects activation of KARs. Confocal immunohistochemistry of rat hippocampus and cultured rat cortex revealed colocalization of the high-affinity KAR subunits with group I mGlu receptors. In hippocampal and cortical cultures, the calcium signal caused by activation of native KARs was potentiated by activation of group I mGlu receptors. In Xenopus laevis oocytes, activation of group I mGlu receptors potentiated heteromeric but not homomeric KAR-mediated currents, with no change in agonist potency. The potentiation of heteromeric KARs by mGlu1 activation was attenuated by GDP?S, blocked by an inhibitor of phospholipase C or the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), prolonged by the phosphatase inhibitor okadaic acid, but unaffected by the tyrosine kinase inhibitor lavendustin A. Protein kinase C (PKC) inhibition reduced the potentiation by mGlu1 of GluK2/GluK5, and conversely, direct activation of PKC by phorbol 12-myristate,13-acetate potentiated GluK2/GluK5. Using site-directed mutagenesis, we identified three serines (Ser833, Ser836, and Ser840) within the membrane proximal region of the GluK5 C-terminal domain that, in combination, are required for mGlu1-mediated potentiation of KARs. Together, these data suggest that phosphorylation of key residues in the C-terminal domain changes the overall charge of this domain, resulting in potentiated agonist responses. PMID:23066089

Rojas, Asheebo; Wetherington, Jonathon; Shaw, Renee; Serrano, Geidy; Swanger, Sharon; Dingledine, Raymond



Regulation of Proteome Maintenance Gene Expression by Activators of Peroxisome Proliferator-Activated Receptor a (PPARa)  

EPA Science Inventory

The nuclear receptor peroxisome proliferator-activated receptor alpha (PPARa) is activated by a large number of xenobiotic and hypolipidemic compounds called peroxisome proliferator chemicals (PPC). One agonist of PPARa (WY-14,643) regulates responses in the mouse liver to chemic...


The Antibodies against the Computationally Designed Mimic of the Glycoprotein Hormone Receptor Transmembrane Domain Provide Insights into Receptor Activation and Suppress the Constitutively Activated Receptor Mutants*  

PubMed Central

The exoloops of glycoprotein hormone receptors (GpHRs) transduce the signal generated by the ligand-ectodomain interactions to the transmembrane helices either through direct hormonal contact and/or by modulating the interdomain interactions between the hinge region (HinR) and the transmembrane domain (TMD). The ligand-induced conformational alterations in the HinRs and the interhelical loops of luteinizing hormone receptor/follicle stimulating hormone receptor/thyroid stimulating hormone receptor were mapped using exoloop-specific antibodies generated against a mini-TMD protein designed to mimic the native exoloop conformations that were created by joining the thyroid stimulating hormone receptor exoloops constrained through helical tethers and library-derived linkers. The antibody against the mini-TMD specifically recognized all three GpHRs and inhibited the basal and hormone-stimulated cAMP production without affecting hormone binding. Interestingly, binding of the antibody to all three receptors was abolished by prior incubation of the receptors with the respective hormones, suggesting that the exoloops are buried in the hormone-receptor complexes. The antibody also suppressed the high basal activities of gain-of-function mutations in the HinRs, exoloops, and TMDs such as those involved in precocious puberty and thyroid toxic adenomas. Using the antibody and point/deletion/chimeric receptor mutants, we demonstrate that changes in the HinR-exoloop interactions play an important role in receptor activation. Computational analysis suggests that the mini-TMD antibodies act by conformationally locking the transmembrane helices by means of restraining the exoloops and the juxta-membrane regions. Using GpHRs as a model, we describe a novel computational approach of generating soluble TMD mimics that can be used to explain the role of exoloops during receptor activation and their interplay with TMDs.

Majumdar, Ritankar; Railkar, Reema; Dighe, Rajan R.



Vitamin A deficiency disturbs collagen IV and laminin composition and decreases matrix metalloproteinase concentrations in rat lung. Partial reversibility by retinoic acid.  


Vitamin A is essential for lung development and pulmonary cell differentiation. Its deficiency leads to altered lung structure and function and to basement membrane architecture and composition disturbances. Previously, we showed that lack of retinoids thickens the alveolar basement membrane and increases collagen IV, which are reversed by retinoic acid, the main biologically active vitamin A form. This study analyzed how vitamin A deficiency affects the subunit composition of collagen IV and laminin of lung basement membranes and pulmonary matrix metalloproteinase content, plus the recovering effect of all-trans-retinoic acid. Male weanling pups were fed a retinol-adequate/-deficient diet until 60 days old. A subgroup of vitamin-A-deficient pups received daily intraperitoneal all-trans-retinoic acid injections for 10 days. Collagen IV and laminin chain composition were modified in vitamin-A-deficient rats. The protein and mRNA contents of chains ?1(IV), ?3(IV) and ?4(IV) increased; those of chains ?2(IV) and ?5(IV) remained unchanged; and the protein and mRNA contents of laminin chains ?5, ?1 and ?1 decreased. The mRNA of laminin chains ?2 and ?4 also decreased. Matrix metalloproteinases 2 and 9 decreased, but the tissue inhibitors of metalloproteinases 1 and 2 did not change. Treating vitamin-A-deficient rats with retinoic acid reversed all alterations, but laminin chains ?2, ?4 and ?5 and matrix metalloproteinase 2 remained low. In conclusion, vitamin A deficiency alters the subunit composition of collagen IV and laminin and the lung's proteolytic potential, which are partly reverted by retinoic acid. These alterations could contribute to impaired lung function and predispose to pulmonary disease. PMID:22832075

Esteban-Pretel, Guillermo; Marín, M Pilar; Renau-Piqueras, Jaime; Sado, Yoshikazu; Barber, Teresa; Timoneda, Joaquín



Spontaneous growth of a laminin-apatite nano-composite in a metastable calcium phosphate solution.  


We have previously reported that a laminin-apatite composite layer is formed on an ethylene-vinyl alcohol copolymer (EVOH) in a laminin-containing calcium phosphate (LCP) solution. In this work, the stability of the LCP solution and growth process of the laminin-apatite composite layer have been investigated. Dynamic light scattering technique revealed that the LCP solution was stable for periods as long as 24 h; it did not induce homogeneous precipitation of laminin or calcium phosphates in the solution. Analysis of the EVOH surface and the LCP solution showed that the laminin-apatite composite layer was formed via coprecipitation of laminin and apatite on the EVOH plate, i.e., spontaneous growing of apatite and simultaneous immobilization of laminin molecules or laminin-calcium phosphate nano-complexes onto its surface. Transmission electron microscopy also revealed that the laminin molecules in the resulting composite layer were not localized or aggregated, but were dispersed on a nano-scale in the entire layer. Because of this nano-composite structure, a large number of laminin molecules were stably immobilized on the EVOH plate. This may be responsible for the excellent cell adhesion properties of this type of composite material. PMID:16024072

Oyane, Ayako; Uchida, Masaki; Onuma, Kazuo; Ito, Atsuo



Metal interactions with voltage- and receptor-activated ion channels.  

PubMed Central

Effects of Pb and several other metal ions on various distinct types of voltage-, receptor- and Ca-activated ion channels have been investigated in cultured N1E-115 mouse neuroblastoma cells. Experiments were performed using the whole-cell voltage clamp and single-channel patch clamp techniques. External superfusion of nanomolar to submillimolar concentrations of Pb causes multiple effects on ion channels. Barium current through voltage-activated Ca channels is blocked by micromolar concentrations of Pb, whereas voltage-activated Na current appears insensitive. Neuronal type nicotinic acetylcholine receptor-activated ion current is blocked by nanomolar concentrations of Pb and this block is reversed at micromolar concentrations. Serotonin 5-HT3 receptor-activated ion current is much less sensitive to Pb. In addition, external superfusion with micromolar concentrations of Pb as well as of Cd and aluminum induces inward current, associated with the direct activation of nonselective cation channels by these metal ions. In excised inside-out membrane patches of neuroblastoma cells, micromolar concentrations of Ca activate small (SK) and big (BK) Ca-activated K channels. Internally applied Pb activates SK and BK channels more potently than Ca, whereas Cd is approximately equipotent to Pb with respect to SK channel activation, but fails to activate BK channels. The results show that metal ions cause distinct, selective effects on the various types of ion channels and that metal ion interaction sites of ion channels may be highly selective for particular metal ions.

Vijverberg, H P; Oortgiesen, M; Leinders, T; van Kleef, R G



Differential Activation of Insulin Receptor Substrates 1 and 2 by Insulin-Like Growth Factor-Activated Insulin Receptors  

Microsoft Academic Search

The insulin-like growth factors (insulin-like growth factor I (IGF-I) and IGF-II) exert important effects on growth, development, and differentiation through the IGF-I receptor (IGF-IR) transmembrane tyrosine kinase. The insulin receptor (IR) is structurally related to the IGF-IR, and at high concentrations, the IGFs can also activate the IR, in spite of their generally low affinity for the latter. Two mechanisms

Adam Denley; Julie M. Carroll; Gemma V. Brierley; Leah Cosgrove; John Wallace; Briony Forbes



Suppression by protease-activated receptor-2 activation of gastric acid secretion in rats  

Microsoft Academic Search

Activation of protease-activated receptor-2 (PAR-2), a receptor activated by trypsin\\/tryptase, induces neurally mediated gastric mucus secretion accompanied by mucosal cytoprotection. In the present study, we investigated whether PAR-2 could modulate gastric acid secretion in rats. Messenger RNAs for PAR-2 and PAR-1 were detected in the gastric mucosa and smooth muscle. The PAR-2-activating peptide SLIGRL-NH2, but not the inactive control peptide,

Hiroyuki Nishikawa; Kenzo Kawai; Sachiyo Nishimura; Shuichi Tanaka; Hiromasa Araki; Bahjat Al-Ani; Morley D Hollenberg; Ryotaro Kuroda; Atsufumi Kawabata



Activated Protein C Prevents Neuronal Apoptosis via Protease Activated Receptors 1 and 3  

Microsoft Academic Search

Activated protein C (APC), a serine protease with anticoagulant and anti-inflammatory activities, exerts direct cytoprotective effects on endothelium via endothelial protein C receptor-dependent activation of protease activated receptor 1 (PAR1). Here, we report that APC protects mouse cortical neurons from two divergent inducers of apoptosis, N-methyl-D-aspartate (NMDA) and staurosporine. APC blocked several steps in NMDA-induced apoptosis downstream to nitric oxide,

Huang Guo; Dong Liu; Harris Gelbard; Tong Cheng; Rae Insalaco; José A Fernández; John H Griffin; Berislav V Zlokovic



Alpha2-adrenergic receptor activation regulates cortical interneuron migration.  


Monoamines such as serotonin and dopamine have been shown to regulate cortical interneuron migration but very little is known regarding noradrenaline. Similarly to other monoamines, noradrenaline is detected during embryonic cortical development and adrenergic receptors are expressed in transient embryonic zones of the pallium that contain migrating neurons. Evidence of a functional role for the adrenergic system in interneuron migration is lacking. In this study we first investigated the expression pattern of adrenergic receptors in mouse cortical interneuron subtypes preferentially derived from the caudal ganglionic eminences, and found that they expressed different subtypes of adrenergic receptors. To directly monitor the effects of adrenergic receptor stimulation on interneuron migration we used time-lapse recordings in cortical slices and observed that alpha2 adrenergic receptors (adra2) receptor activation inhibits the migration of cortical interneurons in a concentration-dependent and reversible manner. Furthermore, we observed that following adra2 activation the directionality of migrating interneurons was significantly modified, suggesting that adra2 stimulation could modulate their responsiveness to guidance cues. Finally the distribution of cortical interneurons was altered in vivo in adra2a/2c-knockout mice. These results support the general hypothesis that adrenergic dysregulation occurring during embryonic development alters cellular processes involved in the formation of cortical circuits. PMID:22805283

Riccio, Orbicia; Hurni, Nicolas; Murthy, Sahana; Vutskits, Laszlo; Hein, Lutz; Dayer, Alexandre



Complement 3 deficiency and oral prednisolone improve strength and prolong survival of laminin alpha2-deficient mice.  


Complement deposition and macrophages are common in biopsies of children with muscular dystrophy. While the presumed roles of complement and macrophages have been those of scavenger to remove and clear necrotic fibers, there is some evidence that they play a primary role in the pathogenesis of these diseases. Here, we explore the role of complement in the pathogenesis of the most severe animal model of congenital dystrophy, the dy-/- mouse, which is laminin alpha2-deficient. We generated animals deficient in both C3 and laminin alpha2. C3 is the third component of the complement cascade and is required for activation of either the classical or alternative pathways. Thirty-three percent of the dy-/-:C3+ mice (n=59) died before 24 weeks while only 14% of the dy-/-:C3-/- (n=29) mice died (p=0.04). Absolute forepaw strength was 25-30% greater for the dy-/-:C3-/- mice up to 20 weeks of age (p<0.05 compared to complement-sufficient). Forepaw strength adjusted for weight also showed significant differences with C3-/- mice being stronger up to 20 weeks (p<0.05). However, by 24 weeks, the two groups did not differ for strength. Next, we treated 20 mice with twice weekly oral prednisolone. Survival at 24 weeks for the prednisolone treated dy-/- mice (C3-/- or C3+) was 90% (p=0.04). This work shows that complement insufficiency and weekly prednisone prolong survival and improve strength of the laminin alpha2-deficient mouse. This work suggests that the complement system may contribute directly to the pathogenesis of this form of dystrophy. Because complement activity may be modified pharmacologically, this work may have implications for treatment of children with congenital muscular dystrophy secondary to laminin alpha2 deficiency. PMID:12044978

Connolly, Anne M; Keeling, Richard M; Streif, Elizabeth M; Pestronk, Alan; Mehta, Shobhna



Fetal Epidermal Differentiation and Barrier Development In Vivo is Accelerated by Nuclear Hormone Receptor Activators1  

Microsoft Academic Search

Nuclear receptors which interact with the retinoid X receptor are involved in the regulation of epidermal differentiation and development. We have recently shown that activators of the peroxisome proliferator-activated receptor and of the farnesoid X-activated receptor accelerate epidermal barrier maturation in fetal rat skin in vitro. In this study we asked whether cutaneous development in utero was affected by peroxisome

Karen Hanley; László G Kömüves; Nathan M Bass; Shan Shan He; Yan Jiang; Debra Crumrine; Mark Friedman; Joseph Bettencourt; Katherine Min; Peter M Elias; Mary L Williams; Kenneth R Feingold



A systematic analytical chemistry\\/cell assay. approach to isolate activators of orphan nuclear receptors from biological extracts: characterization of peroxisome proliferator-activated receptor activators in plasma  

Microsoft Academic Search

Using a novel combination of analytical chemical and molecular biological techniques, lipophilic components of human plasma separated according to their physico-chemical properties were screened for their ability to activate the rat peroxisome proliferator-activated receptor (rPPAR). Activation of an rPPAWglucocorticoid receptor chimera stably expressed in CHO cells by fractions in the initial screening guided further subfractionation. Characterization of an active subfraction

Martin Gottlicher; Eva Widmark; Jan Sjovall; Joseph J. Rafter


NMDA receptor activation induces translocation and activation of Rac in mouse hippocampal area CA1  

PubMed Central

Neuronal development requires several discrete morphological steps that are believed to involve the small GTPase Rac. For example, neural activity, through NMDA receptors and/or AMPA receptors, activates Rac leading to elaboration of dendritic arbors. In the current study, we have conducted studies which indicate that Rac might be an important molecule involved in morphological plasticity in the adult mouse. We demonstrate that Rac is expressed at synapses in the adult mouse hippocampus. We also demonstrate that treatment of hippocampal slices with NMDA induces membrane translocation and activation of Rac in area CA1. Interestingly, we also find that there is an increase in Rac that is associated with NMDA receptor complexes following NMDA receptor activation. Taken together, our data are consistent with the idea that Rac could be participating in NMDA receptor-dependent changes in morphology that occur during synaptic plasticity and memory formation in the adult mouse hippocampus.

Tejada-Simon, Maria V.; Villasana, Laura E.; Serrano, Faridis; Klann, Eric



Dopamine receptor activation by honey bee queen pheromone.  


Queen mandibular pheromone (QMP) is produced by honey bee queens and used to regulate the behavior and physiology of their nestmates. QMP has recently been shown to block aversive learning in young worker bees, an effect that can be mimicked by treating bees with one of QMP's key components, homovanillyl alcohol (HVA). Although the mechanisms underlying this blockade remain unclear, HVA has been found to lower brain dopamine levels and to alter intracellular levels of cAMP in brain centers involved in learning and memory. These findings led to the hypothesis that HVA targets dopamine pathways in the brain, which are known to play a critical role in the formation of aversive olfactory memories. Here, we investigate the possibility that HVA interacts directly with dopamine receptors in the bee. We show that HVA selectively activates the D2-like dopamine receptor AmDOP3 but has neither agonist nor antagonist activity on the D1-like receptors AmDOP1 or AmDOP2 nor agonist activity on the octopamine receptor AmOA1. These results suggest a direct molecular mechanism by which queen pheromone can modulate dopamine signaling pathways. They also implicate the dopamine receptor AmDOP3 in HVA-induced blockade of aversive learning in young worker bees. PMID:19523830

Beggs, Kyle T; Mercer, Alison R



Laminin Mediates Tissue-specific Gene Expression in Mammary Epithelia  

Microsoft Academic Search

Tissue-specific gene expression in mammary epithelium is dependent on the extracellular matrix as well as hormones. There is good evidence that the basement membrane provides signals for regulating beta-casein expression, and that integrins are involved in this process. Here, we demonstrate that in the presence of lactogenic hormones, laminin can direct expression of the beta-casein gene. Mouse mammary epithelial cells

Charles H. Streuli; Christian Schmidhauser; Nina Bailey; Peter Yurchenco; Amy P. N. Skubitz; Calvin Roskelley; Mina J. Bissell



Expression of laminin subunits in human fetal skeletal muscle  

Microsoft Academic Search

Summary  The laminin variant of adult skeletal muscle fibres and Schwann cells is known as merosin, and is composed of M-B1-B2 chains. Blood vessels and immature fibres express the A chain in association with B1 or S, and B2. The importance of merosin has recently been shown by its absence in one form of congenital muscular dystrophy and in the mutant

C. A. Sewry; M. Chevallay; F. M. S. Tomé



Expression of laminin subunits in human fetal skeletal muscle  

Microsoft Academic Search

Summary  The laminin variant of adult skeletal muscle fibres and Schwann cells is known as merosin, and is composed of M-B1-B2 chains.\\u000a Blood vessels and immature fibres express the A chain in association with B1 or S, and B2. The importance of merosin has recently\\u000a been shown by its absence in one form of congenital muscular dystrophy and in the mutantdy\\/dy

C. A. Sewry; M. Chevallay; F. M. S. Tomé



Galectin-1 Modulates Human Melanoma Cell Adhesion to Laminin  

Microsoft Academic Search

Galectins constitute a gene family of ?-galactoside-specific lectins that show high homology in their carbohydrate-binding site. They have been postulated to be involved in many biological events, but their specific functions are not yet well defined. Galectin-1 is a laminin binding protein that recognizes poly N-acetyllactosamine chains on this major basement membrane glycoprotein. In this study, we analyzed the possibility

F. A. Vandenbrule; C. Buicu; M. Baldet; M. E. Sobel; D. N. W. Cooper; P. Marschal; V. Castronovo



Laminin Promotes NeuriticRegenerationfrom Cultured Peripheraland CentralNeurons  

Microsoft Academic Search

The abilityof axons to grow through tissuein vivo during development or regenerationmay be regulatedby the availability of specificneurite-promotingmacromole- culeslocatedwithintheextracellular matrix.We have used tissueculturemethods toexamine the relativeabilityofvariousextracellular matrixcomponents toelicitneuriteoutgrowth from dissociatedchickembryo parasympathetic(ciliary ganglion)neurons inserum-freemonolayer culture. Purifiedlaminin from both mouse and ratsources,as well as a partiallypurified polyornithine-bindingneuritepromoting factor(PNPF-1)from ratSchwannoma cellsallstim- ulateneuriteproduction from these neurons.Laminin and PNPF-1 arealsopotent stimulators of neuritegrowth from culturedneurons




Distribution of ten laminin chains in dystrophic and regenerating muscles  

Microsoft Academic Search

Using immunohistochemical methods, we assessed the distribution of all 10 known laminin chains (?1-5, ?1-3, ?1 and ?2) in skeletal muscles from patients with Duchenne, congenital, limb girdle, or Emery–Dreifuss muscular dystrophies. The ?2, ?1 and ?1 chains were abundant in the basal lamina surrounding muscle fibers in normal controls; ?1, ?3–?5, ?3, and ?2 were undetectable; and ?2 was

Bruce L. Patton; Anne M. Connolly; Paul T. Martin; Jeanette M. Cunningham; Shobhna Mehta; Alan Pestronk; Jeffrey H. Miner; Joshua R. Sanes



The role of LamininB2 (LanB2) during mesoderm differentiation in Drosophila.  


In Drosophila, four genes encode for laminin subunits and the formation of two laminin heterotrimers has been postulated. We report the identification of mutations in the Drosophila LamininB2 (LanB2) gene that encodes for the only laminin ? subunit and is found in both heterotrimers. We describe their effects on embryogenesis, in particular the differentiation of visceral tissues with respect to the ECM. Analysis of mesoderm endoderm interaction indicates disrupted basement membranes and defective endoderm migration, which finally interferes with visceral myotube stretching. Extracellular deposition of laminin is blocked due to the loss of the LanB2 subunit, resulting in an abnormal distribution of ECM components. Our data, concerning the different function of both trimers during organogenesis, suggest that these trimers might act in a cumulative way and probably at multiple steps during ECM assembly. We also observed genetic interactions with kon-tiki and thrombospondin, indicating a role for laminin during muscle attachment. PMID:21387145

Wolfstetter, Georg; Holz, Anne



Ecdysone receptor expression and activity in adult Drosophila melanogaster.  


Disrupting components of the ecdysone/EcR/USP signaling pathway in insects leads to morphological defects and developmental arrest. In adult Drosophila melanogaster decreased EcR function affects fertility, lifespan, behavior, learning, and memory; however we lack a clear understanding of how EcR/USP expression and activity impacts these phenotypes. To shed light on this issue, we characterized the wild-type expression patterns and activity of EcR/USP in individual tissues during early adult life. EcR and usp were expressed in numerous adult tissues, but receptor activity varied depending on tissue type and adult age. Receptor activity did not detectably change in response to mating status, environmental stress, ecdysone treatment or gender but is reduced when a constitutively inactive ecdysone receptor is present. Since only a subset of adult tissues expressing EcR and usp contain active receptors, it appears that an important adult function of EcR/USP in some tissues may be repression of genes containing EcRE's. PMID:21507325

Schwedes, Christoph; Tulsiani, Siddharth; Carney, Ginger E



Modulation of Opioid Receptor Ligand Affinity and Efficacy Using Active and Inactive State Receptor Models  

PubMed Central

Mu opioid receptor (MOR) agonists are widely used for the treatment of pain; however chronic use results in the development of tolerance and dependence. It has been demonstrated that co-administration of a MOR agonist with a delta opioid receptor (DOR) antagonist maintains the analgesia associated with MOR agonists, but with reduced negative side effects. Using our newly refined opioid receptor models for structure-based ligand design, we have synthesized several pentapeptides with tailored affinity and efficacy profiles. In particular, we have obtained pentapeptides 8, Tyr-c(S-S)[DCys-1Nal-Nle-Cys]NH2, and 12, Tyr-c(S-S)[DCys-1Nal-Nle-Cys]OH, which demonstrates high affinity and full agonist behavior at MOR, high affinity but very low efficacy for DOR, and minimal affinity for the kappa opioid receptor (KOR). Functional properties of these peptides as MOR agonists/DOR antagonists lacking undesired KOR activity make them promising candidates for future in vivo studies of MOR/DOR interactions. Subtle structural variation of 12, by substituting D-Cys5 for L-Cys5, generated analog 13 which maintains low nanomolar MOR and DOR affinity, but which displays no efficacy at either receptor. These results demonstrate the power and utility of accurate receptor models for structure-based ligand design, as well as the profound sensitivity of ligand function on its structure.

Anand, Jessica P.; Purington, Lauren C.; Pogozheva, Irina D.; Traynor, John R.; Mosberg, Henry I.



An Estrogen Receptor Alpha Activity Indicator Model in Mice  

PubMed Central

Estrogen Receptor alpha (ER?), plays essential roles in the female reproduction. To investigate the dynamic changes in ER? activity in vivo, we have developed an ER Alpha Activity Indicator (ERAAI) mouse. This ERAAI mouse harbors both a modified ER? Bacterial Artificial Chromosome (BAC) clone and a reporter gene which is regulated specifically by the modified receptor. The ER? modification (Gal4-ER?) consists replacing the DNA binding domain (DBD) of ER? with the DBD of yeast Gal4 transcription factor. This reporter transgene consisting of a humanized renilla Green Fluorescent Protein (hrGFP) sequence controlled by the Upstream Activating Sequences for the Gal4 gene (UASG) was inserted into the modified ER? BAC clone. Expression of Gal4-ER? and hrGFP reliably recapitulates endogenous ER? expression and activity in the estrogen target tissues in response to estrogen stimulation. Therefore, the ERAAI mouse represents a novel animal model to investigate dynamic ER? activity in vivo.

Han, Sang Jun; O'Malley, Bert W.; DeMayo, Francesco J.



The zebrafish candyfloss mutant implicates extracellular matrix adhesion failure in laminin ?2-deficient congenital muscular dystrophy  

PubMed Central

Mutations in the human laminin ?2 (LAMA2) gene result in the most common form of congenital muscular dystrophy (MDC1A). There are currently three models for the molecular basis of cellular pathology in MDC1A: (i) lack of LAMA2 leads to sarcolemmal weakness and failure, followed by cellular necrosis, as is the case in Duchenne muscular dystrophy (DMD); (ii) loss of LAMA2-mediated signaling during the development and maintenance of muscle tissue results in myoblast proliferation and fusion defects; (iii) loss of LAMA2 from the basement membrane of the Schwann cells surrounding the peripheral nerves results in a lack of motor stimulation, leading to effective denervation atrophy. Here we show that the degenerative muscle phenotype in the zebrafish dystrophic mutant, candyfloss (caf) results from mutations in the laminin ?2 (lama2) gene. In vivo time-lapse analysis of mechanically loaded fibers and membrane permeability assays suggest that, unlike DMD, fiber detachment is not initially associated with sarcolemmal rupture. Early muscle formation and myoblast fusion are normal, indicating that any deficiency in early Lama2 signaling does not lead to muscle pathology. In addition, innervation by the primary motor neurons is unaffected, and fiber detachment stems from muscle contraction, demonstrating that muscle atrophy through lack of motor neuron activity does not contribute to pathology in this system. Using these and other analyses, we present a model of lama2 function where fiber detachment external to the sarcolemma is mechanically induced, and retracted fibers with uncompromised membranes undergo subsequent apoptosis.

Hall, Thomas E.; Bryson-Richardson, Robert J.; Berger, Silke; Jacoby, Arie S.; Cole, Nicholas J.; Hollway, Georgina E.; Berger, Joachim; Currie, Peter D.



Structural basis for selective activation of ABA receptors  

SciTech Connect

Changing environmental conditions and lessening fresh water supplies have sparked intense interest in understanding and manipulating abscisic acid (ABA) signaling, which controls adaptive responses to drought and other abiotic stressors. We recently discovered a selective ABA agonist, pyrabactin, and used it to discover its primary target PYR1, the founding member of the PYR/PYL family of soluble ABA receptors. To understand pyrabactin's selectivity, we have taken a combined structural, chemical and genetic approach. We show that subtle differences between receptor binding pockets control ligand orientation between productive and nonproductive modes. Nonproductive binding occurs without gate closure and prevents receptor activation. Observations in solution show that these orientations are in rapid equilibrium that can be shifted by mutations to control maximal agonist activity. Our results provide a robust framework for the design of new agonists and reveal a new mechanism for agonist selectivity.

Peterson, Francis C.; Burgie, E. Sethe; Park, Sang-Youl; Jensen, Davin R.; Weiner, Joshua J.; Bingman, Craig A.; Chang, Chia-En A.; Cutler, Sean R.; Phillips, Jr., George N.; Volkman, Brian F. (MCW); (UW); (UCR)



Molecular Imaging of Epidermal Growth Factor Receptor Kinase Activity  

PubMed Central

Epidermal growth factor (EGF) receptor (EGFR), a receptor tyrosine kinase, is commonly altered in different tumor types leading to abnormally regulated kinase activity and excessive activation of downstream signaling cascades including cell proliferation, differentiation and migration. To investigate the EGFR signaling events in real time and in living cells and animals, we here describe a multidomain chimeric reporter whose bioluminescence can be used as a surrogate for EGFR kinase activity. This luciferase-based reporter was developed in squamous cell carcinoma cells (UMSCC-1) to generate a cancer therapy model for imaging EGFR. The reporter is designed to act as a phosphorylated substrate of EGFR and reconstitutes luciferase activity when it is not phosphorylated, thus providing a robust indication of EGFR inhibition. We validated the reporter in vitro and demonstrated that its activity could be differentially modulated by EGFR tyrosine kinase inhibition with erlotonib or receptor activation with EGF. Further experiments in vivo demonstrated quantitative and dynamic monitoring of EGFR tyrosine kinase activity in xenograft. Results obtained from these studies provide unique insight into pharmacokinetics and pharmacodynamics of agents that modulate EGFR activity, revealing the usefulness of this reporter in evaluating drug availability and cell targeting in both living cells and mouse models.

Khana, Amjad P.; Contessa, Joseph N.; Nyatia, Mukesh K.; Ross, Brian D.; Rehemtulla, Alnawaz



Cofactoring and dimerization of proteinase-activated receptors.  


Proteinase-activated receptors (PARs) are G protein-coupled receptors that transmit cellular responses to extracellular proteases and have important functions in vascular physiology, development, inflammation, and cancer progression. The established paradigm for PAR activation involves proteolytic cleavage of the extracellular N terminus, which reveals a new N terminus that functions as a tethered ligand by binding intramolecularly to the receptor to trigger transmembrane signaling. Most cells express more than one PAR, which can influence the mode of PAR activation and signaling. Clear examples include murine PAR3 cofactoring of PAR4 and transactivation of PAR2 by PAR1. Thrombin binds to and cleaves murine PAR3, which facilitates PAR4 cleavage and activation. This process is essential for thrombin signaling and platelet activation, since murine PAR3 cannot signal alone. Although PAR1 and PAR4 are both competent to signal, PAR1 is able to act as a cofactor for PAR4, facilitating more rapid cleavage and activation by thrombin. PAR1 can also facilitate PAR2 activation through a different mechanism. Cleavage of the PAR1 N terminus by thrombin generates a tethered ligand domain that can bind intermolecularly to PAR2 to activate signaling. Thus, PARs can regulate each other's activity by localizing thrombin when in complex with PAR3 and PAR4 or by cleaved PAR1, providing its tethered ligand domain for PAR2 activation. The ability of PARs to cofactor or transactivate other PARs would necessitate that the two receptors be in close proximity, likely in the form of a heterodimer. Here, we discuss the cofactoring and dimerization of PARs and the functional consequences on signaling. PMID:24064459

Lin, Huilan; Liu, Allen P; Smith, Thomas H; Trejo, Joann



Functional Consequences of Cell Type-Restricted Expression of Laminin ?5 in Mouse Placental Labyrinth and Kidney Glomerular Capillaries  

PubMed Central

The labyrinth is the highly vascularized part of the rodent placenta that allows efficient transfer of gases, nutrients, wastes, and other molecules between the maternal and embryonic circulations. These two blood compartments are separated by blastocyst-derived trophoblasts and endothelial cells with an intervening basement membrane that contains laminin and other typical basement membrane components. Previously we reported that the labyrinth of laminin ?5 knockout (LM?5?/?) embryos exhibits reduced vascularization and detachment of endothelial cells from the basement membrane, which normally contains LM?5. As very little is known about the origin of this vascular basement membrane, we investigated the cellular requirements for LM?5 expression in the mouse placental labyrinth. By fluorescence-activated cell sorting and RT-PCR we confirmed that both endothelial cells and trophoblasts normally express LM?5. Using Cre-loxP technology and doxycycline-mediated gene expression, we generated genetically mosaic placentas in which either the trophoblasts or the endothelial cells, but not both, expressed LM?5. We found that the overall architecture of the labyrinth was normal as long as one of these two cell types expressed LM?5, even if it was transgene-derived human laminin ?5. These results suggest that laminin trimers containing ?5 that are synthesized and secreted by endothelium or by trophoblasts are capable of integrating into the basement membrane and promoting normal vascularization of the placenta. Additional studies showed that endothelium-expressed human LM?5 can support vascularization of the kidney glomerulus, consistent with previous studies using a tissue grafting approach.

Kim, Sung Tae; Adair-Kirk, Tracy L.; Senior, Robert M.; Miner, Jeffrey H.



Laminin Terminates the Netrin/DCC Mediated Attraction of Vagal Sensory Axons  

PubMed Central

Vagal sensory axons navigate to specific sites in the bowel during fetal life. Netrin/deleted in colorectal cancer (DCC) were found to mediate the attraction of vagal sensory axons to the fetal mouse gut. We tested the hypothesis that laminin-111 can reverse the chemoattractive effects of netrin and act as a stop signal for vagal sensory axons. Laminin-111-expressing cells were located in the E12 and E16 mouse bowel by in situ hybridization. At E12, these cells extended centrifugally from the endoderm; by E16, laminin-111 expressing cells were found in the mucosa and outer gut mesenchyme. A similar pattern was seen in preparations of E13 and E15 mouse gut labeled with antibodies to laminin. Application of DiI to nodose ganglia identified vagal sensory axons growing into the fetal bowel. These terminals were found to avoid concentrations of laminin or to terminate at laminin-delimited boundaries. Soluble laminin inhibited the preferential growth of nodose neurites toward netrin-secreting cells (p < 0.01). This effect was mimicked by a peptide, YIGSR, a sequence within the ?1 chain of laminin-111 (p < 0.004) and antagonized by a peptide, IKVAV, a sequence within the ?1 chain of laminin-111. Antibodies to ?1-integrins were also able to reverse the inhibitive effects of laminin and restore the attraction of nodose neurites towards netrin-1-secreting cells. Soluble laminin inhibited the preferential growth of nodose neurites toward a cocultured explant of foregut. These findings suggest that laminin terminates the attraction of vagal sensory axons towards sources of netrin in the developing bowel.

Ratcliffe, Elyanne M.; D'Autreaux, Fabien; Gershon, Michael D.



Comparative study on transcriptional activity of 17 parabens mediated by estrogen receptor ? and ? and androgen receptor.  


The structure-activity relationships of parabens which are widely used as preservatives for transcriptional activities mediated by human estrogen receptor ? (hER?), hER? and androgen receptor (hAR) were investigated. Fourteen of 17 parabens exhibited hER? and/or hER? agonistic activity at concentrations of ? 1 × 10(-5)M, whereas none of the 17 parabens showed AR agonistic or antagonistic activity. Among 12 parabens with linear alkyl chains ranging in length from C? to C??, heptylparaben (C?) and pentylparaben (C?) showed the most potent ER? and ER? agonistic activity in the order of 10(-7)M and 10(-8)M, respectively, and the activities decreased in a stepwise manner as the alkyl chain was shortened to C? or lengthened to C??. Most parabens showing estrogenic activity exhibited ER?-agonistic activity at lower concentrations than those inducing ER?-agonistic activity. The estrogenic activity of butylparaben was markedly decreased by incubation with rat liver microsomes, and the decrease of activity was blocked by a carboxylesterase inhibitor. These results indicate that parabens are selective agonists for ER? over ER?; their interactions with ER?/? are dependent on the size and bulkiness of the alkyl groups; and they are metabolized by carboxylesterases, leading to attenuation of their estrogenic activity. PMID:23567241

Watanabe, Yoko; Kojima, Hiroyuki; Takeuchi, Shinji; Uramaru, Naoto; Ohta, Shigeru; Kitamura, Shigeyuki



Liver X Receptor and Peroxisome Proliferator-Activated Receptor Agonist from Cornus alternifolia  

PubMed Central

Background Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptors superfamily and are transcription factors activated by specific ligands. Liver X receptors (LXR) belong to the nuclear hormone receptors and have been shown to play an important role in cholesterol homeostasis. From the previous screening of several medicinal plants for potential partial PPAR? agonists, the extracts of Cornus alternifolia were found to exhibit promising bioactivity. In this paper, we report the isolation and structural elucidation of four new compounds and their potential as ligands for PPAR. Methods The new compounds were extracted from the leaves of Cornus alternifolia and fractionated by high-performance liquid chromatography. Their structures were elucidated on the basis of spectroscopic evidence and analysis of their hydrolysis products. Results Three new iridoid glycosides including an iridolactone, alternosides A-C (1–3), a new megastigmane glycoside, cornalternoside (4) and 10 known compounds, were obtained from the leaves of Cornus alternifolia. Kaempferol-3-O-?-glucopyranoside (5) exhibited potent agonistic activities for PPAR?, PPAR? and LXR with EC50 values of 0.62, 3.0 and 1.8 ? M, respectively. Conclusions We isolated four new and ten known compounds from Cornus alternifolia, and one known compound showed agonistic activities for PPAR?, PPAR? and LXR. General significance Compound 1 is the first example of a naturally occurring iridoid glycoside containing a ?-glucopyranoside moiety at C-6.

He, Yang-Qing; Ma, Guo-Yi; Peng, Jiang-nan; Ma, Zhan-Ying; Hamann, Mark T.



Cinnamaldehyde suppresses Toll-like receptor 4 activation mediated through the inhibition of receptor oligomerization  

Technology Transfer Automated Retrieval System (TEKTRAN)

Toll-like receptors (TLRs) play a critical role in induction of innate immune and inflammatory responses by recognizing invading pathogens. TLRs have two major downstream signaling pathways, MyD88- and TRIF-dependent pathways leading to the activation of NF'B and IRF3 and the expression of inflammat...


Transcription Activation by the Ecdysone Receptor (EcR\\/USP): Identification of Activation Functions  

Microsoft Academic Search

The ecdysone receptor is a heterodimer of the two nuclear receptors EcR and ultraspiracle (USP). We have identified the regions of Drosophila EcR and USP responsible for transcriptional activation of a semisynthetic Eip71CD promoter in Kc cells. The isoform-specific A\\/B domains of EcR-B1 and B2, but not those of EcR-A or USP, exhibit strong ac- tivation activity (activation function 1




OX1 orexin/hypocretin receptor activation of phospholipase D  

PubMed Central

BACKGROUND AND PURPOSE Orexin receptors potently signal to lipid messenger systems, and our previous studies have suggested that PLD would be one of these. We thus wanted to verify this by direct measurements and clarify the molecular mechanism of the coupling. EXPERIMENTAL APPROACH Orexin receptor-mediated PLD activation was investigated in CHO cells stably expressing human OX1 orexin receptors using [14C]-oleic acid-prelabelling and the transphosphatidylation assay. KEY RESULTS Orexin stimulation strongly increased PLD activity – even more so than the phorbol ester TPA (12-O-tetradecanoyl-phorbol-13-acetate), a highly potent activator of PLD. Both orexin and TPA responses were mediated by PLD1. Orexin-A and -B showed approximately 10-fold difference in potency, and the concentration–response curves were biphasic. Using pharmacological inhibitors and activators, both orexin and TPA were shown to signal to PLD1 via the novel PKC isoform, PKC?. In contrast, pharmacological or molecular biological inhibitors of Rho family proteins RhoA/B/C, cdc42 and Rac did not inhibit the orexin (or the TPA) response, nor did the molecular biological inhibitors of PKD. In addition, neither cAMP elevation, G?i/o nor G?? seemed to play an important role in the orexin response. CONCLUSIONS AND IMPLICATIONS Stimulation of OX1 receptors potently activates PLD (probably PLD1) in CHO cells and this is mediated by PKC? but not other PKC isoforms, PKDs or Rho family G-proteins. At present, the physiological significance of orexin-induced PLD activation is unknown, but this is not the first time we have identified PKC? in orexin signalling, and thus some specific signalling cascade may exist between orexin receptors and PKC?.

Jantti, MH; Putula, J; Somerharju, P; Frohman, MA; Kukkonen, JP



Factors regulating the activities of the low density lipoprotein receptor and the scavenger receptor on human monocyte-macrophages  

Microsoft Academic Search

Improved techniques of cell isolation resulted in 90 to 100 million monocytes from a single donor. Addi- tion of low density lipoprotein (LDL) to cultures of these cells resulted in the down regulation of LDL receptor activity. Addition of malondialdehyde-altered LDL, which enters the cell through a receptor for negatively charged proteins (the scavenger receptor), produced an even greater down

Alan M. Fogelman; Margaret E. Haberland; Martha Hokom; Peter A. Edwards


A sequential dimerization mechanism for erythropoietin receptor activation.  

PubMed Central

We have probed the interaction of human erythropoietin (EPO) with its receptor (EPO-R) by analyzing a panel of 17 EPO mutants in a variety of in vitro assays. Mutant proteins were expressed in 293s cells and quantified by using an N-terminal epitope tag in conjunction with a surface plasmon resonance assay. Receptor binding was studied using both a soluble form of the EPO-R extracellular domain in an ELISA-format binding competition assay and full-length EPO-R in transfected BaF3 cells. Proliferative activity of the mutants was also determined in the BaF3-derived cell line and was correlated with the results from binding assays. Based on the results of these assays, we identified two distinct receptor binding sites on the EPO molecule. We propose that one site, containing residues Arg-150 and Lys-152, binds initially to EPO receptor on the cell surface. A second site, containing Arg-103 and Ser-104 (and possibly Arg-14), is involved in binding a second EPO-R at the cell surface, thus forming a homodimeric receptor complex. Furthermore, we demonstrate that one EPO mutant (R103A), which has previously been shown to lack proliferative function, is in fact an EPO antagonist. Taken together, these data support a sequential dimerization mechanism of EPO-R activation.

Matthews, D J; Topping, R S; Cass, R T; Giebel, L B



Peroxisome proliferator-activated receptors in the cardiovascular system  

PubMed Central

Peroxisome proliferator-activated receptor (PPAR)s are a family of three nuclear hormone receptors, PPAR?, -?, and -?, which are members of the steriod receptor superfamily. The first member of the family (PPAR?) was originally discovered as the mediator by which a number of xenobiotic drugs cause peroxisome proliferation in the liver. Defined functions for all these receptors, until recently, mainly concerned their ability to regulate energy balance, with PPAR? being involved in ?-oxidation pathways, and PPAR? in the differentiation of adipocytes. Little is known about the functions of PPAR?, though it is the most ubiquitously expressed. Since their discovery, PPARs have been shown to be expressed in monocytes/macrophages, the heart, vascular smooth muscle cells, endothelial cells, and in atherosclerotic lesions. Furthermore, PPARs can be activated by a vast number of compounds including synthetic drugs, of the clofibrate, and anti-diabetic thiazoldinedione classes, polyunsaturated fatty acids, and a number of eicosanoids, including prostaglandins, lipoxygenase products, and oxidized low density lipoprotein. This review will aim to introduce the field of PPAR nuclear hormone receptors, and discuss the discovery and actions of PPARs in the cardiovascular system, as well as the source of potential ligands.

Bishop-Bailey, David



Inhibition of the Androgen Receptor Activity by Coprinus comatus Substances  

Microsoft Academic Search

Prostatic adenocarcinoma is the second leading cause of death from cancer in Western men. The common prostate cancer treatments are effective in the early stages; however, advanced prostate cancer is resilient to most of these treatments. Altered androgen receptor (AR) activity caused by point mutations or signaling mechanisms that regulate AR function has been proposed as a key mechanism in

Nesly Dotan; Solomon P. Wasser; Jamal Mahajna



Compositions and Methods for Activating Toll-Like Receptor 4.  

National Technical Information Service (NTIS)

Methods for activating Toll-like receptor 4 via cholesterol-dependent cytolysins isolated from a Gram-positive bacteria are provided. In addition compositions containing an isolated cholesterol-dependent cytolysin or a fragment thereof or a mimetic of the...

J. M. Park M. Karin R. Rest



Profiling receptor tyrosine kinase activation by using Ab microarrays  

Microsoft Academic Search

sensitive to the amounts and modification states of signal trans- duction proteins in crude cell lysates and the integration of these arrays with 96-well microtiter plate technology to create microar- rays in microplates. We apply the Ab arrays to monitoring the activation, uptake, and signaling of ErbB receptor tyrosine kinases in human tumor cell lines. Data obtained from multicolor ratio-

Ulrik B. Nielsen; Mike H. Cardone; Anthony J. Sinskey; Gavin MacBeath; Peter K. Sorger



Activation of Estrogen Receptor by Bavachin from Psoralea corylifolia  

PubMed Central

In this study, we examined the estrogenic activity of bavachin, a component of Psoralea corylifolia that has been used as a traditional medicine in Asia. Bavachin was purified from ethanolic extract of Psoralea corylifolia and characterized its estrogenic activity by ligand binding, reporter gene activation, and endogenous estrogen receptor (ER) target gene regulation. Bavachin showed ER ligand binding activity in competitive displacement of [3H] E2 from recombinant ER. The estrogenic activity of bavachin was characterized in a transient transfection system using ER? or ER? and estrogen-responsive luciferase plasmids in CV-1 cells with an EC50 of 320 nM and 680 nM, respectively. Bavachin increased the mRNA levels of estrogen-responsive genes such as pS2 and PR, and decreased the protein level of ER? by proteasomal pathway. However, bavachin failed to activate the androgen receptor in CV-1 cells transiently transfected with the corresponding receptor and hormone responsive reporter plasmid. These data indicate that bavachin acts as a weak phytoestrogen by binding and activating the ER.

Park, Joonwoo; Kim, Do Hee; Ahn, Hye-Na; Song, Yun Seon; Lee, Young Joo; Ryu, Jae-Ha



Laminin network formation studied by reconstitution of ternary nodes in solution.  


The polymerization of laminins into a cell-associated network is a key process in basement membrane assembly. Network formation is mediated by the homologous short arm tips of the laminin heterotrimer, each consisting of a globular laminin N-terminal (LN) domain followed by a tandem of laminin-type epidermal growth factor-like (LEa) domains. How the short arms interact in the laminin network is unclear. Here, we have addressed this question by reconstituting laminin network nodes in solution and analyzing them by size exclusion chromatography and light scattering. Recombinant LN-LEa1-4 fragments of the laminin ?1, ?2, ?5, ?1, and ?1 chains were monomeric in solution. The ?1 and ?1 fragments formed the only detectable binary complex and ternary complexes of 1:1:1 stoichiometry with all ? chain fragments. Ternary complex formation required calcium and did not occur at 4 °C, like the polymerization of full-length laminins. Experiments with chimeric short arm fragments demonstrated that the LEa2-4 regions of the ?1 and ?1 fragments are dispensable for ternary complex formation, and an engineered glycan in the ?1 LEa1 domain was also tolerated. In contrast, mutation of Ser-68 in the ?1 LN domain (corresponding to a Pierson syndrome mutation in the closely related ?2 chain) abolished ternary complex formation. We conclude that authentic ternary nodes of the laminin network can be reconstituted for structure-function studies. PMID:23166322

Purvis, Alan; Hohenester, Erhard



Platelet-activating factor (PAF) receptor and genetically engineered PAF receptor mutant mice  

Microsoft Academic Search

Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a biologically active phospholipid mediator. Although PAF was initially recognized for its potential to induce platelet aggregation and secretion, intense investigations have elucidated potent biological actions of PAF in a broad range of cell types and tissues, many of which also produce the molecule. PAF acts by binding to a unique G-protein-coupled seven transmembrane receptor.

Satoshi Ishii; Takao Shimizu



Cinnamaldehyde suppresses toll-like receptor 4 activation mediated through the inhibition of receptor oligomerization  

Microsoft Academic Search

Toll-like receptors (TLRs) play a critical role in induction of innate immune and inflammatory responses by recognizing invading pathogens or non-microbial endogenous molecules. TLRs have two major downstream signaling pathways, MyD88- and TRIF-dependent pathways leading to the activation of NF?B and IRF3 and the expression of inflammatory mediators. Deregulation of TLR activation is known to be closely linked to the

Hyung S. Youn; Jun K. Lee; Yong J. Choi; Shin I. Saitoh; Kensuke Miyake; Daniel H. Hwang; Joo Y. Lee



Facilitation of neocortical presynaptic terminal development by NMDA receptor activation  

PubMed Central

Background Neocortical circuits are established through the formation of synapses between cortical neurons, but the molecular mechanisms of synapse formation are only beginning to be understood. The mechanisms that control synaptic vesicle (SV) and active zone (AZ) protein assembly at developing presynaptic terminals have not yet been defined. Similarly, the role of glutamate receptor activation in control of presynaptic development remains unclear. Results Here, we use confocal imaging to demonstrate that NMDA receptor (NMDAR) activation regulates accumulation of multiple SV and AZ proteins at nascent presynaptic terminals of visual cortical neurons. NMDAR-dependent regulation of presynaptic assembly occurs even at synapses that lack postsynaptic NMDARs. We also provide evidence that this control of presynaptic terminal development is independent of glia. Conclusions Based on these data, we propose a novel NMDAR-dependent mechanism for control of presynaptic terminal development in excitatory neocortical neurons. Control of presynaptic development by NMDARs could ultimately contribute to activity-dependent development of cortical receptive fields.



Regulation of native GABA A receptors by PKC and protein phosphatase activity  

Microsoft Academic Search

Rationale and objective: Protein kinase C (PKC) modulation of ionotropic receptors is a common mechanism for regulation of channel function. The effects of PKC and phosphatase activation on native gamma-amino- butyric acid (GABAA) receptors in adult brain are unknown. Previous studies of recombinant GABAA receptors have provided evidence that PKC activation inhibits receptor function, whereas other studies suggest that PKC

Sandeep Kumar; Rahul T. Khisti; A. Leslie Morrow



Micropatterned immobilization of a G protein–coupled receptor and direct detection of G protein activation  

Microsoft Academic Search

G protein–coupled receptors (GPCRs) constitute an abundant family of membrane receptors of high pharmacological interest. Cell-based assays are the predominant means of assessing GPCR activation, but are limited by their inherent complexity. Functional molecular assays that directly and specifically report G protein activation by receptors could offer substantial advantages. We present an approach to immobilize receptors stably and with defined

Christoph Bieri; Oliver P. Ernst; Stephan Heyse; Klaus Peter Hofmann; Horst Vogel



Insulin receptor substrates 1 and 2 but not Shc can activate the insulin receptor independent of insulin and induce proliferation in CHOIR cells  

Microsoft Academic Search

Ligand-activated insulin receptor (IR) attracts and phosphorylates various substrates such as insulin receptor substrates 1–4 (IRS) and Shc. To investigate how binding affinity for substrate affects signalling we generated chimeric receptors with the ?-chain of the insulin receptor containing NPXY motives with different affinities for receptor substrates. We found that the extent of receptor tyrosine phosphorylation positively correlates with binding

Markus. Niessen; Frank Jaschinski; Flurin Item; Morgan P. McNamara; Giatgen A. Spinas; Thomas Trueb



The Mu-Opioid Receptor Gene Polymorphism (A118G) Alters HPA Axis Activation Induced by Opioid Receptor Blockade  

Microsoft Academic Search

An A118G nucleotide exchange in exon 1 of the mu-opioid receptor causes an Asn40Asp substitution polymorphism in the receptor's extracellular domain. In vitro studies show that the Asp40 variant of the mu-opioid receptor binds ?-endorphin three times more avidly than the more common Asn40 variant. Paraventricular corticotropin releasing hormone neurons, which activate the HPA axis, express mu-opioid receptors and are

Gary S Wand; Mary McCaul; Xioaju Yang; Joanna Reynolds; Deidre Gotjen; Shing Lee; Ahmed Ali



Divergent Immunoglobulin G Subclass Activity Through Selective Fc Receptor Binding  

Microsoft Academic Search

Subclasses of immunoglobulin G (IgG) display substantial differences in their ability to mediate effector responses, contributing to variable activity of antibodies against microbes and tumors. We demonstrate that the mechanism underlying this long-standing observation of subclass dominance in function is provided by the differential affinities of IgG subclasses for specific activating IgG Fc receptors compared with their affinities for the

Falk Nimmerjahn; Jeffrey V. Ravetch



Protease-activated Receptor2 (PAR2) in Human Periodontitis  

Microsoft Academic Search

No evidence for the role of protease-activated receptor-2 (PAR2) in human periodontal disease has been demonstrated so far. Thus, we sought to investigate the expression of PAR2 mRNA in chronic periodontitis, and to examine whether its expression is related to the presence of PAR2 potential activators. Microbiological and gingival crevicular fluid samples were collected from individuals with chronic periodontitis and

M. Holzhausen; J. R. Cortelli; V. Araújo da Silva; G. C. Nobre Franco; S. Cavalca Cortelli; N. Vergnolle



Ligand binding and activation of the Ah receptor  

Microsoft Academic Search

The Ah receptor (AhR) is a ligand-dependent transcription factor that can be activated by structurally diverse synthetic and naturally-occurring chemicals. Although a significant amount of information is available with respect to the planar aromatic hydrocarbon AhR ligands, the actual spectrum of chemicals that can bind to and activate the AhR is only now being elucidated. In addition, the lack of

Michael S Denison; Alessandro Pandini; Scott R Nagy; Enoch P Baldwin; Laura Bonati



EphB receptor activity suppresses colorectal cancer progression  

Microsoft Academic Search

Most sporadic colorectal cancers are initiated by activating Wnt pathway mutations, characterized by the stabilization of beta-catenin and constitutive transcription by the beta-catenin\\/T cell factor-4 (Tcf-4) complex. EphB guidance receptors are Tcf4 target genes that control intestinal epithelial architecture through repulsive interactions with Ephrin-B ligands. Here we show that, although Wnt signalling remains constitutively active, most human colorectal cancers lose

Eduard Batlle; Julinor Bacani; Harry Begthel; Suzanne Jonkeer; Alexander Gregorieff; Maaike van de Born; Núria Malats; Elena Sancho; Elles Boon; Tony Pawson; Steven Gallinger; Steven Pals; Hans Clevers



Specific block of androgen receptor activity by antisense oligonucleotides  

Microsoft Academic Search

Claims about molecular mechanisms underlying the resistance to anti-hormones of prostate cancer cells find support in biological experiments, which involve hormone-independent activation of the androgen receptor's (AR) transcriptional activity. In order to test this hypothesis, we attempted to shut down the expression of AR by the means of target-directed antisense oligonucleotides. A set of 49 oligonucleotides matching sequences of the

F Hamy; V Brondani; R Spoerri; S Rigo; C Stamm; T Klimkait



Mechanisms of NOD-like Receptor-Associated Inflammasome Activation.  


A major function of a subfamily of NLR (nucleotide-binding domain, leucine-rich repeat containing, or NOD-like receptor) proteins is in inflammasome activation, which has been implicated in a multitude of disease models and human diseases. This work will highlight key progress in understanding the mechanisms that activate the best-studied NLRs (NLRP3, NLRC4, NAIP, and NLRP1) and in uncovering inflammasome NLRs. PMID:24054327

Wen, Haitao; Miao, Edward A; Ting, Jenny P-Y



? and k opiate receptors in primary astroglial cultures part II: Receptor sets in cultures from various brain regions and interactions with ß-receptor activated cyclic AMP  

Microsoft Academic Search

In a previous paper, and opiate receptors were shown to be co-localized on the same cell in enriched primary cultures of astroglia from neonatal rat cerebral cortex. Activation of the receptors inhibited adenylate cyclase. In this work, the presence of opiate receptors was investigated in astroglial primary cultures from neonatal rat striatum and brain stem. Cyclic adenosine 3, 5-monophosphate accumulation

Peter S. Eriksson; Elisabeth Hansson; Lars Rönnbäck



Activation of Axonal Receptors by GABA Spillover Increases Somatic Firing.  


Axons can be depolarized by ionotropic receptors and transmit subthreshold depolarizations to the soma by passive electrical spread. This raises the possibility that axons and axonal receptors can participate in integration and firing in neurons. Previously, we have shown that exogenous GABA depolarizes cerebellar granule cell axons through local activation of GABAA receptors (GABAARs) and the soma through electrotonic spread of the axonal potential resulting in increased firing. We show here that excitability of granule cells is also increased by release of endogenous GABA from molecular layer interneurons (MLIs) and spillover activation of parallel fiber GABAARs in mice and rats. Changes in granule cell excitability were assessed by excitability testing after activation of MLIs with channelrhodopsin or electrical stimulation in the molecular layer. In granule cells lacking an axon, excitability was not changed, suggesting that axonal receptors are required. To determine the distance over which subthreshold potentials may spread, we estimated the effective axonal electrical length constant (520 ?m) by excitability testing and focal uncaging of RuBi-GABA on the axon at varying distances from the soma. These data suggest that GABAAR-mediated axonal potentials can participate in integration and firing of cerebellar granule cells. PMID:24155298

Pugh, Jason R; Jahr, Craig E



Biological Signaling: the Role of ``Electrostatic Epicenter'' in ``Protein Quake'' and Receptor Activation  

NASA Astrophysics Data System (ADS)

Activation of a receptor protein during biological signaling is often characterized by a two state model: a receptor state (also called ``off state'') for detection of a stimuli, and a signaling state (``on state'') for signal relay. Receptor activation is a process that a receptor protein is structurally transformed from its receptor state to its signaling state through substantial conformational changes that are recognizable by its downstream signal relay partner. What are the structural and energetic origins for receptor activation in biological signaling? We report extensive evidence that further support the role of ``electrostatic epicenter'' in driving ``protein quake'' and receptor activation. Photoactive yellow protein (PYP), a bacterial blue light photoreceptor protein for the negative phototaxis of a salt loving Halorhodospira halophia, is employed as a model system in this study. We will discuss potential applications of this receptor activation mechanism to other receptor proteins, including B-RAF receptor protein that is associated with many cancers.

Xie, Aihua; Kaledhonkar, Sandip; Kang, Zhouyang; Hendriks, Johnny; Hellingwerf, Klaas



Protein kinase C?/? inhibitor Gö6976 promotes PC12 cell adhesion and spreading through membrane recruitment and activation of protein kinase C?.  


Gö6976 is a nonglycosidic indolocarbazole compound widely used as a specific inhibitor of PKC?/?. In experiments probing for a role of PKC? in human laminin-2-integrin-mediated cell adhesion and spreading of PC12 cells, we observed unexpected enhancements of adhesion, spreading and stress fiber formation to 1 ?M Gö6976 with concomitant increase in membrane translocation of PKC? and autophosphorylation of focal adhesion kinase (FAK). Importantly, enhanced cellular behavior and membrane translocation of PKC? induced by Gö6976 was retained in siRNA-transfected PC12 cells to knockdown PKC? expression. Gö6976 also induced laminin-dependent cell adhesion in NIH/3T3 and CV-1 fibroblasts, suggesting of a mechanism that may be common to multiple cell-types. A specific inhibitor of PKC?, rottlerin, completely abrogated Gö6976-dependent increase in PC12 cell adhesion to laminin as well as the activation of small GTPases, Rac1 and Cdc42, that are downstream of PKC? in adhesion receptor signaling. siRNA knockdown of Rac1 and Cdc42 expression inhibited cell spreading and lamellipodia formation in PC12 cells. Overall, these results suggest that Gö6976 may stimulate membrane recruitment of PKC? through a mechanism that is independent of PKC?/? signaling. In addition, the activation of Rac1 and Cdc42 by human laminin-2-integrin-dependent activation of PKC?/FAK signaling mediates cell spreading and lamellipodia formation in PC12 cells. PMID:23063429

Jung, Sung Youn; Kim, O Bok; Kang, Hyun Ki; Jang, Da Hyun; Min, Byung-Moo; Yu, Frank H



Progesterone, but Not Progesterone Independent Activation of Progestin Receptors by a Mating Stimulus, Rapidly Decreases Progestin Receptor Immunoreactivity in Female Rat Brain  

Microsoft Academic Search

Recent studies suggest that progestin receptors may be activated in vivo by neurotransmitters in the absence of ligand. More specifically, vaginal–cervical stimulation (VCS) can influence sexual behavior by activating progestin receptors in the absence of progesterone. Another way to test if progestin receptors are influenced by particular stimuli is to examine progestin receptor immunostaining. We report that progestin receptor immunoreactivity

A. P. Auger; L. M. LaRiccia; C. A. Moffatt; J. D. Blaustein



Regulation of ligands for the NKG2D activating receptor.  


NKG2D is an activating receptor expressed by all NK cells and subsets of T cells. It serves as a major recognition receptor for detection and elimination of transformed and infected cells and participates in the genesis of several inflammatory diseases. The ligands for NKG2D are self-proteins that are induced by pathways that are active in certain pathophysiological states. NKG2D ligands are regulated transcriptionally, at the level of mRNA and protein stability, and by cleavage from the cell surface. In some cases, ligand induction can be attributed to pathways that are activated specifically in cancer cells or infected cells. We review the numerous pathways that have been implicated in the regulation of NKG2D ligands, discuss the pathologic states in which those pathways are likely to act, and attempt to synthesize the findings into general schemes of NKG2D ligand regulation in NK cell responses to cancer and infection. PMID:23298206

Raulet, David H; Gasser, Stephan; Gowen, Benjamin G; Deng, Weiwen; Jung, Heiyoun



Peroxisome Proliferator-Activated Receptor Alpha Target Genes  

PubMed Central

The peroxisome proliferator-activated receptor alpha (PPAR?) is a ligand-activated transcription factor involved in the regulation of a variety of processes, ranging from inflammation and immunity to nutrient metabolism and energy homeostasis. PPAR? serves as a molecular target for hypolipidemic fibrates drugs which bind the receptor with high affinity. Furthermore, PPAR? binds and is activated by numerous fatty acids and fatty acid-derived compounds. PPAR? governs biological processes by altering the expression of a large number of target genes. Accordingly, the specific role of PPAR? is directly related to the biological function of its target genes. Here, we present an overview of the involvement of PPAR? in lipid metabolism and other pathways through a detailed analysis of the different known or putative PPAR? target genes. The emphasis is on gene regulation by PPAR? in liver although many of the results likely apply to other organs and tissues as well.

Rakhshandehroo, Maryam; Knoch, Bianca; Muller, Michael; Kersten, Sander



Pharmacological activation of kainate receptors drives endocannabinoid mobilization.  


Activation of both presynaptic metabotropic cannabinoid type 1 receptors (CB(1)s) and ionotropic kainate receptors (KARs) can efficiently modulate GABA release at many synapses of the CNS. The inhibitory effect of kainic acid (KA) has been ascribed to metabotropic actions, and KAR-induced release of secondary neuromodulatory agents may partly mediate these actions. Here, we investigated the involvement of the endocannabinoid system in the modulation of GABAergic synaptic transmission by pharmacological activation of KARs with KA in CA1 pyramidal neurons of the mouse hippocampus. We show that the depression of GABAergic synaptic transmission induced by KA (3 ?m) is strongly inhibited by the simultaneous blockade of CB(1) and GABA(B) receptors with SR141716A (5 ?m) and CGP55845 (5 ?m), respectively. KA induces a calcium-dependent mobilization of the endocannabinoid anandamide (AEA) by activation of GluK2-containing KARs in postsynaptic pyramidal neurons. Consistently, the effect of KA is prolonged by the inhibitor of AEA degradation URB597 (1 ?m) in a CB(1)-dependent manner, but it is not altered by blockade of degradation or synthesis of the other main endocannabinoid 2-arachidonoylglycerol (2AG). Hence, our work reveals that the pharmacological activation of KARs leads to the stimulation of secondary metabotropic signaling systems. In addition, these data further underline the profound mechanistic differences between exogenous and endogenous activation of KARs in the hippocampus. PMID:21368036

Lourenço, Joana; Matias, Isabel; Marsicano, Giovanni; Mulle, Christophe



Endogenous laminin is required for human airway smooth muscle cell maturation  

PubMed Central

Background Airway smooth muscle (ASM) contraction underlies acute bronchospasm in asthma. ASM cells can switch between a synthetic-proliferative phenotype and a contractile phenotype. While the effects of extracellular matrix (ECM) components on modulation of ASM cells to a synthetic phenotype have been reported, the role of ECM components on maturation of ASM cells to a contractile phenotype in adult lung is unclear. As both changes in ECM components and accumulation of contractile ASM are features of airway wall remodelling in asthma, we examined the role of the ECM protein, laminin, in the maturation of contractile phenotype in human ASM cells. Methods Human ASM cells were made senescence-resistant by stable expression of human telomerase reverse transcriptase. Maturation to a contractile phenotype was induced by 7-day serum deprivation, as assessed by immunoblotting for desmin and calponin. The role of laminin on ASM maturation was investigated by comparing the effects of exogenous laminin coated on culture plates, and of soluble laminin peptide competitors. Endogenous expression of laminin chains during ASM maturation was also measured. Results Myocyte binding to endogenously expressed laminin was required for ASM phenotype maturation, as laminin competing peptides (YIGSR or GRGDSP) significantly reduced desmin and calponin protein accumulation that otherwise occurs with prolonged serum deprivation. Coating of plastic cell culture dishes with different purified laminin preparations was not sufficient to further promote accumulation of desmin or calponin during 7-day serum deprivation. Expression of ?2, ?1 and ?1 laminin chains by ASM cells was specifically up-regulated during myocyte maturation, suggesting a key role for laminin-2 in the development of the contractile phenotype. Conclusion While earlier reports suggest exogenously applied laminin slows the spontaneous modulation of ASM to a synthetic phenotype, we show for the first time that endogenously expressed laminin is required for ASM maturation to the contractile phenotype. As endogenously expressed laminin chains ?2, ?1 and ?1 are uniquely increased during myocyte maturation, these laminin chains may be key in this process. Thus, human ASM maturation appears to involve regulated endogenous expression of a select set of laminin chains that are essential for accumulation of contractile phenotype myocytes.

Tran, Thai; McNeill, Karol D; Gerthoffer, William T; Unruh, Helmut; Halayko, Andrew J



Partial rescue of glomerular laminin alpha5 mutations by wild-type endothelia produce hybrid glomeruli.  


Both endothelial cells and podocytes are sources for laminin alpha1 at the inception of glomerulogenesis and then for laminin alpha5 during glomerular maturation. Why glomerular basement membranes (GBM) undergo laminin transitions is unknown, but this may dictate glomerular morphogenesis. In mice that genetically lack laminin alpha5, laminin alpha5beta2gamma1 is not assembled, vascularized glomeruli fail to form, and animals die at midgestation with neural tube closure and placental deficits. It was previously shown that renal cortices of newborn mice contain endothelial progenitors (angioblasts) and that when embryonic day 12 kidneys are transplanted into newborn kidney, hybrid glomeruli (host-derived endothelium and donor-derived podocytes) result. Reasoning that host endothelium may correct the glomerular phenotype that is seen in laminin alpha5 mutants, alpha5 null embryonic day 12 metanephroi were grafted into wild-type newborn kidney. Hybrid glomeruli were identified in grafts by expression of a host-specific LacZ lineage marker. Labeling of glomerular hybrid GBM with chain-specific antibodies showed a markedly stratified distribution of laminins: alpha5 was found only on the inner endothelial half of GBM, whereas alpha1 located to outer layers beneath mutant podocytes. For measurement of the contribution of host endothelium to hybrid GBM, immunofluorescent signals for laminin alpha5 were quantified: Hybrid GBM contained approximately 50% the normal alpha5 complement as wild-type GBM. Electron microscopy of glomerular hybrids showed vascularization, but podocyte foot processes were absent. It was concluded that (1) endothelial and podocyte-derived laminins remain tethered to their cellular origin, (2) developing endothelial cells contribute large amounts of GBM laminins, and (3) podocyte foot process differentiation may require direct exposure to laminin alpha5. PMID:17599968

Abrahamson, Dale R; St John, Patricia L; Isom, Kathryn; Robert, Barry; Miner, Jeffrey H



Laminin drives survival signals to promote a contractile smooth muscle phenotype and airway hyperreactivity.  


Increased airway smooth muscle (ASM) mass is believed to underlie the relatively fixed airway hyperresponsiveness (AHR) in asthma. Developments of therapeutic approaches to reverse airway remodeling are impeded by our lack of insight on the mechanisms behind the increase in mass of contractile ASM cells. Increased expression of laminin, an extracellular matrix protein, is associated with asthma. Our studies investigate the role of laminin-induced ASM survival signals in the development of increased ASM and AHR. Antagonizing laminin integrin binding using the laminin-selective competing peptide, YIGSR, and mimicking laminin with exogenous ?2-chain laminin, we show that laminin is both necessary and sufficient to induce ASM cell survival, concomitant with the induction of ASM contractile phenotype. Using siRNA, we show that the laminin-binding integrin ?7?1 mediates this process. Moreover, in laminin-211-deficient mice, allergen-induced AHR was not observed. Notably, ASM cells from asthmatic airways express a higher abundance of intracellular cell survival proteins, consistent with a role for reduced rates of cell apoptosis in development of ASM hyperplasia. Targeting the laminin-integrin ?7?1 signaling pathway may offer new avenues for the development of therapies to reduce the increase in mass of contractile phenotype ASM cells that underlie AHR in asthma.-Tran, T., Teoh, C. M., Tam, J. K. C., Qiao, Y., Chin, C. Y., Chong, O. K., Stewart, A. G., Harris, T., Wong, W. S. F., Guan, S. P., Leung, B. P., Gerthoffer, W. T., Unruh, H., and Halayko, A. J. Laminin drives survival signals to promote a contractile smooth muscle phenotype and airway hyperreactivity. PMID:23756649

Tran, Thai; Teoh, Chun Ming; Tam, John Kit Chung; Qiao, Yongkang; Chin, Chin Yein; Chong, Oi Khuan; Stewart, Alastair G; Harris, Trudi; Wong, Wai Shiu Fred; Guan, Shou Ping; Leung, Bernard P; Gerthoffer, William T; Unruh, Helmut; Halayko, Andrew J



Cannabinoids Induce Pancreatic ?-Cell Death by Directly Inhibiting Insulin Receptor Activation  

PubMed Central

Cannabinoid 1 (CB1) receptors have been previously detected in pancreatic ? cells, where they influence insulin action. We now report that CB1 receptors form a heteromeric complex with insulin receptors and G?i, which inhibits insulin receptor kinase activity in ? cells by directly binding to the activation loop in the tyrosine kinase domain of the insulin receptor. Consequently, phosphorylation of pro-apoptotic protein Bad was reduced, leading to activation of Bad and induction of ?-cell death. Pharmacological blockade or genetic deficiency of CB1 receptors led to reduced blood glucose and increased ?-cell survival after injury due to enhanced insulin receptor signaling and reduced activation of Bad. These findings provide direct evidence of physical and functional interactions between CB1 and insulin receptors and provide a mechanism whereby peripherally acting CB1 receptor antagonists improve insulin action in insulin-sensitive tissues independent of the other metabolic effects of CB1 receptors.

Kim, Wook; Lao, Qizong; Shin, Yu-Kyong; Carlson, Olga D.; Lee, Eun Kyung; Gorospe, Myriam; Kulkarni, Rohit N.; Egan, Josephine M.



Different phenolic compounds activate distinct human bitter taste receptors.  


Bitterness is a major sensory attribute of several common foods and beverages rich in polyphenol compounds. These compounds are reported as very important for health as chemopreventive compounds, but they are also known to taste bitter. In this work, the activation of the human bitter taste receptors, TAS2Rs, by six polyphenol compounds was analyzed. The compounds chosen are present in a wide range of plant-derived foods and beverages, namely, red wine, beer, tea, and chocolate. Pentagalloylglucose (PGG) is a hydrolyzable tannin, (-)-epicatechin is a precursor of condensed tannins, procyanidin dimer B3 and trimer C2 belong to the condensed tannins, and malvidin-3-glucoside and cyanidin-3-glucoside are anthocyanins. The results show that the different compounds activate different combinations of the ~25 TAS2Rs. (-)-Epicatechin activated three receptors, TAS2R4, TAS2R5, and TAS2R39, whereas only two receptors, TAS2R5 and TAS2R39, responded to PGG. In contrast, malvidin-3-glucoside and procyanidin trimer stimulated only one receptor, TAS2R7 and TAS2R5, respectively. Notably, tannins are the first natural agonists found for TAS2R5 that display high potency only toward this receptor. The catechol and/or galloyl groups appear to be important structural determinants that mediate the interaction of these polyphenolic compounds with TAS2R5. Overall, the EC(50) values obtained for the different compounds vary 100-fold, with the lowest values for PGG and malvidin-3-glucoside compounds, suggesting that they could be significant polyphenols responsible for the bitterness of fruits, vegetables, and derived products even if they are present in very low concentrations. PMID:23311874

Soares, Susana; Kohl, Susann; Thalmann, Sophie; Mateus, Nuno; Meyerhof, Wolfgang; De Freitas, Victor



Bisphenol A and Its Analogues Activate Human Pregnane X Receptor  

PubMed Central

Background: Bisphenol A (BPA) is a base chemical used extensively in many consumer products. BPA and its analogues are present in environmental and human samples. Many endocrine-disrupting chemicals, including BPA, have been shown to activate the pregnane X receptor (PXR), a nuclear receptor that functions as a master regulator of xenobiotic metabolism. However, the detailed mechanism by which these chemicals activate PXR remains unknown. Objective: We investigated the mechanism by which BPA interacts with and activates PXR and examined selected BPA analogues to determine whether they bind to and activate PXR. Methods: Cell-based reporter assays, in silico ligand–PXR docking studies, and site-directed mutagenesis were combined to study the interaction between BPA and PXR. We also investigated the influence of BPA and its analogues on the regulation of PXR target genes in human LS180 cells. Results: We found that BPA and several of its analogues are potent agonists for human PXR (hPXR) but do not affect mouse PXR activity. We identified key residues within hPXR’s ligand-binding pocket that constitute points of interaction with BPA. We also deduced the structural requirements of BPA analogues that activate hPXR. BPA and its analogues can also induce PXR target gene expression in human LS180 cells. Conclusions: The present study advances our understanding of the mechanism by which BPA interacts with and activates human PXR. Activation of PXR by BPA may explain some of the adverse effects of BPA in humans.

Sui, Yipeng; Ai, Ni; Park, Se-Hyung; Rios-Pilier, Jennifer; Perkins, Jordan T.; Welsh, William J.



Lipids, lipoproteins, and peroxisome proliferator activated receptor-delta.  


Peroxisome proliferator activated receptors (PPARs) are nuclear receptors activated by small, lipophilic compounds. Typically resident on nuclear DNA, full activation requires heterodimer formation with retinoid X receptor and ligand binding, leading to modulation in the expression of hundreds of genes. Of the 3 described forms, (PPAR-alpha, PPAR-gamma, and PPAR-delta), PPAR-delta has been the least investigated. Preclinical in vitro data show that activation of PPAR-delta, like PPAR-alpha, results in enhancement of fatty acid oxidation, leading to increased energy production in the form of adenosine triphosphate and of energy uncoupling. Microarray data in preclinical models suggest substantial PPAR-delta expression in skeletal muscle. Exercise, which induces upregulation of PPAR-delta in muscle tissue, leads to an increased requirement for an external or serum derived triacylglycerol energy source. This suggests that upregulation of skeletal muscle PPAR-delta would influence lipoprotein composition, this being the major source of combustible substrate. In the first human study using a PPAR-delta agonist, experimental data obtained with GW 501516 (a highly specific PPAR-delta agonist) suggested that upregulated enzymes critical to fatty acid oxidation in human cells enhanced fatty acid and beta-oxidation in skeletal muscle. PMID:18047848

Sprecher, Dennis L



Models for the activation pathway of epidermal growth factor receptor protein-tyrosine kinase  

SciTech Connect

Activation of the epidermal growth factor (EGF) receptor's intrinsic protein-tyrosine kinase activity, which occurs upon formation of the receptor-ligand complex, is the critical regulatory event affecting the subsequent EGF-dependent cellular responses leading to DNA synthesis and cell proliferation. The molecular mechanism by which EGF-dependent activation of receptor kinase activity takes place is not clearly understood. In this study, the growth factor-dependent activation of the EGF receptor tyrosine kinase was examined in vitro using detergent-solubilized, partially purified GEF receptors from A5431 human epidermoid carcinoma cells. Evaluation of the cooperativity observed in the EGF-dependent activation of soluble receptor tyrosine kinase would suggest a mechanism requiring the binding of the EGF peptide to both ligand binding sites on a receptor dimer to induce full receptor kinase activity. Equations describing potential cooperative kinase activation pathways have been examined. The theoretical system which best simulates the allosteric regulation observed in the experimental kinase activation data is that describing multiple essential activation. In addition, studies using mutant analogs of the EGF peptide ligand appear to confirm the requirement for an essential conformational change in the receptor-ligand complex to activate the receptor kinase activity. Several mutant growth factor analogues are able to occupy the ligand binding sites on the receptor without inducing the fully active receptor conformation.

Campion, S.R.; Niyogi, S.K. (Oak Ridge National Lab., TN (United States))



Activation of Penile Proadipogenic Peroxisome Proliferator-Activated Receptor ? with an Estrogen: Interaction with Estrogen Receptor Alpha during Postnatal Development  

PubMed Central

Exposure to the estrogen receptor alpha (ER?) ligand diethylstilbesterol (DES) between neonatal days 2 to 12 induces penile adipogenesis and adult infertility in rats. The objective of this study was to investigate the in vivo interaction between DES-activated ER? and the proadipogenic transcription factor peroxisome proliferator-activated receptor gamma (PPAR?). Transcripts for PPARs ?, ?, and ? and ?1a splice variant were detected in Sprague-Dawley normal rat penis with PPAR? predominating. In addition, PPAR?1b and PPAR?2 were newly induced by DES. The PPAR? transcripts were significantly upregulated with DES and reduced by antiestrogen ICI 182, 780. At the cellular level, PPAR? protein was detected in urethral transitional epithelium and stromal, endothelial, neuronal, and smooth muscular cells. Treatment with DES activated ER? and induced adipocyte differentiation in corpus cavernosum penis. Those adipocytes exhibited strong nuclear PPAR? expression. These results suggest a biological overlap between PPAR? and ER? and highlight a mechanism for endocrine disruption.

Mansour, Mahmoud M.; Goyal, Hari O.; Braden, Tim D.; Dennis, John C.; Schwartz, Dean D.; Judd, Robert L.; Bartol, Frank F.; Coleman, Elaine S.; Morrison, Edward E.



Cannabinoids go nuclear: evidence for activation of peroxisome proliferator-activated receptors  

PubMed Central

Cannabinoids act at two classical cannabinoid receptors (CB1 and CB2), a 7TM orphan receptor and the transmitter-gated channel transient receptor potential vanilloid type-1 receptor. Recent evidence also points to cannabinoids acting at members of the nuclear receptor family, peroxisome proliferator-activated receptors (PPARs, with three subtypes ?, ? (?) and ?), which regulate cell differentiation and lipid metabolism. Much evidence now suggests that endocannabinoids are natural activators of PPAR?. Oleoylethanolamide regulates feeding and body weight, stimulates fat utilization and has neuroprotective effects mediated through activation of PPAR?. Similarly, palmitoylethanolamide regulates feeding and lipid metabolism and has anti-inflammatory properties mediated by PPAR?. Other endocannabinoids that activate PPAR? include anandamide, virodhamine and noladin. Some (but not all) endocannabinoids also activate PPAR?; anandamide and 2-arachidonoylglycerol have anti-inflammatory properties mediated by PPAR?. Similarly, ajulemic acid, a structural analogue of a metabolite of ?9-tetrahydrocannabinol (THC), causes anti-inflammatory effects in vivo through PPAR?. THC also activates PPAR?, leading to a time-dependent vasorelaxation in isolated arteries. Other cannabinoids which activate PPAR? include N-arachidonoyl-dopamine, HU210, WIN55212-2 and CP55940. In contrast, little research has been carried out on the effects of cannabinoids at PPAR?. In this newly emerging area, a number of research questions remain unanswered; for example, why do cannabinoids activate some isoforms and not others? How much of the chronic effects of cannabinoids are through activation of nuclear receptors? And importantly, do cannabinoids confer the same neuro- and cardioprotective benefits as other PPAR? and PPAR? agonists? This review will summarize the published literature implicating cannabinoid-mediated PPAR effects and discuss the implications thereof.

O'Sullivan, S E



Visualising Androgen Receptor Activity in Male and Female Mice  

PubMed Central

Androgens, required for normal development and fertility of males and females, have vital roles in the reproductive tract, brain, cardiovascular system, smooth muscle and bone. Androgens function via the androgen receptor (AR), a ligand-dependent transcription factor. To assay and localise AR activity in vivo we generated the transgenic “ARE-Luc” mouse, expressing a luciferase reporter gene under the control of activated endogenous AR. In vivo imaging of androgen-mediated luciferase activity revealed several strongly expressing tissues in the male mouse as expected and also in certain female tissues. In males the testes, prostate, seminal vesicles and bone marrow all showed high AR activity. In females, strong activity was seen in the ovaries, uterus, omentum tissue and mammary glands. In both sexes AR expression and activity was also found in salivary glands, the eye (and associated glands), adipose tissue, spleen and, notably, regions of the brain. Luciferase protein expression was found in the same cell layers as androgen receptor expression. Additionally, mouse AR expression and activity correlated well with AR expression in human tissues. The anti-androgen bicalutamide reduced luciferase signal in all tissues. Our model demonstrates that androgens can act in these tissues directly via AR, rather than exclusively via androgen aromatisation to estrogens and activation of the estrogen receptor. Additionally, it visually demonstrates the fundamental importance of AR signalling outside the normal role in the reproductive organs. This model represents an important tool for physiological and developmental analysis of androgen signalling, and for characterization of known and novel androgenic or antiandrogenic compounds.

Dart, D. Alwyn; Waxman, Jonathan; Aboagye, Eric O.; Bevan, Charlotte L.



Defective Muscle Basement Membrane and Lack of M-Laminin in the Dystrophic dy\\/dy Mouse  

Microsoft Academic Search

M-laminin is a major member of the laminin family of basement membrane proteins. It is prominently expressed in striated muscle and peripheral nerve. M-laminin is deficient in patients with the autosomal recessive Fukuyama congenital muscular dystrophy but is normal in patients with the sex-linked Duchenne and Becker muscular dystrophies. We have examined M-laminin expression in mice with autosomal recessive muscular

Hong Xu; Peter Christmas; Xiao-Rong Wu; Ulla M. Wewer; Eva Engvall



Mechanisms of Proliferation Synergy by Receptor Tyrosine Kinase and G Protein-Coupled Receptor Activation in Human Airway Smooth Muscle  

Microsoft Academic Search

Despite recent studies depicting the capacity of G protein-cou- pled receptors (GPCRs) to activate mitogenic signaling pathways more commonly associated with receptor tyrosine kinases (RTKs), little is known regarding the interactive effects of GPCR and RTK activation on cell growth and signal transduction. Such interactions likely mediate the physiologic growth in most cells in vivo as well as the aberrant,

Vera P. Krymskaya; Michael J. Orsini; Andrew J. Eszterhas; Kristin C. Brodbeck; Jeffrey L. Benovic; Reynold A. Panettieri; Raymond B. Penn



Acute 5-HT7 receptor activation increases NMDA-evoked currents and differentially alters NMDA receptor subunit phosphorylation and trafficking in hippocampal neurons  

PubMed Central

Background N-methyl-D-aspartate (NMDA) receptors are regulated by several G protein-coupled receptors (GPCRs) as well as receptor tyrosine kinases. Serotonin (5-HT) type 7 receptors are expressed throughout the brain including the thalamus and hippocampus. Long-term (2–24 h) activation of 5-HT7 receptors promotes the expression of neuroprotective growth factor receptors, including the platelet-derived growth factor (PDGF) ? receptors which can protect neurons against NMDA-induced neurotoxicity. Results In contrast to long-term activation of 5-HT7 receptors, acute (5 min) treatment of isolated hippocampal neurons with the 5-HT7 receptor agonist 5-carboxamidotryptamine (5-CT) enhances NMDA-evoked peak currents and this increase in peak currents is blocked by the 5-HT7 receptor antagonist, SB 269970. In hippocampal slices, acute 5-HT7 receptor activation increases NR1 NMDA receptor subunit phosphorylation and differentially alters the phosphorylation state of the NR2B and NR2A subunits. NMDA receptor subunit cell surface expression is also differentially altered by 5-HT7 receptor agonists: NR2B cell surface expression is decreased whereas NR1 and NR2A surface expression are not significantly altered. Conclusions In contrast to the negative regulatory effects of long-term activation of 5-HT7 receptors on NMDA receptor signaling, acute activation of 5-HT7 receptors promotes NMDA receptor activity. These findings highlight the potential for temporally differential regulation of NMDA receptors by the 5-HT7 receptor.



Activation of GABA-A Receptor Ameliorates Homocysteine-Induced MMP-9 Activation by ERK Pathway  

PubMed Central

Hyperhomocysteinemia (HHcy) is a risk factor for neuroinflammatory and neurodegenerative diseases. Homocysteine (Hcy) induces redox stress, in part, by activating matrix metalloproteinase-9 (MMP-9), which degrades the matrix and leads to blood–brain barrier dysfunction. Hcy competitively binds to ?-aminbutyric acid (GABA) receptors, which are excitatory neurotransmitter receptors. However, the role of GABA-A receptor in Hcy-induced cerebrovascular remodeling is not clear. We hypothesized that Hcy causes cerebrovascular remodeling by increasing redox stress and MMP-9 activity via the extracellular signal-regulated kinase (ERK) signaling pathway and by inhibition of GABA-A receptors, thus behaving as an inhibitory neurotransmitter. Hcy-induced reactive oxygen species production was detected using the fluorescent probe, 2?–7?-dichlorodihydrofluorescein diacetate. Hcy increased nicotinamide adenine dinucleotide phosphate-oxidase-4 concomitantly suppressing thioredoxin. Hcy caused activation of MMP-9, measured by gelatin zymography. The GABA-A receptor agonist, muscimol ameliorated the Hcy-mediated MMP-9 activation. In parallel, Hcy caused phosphorylation of ERK and selectively decreased levels of tissue inhibitors of metalloproteinase-4 (TIMP-4). Treatment of the endothelial cell with muscimol restored the levels of TIMP-4 to the levels in control group. Hcy induced expression of iNOS and decreased eNOS expression, which lead to a decreased NO bioavailability. Furthermore muscimol attenuated Hcy-induced MMP-9 via ERK signaling pathway. These results suggest that Hcy competes with GABA-A receptors, inducing the oxidative stress transduction pathway and leading to ERK activation.




The influence of laminin on the initial differentiation of cultured neural tube neurons.  


Portions of the metencephalic neural tube containing the trigeminal (V) motor nucleus from 40-hr chick embryos were excised and held freely floating in culture medium for 36-40 hr, so that neuronal generation within motor V could be completed, but precluding neuronal differentiation. The explants were then dissociated and plated either on 1) glass coverslips that had been coated with the extracellular matrix (ECM) glycoprotein, laminin, and subsequently irradiated to produce a grid pattern; or 2) coverslips, one-half of which had been coated with laminin, and the other one-half with collagen, another component of the ECM. The purpose of these studies was to assess possible laminin influences on neuronal adhesion and nerve fiber expression and extension during these periods of initial neuronal differentiation. The early neural tube neurons selectively adhered to the established laminin grid pattern; neuronal survival, elaboration of neurites, and extent of neurites were significantly enhanced on the laminin side of the laminin/collagen preparations. These latter effects were specifically blocked by the application of anti-laminin. In demonstrating these influences during stages of initial neuronal differentiation, the results support the hypothesis that laminin may play a role in normal neurogenesis, presumably by providing an adhesive surface for outgrowing growth cones. PMID:3367395

Heaton, M B; Swanson, D J



Pattern of laminin expression during kidney morphogenesis in Balb/c mice.  


Basement membrane of glomerular mesangium (BMG) is one of important components which play a key role to support of the capillary loops in a renal glomerulus and completeness of BMG due to interaction of ureteric bud and metanephric mesenchyme during glomerulogenesis. As laminin contribute in extra cellular matrix and especially in basement membrane, the aim of the present study was to demonstrate the distribution of this molecule so, in this investigation specific antibody against laminin have been used in light microscopy to study development of BMG of fetal and postnatal mouse glomerular mesangium. Female inbred Balb/c mice were selected and were kept under normal condition and finding vaginal plug was assumed as day zero of pregnancy. Two pregnant mice were sacrificed by cervical dislocation in one of gestational days 13-18, respectively and their fetuses were fixed, serially sectioned and by using antibody against laminin in BMG were carried out. The same process was used for kidneys preparation at 15 postnatal days. Present data revealed that laminin showed weak reaction on day 14 of gestation. The amount of laminin increased continuously until next days of fetal life and primary of 10 days postnatal in BMG. After this period, laminin reaction did not show significant change in newborns. These data indicate that laminin appears just during the glomerulogenesis and because of continuity with vasculature which is required for Extra Cellular Matrix (ECM) and glomerular endothelial cell differentiation, laminin, is the one of major structural proteins in BMG. PMID:21313920

Houshang, Rafighdost; Reza, Nikravesh Mohammad; Mehdi, Jalali



Laminin terminates the Netrin\\/DCC mediated attraction of vagal sensory axons  

Microsoft Academic Search

Vagal sensory axons navigate to spe- cific sites in the bowel during fetal life. Netrin\\/deleted in colorectal cancer (DCC) were found to mediate the attraction of vagal sensory axons to the fetal mouse gut. We tested the hypothesis that laminin-111 can reverse the chemoattractive effects of netrin and act as a stop signal for vagal sensory axons. Laminin-111- expressing cells

Elyanne M. Ratcliffe; Fabien D'Autréaux; Michael D. Gershon



Immunoreactivity for laminin in the developing ventral longitudinal pathway of the brain.  


The first long tract to form in the brain of a vertebrate embryo is the ventral longitudinal pathway. In order to investigate what chemical cues may guide nerve growth cones along this pathway, affinity-purified antibodies to laminin and collagen type IV were used to stain sections of mouse embryos from Embryonic Days 8 through 17. A monoclonal anti-neurofilament antibody was used to show the development of the ventral longitudinal pathway in relationship to immunoreactivity for laminin and collagen type IV. At Day 8 fluorescent immunoreactivity for laminin is bright in the external limiting membrane of the neural tube, but the neuroepithelium does not show bright laminin or neurofilament immunoreactivity. At E9 the ventral longitudinal pathway is forming and punctate immunoreactivity for laminin is present on the surfaces of neuroepithelial cells in the marginal zone, through which axons of the ventral pathway extend. Punctate immunofluorescence for laminin remains concentrated in the marginal zone on Days E10 through E14, but on E16 punctate immunofluorescence was much reduced, although immunoreactivity for laminin remained bright in the maturing pial and arachnoid membranes and on blood vessels in the brain. Immunoreactivity for collagen type IV was strong in the external limiting membrane and on blood vessels, but never showed concentrated punctate immunofluorescence in the marginal zone. These results indicate that laminin may be available on cell surfaces and in extracellular spaces as an adhesive ligand for growth cones during the formation of the ventral longitudinal pathway. PMID:3334714

Letourneau, P C; Madsen, A M; Palm, S L; Furcht, L T



The repair of brain lesion by implantation of hyaluronic acid hydrogels modified with laminin  

Microsoft Academic Search

The hyaluronic acid (HA) hydrogels modified with laminin were used for implantation in rat brain in present study, in order to investigate its effects in reparation of injury in the CNS. Cross-linked HA hydrogels were synthesized and their characteristics were analyzed. Laminin, an extracellular matrix protein, which participates in neuronal development and survival, was immobilized on the backbone of the

Shaoping Hou; Qunyuan Xu; Weiming Tian; Fuzhai Cui; Qing Cai; Jun Ma; In-Soup Lee



Essential and overlapping roles for laminin ? chains in notochord and blood vessel formation  

Microsoft Academic Search

Laminins are major constituents of basement membranes and have wide ranging functions during development and in the adult. They are a family of heterotrimeric molecules created through association of an ?, ? and ? chain. We previously reported that two zebrafish loci, grumpy (gup) and sleepy (sly), encode laminin ?1 and ?1, which are important both for notochord differentiation and

Steven M. Pollard; Michael J. Parsons; Makoto Kamei; Ross N. W. Kettleborough; Kevin A. Thomas; Van N. Pham; Moon-Kyoung Bae; Annabelle Scott; Brant M. Weinstein; Derek L. Stemple



Immunohistochemical localization of laminin and type IV collagen in human cutaneous sensory nerve formations  

Microsoft Academic Search

We used immunohistochemical techniques and monoclonal antibodies to localize two basement membrane components (laminin and type IV collagen) in the nerves and sensory nerve formations, or corpuscles, supplying human digital skin. Furthermore, neurofilament proteins, S-100 protein and epithelial membrane antigen were studied in parallel. In dermal nerve trunks, immunostaining for laminin and type IV collagen was found to be co-localized

J. A. Vega; I. Esteban; F. J. Naves; M. E. Valle; L. Malinovsky



Lung-specific loss of the laminin ?3 subunit confers resistance to mechanical injury  

PubMed Central

Laminins are heterotrimeric glycoproteins of the extracellular matrix that are secreted by epithelial cells and which are crucial for the normal structure and function of the basement membrane. We have generated a mouse harboring a conditional knockout of ?3 laminin (Lama3fl/fl), one of the main laminin subunits in the lung basement membrane. At 60 days after intratracheal treatment of adult Lama3fl/fl mice with an adenovirus encoding Cre recombinase (Ad-Cre), the protein abundance of ?3 laminin in whole lung homogenates was more than 50% lower than that in control-treated mice, suggesting a relatively long half-life for the protein in the lung. Upon exposure to an injurious ventilation strategy (tidal volume of 35 ml per kg of body weight for 2 hours), the mice with a knockdown of the ?3 laminin subunit had less severe injury, as shown by lung mechanics, histology, alveolar capillary permeability and survival when compared with Ad-Null-treated mice. Knockdown of the ?3 laminin subunit resulted in evidence of lung inflammation. However, this did not account for their resistance to mechanical ventilation. Rather, the loss of ?3 laminin was associated with a significant increase in the collagen content of the lungs. We conclude that the loss of ?3 laminin in the alveolar epithelium results in an increase in lung collagen, which confers resistance to mechanical injury.

Urich, Daniela; Eisenberg, Jessica L.; Hamill, Kevin J.; Takawira, Desire; Chiarella, Sergio E.; Soberanes, Saul; Gonzalez, Angel; Koentgen, Frank; Manghi, Tomas; Hopkinson, Susan B.; Misharin, Alexander V.; Perlman, Harris; Mutlu, Gokhan M.; Budinger, G. R. Scott; Jones, Jonathan C. R.



Laminin-apatite composite coating to enhance cell adhesion to ethylene-vinyl alcohol copolymer.  


A laminin-apatite composite layer with enhanced cell adhesive properties was successfully formed on the surface of an ethylene-vinyl alcohol copolymer (EVOH) by a liquid phase coating process. The coating process was carried out with the following procedure. First, an EVOH plate was alternately dipped in 200 mM calcium and 200 mM phosphate solutions to introduce nuclei or precursors of apatite to its surface. Second, the surface-modified EVOH was immersed in a calcium phosphate solution, which was supersaturated with respect to apatite and containing laminin. As a result of this procedure, a laminin-apatite composite layer with a thickness of 2.5-3.0 microm was formed on the EVOH surface. Epithelial-like cells (BSCC93) adhered to the laminin-apatite composite layer showed enhanced cell spreading, which was due to the biological effect of laminin. The number of cells adhered to the laminin-apatite composite layer on EVOH was approximately 10 times as large as that adhered to the surface of the untreated, laminin-adsorbed, or apatite-coated EVOH. Therefore, this type of composite material consisting of a synthetic polymer, apatite, and laminin has great potential as a skin terminal, with improved adhesiveness to skin tissue, as well as good biocompatibility. PMID:15578653

Oyane, Ayako; Uchida, Masaki; Ito, Atsuo



Peroxisome proliferator activated receptor-? and traumatic brain injury  

PubMed Central

Traumatic brain injury (TBI) represents a major health care problem and a significant socioeconomic challenge worldwide. No specific therapy for TBI is available. The peroxisome proliferator activated receptor-? (PPAR-?) belongs to the nuclear receptor superfamily. Although PPAR-? was originally characterized in adipose tissue as a regulator of lipid and glucose metabolism, recent studies showed that PPAR-? is present in most cell types and plays a central role in the regulation of adipogenesis, glucose homeostasis, cellular differentiation, apoptosis and inflammation. Here, we reviewed the current literature on the molecular mechanisms of PPAR-?-related neuroprotection after TBI. Growing evidence has indicated that the beneficial effects of PPAR-? activation in TBI appear to be mediated through downregulation of inflammatory responses, reduction of oxidative stress, inhibition of apoptosis, and promotion of neurogenesis. A thorough understanding of the PPAR-? pathway will be critical to the development of therapeutic interventions for the treatment of patients with TBI.

Qi, Lei; Jacob, Asha; Wang, Ping; Wu, Rongqian



Peroxisome Proliferator-Activated Receptors and Acute Lung Injury  

PubMed Central

Peroxisome proliferator-activated receptors are ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily. PPARs regulate several metabolic pathways by binding to sequence-specific PPAR response elements in the promoter region of target genes, including lipid biosynthesis and glucose metabolism. Recently, PPARs and their respective ligands have been implicated as regulators of cellular inflammatory and immune responses. These molecules are thought to exert anti-inflammatory effects by negatively regulating the expression of proinflammatory genes. Several studies have demonstrated that PPAR ligands possess anti-inflammatory properties and that these properties may prove helpful in the treatment of inflammatory diseases of the lung. This review will outline the anti-inflammatory effects of PPARs and PPAR ligands and discuss their potential therapeutic effects in animal models of inflammatory lung disease.

Paola, Rosanna Di; Cuzzocrea, Salvatore



Characterization of Peroxisome Proliferator-Activated Receptor ?--Independent Effects of PPAR? Activators in the Rodent Liver: Di-(2-ethylhexyl) phthalate also Activates the Constitutive-Activated Receptor  

PubMed Central

Peroxisome proliferator chemicals (PPC) are thought to mediate their effects in rodents on hepatocyte growth and liver cancer through the nuclear receptor peroxisome proliferator–activated receptor (PPAR) ?. Recent studies indicate that the plasticizer di-(2-ethylhexyl) phthalate (DEHP) increased the incidence of liver tumors in PPAR?-null mice. We hypothesized that some PPC, including DEHP, induce transcriptional changes independent of PPAR? but dependent on other nuclear receptors, including the constitutive-activated receptor (CAR) that mediates phenobarbital (PB) effects on hepatocyte growth and liver tumor induction. To determine the potential role of CAR in mediating effects of PPC, a meta-analysis was performed on transcript profiles from published studies in which rats and mice were exposed to PPC and compared the profiles to those produced by exposure to PB. Valproic acid, clofibrate, and DEHP in rat liver and DEHP in mouse liver induced genes, including Cyp2b family members that are known to be regulated by CAR. Examination of transcript changes by Affymetrix ST 1.0 arrays and reverse transcription-PCR in the livers of DEHP-treated wild-type, PPAR?-null, and CAR-null mice demonstrated that (1) most (?94%) of the transcriptional changes induced by DEHP were PPAR?-dependent, (2) many PPAR?-independent genes overlapped with those regulated by PB, (3) induction of genes Cyp2b10, Cyp3a11, and metallothionine-1 by DEHP was CAR dependent but PPAR?-independent, and (4) induction of a number of genes (Cyp8b1, Gstm4, and Gstm7) was independent of both CAR and PPAR?. Our results indicate that exposure to PPAR? activators including DEHP leads to activation of multiple nuclear receptors in the rodent liver.

Ren, Hongzu; Aleksunes, Lauren M.; Wood, Carmen; Vallanat, Beena; George, Michael H.; Klaassen, Curtis D.; Corton, J. Christopher



The insulin receptor changes conformation in unforeseen ways on ligand binding: Sharpening the picture of insulin receptor activation.  


Unraveling the molecular detail of insulin receptor activation has proved challenging, but a major advance is the recent determination of crystallographic structures of insulin in complex with its primary binding site on the receptor. The current model for insulin receptor activation is that two distinct surfaces of insulin monomer engage sequentially with two distinct binding sites on the extracellular surface of the insulin receptor, which is itself a disulfide-linked (??)2 homodimer. In the process, conformational changes occur both within the hormone and the receptor, the latter resulting in the disruption of the intracellular interactions that hold the kinase domains in their basal state and in the initiation of the phosphorylation events that drive insulin signaling. The purpose of this paper is to summarize the extant structural data relating to hormone binding and how it effects receptor activation, as well as to discuss the issues that remain unresolved. PMID:24037759

Ward, Colin W; Menting, John G; Lawrence, Michael C



Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators  

Microsoft Academic Search

We have cloned a member of the steroid hormone receptor superfamily of ligand-activated transcription factors. The receptor homologue is activated by a diverse class of rodent hepatocarcinogens that causes proliferation of peroxisomes. Identification of a peroxisome proliferator-activated receptor should help elucidate the mechanism of the hypolipidaemic effect of these hepatocarcinogens and aid evaluation of their potential carcinogenic risk to man.

Isabelle Issemann; Stephen Green



Peroxisome proliferator-activated receptors: a therapeutic target in COPD?  

Microsoft Academic Search

Extrapulmonary pathology significantly impairs clinical outcome in chronic obstructive pulmonary disease (COPD). The peroxisome proliferator-activated receptors (PPARs) are implicated in the regulation of several hallmarks of systemic COPD pathology, including cachexia, decreased oxidative muscle metabolism, oxidative stress and systemic inflammation. Recently, expression of PPARs and related cofactors was shown to be reduced in peripheral skeletal muscle of patients with moderate-to-severe

A. H. Remels; H. R. Gosker; P. Schrauwen; R. C. Langen; A. M. Schols



Progress in the understanding of protease-activated receptors  

Microsoft Academic Search

Thrombin results from the activation of the blood coagulation system. It is a multifunctional protein that has, besides its\\u000a function in hemostasis and thrombosis, several cellular effects that link the coagulation system with the inflammatory response.\\u000a Many years of investigations were necessary for the discovery of the first functional thrombin receptor, which was found to\\u000a have a unique mechanism of

Esteban C. Gabazza; Osamu Taguchi; Haruhito Kamada; Tatsuya Hayashi; Yukihiko Adachi; Koji Suzuki



Activation of synaptic receptors and its allosteric regulation  

Microsoft Academic Search

This review considers activation of synaptic receptors (SR) as a process of transduction of the energy of interaction between\\u000a an agonist (A) and an agonist-recognizing module (R) toward an ion-conducting module (channel,Ch) orG protein within respective complexes (ARCh orARG). The agonist provides functional and, in the case of metabotropic SR, spatial association of protein components in these\\u000a complexes. Conformational transformations

I. V. Komissarov



Repressive Effects of Resveratrol on Androgen Receptor Transcriptional Activity  

Microsoft Academic Search

BackgroundThe chemopreventive effects of resveratrol (RSV) on prostate cancer have been well established; the androgen receptor (AR) plays pivotal roles in prostatic tumorigenesis. However, the exact underlying molecular mechanisms about the effects of RSV on AR have not been fully elucidated. A model system is needed to determine whether and how RSV represses AR transcriptional activity.MethodologyThe AR cDNA was first

Wen-Feng Shi; Melanie Leong; Ellen Cho; Joseph Farrell; Han-Chun Chen; Jun Tian; Dianzheng Zhang; Mikhail V. Blagosklonny