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1

Laminin receptor activation inhibits endothelial tissue factor expression.  

PubMed

Tissue factor (TF) is an important trigger of arterial thrombosis. The green tea catechin epigallocatechin-3-gallate (EGCG) is a ligand of the 67-kDa laminin receptor (67LR) and exhibits cardioprotective effects. This study investigates whether 67LR regulates TF expression in human endothelial cells. Immunofluorescence demonstrated that human aortic endothelial cells expressed 67LR. Cells grown on laminin expressed 35% less TF in response to TNF-alpha (TNF-alpha) than those grown on fibronectin (n=6; p<0.001). EGCG (1-30 microM) inhibited TNF-alpha and histamine induced endothelial TF expression and activity in a concentration dependent manner resulting in 87% reduction of TF expression (n=5; p<0.001); in contrast, expression of tissue factor pathway inhibitor was not affected (n=4; p=NS). In vivo administration of EGCG (30 mg/kg/day) inhibited TF activity in carotid arteries of C57BL6 mice. Real-time PCR and promoter studies revealed that EGCG decreased TF expression at the transcriptional level and impaired activation of the mitogen activated protein (MAP) kinase JNK 1/2, but not ERK or p38. Similarly, the JNK 1/2 inhibitor SP600125 (1 microM) impaired TF promoter activity (n=4; p<0.001) and protein expression (n=4; p<0.001). 67LR blocking antibodies blunted the inhibitory effect of EGCG on both TF protein expression and JNK activation. In contrast, vascular cell adhesion molecule 1 (VCAM-1) was not affected by laminin nor EGCG, and its expression was not regulated by JNK. EGCG did not affect TNF-alpha stimulated NFkB activation. Laminin receptor activation inhibits endothelial TF expression by impairing JNK phosphorylation. Thus, 67LR may be a potential target for the development of novel anti-thrombotic therapies. PMID:19712679

Holy, Erik W; Stämpfli, Simon F; Akhmedov, Alexander; Holm, Niels; Camici, Giovanni G; Lüscher, Thomas F; Tanner, Felix C

2010-06-01

2

Structure-guided identification of a laminin binding site on the laminin receptor precursor.  

PubMed

The 37/ 67-kDa human laminin receptor (LamR) is a cell surface receptor for laminin, prion protein, and a variety of viruses. Because of its wide range of ligands, LamR plays a role in numerous pathologies. LamR overexpression correlates with a highly invasive cell phenotype and increased metastatic ability, mediated by interactions between LamR and laminin. In addition, the specific targeting of LamR with small interfering RNAs, blocking antibodies, and Sindbis viral vectors confers anti-tumor effects. We adopted a structure-based approach to map a laminin binding site on human LamR by comparing the sequences and crystal structures of LamR and Archaeoglobus fulgidus S2p, a non-laminin-binding ortholog. Here, we identify a laminin binding site on LamR, comprising residues Phe32, Glu35, and Arg155, which are conserved among mammalian species. Mutation of these residues results in a significant loss of laminin binding. Further, recombinant wild-type LamR is able to act as a soluble decoy to inhibit cellular migration towards laminin. Mutation of this laminin binding site results in loss of migration inhibition, which demonstrates the physiological role of Phe32, Glu35, and Arg155 for laminin binding activity. Mapping of the LamR binding site should contribute to the development of therapeutics that inhibit LamR interactions with laminin and may aid in the prevention of tumor growth and metastasis. PMID:21040730

Jamieson, Kelly V; Hubbard, Stevan R; Meruelo, Daniel

2011-01-01

3

Binding sites on laminin receptors as components for antibiotics.  

PubMed

Bacteria use the receptor-adhesion-like interaction between laminin and the laminin receptor in the process of infection. We determined that bacteria do not interact with the receptor-binding site on laminin which could be expected for the bacterial laminin receptor. Rather, binding occurs via the laminin-binding site on the 67-kDa laminin receptor, which has a function similar to the one the bacterial laminin receptor possesses. This finding has implications for the effective use of antimicrobial peptides. PMID:17266648

Kobayashi, Nahoko; Yoshida, Tetsuhiko

2007-01-01

4

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.

1985-07-01

5

Laminin  

PubMed Central

Laminin-1 is emerging as the key molecule in early embryonic basement membrane assembly. Here we review recent insights into its functions gained from the synergistic application of genetic and structural methods.

Sasaki, Takako; Fassler, Reinhard; Hohenester, Erhard

2004-01-01

6

Laminin receptor on platelets is the integrin VLA6  

Microsoft Academic Search

Adhesion of platelets to the subendothelial matrix of an injured vessel wall is an essential step in triggering the formation of a haemostatic plug. Fibronectin, collagen and laminin are three major components of the subendothelial matrix which support platelet adhesion1. Receptors for fibronectin and collagen have been identified on platelets and are included in the integrin family2-11. Here we report

Arnoud Sonnenberg; Piet W. Modderman; Frans Hogervorst

1988-01-01

7

Mechanistic distinctions between agrin and laminin-1 induced aggregation of acetylcholine receptors  

PubMed Central

Background One of the earliest steps in synaptogenesis at the neuromuscular junction is the aggregation of nicotinic acetylcholine receptors at the postsynaptic membrane. This study presents quantitative analyses of receptor and ?-Dystroglycan aggregation in response to agrin and laminin-1, alone or in combination. Results Both laminin and agrin increased overall expression of receptors on the plasma membrane. Following a 24 hour exposure, agrin increased the number of receptor aggregates but did not affect the number of ?-Dystroglycan aggregates, while the reverse was true of laminin-1. Laminin also increased receptor concentration within aggregates, while agrin had no such effect. Finally, the spatial distribution of aggregates was indistinguishable from random in the case of laminin, while agrin induced aggregates were closer together than predicted by a random model. Conclusions Agrin and laminin-1 both increase acetylcholine receptor aggregate size after 24 hours, but several lines of evidence indicate that this is achieved via different mechanisms. Agrin and laminin had different effects on the number and density of receptor and ?-Dystroglycan aggregates. Moreover the random distribution of laminin induced (as opposed to agrin induced) receptor aggregates suggests that the former may influence aggregate size by simple mass action effects due to increased receptor expression.

Lee, Lara K; Kunkel, Dennis D; Stollberg, Jes

2002-01-01

8

The Human Integrin VLA-2 is a Collagen Receptor on Some Cells and a Collagen/Laminin Receptor on Others  

NASA Astrophysics Data System (ADS)

The integrin heterodimer VLA-2, previously known as a collagen receptor, is now shown also to be a laminin receptor. Adhesion of the human melanoma cell line LOX to laminin was inhibited by anti-VLA ? 2 antibodies. Because VLA-2-mediated LOX cell attachment to laminin was not inhibited by digestion with collagenase, collagen contamination of laminin was not a factor. In addition, VLA-2 from LOX cells bound to immobilized laminin, and binding was disrupted by EDTA but not by Arg-Gly-Asp (RGD) peptides. VLA-3 also bound to laminin-Sepharose, although less avidly than VLA-2. Thus, at least four separate members of the integrin ? 1 subfamily serve as laminin receptors--i.e., VLA-2 and VLA-3 (this study) together with VLA-1 and VLA-6 (other reports). Whereas LOX and other cell lines used VLA-2 as both a laminin and collagen receptor, fibroblast VLA-2 mediated collagen but not laminin binding. Likewise, VLA-2 from platelets did not interact with laminin. Despite this functional discordancy, VLA-2 from laminin-binding and nonbinding sources was indistinguishable by all immunochemical and biochemical criteria examined. Thus, functional differences in VLA-2 may be due to cell type-specific modulation.

Elices, Mariano J.; Hemler, Martin E.

1989-12-01

9

Development of laminin receptor agonists: identification of important functional residues by alanine scanning.  

PubMed

An antagonist of cellular adhesion and motility, acetyl-C-[S-Acm]-VIGYSGDRC-[S-Acm]-NH(2) (mEGF(33-42)), shares homology with the agonist sequence CDPGYIGSR-NH(2). It has been proposed that the latter peptide binds to the high affinity 67 kDa laminin receptor. Both peptides have equal affinities for the receptor and similar conformations have been derived for both. We have examined the importance of individual non-homologous residues with respect to receptor binding and antagonistic properties of mEGF(33-42). Alanine scanning of non-conserved residues in the N-terminal half of mEGF(33-42) caused loss of biological activity with respect to cell attachment, receptor binding and migratory response. Substitution of alanine for serine (position 6) caused loss of laminin-specific cell attachment and receptor binding activities. However, the peptide did stimulate migration suggesting that this peptide may be a non-specific stimulator of migration. In contrast, alanine substitution for the C-terminal Cys-S-Acm had no apparent effect on the attachment or receptor binding activities of the peptide but generated an agonist from the antagonist parent. Comparison of the modelled folds of the alanine containing peptides revealed the presence of significant helical content in those peptides capable of stimulating migration and suggests that a reduction in bulk in the N-terminal residues is not conducive to adopting a productive binding conformation. PMID:10962089

Scott, W N; McFerran, N V; Harriott, P; Walker, B; Nelson, J

2000-08-31

10

Identification of oncofetal antigen/immature laminin receptor protein epitopes that activate BALB/c mouse OFA/iLRP-specific effector and regulatory T cell clones.  

PubMed

During tumor development in mice and humans, oncofetal Ag/immature laminin receptor (OFA/iLRP)-specific Th1, CTL, and IL-10-secreting T (Ts) cells are induced. The presence of too many Ts or too few effector T cells appears to predict a poor prognosis. We established clones of OFA/iLRP-specific splenic Th1, CTL, and Ts cells from the OFA/iLRP+ MCA1315 fibrosarcoma-bearing BALB/c mice or from BALB/c mice vaccinated with 1 or 10 microg of rOFA/iLRP. The MCA1315 tumor cell-reactive T cell clones were characterized as to surface Ag phenotype, cytokine secretion profile, and specificity for OFA/iLRP presented by syngeneic splenic APC. OFA/iLRP-specific Th1 and Ts clones were established from all mice. OFA/iLRP-specific CTL could be established from all mice except for mice immunized with 10 microg of rOFA/iLRP. Analysis of the proliferation profile of the OFA/iLRP-specific clones to overlapping OFA/iLRP 12-mer peptides that spanned the OFA/iLRP protein sequence defined the epitopes to which the T cell clones responded. There was a similar spatial distribution of the epitopes to which the two types of CD8 T cell clones responded. The nonapeptide epitopes of the Ts clones were located between aa 36 and 147 of OFA/iLRP, while the epitopes of the CTL clones were located between aa 52 and 163. Even though the CTL and Ts epitopes shared part of the protein, all of the CD8 CTL epitopes were distinct and separable from those of CD8 Ts cells. PMID:16493041

Rohrer, James W; Barsoum, Adel L; Coggin, Joseph H

2006-03-01

11

Differential expression of laminin chains and their integrin receptors in human gastric mucosa.  

PubMed Central

The proliferating cells of the gastric mucosa are found among the pit and mucous neck cells. These cells migrate upward to renew the surface epithelium and downward to restitute the glandular cells. As the epithelial basement membranes (BMs) function as substrate for cell adhesion and migration as well as signals for their differentiation, we studied, by indirect immunofluorescence microscopy, the distribution of different laminin chains and their integrin receptors in adult human stomach. The immunoreactivity for laminin alpha 2 chain localized to the BMs of glands and the lower parts of the gastric pits whereas the laminin alpha 3 chain (laminin-5/kalinin) immunoreactivity was strictly confined to BMs underneath the surface epithelium and the upper parts of the pits. Proliferating mucosal epithelial cells, identified by Ki-67 antibodies, were confined to the areas containing both alpha 2 and alpha 3 laminin chains. The alpha 1, beta 1, and gamma 1 laminin chains were found in all BMs of the mucosa whereas the beta 2 chain was prominent in mucosal blood vessels and also detectable in some glands. Among the laminin integrin receptors, the alpha 3 and beta 4 subunits were seen to be expressed in cells along the BMs with the alpha 3 laminin chain. The alpha 6 integrin, on the other hand, was seen in all gastric epithelia. The present results demonstrate that in the adult human stomach laminin alpha 2 and alpha 3 chains show zonal distribution in BM underlying gastric mucosal epithelium whereas other laminin chains show a more general distribution. Images Figure 1 Figure 2 Figure 3 Figure 4

Virtanen, I.; Tani, T.; Back, N.; Happola, O.; Laitinen, L.; Kiviluoto, T.; Salo, J.; Burgeson, R. E.; Lehto, V. P.; Kivilaakso, E.

1995-01-01

12

Role of the 37 kDa laminin receptor precursor in the life cycle of prions.  

PubMed

Prions are thought to consist of infectious proteins that cause, in the absence of detectable nucleic acid, a group of fatal neurodegenerative diseases, called transmissible spongiform encephalopathies (TSE). Among these diseases are bovine spongiform encephalopathy (BSE), scrapie of sheep and Creutzfeldt-Jakob disease (CJD) in humans. They occur as sporadic, infectious or genetic disorders and have in common the accumulation of an abnormal, pathogenic isoform of the cellular prion protein PrPc which is converted in a post-translational process into PrPSc concomitant with conformational changes of the protein. During this process PrPc acquires a high beta-sheet content and becomes partially resistant to proteases. The mechanism of this conversion as well as the physiological function of the cellular prion protein PrPc are poorly understood, but studies employing PrP knock-out mice demonstrated that PrPc is required for the development of prion diseases. The involvement of co-factors such as chaperones, receptors or an unknown protein, designated "protein X" in the conversion process are discussed. In a yeast two-hybrid screen we have identified the 37 kDa laminin receptor precursor (LRP) as an interactor of the cellular prion protein and this interaction could be confirmed by co-infection and co-transfection studies in mammalian and insect cells. LRP evolved from the ribosomal protein p40 essential for protein synthesis lacking any laminin binding activity to a cell surface receptor binding laminin, elastin and carbohydrates. The gene encoding 37 kDa LRP/p40 has been identified in a variety of species including the sea urchin Urechis caupo, Chlorohydra viridissima, the archaebacterium Haloarcula marismortui, the yeast Saccharomyces cerevisiae as well as in mammals where it is highly conserved. LRP works as a receptor for alphaviruses and is associated with the metastatic potential of solid tumors where it was first identified. The 37 kDa LRP forms its mature 67 kDa isoform with high laminin binding capacity by an unknown mechanism involving acylation. The multifunctionality of LRP as a ribosomal protein and a cell surface receptor for infectious agents such as viruses and prions might be extended by additional properties. PMID:10188208

Rieger, R; Lasmézas, C I; Weiss, S

1999-02-01

13

Laminin stimulates spreading of platelets through integrin ?6?1-dependent activation of GPVI  

PubMed Central

The extracellular matrix protein, laminin, supports platelet adhesion through binding to integrin ?6?1 In the present study, we demonstrate that human laminin, purified from placenta, also stimulates formation of filopodia and lamellipodia in human and mouse platelets through a pathway that is dependent on ?6?1 and the collagen receptor GPVI. The integrin ?6?1 is essential for adhesion to laminin, as demonstrated using an ?6-blocking antibody, whereas GPVI is dispensable for this response, as shown using “knockout” mouse platelets. On the other hand, lamellipodia formation on laminin is completely inhibited in the absence of GPVI, although filopodia formation remains and is presumably mediated via ?6?1 Lamellipodia and filopodia formation are inhibited in Syk-deficient platelets, demonstrating a key role for the kinase in signaling downstream of GPVI and integrin ?6?1 GPVI was confirmed as a receptor for laminin using surface plasmon resonance spectroscopy and by demonstration of lamellipodia formation on laminin in the presence of collagenase. These results identify GPVI as a novel receptor for laminin and support a model in which integrin ?6?1 brings laminin to GPVI, which in turn mediates lamellipodia formation. We speculate that laminin contributes to platelet spreading in vivo through a direct interaction with GPVI.

Inoue, Osamu; Suzuki-Inoue, Katsue; McCarty, Owen J. T.; Moroi, Masaaki; Ruggeri, Zaverio M.; Kunicki, Thomas J.; Ozaki, Yukio; Watson, Steve P.

2006-01-01

14

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

2001-01-01

15

Chemical inhibition of prometastatic lysyl-tRNA synthetase-laminin receptor interaction  

PubMed Central

Lysyl-tRNA synthetase (KRS), a protein synthesis enzyme in the cytosol, relocates to the plasma membrane after a laminin signal and stabilizes a 67-kDa laminin receptor (67LR) that is implicated in cancer metastasis; however, its potential as an antimetastatic therapeutic target has not been explored. We found that the small compound BC-K-YH16899, which binds to KRS, impinged on interaction of KRS with 67LR and suppressed metastasis in 3 different mouse models. The compound inhibited KRS–67LR interaction in two ways. First, it directly blocked the association between KRS and 67LR. Second, it suppressed the dynamic movement of the N-terminal extension of KRS and reduced membrane localization of KRS. However, it did not affect the catalytic activity of KRS. Our results suggest that specific modulation of a cancer-related KRS–67LR interaction may offer a way to control metastasis while avoiding the toxicities associated with inhibition of the normal functions of KRS.

Kim, Dae Gyu; Lee, Jin Young; Kwon, Nam Hoon; Fang, Pengfei; Zhang, Qian; Wang, Jing; Young, Nicolas L.; Guo, Min; Cho, Hye Young; Mushtaq, AmeeqUl; Jeon, Young Ho; Choi, Jin Woo; Han, Jung Min; Kang, Ho Woong; Joo, Jae Eun; Hur, Youn; Kang, Wonyoung; Yang, Heekyoung; Nam, Do-Hyun; Lee, Mi-Sook; Lee, Jung Weon; Kim, Eun-Sook; Moon, Aree; Kim, Kibom; Kim, Doyeun; Kang, Eun Joo; Moon, Youngji; Rhee, Kyung Hee; Han, Byung Woo; Yang, Jee Sun; Han, Gyoonhee; Yang, Won Suk; Lee, Cheolju; Wang, Ming-Wei; Kim, Sunghoon

2014-01-01

16

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

2012-01-01

17

Retinoic acid enhances adhesiveness, laminin and integrin beta 1 synthesis, and retinoic acid receptor expression in F9 teratocarcinoma cells.  

PubMed

The teratocarcinoma-derived F9 cells respond to retinoic acid (RA) and RA plus dibutyrylcyclic adenosine monophosphate (dcAMP) by differentiating into endoderm cells, which elaborate a laminin and type IV collagen-rich matrix. We found that the induction of differentiation is accompanied by a small but consistent increase in cell adhesiveness to a variety of substrates, including laminin. Therefore we investigated biochemical mechanisms involved in this phenomenon. Endoglycosidase treatment showed that laminin contains complex and hybrid oligosaccharide structures. RA enhanced general biosynthesis of laminin without a specific increase in galactose incorporation: this sugar was mainly in polylactosamine structures in the A chain of laminin and as terminal galactose alpha 1,3 galactose in the B chain. Laminin receptor analysis showed that RA decreased laminin binding protein-37 (LBP-37) but increased the amount of beta 1 integrin, suggesting the involvement of beta 1 integrin in the attachment process. Northern blot analysis showed increased expression of retinoid receptors within hours of RA exposure. These studies demonstrate that RA increases cell to substrate interactions by increasing the biosynthesis of laminin and beta 1 integrin. These effects are most likely subsequent to the RA-induced biosynthesis of the retinoid receptors. PMID:7512975

Ross, S A; Ahrens, R A; De Luca, L M

1994-05-01

18

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

2010-01-01

19

Biological Activities of the Homologous Loop Regions in the Laminin ? Chain LG Modules.  

PubMed

Each laminin ? chain (?1-?5 chains) has chain-specific diverse biological functions. The C-terminal globular domain of the ? chain consists of five laminin-like globular (LG1-5) modules and plays a critical role in biological activities. The LG modules consist of a 14-stranded ?-sheet (A-N) sandwich structure. Previously, we described the chain-specific biological activities of the loop regions between the E and F strands in the LG4 modules using five homologous peptides (G4EF1-G4EF5). Here, we further analyze the biological activities of the E-F strands loop regions in the rest of LG modules. We designed 20 homologous peptides (approximately 20 amino acid length), and 17 soluble peptides were used for the cell attachment assay. Thirteen peptides promoted cell attachment activity with different cell morphologies. Cell attachment to peptides G1EF1, G1EF2, G2EF1, G3EF4, and G5EF4 was inhibited by heparin, and peptides G1EF1, G1EF2, and G2EF1 specifically bound to syndecan-overexpressing cells. Cell attachment to peptides G2EF3, G3EF1, G3EF3, G5EF1, G5EF3, and G5EF5 was inhibited EDTA. Further, cell attachment to peptides G3EF3, G5EF1, and G5EF5 was inhibited by both anti-integrin ?2 and ?1 antibodies, whereas cell attachment to peptide G5EF3 was inhibited by only anti-integrin ?1 antibody. Cell attachment to peptides G1EF4, G3EF4, and G5EF4 was inhibited by both heparin and EDTA and was not inhibited by anti-integrin antibodies. The active peptide sequence alignments suggest that the syndecan-binding peptides contain a "basic amino acid (BAA)-Gly-BAA" motif in the middle of the molecule and that the integrin-binding peptides contain an "acidic amino acid (AAA)"-Gly-BAA motif. Core-switched peptide analyses suggested that the "BAA-Gly-BAA" motif is critical for binding to syndecans and that the "AAA-Gly-BAA" motif has potential to recognize integrins. These findings are useful for understanding chain-specific biological activities of laminins and to evaluate receptor-specific binding mechanisms. PMID:24850085

Katagiri, Fumihiko; Hara, Toshihiro; Yamada, Yuji; Urushibata, Shunsuke; Hozumi, Kentaro; Kikkawa, Yamato; Nomizu, Motoyoshi

2014-06-10

20

The major laminin receptor of mouse embryonic stem cells is a novel isoform of the alpha 6 beta 1 integrin  

PubMed Central

Laminin is the first extracellular matrix protein expressed in the developing mouse embryo. It is known to influence morphogenesis and affect cell migration and polarization. Several laminin receptors are included in the integrin family of extracellular matrix receptors. Ligand binding by integrin heterodimers results in signal transduction events controlling cell motility. We report that the major laminin receptor on murine embryonic stem (ES) cells is the integrin heterodimer alpha 6 beta 1, an important receptor for laminin in neurons, lymphocytes, macrophages, fibroblasts, platelets and other cell types. However, the cytoplasmic domain of the ES cell alpha 6 (alpha 6 B) differs totally from the reported cytoplasmic domain amino acid sequence of alpha 6 (alpha 6 A). Comparisons of alpha 6 cDNAs from ES cells and other cells suggest that the alpha 6 A and alpha 6 B cytoplasmic domains derive from alternative mRNA splicing. Anti-peptide antibodies to alpha 6 A are unreactive with ES cells, but react with mouse melanoma cells and embryonic fibroblasts. When ES cells are cultured under conditions that permit their differentiation, they become positive for alpha 6 A, concurrent with the morphologic appearance of differentiated cell types. Thus, expression of the alpha 6 B beta 1 laminin receptor may be favored in undifferentiated, totipotent cells, while the expression of alpha 6 A beta 1 receptor occurs in committed lineages. While the functions of integrin alpha chain cytoplasmic domains are not understood, it is possible that they contribute to transferring signals to the cell interior, e.g., by delivering cytoskeleton organizing signals in response to integrin engagement with extracellular matrix ligands. It is therefore reasonable to propose that the cellular responses to laminin may vary, according to what alpha subunit isoform (alpha 6 A or alpha 6 B) is expressed as part of the alpha 6 beta 1 laminin receptor. The switch from alpha 6 B to alpha 6 A, if confirmed in early embryos, could then be of striking potential relevance to the developmental role of laminin.

1991-01-01

21

P-cadherin signals through the laminin receptor ?6?4 integrin to induce stem cell and invasive properties in basal-like breast cancer cells.  

PubMed

P-cadherin is a classical cell-cell adhesion molecule that, in contrast to E-cadherin, has a positive role in breast cancer progression, being considered a poor prognostic factor in this disease. In previous reports, we have shown that this protein induces cancer stem cell and invasive properties to basal-like breast cancer cells. Here, we clarify the downstream signaling pathways that are triggered by P-cadherin to mediate these effects. We demonstrated that P-cadherin inhibition led to a significant decreased adhesion of cancer cells to the basement membrane substrate laminin, as well as to a major reduction in the expression of the laminin receptor ?6?4 integrin. Remarkably, the expression of this heterodimer was required for the invasive capacity and increased mammosphere forming efficiency induced by P-cadherin expression. Moreover, we showed that P-cadherin transcriptionally up-regulates the ?6 integrin subunit expression and directly interacts with the ?4 integrin subunit. We still showed that P-cadherin downstream signaling, in response to laminin, involves the activation of focal adhesion (FAK), Src and AKT kinases. The association between the expression of P-cadherin, ?6?4 heterodimer and the active FAK and Src phosphorylated forms was validated in vivo. Our data establish that there is a crosstalk between P-cadherin and the laminin receptor ?6?4 integrin signaling pathway, which link has never been previously described. The activation of this heterodimer explains the stem cell and invasive properties induced by P-cadherin to breast cancer cells, pointing to a new molecular mechanism that may be targeted to counteract the effects induced by this adhesion molecule. PMID:24553076

Vieira, André Filipe; Ribeiro, Ana Sofia; Dionísio, Maria Rita; Sousa, Bárbara; Nobre, Ana Rita; Albergaria, André; Santiago-Gómez, Angélica; Mendes, Nuno; Gerhard, Renê; Schmitt, Fernando; Clarke, Robert B; Paredes, Joana

2014-02-15

22

Ligand affinity of the 67-kD elastin/laminin binding protein is modulated by the protein's lectin domain: visualization of elastin/laminin-receptor complexes with gold-tagged ligands  

PubMed Central

Video-enhanced microscopy was used to examine the interaction of elastin- or laminin-coated gold particles with elastin binding proteins on the surface of live cells. By visualizing the binding events in real time, it was possible to determine the specificity and avidity of ligand binding as well as to analyze the motion of the receptor-ligand complex in the plane of the plasma membrane. Although it was difficult to interpret the rates of binding and release rigorously because of the possibility for multiple interactions between particles and the cell surface, relative changes in binding have revealed important aspects of the regulation of affinity of ligand-receptor interaction in situ. Both elastin and laminin were found to compete for binding to the cell surface and lactose dramatically decreased the affinity of the receptor(s) for both elastin and laminin. These findings were supported by in vitro studies of the detergent-solubilized receptor. Further, immobilization of the ligand-receptor complexes through binding to the cytoskeleton dramatically decreased the ability of bound particles to leave the receptor. The changes in the kinetics of ligand-coated gold binding to living cells suggest that both laminin and elastin binding is inhibited by lactose and that attachment of receptor to the cytoskeleton increases its affinity for the ligand.

1991-01-01

23

Laminin5 Is a Marker of Invading Cancer Cells in Some Human Carcinomas and Is Coexpressed with the Receptor for Urokinase Plasminogen Activator in Budding Cancer Cells in Colon Adenocarcinomas1  

Microsoft Academic Search

Recombinant human y 2 chain of laminin-5 was expressed in Esche- ni hut coli, and used to generate specific polyclonal antibodies which were used to study the distribution of the protein in human cancers. A total of 72 biopsies of human cancers were stained, including 23 cases of colon adenocarcinomas, 16 ductal breast carcinomas, 9 malignant melanomas, 14 squamous cell

Charles Pyke; Sirpa Salo; Karl Tryggvason

1995-01-01

24

Structural and functional analysis of the ovine laminin receptor gene ( RPSA ): Possible involvement of the LRP\\/LR protein in scrapie response  

Microsoft Academic Search

Scrapie is a prion disease affecting sheep and goats. Susceptibility to this neurodegenerative disease shows polygenic variance.\\u000a The involvement of the laminin receptor (LRP\\/LR) in the metabolism and propagation of prions has previously been demonstrated.\\u000a In the present work, the ovine laminin receptor gene (RPSA) was isolated, characterized, and mapped to ovine chromosome OAR19q13. Real-time RT-PCR revealed a significant decrease

Ane Marcos-Carcavilla; Jorge H. Calvo; Carmen González; Carmen Serrano; Katayoun Moazami-Goudarzi; Pascal Laurent; Maud Bertaud; Hélène Hayes; Anne E. Beattie; Jaber Lyahyai; Inmaculada Martín-Burriel; Juan María Torres; Magdalena Serrano

2008-01-01

25

Molecular cloning and characterization of a highly conserved human 67-kDa laminin receptor pseudogene mapping to Xq21.3  

Microsoft Academic Search

A highly conserved laminin receptor processed pseudogene (LAMRL5) that has been isolated from a fetal brain cDNA library is described. The pseudogene is a complete copy (97.9% identical) of the transcribed laminin receptor (LAMR1) with all the introns precisely removed. The sequence has direct repeats of 18bp at either end. It has an 885 nucleotide open reading frame from the

Michael P Richardson; Claire Braybrook; Muly Tham; Gudrun E Moore; Philip Stanier

1998-01-01

26

Laminin chain expression suggests that laminin-10 is a major isoform in the mouse hippocampus and is degraded by the tissue plasminogen activator/plasmin protease cascade during excitotoxic injury.  

PubMed

Laminins are important components of the extracellular matrix, and participate in neuronal development, survival and regeneration. The tissue plasminogen activator/plasmin extracellular protease cascade and downstream laminin degradation are implicated in excitotoxin-induced neuronal degeneration. To determine which specific laminin chains are involved, we investigated the expression of laminins in the hippocampus, and the cell types expressing them. Reverse transcription-PCR demonstrated that the messenger RNAs for all laminin chains could be detected in the hippocampus. To determine the localization of laminin chain expression, immunostaining was used. This method showed that alpha5, beta1 and gamma1 are most highly expressed in the neuronal cell layers. Immunoblotting confirmed the hippocampal expression of the chains alpha5, beta1 and gamma1, and RNA in situ hybridization showed a neuronal expression pattern of alpha5, beta1 and gamma1. At early time points following intrahippocampal injection of kainate, alpha5, beta1 and gamma1 chain immunoreactivities were lost. In addition, tissue plasminogen activator-deficient mice, which are resistant to kainate-induced neuronal death, show no significant change in laminins alpha5, beta1 and gamma1 after intrahippocampal kainate injection. Taken together, these results suggest that laminin-10 (alpha5-beta1-gamma1) comprises a major neuronal laminin in the mouse hippocampus, and is degraded before neuronal death during excitotoxic injury by the tissue plasminogen activator/plasmin protease cascade. By identifying a neuronal laminin (laminin-10) that participates in neuronal degeneration after excitotoxic injury, this study clarifies the molecular definition of the extracellular matrix in the hippocampus and further defines a pathway for mechanisms of neuronal death. PMID:12559092

Indyk, J A; Chen, Z L; Tsirka, S E; Strickland, S

2003-01-01

27

Laminin ?2-Mediated Focal Adhesion Kinase Activation Triggers Alport Glomerular Pathogenesis  

PubMed Central

It has been known for some time that laminins containing ?1 and ?2 chains, which are normally restricted to the mesangial matrix, accumulate in the glomerular basement membranes (GBM) of Alport mice, dogs, and humans. We show that laminins containing the ?2 chain, but not those containing the ?1 chain activates focal adhesion kinase (FAK) on glomerular podocytes in vitro and in vivo. CD151-null mice, which have weakened podocyte adhesion to the GBM rendering these mice more susceptible to biomechanical strain in the glomerulus, also show progressive accumulation of ?2 laminins in the GBM, and podocyte FAK activation. Analysis of glomerular mRNA from both models demonstrates significant induction of MMP-9, MMP-10, MMP-12, MMPs linked to GBM destruction in Alport disease models, as well as the pro-inflammatory cytokine IL-6. SiRNA knockdown of FAK in cultured podocytes significantly reduced expression of MMP-9, MMP-10 and IL-6, but not MMP-12. Treatment of Alport mice with TAE226, a small molecule inhibitor of FAK activation, ameliorated fibrosis and glomerulosclerosis, significantly reduced proteinuria and blood urea nitrogen levels, and partially restored GBM ultrastructure. Glomerular expression of MMP-9, MMP-10 and MMP-12 mRNAs was significantly reduced in TAE226 treated animals. Collectively, this work identifies laminin ?2-mediated FAK activation in podocytes as an important early event in Alport glomerular pathogenesis and suggests that FAK inhibitors, if safe formulations can be developed, might be employed as a novel therapeutic approach for treating Alport renal disease in its early stages.

Delimont, Duane; Dufek, Brianna M.; Meehan, Daniel T.; Zallocchi, Marisa; Gratton, Michael Anne; Phillips, Grady; Cosgrove, Dominic

2014-01-01

28

Laminin ?2-Mediated Focal Adhesion Kinase Activation Triggers Alport Glomerular Pathogenesis.  

PubMed

It has been known for some time that laminins containing ?1 and ?2 chains, which are normally restricted to the mesangial matrix, accumulate in the glomerular basement membranes (GBM) of Alport mice, dogs, and humans. We show that laminins containing the ?2 chain, but not those containing the ?1 chain activates focal adhesion kinase (FAK) on glomerular podocytes in vitro and in vivo. CD151-null mice, which have weakened podocyte adhesion to the GBM rendering these mice more susceptible to biomechanical strain in the glomerulus, also show progressive accumulation of ?2 laminins in the GBM, and podocyte FAK activation. Analysis of glomerular mRNA from both models demonstrates significant induction of MMP-9, MMP-10, MMP-12, MMPs linked to GBM destruction in Alport disease models, as well as the pro-inflammatory cytokine IL-6. SiRNA knockdown of FAK in cultured podocytes significantly reduced expression of MMP-9, MMP-10 and IL-6, but not MMP-12. Treatment of Alport mice with TAE226, a small molecule inhibitor of FAK activation, ameliorated fibrosis and glomerulosclerosis, significantly reduced proteinuria and blood urea nitrogen levels, and partially restored GBM ultrastructure. Glomerular expression of MMP-9, MMP-10 and MMP-12 mRNAs was significantly reduced in TAE226 treated animals. Collectively, this work identifies laminin ?2-mediated FAK activation in podocytes as an important early event in Alport glomerular pathogenesis and suggests that FAK inhibitors, if safe formulations can be developed, might be employed as a novel therapeutic approach for treating Alport renal disease in its early stages. PMID:24915008

Delimont, Duane; Dufek, Brianna M; Meehan, Daniel T; Zallocchi, Marisa; Gratton, Michael Anne; Phillips, Grady; Cosgrove, Dominic

2014-01-01

29

Evidence for the Participation of the Neuron-Specific CDK5 Activator P35 during Laminin-Enhanced Axonal Growth  

Microsoft Academic Search

Cultures of cerebellar macroneurons were used to study the pattern of expression, subcellular localization, and function of the neuronal cdk5 activator p35 during laminin-enhanced ax- onal growth. The results obtained indicate that laminin, an extracellular matrix molecule capable of selectively stimulating axonal extension and promoting MAP1B phosphorylation at a proline-directed protein kinase epitope, selectively stimulates p35 expression, increases its association

Gabriela Paglini; Gustavo Pigino; Patricia Kunda; Gerardo Morfini; Ricardo Maccioni; Santiago Quiroga; Adriana Ferreira; Alfredo Caceres

1998-01-01

30

The 37kDa/67kDa Laminin Receptor acts as a receptor for A?42 internalization.  

PubMed

Neuronal loss is a major neuropathological hallmark of Alzheimer's disease (AD). The associations between soluble A? oligomers and cellular components cause this neurotoxicity. The 37?kDa/67?kDa laminin receptor (LRP/LR) has recently been implicated in A? pathogenesis. In this study the mechanism underlying the pathological role of LRP/LR was elucidated. Försters Resonance Energy Transfer (FRET) revealed that LRP/LR and A? form a biologically relevant interaction. The ability of LRP/LR to form stable associations with endogenously shed A? was confirmed by pull down assays and A?-ELISAs. Antibody blockade of this association significantly lowered A?42 induced apoptosis. Furthermore, antibody blockade and shRNA mediated downregulation of LRP/LR significantly hampered A?42 internalization. These results suggest that LRP/LR is a receptor for A?42 internalization, mediating its endocytosis and contributing to the cytotoxicity of the neuropeptide by facilitating intra-cellular A?42 accumulation. These findings recommend anti-LRP/LR specific antibodies and shRNAs as potential therapeutic tools for AD treatment. PMID:24990253

Da Costa Dias, Bianca; Jovanovic, Katarina; Gonsalves, Danielle; Moodley, Kiashanee; Reusch, Uwe; Knackmuss, Stefan; Weinberg, Marc S; Little, Melvyn; Weiss, Stefan F T

2014-01-01

31

The 37kDa/67kDa Laminin Receptor acts as a receptor for A?42 internalization  

PubMed Central

Neuronal loss is a major neuropathological hallmark of Alzheimer's disease (AD). The associations between soluble A? oligomers and cellular components cause this neurotoxicity. The 37?kDa/67?kDa laminin receptor (LRP/LR) has recently been implicated in A? pathogenesis. In this study the mechanism underlying the pathological role of LRP/LR was elucidated. Försters Resonance Energy Transfer (FRET) revealed that LRP/LR and A? form a biologically relevant interaction. The ability of LRP/LR to form stable associations with endogenously shed A? was confirmed by pull down assays and A?-ELISAs. Antibody blockade of this association significantly lowered A?42 induced apoptosis. Furthermore, antibody blockade and shRNA mediated downregulation of LRP/LR significantly hampered A?42 internalization. These results suggest that LRP/LR is a receptor for A?42 internalization, mediating its endocytosis and contributing to the cytotoxicity of the neuropeptide by facilitating intra-cellular A?42 accumulation. These findings recommend anti-LRP/LR specific antibodies and shRNAs as potential therapeutic tools for AD treatment.

Da Costa Dias, Bianca; Jovanovic, Katarina; Gonsalves, Danielle; Moodley, Kiashanee; Reusch, Uwe; Knackmuss, Stefan; Weinberg, Marc S.; Little, Melvyn; Weiss, Stefan F. T.

2014-01-01

32

Identification of interaction domains of the prion protein with its 37-kDa/67-kDa laminin receptor  

PubMed Central

Cell-binding and internalization studies on neuronal and non-neuronal cells have demonstrated that the 37-kDa/67-kDa laminin receptor (LRP/LR) acts as the receptor for the cellular prion protein (PrP). Here we identify direct and heparan sulfate proteoglycan (HSPG)-dependent interaction sites mediating the binding of the cellular PrP to its receptor, which we demonstrated in vitro on recombinant proteins. Mapping analyses in the yeast two-hybrid system and cell-binding assays identified PrPLRPbd1 [amino acids (aa) 144–179] as a direct and PrPLRPbd2 (aa 53–93) as an indirect HSPG-dependent laminin receptor precursor (LRP)-binding site on PrP. The yeast two-hybrid system localized the direct PrP-binding domain on LRP between aa 161 and 179. Expression of an LRP mutant lacking the direct PrP-binding domain in wild-type and mutant HSPG-deficient Chinese hamster ovary cells by the Semliki Forest virus system demonstrates a second HSPG-dependent PrP-binding site on LRP. Considering the absence of LRP homodimerization and the direct and indirect LRP–PrP interaction sites, we propose a comprehensive model for the LRP–PrP–HSPG complex.

Hundt, Christoph; Peyrin, Jean-Michel; Haik, Stephane; Gauczynski, Sabine; Leucht, Christoph; Rieger, Roman; Riley, Maria Louise; Deslys, Jean-Philippe; Dormont, Dominique; Lasmezas, Corinne Ida; Weiss, Stefan

2001-01-01

33

The cell substrate attachment (CSAT) antigen has properties of a receptor for laminin and fibronectin  

Microsoft Academic Search

The cell substrate attachment (CSAT) antigen is an integral membrane glycoprotein complex that participates in the adhesion of cells to extracellular molecules. The CSAT monoclonal antibody, directed against this complex, inhibited adhesion of cardiac and tendon fibroblasts and ske(etat myoblasts to both laminin and fibronectin, thus implicating the CSAT antigen in adhesion to these extracellular molecules. Equilibrium gel filtration was

A. Horwitz; K. DUGGAN; R. GREGGS; C. DECKER; C. BUCK

1985-01-01

34

Presynaptic calcium channels and ?3-integrins are complexed with synaptic cleft laminins, cytoskeletal elements and active zone components.  

PubMed

At chemical synapses, synaptic cleft components interact with elements of the nerve terminal membrane to promote differentiation and regulate function. Laminins containing the ?2 subunit are key cleft components, and they act in part by binding the pore-forming subunit of a pre-synaptic voltage-gated calcium channel (Ca(v)?) (Nishimune et al. 2004). In this study, we identify Ca(v)?-associated intracellular proteins that may couple channel-anchoring to assembly or stabilization of neurotransmitter release sites called active zones. Using Ca(v)?-antibodies, we isolated a protein complex from Torpedo electric organ synapses, which resemble neuromuscular junctions but are easier to isolate in bulk. We identified 10 components of the complex: six cytoskeletal proteins (?2/?2 spectrins, plectin 1, AHNAK/desmoyokin, dystrophin, and myosin 1), two active zone components (bassoon and piccolo), synaptic laminin, and a calcium channel ? subunit. Immunocytochemistry confirmed these proteins in electric organ synapses, and PCR analysis revealed their expression by developing mammalian motor neurons. Finally, we show that synaptic laminins also interact with pre-synaptic integrins containing the ?3 subunit. Together with our previous finding that a distinct synaptic laminin interacts with SV2 on nerve terminals (Son et al. 2000), our results identify three paths by which synaptic cleft laminins can send developmentally important signals to nerve terminals. PMID:20731762

Carlson, Steven S; Valdez, Gregorio; Sanes, Joshua R

2010-11-01

35

Laminin 511 partners with laminin 332 to mediate directional migration of Madin-Darby canine kidney epithelial cells.  

PubMed

Sustained directional migration of epithelial cells is essential for regeneration of injured epithelia. Front-rear polarity of migrating cells is determined by local activation of a signaling network involving Cdc42 and other factors in response to spatial cues from the environment, the nature of which are obscure. We examined the roles of laminin (LM)-511 and LM-332, two structurally different laminin isoforms, in the migration of Madin-Darby canine kidney cells by suppressing expression of their ? subunits using RNA interference. We determined that knockdown of LM-511 inhibits directional migration and destabilizes cell-cell contacts, in part by disturbing the localization and activity of the polarization machinery. Suppression of integrin ?3, a laminin receptor subunit, in cells synthesizing normal amounts of both laminins has a similar effect as knockdown of LM-511. Surprisingly, simultaneous suppression of both laminin ?5 and laminin ?3 restores directional migration and cell-cell contact stability, suggesting that cells recognize a haptotactic gradient formed by a combination of laminins. PMID:22031290

Greciano, Patricia G; Moyano, Jose V; Buschmann, Mary M; Tang, Jun; Lu, Yue; Rudnicki, Jean; Manninen, Aki; Matlin, Karl S

2012-01-01

36

Laminin-511, inducer of hair growth, is down-regulated and its suppressor in hair growth, laminin-332 up-regulated in chemotherapy-induced alopecia  

PubMed Central

Background Chemotherapy-induced alopecia (CIA) has a devastating cosmetic effect, especially in the young. Recent data indicate that two major basement membrane components (laminin-332 and -511) of the skin have opposing effects on hair growth. Objective In this study, we examined the role and localization of laminin-332 and -511 in CIA. Methods We examined the expression of laminin-332 and -511 during the dystrophic catagen form of CIA induced in C57BL/6 mice by cyclophosphamide (CYP) treatment. Results Our data indicate that both laminin-332 and its receptor ?6?4 integrin are up-regulated (both quantitatively and spatially) after mid to late dystrophic catagen around the outer root sheath (ORS) in the lower third of hair follicles in CIA. This up-regulation also occurs at the transcriptional level. In contrast, laminin-511 is down-regulated after mid dystrophic catagen at the protein level, with transcriptional inactivation of laminin-511 occurring transiently at the early dystrophic catagen stage in both epidermal and ORS keratinocytes. Laminin-511 expression correlates with expression of ?3 integrin in CIA and we also demonstrate that laminin-511 can up-regulate the activity of the ?3 integrin promoter in cultured keratinocytes. Injection of a laminin-511 rich protein extract, but not recombinant laminin-332, in the back skin of mice delays hair loss in CYP-induced CIA. Conclusions We propose that abrupt hair loss in CIA is, at least in part, caused by down-regulation of laminin-511 and up-regulation of laminin-332 at the transcriptional and translational levels.

Imanishi, Hisayoshi; Tsuruta, Daisuke; Tateishi, Chiharu; Sugawara, Koji; Paus, Ralf; Tsuji, Tsutomu; Ishii, Masamitsu; Ikeda, Kazuo; Kunimoto, Hiroyuki; Nakajima, Koichi; Jones, Jonathan C.R.; Kobayashi, Hiromi

2010-01-01

37

Laminin cleavage by activated human neutrophils yields proteolytic fragments with selective migratory properties  

Microsoft Academic Search

We studied the interactions between human neutrophils, as well as the purified human neutrophil serine proteases elastase (HNE) and cathepsin G (HNCG), and laminin. Our results show that intact laminin and two proteolytic fragments generated by HNE bind to neutrophils and stimulate cell migration. Domain- specific antilaminin monoclonal antibodies, rotary shadowing electron microscopy, and Western blotting mapped the two promigratory

Robert Steadman; Michael H. Irwin; Warren D. Blackburn

1993-01-01

38

The 37-kDa/67-kDa laminin receptor acts as the cell-surface receptor for the cellular prion protein  

PubMed Central

Recently, we identified the 37-kDa laminin receptor precursor (LRP) as an interactor for the prion protein (PrP). Here, we show the presence of the 37-kDa LRP and its mature 67-kDa form termed high-affinity laminin receptor (LR) in plasma membrane fractions of N2a cells, whereas only the 37-kDa LRP was detected in baby hamster kidney (BHK) cells. PrP co-localizes with LRP/LR on the surface of N2a cells and Semliki Forest virus (SFV) RNA transfected BHK cells. Cell-binding assays reveal the LRP/LR-dependent binding of cellular PrP by neuronal and non-neuronal cells. Hyperexpression of LRP on the surface of BHK cells results in the binding of exogenous PrP. Cell binding is similar in PrP+/+ and PrP0/0 primary neurons, demonstrating that PrP does not act as a co-receptor of LRP/LR. LRP/LR-dependent internalization of PrP is blocked at 4°C. Secretion of an LRP mutant lacking the transmembrane domain (aa 86–101) from BHK cells abolishes PrP binding and internalization. Our results show that LRP/LR acts as the receptor for cellular PrP on the surface of mammalian cells.

Gauczynski, Sabine; Peyrin, Jean-Michel; Haik, Stephane; Leucht, Christoph; Hundt, Christoph; Rieger, Roman; Krasemann, Susanne; Deslys, Jean-Philippe; Dormont, Dominique; Lasmezas, Corinne Ida; Weiss, Stefan

2001-01-01

39

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

2009-01-01

40

Synthetic Peptides Interacting with the 67-kd Laminin Receptor Can Reduce Retinal Ischemia and Inhibit Hypoxia-Induced Retinal Neovascularization  

PubMed Central

The high-affinity 67-kd laminin receptor (67LR) is expressed by proliferating endothelial cells during retinal neovascularization. The role of 67LR has been further examined experimentally by administration of selective 67LR agonists and antagonists in a murine model of proliferative retinopathy. These synthetic 67LR ligands have been previously shown to stimulate or inhibit endothelial cell motility in vitro without any direct effect on proliferation. In the present study, a fluorescently labeled 67LR antagonist (EGF33–42) was injected intraperitoneally into mice and its distribution in the retina was assessed by confocal scanning laser microscopy. Within 2 hours this peptide was localized to the retinal vasculature, including preretinal neovascular complexes, and a significant amount had crossed the blood retinal barrier. For up to 24 hours postinjection, the peptide was still present in the retinal vascular walls and, to a lesser extent, in the neural retina. Non-labeled EGF33–42 significantly inhibited pre-retinal neovascularization in comparison to controls treated with phosphate-buffered saline or scrambled peptide (P < 0.0001). The agonist peptide (Lam?1925–933) also significantly inhibited proliferative retinopathy; however, it caused a concomitant reduction in retinal ischemia in this model by promoting significant revascularization of the central retina (P < 0.001). Thus, 67LR appears to be an important target receptor for the modulation of retinal neovascularization. Agonism of this receptor may be valuable in reducing the hypoxia-stimulated release of angiogenic growth factors which drives retinal angiogenesis.

Gebarowska, Dorota; Stitt, Alan W.; Gardiner, Thomas A.; Harriott, Patrick; Greer, Brett; Nelson, John

2002-01-01

41

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

PubMed Central

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.

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

2012-01-01

42

The 67-kd laminin receptor is preferentially expressed by proliferating retinal vessels in a murine model of ischemic retinopathy.  

PubMed

Endothelial cell association with vascular basement membranes is complex and plays a critical role in regulation of cell adhesion and proliferation. The interaction between the membrane-associated 67-kd receptor (67LR) and the basement membrane protein laminin has been studied in several cell systems where it was shown to be crucial for adhesion and attachment during angiogenesis. As angiogenesis in the pathological setting of proliferative retinopathy is a major cause of blindness in the Western world we examined the expression of 67LR in a murine model of hyperoxia-induced retinopathy that exhibits retinal neovascularization. Mice exposed to hyperoxia for 5 days starting at postnatal day 7 (P7) and returned to room air (at P12) showed closure of the central retinal vasculature. In response to the ensuing retinal ischemia, there was consistent preretinal neovascularization starting around P17, which persisted until P21, after which the new vessels regressed. Immunohistochemistry was performed on these retinas using an antibody specific for 67LR. At P12, immunoreactivity for 67LR was absent in the retina, but by P17 it was observed in preretinal proliferating vessels and also within the adjacent intraretinal vasculature. Intraretinal 67LR immunoreactivity diminished beyond P17 until by P21 immunoreactivity was almost completely absent, although it persisted in the preretinal vasculature. Control P17 mice (not exposed to hyperoxia) failed to demonstrate any 67LR immunoreactivity in their retinas. Parallel in situ hybridization studies demonstrated 67LR gene expression in the retinal ganglion cells of control and hyperoxia-exposed mice. In addition, the neovascular intra- and preretinal vessels of hyperoxia-treated P17 and P21 mice labeled strongly for 67LR mRNA. This study has characterized 67LR immunolocalization and gene expression in a murine model of ischemic retinopathy. Results suggest that, although the 67LR gene is expressed at high levels in the retinal ganglion cells, the mature receptor protein is preferentially localized to the proliferating retinal vasculature and is almost completely absent from quiescent vessels. The differential expression of 67LR between proliferating and quiescent retinal vessels suggests that this laminin receptor is an important and novel target for future chemotherapeutic intervention during proliferative vasculopathies. PMID:9588904

Stitt, A W; McKenna, D; Simpson, D A; Gardiner, T A; Harriott, P; Archer, D B; Nelson, J

1998-05-01

43

The 67-kd laminin receptor is preferentially expressed by proliferating retinal vessels in a murine model of ischemic retinopathy.  

PubMed Central

Endothelial cell association with vascular basement membranes is complex and plays a critical role in regulation of cell adhesion and proliferation. The interaction between the membrane-associated 67-kd receptor (67LR) and the basement membrane protein laminin has been studied in several cell systems where it was shown to be crucial for adhesion and attachment during angiogenesis. As angiogenesis in the pathological setting of proliferative retinopathy is a major cause of blindness in the Western world we examined the expression of 67LR in a murine model of hyperoxia-induced retinopathy that exhibits retinal neovascularization. Mice exposed to hyperoxia for 5 days starting at postnatal day 7 (P7) and returned to room air (at P12) showed closure of the central retinal vasculature. In response to the ensuing retinal ischemia, there was consistent preretinal neovascularization starting around P17, which persisted until P21, after which the new vessels regressed. Immunohistochemistry was performed on these retinas using an antibody specific for 67LR. At P12, immunoreactivity for 67LR was absent in the retina, but by P17 it was observed in preretinal proliferating vessels and also within the adjacent intraretinal vasculature. Intraretinal 67LR immunoreactivity diminished beyond P17 until by P21 immunoreactivity was almost completely absent, although it persisted in the preretinal vasculature. Control P17 mice (not exposed to hyperoxia) failed to demonstrate any 67LR immunoreactivity in their retinas. Parallel in situ hybridization studies demonstrated 67LR gene expression in the retinal ganglion cells of control and hyperoxia-exposed mice. In addition, the neovascular intra- and preretinal vessels of hyperoxia-treated P17 and P21 mice labeled strongly for 67LR mRNA. This study has characterized 67LR immunolocalization and gene expression in a murine model of ischemic retinopathy. Results suggest that, although the 67LR gene is expressed at high levels in the retinal ganglion cells, the mature receptor protein is preferentially localized to the proliferating retinal vasculature and is almost completely absent from quiescent vessels. The differential expression of 67LR between proliferating and quiescent retinal vessels suggests that this laminin receptor is an important and novel target for future chemotherapeutic intervention during proliferative vasculopathies. Images Figure 1 Figure 2 Figure 3 Figure 4

Stitt, A. W.; McKenna, D.; Simpson, D. A.; Gardiner, T. A.; Harriott, P.; Archer, D. B.; Nelson, J.

1998-01-01

44

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

SciTech Connect

Highlights: Black-Right-Pointing-Pointer Expressions of CD80, CD86, and MHC class I/II were inhibited by EGCG via 67LR. Black-Right-Pointing-Pointer EGCG-treated DCs inhibited LPS-induced pro-inflammatory cytokines via 67LR. Black-Right-Pointing-Pointer EGCG-treated DCs inhibited MAPKs activation and NF-{kappa}B p65 translocation via 67LR. Black-Right-Pointing-Pointer EGCG elevated the expression of the Tollip protein through 67LR in DCs. -- Abstract: 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]-{alpha}, interleukin [IL]-1{beta}, 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 {kappa}B (NF-{kappa}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.

Byun, Eui-Baek [Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of)] [Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of); Choi, Han-Gyu [Department of Microbiology and Research Institute for Medical Sciences, College of Medicine, Chungnam National University, Daejeon 301-747 (Korea, Republic of)] [Department of Microbiology and Research Institute for Medical Sciences, College of Medicine, Chungnam National University, Daejeon 301-747 (Korea, Republic of); Sung, Nak-Yun [Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of)] [Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of); Byun, Eui-Hong, E-mail: ehbyun80@gmail.com [Department of Microbiology and Research Institute for Medical Sciences, College of Medicine, Chungnam National University, Daejeon 301-747 (Korea, Republic of)] [Department of Microbiology and Research Institute for Medical Sciences, College of Medicine, Chungnam National University, Daejeon 301-747 (Korea, Republic of)

2012-10-05

45

Laminins in basement membrane assembly  

PubMed Central

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.

Hohenester, Erhard; Yurchenco, Peter D.

2013-01-01

46

Detection of the 67–kD Laminin Receptor in Prostate Cancer Biopsies as a Predictor of Recurrence after Radical Prostatectomy  

Microsoft Academic Search

Objectives: Reliable prognostic indicators are needed for a better pretherapeutic assessment of the agressiveness of organ–confined prostate cancer (PC) lesions. The 67–kD laminin receptor (67LR) is a cell–surface–associated protein involved in the acquisition of the invasive and metastatic phenotype of a variety of human cancer cell types. We have previously shown that 67LR detection in PC tissues from radical prostatectomy

David Waltregny; Laurence de Leval; Luc Coppens; Enis Youssef; Jean de Leval; Vincent Castronovo

2001-01-01

47

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

Microsoft Academic Search

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-express- ing hematologic targets, CTLs were gen- erated

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

2003-01-01

48

Synthetic peptides interacting with the 67-kd laminin receptor can reduce retinal ischemia and inhibit hypoxia-induced retinal neovascularization.  

PubMed

The high-affinity 67-kd laminin receptor (67LR) is expressed by proliferating endothelial cells during retinal neovascularization. The role of 67LR has been further examined experimentally by administration of selective 67LR agonists and antagonists in a murine model of proliferative retinopathy. These synthetic 67LR ligands have been previously shown to stimulate or inhibit endothelial cell motility in vitro without any direct effect on proliferation. In the present study, a fluorescently labeled 67LR antagonist (EGF(33-42)) was injected intraperitoneally into mice and its distribution in the retina was assessed by confocal scanning laser microscopy. Within 2 hours this peptide was localized to the retinal vasculature, including preretinal neovascular complexes, and a significant amount had crossed the blood retinal barrier. For up to 24 hours postinjection, the peptide was still present in the retinal vascular walls and, to a lesser extent, in the neural retina. Non-labeled EGF(33-42) significantly inhibited pre-retinal neovascularization in comparison to controls treated with phosphate-buffered saline or scrambled peptide (P < 0.0001). The agonist peptide (Lam beta 1(925-933)) also significantly inhibited proliferative retinopathy; however, it caused a concomitant reduction in retinal ischemia in this model by promoting significant revascularization of the central retina (P < 0.001). Thus, 67LR appears to be an important target receptor for the modulation of retinal neovascularization. Agonism of this receptor may be valuable in reducing the hypoxia-stimulated release of angiogenic growth factors which drives retinal angiogenesis. PMID:11786424

Gebarowska, Dorota; Stitt, Alan W; Gardiner, Thomas A; Harriott, Patrick; Greer, Brett; Nelson, John

2002-01-01

49

Biologically-active laminin-111 fragment that modulates the epithelial-to-mesenchymal transition in embryonic stem cells  

PubMed Central

The dynamic interplay between the extracellular matrix and embryonic stem cells (ESCs) constitutes one of the key steps in understanding stem cell differentiation in vitro. Here we report a biologically-active laminin-111 fragment generated by matrix metalloproteinase 2 (MMP2) processing, which is highly up-regulated during differentiation. We show that the ?1-chain–derived fragment interacts via ?3?1-integrins, thereby triggering the down-regulation of MMP2 in mouse and human ESCs. Additionally, the expression of MMP9 and E-cadherin is up-regulated in mouse ESCs—key players in the epithelial-to-mesenchymal transition. We also demonstrate that the fragment acts through the ?3?1-integrin/extracellular matrix metalloproteinase inducer complex. This study reveals a previously unidentified role of laminin-111 in early stem cell differentiation that goes far beyond basement membrane assembly and a mechanism by which an MMP2-cleaved laminin fragment regulates the expression of E-cadherin, MMP2, and MMP9.

Horejs, Christine-Maria; Serio, Andrea; Purvis, Alan; Gormley, Adam J.; Bertazzo, Sergio; Poliniewicz, Anna; Wang, Alex J.; DiMaggio, Peter; Hohenester, Erhard; Stevens, Molly M.

2014-01-01

50

Biologically-active laminin-111 fragment that modulates the epithelial-to-mesenchymal transition in embryonic stem cells.  

PubMed

The dynamic interplay between the extracellular matrix and embryonic stem cells (ESCs) constitutes one of the key steps in understanding stem cell differentiation in vitro. Here we report a biologically-active laminin-111 fragment generated by matrix metalloproteinase 2 (MMP2) processing, which is highly up-regulated during differentiation. We show that the ?1-chain-derived fragment interacts via ?3?1-integrins, thereby triggering the down-regulation of MMP2 in mouse and human ESCs. Additionally, the expression of MMP9 and E-cadherin is up-regulated in mouse ESCs--key players in the epithelial-to-mesenchymal transition. We also demonstrate that the fragment acts through the ?3?1-integrin/extracellular matrix metalloproteinase inducer complex. This study reveals a previously unidentified role of laminin-111 in early stem cell differentiation that goes far beyond basement membrane assembly and a mechanism by which an MMP2-cleaved laminin fragment regulates the expression of E-cadherin, MMP2, and MMP9. PMID:24706882

Horejs, Christine-Maria; Serio, Andrea; Purvis, Alan; Gormley, Adam J; Bertazzo, Sergio; Poliniewicz, Anna; Wang, Alex J; DiMaggio, Peter; Hohenester, Erhard; Stevens, Molly M

2014-04-22

51

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.

2010-01-01

52

Laminin receptor specific therapeutic gold nanoparticles (198AuNP-EGCg) show efficacy in treating prostate cancer  

PubMed Central

Systemic delivery of therapeutic agents to solid tumors is hindered by vascular and interstitial barriers. We hypothesized that prostate tumor specific epigallocatechin-gallate (EGCg) functionalized radioactive gold nanoparticles, when delivered intratumorally (IT), would circumvent transport barriers, resulting in targeted delivery of therapeutic payloads. The results described herein support our hypothesis. We report the development of inherently therapeutic gold nanoparticles derived from the Au-198 isotope; the range of the 198Au ?-particle (approximately 11 mm in tissue or approximately 1100 cell diameters) is sufficiently long to provide cross-fire effects of a radiation dose delivered to cells within the prostate gland and short enough to minimize the radiation dose to critical tissues near the periphery of the capsule. The formulation of biocompatible 198AuNPs utilizes the redox chemistry of prostate tumor specific phytochemical EGCg as it converts gold salt into gold nanoparticles and also selectively binds with excellent affinity to Laminin67R receptors, which are over expressed in prostate tumor cells. Pharmacokinetic studies in PC-3 xenograft SCID mice showed approximately 72% retention of 198AuNP-EGCg in tumors 24 h after intratumoral administration. Therapeutic studies showed 80% reduction of tumor volumes after 28 d demonstrating significant inhibition of tumor growth compared to controls. This innovative nanotechnological approach serves as a basis for designing biocompatible target specific antineoplastic agents. This novel intratumorally injectable 198AuNP-EGCg nanotherapeutic agent may provide significant advances in oncology for use as an effective treatment for prostate and other solid tumors.

Shukla, Ravi; Chanda, Nripen; Zambre, Ajit; Upendran, Anandhi; Katti, Kavita; Kulkarni, Rajesh R.; Nune, Satish Kumar; Casteel, Stan W.; Smith, Charles Jeffrey; Vimal, Jatin; Boote, Evan; Robertson, J. David; Kan, Para; Engelbrecht, Hendrik; Watkinson, Lisa D.; Carmack, Terry L.; Lever, John R.; Cutler, Cathy S.; Caldwell, Charles; Kannan, Raghuraman; Katti, Kattesh V.

2012-01-01

53

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.

1999-01-01

54

Interactions of laminin ?3 fragment with ?1-integrin receptor: A revisit of the apical ectoplasmic specialization-blood-testis-barrier-hemidesmosome functional axis in the testis.  

PubMed

Recent studies have demonstrated the presence of a functional axis that coordinates the events of spermiation and blood-testis barrier (BTB) restructuring which take place simultaneously at the opposite ends of the seminiferous epithelium at stage VIII of the epithelial cycle of spermatogenesis in the rat testis. In short, the disruption of the apical ectoplasmic specialization (apical ES) at the Sertoli cell-elongated spermatid interface, which facilitates the release of sperm at spermiation near the tubule lumen, is coordinated with restructuring at the BTB to accommodate the transit of preleptotene spermatocytes across the immunological barrier near the basement membrane. These two events are likely coordinated by a functional axis involving hemidesmosome at the Sertoli cell-basement membrane interface, and it was designated the apical ES-BTB-hemidesmosome axis. It was demonstrated that fragments of laminin chains (e.g., laminin ?3 or ?3 chains) derived from the ?6?1-integrin-laminin333 protein complex at the apical ES, which were likely generated via the action of MMP-2 (matrix metalloprotease-2, MMP2) prior to spermiation, acted as biologically active peptides to perturb the BTB permeability function by accelerating protein endocytosis (e.g., occludin) at the site, thereby destabilizing the BTB integrity to facilitate the transit of preleptotene spermatocytes. These laminin fragments also perturbed hemidesmosome function via their action on ?1-integrin, a component of hemidesmosome in the testis, which in turn, sent a signal to further destabilize the BTB function. As such, the events of spermiation and BTB restructuring are coordinated via this functional axis. Recent studies using animal models treated with toxicants, such as mono-(2-ethylhexyl) phthalate (MEHP), or adjudin, a male contraceptive under investigation, have also supported the presence of this functional axis in the mouse. In this short review, we critically evaluate the role of this local functional axis in the seminiferous epithelium in spermatogenesis. We also provide molecular modeling information on the interactions between biologically active laminin fragments and ?1-integrin, which will be important to assist in the design of more potent laminin-based peptides to disrupt this axis, thereby perturbing spermatogenesis for male contraception and to understand the underlying biology that coordinates spermiation and BTB restructuring during spermatogenesis. PMID:22319666

Cheng, C Yan; Lie, Pearl Py; Mok, Ka-Wai; Cheng, Yan-Ho; Wong, Elissa Wp; Mannu, Jayakanthan; Mathur, Premendu P; Yan, Helen H N; Mruk, Dolores D

2011-07-01

55

The 37 kDa\\/67 kDa laminin receptor is required for PrPSc propagation in scrapie-infected neuronal cells  

Microsoft Academic Search

The accumulation of PrPSc in scrapie-infected neuronal cells has been prevented by three approaches: (i) transfection of ScMNB cells with an antisense laminin receptor precursor (LRP) RNA-expression plasmid, (ii) transfection of ScN2a cells and ScGT1 cells with small interfering RNAs (siRNAs) specific for the LRP mRNA, and (iii) incubation of ScN2a cells with an anti-LRP\\/LR antibody. LRP antisense RNA and

Christoph Leucht; Steve Simoneau; Clémence Rey; Karen Vana; Roman Rieger; Corinne Ida Lasmézas; Stefan Weiss

2003-01-01

56

Laminin-dependent and laminin-independent adhesion of human melanoma cells to sulfatides.  

PubMed

Sulfatides (galactosylceramide-I3-sulfate) but not neutral glycolipids or gangliosides adsorbed on plastic promote adhesion of the human melanoma cell line G361. Direct adhesion of G361 cells requires densities of sulfatide greater than 1 pmol/mm2. In the presence of laminin, however, specific adhesion of G361 cells to sulfatide or seminolipid (galactosylalkylacyl-glycerol-I3-sulfate) but not to other lipids is strongly stimulated and requires only 25 fmol/mm2 of adsorbed lipid. The effects of laminin and sulfatide on adhesion are synergistic, suggesting that laminin is mediating adhesion by cross-linking receptors on the melanoma cell surface to sulfatide adsorbed on the plastic. Although thrombospondin binds to sulfatides and G361 cells, it does not enhance, but rather inhibits direct and laminin-dependent G361 cell adhesion to sulfatide. In contrast, C32 melanoma cells also adhere specifically to sulfatide, but adhesion of these cells is not enhanced by laminin or inhibited by antibodies to laminin that block laminin-dependent adhesion of G361 cells. Thrombospondin is a potent inhibitor of C32 cell adhesion to sulfatide. Fucoidan, which inhibits laminin binding to sulfatide, inhibits laminin-dependent adhesion of G361 cells by 50% at 0.2 micrograms/ml. Several other tumor cell lines also attach directly on sulfatide-coated surfaces. Laminin stimulates adhesion to sulfatide of three of the six cell lines tested. The ability of laminin to promote adhesion of tumor cells to sulfatide suggests that binding to sulfatide could participate in laminin-mediated cell-cell adhesion. Thus, many tumor cell lines can attach on sulfatide substrates using endogenous sulfatide binding proteins, and in some cells laminin but not thrombospondin can promote tumor cell adhesion to sulfatide. PMID:2967105

Roberts, D D; Wewer, U M; Liotta, L A; Ginsburg, V

1988-06-15

57

Laminin peptides stimulate human neutrophil motility.  

PubMed

Laminin, isolated from Engelbreth-Holm-Swarm tumor, and 10 chemically synthesized peptides, corresponding to various regions of the laminin A and B1 chains, were compared for their abilities to stimulate human peripheral blood polymorphonuclear leukocyte (PMN) chemotaxis and chemokinesis through polycarbonate membrane filters in a 48-well microchemotaxis assay. Peptides F-9, F-11, F-12, and F-13 were derived from the B1 chain of laminin at the intersection of the cross, and six peptides were derived from the laminin A chain: peptide TG-1 from the amino-terminal top globule; peptides GD-1, GD-3, GD-6, and GD-7 from the carboxyl-terminal globular domain; and peptide AG-1 from above the carboxyl-terminal globular domain. Laminin and the peptides were evaluated over a concentration range of 1 to 200 micrograms/ml in motility assays. Six of the peptides, F-9, F-12, GD-1, GD-3, GD-6, and TG-1, stimulated human PMN migration in the absence of a gradient (chemokinesis). A fluorescein conjugate of the most active laminin peptide, GD-1, exhibited nonspecific, nonsaturable binding to PMN. Intact laminin and the other peptides failed to stimulate human PMN migration. In contrast, intact Engelbreth-Holm-Swarm laminin stimulated rabbit peripheral blood PMN chemokinesis. These results demonstrate that rabbit and human peripheral blood PMNs have divergent migratory responses to intact laminin. These findings suggest that intact basement membrane laminin does not directly stimulate human blood PMN motility in vivo, but that selected laminin peptide sequences, which may be generated during proteolytic digestion of laminin, can activate human PMN migration. PMID:8189064

Harvath, L; Brownson, N E; Fields, G B; Skubitz, A P

1994-06-01

58

Angiotensin II type 1 receptor antagonists alleviate muscle pathology in the mouse model for laminin-?2-deficient congenital muscular dystrophy (MDC1A)  

PubMed Central

Background Laminin-?2-deficient congenital muscular dystrophy (MDC1A) is a severe muscle-wasting disease for which no curative treatment is available. Antagonists of the angiotensin II receptor type 1 (AT1), including the anti-hypertensive drug losartan, have been shown to block also the profibrotic action of transforming growth factor (TGF)-? and thereby ameliorate disease progression in mouse models of Marfan syndrome. Because fibrosis and failure of muscle regeneration are the main reasons for the severe disease course of MDC1A, we tested whether L-158809, an analog derivative of losartan, could ameliorate the dystrophy in dyW/dyW mice, the best-characterized model of MDC1A. Methods L-158809 was given in food to dyW/dyW mice at the age of 3?weeks, and the mice were analyzed at the age of 6 to 7?weeks. We examined the effect of L-158809 on muscle histology and on muscle regeneration after injury as well as the locomotor activity and muscle strength of the mice. Results We found that TGF-? signaling in the muscles of the dyW/dyW mice was strongly increased, and that L-158809 treatment suppressed this signaling. Consequently, L-158809 reduced fibrosis and inflammation in skeletal muscle of dyW/dyW mice, and largely restored muscle regeneration after toxin-induced injury. Mice showed improvement in their locomotor activity and grip strength, and their body weight was significantly increased. Conclusion These data provide evidence that AT1 antagonists ameliorate several hallmarks of MDC1A in dyW/dyW mice, the best-characterized mouse model for this disease. Because AT1 antagonists are well tolerated in humans and widely used in clinical practice, these results suggest that losartan may offer a potential future treatment of patients with MDC1A.

2012-01-01

59

Screening of integrin-binding peptides in a laminin peptide library derived from the mouse laminin ? chain short arm regions.  

PubMed

Laminins, major components of basement membrane, consist of three different subunits, ?, ?, and ? chains, and so far, five ?, three ?, and three ? chains have been identified. We have constructed synthetic peptide libraries derived from the laminin sequences and identified various cell-adhesive peptides. Ten active peptides from the laminin ? chain sequences (?1-?5) were found to promote integrin-mediated cell adhesion. Previously, we found fourteen cell-adhesive peptides from the ?1 chain sequence but their receptors have not been analyzed. Here, we expanded the synthetic peptide library to add peptides from the short arm regions of the laminin ?2 and ?3 chains and screened for integrin-binding peptides. Twenty-seven peptides promoted human dermal fibroblast (HDF) attachment in a peptide-coated plate assay. The morphological appearance of HDFs on the peptide-coated plates differed depending on the peptides. B34 (REKYYYAVYDMV, mouse laminin ?1 chain, 255-266), B67 (IPYSMEYEILIRY, mouse laminin ?1 chain, 604-616), B2-105 (APNFWNFTSGRG, mouse laminin ?2 chain, 1081-1092), and B3-19 (GHLTGGKVQLNL, mouse laminin ?3 chain, 182-193) promoted HDF spreading and HDF attachment was inhibited by EDTA, suggesting that the peptides interact with integrins. Immunostaining analyses revealed that B67 induced well-organized actin stress fibers and focal contacts containing vinculin, however, B34, B2-105, and B3-19 did not exhibit stress fiber formation or focal contacts. The inhibition assay using anti-integrin antibodies indicated that B67 interacts with ?3, ?6, and ?1 integrins, and B34 and B3-19 interact with ?1 integrin. Based on adhesion analysis of peptides modified with an alanine scan and on switching analysis with the homologous inactive sequence B2-64 (LPRAMDYDLLLRW, mouse laminin ?2 chain, 618-630), the Glu(8) residue in the B67 peptide was critical for HDF adhesion. These findings are useful for identifying an integrin binding motif. The B67 peptide has potential for use as a molecular probe for integrins. PMID:24785228

Katagiri, Fumihiko; Takagi, Masaharu; Nakamura, Minako; Tanaka, Yoichiro; Hozumi, Kentaro; Kikkawa, Yamato; Nomizu, Motoyoshi

2014-05-15

60

Laminin-332 coordinates mechanotransduction and growth cone bifurcation in sensory neurons.  

PubMed

Laminin-332 is a major component of the dermo-epidermal skin basement membrane and maintains skin integrity. The transduction of mechanical force into electrical signals by sensory endings in the skin requires mechanosensitive channels. We found that mouse epidermal keratinocytes produce a matrix that is inhibitory for sensory mechanotransduction and that the active molecular component is laminin-332. Substrate-bound laminin-332 specifically suppressed one type of mechanosensitive current (rapidly adapting) independently of integrin-receptor activation. This mechanotransduction suppression could be exerted locally and was mediated by preventing the formation of protein tethers necessary for current activation. We also found that laminin-332 could locally control sensory axon branching behavior. Loss of laminin-332 in humans led to increased sensory terminal branching and may lead to a de-repression of mechanosensitive currents. These previously unknown functions for this matrix molecule may explain some of the extreme pain experienced by individuals with epidermolysis bullosa who are deficient in laminin-332. PMID:21725315

Chiang, Li-Yang; Poole, Kate; Oliveira, Beatriz E; Duarte, Neuza; Sierra, Yinth Andrea Bernal; Bruckner-Tuderman, Leena; Koch, Manuel; Hu, Jing; Lewin, Gary R

2011-08-01

61

The laminin family  

PubMed Central

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.

2013-01-01

62

Laminin molecular domains which alter metastasis in a murine model.  

PubMed Central

Human and murine tumor cells contain cell surface receptors for the basement membrane glycoprotein laminin. Since a biologic role for the receptor had not previously been demonstrated, we explored the possibility that the laminin receptor may be involved in hematogenous metastases formation. Preincubation of metastatic murine melanoma cells with syngeneic whole laminin followed by tail vein injection increased tumor cell retention in the lung and strongly stimulated metastases formation. The domain of the laminin molecule responsible for stimulating metastases was identified. Laminin is a cross-shaped molecule with three short arms and one long arm. All arms have globular end regions. Purified protease-derived fragments of laminin were prepared which (a) lacked only the long arm of the molecule (alpha fragment) or, (b) lacked both the long arm and the globular end regions of the short arms (C1 fragment). Both types of fragments contained the laminin receptor binding region. The fragments had opposite effects on metastases. The alpha fragment stimulated metastases formation to the same extent as whole laminin. In contrast, the C1 fragment greatly reduced or abolished metastases formation in a dose-dependent manner. The C1 fragment also inhibited tumor cell attachment to whole amnion basement membrane in vitro. We conclude that intact globular end regions on the short arms (but not the long arm) of the cell surface receptor-bound laminin molecule are necessary for stimulating metastases by the intravenous route. Images

Barsky, S H; Rao, C N; Williams, J E; Liotta, L A

1984-01-01

63

Ex vivo pathogenicity of anti-laminin ?1 autoantibodies.  

PubMed

Autoimmunity against laminins has been described in several autoimmune diseases (including mucous membrane pemphigoid, anti-laminin ?1 pemphigoid, and connective tissue diseases), in pregnancy loss, and in infections such as Chagas disease. Except for anti-laminin-332 mucous membrane pemphigoid, adequate evidence has been lacking for the tissue injury potential of laminin-specific antibodies and the pathogenic epitopes. We evaluated the pathogenic potential of antibodies targeting laminin ?1, a major constituent of basement membranes and the main antigen in anti-laminin ?1 pemphigoid. Rabbit antibodies were generated against fragments of the N-terminus and C-terminus of murine laminin ?1, and their ability to disrupt ligand interactions and/or to activate complement and granulocytes was assessed using previously established ex vivo assays. Our findings document a pathogenic potential of antibodies targeting the laminin ?1 N-terminus. These antibodies interfere with the binding of nidogen to laminin and can activate granulocytes and the complement cascade. We detected antibodies with different degrees of reactivity with laminin ?1 N-terminus in patients with anti-laminin ?1 pemphigoid, cutaneous lupus erythematosus, and scleroderma. Our results provide mechanistic insights into the tissue damage associated with laminin autoimmunity and could facilitate development of appropriate diagnostic tools and therapeutic strategies. PMID:24300951

Florea, Florina; Bernards, Claudia; Caproni, Marzia; Kleindienst, Jessika; Hashimoto, Takashi; Koch, Manuel; Sitaru, Cassian

2014-02-01

64

Laminin deposition in the extracellular matrix: a complex picture emerges  

PubMed Central

Summary Laminins are structural components of basement membranes. In addition, they are key extracellular-matrix regulators of cell adhesion, migration, differentiation and proliferation. This Commentary focuses on a relatively understudied aspect of laminin biology: how is laminin deposited into the extracellular matrix? This topic has fascinated researchers for some time, particularly considering the diversity of patterns of laminin that can be visualized in the matrix of cultured cells. We discuss current ideas of how laminin matrices are assembled, the role of matrix receptors in this process and how laminin-associated proteins modulate matrix deposition. We speculate on the role of signaling pathways that are involved in laminin-matrix deposition and on how laminin patterns might play an important role in specifying cell behaviors, especially directed migration. We conclude with a description of new developments in the way that laminin deposition is being studied, including the use of tagged laminin subunits that should allow the visualization of laminin-matrix deposition and assembly by living cells.

Hamill, Kevin J.; Kligys, Kristina; Hopkinson, Susan B.; Jones, Jonathan C. R.

2009-01-01

65

Laminin enhances beta(2)-adrenergic receptor stimulation of L-type Ca(2+) current via cytosolic phospholipase A(2) signalling in cat atrial myocytes.  

PubMed

We previously reported that attachment of atrial myocytes to the extracellular matrix protein laminin (LMN), decreases adenylate cyclase (AC)/cAMP and increases beta(2)-adrenergic receptor (AR) stimulation of L-type Ca(2+) current (I(Ca,L)). This study therefore sought to determine whether LMN enhances beta(2)-AR signalling via a cAMP-independent mechanism, i.e. cytosolic phospholipase A(2) (cPLA(2)) 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 microm zinterol (zint-beta(2)-AR) stimulation of I(Ca,L) was larger in +LMN than LMN myocytes. In +LMN myocytes, zint-beta(2)-AR stimulation of I(Ca,L) was inhibited by inhibition of cPLA(2) by arachidonyltrifluoromethyl ketone (AACOCF(3); 10 microm), inhibition of G(i) by pertussis toxin and chelation of intracellular Ca(2+) by 10 microm BAPTA-AM. In contrast to zinterol, stimulation of I(Ca,L) by fenoterol (fen-beta(2)-AR), a beta(2)-AR agonist that acts exclusively via G(s) signalling, was smaller in +LMN than LMN myocytes. Arachidonic acid (AA; 5 microm) stimulated I(Ca,L) to a similar extent in LMN and +LMN myocytes. Inhibition of cAMP-dependent protein kinase A (cAMP/PKA) by either 5 mum H89 or 1 microm KT5720 in LMN myocytes mimicked the effects of +LMN myocytes to enhance zint-beta(2)-AR stimulation of I(Ca,L), which was blocked by 10 microm AACOCF(3). In contrast, H89 inhibited fen-beta(2)-AR stimulation of I(Ca,L), which was unchanged by AACOCF(3). Inhibition of ERK1/2 by 1 microm U0126 inhibited zint-beta(2)-AR stimulation of I(Ca,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-beta(2)-AR stimulation of I(Ca,L). We conclude that LMN enhances zint-beta(2)-AR stimulation of I(Ca,L) via G(i)/ERK1/2/cPLA(2)/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 beta(2)-AR signalling in atrial muscle. PMID:19703961

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

2009-10-15

66

The Hippocampal Laminin Matrix Is Dynamic and Critical for Neuronal Survival  

PubMed Central

Laminins are extracellular matrix proteins that participate in neuronal development, survival, and regeneration. During excitotoxin challenge in the mouse hippocampus, neuron interaction with laminin-10 (?5,?1,?1) protects against neuronal death. To investigate how laminin is involved in neuronal viability, we infused laminin-1 (?1,?1,?1) into the mouse hippocampus. This infusion specifically disrupted the endogenous laminin layer. This disruption was at least partially due to the interaction of the laminin-1 ?1 chain with endogenous laminin-10, because infusion of anti-laminin ?1 antibody had the same effect. The disruption of the laminin layer by laminin-1 1) did not require the intact protein because infusion of plasmin-digested laminin-1 gave similar results; 2) was posttranscriptional, because there was no effect on laminin mRNA expression; and 3) occurred in both tPA–/– and plasminogen–/– mice, indicating that increased plasmin activity was not responsible. Finally, although tPA–/– mice are normally resistant to excitotoxin-induced neurodegeneration, disruption of the endogenous laminin layer by laminin-1 or anti-laminin ?1 antibody renders the tPA–/– hippocampal neurons sensitive to kainate. These results demonstrate that neuron interactions with the deposited matrix are not necessarily recapitulated by interactions with soluble components and that the laminin matrix is a dynamic structure amenable to modification by exogenous molecules.

Chen, Zu-Lin; Indyk, Justin A.; Strickland, Sidney

2003-01-01

67

The hippocampal laminin matrix is dynamic and critical for neuronal survival.  

PubMed

Laminins are extracellular matrix proteins that participate in neuronal development, survival, and regeneration. During excitotoxin challenge in the mouse hippocampus, neuron interaction with laminin-10 (alpha5,beta1,gamma1) protects against neuronal death. To investigate how laminin is involved in neuronal viability, we infused laminin-1 (alpha1,beta1,gamma1) into the mouse hippocampus. This infusion specifically disrupted the endogenous laminin layer. This disruption was at least partially due to the interaction of the laminin-1 gamma1 chain with endogenous laminin-10, because infusion of anti-laminin gamma1 antibody had the same effect. The disruption of the laminin layer by laminin-1 1) did not require the intact protein because infusion of plasmin-digested laminin-1 gave similar results; 2) was posttranscriptional, because there was no effect on laminin mRNA expression; and 3) occurred in both tPA(-/-) and plasminogen(-/-) mice, indicating that increased plasmin activity was not responsible. Finally, although tPA(-/-) mice are normally resistant to excitotoxin-induced neurodegeneration, disruption of the endogenous laminin layer by laminin-1 or anti-laminin gamma1 antibody renders the tPA(-/-) hippocampal neurons sensitive to kainate. These results demonstrate that neuron interactions with the deposited matrix are not necessarily recapitulated by interactions with soluble components and that the laminin matrix is a dynamic structure amenable to modification by exogenous molecules. PMID:12857855

Chen, Zu-Lin; Indyk, Justin A; Strickland, Sidney

2003-07-01

68

Two dimensional VOPBA reveals laminin receptor (LAMR1) interaction with dengue virus serotypes 1, 2 and 3  

Microsoft Academic Search

BACKGROUND: The search for the dengue virus receptor has generated many candidates often identified only by molecular mass. The wide host range of the viruses in vitro combined with multiple approaches to identifying the receptor(s) has led to the notion that many receptors or attachment proteins may be involved and that the different dengue virus serotypes may utilize different receptors

Phaik Hooi Tio; Wan Wui Jong; Mary Jane Cardosa

2005-01-01

69

Plasmin-mediated degradation of laminin ?-1 is critical for ethanol-induced neurodegeneration  

PubMed Central

Background Alcoholism may result in severe neurological deficits and cognitive impairments. Many of central effects of ethanol (EtOH) can be explained by upregulation of NMDA and downregulation of GABA(A) receptors in response to a long-term EtOH consumption. Abrupt ethanol withdrawal (EW) may result in neuronal hyperexcitability leading to hallucinations, seizures, neurodegeneration and sometimes death. Methods Using a multidisciplinary approach in wild-type and genetically modified mice we have examined the contribution of the tissue-plasminogen activator, plasminogen and laminin to EW-induced cell death. Results Here we show that EW-induced neurodegeneration is mediated by the tissue plasminogen activator (tPA)/plasmin system. During EW, tPA is upregulated in the hippocampus, converts plasminogen to plasmin, which in turn degrades an extracellular matrix component laminin, leading to caspase-3-dependent cell death. Consequently, mice in which the tPA or plasminogen genes have been deleted do not show EW-induced laminin degradation, mitochondial dysfunction, and neurodegeneration. Finally we demonstrate, that disruption of the hippocampal laminin ?-1 renders the mice resistant to neurotoxic effects of EW. Conclusions Our data identify laminin ?-1 as a novel target to combat neurodegeneration.

Skrzypiec, Anna; Maiya, Rajani; Chen, Zulin; Pawlak, Robert; Strickland, Sidney

2009-01-01

70

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

2001-01-01

71

Dystroglycan loss disrupts polarity and beta-casein induction inmammary epithelial cells by perturbing laminin anchoring  

SciTech Connect

Precise contact between epithelial cells and their underlying basement membrane is critical to the maintenance of tissue architecture and function. To understand the role that the laminin receptor dystroglycan (DG) plays in these processes, we assayed cell responses to laminin-111 following conditional ablation of DG expression in cultured mammary epithelial cells (MECs). Strikingly, DG loss disrupted laminin-111-induced polarity and {beta}-casein production, and abolished laminin assembly at the step of laminin binding to the cell surface. DG re-expression restored these deficiencies. Investigations of mechanism revealed that DG cytoplasmic sequences were not necessary for laminin assembly and signaling, and only when the entire mucin domain of extracellular DG was deleted did laminin assembly not occur. These results demonstrate that DG is essential as a laminin-111 co-receptor in MECs that functions by mediating laminin anchoring to the cell surface, a process that allows laminin polymerization, tissue polarity, and {beta}-casein induction. The observed loss of laminin-111 assembly and signaling in DG-/-MECs provides insights into the signaling changes occurring in breast carcinomas and other cancers, where DG's laminin-binding function is frequently defective.

Weir, M. Lynn; Oppizzi, Maria Luisa; Henry, Michael D.; Onishi,Akiko; Campbell, Kevin P.; Bissell, Mina J.; Muschler, John L.

2006-02-17

72

Electric field-directed cell motility involves up-regulated expression and asymmetric redistribution of the epidermal growth factor receptors and is enhanced by fibronectin and laminin.  

PubMed

Wounding corneal epithelium establishes a laterally oriented, DC electric field (EF). Corneal epithelial cells (CECs) cultured in similar physiological EFs migrate cathodally, but this requires serum growth factors. Migration depends also on the substrate. On fibronectin (FN) or laminin (LAM) substrates in EF, cells migrated faster and more directly cathodally. This also was serum dependent. Epidermal growth factor (EGF) restored cathodal-directed migration in serum-free medium. Therefore, the hypothesis that EGF is a serum constituent underlying both field-directed migration and enhanced migration on ECM molecules was tested. We used immunofluorescence, flow cytometry, and confocal microscopy and report that 1) EF exposure up-regulated the EGF receptor (EGFR); so also did growing cells on substrates of FN or LAM; and 2) EGFRs and actin accumulated in the cathodal-directed half of CECs, within 10 min in EF. The cathodal asymmetry of EGFR and actin staining was correlated, being most marked at the cell-substrate interface and showing similar patterns of asymmetry at various levels through a cell. At the cell-substrate interface, EGFRs and actin frequently colocalized as interdigitated, punctate spots resembling tank tracks. Cathodal accumulation of EGFR and actin did not occur in the absence of serum but were restored by adding ligand to serum-free medium. Inhibition of MAPK, one second messenger engaged by EGF, significantly reduced EF-directed cell migration. Transforming growth factor beta and fibroblast growth factor also restored cathodal-directed cell migration in serum-free medium. However, longer EF exposure was needed to show clear asymmetric distribution of the receptors for transforming growth factor beta and fibroblast growth factor. We propose that up-regulated expression and redistribution of EGFRs underlie cathodal-directed migration of CECs and directed migration induced by EF on FN and LAM. PMID:10198071

Zhao, M; Dick, A; Forrester, J V; McCaig, C D

1999-04-01

73

Laminin-111 Stimulates Proliferation of Mouse Embryonic Stem Cells Through a Reduction of Gap Junctional Intercellular Communication via RhoA-Mediated Cx43 Phosphorylation and Dissociation of Cx43/ZO-1/Drebrin Complex  

PubMed Central

Abstract Gap junctions within extracellular matrix (ECM)-defined boundaries ensure synchronous activity between cells destined to become functional mediators that regulate cell behavior. However, the role of ECM in connexin (Cx) function in mouse embryonic stem cells (mESCs) has not been elucidated. Therefore, we examined the role of laminin-111 in the control of Cx43 functions and related signal pathways in mESCs. ECM components (laminin-111, fibronectin, and collagen I) increased Cx43 phosphorylation and decreased Lucifer yellow (Ly) diffusion. In addition, laminin-111 increased the proliferation index through reduction of gap junctional intercellular communication (GJIC), which was confirmed by 18?-glycyrrhetinic acid (18?-GA). Laminin-111 increased phosphorylation of focal adhesion kinase (FAK)/Src and protein kinase C (PKC), which were inhibited by integrin ?1 antibody (Ab) and laminin receptor-1 (LR-1) Ab, respectively. In addition, inhibition of both FAK/Src and PKC blocked Cx43 phosphorylation. Laminin-111 increased the Ras homolog gene family, member A (RhoA) activation, which was blocked by FAK/Src and PKC inhibitors, suggesting the existence of parallel pathways that merge at RhoA. Inhibition of RhoA reversed the laminin-111-induced increase of Cx43 phosphorylation and reduction of GJIC. Laminin-111 also stimulated the dissociation of Cx43/ZO-1 complex followed by disruption of Cx43/drebrin and Cx43/F-actin complexes, which were reversed by C3 (RhoA inhibitor). ZO-1 small interfering (si) RNA significantly decreased Ly diffusion. Moreover, laminin-111 decreased Cx43 labeling at the intercellular junction, whereas pretreatment with degradation inhibitors (lysosomal protease inhibitor, chloroquine; proteasome inhibitor, lactacystin) increased Cx43 expression, reversely. In conclusion, laminin-111 stimulated mESC proliferation through a reduction of GJIC via RhoA-mediated Cx43 phosphorylation and Cx43/ZO-1/drebrin complex instability-mediated Cx43 degradation.

Suh, Han Na; Kim, Mi Ok

2012-01-01

74

Laminin-111 stimulates proliferation of mouse embryonic stem cells through a reduction of gap junctional intercellular communication via RhoA-mediated Cx43 phosphorylation and dissociation of Cx43/ZO-1/drebrin complex.  

PubMed

Gap junctions within extracellular matrix (ECM)-defined boundaries ensure synchronous activity between cells destined to become functional mediators that regulate cell behavior. However, the role of ECM in connexin (Cx) function in mouse embryonic stem cells (mESCs) has not been elucidated. Therefore, we examined the role of laminin-111 in the control of Cx43 functions and related signal pathways in mESCs. ECM components (laminin-111, fibronectin, and collagen I) increased Cx43 phosphorylation and decreased Lucifer yellow (Ly) diffusion. In addition, laminin-111 increased the proliferation index through reduction of gap junctional intercellular communication (GJIC), which was confirmed by 18?-glycyrrhetinic acid (18?-GA). Laminin-111 increased phosphorylation of focal adhesion kinase (FAK)/Src and protein kinase C (PKC), which were inhibited by integrin ?1 antibody (Ab) and laminin receptor-1 (LR-1) Ab, respectively. In addition, inhibition of both FAK/Src and PKC blocked Cx43 phosphorylation. Laminin-111 increased the Ras homolog gene family, member A (RhoA) activation, which was blocked by FAK/Src and PKC inhibitors, suggesting the existence of parallel pathways that merge at RhoA. Inhibition of RhoA reversed the laminin-111-induced increase of Cx43 phosphorylation and reduction of GJIC. Laminin-111 also stimulated the dissociation of Cx43/ZO-1 complex followed by disruption of Cx43/drebrin and Cx43/F-actin complexes, which were reversed by C3 (RhoA inhibitor). ZO-1 small interfering (si) RNA significantly decreased Ly diffusion. Moreover, laminin-111 decreased Cx43 labeling at the intercellular junction, whereas pretreatment with degradation inhibitors (lysosomal protease inhibitor, chloroquine; proteasome inhibitor, lactacystin) increased Cx43 expression, reversely. In conclusion, laminin-111 stimulated mESC proliferation through a reduction of GJIC via RhoA-mediated Cx43 phosphorylation and Cx43/ZO-1/drebrin complex instability-mediated Cx43 degradation. PMID:22150760

Suh, Han Na; Kim, Mi Ok; Han, Ho Jae

2012-07-20

75

Differential Modulation of Integrin Receptors and Extracellular Matrix Laminin by Transforming Growth Factor-? 1 in Rat Alveolar Epithelial Cells  

Microsoft Academic Search

The transforming growth factors-? (TGFs-?) family of genes plays important roles in cell growth and differentiation in many cell types. TGF? modulates the synthesis and accumulation of extracellular matrix (ECM) components and the expression of cell surface receptors for ECM components. TGF? is increased in alveolar lining fluid during inflammatory reactions of the lung and has been identified in alveolar

Niranjan M. Kumar; S. L. Sigurdson; D. Sheppard; Jamson S. Lwebuga-Mukasa

1995-01-01

76

Simplified Purification Procedure of Laminin-332 and Laminin-511 from Human Cell Lines  

PubMed Central

Laminins are glycoproteins expressed in the basement membrane of multiple epithelial tissues. Previously described purification procedures for the human laminin variants laminin-5 (LN-332) and laminin-10 (LN-511) use tissue as starting material and have multiple steps. We demonstrate a two-step laminin immunoaffinity purification method to produce consistent quantities of intact and biologically active LN-332 and LN-511 from human keratinocyte (HaCaT) and human lung carcinoma (A549) cell lines, respectively. The purification of LN-332 and LN-551 was demonstrated by PAGE analysis, silver staining and Western blot analysis. The purification procedure includes instruction on removing a cell adhesion contaminant known as galectin-3 binding protein from purified LN-511. The biological activity of purified laminin was tested in a standard cell adhesion assay and compared to commercially available LN-111. This rapid and reproducible purification method will contribute to understanding the role of LN-332 and LN-511 in cell behavior, signaling and gene expression.

Sroka, Isis C.; Chen, Man Ling; Cress, Anne E.

2008-01-01

77

Seventeen copies of the human 37 kDa laminin receptor precursor/p40 ribosome-associated protein gene are processed pseudogenes arisen from retropositional events.  

PubMed

A cDNA coding for a 37 kDa polypeptide has been identified in several species as both the potential precursor of the 67 kDa laminin receptor (37LRP) and a putative ribosome-associated protein (p40). Interestingly, increased expression of this polypeptide (37LRP/p40) is consistently observed in invasive and metastatic cancer cells and is associated with poor prognosis. Southern-blot analysis of human genomic DNA predicted multiple copies of the 37LRP/p40 gene. In this study, we report that the number of copies of this sequence in the human genome is 26 +/- 2. We have sequenced and analyzed 19 genomic clones corresponding to the 37LRP/p40 gene and found that they were all processed pseudogenes. They all lack intronic sequences and show multiple genetic alterations leading in some cases to the appearance of stop codons. Moreover, they all bear characteristic features of retroposons as the presence of a poly(A)-tail at their 3' end and short direct repeated flanking DNA sequences. None of the pseudogenes analyzed present cis-elements in their 5' flanking region such as TATA or GC boxes. Our date reveal that over 50% of the 37LRP/p40 gene copies are pseudogenes most probably generated by retropositional events. The finding of multiple pseudogenes for the 37LRP/p40 suggests that the accumulation of several copies of this gene might have given a survival advantage to the cell in the course of evolution. PMID:8605257

Jackers, P; Clausse, N; Fernandez, M; Berti, A; Princen, F; Wewer, U; Sobel, M E; Castronovo, V

1996-02-01

78

Sarcolemma instability during mechanical activity in Largemyd cardiac myocytes with loss of dystroglycan extracellular matrix receptor function  

PubMed Central

The abnormal glycosylation and loss of extracellular matrix receptor function of the protein dystroglycan (DG) lead to the development of muscular dystrophy and cardiomyopathy. Dystroglycan is an important receptor for extracellular matrix proteins, such as laminin, in the basement membrane surrounding muscle. Largemyd mice have a null mutation in a gene encoding the glycosyltransferase LARGE that results in abnormal glycosylation of ?-DG and phenotypes similar to those in human ?-DG glycosylation-deficient muscular dystrophy. Here, we show that Largemyd hearts with the loss of DG extracellular matrix receptor function display a cardiomyopathy characterized by myocyte damage in patches of cells positive for membrane impermeant dyes. To examine the cellular mechanisms, we show that isolated adult cardiac myocytes from Largemyd mice retain normal laminin-dependent cell adhesion, cell surface laminin deposition and basement membrane assembly. However, although isolated adult cardiac myocytes with the loss of ?-DG glycosylation adhere normally to laminin substrates both passively and in the presence of mechanical activity, Largemyd myocytes rapidly take up membrane impermeant dye following cyclical cell stretching. Therefore, while other cell surface laminin receptors are likely responsible for myocardial cell adhesion to the basement membrane, DG has a unique function of stabilizing the cardiac myocyte plasma membrane during repetitive mechanical activity by tightly binding the transmembrane dystrophin–glycoprotein complex to the extracellular matrix. This function of DG to stabilize the myocyte membrane during normal physiologic cell length changes is likely critical for the prevention of the myocardial damage and subsequent remodeling observed in ?-DG glycosylation-deficient muscular dystrophies.

Kabaeva, Zhyldyz; Meekhof, Kailyn E.; Michele, Daniel E.

2011-01-01

79

A new role for laminins as modulators of protein toxicity in Caenorhabditis elegans.  

PubMed

Protein misfolding is a common theme in aging and several age-related diseases such as Alzheimer's and Parkinson's disease. The processes involved in the development of these diseases are many and complex. Here, we show that components of the basement membrane (BM), particularly laminin, affect protein integrity of the muscle cells they support. We knocked down gene expression of epi-1, a laminin ?-chain, and found that this resulted in increased proteotoxicity in different Caenorhabditis elegans transgenic models, expressing aggregating proteins in the body wall muscle. The effect could partially be rescued by decreased insulin-like signaling, known to slow the aging process and the onset of various age-related diseases. Our data points to an underlying molecular mechanism involving proteasomal degradation and HSP-16 chaperone activity. Furthermore, epi-1-depleted animals had altered synaptic function and displayed hypersensitivity to both levamisole and aldicarb, an acetylcholine receptor agonist and an acetylcholinesterase inhibitor, respectively. Our results implicate the BM as an extracellular modulator of protein homeostasis in the adjacent muscle cells. This is in agreement with previous research showing that imbalance in neuromuscular signaling disturbs protein homeostasis in the postsynaptic cell. In our study, proteotoxicity may indeed be mediated by the neuromuscular junction which is part of the BM, where laminins are present in high concentration, ensuring the proper microenvironment for neuromuscular signaling. Laminins are evolutionarily conserved, and thus the BM may play a much more causal role in protein misfolding diseases than currently recognized. PMID:22051349

Jensen, Louise T; Møller, Tine H; Larsen, Simon A; Jakobsen, Helle; Olsen, Anders

2012-02-01

80

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.

1997-01-01

81

Identification of Cell Adhesive Sequences in the N-terminal Region of the Laminin ?2 Chain*  

PubMed Central

The laminin ?2 chain is specifically expressed in the basement membrane surrounding muscle and nerve. We screened biologically active sequences in the mouse laminin N-terminal region of ?2 chain using 216 soluble peptides and three recombinant proteins (rec-a2LN, rec-a2LN+, and rec-a2N) by both the peptide- or protein-coated plate and the peptide-conjugated Sepharose bead assays. Ten peptides showed cell attachment activity in the plate assay, and 8 peptides were active in the bead assay. Seven peptides were active in the both assays. Five peptides promoted neurite outgrowth with PC12 cells. To clarify the cellular receptors, we examined the effects of heparin and EDTA on cell attachment to 11 active peptides. Heparin inhibited cell attachment to 10 peptides, and EDTA significantly affected only A2-8 peptide (YHYVTITLDLQQ, mouse laminin ?2 chain, 117–128)-mediated cell attachment. Cell attachment to A2-8 was also specifically inhibited by anti-integrin ?1 and anti-integrin ?2?1 antibodies. These results suggest that A2-8 promotes an integrin ?2?1-mediated cell attachment. The rec-a2LN protein, containing the A2-8 sequence, bound to integrin ?2?1 and cell attachment to rec-a2LN was inhibited by A2-8 peptide. Further, alanine substitution analysis of both the A2-8 peptide and the rec-a2LN+ protein revealed that the amino acids Ile-122, Leu-124, and Asp-125 were involved in integrin ?2?1-mediated cell attachment, suggesting that the A2-8 site plays a functional role as an integrin ?2?1 binding site in the LN module. These active peptides may provide new insights on the molecular mechanism of laminin-receptor interactions.

Hozumi, Kentaro; Ishikawa, Masaya; Hayashi, Takemitsu; Yamada, Yuji; Katagiri, Fumihiko; Kikkawa, Yamato; Nomizu, Motoyoshi

2012-01-01

82

Electron microscopy of an ?-dystroglycan fragment containing receptor sites for lymphocytic choriomeningitis virus and laminin, and use of the receptoid body as a reagent to neutralize virus  

Microsoft Academic Search

We report the electron microscopic structure of an ?-dystroglycan (?-DG) fragment (DGEKFc4) that contains binding sites for lymphocytic choriomeningitis virus (LCMV) and the extracellular matrix (ECM) molecule laminin. In electron microscopic images, DGEKFc4 appears as dumbbell-shaped rods with a length of 7.5 ± 0.5 nM and width of 3 ± 0.3 nM. The C-terminal human Fc allows binding of anti-human

Stefan Kunz; Lesley Calder; Michael B. A Oldstone

2004-01-01

83

Serum laminin in Graves' disease.  

PubMed

We determined serum levels of laminin in 23 patients with Graves' disease (GD) and in 24 patients with toxic nodular goiter (TNG). Elevated levels of soluble laminin were observed in patients with GD prior to treatment (median concentration 1376 ng mL-1 [range 712-2402]), compared to patients with TNG (median 442 ng mL-1 [284-891]), and normal controls (median 492 ng mL-1 [range 235-675], n = 26), respectively. In GD patients serum laminin levels decreased during thiamazole treatment and normalized within 8 weeks of therapy. There was no correlation between serum laminin levels and serum levels of thyroid hormones and/or auto-antibodies, respectively. Whether serum laminin is a marker for alterations of extracellular matrix during GD and release of basement membrane components in the circulation and/or reflects an impaired clearance remains to be elucidated. PMID:7656921

Wenisch, C; Myskiw, D; Narzt, E; Presterl, E; Graninger, W

1995-06-01

84

Neuronal migration on laminin in vitro.  

PubMed

Chick sympathetic (E-9) or telencephalic (E-7) neurons were cultured at low density on poly-DL-ornithine (PORN), poly-L-lysine (POLS), laminin or laminin-covered PORN or POLS and monitored with time-lapse videomicroscopy. Neurons migrated on laminin, or laminin-covered PORN or POLS, but not on PORN or POLS alone. Neuronal migration did not involve interactions with other cells indicating that neurons are capable of independent migration when exposed to a laminin substrate. PMID:1600626

Liang, S; Crutcher, K A

1992-03-20

85

Agrin and Synaptic Laminin Are Required to Maintain Adult Neuromuscular Junctions  

PubMed Central

As synapses form and mature the synaptic partners produce organizing molecules that regulate each other’s differentiation and ensure precise apposition of pre- and post-synaptic specializations. At the skeletal neuromuscular junction (NMJ), these molecules include agrin, a nerve-derived organizer of postsynaptic differentiation, and synaptic laminins, muscle-derived organizers of presynaptic differentiation. Both become concentrated in the synaptic cleft as the NMJ develops and are retained in adulthood. Here, we used mutant mice to ask whether these organizers are also required for synaptic maintenance. Deletion of agrin from a subset of adult motor neurons resulted in the loss of acetylcholine receptors and other components of the postsynaptic apparatus and synaptic cleft. Nerve terminals also atrophied and eventually withdrew from muscle fibers. On the other hand, mice lacking the presynaptic organizer laminin-?4 retained most of the synaptic cleft components but exhibited synaptic alterations reminiscent of those observed in aged animals. Although we detected no marked decrease in laminin or agrin levels at aged NMJs, we observed alterations in the distribution and organization of these synaptic cleft components suggesting that such changes could contribute to age-related synaptic disassembly. Together, these results demonstrate that pre- and post-synaptic organizers actively function to maintain the structure and function of adult NMJs.

Samuel, Melanie A.; Valdez, Gregorio; Tapia, Juan C.; Lichtman, Jeff W.; Sanes, Joshua R.

2012-01-01

86

Laminin promotes neuritic regeneration from cultured peripheral and central neurons  

PubMed Central

The ability of axons to grow through tissue in vivo during development or regeneration may be regulated by the availability of specific neurite-promoting macromolecules located within the extracellular matrix. We have used tissue culture methods to examine the relative ability of various extracellular matrix components to elicit neurite outgrowth from dissociated chick embryo parasympathetic (ciliary ganglion) neurons in serum-free monolayer culture. Purified laminin from both mouse and rat sources, as well as a partially purified polyornithine-binding neurite promoting factor (PNPF-1) from rat Schwannoma cells all stimulate neurite production from these neurons. Laminin and PNPF-1 are also potent stimulators of neurite growth from cultured neurons obtained from other peripheral as well as central neural tissues, specifically avian sympathetic and sensory ganglia and spinal cord, optic tectum, neural retina, and telencephalon, as well as from sensory ganglia of the neonatal mouse and hippocampal, septal, and striatal tissues of the fetal rat. A quantitative in vitro bioassay method using ciliary neurons was used to (a) measure and compare the specific neurite-promoting activities of these agents, (b) confirm that during the purification of laminin, the neurite-promoting activity co- purifies with the laminin protein, and (c) compare the influences of antilaminin antibodies on the neurite-promoting activity of laminin and PNPF-1. We conclude that laminin and PNPF-1 are distinct macromolecules capable of expressing their neurite-promoting activities even when presented in nanogram amounts. This neurite-promoting bioassay currently represents the most sensitive test for the biological activity of laminin.

1983-01-01

87

Mechanism of erythropoietin receptor activation  

Microsoft Academic Search

Erythropoietin receptor (EPOR) is a founding member of the cytokine receptor superfamily [1]. A type I transmembrane protein that binds the ligand erythropoietin (EPO) with high affinity (kDa approximately 400 pM)\\u000a on the surface of erythroid progenitors, EPOR is devoid of catalytic activity. Given that the extracellular domain of EPOR\\u000a only contains two cytokine receptor homology modules (D1 and D2),

Stefan N. Constantinescu

88

Plasminogen Enhances Neuritogenesis on Laminin-1  

PubMed Central

Proteins of the plasminogen activation system are broadly expressed throughout the nervous system and key roles for these proteins in neuronal function have been demonstrated. Recent reports have established that plasminogen is synthesized in neuroendocrine tissues, making this protein and the proteolytic activity of the product of its activation, plasmin, available at sites separated anatomically from circulating, hepatocyte-derived plasminogen. Results with plasminogen deficient humans and mice suggest a role for plasminogen in neuritogenesis. To elucidate the role of the plasminogen activation system in these processes, the function of plasminogen during neuritogenesis and neurite outgrowth was studied. It is shown herein that plasminogen participates in neuritogenesis, as plasmin inhibitors reduced both neurite outgrowth and neurite length in PC-12 cells. Addition of exogenous plasminogen enhanced neurite outgrowth and neurite length in both PC-12 cells and primary cortical neurons. The proteolytic activity of plasmin was required, since mutation of the catalytic serine residue completely abolished the stimulatory activity. Furthermore, mutation of the lysine binding site within kringle 5 of the plasminogen molecule also reduced the neuritogenic activity of plasminogen. Additionally, we demonstrate that plasminogen specifically bound to laminin-1, the interaction resulted in increased plasminogen activation by tissue-type plasminogen activator, and was dependent on a functional lysine binding site within plasminogen kringle 5. Moreover, during NGF-induced neuritogenesis, laminin-1 was degraded and this cleavage was catalyzed by plasmin. This study provides the first direct evidence that plasminogen participates in neurite outgrowth and further suggests that laminin-1 degradation by plasmin contributes to the process of neuritogenesis.

Gutierrez-Fernandez, Ana; Gingles, Neill A.; Bai, Hongdong; Castellino, Francis J.; Parmer, Robert J.; Miles, Lindsey A.

2009-01-01

89

Loss of cell surface laminin anchoring promotes tumor growth and is associated with poor clinical outcomes  

PubMed Central

Perturbations in the composition and assembly of extracellular matrices (ECMs) contribute to progression of numerous diseases, including cancers. Anchoring of laminins at the cell surface enables assembly and signaling of many ECMs, but the possible contributions of altered laminin anchoring to cancer progression remain undetermined. In this study, we investigated the prominence and origins of defective laminin anchoring in cancer cells and its association with cancer subtypes and clinical outcomes. We found loss of laminin anchoring to be widespread in cancer cells. Perturbation of laminin anchoring originated from several distinct defects which all led to dysfunctional glycosylation of the ECM receptor dystroglycan. In aggressive breast and brain cancers, defective laminin anchoring was often due to suppressed expression of the glycosyltransferase LARGE. Reduced expression of LARGE characterized a broad array of human tumors where it was associated with aggressive cancer subtypes and poor clinical outcomes. Notably, this defect robustly predicted poor survival in patients with brain cancers. Restoring LARGE expression repaired anchoring of exogenous and endogenous laminin and modulated cell proliferation and tumor growth. Together, our findings suggest that defects in laminin anchoring occur commonly in cancer cells, are characteristic of aggressive cancer subtypes, and are important drivers of disease progression.

Akhavan, Armin; Griffith, Obi L.; Soroceanu, Liliana; Leonoudakis, Dmitri; Luciani-Torres, Maria Gloria; Daemen, Anneleen; Gray, Joe W.; Muschler, John L.

2012-01-01

90

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

2012-01-01

91

Sulfatide-binding domain of the laminin A chain.  

PubMed

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

1990-07-25

92

Laminin ?2 Chain-Positive Vessels and Epidermal Growth Factor in Lung Neuroendocrine Carcinoma  

PubMed Central

Capillaries expressing the laminin ?2 chain in basement membranes may be considered early developing vessels in normal and neoplastic human tissues. Therefore, we investigated whether up-regulation of this extracellular matrix protein favors transendothelial migration of neoplastic cells and then metastasis. In lung small and large cell neuroendocrine carcinomas, which exhibit a stronger metastatic tendency among carcinomas, laminin ?2 chain-positive vessels were more numerous than in carcinoid tumors and supraglottis, breast, and lung non-small cell carcinomas, suggesting a direct relationship between these vessels and metastasis. In vitro studies showed that epidermal growth factor (EGF) induced a more efficient migration of the AE-2 lung neuroendocrine carcinoma cell line through the purified laminin ?2 chain rather than through the laminin ?1 chain and fibronectin. AE-2 cells constitutively expressed all EGF receptors and the ?6?1 integrin, which is one of the laminin ?2 chain receptors. EGF up-regulated ?6?1 expression in several tumors. In this regard, we show that EGF increased the chemo-kinetic migration of AE-2 cells through EAHY endothelial monolayers, which was inhibited by the anti-?6 integrin chain monoclonal antibody. These data indicate that laminin ?2 chain and ?6?1 may be mutually involved in EGF-dependent migration of AE-2 cells and that laminin ?2 chain-positive vessels may favor metastasis of EGF-dependent tumors.

Vitolo, Domenico; Ciocci, Luciano; Deriu, Gloria; Spinelli, Silvia; Cortese, Stefania; Masuelli, Laura; Morrone, Stefania; Filice, Mary Jo; Coloni, Giorgio Furio; Natali, Pier Giorgio; Baroni, Carlo Davide

2006-01-01

93

Laminin-based cell adhesion anchors microtubule plus ends to the epithelial cell basal cortex through LL5?/?  

PubMed Central

LL5? has been identified as a microtubule-anchoring factor that attaches EB1/CLIP-associating protein (CLASP)–bound microtubule plus ends to the cell cortex. In this study, we show that LL5? and its homologue LL5? (LL5s) colocalize with autocrine laminin-5 and its receptors, integrins ?3?1 and ?6?4, at the basal side of fully polarized epithelial sheets. Depletion of both laminin receptor integrins abolishes the cortical localization of LL5s, whereas LL5 depletion reduces the amount of integrin ?3 at the basal cell cortex. Activation of integrin ?3 is sufficient to initiate LL5 accumulation at the cell cortex. LL5s form a complex with the cytoplasmic tails of these integrins, but their interaction might be indirect. Analysis of the three-dimensional distribution of microtubule growth by visualizing EB1-GFP in epithelial sheets in combination with RNA interference reveals that LL5s are required to maintain the density of growing microtubules selectively at the basal cortex. These findings reveal that signaling from laminin–integrin associations attaches microtubule plus ends to the epithelial basal cell cortex.

Hotta, Azusa; Kawakatsu, Tomomi; Nakatani, Tomoya; Sato, Toshitaka; Matsui, Chiyuki; Sukezane, Taiko; Akagi, Tsuyoshi; Hamaji, Tomoko; Grigoriev, Ilya; Akhmanova, Anna; Takai, Yoshimi

2010-01-01

94

Prostate specific membrane antigen produces pro-angiogenic laminin peptides downstream of matrix metalloprotease-2.  

PubMed

Prostate specific membrane antigen (PSMA) is a pro-angiogenic cell-surface protease that we previously demonstrated regulates blood vessel formation in a laminin and integrin ?1-dependent manner. Here, we examine the principal mechanism of PSMA activation of integrin ?1. We show that digesting laminin sequentially with recombinant matrix metalloprotease-2 (MMP-2) and PSMA generates small peptides that enhance endothelial cell adhesion and migration in vitro. We also provide evidence that these laminin peptides activate adhesion via integrin ?6?1 and focal adhesion kinase. Using an in vivo Matrigel implant assay, we show that these MMP/PSMA-derived laminin peptides also increase angiogenesis in vivo. Together, our results reveal a novel mechanism of PSMA activation of angiogenesis by processing laminin downstream of MMP-2. PMID:23775497

Conway, Rebecca E; Joiner, Kyle; Patterson, Alex; Bourgeois, David; Rampp, Robert; Hannah, Benjamin C; McReynolds, Samantha; Elder, John M; Gilfilen, Hannah; Shapiro, Linda H

2013-10-01

95

A Novel Monoclonal Antibody to Human Laminin ?5 Chain Strongly Inhibits Integrin-Mediated Cell Adhesion and Migration on Laminins 511 and 521  

PubMed Central

Laminins, a large family of ??? heterotrimeric proteins mainly found in basement membranes, are strong promoters of adhesion and migration of multiple cell types, such as tumor and immune cells, via several integrin receptors. Among laminin ? (LM?) chains, ?5 displays the widest tissue distribution in adult life and is synthesized by most cell types. Here, we have generated and characterized five novel monoclonal antibodies (mAbs) to the human LM?5 chain to further study the biological relevance of ?5 laminins, such as laminins 511 (?5?1?1) and 521 (?5?2?1). As detected by ELISA, immunohistochemistry, immunoprecipitation and Western blotting, each antibody displayed unique properties when compared to mAb 4C7, the prototype LM?5 antibody. Of greatest interest, mAb 8G9, but not any other antibody, strongly inhibited ?3?1/?6?1 integrin-mediated adhesion and migration of glioma, melanoma, and carcinoma cells on laminin-511 and, together with mAb 4C7, on laminin-521. Accordingly, mAb 8G9 abolished the interaction of soluble ?3?1 integrin with immobilized laminins 511 and 521. Binding of mAb 8G9 to laminin-511 was unaffected by the other mAbs to the LM?5 chain but largely hindered by mAb 4E10 to a LM?1 chain epitope near the globular domain of laminin-511. Thus, mAb 8G9 defines a novel epitope localized at or near the integrin-binding globular domain of the LM?5 chain, which is essential for cell adhesion and migration, and identifies a potential therapeutic target in malignant and inflammatory diseases.

Wondimu, Zenebech; Omrani, Shahin; Ishikawa, Taichi; Javed, Fawad; Oikawa, Yuko; Juronen, Erkki; Ingerpuu, Sulev; Patarroyo, Manuel

2013-01-01

96

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

PubMed

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

1985-11-01

97

High Resolution Imaging Study of Interactions between the 37 kDa/67 kDa Laminin Receptor and APP, Beta-Secretase and Gamma-Secretase in Alzheimer's Disease  

PubMed Central

Alzheimer's disease (AD) is the most prevalent form of dementia affecting the elderly. Neurodegeneration is caused by the amyloid beta (A?) peptide which is generated from the sequential proteolytic cleavage of the Amyloid Precursor Protein (APP) by the ?– and ?- secretases. Previous reports revealed that the 37 kDa/67 kDa laminin receptor (LRP/LR) is involved in APP processing, however, the exact mechanism by which this occurs remains largely unclear. This study sought to assess whether LRP/LR interacted with APP, ?- or ?-secretase. Detailed confocal microscopy revealed that LRP/LR showed a strong co-localisation with APP, ?- and ?-secretase, respectively, at various sub-cellular locations. Superresolution Structured Illumination Microscopy (SR-SIM) showed that interactions were unlikely between LRP/LR and APP and ?-secretase, respectively, while there was strong co-localisation between LRP/LR and ?-secretase at this 80 nm resolution. FRET was further employed to assess the possibility of protein-protein interactions and only an interaction between LRP/LR and ?-secretase was found. FLAG co-immunoprecipitation confirmed these findings as LRP/LR co-immunoprecipitated with ?-secretase, but failed to do so with APP. These findings indicate that LRP/LR exerts its influence on A? shedding via a direct interaction with the ?-secretase and possibly an indirect interaction with the ?-secretase.

Jovanovic, Katarina; Loos, Ben; Da Costa Dias, Bianca; Penny, Clement; Weiss, Stefan F. T.

2014-01-01

98

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.

2013-01-01

99

Expression and localization of laminin 5, laminin 10, type IV collagen, and amelotin in adult murine gingiva.  

PubMed

The biochemical composition of the internal and external basal laminae in the junctional epithelium differs significantly, and the precise cellular origin of their respective molecules remains to be determined. In the present study, the expression and localization of three basement membrane-specific molecules-laminin 5 (?2 chain), type IV collagen (?1 chain), and laminin 10 (?5 chain)-and one tooth-specific molecule, amelotin, was analyzed in adult murine gingiva by using in situ hybridization and immunohistochemistry. The results showed that the outermost cells in junctional epithelium facing the tooth enamel strongly expressed laminin 5 mRNA, supporting the immunohistochemical staining data. This suggests that laminin 5 is actively synthesized in junctional epithelial cells and that the products are incorporated into the internal basal lamina to maintain firm epithelial adhesion to the tooth enamel throughout life. Conversely, no amelotin mRNA signals were detected in the junctional epithelial cells, suggesting that the molecules localized on the internal basal lamina are mainly derived from maturation-stage ameloblasts. Weak and sporadic expression of type IV collagen in addition to laminin 10 in the gingiva indicates that these molecules undergo turnover less frequently in adult animals. PMID:24338356

Sawada, Takashi; Yamazaki, Takaki; Shibayama, Kazuko; Kumazawa, Kaido; Yamaguchi, Yoko; Ohshima, Mitsuhiro

2014-06-01

100

Sonic hedgehog-dependent synthesis of laminin ?1 controls basement membrane assembly in the myotome  

PubMed Central

Summary Basement membranes have essential structural and signalling roles in tissue morphogenesis during embryonic development, but the mechanisms that control their formation are still poorly understood. Laminins are key components of basement membranes and are thought to be essential for initiation of basement membrane assembly. Here, we report that muscle progenitor cells populating the myotome migrate aberrantly in the ventral somite in the absence of sonic hedgehog (Shh) signalling, and we show that this defect is due to the failure to form a myotomal basement membrane. We reveal that expression of Lama1, which encodes laminin ?1, a subunit of laminin-111, is not activated in Shh-/- embryos. Recovery of Lama1 expression or addition of exogenous laminin-111 to Shh-/-;Gli3-/- embryos restores the myotomal basement membrane, demonstrating that laminin-111 is necessary and sufficient to initiate assembly of the myotomal basement membrane. This study uncovers an essential role for Shh signalling in the control of laminin-111 synthesis and in the initiation of basement membrane assembly in the myotome. Furthermore, our data indicate that laminin-111 function cannot be compensated by laminin-511.

Anderson, Claire; Thorsteinsdottir, Solveig; Borycki, Anne-Gaelle

2009-01-01

101

Proteolytically Activated Receptor3  

Microsoft Academic Search

Macromolecular assembly and generation of serine proteases on cellular surfaces is critically invloved in regulation of hemostatic, inflammatory, or fibrinolytic pathways. The concept that a number of these serine proteases may effect cellular activation and proliferative responses has engendered an emerging paradigm focusing on the molecular mechanisms regulating cellular\\/protease interactions. Previous data suggest that some of these cellular responses are

Lisa D Cupit; Valentina A Schmidt; Wadie F Bahou

1999-01-01

102

Laminin 332 expression in breast carcinoma.  

PubMed

Laminin 332 (LN332) is a basally expressed extracellular matrix protein that enhances the migration and invasion of breast carcinoma cells. The goal of this study was to examine LN332 expression breast carcinoma. Triple negative breast carcinomas lack estrogen receptor (ER), progesterone receptor (PR) expression and HER2 positivity. Immunohistochemistry for ER, PR, HER2, and dual silver in situ hybridization for the HER2 gene were used to define the phenotype of 243 breast cancers in biopsies or arrays. Immunohistochemistry for LN332 revealed that 70% of triple negative carcinomas stained for LN332. Cytokeratins 5/6 (CK5/6), epidermal growth factor receptor and p63 alone stained fewer triple negative breast carcinomas each, but the combination of LN332 and CK5/6 or epidermal growth factor receptor identified 92% of triple negative breast carcinoma. Of the 163 non-triple negative cases, LN332 was expressed in only 15%. The identification of LN332 in triple negative breast carcinomas is consistent with gene profiling studies showing its expression among breast carcinomas with a basal phenotype. The observation that a proinvasive protein such as LN332 is expressed in breast cancer suggests another mechanism by which the triple negative phenotype could be aggressive. PMID:22427740

Kwon, Soon-Young; Chae, Seoung W; Wilczynski, Sharon P; Arain, Ahmad; Carpenter; Philip M

2012-03-01

103

Laminin peptide YIGSR induces collagen synthesis in Hs27 human dermal fibroblasts  

SciTech Connect

Highlights: Black-Right-Pointing-Pointer We identify a function of the YIGSR peptide to enhance collagen synthesis in Hs27. Black-Right-Pointing-Pointer YIGSR peptide enhanced collagen type 1 synthesis both of gene and protein levels. Black-Right-Pointing-Pointer There were no changes in cell proliferation and MMP-1 level in YIGSR treatment. Black-Right-Pointing-Pointer The YIGSR effect on collagen synthesis mediated activation of FAK, pyk2 and ERK. Black-Right-Pointing-Pointer The YIGSR-induced FAK and ERK activation was modulated by FAK and MEK inhibitors. -- Abstract: 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 {beta}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 67 kDa 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.

Yoon, Jong Hyuk; Kim, Jaeyoon; Lee, Hyeongjoo [NovaCell Technology Inc., Pohang, Kyungbuk 790-784 (Korea, Republic of)] [NovaCell Technology Inc., Pohang, Kyungbuk 790-784 (Korea, Republic of); Kim, So Young [Department of Dermatology, Chung-Ang University College of Medicine, Seoul 156-756 (Korea, Republic of) [Department of Dermatology, Chung-Ang University College of Medicine, Seoul 156-756 (Korea, Republic of); Department of Convergence Medicine and Pharmaceutical Biosciences, Graduate School, Chung-Ang University, Seoul 156-756 (Korea, Republic of); Jang, Hwan-Hee [Functional Food and Nutrition Division, Department of Agrofood Resources, Rural Development Administration, Suwon 441-853 (Korea, Republic of)] [Functional Food and Nutrition Division, Department of Agrofood Resources, Rural Development Administration, Suwon 441-853 (Korea, Republic of); Ryu, Sung Ho [Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology (POSTECH), Pohang, Kyungbuk 790-784 (Korea, Republic of)] [Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology (POSTECH), Pohang, Kyungbuk 790-784 (Korea, Republic of); Kim, Beom Joon [Department of Dermatology, Chung-Ang University College of Medicine, Seoul 156-756 (Korea, Republic of) [Department of Dermatology, Chung-Ang University College of Medicine, Seoul 156-756 (Korea, Republic of); Department of Convergence Medicine and Pharmaceutical Biosciences, Graduate School, Chung-Ang University, Seoul 156-756 (Korea, Republic of); Lee, Taehoon G., E-mail: taehoon@novacelltech.com [NovaCell Technology Inc., Pohang, Kyungbuk 790-784 (Korea, Republic of)

2012-11-23

104

Modulation of matrix metalloproteinase-9 secretion from tumor-associated macrophage-like cells by proteolytically processed laminin-332 (laminin-5).  

PubMed

Macrophages infiltrating tumor tissues (tumor-associated macrophages, TAM) affect the malignant behaviors of tumor cells. We previously reported that monocytes were differentiated into TAM-like cells secreting matrix metalloproteinase (MMP)-9 by co-culture with tumor cells, and that cell adhesion to extracellular matrix (ECM) proteins played a critical role in the differentiation. In this study, we found that the monocyte differentiation was promoted by laminin-332 (laminin-5), a major epithelial ECM component. We also demonstrated that the proteolytic processing of the ?2 chain of laminin-332 was essential for its activity but that the N-terminal short arm of the ?2 chain inhibited MMP-9 secretion. These results indicate that the activity of laminin-332 for monocyte differentiation is dynamically regulated by the proteolytic processing of the ?2 chain. PMID:24292405

Kamoshida, Go; Ogawa, Takashi; Oyanagi, Jun; Sato, Hiroki; Komiya, Eriko; Higashi, Shouichi; Miyazaki, Kaoru; Tsuji, Tsutomu

2014-03-01

105

Laminins and retinal vascular development  

PubMed Central

The mechanisms controlling vascular development, both normal and pathological, are not yet fully understood. Many diseases, including cancer and diabetic retinopathy, involve abnormal blood vessel formation. Therefore, increasing knowledge of these mechanisms may help develop novel therapeutic targets. The identification of novel proteins or cells involved in this process would be particularly useful. The retina is an ideal model for studying vascular development because it is easy to access, particularly in rodents where this process occurs post-natally. Recent studies have suggested potential roles for laminin chains in vascular development of the retina. This review will provide an overview of these studies, demonstrating the importance of further research into the involvement of laminins in retinal blood vessel formation.

Edwards, Malia M.; Lefebvre, Olivier

2013-01-01

106

Laminin 332 processing impacts cellular behavior.  

PubMed

Laminin 332, composed of the ?3, ?3 and ?2 chains, is an epithelial-basement membrane specific laminin variant. Its main role in normal tissues is the maintenance of epithelial-mesenchymal cohesion in tissues exposed to external forces, including skin and stratified squamous mucosa. After being secreted and deposited in the extracellular matrix, laminin 332 undergoes physiological maturation processes consisting in the proteolytic processing of domains located within the ?3 and the ?2 chains. These maturation events are essential for laminin 332 integration into the basement membrane where it plays an important function in the nucleation and maintenance of anchoring structures. Studies in normal and pathological situations have revealed that laminin 332 can trigger distinct cellular events depending on the level of its proteolytic cleavages. In this review, the biological and structural characteristics of laminin 332 domains are presented and we discuss whether they trigger specific functions. PMID:23263634

Rousselle, Patricia; Beck, Konrad

2013-01-01

107

Native Chick Laminin4 Containing the 132 Chain (s-Laminin) Promotes Motor Axon Growth  

Microsoft Academic Search

After denervation of muscle, motor axons reinnervate original synaptic sites. A recombinant frag- ment of the synapse specific laminin 132 chain (s-laminin) was reported to inhibit motor axon growth. Conse- quently, a specific sequence (leucine-arginine- glutamate, LRE) of the laminin 132 chain was proposed to act as a stop signal and to mediate specific reinnerva- tion at the neuromuscular junction

Ralph Brandenberger; Richard A. Kammerer; Jiirgen Engel; Matthias Chiquet

1996-01-01

108

Laminin alpha2 chain-positive vessels and epidermal growth factor in lung neuroendocrine carcinoma: a model of a novel cooperative role of laminin-2 and epidermal growth factor in vessel neoplastic invasion and metastasis.  

PubMed

Capillaries expressing the laminin alpha2 chain in basement membranes may be considered early developing vessels in normal and neoplastic human tissues. Therefore, we investigated whether up-regulation of this extracellular matrix protein favors transendothelial migration of neoplastic cells and then metastasis. In lung small and large cell neuroendocrine carcinomas, which exhibit a stronger metastatic tendency among carcinomas, laminin alpha2 chain-positive vessels were more numerous than in carcinoid tumors and supraglottis, breast, and lung non-small cell carcinomas, suggesting a direct relationship between these vessels and metastasis. In vitro studies showed that epidermal growth factor (EGF) induced a more efficient migration of the AE-2 lung neuroendocrine carcinoma cell line through the purified laminin alpha2 chain rather than through the laminin beta1 chain and fibronectin. AE-2 cells constitutively expressed all EGF receptors and the alpha6beta1 integrin, which is one of the laminin alpha2 chain receptors. EGF up-regulated alpha6beta1 expression in several tumors. In this regard, we show that EGF increased the chemo-kinetic migration of AE-2 cells through EAHY endothelial monolayers, which was inhibited by the anti-alpha6 integrin chain monoclonal antibody. These data indicate that laminin alpha2 chain and alpha6beta1 may be mutually involved in EGF-dependent migration of AE-2 cells and that laminin alpha2 chain-positive vessels may favor metastasis of EGF-dependent tumors. PMID:16507913

Vitolo, Domenico; Ciocci, Luciano; Deriu, Gloria; Spinelli, Silvia; Cortese, Stefania; Masuelli, Laura; Morrone, Stefania; Filice, Mary Jo; Coloni, Giorgio Furio; Natali, Pier Giorgio; Baroni, Carlo Davide

2006-03-01

109

Laminin and Fibronectin in Cell Adhesion: Enhanced Adhesion of Cells from Regenerating Liver to Laminin  

NASA Astrophysics Data System (ADS)

Laminin, a basement membrane glycoprotein isolated from cultures of mouse endodermal cells and rat yolk sac carcinoma cells, promoted the attachment of liver cells obtained from regenerating mouse liver. Cells from normal mouse liver attached readily to dishes coated with fibronectin but attached poorly to surfaces coated with laminin. Both proteins efficiently promoted the attachment of cells from livers undergoing regeneration. After regeneration, the attachment to laminin returned to the low levels found in animals not subjected to partial hepatectomy but attachment to fibronectin remained high. Immunofluorescent staining of sections of normal liver with antilaminin revealed the presence of laminin in or adjacent to the walls of the bile ducts and blood vessels. After induction of regeneration by partial hepatectomy, increased amounts of laminin appeared in the sinusoidal areas. After carbon tetrachloride poisoning, staining for laminin was especially pronounced in the necrotic and postnecrotic areas around the central veins. This additional expression of laminin was transient. It reached a maximum around 5-6 days after the injury and then gradually disappeared. These findings show that laminin is an adhesive protein. The increase of laminin in regenerating liver and the adhesiveness of cells from such livers to laminin suggest a role for laminin in the maintenance of a proper tissue organization during liver regeneration.

Carlsson, Roland; Engvall, Eva; Freeman, Aaron; Ruoslahti, Erkki

1981-04-01

110

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

2010-01-01

111

Matrix metalloproteinase-2 is involved in A549 cell migration on laminin-10/11.  

PubMed

We have reported that laminin-10/11 strongly promotes migration of A549 human lung carcinoma cells by activating the alpha3beta1 integrin-dependent signaling pathway. To elucidate the mechanism involved, we investigated whether matrix metalloproteinases (MMPs) are involved in cell migration on laminin-10/11. Here, we demonstrate that laminin-10/11, but not fibronectin which does not greatly promote A549 cell movement, stimulated MMP-2 secretion approximately 3-fold. The cell migration-promoting activity of laminin-10/11 was down-regulated by an MMP inhibitor. In addition, cell motility was significantly increased when cells adhered to a mixture of fibronectin and laminin-10/11 with a concomitant decrease of focal contacts, compared with those adhering to fibronectin alone. The enhanced cell migration was partially suppressed by the MMP inhibitor. Furthermore, an anti-alpha3 integrin, but not an anti-alpha5 integrin, antibody induced the activated form of MMP-2. These data suggest that MMP-2 may play an important role in A549 cell migration on laminin-10/11 through an alpha3beta1 integrin-dependent pathway. PMID:12147229

Gu, J; Nishiuchi, R; Sekiguchi, K

2002-08-01

112

Molecular basis of cytokine receptor activation.  

PubMed

Cytokines are secreted soluble peptides that precisely regulate multiple cellular functions. Amongst these the GM-CSF/IL-3/IL-5 family of cytokines controls whether hematopoietic cells will survive or apoptose, proliferate, differentiate, migrate, or perform effector functions such as phagocytosis or reactive oxygen species release. Their potent and pleiotropic activities are mediated through binding to high affinity membrane receptors at surprisingly low numbers per cell. Receptor binding triggers a cascade of intracellular signaling events, including reversible phosphorylation of receptor subunits and associated signaling molecules, leading to multiple biological responses, with the prevention of apoptosis or "cell survival" being a key cellular function that underpins all others. Many chronic inflammatory diseases and a number of haematological malignancies are driven by deregulated GM-CSF, IL-3, or IL-5 cytokine receptor signaling, highlighting their importance in disease. A major step in understanding how these cytokine receptors function is to elucidate their three dimensional structure and to relate this to the many signaling pathways emanating from their receptors. We have recently solved the structure of the human GM-CSF receptor complexed to GM-CSF which revealed distinct forms of receptor assembly: a hexamer that comprises two molecules each of GM-CSF, GM-CSF receptor alpha chain and GM-CSF receptor beta chain; and an unexpected dodecamer in which two hexameric complexes associate through a novel site 4. This latter form is necessary to bring JAK2 molecules sufficiently close together to enable full receptor activation. In this review we focus on the most recent insights in cytokine receptor signaling, and in receptor assembly. The stage is now set to link distinct forms of cytokine receptor assembled structures to specific forms of cytokine receptor signaling and function. Armed with this knowledge it may be possible to map distinct cytokine receptor signaling pathways from the cell surface to the cell nucleus which may themselves become new therapeutic targets. PMID:20540154

Lopez, Angel F; Hercus, Timothy R; Ekert, Paul; Littler, Dene R; Guthridge, Mark; Thomas, Daniel; Ramshaw, Hayley S; Stomski, Frank; Perugini, Michelle; D'Andrea, Richard; Grimbaldeston, Michele; Parker, Michael W

2010-07-01

113

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

114

Laminin5 Expression Is Independent of the Injury and the Microenvironment During Reepithelialization of Wounds  

Microsoft Academic Search

SUMMARY We examined the expression of laminin-5 and its integrin receptors during reepithelialization of human wounds. We used suction blisters of skin as a model of kerati- nocyte migration on a basement membrane matrix and mucosal full-thickness wounds as a model in which keratinocytes migrate in a provisional matrix. An animal model, in which human epidermal keratinocytes were injected into

Tiina Kainulainen; Lari Häkkinen; Sara Hamidi; Kirsi Larjava; Matti Kallioinen; Juha Peltonen; Tuula Salo; Hannu Larjava; Aarne Oikarinen

115

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.

1986-05-01

116

Amino-terminal fragments of laminin ?2 chain retract vascular endothelial cells and increase vascular permeability.  

PubMed

Laminin ?2 (Lm?2) chain, a subunit of laminin-332, is a typical molecular marker of invading cancer cells, and its expression correlates with poor prognosis of cancer patients. It was previously found that forced expression of Lm?2 in cancer cells promotes their invasive growth in nude mice. However, the mechanism of the tumor-promoting activity of Lm?2 remains unknown. Here we investigated the interaction between Lm?2 and vascular endothelial cells. When treated with an N-terminal proteolytic fragment of ?2 (?2pf), HUVECs became markedly retracted or shrunken. The overexpression of Lm?2 or treatment with ?2pf stimulated T-24 bladder carcinoma cells to invade into the HUVEC monolayer and enhanced their transendothelial migration in vitro. Moreover, ?2pf increased endothelial permeability in vitro and in vivo. As the possible mechanisms, ?2pf activated ERK and p38 MAPK but inactivated Akt in HUVECs. Such effects of ?2pf led to prominent actin stress fiber formation in HUVECs, which was blocked by a ROCK inhibitor. In addition, ?2pf induced delocalization of VE-cadherin and ?-catenin from the intercellular junction. As possible receptors, ?2pf interacted with heparan sulfate proteoglycans on the surface of HUVECs. Moreover, we localized the active site of ?2pf to the N-terminal epidermal growth factor-like repeat. These data suggest that the interaction between ?2pf and heparan sulfate proteoglycans induces cytoskeletal changes of endothelial cells, leading to the loss of endothelial barrier function and the enhanced transendothelial migration of cancer cells. These activities of Lm?2 seem to support the aberrant growth of cancer cells. PMID:24238220

Sato, Hiroki; Oyanagi, Jun; Komiya, Eriko; Ogawa, Takashi; Higashi, Shouichi; Miyazaki, Kaoru

2014-02-01

117

Laminin ?1 Regulates Age-Related Mesangial Cell Proliferation and Mesangial Matrix Accumulation through the TGF-? Pathway.  

PubMed

Laminin ?1 (LAMA1), a subunit of the laminin-111 basement membrane component, has been implicated in various biological functions in vivo and in vitro. Although LAMA1 is present in kidney, its roles in the kidney are unknown because of early embryonic lethality. Herein, we used a viable conditional knockout mouse model with a deletion of Lama1 in the epiblast lineage (Lama1(CKO)) to study the role of LAMA1 in kidney development and function. Adult Lama1(CKO) mice developed focal glomerulosclerosis and proteinuria with age. In addition, mesangial cell proliferation was increased, and the mesangial matrix, which normally contains laminin-111, was greatly expanded. In vitro, mesangial cells from Lama1(CKO) mice exhibited significantly increased proliferation compared with those from controls. This increased proliferation was inhibited by the addition of exogenous LAMA1-containing laminin-111, but not by laminin-211 or laminin-511, suggesting a specific role for LAMA1 in regulating mesangial cell behavior. Moreover, the absence of LAMA1 increased transforming growth factor (TGF)-?1-induced Smad2 phosphorylation, and inhibitors of TGF-?1 receptor I kinase blocked Smad2 phosphorylation in both control and Lama1(CKO) mesangial cells, indicating that the increased Smad2 phosphorylation occurred in the absence of LAMA1 via the TGF-?1 receptor. These findings suggest that LAMA1 plays a critical role in kidney function and kidney aging by regulating the mesangial cell population and mesangial matrix deposition through TGF-?/Smad signaling. PMID:24720953

Ning, Liang; Kurihara, Hidetake; de Vega, Susana; Ichikawa-Tomikawa, Naoki; Xu, Zhuo; Nonaka, Risa; Kazuno, Saiko; Yamada, Yoshihiko; Miner, Jeffrey H; Arikawa-Hirasawa, Eri

2014-06-01

118

Constitutive Activity of Neural Melanocortin Receptors  

Microsoft Academic Search

The two neural melanocortin receptors (MCRs), melanocortin-3 and -4 receptors (MC3R and MC4R), are G protein-coupled receptors expressed primarily in the brain that regulate different aspects of energy homeostasis. The MCRs are unique in having endogenous antagonists, agouti and agouti-related protein (AgRP). These antagonists were later shown to be inverse agonists. The MC3R has little or no constitutive activity, whereas

Ya-Xiong Tao; Hui Huang; Zhi-Qiang Wang; Fan Yang; Jessica N. Williams; Gregory V. Nikiforovich

2010-01-01

119

Protease activated receptors modulate aortic vascular tone  

Microsoft Academic Search

The effect of agonists of the known protease activated receptors (PAR), the thrombin and the PAR-2 receptors, on vasoactive mediator release and vascular tone were studied using rings of rat aorta. Stimulation of aortic rings with the thrombin receptor agonist, Trap-14, or the PAR-2 agonist, SLIGRL, resulted in a rapid release of nitric oxide. Trap-14 and SLIGRL-induced nitric oxide release

Harold I. Magazine; Jonathan M. King; Kamal D. Srivastava

1996-01-01

120

Laminin and Fibronectin in Cell Adhesion: Enhanced Adhesion of Cells from Regenerating Liver to Laminin  

Microsoft Academic Search

Laminin, a basement membrane glycoprotein isolated from cultures of mouse endodermal cells and rat yolk sac carcinoma cells, promoted the attachment of liver cells obtained from regenerating mouse liver. Cells from normal mouse liver attached readily to dishes coated with fibronectin but attached poorly to surfaces coated with laminin. Both proteins efficiently promoted the attachment of cells from livers undergoing

Roland Carlsson; Eva Engvall; Aaron Freeman; Erkki Ruoslahti

1981-01-01

121

Artificial laminin polymers assembled in acidic pH mimic basement membrane organization.  

PubMed

Natural laminin matrices are formed on cell membranes by a cooperative process involving laminin self-polymerization and binding to cognate cellular receptors. In a cell-free system, laminin can self-polymerize, given that a minimal critical concentration is achieved. We have previously described that pH acidification renders self-polymerization independent of protein concentration. Here we studied the ultrastructure of acid-induced laminin polymers using electron and atomic force microscopies. Polymers presented the overall appearance of natural matrices and could be described as homogeneous polygonal sheets, presenting struts of 21 +/- 5 and 86 +/- 3 nm of height, which approximately correspond to the sizes of the short and the long arms of the molecule, respectively. The addition of fragment E3 (the distal two domains of the long arm) did not affect the polymerization in solution nor the formation of adsorbed matrices. On the other hand, the addition of fragment E1', which contains two intact short arms, completely disrupted polymerization. These results indicate that acid-induced polymers, like natural ones, involve only interactions between the short arms. The electrostatic surface map of laminin alpha1 LG4-5 shows that acidification renders the distal end in the long arms exclusively positive, precluding homophylic interactions between them. Therefore, acidification reproduces in vitro, and at a physiological protein concentration, what receptor interaction does in the cellular context, namely, it prevents the long arm from disturbing formation of the homogeneous matrix involving the short arms only. We propose that acid-induced polymers are the best tool to study cellular response to laminin in the future. PMID:18276595

Barroso, Madalena Martins Sant'ana; Freire, Elisabete; Limaverde, Gabriel S C S; Rocha, Gustavo Miranda; Batista, Evander J O; Weissmüller, Gilberto; Andrade, Leonardo Rodrigues; Coelho-Sampaio, Tatiana

2008-04-25

122

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.

2010-01-01

123

Constitutive activity of neural melanocortin receptors.  

PubMed

The two neural melanocortin receptors (MCRs), melanocortin-3 and -4 receptors (MC3R and MC4R), are G protein-coupled receptors expressed primarily in the brain that regulate different aspects of energy homeostasis. The MCRs are unique in having endogenous antagonists, agouti and agouti-related protein (AgRP). These antagonists were later shown to be inverse agonists. The MC3R has little or no constitutive activity, whereas the MC4R has significant constitutive activity that can easily be detected. We describe herein methods for detecting constitutive activities in these receptors and small molecule ligands as inverse agonists. AgRP is an inverse agonist for both MC3R and MC4R. We also provide models for the constitutively active MC4R mutants. PMID:21036237

Tao, Ya-Xiong; Huang, Hui; Wang, Zhi-Qiang; Yang, Fan; Williams, Jessica N; Nikiforovich, Gregory V

2010-01-01

124

Monoacylglycerols Activate Capsaicin Receptor, TRPV1  

Microsoft Academic Search

Transient receptor potential vanilloid subtype 1 (TRPV1) is known as capsaicin (CAP) receptor and activated by CAP. Activation\\u000a of TRPV1 by CAP increases energy expenditure and thermogenesis in rodents or human. Therefore, TRPV1 may be target for energy\\u000a expenditure enhancement and thermogenesis. To search for novel TRPV1 agonist, we screened 19 types of foods by using TRPV1-expressing\\u000a HEK293 cells. TRPV1

Yusaku Iwasaki; Orine Saito; Manabu Tanabe; Kimiko Inayoshi; Kenji Kobata; Shuichi Uno; Akihito Morita; Tatsuo Watanabe

2008-01-01

125

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

PubMed Central

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.

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

2012-01-01

126

Interdomain movements in metabotropic glutamate receptor activation.  

PubMed

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

2011-09-13

127

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

2011-01-01

128

Expression of laminin chains during myogenic differentiation.  

PubMed

Expression of two Ae-related chains of the extracellular matrix glycoprotein laminin was induced as multipotent C3H10T1/2 mouse embryo fibroblasts differentiated into myoblasts and myofibers. C3H10T1/2 fibroblasts expressed the B1e (M(r) = 215,000) and B2e (M(r) = 205,000) laminin chains based on metabolic radiolabeling, immunoprecipitation, peptide mapping, and mRNA analysis. In contrast, myoblasts derived from C3H10T1/2 fibroblasts treated with DNA demethylating agents or transfected with the cDNA encoding MyoD expressed the Ae (M(r) = 400,000) and a novel Ae-related laminin chain (designated Ac3h, M(r) = 350,000) in addition to the B1e and B2e chains. Expression of the Ae and Ac3h chains paralleled the capacity for myofiber formation in six additional C3H10T1/2 myoblast clones with varied potentials for terminal differentiation and coincided with a switch in laminin isoforms from those of M(r) = 850,000 synthesized by C3H10T1/2 fibroblasts to those of M(r) = 900,000-950,000 synthesized by C3H10T1/2 myoblasts and myofibers. Cultures of mouse C2C12, mouse BC3H1, rat L6, and primary mouse myoblasts also synthesized the Ae, Ac3h, B1e, and B2e laminin chains. The results demonstrate that expression of the Ae and Ac3h laminin chains is associated with expression of MyoD and the mammalian myogenic differentiation program. PMID:7510707

Kroll, T G; Peters, B P; Hustad, C M; Jones, P A; Killen, P D; Ruddon, R W

1994-03-25

129

Regeneration of Aplysia Bag Cell Neurons is Synergistically Enhanced by Substrate-Bound Hemolymph Proteins and Laminin  

PubMed Central

We have investigated Aplysia hemolymph as a source of endogenous factors to promote regeneration of bag cell neurons. We describe a novel synergistic effect between substrate-bound hemolymph proteins and laminin. This combination increased outgrowth and branching relative to either laminin or hemolymph alone. Notably, the addition of hemolymph to laminin substrates accelerated growth cone migration rate over ten-fold. Our results indicate that the active factor is either a high molecular weight protein or protein complex and is not the respiratory protein hemocyanin. Substrate-bound factor(s) from central nervous system-conditioned media also had a synergistic effect with laminin, suggesting a possible cooperation between humoral proteins and nervous system extracellular matrix. Further molecular characterization of active factors and their cellular targets is warranted on account of the magnitude of the effects reported here and their potential relevance for nervous system repair.

Hyland, Callen; Dufrense, Eric R.; Forscher, Paul

2014-01-01

130

Regeneration of Aplysia Bag Cell Neurons is Synergistically Enhanced by Substrate-Bound Hemolymph Proteins and Laminin  

NASA Astrophysics Data System (ADS)

We have investigated Aplysia hemolymph as a source of endogenous factors to promote regeneration of bag cell neurons. We describe a novel synergistic effect between substrate-bound hemolymph proteins and laminin. This combination increased outgrowth and branching relative to either laminin or hemolymph alone. Notably, the addition of hemolymph to laminin substrates accelerated growth cone migration rate over ten-fold. Our results indicate that the active factor is either a high molecular weight protein or protein complex and is not the respiratory protein hemocyanin. Substrate-bound factor(s) from central nervous system-conditioned media also had a synergistic effect with laminin, suggesting a possible cooperation between humoral proteins and nervous system extracellular matrix. Further molecular characterization of active factors and their cellular targets is warranted on account of the magnitude of the effects reported here and their potential relevance for nervous system repair.

Hyland, Callen; Dufrense, Eric R.; Forscher, Paul

2014-04-01

131

Roles for Laminin in Embryogenesis: Exencephaly, Syndactyly, and Placentopathy in Mice Lacking the Laminin a 5 Chain  

Microsoft Academic Search

Laminins are the major noncollagenous gly- coproteins of all basal laminae (BLs). They are a \\/ b \\/ g heterotrimers assembled from 10 known chains, and they subserve both structural and signaling roles. Previ- ously described mutations in laminin chain genes result in diverse disorders that are manifested postnatally and therefore provide little insight into laminin's roles in embryonic development.

Jeffrey H. Miner; Jeanette Cunningham; Joshua R. Sanes

132

Co-Activation of Epidermal Growth Factor Receptor and c-MET Defines a Distinct Subset of Lung Adenocarcinomas  

PubMed Central

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.

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

2010-01-01

133

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

2013-01-01

134

The physiology of vitamin D receptor activation.  

PubMed

Vitamin D is a steroid hormone that has long been known for its important role in regulating body levels of calcium and phosphorus, and in mineralization of bone. In addition to its endocrine effects, vitamin D has important autocrine/paracrine roles. The last step in the activation of vitamin D, the hydroxylation on carbon 1, takes place mainly in the kidney. However, extrarenal sites showing 1alpha-hydroxylase activity have been also found. The hormonally active form of vitamin D (1,25(OH)-D(3) or calcitriol) mediates its biological effects by binding to the vitamin D receptor, which then translocates to the nuclei of the cell and binds to specific DNA sites to modify the expression of target genes. After activation of the receptor, the protein changes its tridimensional conformation, this change being the key process in order to exert its nuclear actions. Several steps take place in order to increase or decrease the transcription rate of a target gene. First, homodimerization of the vitamin D receptor or heterodimerization with the retinoic X receptor allows the complex to go into the nucleus and bind to the DNA. Then several proteins are recruited to the complex that either increase or decrease chromatin condensation acting then as corepresors or coactivators, respectively, and decreasing or increasing the target gene transcription. The coactivators bind several extra proteins that build a bridge to the basal transcription machinery. Therefore, little changes in the receptor's tridimensional change elicted by the activator can lead to differences in protein recruitment and, thus, in gene transactivation. Furthermore, differences in the cellular environment can yield different responses to the same activator. This characteristic of the nuclear receptors makes them a good candidate as a valuable therapeutic target. PMID:19494615

Valdivielso, Jose M

2009-01-01

135

Endosomal Signaling by Protease-activated Receptors  

PubMed Central

Protease-activated receptors (PARs) are a family of G protein-coupled receptors (GPCRs) that are uniquely activated by proteolysis. There are four members of the PAR family including: PAR1, PAR2, PAR3 and PAR4. PARs are expressed primarily in cells of the vasculature and elicit cellular responses to coagulant and anti-coagulant proteases. PAR1 exemplifies the unusual proteolytic mechanism of receptor activation. Thrombin binds to and cleaves the N-terminal exodomain of PAR1 generating a new N-terminus that functions as a tethered ligand. The N-terminal tethered ligand domain of PAR1 binds intramolecularly to the receptor to trigger transmembrane signaling and cannot diffuse away. Similar to other GPCRs, activation of PARs promotes coupling to heterotrimeric G proteins at the plasma membrane. After activation, PARs are rapidly internalized to endosomes and then sorted to lysosomes and degraded. Internalization functions to uncouple PARs from heterotrimeric G proteins at the cell surface. However, recent studies indicate that activated internalized PARs signal from endosomes through the recruitment of ?-arrestins and potentially other pathways. Here we provide an overview of methods and strategies used to examine endosomal signaling by PARs.

Grimsey, Neil; Lin, Huilan; Trejo, JoAnn

2014-01-01

136

Keratinocyte-targeted expression of human laminin ?2 rescues skin blistering and early lethality of laminin ?2 deficient mice.  

PubMed

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. PMID:23029085

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

2012-01-01

137

Myofibrillar and cytoskeletal assembly in neonatal rat cardiac myocytes cultured on laminin and collagen  

Microsoft Academic Search

Neonatal rat cardiomyocytes were cultured on extracellular matrix components laminin and collagens I+III to examine effects of extracellular matrix on the assembly of cytoskeletal proteins during myofibrillogenesis. Myofibril assembly was visualized by immunofluorescence of marker proteins for myofibrils (f-actin for I bands and a-actinin for Z bands), focal adhesions (vinculin), and transmembrane extracellular matrix receptors (ß1 integrin) as cells spread

Lula L. Hilenski; Louis Terracio; Thomas K. Borg

1991-01-01

138

Characterization of a Tight Molecular Complex between Integrin ?6?4 and Laminin5 Extracellular Matrix  

Microsoft Academic Search

In many adult epithelia, e.g., epidermis or intestine, adhesion of epithelial cells to basement membrane requires the integrin ?6?4 and laminin-5 (Ln-5). In the absence of one or the other, extensive blistering and exfoliation occur. While ?6?4 was reported to be a receptor for Ln-5, this interaction is poorly understood. We characterize complexes between ?6?4 and Ln-5 in cell-free preparations

Jutta Falk-Marzillier; Susan Z. Domanico; Anthony Pelletier; Lina Mullen; Vito Quaranta

1998-01-01

139

Vimentin and laminin expression is associated with basal-like phenotype in both sporadic and BRCA1-associated breast carcinomas  

PubMed Central

Aims To determine whether basal?like phenotype and vimentin and/or laminin are related in both sporadic/familial (BRCA1 or BRCA2 mutated) tumours. Methods 230 non?familial and 28 hereditary node?negative invasive breast carcinomas were immunohistochemically analysed for oestrogen receptors (ER), progesterone receptors (PR), cytokeratin 5/6 (CK5/6), epidermal growth factor receptors (EGFR), Ki67, p53, vimentin and laminin, using tissue microarrays. Tumours were considered to have basal?like phenotype if they were ER negative and HER2 negative, but positive for CK5/6 and/or EGFR. Results In sporadic tumours, vimentin expression was found in 77.8% cases with basal?like phenotype and 15.5% of non?basal cases (p<0.001). In familial cases, vimentin was expressed in 83.3% basal?like cancers and 16.7% of non?basal tumours (p<0.001). Vimentin expression was more frequent in BRCA1 than BRCA2 mutation carriers. Vimentin expressing tumours were associated with poor prognosis (p?=?0.012) among patients not receiving adjuvant chemotherapy and showed a trend for local recurrence or visceral but not bone metastasis (p?=?0.021). Laminin expression was also related to basal?like phenotype in both sporadic/familial cases (p<0.001 and p?=?0.007, respectively), but neither with prognosis nor recurrence pattern in sporadic cancers. Conclusions Vimentin and laminin expression is associated with basal?like phenotype in breast cancer. Expression of vimentin and laminin is characteristic of BRCA1 associated tumours. Since vimentin and laminin staining is widely used by pathologists for diagnostic purposes, thus demonstrating the robustness of their specific antibodies, the immunohistochemical evaluation of these two molecules could be used in identification of basal?like breast tumours in both sporadic/familial cases.

Rodriguez-Pinilla, Socorro Maria; Sarrio, David; Honrado, Emiliano; Moreno-Bueno, Gema; Hardisson, David; Calero, Francisco; Benitez, Javier; Palacios, Jose

2007-01-01

140

Automated Methods of Detecting Receptor Activity.  

National Technical Information Service (NTIS)

Methods of detecting G protein-coupled receptor (GPCR) activity in vitro and in vivo are provided. In one embodiment, the method includes providing at least one cell that expresses a GPCR and a plurality of conjugated proteins. Each of the plurality of co...

L. S. Barak R. H. Oakley

2005-01-01

141

Using Nuclear Receptor Activity to Stratify Hepatocarcinogens  

EPA Science Inventory

Nuclear receptors (NR) are a superfamily of ligand-activated transcription factors that control a range of cellular processes. Persistent stimulation of some NR is a non-genotoxic mechanism of rodent liver cancer with unclear relevance to humans. Here we report on a systematic an...

142

Glycoconjugates and cell adhesion: the adhesive proteins laminin, thrombospondin and von Willebrand's factor bind specifically to sulfated glycolipids.  

PubMed

The adhesive glycoproteins laminin, thrombospondin and von Willebrand's factor bind specifically and with high affinity to sulfated glycolipids, and it is this binding that probably accounts for their ability to agglutinate glutaraldehyde-fixed erythrocytes. The 3 proteins differ, however, in the effect of sulfated polysaccharides on their binding to sulfatides. Fucoidan strongly inhibits binding of both laminin and thrombospondin, but not of von Willebrand's factor, suggesting the involvement of laminin or thrombospondin or other unknown sulfatide-binding proteins in specific cell interactions that are also inhibited by fucoidan. Thrombospondin adsorbed onto plastic promotes the attachment and spreading of G361 melanoma cells. Interestingly, fucoidan and an antibody directed against the sulfatide-binding domain of thrombospondin selectively inhibit spreading but not attachment. Sulfatides, but not neutral glycolipids or gangliosides, when adsorbed onto plastic also promote attachment and spreading of G361 melanoma cells. Direct adhesion of G361 cells requires high densities of sulfatide. In the presence of laminin, however, specific adhesion of G361 cells to sulfatide is strongly stimulated and requires only low densities of adsorbed lipid, suggesting that laminin mediates adhesion by cross-linking receptors on the melanoma cell surface to sulfatide adsorbed onto the plastic. Although thrombospondin binds to sulfatide and to G361 cells, it does not enhance but rather inhibits direct and laminin-dependent G361 cell adhesion to sulfatide, presumably because it is unable to bind simultaneously to ligands on opposing surfaces. Thus, sulfated glycoconjugates participate in both laminin- and thrombospondin-mediated cell adhesion, but their mechanisms of interaction are different. PMID:3149529

Ginsburg, V; Roberts, D D

1988-11-01

143

Laminin in the male germ cells of Drosophila  

PubMed Central

To study genes that may be crucial for the male germ cell development of Drosophila we screened a cDNA expression library with a polyclonal antiserum against testis proteins of Drosophila hydei. We identified a cDNA fragment that exhibited a complete sequence similarity with the cDNA of the laminin B2 chain, an important component of the extracellular matrix. Transcripts of laminin B2 were detected in the RNA of male germ cells with the polymerase chain reaction and by in situ hybridization. We studied the reaction of different polyclonal antibodies including those against a Drosophila laminin B2-lac fusion protein, the entire Drosophila laminin complex, or against the mouse laminin complex and against laminin A and B1 chains with specific structures in developing male germ cells of Drosophila. Antigenic sites against laminin B2 were found in the lampbrush loops in primary spermatocyte nuclei, in nuclei of spermatids, and in heads of spermatozoa. The axonemes of elongating spermatids react with antibodies against the Drosophila laminin B1, B2 and laminin A chains. The possible biological functions of the laminin in the male germ cells of Drosophila are discussed.

1992-01-01

144

Pyrazolone compounds and thrombopoietin receptor activator  

US Patent & Trademark Office Database

A preventive, therapeutic or improving agent for diseases against which activation of the thrombopoietin receptor is effective or a platelet increasing agent, which contains a thrombopoietin receptor activator represented by the formula (1): wherein A is a C?2-14#191 aryl group, B is a hydrogen atom, a C?1-6#191 alkyl group, a C?1-3#191 alkyl group substituted with one or more fluorine atoms or a C?2-14#191 aryl group, D is a hydrogen atom, a C?1-6#191 alkyl group, a C?1-3#191 alkyl group substituted with one or more fluorine atoms or a C?2-14#191 aryl group, and E is a C?2-14#191 aryl group, a tautomer, prodrug or pharmaceutically acceptable salt of the activator or a solvate thereof, as an active ingredient. ##STR00001##

2011-11-08

145

The Heterotrimeric Laminin Coiled-Coil Domain Exerts Anti-Adhesive Effects and Induces a Pro-Invasive Phenotype  

PubMed Central

Laminins are large heterotrimeric cross-shaped extracellular matrix glycoproteins with terminal globular domains and a coiled-coil region through which the three chains are assembled and covalently linked. Laminins are key components of basement membranes, and they serve as attachment sites for cell adhesion, migration and proliferation. In this work, we produced a recombinant fragment comprising the entire laminin coiled-coil of the ?1-, ?1-, and ?1-chains that assemble into a stable heterotrimeric coiled-coil structure independently of the rest of the molecule. This domain was biologically active and not only failed to serve as a substrate for cell attachment, spreading and focal adhesion formation but also inhibited cell adhesion to laminin when added to cells in a soluble form at the time of seeding. Furthermore, gene array expression profiling in cells cultured in the presence of the laminin coiled-coil domain revealed up-regulation of genes involved in cell motility and invasion. These findings were confirmed by real-time quantitative PCR and zymography assays. In conclusion, this study shows for the first time that the laminin coiled-coil domain displays anti-adhesive functions and has potential implications for cell migration during matrix remodeling.

Santos-Valle, Patricia; Guijarro-Munoz, Irene; Cuesta, Angel M.; Alonso-Camino, Vanesa; Villate, Maider; Alvarez-Cienfuegos, Ana; Blanco, Francisco J.; Sanz, Laura; Alvarez-Vallina, Luis

2012-01-01

146

Epigenetic regulation of pregnane X receptor activity.  

PubMed

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

2013-05-01

147

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

1992-01-01

148

Laminin 332 in junctional epidermolysis bullosa.  

PubMed

Laminin 332 is an essential component of the dermal-epidermal junction, a highly specialized basement membrane zone that attaches the epidermis to the dermis and thereby provides skin integrity and resistance to external mechanical forces. Mutations in the LAMA3, LAMB3 and LAMC2 genes that encode the three constituent polypeptide chains, ?3, ?3 and ?2, abrogate or perturb the functions of laminin 332. The phenotypic consequences are diminished dermal-epidermal adhesion and, as clinical symptoms, skin fragility and mechanically induced blistering. The disorder is designated as junctional epidermolysis bullosa (JEB). This article delineates the signs and symptoms of the different forms of JEB, the mutational spectrum, genotype-phenotype correlations as well as perspectives for future molecular therapies. PMID:23076207

Kiritsi, Dimitra; Has, Cristina; Bruckner-Tuderman, Leena

2013-01-01

149

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))

1988-11-01

150

Biased Signaling of Protease-Activated Receptors  

PubMed Central

In addition to their role in protein degradation and digestion, proteases can also function as hormone-like signaling molecules that regulate vital patho-physiological processes, including inflammation, hemostasis, pain, and repair mechanisms. Certain proteases can signal to cells by cleaving protease-activated receptors (PARs), a family of four G protein-coupled receptors. PARs are expressed by almost all cell types, control important physiological and disease-relevant processes, and are an emerging therapeutic target for major diseases. Most information about PAR activation and function derives from studies of a few proteases, for example thrombin in the case of PAR1, PAR3, and PAR4, and trypsin in the case of PAR2 and PAR4. These proteases cleave PARs at established sites with the extracellular N-terminal domains, and expose tethered ligands that stabilize conformations of the cleaved receptors that activate the canonical pathways of G protein- and/or ?-arrestin-dependent signaling. However, a growing number of proteases have been identified that cleave PARs at divergent sites to activate distinct patterns of receptor signaling and trafficking. The capacity of these proteases to trigger distinct signaling pathways is referred to as biased signaling, and can lead to unique patho-physiological outcomes. Given that a different repertoire of proteases are activated in various patho-physiological conditions that may activate PARs by different mechanisms, signaling bias may account for the divergent actions of proteases and PARs. Moreover, therapies that target disease-relevant biased signaling pathways may be more effective and selective approaches for the treatment of protease- and PAR-driven diseases. Thus, rather than mediating the actions of a few proteases, PARs may integrate the biological actions of a wide spectrum of proteases in different patho-physiological conditions.

Zhao, Peishen; Metcalf, Matthew; Bunnett, Nigel W.

2014-01-01

151

Laminin stimulates protein tyrosine dephosphorylation in PC12 cells.  

PubMed

Laminin stimulates neurite outgrowth in rat pheochromocytoma cells (PC12 cells). Here, we investigated laminin signal transduction mechanisms by adding the tyrosine kinase/phosphatase modulators, genistein, quercetin, aurin tricarboxylic acid (ATA), and vanadate to PC12 cells. At 10 microM both genistein and quercetin enhanced laminin-mediated neurite outgrowth by 1.7- and 2.3-fold, respectively, while at 10 microM, ATA inhibited laminin-mediated neurite outgrowth by 92%. Vanadate inhibited neurite outgrowth by 63% at 10 microM. Immunoblot analysis revealed four proteins of approximately 240, 22, 110, and 35 kDa, which were dephosphorylated on tyrosine residues in laminin-treated PC12 cells, but not in NIH 3T3 cells. These results demonstrate that laminin-mediated neurite outgrowth involves protein tyrosine dephosphorylation and suggests that this mechanism may have specificity to neuronal cells. PMID:10471391

Weeks, B S; Wilson, P J; Heffernan, C C; Gorra, V A; White, L E; Ahmad, A

1999-09-01

152

Coactivator Selective Regulation of Androgen Receptor Activity  

PubMed Central

The androgen receptor (AR) is a ligand activated nuclear receptor, which regulates transcription and stimulates growth of androgen dependent prostate cancer. To regulate transcription, AR recruits a series of coactivators that modify chromatin and facilitate transcription. However, information on ligand and target gene specific requirements for coactivators is limited. We compared the actions of the p160 coactivators SRC-1 and SRC-3/RAC3 with SRA (steroid receptor RNA activator). All three coactivate AR in the presence of agonist as expected. However, overexpression of either SRC-1 or SRC-3 increased AR activity in response to the partial antagonist, cyproterone acetate, whereas SRA was unable to stimulate AR activity under these conditions. Using siRNA to reduce expression of these coactivators in LNCaP cells, we also found promoter specific requirement for these coactivators. SRC-3 is required for optimal androgen dependent induction of PSA, TMPRSS2, and PMEPA1 whereas SRA is required only for optimal induction of the TMPRSS2 gene. These data indicate that different groups of AR target genes have distinct requirements for coactivators and response to AR ligands.

Agoulnik, Irina U; Weigel, Nancy L

2009-01-01

153

Origin and evolution of laminin gene family diversity.  

PubMed

Laminins are a family of multidomain glycoproteins that are important contributors to the structure of metazoan extracellular matrices. To investigate the origin and evolution of the laminin family, we characterized the full complement of laminin-related genes in the genome of the sponge, Amphimedon queenslandica. As a representative of the Demospongiae, a group consistently placed within the earliest diverging branch of animals by molecular phylogenies, Amphimedon is uniquely placed to provide insight into early steps in the evolution of metazoan gene families. Five Amphimedon laminin-related genes possess the conserved molecular features, and most of the domains found in bilaterian laminins, but all display domain architectures distinct from those of the canonical laminin chain types known from model bilaterians. This finding prompted us to perform a comparative genomic analysis of laminins and related genes from a choanoflagellate and diverse metazoans and to conduct phylogenetic analyses using the conserved Laminin N-terminal domain in order to explore the relationships between genes with distinct architectures. Laminin-like genes appear to have originated in the holozoan lineage (choanoflagellates + metazoans + several other unicellular opisthokont taxa), with several laminin domains originating later and appearing only in metazoan (animal) or eumetazoan (placozoans + ctenophores + cnidarians + bilaterians) laminins. Typical bilaterian ?, ?, and ? laminin chain forms arose in the eumetazoan stem and another chain type that is conserved in Amphimedon, the cnidarian, Nematostella vectensis, and the echinoderm, Strongylocentrotus purpuratus, appears to have been lost independently from the placozoan, Trichoplax adhaerens, and from multiple bilaterians. Phylogenetic analysis did not clearly reconstruct relationships between the distinct laminin chain types (with the exception of the ? chains) but did reveal how several members of the netrin family were generated independently from within the laminin family by duplication and domain shuffling and by domain loss. Together, our results suggest that gene duplication and loss and domain shuffling and loss all played a role in the evolution of the laminin family and contributed to the generation of lineage-specific diversity in the laminin gene complements of extant metazoans. PMID:22319142

Fahey, Bryony; Degnan, Bernard M

2012-07-01

154

Immunogold localisation of laminin in normal and exfoliative iris.  

PubMed Central

Immunoelectron microscopic studies of exfoliative iris tissue (seven specimens) revealed the presence of laminin in the fibrillar component of exfoliation material. The immunogold label was uniformly distributed on the exfoliation fibres. Deposition of laminin labelled exfoliation material in the dilator muscle was a noteworthy feature, as was an apparent depletion of laminin in the basement membranes of ostensibly unaffected vessels. In control iris tissue (five enucleated eyes) laminin was identified in the basement membrane round vascular contractile cells, but not beneath the endothelium. Images

Konstas, A. G.; Marshall, G. E.; Lee, W. R.

1990-01-01

155

On guard—activating NK cell receptors  

Microsoft Academic Search

Although natural killer (NK) cells are known to preferentially kill cells that lack major histocompatibility complex class I antigens, we do not know what signals the attack of these targets. Several membrane receptors have recently been implicated in this process and include molecules with immunoreceptor tyrosine-based activation motifs (ITAM) and motifs that bind phosphoinositide-3 kinase (PI3K). Evidence is emerging that

Lewis L. Lanier

2001-01-01

156

Adipogenic effect of calcium sensing receptor activation.  

PubMed

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

2013-12-01

157

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

2008-01-01

158

Beta 8 integrins mediate interactions of chick sensory neurons with laminin-1, collagen IV, and fibronectin.  

PubMed Central

Integrins are major receptors used by cells to interact with extracellular matrices. In this paper, we identify the first ligands for the beta 8 family of integrins, presenting evidence that integrin heterodimers containing the beta 8 subunit mediate interactions of chick sensory neurons with laminin-1, collagen IV, and fibronectin. A polyclonal antibody, anti-beta 8-Ex, was prepared to a bacterial fusion protein expressing an extracellular portion of the chicken beta 8 subunit. In nonreducing conditions, this antibody immunoprecipitated from surface-labeled embryonic dorsal root ganglia neurons a M(r) 100 k protein, the expected M(r) of the beta 8 subunit, and putative alpha subunit(s) of M(r) 120 k. Affinity-purified anti-beta 8-Ex strongly inhibited sensory neurite outgrowth on laminin-1, collagen IV, and fibronectin-coated substrata. Binding sites were identified in a heat-resistant domain in laminin-1 and in the carboxyl terminal, 40-kDa fibronectin fragment. On substrates coated with the carboxyl terminal fragment of fibronectin, antibodies to beta 1 and beta 8 were only partially effective alone, but were completely effective in combination, at inhibiting neurite outgrowth. Results thus indicate that the integrin beta 8 subunit in association with one or more alpha subunits forms an important set of extracellular matrix receptors on sensory neurons. Images

Venstrom, K; Reichardt, L

1995-01-01

159

Proteinase-activated receptors (PARs) - focus on receptor-receptor-interactions and their physiological and pathophysiological impact  

PubMed Central

Proteinase-activated receptors (PARs) are a subfamily of G protein-coupled receptors (GPCRs) with four members, PAR1, PAR2, PAR3 and PAR4, playing critical functions in hemostasis, thrombosis, embryonic development, wound healing, inflammation and cancer progression. PARs are characterized by a unique activation mechanism involving receptor cleavage by different proteinases at specific sites within the extracellular amino-terminus and the exposure of amino-terminal “tethered ligand“ domains that bind to and activate the cleaved receptors. After activation, the PAR family members are able to stimulate complex intracellular signalling networks via classical G protein-mediated pathways and beta-arrestin signalling. In addition, different receptor crosstalk mechanisms critically contribute to a high diversity of PAR signal transduction and receptor-trafficking processes that result in multiple physiological effects. In this review, we summarize current information about PAR-initiated physical and functional receptor interactions and their physiological and pathological roles. We focus especially on PAR homo- and heterodimerization, transactivation of receptor tyrosine kinases (RTKs) and receptor serine/threonine kinases (RSTKs), communication with other GPCRs, toll-like receptors and NOD-like receptors, ion channel receptors, and on PAR association with cargo receptors. In addition, we discuss the suitability of these receptor interaction mechanisms as targets for modulating PAR signalling in disease.

2013-01-01

160

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

PubMed

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

2013-10-01

161

Adrenergic receptor activation involves ATP release and feedback through purinergic receptors  

PubMed Central

Formyl peptide receptor-induced chemotaxis of neutrophils depends on the release of ATP and autocrine feedback through purinergic receptors. Here, we show that adrenergic receptor signaling requires similar purinergic feedback mechanisms. Real-time RT-PCR analysis revealed that human embryonic kidney (HEK)-293 cells express several subtypes of adrenergic (?1-, ?2-, and ?-receptors), adenosine (P1), and nucleotide receptors (P2). Stimulation of Gq-coupled ?1-receptors caused release of cellular ATP and MAPK activation, which was blocked by inhibiting P2 receptors with suramin. Stimulation of Gi-coupled ?2-receptors induced weak ATP release, while Gs-coupled ?-receptors caused accumulation of extracellular ADP and adenosine. ?-Receptors triggered intracellular cAMP signaling, which was blocked by scavenging extracellular adenosine with adenosine deaminase or by inhibiting A2a adenosine receptors with SCH58261. These findings suggest that adrenergic receptors require purinergic receptors to elicit downstream signaling responses in HEK-293 cells. We evaluated the physiological relevance of these findings using mouse aorta tissue rings. Stimulation of ?1-receptors induced ATP release and tissue contraction, which was reduced by removing extracellular ATP with apyrase or in the absence of P2Y2 receptors in aorta rings from P2Y2 receptor knockout mice. We conclude that, like formyl peptide receptors, adrenergic receptors require purinergic feedback mechanisms to control complex physiological processes such as smooth muscle contraction and regulation of vascular tone.

Sumi, Yuka; Woehrle, Tobias; Chen, Yu; Yao, Yongli; Li, Andrew

2010-01-01

162

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.

2008-01-01

163

Aminoglycoside neurotoxicity involves NMDA receptor activation.  

PubMed

Previous studies have led to the hypothesis that the ototoxicity produced by aminoglycoside antibiotics involves the excitotoxic activation of cochlear NMDA receptors. If this hypothesis is correct, then these antibiotics should also injure neurons within the brain. Because aminoglycosides do not readily penetrate the blood brain barrier, we examined the effects of the aminoglycoside neomycin following intrastriatal injection. Neomycin (10-250 nmol) produced dose-dependent striatal damage manifested as an increased gliosis as measured by: (1) [3H]PK-11195 binding, (2) staining for the astrocytic marker glial fibrillary acidic protein (GFAP) and (3) staining for OX-6, an MHC class II antigen expressed by microglia and macrophages. Co-injection of subthreshhold doses of NMDA potentiates the striatal damage produced by neomycin (10 nmol). Moreover, neomycin-induced striatal damage is attenuated by a combination of the NMDA antagonists ifenprodil and 5, 7-dichlorokynurenic acid. Intrastriatal administration of compounds structurally related to neomycin, but devoid of modulatory actions at NMDA receptors (paromamine and 2-deoxystreptamine), fail to produce neuronal damage. These data support the hypothesis that aminoglycoside-induced ototoxicity is, in part, an excitotoxic process involving the activation of NMDA receptors. Moreover, aminoglycosides may damage the central nervous system in individuals with compromised blood brain barriers. PMID:9878779

Segal, J A; Harris, B D; Kustova, Y; Basile, A; Skolnick, P

1999-01-01

164

Laminins affect T cell trafficking and allograft fate.  

PubMed

Lymph nodes (LNs) are integral sites for the generation of immune tolerance, migration of CD4? T cells, and induction of Tregs. Despite the importance of LNs in regulation of inflammatory responses, the LN-specific factors that regulate T cell migration and the precise LN structural domains in which differentiation occurs remain undefined. Using intravital and fluorescent microscopy, we found that alloreactive T cells traffic distinctly into the tolerant LN and colocalize in exclusive regions with alloantigen-presenting cells, a process required for Treg induction. Extracellular matrix proteins, including those of the laminin family, formed regions within the LN that were permissive for colocalization of alloantigen-presenting cells, alloreactive T cells, and Tregs. We identified unique expression patterns of laminin proteins in high endothelial venule basement membranes and the cortical ridge that correlated with alloantigen-specific immunity or immune tolerance. The ratio of laminin ?4 to laminin ?5 was greater in domains within tolerant LNs, compared with immune LNs, and blocking laminin ?4 function or inducing laminin ?5 overexpression disrupted T cell and DC localization and transmigration through tolerant LNs. Furthermore, reducing ?4 laminin circumvented tolerance induction and induced cardiac allograft inflammation and rejection in murine models. This work identifies laminins as potential targets for immune modulation. PMID:24691446

Warren, Kristi J; Iwami, Daiki; Harris, Donald G; Bromberg, Jonathan S; Burrell, Bryna E

2014-05-01

165

Laminins affect T cell trafficking and allograft fate  

PubMed Central

Lymph nodes (LNs) are integral sites for the generation of immune tolerance, migration of CD4+ T cells, and induction of Tregs. Despite the importance of LNs in regulation of inflammatory responses, the LN-specific factors that regulate T cell migration and the precise LN structural domains in which differentiation occurs remain undefined. Using intravital and fluorescent microscopy, we found that alloreactive T cells traffic distinctly into the tolerant LN and colocalize in exclusive regions with alloantigen-presenting cells, a process required for Treg induction. Extracellular matrix proteins, including those of the laminin family, formed regions within the LN that were permissive for colocalization of alloantigen-presenting cells, alloreactive T cells, and Tregs. We identified unique expression patterns of laminin proteins in high endothelial venule basement membranes and the cortical ridge that correlated with alloantigen-specific immunity or immune tolerance. The ratio of laminin ?4 to laminin ?5 was greater in domains within tolerant LNs, compared with immune LNs, and blocking laminin ?4 function or inducing laminin ?5 overexpression disrupted T cell and DC localization and transmigration through tolerant LNs. Furthermore, reducing ?4 laminin circumvented tolerance induction and induced cardiac allograft inflammation and rejection in murine models. This work identifies laminins as potential targets for immune modulation.

Warren, Kristi J.; Iwami, Daiki; Harris, Donald G.; Bromberg, Jonathan S.; Burrell, Bryna E.

2014-01-01

166

LG4-5 domains of laminin-2 binds ?-dystroglycan to allow myotube attachment and prevent anoikis  

PubMed Central

Poly(2-hydroxyethyl methacrylate) (PolyHEMA) prevents cell attachment was used here to study anoikis, the process where cells die when unattached or attached to an inappropriate matrix, in mouse C2C12 myotubes. A method was developed to efficiently embed proteins into PolyHEMA and the effect on cultured myotubes was determined. Myotubes grown on PolyHEMA-coated plates fail to attach to the surface and remain as rounded, suspended cells, undergo dramatic increases in apoptosis and necrosis, and the number of viable cells decreases,. Incorporation of merosin (laminin-211) or the short laminin globular (LG4-5) modules of the laminin ?2 chain C-terminus (called 2E3) that binds ?-dystroglycan diminishes both apoptosis and necrosis and increases viability while bovine serum albumin had a much lesser effect, showing the specificity of this effect for these matrix proteins. One sarcolemma receptor for laminin-binding is ?-dystroglycan. An antibody which binds ?-dystroglycan but which does not block laminin-binding (VIA4) had little effect on apoptosis or viability on merosin or 2E3 embedded plates while another antibody (IIH6) which specifically blocks binding dramatically decreased viability and increased apoptosis. When merosin or 2E3 are added to culture media rather than embedded on plates these can also increase viability and decrease apoptosis even though the cells remain in suspension, though the effect is not as great as found for the embedded proteins where the cells attach. Thus, we conclude that the binding of a small LG4-5 modules of laminin-211 to ?-dystroglycan is important in preventing anoikis and that attachment plus binding is necessary for maximal cell survival.

Munoz, Jesus; Zhou, YanWen; Jarrett, Harry W.

2010-01-01

167

Laminin Nanofiber Meshes That Mimic Morphological Properties and Bioactivity of Basement Membranes  

PubMed Central

The basement membrane protein, laminin I, has been used broadly as a planar two-dimensional film or in a three-dimensional form as a reconstituted basement membrane gel such as Matrigel to support cellular attachment, growth, and differentiation in vitro. In basement membranes in vivo, laminin exhibits a fibrillar morphology, highlighting the electrospinning process as an ideal method to recreate such fibrous substrates in vitro. Electrospinning was employed to fabricate meshes of murine laminin I nanofibers (LNFs) with fiber size, geometry, and porosity of authentic basement membranes. Purified laminin I was solubilized and electrospun in parametric studies of fiber diameters as a function of polymer solution concentration, collecting distance, and flow rate. Resulting fiber diameters ranged from 90 to 300?nm with mesh morphologies containing beads. Unlike previously described nanofibers (NFs) synthesized from proteins such as collagen, meshes of LNFs retain their structural features when wetted and do not require fixation by chemical crosslinking, which often destroys cell attachment and other biological activity. The LNF meshes maintained their geometry for at least 2 days in culture without chemical crosslinking. PC12 cells extended neurites without nerve growth factor stimulation on LNF substrates. Additionally, LNFs significantly enhance both the rate and quantity of attachment of human adipose stem cells (ASCs) compared to laminin films. ASCs were viable and maintained attachment to LNF meshes in serum-free media for at least 3 days in culture and extended neurite-like processes after 24?h in serum-free media conditions without media additives to induce differentiation. LNF meshes are a novel substrate for cell studies in vitro, whose properties may be an excellent scaffold material for delivering cells in tissue engineering applications in vivo.

Neal, Rebekah A.; McClugage, Samuel G.; Link, Mia C.; Sefcik, Lauren S.; Ogle, Roy C.

2009-01-01

168

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

PubMed

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

2001-01-01

169

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.

1989-01-01

170

A synthetic peptide deduced from the sequence in the cross-region of laminin A chain mediates neurite outgrowth, cell attachment and heparin binding.  

PubMed Central

Synthetic peptides from the cross-region of the laminin A chain were prepared and tested for their biological activities, especially for neurite outgrowth. A synthetic 8-mer peptide (designated LMA-5) in the cross-region of the laminin A chain was found to promote neurite outgrowth in PC12 cells and cerebellar microexplant cultures. Furthermore, this peptide mediated cell attachment and heparin binding. In addition, an antibody against peptide LMA-5 inhibited laminin- and LMA-5-mediated cell attachment. This antibody also inhibited more than half of the LMA-5-promoted neurite outgrowth in cerebellar microexplant cultures. These data suggest that peptide LMA-5 is one of the active sites in laminin that regulate cell behaviour including neurite outgrowth, cell attachment and heparin binding. Images Figure 4

Tashiro, K; Nagata, I; Yamashita, N; Okazaki, K; Ogomori, K; Tashiro, N; Anai, M

1994-01-01

171

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

1998-01-01

172

Fibrates, glitazones, and peroxisome proliferator-activated receptors  

PubMed Central

Several decades ago, fibrates were approved for the treatment of dyslipidemia, whereas thiazolidinediones were screened in animal models to improve glucose homeostasis and subsequently developed for the treatment of type 2 diabetes. Relatively recently, these drugs were found to act via peroxisome proliferator-activated receptors, nuclear receptors which control lipid metabolism and glucose homeostasis. In this historical perspective, we discuss the history of discovery of the peroxisome proliferator-activated receptors, from the clinical development of their agonists to the subsequent discovery of these receptors and their mechanisms of action, to finally evoke possibilities of targeted pharmacology for future development of selective peroxisome proliferator-activated receptors modulators.

Lalloyer, Fanny; Staels, Bart

2010-01-01

173

Plant Immunity: The Origami of Receptor Activation  

Microsoft Academic Search

Mutations in plant cytosolic HSP90 genes have been found to impair the immune responses triggered by host pathogen receptors. HSP90 links the plant receptors to other components essential for receptor function. The new findings suggest mechanistic parallels with steroid receptor regulation in animals.

Paul Schulze-Lefert

2004-01-01

174

Differential effects of phthalate esters on transcriptional activities via human estrogen receptors ? and ?, and androgen receptor  

Microsoft Academic Search

Some phthalates are suspected to disrupt the endocrine system, especially by mimicking estrogens. In this study, we characterized the activities of human estrogen receptor ? (hER?), human estrogen receptor ? (hER?), and human androgen receptor (hAR) in the presence of 22 phthalates including 3 of their metabolites using highly sensitive reporter gene assays. Of the 22 compounds tested, several phthalate

Shinji Takeuchi; Mitsuru Iida; Satoshi Kobayashi; Kazuo Jin; Tadashi Matsuda; Hiroyuki Kojima

2005-01-01

175

Activation of neurotensin receptor type 1 attenuates locomotor activity.  

PubMed

Intracerebroventricular administration of neurotensin (NT) suppresses locomotor activity. However, the brain regions that mediate the locomotor depressant effect of NT and receptor subtype-specific mechanisms involved are unclear. Using a brain-penetrating, selective NT receptor type 1 (NTS1) agonist PD149163, we investigated the effect of systemic and brain region-specific NTS1 activation on locomotor activity. Systemic administration of PD149163 attenuated the locomotor activity of C57BL/6J mice both in a novel environment and in their homecage. However, mice developed tolerance to the hypolocomotor effect of PD149163 (0.1 mg/kg, i.p.). Since NTS1 is known to modulate dopaminergic signaling, we examined whether PD149163 blocks dopamine receptor-mediated hyperactivity. Pretreatment with PD149163 (0.1 or 0.05 mg/kg, i.p.) inhibited D2R agonist bromocriptine (8 mg/kg, i.p.)-mediated hyperactivity. D1R agonist SKF-81297 (8 mg/kg, i.p.)-induced hyperlocomotion was only inhibited by 0.1 mg/kg of PD149163. Since the nucleus accumbens (NAc) and medial prefrontal cortex (mPFC) have been implicated in the behavioral effects of NT, we examined whether microinjection of PD149163 into these regions reduces locomotion. Microinjection of PD149163 (2 pmol) into the NAc, but not the mPFC suppressed locomotor activity. In summary, our results indicate that systemic and intra-NAc activation of NTS1 is sufficient to reduce locomotion and NTS1 activation inhibits D2R-mediated hyperactivity. Our study will be helpful to identify pharmacological factors and a possible therapeutic window for NTS1-targeted therapies for movement disorders. PMID:24929110

Vadnie, Chelsea A; Hinton, David J; Choi, Sun; Choi, YuBin; Ruby, Christina L; Oliveros, Alfredo; Prieto, Miguel L; Park, Jun Hyun; Choi, Doo-Sup

2014-10-01

176

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

2006-01-01

177

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

1995-01-01

178

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

2012-01-01

179

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

2006-01-01

180

Identification of Gene Markers for Activation of the Nuclear Receptor Pregnane X Receptor  

EPA Science Inventory

Many environmentally-relevant chemicals and drugs activate the nuclear receptor pregnane X receptor (PXR). Activation of PXR in the mouse liver can lead to increases in liver weight in part through increased hepatocyte replication similar to chemicals that activate other nuclear ...

181

Laminin alpha 2 enables glioblastoma stem cell growth  

PubMed Central

Objective Glioblastomas (GBMs) are lethal cancers that display cellular hierarchies parallel to normal brain. At the apex are GBM stem cells (GSCs), which are relatively resistant to conventional therapy. An important driver of malignancy and self-renewal in GSCs are interactions with the adjacent perivascular niche. Extracellular matrix (ECM) cues instruct neural stem/progenitor cell-niche interactions and the objective of our study was to elucidate its composition and contribution to GSC maintenance in the perivascular niche. Methods We interrogated human tumor tissue for immunofluorescence analysis and derived GSC from tumor tissues for functional studies. Bioinformatics analyses were conducted by mining publicly available databases. Results We find that laminin ECM proteins are localized to the perivascular GBM niche and inform negative patient prognosis. To identify the source of laminins, we characterized cellular elements within the niche and found that laminin ? chains were expressed by non-stem tumor cells and tumor associated endothelial cells (ECs). RNA interference targeting laminin ?2 inhibited GSC growth and self-renewal. In co-culture studies of GSCs and ECs, laminin ?2 knockdown in ECs resulted in decreased tumor growth. Interpretation Our studies highlight the contribution of non-stem tumor cell-derived laminin juxtracrine signaling. As laminin ?2 has recently been identified as a molecular marker of aggressive ependymoma, we propose that the brain vascular ECM promotes tumor malignancy through maintenance of the GSC compartment providing not only a molecular fingerprint but also a possible therapeutic target.

Lathia, Justin D.; Li, Meizhang; Hall, Peter E.; Gallagher, Joseph; Hale, James S.; Wu, Qiulian; Venere, Monica; Levy, Emily; Rani, MR Sandhya; Huang, Ping; Bae, Eunnyung; Selfridge, Julia; Cheng, Lin; Guvenc, Hacer; McLendon, Roger E.; Nakano, Ichiro; Sloan, Andrew E.; Phillips, Heidi S.; Lai, Albert; Gladson, Candece; Bredel, Markus; Bao, Shideng; Hjelmeland, Anita B.; Rich, Jeremy N.

2013-01-01

182

Model for growth hormone receptor activation based on subunit rotation within a receptor dimer  

SciTech Connect

Growth hormone is believed to activate the growth hormone receptor (GHR) by dimerizing two identical receptor subunits, leading to activation of JAK2 kinase associated with the cytoplasmic domain. However, we have reported previously that dimerization alone is insufficient to activate full-length GHR. By comparing the crystal structure of the liganded and unliganded human GHR extracellular domain, we show here that there is no substantial change in its conformation on ligand binding. However, the receptor can be activated by rotation without ligand by inserting a defined number of alanine residues within the transmembrane domain. Fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) and coimmunoprecipitation studies suggest that receptor subunits undergo specific transmembrane interactions independent of hormone binding. We propose an activation mechanism involving a relative rotation of subunits within a dimeric receptor as a result of asymmetric placement of the receptor-binding sites on the ligand.

Brown, Richard J.; Adams, Julian J.; Pelekanos, Rebecca A.; Wan, Yu; McKinstry, William J.; Palethorpe, Kathryn; Seeber, Ruth M.; Monks, Thea A.; Eidne, Karin A.; Parker, Michael W.; Waters, Michael J. (UWA); (St. Vincent); (Queensland)

2010-07-13

183

Complex interactions between the laminin 4 subunit and integrins regulate endothelial cell behavior in vitro and angiogenesis in vivo  

NASA Astrophysics Data System (ADS)

The 4 laminin subunit is a component of the basement membrane of blood vessels where it codistributes with the integrins v3, 31, and 61. An antibody against the G domain (residues 919-1207; G919-1207) of the 4 laminin subunit inhibits angiogenesis in a mouse-human chimeric model, indicating the functional importance of this domain. Additional support for the latter derives from the ability of recombinant G919-1207 to support endothelial cell adhesion. In particular, endothelial cell adhesion to G919-1207 is half-maximal at 1.4 nM, whereas residues 919-1018 and 1016-1207 of the G domain are poor cellular ligands. Function blocking antibodies against integrins v3 and 1 and a combination of antibodies against 3 and ?6 integrin subunits inhibit endothelial cell attachment to G919-1207. Moreover, both ?v?3 and ?3?1 integrin bind with high affinity to G919-1207. Together, our studies demonstrate that the G domain of laminin ?4 chain is a specific, high affinity ligand for the ?v?3 and ?3?1 integrin heterodimers and that these integrins, together with ?6?1, function cooperatively to mediate endothelial cell-?4 laminin interaction and hence blood vessel development. We propose a model based on these data that reconcile apparent discrepancies in the recent literature with regard to the role of the ?v?3 integrin in angiogenesis. matrix | matrix receptor | blood vessels

Gonzalez, Annette M.; Gonzales, Meredith; Herron, G. Scott; Nagavarapu, Usha; Hopkinson, Susan B.; Tsuruta, Daisuke; Jones, Jonathan C. R.

2002-12-01

184

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.

2009-06-03

185

Angiogenic activitiy of syndecan-binding laminin peptide AG73 (RKRLQVQLSIRT)  

Microsoft Academic Search

The AG73 peptide (RKRLQVQLSIRT, mouse laminin alpha 1 chain 2719–2730) promotes cell adhesion and tumor metastasis, and interacts with transmembrane syndecan proteoglycans. Here, we demonstrate AG73 peptide angiogenic activity using in vitro, ex vivo, and in vivo models. AG73 induced murine endothelial cell (SVEC4-10) tube formation on Cultrex Basement Membrane Extract (Cultrex BME) and stimulated sprouting of aortic rings. None

Mayumi Mochizuki; Deborah Philp; Kentaro Hozumi; Nobuharu Suzuki; Yoshihiko Yamada; Hynda K. Kleinman; Motoyoshi Nomizu

2007-01-01

186

Fatty Acids and Retinoids Control Lipid Metabolism through Activation of Peroxisome Proliferator-Activated Receptor-Retinoid X Receptor Heterodimers  

Microsoft Academic Search

The nuclear hormone receptors called PPARs (peroxisome proliferator-activated receptors alpha, beta, and gamma) regulate the peroxisomal beta-oxidation of fatty acids by induction of the acyl-CoA oxidase gene that encodes the rate-limiting enzyme of the pathway. Gel retardation and cotransfection assays revealed that PPARalpha heterodimerizes with retinoid X receptor beta (RXRbeta RXR is the receptor for 9-cis-retinoic acid) and that the

Hansjorg Keller; Christine Dreyer; Jeffrey Medin; Abderrahim Mahfoudi; Keiko Ozato; Walter Wahli

1993-01-01

187

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

2008-01-01

188

Ovarian steroids and selective estrogen receptor modulators activity on rat brain NMDA and AMPA receptors.  

PubMed

Glutamate and glutamate receptors are well known to play a major excitatory role in the brain. Recent findings on ovarian steroids and selective estrogen receptor modulators (SERMs) activity on rat brain AMPA and NMDA receptors are reviewed. Ovarian steroid withdrawal by ovariectomy is without effect on NMDA and AMPA receptors in most brain regions, except in hippocampus, where it decreases NMDA receptor specific binding, compared to intact rat values. Estradiol treatment increases hippocampal NMDA receptor specific binding of ovariectomized rats while it decreases this binding in frontal cortex and striatum. Estradiol treatment has no effect on AMPA receptor specific binding in hippocampus, but decreases binding in frontal cortex, striatum and nucleus accumbens. Progesterone and estradiol+progesterone treatments decrease NMDA, but not AMPA receptors specific binding in frontal cortex compared to ovariectomized rats. No effect was observed in other brain regions. Tamoxifen and raloxifene are SERMs with varying effects on estrogen responses in mammary, bone and uterine tissues. Tamoxifen and raloxifene have estrogenic activity upon modulation of brain NMDA and AMPA receptors. Using specific ligands for binding autoradiography of NMDA receptor subunits and specific probes for subunits measured by in situ hybridization, it was shown that estradiol and SERMs modulate NR1 and NR2B subunits whereas the NR1/2A subunit remains unchanged. In summary, regional agonist estrogenic activity on brain AMPA and NMDA receptors of tamoxifen and raloxifene, like that of estradiol, is observed, whereas progesterone has limited effects or opposes the estradiol effect. PMID:11744083

Cyr, M; Ghribi, O; Thibault, C; Morissette, M; Landry, M; Di Paolo, T

2001-11-01

189

Synthetic D-amino acid peptide inhibits tumor cell motility on laminin-5  

PubMed Central

Cell motility is partially dependent on interactions between the integrins and the extracellular matrix. Our previous studies have identified synthetic D-amino acid cell adhesion peptides using a combinatorial screening approach. In this study, we demonstrate that HYD1 (kikmviswkg) completely blocks random haptotactic migration and inhibits invasion of prostate carcinoma cells on laminin-5. This effect is adhesion independent and reversible. The inhibition of migration by HYD1 involves a dramatic remodeling of the actin cytoskeleton resulting in increased stress fiber formation and actin colocalization with cortactin at the cell membrane. HYD1 interacts with ?6?1 (not ?6?4) and ?3?1 integrins and surprisingly elevates laminin-5-dependent intracellular signals including focal adhesion kinase, mitogen-activated protein kinase kinase and extracellular signal-regulated kinase. HYD1 does not contain a previously characterized binding sequence for integrins. A scrambled derivative of HYD1, called HYDS (wiksmkivkg), does not interact with the ?6 or ?3 integrin subunits and is not biologically active. Taken together, these results indicate that HYD1 is a biologically active integrin-targeting peptide that reversibly inhibits tumor cell migration on laminin-5 and uncouples phosphotyrosine signaling from cytoskeletal-dependent migration.

Sroka, Thomas C.; Pennington, Michael E.; Cress, Anne E.

2009-01-01

190

Structural insights into G-protein-coupled receptor activation?  

PubMed Central

G-protein-coupled receptors (GPCRs) are the largest family of eukaryotic plasma membrane receptors, and are responsible for the majority of cellular responses to external signals. GPCRs share a common architecture comprising seven transmembrane (TM) helices. Binding of an activating ligand enables the receptor to catalyze the exchange of GTP for GDP in a heterotrimeric G protein. GPCRs are in a conformational equilibrium between inactive and activating states. Crystallographic and spectroscopic studies of the visual pigment rhodopsin and two b-adrenergic receptors have defined some of the conformational changes associated with activation.

Weis, William I; Kobilka, Brian K

2014-01-01

191

Local Kappa Opioid Receptor Activation Decreases Temporomandibular Joint Inflammation  

Microsoft Academic Search

In an attempt to decrease central side effects associated with the use of opioids, some strategies have been developed by\\u000a targeting peripheral opioid receptors. In this context, kappa receptors are of major interest, since, in contrast to other\\u000a opioid receptors, their activation is not associated with potent peripheral side effects. We have recently demonstrated that\\u000a local activation of kappa opioid

Tânia C. Chicre-Alcântara; Karla E. Torres-Chávez; Luana Fischer; Juliana T. Clemente-Napimoga; Vilma Melo; Carlos Amílcar Parada; Claudia Herrera Tambeli

192

Immobilization and therapeutic passive stretching generate thickening and increase the expression of laminin and dystrophin in skeletal muscle  

PubMed Central

Extracellular matrix and costamere proteins transmit the concentric, isometric, and eccentric forces produced by active muscle contraction. The expression of these proteins after application of passive tension stimuli to muscle remains unknown. This study investigated the expression of laminin and dystrophin in the soleus muscle of rats immobilized with the right ankle in plantar flexion for 10 days and subsequent remobilization, either by isolated free movement in a cage or associated with passive stretching for up to 10 days. The intensity of the macrophage response was also evaluated. One hundred and twenty-eight female Wistar rats were divided into 8 groups: free for 10 days; immobilized for 10 days; immobilized/free for 1, 3, or 10 days; or immobilized/stretched/free for 1, 3, or 10 days. After the experimental procedures, muscle tissue was processed for immunofluorescence (dystrophin/laminin/CD68) and Western blot analysis (dystrophin/laminin). Immobilization increased the expression of dystrophin and laminin but did not alter the number of macrophages in the muscle. In the stretched muscle groups, there was an increase in dystrophin and the number of macrophages after 3 days compared with the other groups; dystrophin showed a discontinuous labeling pattern, and laminin was found in the intracellular space. The amount of laminin was increased in the muscles treated by immobilization followed by free movement for 10 days. In the initial stages of postimmobilization (1 and 3 days), an exacerbated macrophage response and an increase of dystrophin suggested that the therapeutic stretching technique induced additional stress in the muscle fibers and costameres.

Cacao-Benedini, L.O.; Ribeiro, P.G.; Prado, C.M.; Chesca, D.L.; Mattiello-Sverzut, A.C.

2014-01-01

193

Immobilization and therapeutic passive stretching generate thickening and increase the expression of laminin and dystrophin in skeletal muscle.  

PubMed

Extracellular matrix and costamere proteins transmit the concentric, isometric, and eccentric forces produced by active muscle contraction. The expression of these proteins after application of passive tension stimuli to muscle remains unknown. This study investigated the expression of laminin and dystrophin in the soleus muscle of rats immobilized with the right ankle in plantar flexion for 10 days and subsequent remobilization, either by isolated free movement in a cage or associated with passive stretching for up to 10 days. The intensity of the macrophage response was also evaluated. One hundred and twenty-eight female Wistar rats were divided into 8 groups: free for 10 days; immobilized for 10 days; immobilized/free for 1, 3, or 10 days; or immobilized/stretched/free for 1, 3, or 10 days. After the experimental procedures, muscle tissue was processed for immunofluorescence (dystrophin/laminin/CD68) and Western blot analysis (dystrophin/laminin). Immobilization increased the expression of dystrophin and laminin but did not alter the number of macrophages in the muscle. In the stretched muscle groups, there was an increase in dystrophin and the number of macrophages after 3 days compared with the other groups; dystrophin showed a discontinuous labeling pattern, and laminin was found in the intracellular space. The amount of laminin was increased in the muscles treated by immobilization followed by free movement for 10 days. In the initial stages of postimmobilization (1 and 3 days), an exacerbated macrophage response and an increase of dystrophin suggested that the therapeutic stretching technique induced additional stress in the muscle fibers and costameres. PMID:24820070

Cação-Benedini, L O; Ribeiro, P G; Prado, C M; Chesca, D L; Mattiello-Sverzut, A C

2014-06-01

194

Peroxisome proliferator-activated receptors-alpha modulate dopamine cell activity through nicotinic receptors  

PubMed Central

Background Modulation of midbrain dopamine neurons by nicotinic acetylcholine receptors (nAChRs) plays an important role in behavior, cognition, motivation and reward. Specifically, nAChRs containing ?2 subunits (?2-nAChRs) switch dopamine cells from a resting to an excited state. However, how ?2-nAChRs can be modulated and thereby dopamine firing activity be affected is still elusive. Because changes in dopamine cell activity are reflected in the dynamics of micro-circuits generating altered responses to stimuli/inputs, factors regulating their state are fundamental. Among these, endogenous ligands to the nuclear receptor-transcription factor peroxisome proliferator-activated receptors type-alpha (PPAR?) have been recently found to suppress nicotine-induced responses of dopamine neurons. Methods We used both in vitro and in vivo electrophysiological techniques together with behavioral analysis to investigate on the effects of modulation of PPAR? in Sprague Dawley rat and C57BLJ/6 mouse dopamine neurons, and their interactions with ?2-nAChRs. To this aim, we took advantage of a selective re-expression of ?2-nAChR exclusively in dopamine cells by stereotaxically injecting a lentiviral vector in the mouse ventral tegmental area. Results We found that activation of PPAR? decreases in vitro both dopamine cell activity and ventral tegmental area net output through negative modulation of ?2-nAChRs. Additionally, PPAR? activation in vivo reduces both the number of spontaneously active dopamine neurons and nicotine-induced increased locomotion. Conclusions Our combined findings suggest PPAR? ligands as important negative modulators of ?2-nAChRs on dopamine neurons. Thus, PPAR? ligands might prove beneficial in treating those disorders where dopamine dysfunction plays a prominent role, such as schizophrenia and nicotine addiction.

Melis, Miriam; Carta, Stefano; Fattore, Liana; Tolu, Stefania; Yasar, Sevil; Goldberg, Steven R.; Fratta, Walter; Maskos, Uwe; Pistis, Marco

2010-01-01

195

Fluorescently tagged laminin subunits facilitate analyses of the properties, assembly and processing of laminins in live and fixed lung epithelial cells and keratinocytes  

PubMed Central

Recent analyses of collagen, elastin and fibronectin matrix assembly, organization and remodeling have been facilitated by the use of tagged proteins that can be visualized without the need for antibody labeling. Here, we report the generation of C-terminal tagged, full-length and “processed” (?3?LG4-5) human ?3 as well as C-terminal tagged, full-length human ?3 laminin subunits in adenoviral vectors. Human epidermal keratinocytes (HEKs) and human bronchial epithelial (BEP2D) cells, which assemble laminin-332-rich matrices, as well as primary rat lung alveolar type II (ATII) cells, which elaborate a fibrous network rich in laminin-311, were infected with adenovirus encoding the tagged human laminin subunits. In HEKs and BEP2D cells, tagged, full-length ?3, ?3?LG4-5 and ?3 laminin subunits incorporate into arrays of matrix organized into patterns that are comparable to those observed when such cells are stained using laminin-332 subunit antibody probes. Moreover, HEKs and BEP2Ds move over these tagged, laminin-332-rich matrix arrays. We have also used the tagged ?3 laminin subunit-containing matrices to demonstrate that assembled laminin-332 arrays influence laminin matrix secretion and/or assembly. In the case of rat ATII cells, although tagged ?3 laminin subunits are not detected in the matrix of rat ATII cells infected with virus encoding full-length human ?3 laminin protein, processed human ?3 laminin subunits are incorporated into an extracellular fibrous array. We discuss how these novel laminin reagents can be used to study the organization, processing and assembly of laminin matrices and how they provide new insights into the potential functional importance of laminin fragments.

Hopkinson, Susan B.; DeBiase, Phillip J.; Kligys, Kristina; Hamill, Kevin; Jones, Jonathan C.R.

2008-01-01

196

Protease activated receptors in cardiovascular function and disease  

Microsoft Academic Search

Recent studies have shown that a novel class of protease activated receptors (PARs), which are composed of seven transmembrane G protein-coupled domains, are activated by serine proteases such as thrombin, trypsin and tryptase. Although four types (PAR 1, PAR 2, PAR 3 and PAR 4) of this class of receptors have been identified, their discrete physiological and pathological roles are

Junor A. Barnes; Shamjeet Singh; Aldrin V. Gomes

2004-01-01

197

Peroxisome proliferator-activated receptor-? in macrophage lipid homeostasis  

Microsoft Academic Search

Peroxisome proliferator-activated receptor-? (PPAR?), a fatty acid receptor, has received particular attention as the molecular target of insulin-sensitizing drugs, and as a regulator of lipid accumulation by the coronary artery macrophages known as foam cells. Controversial results have been reported regarding the consequences of PPAR? activation in the inflammatory response, the progression or improvement of the atherosclerotic lesion, and the

Chih-Hao Lee; Ronald M Evans

2002-01-01

198

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

2012-01-01

199

Rat preputial sebocyte differentiation involves peroxisome proliferator-activated receptors.  

PubMed

The hallmark of sebaceous epithelial cell (sebocyte) differentiation is the accumulation of fused neutral fat droplets. Very little sebocyte differentiation occurs, however, in primary or organ culture, even upon incubating with androgens, which are required for maturation in vivo. We hypothesized that sebocyte cell culture systems lack activators of the peroxisome proliferator-activated receptors that are involved in adipocyte differentiation. We here report that activation of peroxisome proliferator-activated receptor gamma and alpha by their respective specific ligands, a thiazolidinedione and a fibrate, induced lipid droplet formation in sebocytes but not epidermal cells. Linoleic acid and carbaprostacyclin, both peroxisome proliferator-activated receptor delta and alpha ligand-activators, were more effective but less specific, stimulating lipid formation in both types of cells. Either was more effective than the combination of peroxisome proliferator-activated receptor gamma and alpha activation, suggesting that peroxisome proliferator-activated receptor delta is involved in this lipid formation. Linoleic acid 0.1 mM stimulated significantly more advanced sebocyte maturation than any other treatment, including carbaprostacyclin, which suggests a distinct role of long chain fatty acids in sebocyte differentiation. Peroxisome proliferator-activated receptor gammal mRNA was demonstrated in sebocytes, but not in epidermal cells; it was more strongly expressed in freshly dispersed than in cultured sebocytes. In contrast, peroxisome proliferator-activated receptor delta mRNA was expressed to a similarly high extent before and after culture in both sebocytes and epidermal cells. These findings are compatible with the concepts that peroxisome proliferator-activated receptor gamma1 gene expression plays a unique role in the differentiation of sebocytes, while peroxisome proliferator-activated receptor delta activation and long chain fatty acids finalize sebocyte maturation and are capable of stimulating epidermal lipid formation. These findings have implications for the development of new modalities of treatment for acne vulgaris. PMID:9989800

Rosenfield, R L; Kentsis, A; Deplewski, D; Ciletti, N

1999-02-01

200

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

2007-01-01

201

Protease-Activated Receptors, Apoptosis and Tumor Growth  

Microsoft Academic Search

Protease-activated receptors (PARs) are G-protein-coupled receptors (GPCRs) that are activated by a unique proteolytic mechanism. Besides the important role of blood coagulation factors in preventing bleeding after vascular injury, these serine proteinases actively engage target cells thereby fulfilling critical functions in cell biology. Cellular responses triggered by coagulation factor-induced PAR activation suggest that PARs play an important role in proliferation,

Keren S. Borensztajn; C. Arnold Spek

2009-01-01

202

Topical Peroxisome Proliferator Activated Receptor-? Activators Reduce Inflammation in Irritant and Allergic Contact Dermatitis Models1  

Microsoft Academic Search

Activators of peroxisome proliferator activated receptor-?, a nuclear hormone receptor that heterodimerizes with retinoid X receptor, stimulate epidermal differentiation and inhibit proliferation. Here we determined the anti-inflammatory effects of peroxisome proliferator activated receptor-? agonists in models of irritant and allergic contact dermatitis produced in mouse ears by topical treatment with 12-O-tetradecanoylphorbol-13-acetate and oxazalone, respectively. As expected, 12-O-tetradecanoylphorbol-13-acetate treatment resulted in

Mary Y. Sheu; Ashley J. Fowler; Jack Kao; Matthias Schmuth; Kristina Schoonjans; Johan Auwerx; Joachim W. Fluhr; Mao-Qiang Man; Peter M. Elias; Kenneth R. Feingold

2002-01-01

203

Frequent Co-Localization of Cox-2 and Laminin-5 ?2 Chain at the Invasive Front of Early-Stage Lung Adenocarcinomas  

PubMed Central

Laminin-5 is an extracellular matrix protein that plays a key role in cell migration and tumor invasion. Cox-2 is an induced isoform of cyclooxygenases that plays an important role in carcinogenesis, suppression of apoptosis, angiogenesis, and metastasis of colon cancer. We report frequent co-expression of cox-2 and laminin-5 at the invasive front of early-stage lung adenocarcinomas. We investigated the expression of cox-2 and laminin-5 immunohistochemically in 102 cases of small-sized lung adenocarcinoma (maximum dimension, 2 cm or less). Cox-2 and laminin-5 were expressed in 97 (95.1%) and 82 (80.4%) cases, respectively. Both were preferentially localized in cancer cells at the cancer-stroma interface, although cox-2 tended to show a diffuse staining pattern in some cases. A comparison of their staining patterns revealed a striking similarity in their distribution in 24 cases, and a partial overlap between their localization in another 20 cases. Moreover, an overall correlation was found between the expression levels of cox-2 and laminin-5 (P = 0.018). To gain insight into the mechanisms that regulate the expression of these proteins, we additionally studied their expression in 58 cases of stage I lung adenocarcinoma, in which p53 status was determined by immunohistochemistry, polymerase chain reaction-single strand conformation polymorphism analysis, and direct sequencing. The results showed that tumors with mutant p53 tended to express more cox-2 than those with wild-type p53 (P = 0.080). Also, tumors that overexpressed p53 had higher levels of cox-2 and laminin-5 than those without p53 overexpression (P = 0.032 and 0.047, respectively). Further immunohistochemical analysis showed that tumors that overexpressed both epidermal growth factor receptor (EGFR) and erbB-2 had higher levels of cox-2 and laminin-5 than those without concomitant overexpression of these proteins (P = 0.014 and P = 0.018, respectively). To see whether EGFR signaling is involved in cox-2 and laminin-5 expression, we further conducted in vitro analyses using six lung adenocarcinoma cell lines (A549, HLC-1, ABC-1, LC-2/ad, VMRC-LCD, and L27). Western blot analyses showed that cox-2 mRNA levels, and to a lesser extent laminin-5 ?2 mRNA levels, correlated with the expression levels of erbB-2 and the phosphorylated form of MAPK/ERK-1/2 protein. The addition of transforming growth factor-? increased both cox-2 and laminin-5 ?2 mRNA levels in A549, ABC-1, and L27 with different kinetics; the induction of cox-2 occurred earlier than that of laminin-5 ?2. Finally, the migration of ABC-1 cells was inhibited by MAP kinase kinase inhibitor PD98059 and a selective cox-2 inhibitor NS-398. In contrast, the migration of A549 cells was inhibited by PD98059, but much less effectively by NS-398. These results suggest that co-stimulatory mechanisms may exist that increase the expression of cox-2 and laminin-5 at the invasive front of lung adenocarcinomas and that EGFR signaling could be one of the mechanisms. Further investigations are warranted concerning the role of cox-2 and laminin-5 in cancer cell invasion and the significance of p53 and EGFR signaling in the regulation of cox-2 and laminin-5 expression.

Niki, Toshiro; Kohno, Takashi; Iba, Sanae; Moriya, Yasumitsu; Takahashi, Yoko; Saito, Miyuki; Maeshima, Arafumi; Yamada, Tesshi; Matsuno, Yoshihiro; Fukayama, Masashi; Yokota, Jun; Hirohashi, Setsuo

2002-01-01

204

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

PubMed

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

2013-08-23

205

Effects of 1,25-dihydroxyvitamin D3 on tumor cell invasion to the extracellular matrix in human fibrosarcoma HT1080 cells and its correlation with laminin.  

PubMed

We investigated the role of the active form of vitamin D, 1,25-dihydroxyvitamin D3[1,25(OH)2D3], in promoting tumor cell invasiveness through the extracellular matrix, and showed that 1,25(OH)2D3-induced reduction of laminin production by the cells was correlated with the inhibitory effect of the hormone on tumor cell invasiveness. 1,25(OH)2D3 significantly inhibited invasiveness through the matrix, type IV collagenolytic and migratory activity, but not cell attachment to the matrix in human fibrosarcoma HT1080 cells. The 1,25(OH)2D3-induced inhibition showed the same dose dependency and magnitude for invasiveness as for the effects on type IV collagenolysis and cell migration. 1,25(OH)2D3 inhibited laminin production from the cells in a dose-dependent manner. The inhibitory effects of 1,25(OH)2D3 on the invasiveness, type IV collagenolysis and cell migration appeared to parallel the hormone-induced reduction of laminin production. Antilaminin monoclonal antibody, blocking the activity of laminin in the culture medium, inhibited HT1080 cell invasiveness. In the presence of exogenous laminin, 1,25(OH)2D3-induced inhibition of invasion was not observed. These findings suggest that 1,25(OH)2D3 acts on HT1080 cells, inhibiting the expression of laminin from the cells, and that the reduced laminin expression leads to the inhibition in the type IV collagenolytic and migratory activity of the cells, and consequently, to the inhibition of invasiveness through the extracellular matrix. PMID:9222304

Yudoh, K; Matsui, H; Tsuji, H

1997-01-01

206

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

207

Activation and dynamic network of the M2 muscarinic receptor.  

PubMed

G-protein-coupled receptors (GPCRs) mediate cellular responses to various hormones and neurotransmitters and are important targets for treating a wide spectrum of diseases. Although significant advances have been made in structural studies of GPCRs, details of their activation mechanism remain unclear. The X-ray crystal structure of the M2 muscarinic receptor, a key GPCR that regulates human heart rate and contractile forces of cardiomyocytes, was determined recently in an inactive antagonist-bound state. Here, activation of the M2 receptor is directly observed via accelerated molecular dynamics simulation, in contrast to previous microsecond-timescale conventional molecular dynamics simulations in which the receptor remained inactive. Receptor activation is characterized by formation of a Tyr206(5.58)-Tyr440(7.53) hydrogen bond and ?6-Å outward tilting of the cytoplasmic end of transmembrane ?-helix 6, preceded by relocation of Trp400(6.48) toward Phe195(5.47) and Val199(5.51) and flipping of Tyr430(7.43) away from the ligand-binding cavity. Network analysis reveals that communication in the intracellular domains is greatly weakened during activation of the receptor. Together with the finding that residue motions in the ligand-binding and G-protein-coupling sites of the apo receptor are correlated, this result highlights a dynamic network for allosteric regulation of the M2 receptor activation. PMID:23781107

Miao, Yinglong; Nichols, Sara E; Gasper, Paul M; Metzger, Vincent T; McCammon, J Andrew

2013-07-01

208

P2 receptors activated by uracil nucleotides--an update.  

PubMed

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

2006-01-01

209

Peroxisome proliferator-activated receptors: insight into multiple cellular functions  

Microsoft Academic Search

Peroxisome proliferator-activated receptors, PPARs, (NR1C) are nuclear hormone receptors implicated in energy homeostasis. Upon activation, these ligand-inducible transcription factors stimulate gene expression by binding to the promoter of target genes. The different structural domains of PPARs are presented in terms of activation mechanisms, namely ligand binding, phosphorylation, and cofactor interaction. The specificity of ligands, such as fatty acids, eicosanoids, fibrates

Pascal Escher; Walter Wahli

2000-01-01

210

An antagonist peptide–EPO receptor complex suggests that receptor dimerization is not sufficient for activation  

Microsoft Academic Search

Dimerization of the erythropoietin (EPO) receptor (EPOR), in the presence of either natural (EPO) or synthetic (EPO-mimetic peptides, EMPs) ligands is the principal extracellular event that leads to receptor activation. The crystal structure of the extracellular domain of EPOR bound to an inactive (antagonist) peptide at 2.7 Å resolution has unexpectedly revealed that dimerization still occurs, but the orientation between

Oded Livnah; Dana L. Johnson; Enrico A. Stura; Francis X. Farrell; Francis P. Barbone; Yun You; Kathleen D. Liu; Mark A. Goldsmith; Wen He; Christopher D. Krause; Sidney Pestka; Linda K. Jolliffe; Ian A. Wilson

1998-01-01

211

Ensemble of G Protein-Coupled Receptor Active States  

PubMed Central

G protein-coupled receptors (GPCRs) play critical roles in cellular signal transduction and are important targets for therapeutics. Although these receptors have been intensely studied for quite some time, our understanding about their mechanism of action is still incomplete. GPCR activity has traditionally been viewed within the context of two-state models where the receptor is in equilibrium between a single inactive state and a single active state. This framework is too simple and restrictive to accommodate more recent observations made on these receptors, which instead point to a situation where the receptor can adopt several different active conformational substates with distinct functional effects. Structural and functional evidence for this emerging view is presented in this review. Implications of this emerging view in rationalizing diseased states and in drug discovery are also discussed.

Park, P.S.-H.

2012-01-01

212

Co-expression of laminin ?3 and ?2 chains and epigenetic inactivation of laminin ?3 chain in gastric cancer.  

PubMed

Laminin-332 (LM-332, formerly termed laminin-5) is a heterotrimeric glycoprotein that regulates cell adhesion and migration. Molecular alterations of LM-332 are involved in cancer progression. The aim of this study was to clarify alterations of LM-332 in gastric carcinoma. The expression of LM-332 subunits in 10 gastric carcinoma cell lines was investigated by RT-PCR, Western blotting, and immuno-cytochemical/immunofluorescent analyses. The promoter methylation status of LM-332-encoding genes (LAMA3, LAMB3 and LAMC2) was analyzed by methylation-specific PCR (MSP). The relationship between cell migration and LM-332 expression was assessed by the scratch assay. The expression of LM-332 was analyzed immunohistochemically in 90 gastric cancer tissues. Co-expression of laminin ?3 and ?2 chains was often observed in gastric carcinoma cell lines at mRNA and protein levels. In contrast, there was no expression of laminin ?3 at either the mRNA or protein levels. Extra-cellular secretion of laminin ?3 and ?2 chains was found in 2 of the 10 cell lines. The LAMA3 gene was transcriptionally silenced by methylation of the promoter CpG islands in all of the cell lines, while the LAMB3 and LAMC2 genes were silenced in several cell lines. Treatment with a demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC), restored expression of the LM-332-encoding genes. Methylation frequency of LAMA3 was higher than those of the LAMB2 and LAMC2 genes in gastric cancer tissues. Migration distances were significantly correlated with cytoplasmic laminin ?2 chain expression. Immunohistochemistry showed frequent co-expression of laminin ?3 and ?2 chains in gastric carcinoma cells, which was significantly correlated with depth of invasion and advanced tumor stage. The results suggest that the laminin ?3 and ?2 chains accumulate intracellularly and play a role in gastric cancer progression, while epigenetic silencing of the laminin ?3 chain may lead to inability to synthesize the basement membrane and may affect cancer cell invasion. Cancer cell motility appears to be associated with the cyto-plasmic laminin ?2 chain in vitro. PMID:21617852

Ii, Masanori; Yamamoto, Hiroyuki; Taniguchi, Hiroaki; Adachi, Yasushi; Nakazawa, Mayumi; Ohashi, Hirokazu; Tanuma, Tokuma; Sukawa, Yasutaka; Suzuki, Hiromu; Sasaki, Shigeru; Imai, Kohzoh; Shinomura, Yasuhisa

2011-09-01

213

The Environmental Estrogen, Nonylphenol, Activates the Constitutive Androstane Receptor  

Microsoft Academic Search

Nonylphenol (NP) and its parent compounds, the nonylphenol ethoxylates are some of the most prevalent chemicals found in U.S. waterways. NP is also resistant to biodegradation and is a known environmental estrogen, which makes NP a chemical of concern. Our data show that NP also activates the constitutive androstane receptor (CAR), an orphan nuclear receptor important in the induction of

Juan P. Hernandez; Wendong Huang; Laura M. Chapman; Steven Chua; David D. Moore; William S. Baldwin

2007-01-01

214

Vitamin E activates gene expression via the pregnane X receptor  

Microsoft Academic Search

Tocopherols and tocotrienols are metabolized by side chain degradation via initial ?-oxidation and subsequent ?-oxidation. ?-Oxidation is performed by cytochrome P450 (CYP) enzymes which are often regulated by their substrates themselves. Results presented here show that all forms of Vitamin E are able to activate gene expression via the pregnane X receptor (PXR), a nuclear receptor regulating a variety of

Nico Landes; Paul Pfluger; Dirk Kluth; Marc Birringer; Ralph Rühl; Gaby-Fleur Böl; Hansruedi Glatt; Regina Brigelius-Flohé

2003-01-01

215

ACTH receptor: Ectopic expression, activity and signaling  

Microsoft Academic Search

Failure in obtaining expression of functional adrenocorticotropic hormone receptor (ACTHR, or melanocortin 2 receptor, MC2R) in non-adrenal cells has hindered molecular analysis of ACTH signaling pathways. Here, we ectopically expressed the mouse ACTHR in Balb\\/c mouse 3T3 fibroblasts to analyze ACTH signaling pathways involved in induction of fos and jun genes. Natural constitutive expression of the MC2R accessory protein (MRAP)

F?bio Lu?s Forti; Matheus H. S. Dias; Hugo Aguirre Armelin

2006-01-01

216

Potentiation of liver X receptor transcriptional activity by peroxisome-proliferator-activated receptor gamma co-activator 1 alpha.  

PubMed Central

Peroxisome-proliferator-activated receptor (PPAR) gamma co-activator 1 alpha (PGC-1 alpha/PPARGC1) plays an important role in energy metabolism by co-ordinating transcriptional programmes of mitochondrial biogenesis, adaptive thermogenesis and fatty acid beta-oxidation. PGC-1 alpha has also been identified to play a role in the intermediary metabolism by co-activating key transcription factors of hepatic gluconeogenesis and glucose uptake in muscles. In the present study, we show that PGC-1 alpha serves as a co-activator for the liver X receptor (LXR) alpha, known to contribute to the regulation of cellular cholesterol homoeostasis. In transient transfection studies, PGC-1 alpha amplified the LXR-mediated autoregulation of the LXR alpha promoter in a human brown adipocyte line and in 3T3-L1 cells via an LXR response element described previously. LXR-mediated transactivation via a natural LXR response element from the cholesteryl ester transfer-protein gene promoter was also enhanced by PGC-1 alpha in a ligand-dependent manner. Mutational analysis showed that the LXXLL signature motif (L2) of PGC-1 alpha was essential for co-activation of LXR-mediated transcriptional responses. This motif is located in the vicinity of the binding region for a putative repressor described previously. The repressor sequesters PGC-1 alpha from PPAR alpha and the glucocorticoid receptor, and this repressor did not interfere with PGC-1 alpha-mediated co-activation of LXR-dependent gene transcription. Moreover, inhibition of p38 mitogen-activated protein kinase signalling, shown to abolish the co-activation of PPAR alpha by PGC-1 alpha, had only a moderate inhibitory effect on the co-activation of LXR. These results identify PGC-1 alpha as a bona fide LXR co-activator and implicate distinct interfaces of PGC-1 alpha and/or additional cofactors in the modulation of LXR and PPAR alpha transcriptional activities.

Oberkofler, Hannes; Schraml, Elisabeth; Krempler, Franz; Patsch, Wolfgang

2003-01-01

217

Metal Binding Is Critical for the Folding and Function of Laminin Binding Protein, Lmb of Streptococcus agalactiae  

PubMed Central

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.

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

2013-01-01

218

T-cell receptor early signalling complex activation in response to interferon-? receptor stimulation  

PubMed Central

Signalling through the IFN?R (interferon-? receptor) and TCR (T-cell receptor) in Jurkat T lymphocytes results in distinct immune responses. Despite this both receptors elicit ERK (extracellular-signal-regulated kinase)/MAPK (mitogen-activated protein kinase) phosphorylation. Vav and Slp76 are shown to be required for IFN? (interferon-?)-stimulated ERK activity. These form a subset of proteins which behave identically on stimulation of both receptors. TCR deletion abrogates IFN?R-stimulated MAPK activity, whereas the canonical JAK/STAT (Janus kinase/signal transducer and activator of transcription) pathway is unaffected. Thus recruitment of the intact TCR ESC (early signalling complex) is necessary for this downstream MAPK response. Despite using a common ESC, stimulation of the IFN?R does not produce the transcriptional response associated with TCR. Up-regulation of the MAPK pathway by IFN?R might be important to ensure that the cell responds to only one stimulant.

Stevens, Claire N.; Simeone, Ann-Marie; John, Susan; Ahmed, Zamal; Lucherini, Orso M.; Baldari, C. Tatiana; Ladbury, John E.

2010-01-01

219

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.

2012-01-01

220

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

2000-01-01

221

Kinetics and mechanism of G protein-coupled receptor activation.  

PubMed

The activation of a G protein-coupled receptor is generally triggered by binding of an agonist to the receptor's binding pocket, or, in the case of rhodopsin, by light-induced changes of the pre-bound retinal. This is followed by a series of a conformational changes towards an active receptor conformation, which is capable of signalling to G proteins and other downstream proteins. In the past few years, a number of new techniques have been employed to analyze the kinetics of this activation process, including X-ray crystallographic three-dimensional structures of receptors in the inactive and the active states, NMR studies of labelled receptors, molecular simulations, and optical analyses with fluorescence resonance energy transfer (FRET). Here we review our current understanding of the activation process of GPCRs as well as open questions in the sequence of events ranging from (sub-)microsecond activation by light or agonist binding to millisecond activation of receptors by soluble ligands and the subsequent generation of an intracellular signal. PMID:24530699

Lohse, Martin J; Maiellaro, Isabella; Calebiro, Davide

2014-04-01

222

Tie2 and Eph receptor tyrosine kinase activation and signaling.  

PubMed

The Eph and Tie cell surface receptors mediate a variety of signaling events during development and in the adult organism. As other receptor tyrosine kinases, they are activated on binding of extracellular ligands and their catalytic activity is tightly regulated on multiple levels. The Eph and Tie receptors display some unique characteristics, including the requirement of ligand-induced receptor clustering for efficient signaling. Interestingly, both Ephs and Ties can mediate different, even opposite, biological effects depending on the specific ligand eliciting the response and on the cellular context. Here we discuss the structural features of these receptors, their interactions with various ligands, as well as functional implications for downstream signaling initiation. The Eph/ephrin structures are already well reviewed and we only provide a brief overview on the initial binding events. We go into more detail discussing the Tie-angiopoietin structures and recognition. PMID:24478383

Barton, William A; Dalton, Annamarie C; Seegar, Tom C M; Himanen, Juha P; Nikolov, Dimitar B

2014-03-01

223

Constitutively active ALK2 receptor mutants require type II receptor cooperation.  

PubMed

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

2013-06-01

224

Constitutively Active ALK2 Receptor Mutants Require Type II Receptor Cooperation  

PubMed Central

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.

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

2013-01-01

225

Constitutive Somatostatin Receptor Activity Determines Tonic Pituitary Cell Response  

PubMed Central

Somatostatin (SRIF) binds G protein-coupled SRIF receptor subtypes (SST1, -2, -3, -4, and -5) to regulate cell secretion and proliferation. Hypothalamic SRIF inhibits pituitary growth hormone, thyroid stimulating hormone, and ACTH secretion. We tested SRIF-independent constitutive SST activity in AtT20 mouse pituitary corticotroph cells in which ACTH secretion is highly sensitive to SRIF action. Stable transfectants expressing SST2 or SST5 were sensitized to selective agonist action, and constitutive SST receptor activity was demonstrated by forskolin and pertussis toxin cAMP cell responses. Persistent constitutive SST activity decreased cell ACTH responses to CRH through decreased expression of CRH receptor subtype 1. Decreased dopamine receptor type 1 expression was associated with attenuated dopamine agonist action, whereas responses to isoproterenol were enhanced through increased ?2-adrenoreceptor expression. Thus, integrated pituitary cell ACTH regulation is determined both by phasic SRIF action, as well as by tonic constitutive SST activity, independently of SRIF.

Ben-Shlomo, Anat; Zhou, Cuiqi; Pichurin, Oxana; Chesnokova, Vera; Liu, Ning-Ai; Culler, Michael D.; Melmed, Shlomo

2009-01-01

226

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

2012-01-01

227

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

1998-01-01

228

Structural and biophysical characterisation of agrin laminin G3 domain constructs.  

PubMed

Agrin mediates accumulation of acetylcholine receptors (AChRs) at the developing neuromuscular junction, but has also been implicated as a regulator of central nervous system (CNS) synapses. A C-terminal region of agrin (Ag-C20) binds to the ?3 subunit of the sodium-potassium ATPase (NKA) in CNS neurons suggesting that ?3NKA is a neuronal agrin receptor, whereas a shorter agrin fragment (Ag-C15) was shown to act as a competitive antagonist. Here, we show that the agrin C22 construct, which represents the naturally occurring neurotrypsin cleavage product, constitutes a well-folded, stable domain, while the deletion of 48 residues that correspond to strands ?1-?4 of the agrin laminin G3 domain imposed by the agrin C15 construct leads to a misfolded protein. PMID:21037280

Tidow, Henning; Mattle, Daniel; Nissen, Poul

2011-01-01

229

Activation of 5-HT7 receptors increases neuronal platelet-derived growth factor ? receptor expression.  

PubMed

Several antipsychotics have a high affinity for 5-HT7 receptors yet despite intense interest in the 5-HT7 receptor as a potential drug target to treat psychosis, the function and signaling properties of 5-HT7 receptors in neurons remain largely uncharacterized. In primary mouse hippocampal and cortical neurons, as well as in the SH-SY5Y cell line, incubation with 5-HT, 5-carboxamidotryptamine (5-CT), or 5-HT7 receptor-selective agonists increases the expression of platelet-derived growth factor (PDGF)? receptors. The increased PDGF? receptor expression is cyclic AMP-dependent protein kinase (PKA)-dependent, suggesting that 5-HT7 receptors couple to G?(s) in primary neurons. Interestingly, up-regulated PDGF? receptors display an increased basal phosphorylation state at the phospholipase C?-activating tyrosine 1021. This novel linkage between the 5-HT7 receptor and the PDGF system may be an important GPCR-neurotrophic factor signaling pathway in neurons. PMID:22285262

Vasefi, Maryam S; Kruk, Jeff S; Liu, Hui; Heikkila, John J; Beazely, Michael A

2012-03-01

230

Spreading of explants of embryonic chick mesenchymes and epithelia on fibronectin and laminin  

Microsoft Academic Search

Tissues from 2.5-day chick embryos were explanted onto glass coated with adsorbed fibronectin or laminin, or extracellular matrices (ECM) of deoxycholate-extracted chick embryo cells. Spreading of somitic and trunk neural-crest mesenchyme cells was equally rapid and extensive on fibronectin, laminin and on the fibronectinrich, laminin-poor ECM produced by mesenchymal cells. No preference for fibronectin over laminin was displayed by these

Don Newgreen

1984-01-01

231

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.

2011-01-01

232

Evidence That a Laminin-Like Insect Protein Mediates Early Events in the Interaction of a Phytoparasite with Its Vector's Salivary Gland  

PubMed Central

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.

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

2012-01-01

233

Activation of the p75 neurotrophin receptor through conformational rearrangement of disulphide-linked receptor dimers  

PubMed Central

SUMMARY Ligand-mediated dimerization has emerged as a universal mechanism of growth factor receptor activation. Recent structural studies have shown that neurotrophins interact with dimers of the p75 neurotrophin receptor (p75NTR), but the actual mechanism of receptor activation has remained elusive. Here we show that p75NTR forms disulphide-linked dimers independently of neurotrophin binding through the highly conserved Cys257 in its transmembrane domain. Mutation of Cys257 abolished neurotrophin-dependent receptor activity but did not affect downstream signaling by the p75NTR/NgR/Lingo-1 complex in response to MAG, indicating the existence of distinct, ligand-specific activation mechanisms for p75NTR. FRET experiments revealed a close association of p75NTR intracellular domains that was transiently disrupted by conformational changes induced upon NGF binding. Although mutation of Cys257 did not alter the oligomeric state of p75NTR, the mutant receptor was no longer able to propagate conformational changes to the cytoplasmic domain upon ligand binding. We propose that neurotrophins activate p75NTR by a novel mechanism involving rearrangement of disulphide-linked receptor subunits.

Vilar, Marcal; Charalampopoulos, Ioannis; Kenchappa, Rajappa S.; Simi, Anastasia; Karaca, Esra; Reversi, Alessandra; Choi, Soyoung; Bothwell, Mark; Mingarro, Ismael; Friedman, Wilma J.; Schiavo, Giampietro; Bastiaens, Philippe I. H.; Verveer, Peter J.; Carter, Bruce D.; Ibanez, Carlos F.

2010-01-01

234

M5 receptor activation produces opposing physiological outcomes in dopamine neurons depending on the receptor's location.  

PubMed

Of the five muscarinic receptor subtypes, the M5 receptor is the only one detectable in midbrain dopaminergic neurons, making it an attractive potential therapeutic target for treating disorders in which dopaminergic signaling is disrupted. However, developing an understanding of the role of M5 in regulating midbrain dopamine neuron function has been hampered by a lack of subtype-selective compounds. Here, we extensively characterize the novel compound VU0238429 and demonstrate that it acts as a positive allosteric modulator with unprecedented selectivity for the M5 receptor. We then used VU0238429, along with M5 knock-out mice, to elucidate the role of this receptor in regulating substantia nigra pars compacta (SNc) neuron physiology in both mice and rats. In sagittal brain slices that isolate the SNc soma from their striatal terminals, activation of muscarinic receptors induced Ca2+ mobilization and inward currents in SNc dopamine neurons, both of which were potentiated by VU0238429 and absent in M5 knock-out mice. Activation of M5 also increased the spontaneous firing rate of SNc neurons, suggesting that activation of somatodendritic M5 increases the intrinsic excitability of SNc neurons. However, in coronal slices of the striatum, potentiation of M5 with VU0238429 resulted in an inhibition in dopamine release as monitored with fast scan cyclic voltammetry. Accordingly, activation of M5 can lead to opposing physiological outcomes depending on the location of the receptor. Although activation of somatodendritic M5 receptors on SNc neurons leads to increased neuronal firing, activation of M5 receptors in the striatum induces an inhibition in dopamine release. PMID:24573284

Foster, Daniel J; Gentry, Patrick R; Lizardi-Ortiz, Jose E; Bridges, Thomas M; Wood, Michael R; Niswender, Colleen M; Sulzer, David; Lindsley, Craig W; Xiang, Zixiu; Conn, P Jeffrey

2014-02-26

235

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

PubMed

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. PMID:21794146

Sanderson, Thomas M; Collingridge, Graham L; Fitzjohn, Stephen M

2011-01-01

236

Peroxisome proliferator-activated receptors: regulation of transcriptional activities and roles in inflammation  

Microsoft Academic Search

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear receptor superfamily. Three PPARs isoforms have been characterized: PPAR?, ?\\/? and ?. As other nuclear receptors, the PPARs are organized in distinct functional domains: A\\/B, C or DNA binding domain (DBD), D, E or ligand binding domain (LBD) and F. The A\\/B domain contains the activation function 1

Christophe Blanquart; Olivier Barbier; Jean Charles Fruchart; Bart Staels; Corine Glineur

2003-01-01

237

Inhibitors for Androgen Receptor Activation Surfaces.  

National Technical Information Service (NTIS)

Our research demonstrated a new protein interaction site on the androgen receptor. We showed that the site called BF3 has the characteristics of functioning in repression and likely binds one or more proteins. We are presently performing experiments to id...

R. J. Fletterick

2008-01-01

238

Induction of a Basement Membrane Glycoprotein in Embryonic Kidney: Possible Role of Laminin in Morphogenesis  

Microsoft Academic Search

The glycoprotein laminin is found exclusively in the basement membranes of adult tissues, not in the mesenchymal stroma. We studied the appearance and distribution of laminin during the early formation of kidney tubules in mouse embryos and in an in vitro transfilter model system. In immunofluorescence using affinity-purified antibodies, the distribution of laminin showed a clear correlation, both spatially and

Peter Ekblom; Kari Alitalo; Antti Vaheri; Rupert Timpl; Lauri Saxen

1980-01-01

239

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

PubMed

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

2013-08-01

240

Proteolytic Processing of Laminin-332 by Hepsin and Matriptase and Its Role in Prostate Cancer Progression.  

National Technical Information Service (NTIS)

Laminin-332 is lost in prostate cancer progression. Laminin-332 is known to be cleaved by various cell surface proteases, including MMPs, and this cleavage facilitates migration of cancer cells. We have found that Laminin-332 is individually cleaved by tw...

M. Tripathi

2010-01-01

241

Proteolytic Processing of Laminin-332 by Hepsin and Matriptase and Its Role in Prostate Cancer Progression.  

National Technical Information Service (NTIS)

Laminin-332 is lost in prostate cancer progression. Laminin-332 is known to be cleaved by various cell surface proteases, including MMPs, and this cleavage facilitates migration of cancer cells. We have found that Laminin-332 is individually cleaved by tw...

M. Tripathi

2011-01-01

242

Design and functional evaluation of an optically active ?-opioid receptor.  

PubMed

The use of opioids, which achieve therapeutic analgesia through activation of ?-opioid receptors, are limited in the management of chronic pain by adverse effects including tolerance and addiction. Optogenetics is an emerging approach of designing molecular targets that can produce cell-specific receptor-mediated analgesia with minimal side effects. Here we report the design and functional characterization of a chimeric ?-opioid receptor that could be photoactivated to trigger intracellular signaling. A prototype optoactive ?-opioid receptor (optoMOR) was designed by replacing the intracellular domains from rhodopsin with those of the native ?-opioid receptor and was transiently expressed in human embryonic kidney (HEK293) cells. Expression and distribution of the protein were confirmed by immunocytochemistry. The signal-transduction mechanisms induced by photoactivation of the optoMOR were evaluated and compared with the native ?-opioid receptor stimulation by an agonist, D-Ala(2), N-MePhe(4), Gly-ol-enkephalin (DAMGO). Cells were depolarized by extracellular potassium and the depolarization-induced calcium (Ca(2+)) influx was quantified by using Fura-2 imaging. The forskolin-stimulated adenylate cyclase/cAMP cascade was evaluated by ELISA or western blotting of brain-derived neurotrophic factor (BDNF) and the phosphorylation of cAMP response element binding protein (CREB). The optoMOR protein distribution was observed intracellularly and on the plasma membrane similar to the native ?-opioid receptor in HEK293 cells. Photoactivation of optoMOR decreased the Ca(2+) influx and inhibited the forskolin-induced cAMP generation, activation of CREB, and BDNF levels in optoMOR-expressing cells similar to the activation of native ?-opioid receptor by DAMGO. Thus the current study has accomplished the design of a prototype optoMOR and characterized the cellular signaling mechanisms activated by light stimulation of this receptor. PMID:23454521

Barish, Philip A; Xu, Ying; Li, Jianxin; Sun, Jiao; Jarajapu, Yagna P R; Ogle, William O

2013-04-01

243

Activation kinetics of single P2X receptors  

PubMed Central

After the primary structure of P2X receptors had been identified, their function had to be characterized on the molecular level. Since these ligand-gated ion channels become activated very quickly after binding of ATP, methods with adequate time resolution have to be applied to investigate the early events induced by the agonist. Single-channel recordings were performed to describe conformational changes on P2X2, P2X4, and P2X7 receptors induced by ATP and also by allosteric receptor modifiers. The main results of these studies and the models of P2X receptor kinetics derived from these observations are reviewed here. The investigation of purinoceptors by means of the patch clamp technique following site-directed mutagenesis will probably reveal more details of P2X receptor function at the molecular level.

2007-01-01

244

Regulation of microglia activation and deactivation by nuclear receptors.  

PubMed

Microglia cells function as sentinels for innate immunity in the central nervous system (CNS). To perform this function, microglia express a diverse set of pattern recognition receptors (PRRs) for pathogen-associated molecular patterns (PAMPs) that include Toll-like receptors (TLRs) and inflammasomes. Several members of the TLR and inflammasome family also recognize endogenously derived molecules that are generated as a consequence of tissue injury or other pathological processes. Recognition of PAMPs or endogenous ligands by PRRs in microglia induces the robust activation of innate immune responses leading to the production of proinflammatory mediators and the activation of adaptive immunity. Activation of microglia is essential for clearance of infection and repair of tissue injury. However, uncontrolled inflammatory responses of microglia are also thought to contribute to the severity of many neurodegenerative diseases. Thus, activation of microglia must be properly and tightly regulated to maintain normal tissue homeostasis. Several mechanisms have been identified that appear to function in the active maintenance of quiescence under normal conditions and/or re-establish this state following resolution of infection or injury. These mechanisms involve communication with neurons and other glia through secreted molecules or surface expressing receptors as well as actions of members of the nuclear receptor (NR) superfamily of transcription factors. Here, we review recent advances in our understanding of the regulation of microglia activation and deactivation with a focus on counter-regulation of microglia activation by nuclear receptors. PMID:22987512

Saijo, Kaoru; Crotti, Andrea; Glass, Christopher K

2013-01-01

245

Activation of spinal GABAB receptors normalizes N-methyl-D-aspartate receptor in diabetic neuropathy.  

PubMed

N-methyl-D-aspartate receptor (NMDAR) activity is increased, while GABAB receptor is downregulated in the spinal cord dorsal horn in diabetic neuropathy. In this study, we determined the interaction of NMDARs and GABAB receptors in streptozotocin (STZ)-induced diabetic neuropathy. The paw withdrawal threshold (PWT) was significantly lower in STZ-treated rats than in vehicle-treated rats. Intrathecal injection of baclofen, a GABAB receptor agonist, significantly increased the PWT in STZ-treated rats, an effect that was abolished by pre-administration of the GABAB receptor specific antagonist CGP55845. Spinal NR2B, an NMDA receptor subunit, protein and mRNA expression levels were significantly higher in STZ-treated rats than in vehicle-treated rats. Intrathecal baclofen significantly reduced the NR2B protein and mRNA expression levels in STZ-treated rats. Intrathecal administration of CGP55845 eliminated baclofen-induced reduction of NR2B protein and mRNA levels in STZ-treated rats. In addition, the phosphorylated cAMP response element-binding (CREB) protein level was significantly higher in the spinal cord dorsal horn in STZ-treated rats compared with vehicle-treated rats. Intrathecal injection of baclofen significantly decreased phosphorylated CREB protein level in STZ-treated rats; an effect was blocked by CGP55845. These data suggest that activation of GABAB receptors in the spinal cord dorsal horn normalizes NMDAR expression level in diabetic neuropathic pain. PMID:24787504

Bai, Hui-Ping; Liu, Peng; Wu, Yu-Ming; Guo, Wen-Ya; Guo, Yue-Xian; Wang, Xiu-Li

2014-06-15

246

Laminin mediates tethering and spreading of colon cancer cells in physiological shear flow  

PubMed Central

Under the physiological shear condition, cultured colon cancer cells bound to laminin (LM), but not to fibronectin or vitronectin. Most of the tethered cells did not roll, but arrested immediately and spread within 10–30 min on LM under the continuous presence of shear flow. The tethering of Colo201 was partially inhibited by monoclonal antibodies (mAbs) to ?6 integrin and a combination of mAbs to ?1 and ?4 integrins, but not by mAb to 67KD laminin receptor. Some Colo201 cells still tethered at 4°C. This suggests that ?6?1 and ?6?4 integrins participate in Colo201 tethering on LM, although other non-integrin molecules play roles. In contrast, the spread of Colo201 was effectively inhibited by the mAbs to integrin ?2, ?6 and ?1 chains. The effect of anti-?2 plus anti-?6 mAbs was almost equal to anti-?1, suggesting that Colo201 cells mainly use ?2?1 and ?6?1 integrins for spreading on LM. When the cells were perfused on subconfluent endothelial cells (HUVEC) cultured on LM, they did not tether on HUVEC but did on coated LM exposed at intercellular gap area. Immunohistochemistry revealed that LM abundantly existed in the cytosol of human portal and hepatic vein endothelial cells. These data suggest that LM can mediate from tethering to spreading of colon cancer cells under the blood flow and plays an essential role in haematogeneous metastasis. © 1999 Cancer Research Campaign

Kitayama, J; Nagawa, H; Tsuno, N; Osada, T; Hatano, K; Sunami, E; Saito, H; Muto, T

1999-01-01

247

Sustained Glutamate Receptor Activation Down-regulates GABAB Receptors by Shifting the Balance from Recycling to Lysosomal Degradation*  

PubMed Central

Metabotropic GABAB receptors are abundantly expressed at glutamatergic synapses where they control excitability of the synapse. Here, we tested the hypothesis that glutamatergic neurotransmission may regulate GABAB receptors. We found that application of glutamate to cultured cortical neurons led to rapid down-regulation of GABAB receptors via lysosomal degradation. This effect was mimicked by selective activation of AMPA receptors and further accelerated by coactivation of group I metabotropic glutamate receptors. Inhibition of NMDA receptors, blockade of L-type Ca2+ channels, and removal of extracellular Ca2+ prevented glutamate-induced down-regulation of GABAB receptors, indicating that Ca2+ influx plays a critical role. We further established that glutamate-induced down-regulation depends on the internalization of GABAB receptors. Glutamate did not affect the rate of GABAB receptor endocytosis but led to reduced recycling of the receptors back to the plasma membrane. Blockade of lysosomal activity rescued receptor recycling, indicating that glutamate redirects GABAB receptors from the recycling to the degradation pathway. In conclusion, the data indicate that sustained activation of AMPA receptors down-regulates GABAB receptors by sorting endocytosed GABAB receptors preferentially to lysosomes for degradation on the expense of recycling. This mechanism may relieve glutamatergic synapses from GABAB receptor-mediated inhibition resulting in increased synaptic excitability.

Maier, Patrick J.; Marin, Isabel; Grampp, Thomas; Sommer, Andrea; Benke, Dietmar

2010-01-01

248

Dynamic regulation of Drosophila nuclear receptor activity in vivo.  

PubMed

Nuclear receptors are a large family of transcription factors that play major roles in development, metamorphosis, metabolism and disease. To determine how, where and when nuclear receptors are regulated by small chemical ligands and/or protein partners, we have used a 'ligand sensor' system to visualize spatial activity patterns for each of the 18 Drosophila nuclear receptors in live developing animals. Transgenic lines were established that express the ligand binding domain of each nuclear receptor fused to the DNA-binding domain of yeast GAL4. When combined with a GAL4-responsive reporter gene, the fusion proteins show tissue- and stage-specific patterns of activation. We show that these responses accurately reflect the presence of endogenous and exogenously added hormone, and that they can be modulated by nuclear receptor partner proteins. The amnioserosa, yolk, midgut and fat body, which play major roles in lipid storage, metabolism and developmental timing, were identified as frequent sites of nuclear receptor activity. We also see dynamic changes in activation that are indicative of sweeping changes in ligand and/or co-factor production. The screening of a small compound library using this system identified the angular psoralen angelicin and the insect growth regulator fenoxycarb as activators of the Ultraspiracle (USP) ligand-binding domain. These results demonstrate the utility of this system for the functional dissection of nuclear receptor pathways and for the development of new receptor agonists and antagonists that can be used to modulate metabolism and disease and to develop more effective means of insect control. PMID:16914501

Palanker, Laura; Necakov, Aleksandar S; Sampson, Heidi M; Ni, Ruoyu; Hu, Chun; Thummel, Carl S; Krause, Henry M

2006-09-01

249

Dynamic regulation of Drosophila nuclear receptor activity in vivo  

PubMed Central

Nuclear receptors are a large family of transcription factors that play major roles in development, metamorphosis, metabolism and disease. To determine how, where and when nuclear receptors are regulated by small chemical ligands and/or protein partners, we have used a ‘ligand sensor’ system to visualize spatial activity patterns for each of the 18 Drosophila nuclear receptors in live developing animals. Transgenic lines were established that express the ligand binding domain of each nuclear receptor fused to the DNA-binding domain of yeast GAL4. When combined with a GAL4-responsive reporter gene, the fusion proteins show tissue- and stage-specific patterns of activation. We show that these responses accurately reflect the presence of endogenous and exogenously added hormone, and that they can be modulated by nuclear receptor partner proteins. The amnioserosa, yolk, midgut and fat body, which play major roles in lipid storage, metabolism and developmental timing, were identified as frequent sites of nuclear receptor activity. We also see dynamic changes in activation that are indicative of sweeping changes in ligand and/or co-factor production. The screening of a small compound library using this system identified the angular psoralen angelicin and the insect growth regulator fenoxycarb as activators of the Ultraspiracle (USP) ligand-binding domain. These results demonstrate the utility of this system for the functional dissection of nuclear receptor pathways and for the development of new receptor agonists and antagonists that can be used to modulate metabolism and disease and to develop more effective means of insect control.

Palanker, Laura; Necakov, Aleksandar S.; Sampson, Heidi M.; Ni, Ruoyu; Hu, Chun; Thummel, Carl S.; Krause, Henry M.

2007-01-01

250

Opioid receptor activation in live cells  

Microsoft Academic Search

Interaction of the -opioid receptor (MOP) with selected ligands was investigated in live cells using advanced imaging by confocal laser scanning microscopy integrated with fluorescence correlation spectroscopy and fluorescence cross-correlation spec- troscopy. In PC12 cells stably transformed to express the fluorescently labeled MOP-enhanced green fluores- cent protein construct, two pools of MOP were identi- fied that could be discriminated by

Vladana Vukojevic; Yu Ming; Claudio D'Addario; Mats Hansen; Ulo Langel; Rudiger Schulz; Bjorn Johansson; Rudolf Rigler; Lars Terenius

2008-01-01

251

HINT1 protein cooperates with cannabinoid 1 receptor to negatively regulate glutamate NMDA receptor activity  

PubMed Central

Background G protein-coupled receptors (GPCRs) are the targets of a large number of drugs currently in therapeutic use. Likewise, the glutamate ionotropic N-methyl-D-aspartate receptor (NMDAR) has been implicated in certain neurological disorders, such as neurodegeration, neuropathic pain and mood disorders, as well as psychosis and schizophrenia. Thus, there is now an important need to characterize the interactions between GPCRs and NMDARs. Indeed, these interactions can produce distinct effects, and whereas the activation of Mu-opioid receptor (MOR) increases the calcium fluxes associated to NMDARs, that of type 1 cannabinoid receptor (CNR1) antagonizes their permeation. Notably, a series of proteins interact with these receptors affecting their responses and interactions, and then emerge as novel therapeutic targets for the aforementioned pathologies. Results We found that in the presence of GPCRs, the HINT1 protein influences the activity of NMDARs, whereby NMDAR activation was enhanced in CNR1+/+/HINT1-/- cortical neurons and the cannabinoid agonist WIN55,212-2 provided these cells with no protection against a NMDA insult. NMDAR activity was normalized in these cells by the lentiviral expression of HINT1, which also restored the neuroprotection mediated by cannabinoids. NMDAR activity was also enhanced in CNR1-/-/HINT1+/+ neurons, although this activity was dampened by the expression of GPCRs like the MOR, CNR1 or serotonin 1A (5HT1AR). Conclusions The HINT1 protein plays an essential role in the GPCR-NMDAR connection. In the absence of receptor activation, GPCRs collaborate with HINT1 proteins to negatively control NMDAR activity. When activated, most GPCRs release the control of HINT1 and NMDAR responsiveness is enhanced. However, cannabinoids that act through CNR1 maintain the negative control of HINT1 on NMDAR function and their protection against glutamate excitotoxic insult persists.

2013-01-01

252

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

2013-01-01

253

Opportunistic activation of TRP receptors by endogenous lipids: Exploiting lipidomics to understand TRP receptor cellular communication  

PubMed Central

Transient receptor potential channels (TRPs) form a large family of ubiquitous non-selective cation channels that function as cellular sensors and in many cases regulate intracellular calcium. Identification of the endogenous ligands that activate these TRP receptors is still under intense investigation with the majority of these channels still remaining “orphans”. That these channels respond to a variety of external stimuli (e.g. plant-derived lipids, changes in temperature, and changes in pH) provides a framework for their abilities as cellular sensors, however, the mechanism of direct activation is still under much debate and research. In the cases where endogenous ligands (predominately lipids) have shown direct activation of a channel, multiple ligands have been shown to activate the same channel suggesting that these receptors are “promiscuous” in nature. Lipidomics of a growing class of endogenous lipids, N-acyl amides, the most famous of which is N-arachidonoyl ethanolamine (the endogenous cannabinoid, Anandamide) is providing a novel set of ligands that have been shown to activate some members of the TRP family and have the potential to deorphanize many more. Here it is argued that activation of TRPV receptors, a subset of the larger family of TRPs, by multiple endogenous lipids that are structurally analogous is a model system to drive our understanding that many TRP receptors are not promiscuous, but are more characteristically “opportunistic” in nature; exploiting the structural similarity and biosynthesis of a narrow range of analogous endogenous lipids. In addition, this manuscript will compare the activation properties of TRPC5 to the activity profile of an “orphan” lipid, N-palmitoyl glycine; further demonstrating that lipidomics aimed at expanding our knowledge of the family of N-acyl amides has the potential to provide novel avenues of research for TRP receptors.

Bradshaw, Heather B.; Raboune, Siham; Hollis, Jennifer L.

2012-01-01

254

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

2002-01-01

255

Dopamine D2 Receptor Activation Modulates Perceived Odor Intensity  

Microsoft Academic Search

Dopaminergic modulation affects odor detection thresholds and olfactory discrimination capabilities in rats. The authors show that dopamine D2 receptor modulation affects odor discrimination capabilities in a manner similar to the modulation of stimulus intensity. Performance in a simultaneous odor discrimination task was systematically altered by manipulations of both odorant concentration and D2 receptor activation (agonist quinpirole, 0.025–0.5 mg\\/kg; antagonist spiperone,

Catherine J. Wei; Christiane Linster; Thomas A. Cleland

2006-01-01

256

Receptor activity modifying proteins (RAMPs) interact with the VPAC2 receptor and CRF1 receptors and modulate their function  

PubMed Central

Background and Purpose Although it is established that the receptor activity modifying proteins (RAMPs) can interact with a number of GPCRs, little is known about the consequences of these interactions. Here the interaction of RAMPs with the glucagon-like peptide 1 receptor (GLP-1 receptor), the human vasoactive intestinal polypeptide/pituitary AC-activating peptide 2 receptor (VPAC2) and the type 1 corticotrophin releasing factor receptor (CRF1) has been examined. Experimental Approach GPCRs were co-transfected with RAMPs in HEK 293S and CHO-K1 cells. Cell surface expression of RAMPs and GPCRs was examined by elisa. Where there was evidence for interactions, agonist-stimulated cAMP production, Ca2+ mobilization and GTP?S binding to Gs, Gi, G12 and Gq were examined. The ability of CRF to stimulate adrenal corticotrophic hormone release in Ramp2+/– mice was assessed. Key Results The GLP-1 receptor failed to enhance the cell surface expression of any RAMP. VPAC2 enhanced the cell surface expression of all three RAMPs. CRF1 enhanced the cell surface expression of RAMP2; the cell surface expression of CRF1 was also increased. There was no effect on agonist-stimulated cAMP production. However, there was enhanced G-protein coupling in a receptor and agonist-dependent manner. The CRF1 : RAMP2 complex resulted in enhanced elevation of intracellular calcium to CRF and urocortin 1 but not sauvagine. In Ramp2+/– mice, there was a loss of responsiveness to CRF. Conclusions and Implications The VPAC2 and CRF1 receptors interact with RAMPs. This modulates G-protein coupling in an agonist-specific manner. For CRF1, coupling to RAMP2 may be of physiological significance.

Wootten, D; Lindmark, H; Kadmiel, M; Willcockson, H; Caron, KM; Barwell, J; Drmota, T; Poyner, DR

2013-01-01

257

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

2013-01-01

258

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

1990-01-01

259

Reversible acetylation regulates vascular endothelial growth factor receptor-2 activity.  

PubMed

The tyrosine kinase receptor vascular endothelial growth factor receptor 2 (VEGFR2) is a key regulator of angiogenesis. Here we show that VEGFR2 is acetylated in endothelial cells both at four lysine residues forming a dense cluster in the kinase insert domain and at a single lysine located in the receptor activation loop. These modifications are under dynamic control of the acetyltransferase p300 and two deacetylases HDAC5 and HDAC6. We demonstrate that VEGFR2 acetylation essentially regulates receptor phosphorylation. In particular, VEGFR2 acetylation significantly alters the kinetics of receptor phosphorylation after ligand binding, allowing receptor phosphorylation and intracellular signaling upon prolonged stimulation with VEGF. Molecular dynamics simulations indicate that acetylation of the lysine in the activation loop contributes to the transition to an open active state, in which tyrosine phosphorylation is favored by better exposure of the kinase target residues. These findings indicate that post-translational modification by acetylation is a critical mechanism that directly affects VEGFR2 function. PMID:24620033

Zecchin, Annalisa; Pattarini, Lucia; Gutierrez, Maria Ines; Mano, Miguel; Mai, Antonello; Valente, Sergio; Myers, Mike P; Pantano, Sergio; Giacca, Mauro

2014-04-01

260

Novel benzopolycyclic amines with NMDA receptor antagonist activity.  

PubMed

A new series of benzopolycyclic amines active as NMDA receptor antagonists were synthesized. Most of them exhibited increased activity compared with related analogues previously published. All the tested compounds were more potent than clinically approved amantadine and one of them displayed a lower IC50 value than memantine, an anti-Alzheimer's approved drug. PMID:24698811

Valverde, Elena; Sureda, Francesc X; Vázquez, Santiago

2014-05-01

261

Regulatory network of inflammation downstream of proteinase-activated receptors  

Microsoft Academic Search

BACKGROUND: Protease-activated receptors (PAR) are present in the urinary bladder, and their expression is altered in response to inflammation. PARs are a unique class of G protein-coupled that carry their own ligands, which remain cryptic until unmasked by proteolytic cleavage. Although the canonical signal transduction pathway downstream of PAR activation and coupling with various G proteins is known and leads

Ricardo Saban; Michael R D'Andrea; Patricia Andrade-Gordon; Claudia K Derian; Igor Dozmorov; Michael A Ihnat; Robert E Hurst; Cindy Simpson; Marcia R Saban

2007-01-01

262

Motogenic and morphogenic activity of epithelial receptor tyrosine kinases  

PubMed Central

Receptor tyrosine kinases play essential roles in morphogenesis and differentiation of epithelia. Here we examined various tyrosine kinase receptors, which are preferentially expressed in epithelia (c-met, c- ros, c-neu, and the keratin growth factor [KGF] receptor), for their capacity to induce cell motility and branching morphogenesis of epithelial cells. We exchanged the ligand-binding domain of these receptors by the ectodomain of trkA and could thus control signaling by the new ligand, NGF. We demonstrate here that the tyrosine kinases of c- met, c-ros, c-neu, the KGF receptor, and trkA, but not the insulin receptor, induced scattering and increased motility of kidney epithelial cells in tissue culture. Mutational analysis suggests that SHC binding is essential for scattering and increased cell motility induced by trkA. The induction of motility in epithelial cells is thus an important feature of various receptor tyrosine kinases, which in vivo play a role in embryogenesis and metastasis. In contrast, only the c-met receptor promoted branching morphogenesis of kidney epithelial cells in three-dimensional matrices, which resemble the formation of tubular epithelia in development. Interestingly, the ability of c-met to induce morphogenesis could be transferred to trkA, when in a novel receptor hybrid COOH-terminal sequences of c-met (including Y14 to Y16) were fused to the trkA kinase domain. These data demonstrate that tubulogenesis of epithelia is a restricted activity of tyrosine kinases, as yet only demonstrated for the c-met receptor. We predict the existence of specific substrates that mediate this morphogenesis signal.

1996-01-01

263

Activation of Group I Metabotropic Glutamate Receptors Potentiates Heteromeric Kainate Receptors  

PubMed Central

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.

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

2013-01-01

264

Localization of a tumor cell adhesion domain of laminin by a monoclonal antibody  

SciTech Connect

Monoclonal antibodies were prepared to localize the domain(s) of laminin to which tumor cells adhere. Rat Y3-Ag 1.2.3 myeloma cells were fused with spleen cells from a rat immunized with a purified 440-kDa fragment of chymotrypsin-digested laminin. Three monoclonal antibodies (AL-1 to AL-3) that bound to intact laminin in a solid-phase radioimmunoassay were chosen for further analysis. The epitopes recognized by these antibodies were characterized by radioimmunoassays, immunoblotting, radioimmunoprecipitation, and immunoaffinity chromatography. In cell adhesion assays, monoclonal antibody AL-2 inhibited the highly metastatic melanoma cell line, K-1735-M4, to both intact laminin and the 440-kDa fragment of laminin. Electron microscopic examination of laminin-monoclonal antibody interactions showed that monoclonal antibody AL-2 reacted with the long arm of laminin directly below the cross-region. Two monoclonal antibodies that failed to inhibit tumor cell adhesion to laminin reacted with epitopes on the lateral short arms or cross-region of laminin as seen by electron microscopy. These results suggest that a new tumor cell binding domain of laminin may be located close to the cross-region on the long arm of laminin.

Skubitz, A.P.N.; Charonis, A.S.; Tsilibary, E.C.; Furcht, L.T. (Univ. of Minnesota, Minneapolis (United States))

1987-12-01

265

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.

2012-01-01

266

Polymerized laminin-332 matrix supports rapid and tight adhesion of keratinocytes, suppressing cell migration.  

PubMed

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. PMID:22563463

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

2012-01-01

267

Neurotrophin receptors expression and JNK pathway activation in human astrocytomas  

PubMed Central

Background Neurotrophins are growth factors that regulate cell growth, differentiation and apoptosis in the nervous system. Their diverse actions are mediated through two different transmembrane – receptor signaling systems: Trk receptor tyrosine kinases (TrkA, TrkB, TrkC) and p75NTR neurotrophin receptor. Trk receptors promote cell survival and differentiation while p75NTR induces, in most cases, the activity of JNK-p53-Bax apoptosis pathway or suppresses intracellular survival signaling cascades. Robust Trk activation blocks p75NTR -induced apoptosis by suppressing the JNK-p53-Bax pathway. The aim of this exploratory study was to investigate the expression levels of neurotrophin receptors, Trks and p75NTR, and the activation of JNK pathway in human astrocytomas and in adjacent non-neoplastic brain tissue. Methods Formalin-fixed paraffin-embedded serial sections from 33 supratentorial astrocytomas (5 diffuse fibrillary astrocytomas, WHO grade II; 6 anaplastic astrocytomas, WHO grade III; 22 glioblastomas multiforme, WHO grade IV) were immunostained following microwave pretreatment. Polyclonal antibodies against TrkA, TrkB, TrkC and monoclonal antibodies against p75NTR and phosphorylated forms of JNK (pJNK) and c-Jun (pc-Jun) were used. The labeling index (LI), defined as the percentage of positive (labeled) cells out of the total number of tumor cells counted, was determined. Results Moderate to strong, granular cytoplasmic immunoreactivity for TrkA, TrkB and TrkC receptors was detected in greater than or equal to 10% of tumor cells in the majority of tumors independently of grade; on the contrary, p75NTR receptor expression was found in a small percentage of tumor cells (~1%) in some tumors. The endothelium of tumor capillaries showed conspicuous immunoreactivity for TrkB receptor. Trk immunoreactivity seemed to be localized in some neurons and astrocytes in non-neoplastic tissue. Phosphorylated forms of JNK (pJNK) and c-Jun (pc-Jun) were significantly co-expressed in a tumor grade-dependent manner (p < 0.05). Interestingly, a statistically significant (p < 0.05) reverse relationship between Trk receptors LIs and pc-Jun/pJNK LIs was noted in some glioblastomas multiforme. Conclusion In the context of astrocytomas, Trk receptors (TrkA, TrkB, TrkC) expression may promote tumor growth independently of grade. Furthermore, activation of JNK pathway may contribute to progression towards malignancy. Considering the fact that regional tumor heterogeneity may be a limiting factor for immunohistochemical studies, the significance of the reverse relationship between Trk receptors and pc-Jun/pJNK LIs with respect to biological behavior of human astrocytomas requires further evaluation.

Assimakopoulou, Martha; Kondyli, Maria; Gatzounis, George; Maraziotis, Theodore; Varakis, John

2007-01-01

268

Activation of Xenobiotic Receptors: Driving into the Nucleus  

PubMed Central

Importance of the field Xenobiotic receptors (XRs) play pivotal roles in regulating the expression of genes that determine the clearance and detoxification of xenobiotics, such as drugs and environmental chemicals. Recently, it has become increasingly evident that most XRs shuttle between the cytoplasm and nucleus, and activation of such receptors is directly associated with xenobiotic-induced nuclear import. Areas covered in this review The scope of this review covers research literature that discusses nuclear translocation and activation of XRs, as well as unpublished data generated from this laboratory. Specific emphasis is given to the constitutive androstane receptor (CAR), the pregnane X receptor, and the aryl hydrocarbon receptor. What the readers will gain A number of molecular chaperons presumably associated with cellular localization of XRs have been identified. Primary hepatocyte cultures have been established as a unique model retaining inactive CAR in the cytoplasm. Moreover, several splicing variants of human CAR exhibit altered cellular localization and chemical activation. Take home message Nuclear accumulation is an essential step in the activation of XRs. Although great strides have been made, much remains to be understood concerning the mechanisms underlying intracellular localization and trafficking of XRs, which involve both direct ligand-binding and indirect pathways.

Li, Haishan; Wang, Hongbing

2010-01-01

269

Steroid signaling activation and intracellular localization of sex steroid receptors  

PubMed Central

In addition to stimulating gene transcription, sex steroids trigger rapid, non-genomic responses in the extra-nuclear compartment of target cells. These events take place within seconds or minutes after hormone administration and do not require transcriptional activity of sex steroid receptors. Depending on cell systems, activation of extra-nuclear signaling pathways by sex steroids fosters cell cycle progression, prevents apoptosis, leads to epigenetic modifications and increases cell migration through cytoskeleton changes. These findings have raised the question of intracellular localization of sex steroid receptors mediating these responses. During the past years, increasing evidence has shown that classical sex steroid receptors localized in the extra-nuclear compartment or close to membranes of target cells induce these events. The emerging picture is that a process of bidirectional control between signaling activation and sex steroid receptor localization regulates the outcome of hormonal responses in target cells. This mechanism ensures cell cycle progression in estradiol-treated breast cancer cells, and its derangement might occur in progression of human proliferative diseases. These findings will be reviewed here together with unexpected examples of the relationship between sex steroid receptor localization, signaling activation and biological responses in target cells. We apologize to scientists whose reports are not mentioned or extensively discussed owing to space limitations.

Giraldi, Tiziana; Giovannelli, Pia; Di Donato, Marzia; Migliaccio, Antimo; Auricchio, Ferdinando

2010-01-01

270

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.

2007-07-01

271

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.

2007-01-01

272

Catabolites of Cholesterol Synthesis Pathways and Forskolin as Activators of the Farnesoid X-Activated Nuclear Receptor  

Microsoft Academic Search

The nuclear receptors are a family of transcriptional mediators that, upon activation, bind DNA and regulate gene transcription. Among these receptors, the farnesoid X-activated receptor (FXR) has recently been identified as one activated by bile acids and farnesol. To investigate the potential of other sterols to activate FXR, as well as to examine relevant relationships among identified activators of FXR,

William R. Howard; John A. Pospisil; Eugenia Njolito; Daniel J. Noonan

2000-01-01

273

Histamine 3 Receptor Activation Reduces the Expression of Neuronal Angiotensin II Type 1 Receptors in the Heart  

PubMed Central

In severe myocardial ischemia, histamine 3 (H3) receptor activation affords cardioprotection by preventing excessive norepinephrine release and arrhythmias; pivotal to this action is the inhibition of neuronal Na+/H+ exchanger (NHE). Conversely, angiotensin II, formed locally by mast cell-derived renin, stimulates NHE via angiotensin II type 1 (AT1) receptors, facilitating norepinephrine release and arrhythmias. Thus, ischemic dysfunction may depend on a balance between the NHE-modulating effects of H3 receptors and AT1 receptors. The purpose of this investigation was therefore to elucidate the H3/AT1 receptor interaction in myocardial ischemia/reperfusion. We found that H3 receptor blockade with clobenpropit increased norepinephrine overflow and arrhythmias in Langendorff-perfused guinea pig hearts subjected to ischemia/reperfusion. This coincided with increased neuronal AT1 receptor expression. NHE inhibition with cariporide prevented both increases in norepinephrine release and AT1 receptor expression. Moreover, norepinephrine release and AT1 receptor expression were increased by the nitric oxide (NO) synthase inhibitor NG-methyl-l-arginine and the protein kinase C activator phorbol myristate acetate. H3 receptor activation in differentiated sympathetic neuron-like PC12 cells permanently transfected with H3 receptor cDNA caused a decrease in protein kinase C activity and AT1 receptor protein abundance. Collectively, our findings suggest that neuronal H3 receptor activation inhibits NHE by diminishing protein kinase C activity. Reduced NHE activity sequentially causes intracellular acidification, increased NO synthesis, and diminished AT1 receptor expression. Thus, H3 receptor-mediated NHE inhibition in ischemia/reperfusion not only opposes the angiotensin II-induced stimulation of NHE in cardiac sympathetic neurons, but also down-regulates AT1 receptor expression. Cardioprotection ultimately results from the combined attenuation of angiotensin II and norepinephrine effects and alleviation of arrhythmias.

Hashikawa-Hobara, Narumi; Chan, Noel Yan-Ki

2012-01-01

274

Chlorotriazines do not activate the aryl hydrocarbon receptor, the oestrogen receptor or the thyroid receptor in in vitro assays.  

PubMed

Atrazine, prometryn, propazine and simazine are chlorotriazines that are commonly employed as herbicides. However, their use is a major cause of concern, due to their reported endocrine disrupting effects in different taxa. Data from studies on the molecular and cellular processes underlying the hormonal action of these substances are contradictory. The ability of these chlorotriazines and the atrazine metabolites, desethyl-s-chlorotriazine and desisopropyl-s-chlorotriazine, to trigger responses mediated by the oestrogen receptor (ER), aryl hydrocarbon receptor (AhR) and thyroid receptor (TR), was studied by using in vitro approaches. Transcriptional activation assays were applied to observe the activation of ER and TR. The induction of ethoxyresorufin-O-deethylase (EROD) activity in the RTG-2 cell line served as an indicator of AhR activation. No responses were found in any of the assays, with any of the six chlorotriazines tested. Our observations indicate that the chlorotriazines tested are unlikely to cause their endocrine effects via these receptors. PMID:24773485

de la Casa-Resino, Irene; Navas, José M; Fernández-Cruz, María L

2014-03-01

275

TGF-?1 activates two distinct type I receptors in neurons  

PubMed Central

Transforming growth factor-?s (TGF-?s) are pleiotropic cytokines involved in development and maintenance of the nervous system. In several neural lesion paradigms, TGF-?1 exerts potent neuroprotective effects. Neurons treated with TGF-?1 activated the canonical TGF-? receptor I/activin-like kinase receptor 5 (ALK5) pathway. The transcription factor nuclear factor-?B (NF-?B) plays a fundamental role in neuroprotection. Treatment with TGF-?1 enhanced NF-?B activity in gelshift and reporter gene analyses. However, ectopic expression of a constitutively active ALK5 failed to mimic these effects. ALK1 has been described as an alternative TGF-? receptor in endothelial cells. Interestingly, we detected significant basal expression of ALK1 and its injury-induced up-regulation in neurons. Treatment with TGF-?1 also induced a pronounced increase in downstream Smad1 phosphorylation. Overexpression of a constitutively active ALK1 mimicked the effect of TGF-?1 on NF-?B activation and neuroprotection. Our data suggest that TGF-?1 simultaneously activates two distinct receptor pathways in neurons and that the ALK1 pathway mediates TGF-?1–induced NF-?B survival signaling.

Konig, Hans-Georg; Kogel, Donat; Rami, Abdelhaq; Prehn, Jochen H.M.

2005-01-01

276

Modulation of Receptor Phosphorylation Contributes to Activation of Peroxisome Proliferator Activated Receptor ? by Dehydroepiandrosterone and Other Peroxisome Proliferators  

PubMed Central

Dehydroepiandrosterone (DHEA), a C19 human adrenal steroid, activates peroxisome proliferator-activated receptor ? (PPAR?) in vivo but does not ligand-activate PPAR? in transient transfection experiments. We demonstrate that DHEA regulates PPAR? action by altering both the levels and phosphorylation status of the receptor. Human hepatoma cells (HepG2) were transiently transfected with the expression plasmid encoding PPAR? and a plasmid containing two copies of fatty acyl coenzyme oxidase (FACO) peroxisome-proliferator activated receptor responsive element consensus oligonucleotide in a luciferase reporter gene. Nafenopin treatment increased reporter gene activity in this system, whereas DHEA treatment did not. Okadaic acid significantly decreased nafenopin-induced reporter activity in a concentration-dependent manner. Okadaic acid treatment of primary rat hepatocytes decreased both DHEA- and nafenopin-induced FACO activity in primary rat hepatocytes. DHEA induced both PPAR? mRNA and protein levels, as well as PP2A message in primary rat hepatocytes. Western blot analysis showed that the serines at positions 12 and 21 were rapidly dephosphorylated upon treatment with DHEA and nafenopin. Results using specific protein phosphatase inhibitors suggested that protein phosphatase 2A (PP2A) is responsible for DHEA action, and protein phosphatase 1 might be involved in nafenopin induction. Mutation of serines at position 6, 12, and 21 to an uncharged alanine residue significantly increased transcriptional activity, whereas mutation to negative charged aspartate residues (mimicking receptor phosphorylation) decreased transcriptional activity. DHEA action involves induction of PPAR? mRNA and protein levels as well as increased PPAR? transcriptional activity through decreasing receptor phosphorylation at serines in the AF1 region.

Tamasi, Viola; Miller, Kristy K. Michael; Ripp, Sharon L.; Vila, Ermin; Geoghagen, Thomas E.; Prough, Russell A.

2008-01-01

277

Mechanisms of Activation of Receptor Tyrosine Kinases: Monomers or Dimers  

PubMed Central

Receptor tyrosine kinases (RTKs) play essential roles in cellular processes, including metabolism, cell-cycle control, survival, proliferation, motility and differentiation. RTKs are all synthesized as single-pass transmembrane proteins and bind polypeptide ligands, mainly growth factors. It has long been thought that all RTKs, except for the insulin receptor (IR) family, are activated by ligand-induced dimerization of the receptors. An increasing number of diverse studies, however, indicate that RTKs, previously thought to exist as monomers, are present as pre-formed, yet inactive, dimers prior to ligand binding. The non-covalently associated dimeric structures are reminiscent of those of the IR family, which has a disulfide-linked dimeric structure. Furthermore, recent progress in structural studies has provided insight into the underpinnings of conformational changes during the activation of RTKs. In this review, I discuss two mutually exclusive models for the mechanisms of activation of the epidermal growth factor receptor, the neurotrophin receptor and IR families, based on these new insights.

Maruyama, Ichiro N.

2014-01-01

278

Mechanisms of activation of receptor tyrosine kinases: monomers or dimers.  

PubMed

Receptor tyrosine kinases (RTKs) play essential roles in cellular processes, including metabolism, cell-cycle control, survival, proliferation, motility and differentiation. RTKs are all synthesized as single-pass transmembrane proteins and bind polypeptide ligands, mainly growth factors. It has long been thought that all RTKs, except for the insulin receptor (IR) family, are activated by ligand-induced dimerization of the receptors. An increasing number of diverse studies, however, indicate that RTKs, previously thought to exist as monomers, are present as pre-formed, yet inactive, dimers prior to ligand binding. The non-covalently associated dimeric structures are reminiscent of those of the IR family, which has a disulfide-linked dimeric structure. Furthermore, recent progress in structural studies has provided insight into the underpinnings of conformational changes during the activation of RTKs. In this review, I discuss two mutually exclusive models for the mechanisms of activation of the epidermal growth factor receptor, the neurotrophin receptor and IR families, based on these new insights. PMID:24758840

Maruyama, Ichiro N

2014-01-01

279

Immunolocalization of protease-activated receptor-2 in skin: receptor activation stimulates interleukin-8 secretion by keratinocytes in vitro.  

PubMed Central

The protease-activated receptor-2 (PAR-2) is a seven transmembrane domain receptor related to the thrombin receptor, which is activated in vitro by cleavage by trypsin. Affinity-purified rabbit IgG raised against a peptide corresponding to the trypsin cleavage site of PAR-2 was used for an immunohistochemical study of skin. The expression of PAR-2 in epidermis was striking, with keratinocytes showing abundant intercellular and cytoplasmic staining. Basal cells showed the strongest staining intensity and the stratum corneum was negative. Staining with control IgG used at the same concentration was consistently negative. The functional expression of PAR-2 by the simian virus transformed human skin keratinocyte cell line SVK14 was demonstrated by Northern blot analysis, flow cytometric analysis and the measurement of intracellular calcium. Treatment of SVK14 with trypsin or a receptor agonist peptide (SLIGKV-NH2) caused a dose-dependent increase in the secretion of the chemokine interleukin-8 (IL-8) in vitro. The effect of the peptide was specific, since control acetylated peptide was without activity. We conclude that PAR-2 is highly expressed by epidermal keratinocytes and receptor activation in vitro leads to increased IL-8 secretion by keratinocytes. These data raise the possibility that PAR-2 may play a role in epidermal homeostasis and inflammatory conditions. Images Figure 2 Figure 3 Figure 5

Hou, L; Kapas, S; Cruchley, A T; Macey, M G; Harriott, P; Chinni, C; Stone, S R; Howells, G L

1998-01-01

280

Immunolocalization of protease-activated receptor-2 in skin: receptor activation stimulates interleukin-8 secretion by keratinocytes in vitro.  

PubMed

The protease-activated receptor-2 (PAR-2) is a seven transmembrane domain receptor related to the thrombin receptor, which is activated in vitro by cleavage by trypsin. Affinity-purified rabbit IgG raised against a peptide corresponding to the trypsin cleavage site of PAR-2 was used for an immunohistochemical study of skin. The expression of PAR-2 in epidermis was striking, with keratinocytes showing abundant intercellular and cytoplasmic staining. Basal cells showed the strongest staining intensity and the stratum corneum was negative. Staining with control IgG used at the same concentration was consistently negative. The functional expression of PAR-2 by the simian virus transformed human skin keratinocyte cell line SVK14 was demonstrated by Northern blot analysis, flow cytometric analysis and the measurement of intracellular calcium. Treatment of SVK14 with trypsin or a receptor agonist peptide (SLIGKV-NH2) caused a dose-dependent increase in the secretion of the chemokine interleukin-8 (IL-8) in vitro. The effect of the peptide was specific, since control acetylated peptide was without activity. We conclude that PAR-2 is highly expressed by epidermal keratinocytes and receptor activation in vitro leads to increased IL-8 secretion by keratinocytes. These data raise the possibility that PAR-2 may play a role in epidermal homeostasis and inflammatory conditions. PMID:9767417

Hou, L; Kapas, S; Cruchley, A T; Macey, M G; Harriott, P; Chinni, C; Stone, S R; Howells, G L

1998-07-01

281

Extracellular loop 2 in the FSH receptor is crucial for ligand mediated receptor activation.  

PubMed

The present study aims to determine the role of the specific residues of the extracellular loops (ELs) of the FSH receptor (FSHR) in hormone binding and receptor activation. By substituting the sequences of each of the ELs of human FSHR with those of the luteinizing hormone/choriogonadotropin receptor (LH/CGR), we generated three mutant constructs where the three ELs were individually replaced. A fourth construct had all the three substituted ELs. The receptor expression and hormone binding ability of the mutants were comparable to that of the wild type. Hormone-induced signaling and internalization were lower in the EL2 substitution mutant (EL2M). In this mutant, the EL2 of FSHR was substituted with the corresponding loop of LH/CGR. Interestingly, homology modeling revealed a change in the orientation of EL2 in the mutant receptor. Thus, disruption of EL2 affected overall receptor function, suggesting the role of FSHR specific residues of the loop in ligand mediated signaling. PMID:22641019

Dupakuntla, Madhavi; Pathak, Bhakti; Roy, Binita Sur; Mahale, Smita D

2012-10-15

282

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.

2012-01-01

283

Protease-activated receptor 1-dependent neuronal damage involves NMDA receptor function  

PubMed Central

Protease-activated receptor 1 (PAR1) is a G-protein coupled receptor that is expressed throughout the central nervous system. PAR1 activation by brain-derived as well as blood-derived proteases has been shown to have variable and complex effects in a variety of animal models of neuronal injury and inflammation. In this study, we have evaluated the effects of PAR1 on lesion volume in wild-type or PAR1?/? C57Bl/6 mice subjected to transient occlusion of the middle cerebral artery or injected with NMDA in the striatum. We found that removal of PAR1 reduced infarct volume following transient focal ischemia to 57% of control. Removal of PAR1 or application of a PAR1 antagonist also reduced the neuronal injury associated with intrastriatal injection of NMDA to 60% of control. To explore whether NMDA receptor potentiation by PAR1 activation contributes to the harmful effects of PAR1, we investigated the effect of NMDA receptor antagonists on the neuroprotective phenotype of PAR1?/? mice. We found that MK801 reduced penumbral but not core neuronal injury in mice subjected to transient middle cerebral artery occlusion or intrastriatal NMDA injection. Lesion volumes in both models were not significantly different between PAR1?/? mice treated with and without MK801. Use of the NMDA receptor antagonist and dissociative anesthetic ketamine also renders NMDA-induced lesion volumes identical in PAR1?/? mice and wild-type mice. These data suggest that the ability of PAR1 activation to potentiate NMDA receptor function may underlie its harmful actions during injury.

Hamill, Cecily E.; Mannaioni, Guido; Lyuboslavsky, Polina; Sastre, Aristide A.; Traynelis, Stephen F.

2009-01-01

284

Coagulation, protease-activated receptors, and viral myocarditis.  

PubMed

The coagulation protease cascade plays an essential role in hemostasis. In addition, a clot contributes to host defense by limiting the spread of pathogens. Coagulation proteases induce intracellular signaling by cleavage of cell surface receptors called protease-activated receptors (PARs). These receptors allow cells to sense changes in the extracellular environment, such as infection. Viruses activate the coagulation cascade by inducing tissue factor expression and by disrupting the endothelium. Virus infection of the heart can cause myocarditis, cardiac remodeling, and heart failure. A recent study using a mouse model have shown that tissue factor, thrombin, and PAR-1 signaling all positively regulate the innate immune during viral myocarditis. In contrast, PAR-2 signaling was found to inhibit interferon-? expression and the innate immune response. These observations suggest that anticoagulants may impair the innate immune response to viral infection and that inhibition of PAR-2 may be a new strategy to reduce viral myocarditis. PMID:24203054

Antoniak, Silvio; Mackman, Nigel

2014-03-01

285

Laminin-database v.2.0: an update on laminins in health and neuromuscular disorders.  

PubMed

The laminin (LM)-database, hosted at http://www.lm.lncc.br, was published in the NAR database 2011 edition. It was the first database that provided comprehensive information concerning a non-collagenous family of extracellular matrix proteins, the LMs. In its first version, this database contained a large amount of information concerning LMs related to health and disease, with particular emphasis on the haemopoietic system. Users can easily access several tabs for LMs and LM-related molecules, as well as LM nomenclatures and direct links to PubMed. The LM-database version 2.0 integrates data from several publications to achieve a more comprehensive knowledge of LMs in health and disease. The novel features include the addition of two new tabs, 'Neuromuscular Disorders' and 'miRNA--LM Relationship'. More specifically, in this updated version, an expanding set of data has been displayed concerning the role of LMs in neuromuscular and neurodegenerative diseases, as well as the putative involvement of microRNAs. Given the importance of LMs in several biological processes, such as cell adhesion, proliferation, differentiation, migration and cell death, this upgraded version expands for users a panoply of information, regarding complex molecular circuitries that involve LMs in health and disease, including neuromuscular and neurodegenerative disorders. PMID:24106090

Golbert, Daiane C F; Santana-van-Vliet, Eliane; Mundstein, Alex S; Calfo, Vicente; Savino, Wilson; de Vasconcelos, Ana Tereza R

2014-01-01

286

Activation of glycine receptors modulates spontaneous epileptiform activity in the immature rat hippocampus.  

PubMed

While the expression of glycine receptors in the immature hippocampus has been shown, no information about the role of glycine receptors in controlling the excitability in the immature CNS is available. Therefore, we examined the effect of glycinergic agonists and antagonists in the CA3 region of an intact corticohippocampal preparation of the immature (postnatal days 4-7) rat using field potential recordings. Bath application of 100 ?m taurine or 10 ?m glycine enhanced the occurrence of recurrent epileptiform activity induced by 20 ?m 4-aminopyridine in low Mg(2+) solution. This proconvulsive effect was prevented by 3 ?m strychnine or after incubation with the loop diuretic bumetanide (10 ?m), suggesting that it required glycine receptors and an active NKCC1-dependent Cl(-) accumulation. Application of higher doses of taurine (?1 mm) or glycine (100 ?m) attenuated recurrent epileptiform discharges. The anticonvulsive effect of taurine was also observed in the presence of the GABAA receptor antagonist gabazine and was attenuated by strychnine, suggesting that it was partially mediated by glycine receptors. Bath application of the glycinergic antagonist strychnine (0.3 ?m) induced epileptiform discharges. We conclude from these results that in the immature hippocampus, activation of glycine receptors can mediate both pro- and anticonvulsive effects, but that a persistent activation of glycine receptors is required to suppress epileptiform activity. In summary, our study elucidated the important role of glycine receptors in the control of neuronal excitability in the immature hippocampus. PMID:24665103

Chen, Rongqing; Okabe, Akihito; Sun, Haiyan; Sharopov, Salim; Hanganu-Opatz, Ileana L; Kolbaev, Sergei N; Fukuda, Atsuo; Luhmann, Heiko J; Kilb, Werner

2014-05-15

287

Chick laminin: isolation by monoclonal antibodies and differential distribution of variants in the embryo.  

PubMed

In order to study the expression and function of laminin variants during chick embryonic development, we have generated monoclonal antibodies against chick heart laminin. One monoclonal antibody (mAb), called 9/F-10, could be used to purify chick laminin to homogeneity. By rotary shadowing, cross-shaped and T-shaped laminin particles as well as aggregates of two laminin molecules crosslinked via their short arms could be observed in this preparation. Purified chick laminin was very potent in mediating neurite growth by chick embryonic neurons. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of reduced chick heart laminin revealed a complex pattern of polypeptides which are immunologically related to several mammalian laminin chains. The two mAbs, 9/F-10 and 3/E-8, recognize two different types of chick laminin subunits. By immunofluorescence, antibody 3/E-8 labels basement membranes, aortic smooth muscle, and mesenchyme of 6-day-old chick embryos. In contrast, staining by mAb 9/F-10 is confined to basement membranes. Therefore, the two antibodies seem to distinguish between two different chick laminin isoforms. PMID:1959563

Brubacher, D; Wehrle-Haller, B; Chiquet, M

1991-12-01

288

Laminins containing the ?2 and ?3 chains regulate astrocyte migration and angiogenesis in the retina  

PubMed Central

Pathologies of retinal blood vessels are among the major causes of blindness worldwide. A key cell type that regulates retinal vascular development is the astrocyte. Generated extrinsically to the retina, astrocytes migrate into the retina through the optic nerve head. Even though there is a strong correlation between astrocyte distribution and retinal vascular development, the factors that guide astrocytes into the retina remain unclear. In this study, we show that astrocytes migrate within a laminin-containing basement membrane - the inner limiting membrane. Genetic deletion of the laminin ?2 and ?3 chains affects astrocyte migration and spatial distribution. We show that laminins act as haptotactic factors in vitro in an isoform-specific manner, inducing astrocyte migration and promoting astrocyte differentiation. The addition of exogenous laminins to laminin-null retinal explants rescues astrocyte migration and spatial patterning. Furthermore, we show that the loss of laminins reduces ?1 integrin expression in astrocytes. Culturing laminin-null retinal astrocytes on laminin substrates restores focal localization of ?1 integrin. Finally, we show that laminins containing ?2 and ?3 chains regulate subsequent retinal blood vessel growth and maintain vascular integrity. These in vivo and in vitro studies demonstrate clearly that laminins containing ?2 and ?3 chains are indispensable for migration and spatial organization of astrocytes and that they play a crucial role during retinal angiogenesis in vivo.

Gnanaguru, Gopalan; Bachay, Galina; Biswas, Saptarshi; Pinzon-Duarte, German; Hunter, Dale D.; Brunken, William J.

2013-01-01

289

Endocannabinoid tone versus constitutive activity of cannabinoid receptors  

PubMed Central

This review evaluates the cellular mechanisms of constitutive activity of the cannabinoid (CB) receptors, its reversal by inverse agonists, and discusses the pitfalls and problems in the interpretation of the research data. The notion is presented that endogenously produced anandamide (AEA) and 2-arachidonoylglycerol (2-AG) serve as autocrine or paracrine stimulators of the CB receptors, giving the appearance of constitutive activity. It is proposed that one cannot interpret inverse agonist studies without inference to the receptors' environment vis-à-vis the endocannabinoid agonists which themselves are highly lipophilic compounds with a preference for membranes. The endocannabinoid tone is governed by a combination of synthetic pathways and inactivation involving transport and degradation. The synthesis and degradation of 2-AG is well characterized, and 2-AG has been strongly implicated in retrograde signalling in neurons. Data implicating endocannabinoids in paracrine regulation have been described. Endocannabinoid ligands can traverse the cell's interior and potentially be stored on fatty acid-binding proteins (FABPs). Molecular modelling predicts that the endocannabinoids derived from membrane phospholipids can laterally diffuse to enter the CB receptor from the lipid bilayer. Considering that endocannabinoid signalling to CB receptors is a much more likely scenario than is receptor activation in the absence of agonist ligands, researchers are advised to refrain from assuming constitutive activity except for experimental models known to be devoid of endocannabinoid ligands. LINKED ARTICLES This article is part of a themed issue on Cannabinoids in Biology and Medicine. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2011.163.issue-7

Howlett, Allyn C; Reggio, Patricia H; Childers, Steven R; Hampson, Robert E; Ulloa, Nadine M; Deutsch, Dale G

2011-01-01

290

The Orphan Nuclear Receptor SHP Utilizes Conserved LXXLL-Related Motifs for Interactions with Ligand-Activated Estrogen Receptors  

Microsoft Academic Search

SHP (short heterodimer partner) is an unusual orphan nuclear receptor consisting only of a ligand-binding domain, and it exhibits unique features of interaction with conventional nuclear receptors. While the mech- anistic basis of these interactions has remained enigmatic, SHP has been suggested to inhibit nuclear receptor activation by at least three alternatives; inhibition of DNA binding via dimerization, direct antagonism

LOTTA JOHANSSON; A. Bavner; J. S. Thomsen; M. Farnegardh; J.-A. Gustafsson; E. Treuter

2000-01-01

291

Ligand binding and co-activator assembly of the peroxisome proliferator-activated receptor-?  

Microsoft Academic Search

The peroxisome proliferator-activated receptor-? (PPAR-?) is a ligand-dependent transcription factor that is important in adipocyte differentiation and glucose homeostasis and which depends on interactions with co-activators, including steroid receptor co-activating factor-1 (SRC-1). Here we present the X-ray crystal structure of the human apo-PPAR-? ligand-binding domain (LBD), at 2.2 ? resolution; this structure reveals a large binding pocket, which may explain

Robert T. Nolte; G. Bruce Wisely; Stefan Westin; Jeffery E. Cobb; Millard H. Lambert; Riki Kurokawa; Michael G. Rosenfeldk; Timothy M. Willson; Christopher K. Glass; Michael V. Milburn

1998-01-01

292

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

2012-01-01

293

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

2000-01-01

294

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

1996-01-01

295

Nuclear Receptor-Mediated Regulation of Carboxylesterase Expression and Activity  

PubMed Central

i) Importance of the field Emerging evidence demonstrates that several nuclear receptor (NR) family members regulate drug-inducible expression and activity of several important carboxylesterase (CES) enzymes in mammalian liver and intestine. Numerous clinically prescribed anticancer prodrugs, carbamate and pyrethroid insecticides, environmental toxicants, and procarcinogens are substrates for CES enzymes. Moreover, a key strategy employed in rational drug design frequently utilizes an ester linkage methodology to selectively target a prodrug, or to improve the water solubility of a novel compound. ii) Areas covered in this review This review will summarize the current state of knowledge regarding NR-mediated regulation of CES enzymes in mammals, and highlight their importance in drug metabolism, drug-drug interactions, and toxicology. iii) What the reader will gain New knowledge regarding the transcriptional regulation of CES enzymes by NR proteins pregnane x receptor (PXR, NR1I2) and constitutive androstane receptor (CAR, NR1I3) has recently come to light through the use of knockout and transgenic mouse models. Novel insights regarding the species-specific cross-regulation of glucocorticoid receptor (GR, NR3C1) and peroxisome proliferator activated receptor alpha (PPAR?, NR1C1) signaling and CES gene expression is discussed. iv) Take home message Elucidation of the role of NR-mediated regulation of CES enzymes in liver and intestine will have a significant impact on rational drug design and the development of novel prodrugs, especially for patients on combination therapy.

Staudinger, Jeff L.; Xu, Chenshu; Cui, Yue J.; Klaassen, Curtis D.

2009-01-01

296

Retinoic acid receptor agonist activity of naturally occurring diterpenes.  

PubMed

Recent accumulating evidence indicates that all-trans retinoic acid (ATRA) may be useful for preventing or treating inflammation, allergy, and autoimmune diseases, despite its severe side effects. In this study, screening of 99 crude drugs for retinoic acid receptor (RAR) ligands by luciferase reporter assay demonstrated that the methanol extract of Aralia cordata Rhizoma most effectively activates the transcriptional activity of RAR?. Pimaradienoic acid (ent-pimara-8(14),15-dien-19-oic acid) was subsequently isolated as the constituent capable of activating RAR. Pimaric acid and abietic acid, which have similar structures to pimaradienoic acid, were also found to be novel RAR agonists, although abietic acid only slightly activated peroxisome proliferator-activated receptor gamma. These three natural RAR agonists with diterpene structures, while structurally different from ATRA, were able to increase the mRNA levels of the constitutive androstane receptor in HepG2 cells, induce F9 cell differentiation followed by Cyp26a1 mRNA expression, and differentiate HL-60 cells via RAR activation in a different manner from ATRA. These results demonstrate that some diterpenes exist as naturally occurring RAR agonists and that the differences in chemical structure between ATRA and these diterpenes may induce distinct gene activation and a specific cellular response. PMID:24799257

Tanabe, Hiroki; Yasui, Tomohiro; Kotani, Hitoshi; Nagatsu, Akito; Makishima, Makoto; Amagaya, Sakae; Inoue, Makoto

2014-06-15

297

Activation of Estrogen Receptor by Bavachin from Psoralea corylifolia.  

PubMed

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 [(3)H] 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. PMID:24116293

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

2012-03-01

298

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

2007-01-01

299

The Search for Endogenous Activators of the Aryl Hydrocarbon Receptor  

PubMed Central

In its simplest aspect, this review is an attempt to describe the major ligand classes of the aryl hydrocarbon receptor (AHR). A grander objective is to provide models that may help define the physiological activator or “endogenous ligand” of the AHR. We begin by presenting evidence that supports a developmental function for the AHR. This is followed by proposing mechanisms by which an endogenous ligand and consequent AHR activation might be important during normal physiology and development. With this background, we then present a survey of the known xenobiotic, endogenous, dietary and “un-conventional” activators of the AHR. When possible, this includes information about their induction potency, receptor binding affinity and potential for exposure. Because of the essential function of the AHR in embryonic development, we discuss the candidacy of each of these compounds as physiologically important activators.

Nguyen, Linh P.; Bradfield, Christopher A.

2008-01-01

300

Protease-activated-receptor-2 affects protease-activated-receptor-1-driven breast cancer.  

PubMed

Mammalian protease-activated-receptor-1 and -2 (PAR1 and PAR2) are activated by proteases found in the flexible microenvironment of a tumor and play a central role in breast cancer. We propose in the present study that PAR1 and PAR2 act together as a functional unit during malignant and physiological invasion processes. This notion is supported by assessing pro-tumor functions in the presence of short hairpin; shRNA knocked-down hPar2 or by the use of a truncated PAR2 devoid of the entire cytoplasmic tail. Silencing of hPar2 by shRNA-attenuated thrombin induced PAR1 signaling as recapitulated by inhibiting the assembly of Etk/Bmx or Akt onto PAR1-C-tail, by thrombin-instigated colony formation and invasion. Strikingly, shRNA-hPar2 also inhibited the TFLLRN selective PAR1 pro-tumor functions. In addition, while evaluating the physiological invasion process of placenta extravillous trophoblast (EVT) organ culture, we observed inhibition of both thrombin or the selective PAR1 ligand; TFLLRNPNDK induced EVT invasion by shRNA-hPar2 but not by scrambled shRNA-hPar2. In parallel, when a truncated PAR2 was utilized in a xenograft mouse model, it inhibited PAR1-PAR2-driven tumor growth in vivo. Similarly, it also attenuated the interaction of Etk/Bmx with the PAR1-C-tail in vitro and decreased markedly selective PAR1-induced Matrigel invasion. Confocal images demonstrated co-localization of PAR1 and PAR2 in HEK293T cells over-expressing YFP-hPar2 and HA-hPar1. Co-immuno-precipitation analyses revealed PAR1-PAR2 complex formation but no PAR1-CXCR4 complex was formed. Taken together, our observations show that PAR1 and PAR2 act as a functional unit in tumor development and placenta-uterus interactions. This conclusion may have significant consequences on future breast cancer therapeutic modalities and improved late pregnancy outcome. PMID:24177339

Jaber, Mohammad; Maoz, Miriam; Kancharla, Arun; Agranovich, Daniel; Peretz, Tamar; Grisaru-Granovsky, Sorina; Uziely, Beatrice; Bar-Shavit, Rachel

2014-07-01

301

UV activates growth factor receptors via reactive oxygen intermediates  

PubMed Central

Exposure of mammalian cells to UV irradiation induces rapid and transient expression of early growth response-1 gene (Egr-1) encoding a transcription factor that plays a role in cell survival. These signals from the irradiated cell surface are likely to involve more than one pathway, and we show here that an essential pathway involves activation of several growth factor receptors by reactive oxygen intermediates (ROI). UVC irradiation causes the tyrosine phosphorylation of EGF receptor (EGFR) in mouse NIH 3T3 fibroblasts and HC11 mouse mammary cells. EGFR activation by irradiation of cells is abrogated by suramin, by antioxidants, and by the presence of a dominant negative EGFR. UV induces the formation of complexes between activated EGFR and SOS, Grb2, PLC gamma, and SHC that can be precipitated with antibodies to EGFR. The activation of EGFR by UV is mimicked by H2O2, suggesting that ROI may function upstream of EGFR activation. Our observations support the hypothesis that ROI and growth factor receptors operate in the early steps of the UV signal that lead to the enhanced expression and activity of Egr-1.

1996-01-01

302

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

2013-03-01

303

Activation of Axonal Receptors by GABA Spillover Increases Somatic Firing  

PubMed Central

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.

Pugh, Jason R.

2013-01-01

304

Apoptotic and antitumor activity of death receptor antibodies require inhibitory Fc? receptor engagement.  

PubMed

By virtue of their ability to induce apoptosis and regulate growth, differentiation, and cytokine responses, the tumor necrosis factor receptor (TNFR) superfamily members have emerged as attractive targets for anticancer therapeutics. Agonistic antibodies to apoptosis-inducing TNFRs, such as death receptor 5 (DR5), although displaying impressive activities against a variety of tumors in preclinical models, appear to be less active in clinical trials. We report that the in vivo apoptotic and antitumor activities of these antibodies have an absolute requirement for the coengagement of an inhibitory Fc? receptor, Fc?RIIB. Anti-DR5 antibodies of the type currently in clinical trials have weak Fc?RIIB binding and thus are compromised in their proapoptotic and antitumor activities in both colon and breast carcinoma models. Enhancing Fc?RIIB engagement increases apoptotic and antitumor potency. Our results demonstrate that Fc domain interactions are critical to the therapeutic activity of anti-DR5 antibodies and, together with previous reports on agonistic anti-CD40 antibodies, establish a common requirement for Fc?RIIB coengagement for optimal biological effects of agonistic anti-TNFR antibodies. PMID:22723355

Li, Fubin; Ravetch, Jeffrey V

2012-07-01

305

Serum concentration of laminin, and course of the disease in patients with various malignancies.  

PubMed

Serum concentration of laminin was measured radioimmunologically in 96 patients suffering from various malignancies. Laminin levels were significantly elevated in patients with carcinomas and leukemias, but not in patients with sarcomas or lymphomas when compared with healthy controls. A good correlation could be found between serum laminin concentration and response to therapy in patients with carcinoma and leukemia. Elevated laminin levels were associated with a progressive course of the tumor condition. Furthermore, a close correlation has been detected between serum concentrations of laminin and carcinoembryonic antigen (CEA) in patients with carcinoma of the colon. The serum laminin level seems to be a valuable parameter for observation of the course of certain malignancies. PMID:3625257

Rochlitz, C; Hasslacher, C; Brocks, D G; Herrmann, R

1987-09-01

306

Short laminin Peptide for improved neural stem cell growth.  

PubMed

Human neural stem/progenitor cells (hNSCs) are very difficult to culture and require human or animal source extracellular matrix molecules, such as laminin or collagen type IV, to support attachment and to regulate their survival and proliferation. These extracellular matrix molecules are difficult to purify from human or animal tissues, have high batch-to-batch variability, and may cause an immune response if used in clinical applications. Although several laminin- and collagen IV-derived peptides are commercially available, they do not support long-term hNSC attachment and growth. To solve this problem, we developed a novel peptide sequence with only 12 amino acids based on the Ile-Lys-Val-Ala-Val, or IKVAV, sequence: Ac-Cys-Cys-Arg-Arg-Ile-Lys-Val-Ala-Val-Trp-Leu-Cys. This short peptide sequence, similar to tissue-derived full laminin molecules, supported hNSCs to attach and proliferate to confluence for continuous passage and subculture. This short peptide also directed hNSCs to differentiate into neurons. When conjugated to poly(ethylene glycol) hydrogels, this short peptide benefited hNSC attachment and proliferation on the surface of hydrogels and promoted cell migration inside the hydrogels with maximum enhancement at a peptide density of 10 ?M. This novel short peptide shows great promise in artificial niche development for supporting hNSC culture in vitro and in vivo and for promoting hNSC transplantation in future clinical therapy. PMID:24692587

Li, Xiaowei; Liu, Xiaoyan; Josey, Benjamin; Chou, C James; Tan, Yu; Zhang, Ning; Wen, Xuejun

2014-05-01

307

Solubilization and characterization of active neurotensin receptors from mouse brain  

SciTech Connect

Neurotensin receptors were solubilized from mouse brain using the zwitterionic detergent 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonic acid (CHAPS). The binding of /sup 125/I-labeled (Tyr/sup 3/)neurotensin to the soluble fraction was time-dependent, saturable, and reversible. Unlabeled neurotensin and its analogues acetylneurotensin (8-13), neurotensin (9-13), and neurotensin (1-12) competitively antagonized the binding of /sup 125/I-labeled (Tyr/sup 3/)neurotensin to CHAPS-solubilized extracts with relative potencies similar to those observed with membrane-bound receptors. Scatchard analysis of equilibrium binding data indicated that the soluble extract contained a single class of neurotensin binding sites with a K/sub d/ of 0.36 nM and a B/sub m/ of 63 fmol/mg. As already observed with membrane-bound receptors, the affinity of neurotensin for the soluble binding activity was decreased by Na/sup +/ ions. By contrast, soluble receptors were no longer sensitive to GTP and the antihistamine drug levocabastine. A molecular weight of about 100,000 was determined for soluble neurotensin receptors both under native conditions by gel filtration on Ultrogel AcA 34 and under denaturating conditions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after photoaffinity labeling.

Mazella, J.; Chabry, J.; Kitabgi, P.; Vincent, J.P.

1988-01-05

308

Expression of laminin gamma 1 cultured hepatocytes involves repeated CTC and GC elements in the LAMC1 promoter.  

PubMed Central

Laminin gamma 1 chain is present in all basement membranes and is expressed at high levels in various diseases, such as hepatic fibrosis. We have identified cis- and trans-acting elements involved in the regulation of this gene in normal rat liver, as well as in hepatocyte primary cultures and hepatoma cell lines. Northern-blot analyses showed that laminin gamma 1 mRNA was barely detectable in freshly isolated hepatocytes and expressed at high levels in hepatocyte primary cultures, as early as 4 h after liver dissociation. Actinomycin D and cycloheximide treatment in vivo and in vitro indicated that laminin gamma 1 overexpression in cultured hepatocytes was under the control of transcriptional mechanisms. Transfection of deletion mutants of the 5' flanking region of murine LAMC1 gene in hepatoma cells that constitutively express laminin gamma 1 indicated that regulatory elements were located between -594 bp and -94 bp. This segment included GC- and CTC-containing motifs. Gel-shift analyses showed that two complexes were resolved with different affinity for the CTC sequence depending on the location of the GC box. The pattern of complex formation with nuclear factors from freshly isolated and cultured hepatocytes was different from that obtained with total liver and similar to that with hepatoma cells. Southwestern analysis indicated that several polypeptides bound the CTC-rich sequence. Affinity chromatography demonstrated that A M(r) 60,000 polypeptide was a major protein binding to the CTC motif. This polypeptide is probably involved in the transcriptional activation of various proto-oncogenes and extracellular matrix genes that are expressed at high levels in both hepatoma cells and early hepatocyte cultures.

Levavasseur, F; Lietard, J; Ogawa, K; Theret, N; Burbelo, P D; Yamada, Y; Guillouzo, A; Clement, B

1996-01-01

309

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

2010-01-01

310

In vivo bradykinin B2 receptor activation reduces renal fibrosis  

PubMed Central

Angiotensin-converting enzyme (ACE) inhibitors reduce the progression of various fibrotic renal diseases both in humans and in animal models. Unilateral ureteral obstruction (UUO) is an animal model of accelerated renal tubulointerstitial fibrosis that is attenuated by ACE inhibition. Although ACE inhibitors increase bradykinin concentrations in addition to their effect on angiotensin II formation, the role of bradykinin in renal fibrosis has not been studied. We show here that genetic ablation (B2–/– mice) or pharmacological blockade of the bradykinin B2 receptor increases UUO-induced interstitial fibrosis in mice, whereas transgenic rats expressing increased endogenous bradykinin show reduced UUO-induced interstitial fibrosis. The increased interstitial fibrosis in B2–/– mice was accompanied by a decreased activity of plasminogen activators (PAs) and metalloproteinase-2 (MMP-2), enzymes involved in ECM degradation, suggesting that the protective effects of bradykinin involve activation of a B2 receptor/PA/MMP-2 cascade. This ability of bradykinin to increase PA activity was confirmed in primary culture proximal tubular cells. Thus, in both mice and rats, bradykinin B2 receptor activation reduces renal tubulointerstitial fibrosis in vivo, most likely by increasing ECM degradation.

Schanstra, Joost P.; Neau, Eric; Drogoz, Pascale; Arevalo Gomez, Miguel A.; Lopez Novoa, Jose Miguel; Calise, Denis; Pecher, Christiane; Bader, Michael; Girolami, Jean-Pierre; Bascands, Jean-Loup

2002-01-01

311

The Novel Leptospiral Surface Adhesin Lsa20 Binds Laminin and Human Plasminogen and Is Probably Expressed during Infection?  

PubMed Central

Leptospirosis is an emerging infectious disease caused by pathogenic species of Leptospira. In this work, we report the cloning, expression, purification, and characterization of two predicted leptospiral outer membrane proteins, LIC11469 and LIC11030. The LIC11469 protein is well conserved among leptospiral strains, while LIC11030 was identified only in Leptospira interrogans. We confirmed by surface proteolysis of intact leptospires with proteinase K that these proteins are most likely new surface leptospiral proteins. The recombinant proteins were evaluated for their capacity to attach to extracellular matrix (ECM) components and to plasminogen. The leptospiral protein encoded by LIC11469, named Lsa20 (leptospiral surface adhesin of 20 kDa), binds to laminin and to plasminogen. The binding with both components was not detected when Lsa20 was previously denatured or blocked with anti-Lsa20 antibodies. Moreover, Lsa20 binding to laminin was also confirmed by surface plasmon resonance (SPR). Laminin competes with plasminogen for binding to Lsa20, suggesting the same ligand-binding site. Lsa20-bound plasminogen could be converted to enzymatically active plasmin, capable of cleaving plasmin substrate d-valyl-leucyl-lysine-p-nitroanilide dihydrochloride. Lsa20 was recognized by antibodies in confirmed-leptospirosis serum samples, suggesting that this protein is expressed during infection. Taken together, our results indicate that Lsa20 is a novel leptospiral adhesin that in concert with the host-derived plasmin may help the bacteria to adhere and to spread through the hosts.

Mendes, Renata Siqueira; Von Atzingen, Marina; de Morais, Zenaide Maria; Goncales, Amane Paldes; Serrano, Solange M. T.; Asega, Amanda F.; Romero, Eliete Calo; Vasconcellos, Silvio Arruda; Nascimento, Ana Lucia Tabet O.

2011-01-01

312

Liver X receptor and peroxisome proliferator-activated receptor as integrators of lipid homeostasis and immunity.  

PubMed

Lipid metabolism has emerged as an important modulator of innate and adaptive immune cell fate and function. The lipid-activated transcription factors peroxisome proliferator-activated receptor (PPAR) ?, ?/?, ? and liver X receptor (LXR) are members of the nuclear receptor superfamily that have a well-defined role in regulating lipid homeostasis and metabolic diseases. Accumulated evidence over the last decade indicates that PPAR and LXR signaling also influence multiple facets of inflammation and immunity, thereby providing important crosstalk between metabolism and immune system. Herein, we provide a brief introduction to LXR and PPAR biology and review recent discoveries highlighting the importance of PPAR and LXR signaling in the modulation of normal and pathologic states of immunity. We also examine advances in our mechanistic understanding of how nuclear receptors impact immune system function and homeostasis. Finally, we discuss whether LXRs and PPARs could be pharmacologically manipulated to provide novel therapeutic approaches for modulation of the immune system under pathologic inflammation or in the context of allergic and autoimmune disease. PMID:22889216

Kidani, Yoko; Bensinger, Steven J

2012-09-01

313

M3-muscarinic receptor promotes insulin release via receptor phosphorylation/arrestin-dependent activation of protein kinase D1  

PubMed Central

The activity of G protein-coupled receptors is regulated via hyper-phosphorylation following agonist stimulation. Despite the universal nature of this regulatory process, the physiological impact of receptor phosphorylation remains poorly studied. To address this question, we have generated a knock-in mouse strain that expresses a phosphorylation-deficient mutant of the M3-muscarinic receptor, a prototypical Gq/11-coupled receptor. This mutant mouse strain was used here to investigate the role of M3-muscarinic receptor phosphorylation in the regulation of insulin secretion from pancreatic islets. Importantly, the phosphorylation deficient receptor coupled to Gq/11-signaling pathways but was uncoupled from phosphorylation-dependent processes, such as receptor internalization and ?-arrestin recruitment. The knock-in mice showed impaired glucose tolerance and insulin secretion, indicating that M3-muscarinic receptors expressed on pancreatic islets regulate glucose homeostasis via receptor phosphorylation-/arrestin-dependent signaling. The mechanism centers on the activation of protein kinase D1, which operates downstream of the recruitment of ?-arrestin to the phosphorylated M3-muscarinic receptor. In conclusion, our findings support the unique concept that M3-muscarinic receptor-mediated augmentation of sustained insulin release is largely independent of G protein-coupling but involves phosphorylation-/arrestin-dependent coupling of the receptor to protein kinase D1.

Kong, Kok Choi; Butcher, Adrian J.; McWilliams, Phillip; Jones, David; Wess, Jurgen; Hamdan, Fadi F.; Werry, Tim; Rosethorne, Elizabeth M.; Charlton, Steven J.; Munson, Sarah E.; Cragg, Hannah A.; Smart, Alison D.; Tobin, Andrew B.

2010-01-01

314

Adiponectin and its receptors are expressed in adult ventricular cardiomyocytes and upregulated by activation of peroxisome proliferator-activated receptor ?  

PubMed Central

Adiponectin is a protein hormone involved in maintaining energy homeostasis in metabolically active tissues. It enhances glucose and lipid metabolism via activation of AMP-dependent kinase (AMPK) in skeletal muscle and liver. Energy homeostasis is vital for the heart to work as a pump. In this study, we investigated whether adiponectin and its receptors are expressed in adult ventricular cardiomyocytes. We observed adiponectin transcript and protein in cultured ventricular cardiomyocytes isolated from adult rat, by quantitative real-time PCR, ELISA assays, Western blots, and immunofluorescent staining. In addition, we detected adiponectin receptor (AdipoR1 and AdipoR2) expression in the heart. AdipoR1 was expressed in rat myocardium at a level of about 50% of that in skeletal muscle; whereas adipoR2 was expressed at a similar level to that in liver. Rosiglitazone, a Peroxisome proliferator activated receptor ? (PPAR?) activator, substantially elevated expression of adiponectin in cultured cardiomyocytes and its secretion into cultured media. Rosiglitazone also increased adipoR1 and adipoR2 expression in cardiomyocytes. Treatment of recombinant globular adiponectin in cultured cardiomyocytes increased fatty acid oxidation and glucose uptake via activation of AMPK, suggesting a role for adiponectin in cardiac energy metabolism. Together, these data establish the existence of a local cardiac-specific adiponectin system that is regulated by PPAR?. Moreover, these findings indicate a role for adiponectin on normal myocardial energy homeostasis, in part, through the activation of AMPK.

Ding, Guoliang; Qin, Qianhong; He, Nu; Francis-David, Sharon C.; Hou, Jie; Ricks, Ernest; Yang., Qinglin

2007-01-01

315

Peroxisome proliferator-activated receptor-? ameliorates pulmonary arterial hypertension by inhibiting 5-hydroxytryptamine 2B receptor.  

PubMed

An elevated plasma level of 5-hydroxytryptamine (5-HT) or upregulation of 5-HT receptor signaling or both is implicated in vascular contraction and remodeling in pulmonary arterial hypertension (PAH). Recently, peroxisome proliferator-activated receptor-? (PPAR?) agonists have been shown to ameliorate PAH. However, their effects on the 5-HT-induced contraction of pulmonary arteries remain unknown. Here, we examined the role of PPAR? in inhibiting 5-HT2B receptor (5-HT2BR) to ameliorate PAH. Pulmonary arteries from PAH rats induced by monocrotaline or chronic hypoxia showed an enhanced vasoconstriction in response to BW723C86, a specific agonist for 5-HT2BR. Expression of 5-HT2BR was also increased in pulmonary arteries from the PAH rats, accompanied by vascular remodeling and right ventricular hypertrophy. Treatment with the PPAR? agonist rosiglitazone in vivo reversed the expression and the vasocontractive effect of 5-HT2BR as well as the thickening of pulmonary arteries. In pulmonary artery smooth muscle cells, 5-HT induced the gene expression of 5-HT2BR, which was inhibited by rosiglitazone, pioglitazone, or adenovirus-mediated overexpression of constitutively activated PPAR?. The pharmacological effect of PPAR? was through the suppression of the 5-HT-induced activator protein-1 activity. These results demonstrated the beneficial effect of PPAR? on 5-HT2BR-mediated vasocontraction, providing a new mechanism for the potential use of PPAR? agonists in PAH. PMID:23108648

Liu, Yahan; Tian, Xiao Yu; Mao, Guangmei; Fang, Xi; Fung, Man Lung; Shyy, John Y-J; Huang, Yu; Wang, Nanping

2012-12-01

316

Human peroxisome proliferator-activated receptor mRNA and protein expression during development  

EPA Science Inventory

The peroxisome proliferator-activated receptors (PPAR) are nuclear hormone receptors that regulate lipid and glucose homeostasis and are important in reproduction and development. PPARs are targets ofpharmaceuticals and are also activated by environmental contaminants, including ...

317

Characterization of Steroid Receptor RNA Activator Protein Function in Modulating the Estrogen Signaling Pathway.  

National Technical Information Service (NTIS)

The core of my hypothesis is that Steroid receptor RNA activator protein, a newly discovered protein from a once thought noncoding RNA, is involved in the modulation of the estrogen signaling pathway. The steroid receptor RNA activator (SRA) differs from ...

C. Shilpa

2006-01-01

318

PACAP-induced ERK activation in HEK cells expressing PAC1 receptors involves both receptor internalization and PKC signaling.  

PubMed

The pituitary adenylate cyclase-activating polypeptide (PACAP)-selective PAC1 receptor (Adcyap1r1) is a G protein-coupled receptor (GPCR) that activates adenylyl cyclase and PLC. Similar to many other GPCRs, our previous studies showed that the PAC1 receptor is internalized after ligand binding to form signaling endosomes, which recruit additional second messenger pathways. Using a human embryonic kidney (HEK 293) PAC1Hop1-EGFP receptor cell line, we have examined how different PAC1 receptor signaling mechanisms contribute to MEK/ERK activation. Unlike PAC1 receptor-stimulated adenylyl cyclase/cAMP production in the plasma membrane, PACAP-mediated ERK phosphorylation was partly dependent on receptor internalization, as determined by treatment with pharmacological inhibitors of endocytosis or temperature reduction, which also suppressed receptor internalization. Stimulation of cAMP generation by forskolin or exposure to the cell-permeable cAMP analogs 8-bromo-cAMP and dibutyryl cAMP had minimal effects on ERK phosphorylation in this system. The ability of reduced temperature (24°C) to consistently suppress ERK activation to a greater extent than the endocytosis inhibitors Pitstop 2 and dynasore indicated that other mechanisms, in addition to PAC1 internalization/endosome activation, were involved. Inhibition of PAC1 receptor-stimulated PLC/diacylglycerol/PKC signaling by bisindoylmaleimide I also attenuated ERK phosphorylation, and direct PKC activation with phorbol ester increased ERK phosphorylation in a temperature-dependent manner. Inhibition of PAC1 receptor endocytosis and PKC activation completely blocked PACAP-stimulated ERK activation. PACAP augmented phosphorylated ERK staining uniformly over the cytoplasm and nucleus, and PKC signaling facilitated nuclear phosphorylated ERK translocation. In sum, our results show that PACAP/PAC1 receptor endocytosis and PLC/diacylglycerol/PKC activation represent two complementary mechanisms contributing to PACAP-induced ERK activation. PMID:24696141

May, Victor; Buttolph, Thomas R; Girard, Beatrice M; Clason, Todd A; Parsons, Rodney L

2014-06-01

319

Defining the Interaction of the Treponema pallidum Adhesin Tp0751 with Laminin  

PubMed Central

Various invasive pathogens attach to host tissues via the extracellular matrix component laminin, the major glycoprotein found within basement membranes. Previous investigations identified the laminin-binding adhesin Tp0751 within the spirochete bacterium Treponema pallidum. In the current study, Tp0751 was shown to attach to a variety of laminin isoforms that are widely distributed throughout the host, including laminins 1, 2, 4, 8, and 10. Such universal attachment is conducive for an adhesin present within a highly invasive pathogen that encounters a variety of tissue sites during the course of infection. Additional studies systematically identified the amino acid residues within Tp0751 that contribute to laminin binding using synthetic peptides designed from the mature protein sequence. The minimum laminin-binding region of the adhesin was localized to 10 amino acids; peptides containing these residues inhibited attachment of Tp0751 and T. pallidum to laminin. Further, Tp0751-specific antibodies inhibited attachment of T. pallidum to laminin. This study furthers our knowledge of the interaction of T. pallidum with laminin, an association that is proposed to facilitate bacterial traversal of basement membranes and subsequent entry into the circulation and tissue invasion. As such, these investigations will reveal new targets for possible prevention of bacterial dissemination and establishment of chronic infection.

Cameron, Caroline E.; Brouwer, Nathan L.; Tisch, Lisa M.; Kuroiwa, Janelle M. Y.

2005-01-01

320

Laminin Network Formation Studied by Reconstitution of Ternary Nodes in Solution*  

PubMed Central

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.

Purvis, Alan; Hohenester, Erhard

2012-01-01

321

Ligand-Regulated Heterodimerization of Peroxisome Proliferator-Activated Receptor ? with Liver X Receptor ?.  

PubMed

Peroxisome proliferator-activated receptor ? (PPAR?) and liver X receptor ? (LXR?) are members of the nuclear receptor superfamily that function to regulate lipid metabolism. Complex interactions between the LXR? and PPAR? pathways exist, including competition for the same heterodimeric partner, retinoid X receptor ? (RXR?). Although data have suggested that PPAR? and LXR? may interact directly, the role of endogenous ligands in such interactions has not been investigated. Using in vitro protein-protein binding assays, circular dichroism, and co-immunoprecipitation of endogenous proteins, we established that full-length human PPAR? and LXR? interact with high affinity, resulting in altered protein conformations. We demonstrated for the first time that the affinity of this interaction and the resulting conformational changes could be altered by endogenous PPAR? ligands, namely long chain fatty acids (LCFA) or their coenzyme A thioesters. This heterodimer pair was capable of binding to PPAR? and LXR? response elements (PPRE and LXRE, respectively), albeit with an affinity lower than that of the respective heterodimers formed with RXR?. LCFA had little effect on binding to the PPRE but suppressed binding to the LXRE. Ectopic expression of PPAR? and LXR? in mammalian cells yielded an increased level of PPRE transactivation compared to overexpression of PPAR? alone and was largely unaffected by LCFA. Overexpression of both receptors also resulted in transactivation from an LXRE, with decreased levels compared to that of LXR? overexpression alone, and LCFA suppressed transactivation from the LXRE. These data are consistent with the hypothesis that ligand binding regulates heterodimer choice and downstream gene regulation by these nuclear receptors. PMID:24713062

Balanarasimha, Madhumitha; Davis, Andrea M; Soman, Frances L; Rider, S Dean; Hostetler, Heather A

2014-04-29

322

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.

2012-01-01

323

Mammalian EGF receptor activation by the rhomboid protease RHBDL2  

PubMed Central

The epidermal growth factor receptor (EGFR) has several functions in mammalian development and disease, particularly cancer. Most EGF ligands are synthesized as membrane-tethered precursors, and their proteolytic release activates signalling. In Drosophila, rhomboid intramembrane proteases catalyse the release of EGF-family ligands; however, in mammals this seems to be primarily achieved by ADAM-family metalloproteases. We report here that EGF is an efficient substrate of the mammalian rhomboid RHBDL2. RHBDL2 cleaves EGF just outside its transmembrane domain, thereby facilitating its secretion and triggering activation of the EGFR. We have identified endogenous RHBDL2 activity in several tumour cell lines.

Adrain, Colin; Strisovsky, Kvido; Zettl, Markus; Hu, Landian; Lemberg, Marius K; Freeman, Matthew

2011-01-01

324

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.

2013-01-01

325

Dopamine-2 receptor activation suppresses PACAP expression in gonadotrophs.  

PubMed

Pituitary adenylate cyclase-activating polypeptide (PACAP) is expressed at a high level in the fetal pituitary and decreases profoundly between embryonic day 19 and postnatal day 1 (PN1), with a further decrease from PN1 to PN4. In this series of experiments, we investigated the hypothesis that dopamine 2 receptor (Drd2) activation interrupts a cAMP-dependent feed-forward loop that maintains PACAP expression at a high level in the fetal pituitary. Using single-cell RT-PCR of pituitary cell cultures from newborn rats, Drd2 mRNA was identified in gonadotrophs that were also positive for PACAP mRNA. PACAP expression in pituitary cultures from embryonic day 19 rats was suppressed by the PACAP6-38 antagonist and by the Drd2 agonist bromocriptine. Increasing concentrations of bromocriptine inhibited cAMP production as well as cAMP signaling based on cAMP response element-luciferase activity, decreased PACAP promoter activity, and decreased PACAP mRNA levels in ?T3-1 gonadotroph cells. Furthermore, blockade of dopamine receptors by injecting haloperidol into newborn rat pups partially reversed the developmental decline in pituitary PACAP mRNA that occurs between PN1 and PN4. These results provide evidence that dopamine receptor signaling regulates PACAP expression under physiological conditions and lend support to the hypothesis that a rise in hypothalamic dopamine at birth abrogates cAMP signaling in fetal gonadotrophs to interrupt a feed-forward mechanism that maintains PACAP expression at a high level in the fetal pituitary. We propose that this perinatal decline in pituitary PACAP reduces pituitary follistatin which permits GnRH receptors and FSH-? to increase to facilitate activation of the neonatal gonad. PMID:24823390

Winters, Stephen J; Ghooray, Dushan T; Yang, Rong Q; Holmes, Joshua B; O'Brien, Andrew Rw; Morgan, Jay; Moore, Joseph P

2014-07-01

326

Neural crest motility on fibronectin is regulated by integrin activation  

PubMed Central

Cell migration is essential for proper development of numerous structures derived from embryonic neural crest cells (NCCs). Although recent work has shown that receptor recycling plays an important role in NCC motility on laminin, the molecular mechanisms regulating NCC motility on fibronectin remain unclear. One mechanism by which cells regulate motility is by modulating the affinity of integrin receptors. Here, we provide evidence that cranial and trunk NCCs rely on functional regulation of integrins to migrate efficiently on fibronectin (FN) in vitro. For NCCs cultured on fibronectin, velocity decreases after Mn2+ application (a treatment that activates all surface integrins) while velocity on laminin (LM) is not affected. The distribution of activated integrin ?1 receptors on the surface of NCCs is also substratum-dependent. Integrin activation affects cranial and trunk NCCs differently when cultured on different concentrations of FN substrata; only cranial NCCs slow in a FN concentration-dependent manner. Furthermore, Mn2+ treatment alters the distribution and number of activated integrin ?1 receptors on the surface of cranial and trunk NCCs in different ways. We provide a hypothesis whereby a combination of activated surface integrin levels and the degree to which those receptors are clustered determines NCC motility on fibronectin.

Strachan, L.R.; Condic, M.L.

2008-01-01

327

Regulation of extracellular matrix proteins and integrin cell substratum adhesion receptors on epithelium during cutaneous human wound healing in vivo.  

PubMed Central

Although changes in extracellular matrix proteins during wound healing have been well documented, little is known about the regulation of corresponding extracellular matrix adhesion receptors (integrins). To study this process in a human in vivo model, full thickness human skin grafts were transplanted onto severe combined immunodeficient mice and deep excisional wounds involving both the epidermal and dermal layers were then made. The changes in the expression of cell matrix proteins and epithelial integrins over time were analyzed with specific antibodies using immunohistochemistry. Wounding was associated with alterations in extracellular matrix proteins, namely, loss of laminin and type IV collagen in the region of the wound and expression of tenascin and fibronectin. Changes were also noted in the integrins on the migrating keratinocytes. There was marked up-regulation of the alpha v subunit and de novo expression of the fibronectin receptor (alpha 5 beta 1) during the stage of active migration (days 1 to 3 after wounding). In the later stages of wound healing, after epithelial integrity had been established, redistribution of the alpha 2, alpha 3, alpha 6, and beta 4 collagen/laminin-binding integrin subunits to suprabasal epidermal layers was noted. Thus, during cutaneous wound healing, keratinocytes up-regulate fibronectin/fibrinogen-binding integrins and redistribute collagen/laminin-binding integrins. This study demonstrates that the human skin/severe combined immunodeficient chimera provides a useful model to study events during human wound repair. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6

Juhasz, I.; Murphy, G. F.; Yan, H. C.; Herlyn, M.; Albelda, S. M.

1993-01-01

328

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

1990-01-01

329

Peroxisome Proliferators-Activated Receptor (PPAR) Modulators and Metabolic Disorders  

PubMed Central

Overweight and obesity lead to an increased risk for metabolic disorders such as impaired glucose regulation/insulin resistance, dyslipidemia, and hypertension. Several molecular drug targets with potential to prevent or treat metabolic disorders have been revealed. Interestingly, the activation of peroxisome proliferator-activated receptor (PPAR), which belongs to the nuclear receptor superfamily, has many beneficial clinical effects. PPAR directly modulates gene expression by binding to a specific ligand. All PPAR subtypes (?, ?, and ?) are involved in glucose metabolism, lipid metabolism, and energy balance. PPAR agonists play an important role in therapeutic aspects of metabolic disorders. However, undesired effects of the existing PPAR agonists have been reported. A great deal of recent research has focused on the discovery of new PPAR modulators with more beneficial effects and more safety without producing undesired side effects. Herein, we briefly review the roles of PPAR in metabolic disorders, the effects of PPAR modulators in metabolic disorders, and the technologies with which to discover new PPAR modulators.

Cho, Min-Chul; Lee, Kyoung; Paik, Sang-Gi; Yoon, Do-Young

2008-01-01

330

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

2010-01-01

331

The ryanodine receptor mediates early zymogen activation in pancreatitis  

PubMed Central

Acute pancreatitis is characterized by the pathologic activation of zymogens within pancreatic acinar cells. The process requires a rise in cytosolic Ca2+ from undefined intracellular stores. We hypothesized that zymogen activation is mediated by ryanodine receptor (RYR)-regulated Ca2+ release, because early zymogen activation takes place in a supranuclear compartment that overlaps in distribution with the RYR. Ca2+ signals in the basolateral, but not apical, region of acinar cells observed during supraphysiologic agonist stimulation were dependent on RYR Ca2+ release. Inhibition of RYR or depletion of RYR-sensitive Ca2+ pools each reduced pathologic zymogen activation in isolated acinar cells, but neither treatment affected amylase secretion. Inhibition of RYR also inhibited zymogen activation in vivo. We propose that Ca2+ release from the RYR mediates zymogen activation but not enzyme secretion. The findings imply a role for the RYR in acute pancreatitis.

Husain, Sohail Z.; Prasad, Priyajit; Grant, Wayne M.; Kolodecik, Thomas R.; Nathanson, Michael H.; Gorelick, Fred S.

2005-01-01

332

Ligand binding and activation of the secretin receptor, a prototypic family B G protein-coupled receptor  

PubMed Central

The secretin receptor is a prototypic member of family B G protein-coupled receptors that binds and responds to a linear 27-residue peptide natural ligand. The carboxyl-terminal region of this peptide assumes a helical conformation that occupies the peptide-binding cleft within the structurally complex disulphide-bonded amino-terminal domain of this receptor. The amino terminus of secretin is directed toward the core helical bundle domain of this receptor that seems to be structurally distinct from the analogous region of family A G protein-coupled receptors. This amino-terminal region of secretin is critical for its biological activity, to stimulate Gs coupling and the agonist-induced cAMP response. While the natural peptide ligand is known to span the two key receptor domains, with multiple residue-residue approximation constraints well established, the orientation of the receptor amino terminus relative to the receptor core helical bundle domain is still unclear. Fluorescence studies have established that the mid-region and carboxyl-terminal end of secretin are protected by the receptor peptide-binding cleft and the amino terminus of secretin is most exposed to the aqueous milieu as it is directed toward the receptor core, with the mid-region of the peptide becoming more exposed upon receptor activation. Like other family B peptide hormone receptors, the secretin receptor is constitutively present in a structurally specific homo-dimeric complex built around the lipid-exposed face of transmembrane segment four. This complex is important for facilitating G protein association and achieving the high affinity state of this receptor. LINKED ARTICLES This article is part of a themed section on Secretin Family (Class B) G Protein-Coupled Receptors. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.166.issue-1

Miller, Laurence J; Dong, Maoqing; Harikumar, Kaleeckal G

2012-01-01

333

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

2009-01-01

334

Sphingosine-1-phosphate receptor 1 reporter mice reveal receptor activation sites in vivo.  

PubMed

Activation of the GPCR sphingosine-1-phosphate receptor 1 (S1P1) by sphingosine-1-phosphate (S1P) regulates key physiological processes. S1P1 activation also has been implicated in pathologic processes, including autoimmunity and inflammation; however, the in vivo sites of S1P1 activation under normal and disease conditions are unclear. Here, we describe the development of a mouse model that allows in vivo evaluation of S1P1 activation. These mice, known as S1P1 GFP signaling mice, produce a S1P1 fusion protein containing a transcription factor linked by a protease cleavage site at the C terminus as well as a ?-arrestin/protease fusion protein. Activated S1P1 recruits the ?-arrestin/protease, resulting in the release of the transcription factor, which stimulates the expression of a GFP reporter gene. Under normal conditions, S1P1 was activated in endothelial cells of lymphoid tissues and in cells in the marginal zone of the spleen, while administration of an S1P1 agonist promoted S1P1 activation in endothelial cells and hepatocytes. In S1P1 GFP signaling mice, LPS-mediated systemic inflammation activated S1P1 in endothelial cells and hepatocytes via hematopoietically derived S1P. These data demonstrate that S1P1 GFP signaling mice can be used to evaluate S1P1 activation and S1P1-active compounds in vivo. Furthermore, this strategy could be potentially applied to any GPCR to identify sites of receptor activation during normal physiology and disease. PMID:24667638

Kono, Mari; Tucker, Ana E; Tran, Jennifer; Bergner, Jennifer B; Turner, Ewa M; Proia, Richard L

2014-05-01

335

Sphingosine-1-phosphate receptor 1 reporter mice reveal receptor activation sites in vivo  

PubMed Central

Activation of the GPCR sphingosine-1-phosphate receptor 1 (S1P1) by sphingosine-1-phosphate (S1P) regulates key physiological processes. S1P1 activation also has been implicated in pathologic processes, including autoimmunity and inflammation; however, the in vivo sites of S1P1 activation under normal and disease conditions are unclear. Here, we describe the development of a mouse model that allows in vivo evaluation of S1P1 activation. These mice, known as S1P1 GFP signaling mice, produce a S1P1 fusion protein containing a transcription factor linked by a protease cleavage site at the C terminus as well as a ?-arrestin/protease fusion protein. Activated S1P1 recruits the ?-arrestin/protease, resulting in the release of the transcription factor, which stimulates the expression of a GFP reporter gene. Under normal conditions, S1P1 was activated in endothelial cells of lymphoid tissues and in cells in the marginal zone of the spleen, while administration of an S1P1 agonist promoted S1P1 activation in endothelial cells and hepatocytes. In S1P1 GFP signaling mice, LPS-mediated systemic inflammation activated S1P1 in endothelial cells and hepatocytes via hematopoietically derived S1P. These data demonstrate that S1P1 GFP signaling mice can be used to evaluate S1P1 activation and S1P1-active compounds in vivo. Furthermore, this strategy could be potentially applied to any GPCR to identify sites of receptor activation during normal physiology and disease.

Kono, Mari; Tucker, Ana E.; Tran, Jennifer; Bergner, Jennifer B.; Turner, Ewa M.; Proia, Richard L.

2014-01-01

336

Acute Activation, Desensitization and Smoldering Activation of Human Acetylcholine Receptors  

PubMed Central

The behavioral effects of nicotine and other nicotinic agonists are mediated by AChRs in the brain. The relative contribution of acute activation versus chronic desensitization of AChRs is unknown. Sustained “smoldering activation” occurs over a range of agonist concentrations at which activated and desensitized AChRs are present in equilibrium. We used a fluorescent dye sensitive to changes in membrane potential to examine the effects of acute activation and chronic desensitization by nicotinic AChR agonists on cell lines expressing human ?4?2, ?3?4 and ?7 AChRs. We examined the effects of acute and prolonged application of nicotine and the partial agonists varenicline, cytisine and sazetidine-A on these AChRs. The range of concentrations over which nicotine causes smoldering activation of ?4?2 AChRs was centered at 0.13 µM, a level found in smokers. However, nicotine produced smoldering activation of ?3?4 and ?7 AChRs at concentrations well above levels found in smokers. The ?4?2 expressing cell line contains a mixture of two stoichiometries, namely (?4?2)2?2 and (?4?2)2?4. The (?4?2)2?2 stoichiometry is more sensitive to activation by nicotine. Sazetidine-A activates and desensitizes only this stoichiometry. Varenicline, cytisine and sazetidine-A were partial agonists on this mixture of ?4?2 AChRs, but full agonists on ?3?4 and ?7 AChRs. It has been reported that cytisine and varenicline are most efficacious on the (?4?2)2?4 stoichiometry. In this study, we distinguish the dual effects of activation and desensitization of AChRs by these nicotinic agonists and define the range of concentrations over which smoldering activation can be sustained.

Campling, Barbara G.; Kuryatov, Alexander; Lindstrom, Jon

2013-01-01

337

Streptokinase-induced platelet activation involves antistreptokinase antibodies and cleavage of protease-activated receptor-1.  

PubMed

Streptokinase activates platelets, limiting its effectiveness as a thrombolytic agent. The role of antistreptokinase antibodies and proteases in streptokinase-induced platelet activation was investigated. Streptokinase induced localization of human IgG to the platelet surface, platelet aggregation, and thromboxane A(2) production. These effects were inhibited by a monoclonal antibody to the platelet Fc receptor, IV.3. The platelet response to streptokinase was also blocked by an antibody directed against the cleavage site of the platelet thrombin receptor, protease-activated receptor-1 (PAR-1), but not by hirudin or an active site thrombin inhibitor, Ro46-6240. In plasma depleted of plasminogen, exogenous wild-type plasminogen, but not an inactive mutant protein, S(741)A plasminogen, supported platelet aggregation, suggesting that the protease cleaving PAR-1 was streptokinase-plasminogen. Streptokinase-plasminogen cleaved a synthetic peptide corresponding to PAR-1, resulting in generation of PAR-1 tethered ligand sequence and selectively reduced binding of a cleavage-sensitive PAR-1 antibody in intact cells. A combination of streptokinase, plasminogen, and antistreptokinase antibodies activated human erythroleukemic cells and was inhibited by pretreatment with IV.3 or pretreating the cells with the PAR-1 agonist SFLLRN, suggesting Fc receptor and PAR-1 interactions are necessary for cell activation in this system also. Streptokinase-induced platelet activation is dependent on both antistreptokinase-Fc receptor interactions and cleavage of PAR-1. (Blood. 2000;95:1301-1308) PMID:10666203

McRedmond, J P; Harriott, P; Walker, B; Fitzgerald, D J

2000-02-15

338

?-Aminobutyric Acid Type A (GABAA) Receptor Activation Modulates Tau Phosphorylation*  

PubMed Central

Abnormal phosphorylation and aggregation of the microtubule-associated protein Tau are hallmarks of various neurodegenerative diseases, such as Alzheimer disease. Molecular mechanisms that regulate Tau phosphorylation are complex and currently incompletely understood. We have developed a novel live cell reporter system based on protein-fragment complementation assay to study dynamic changes in Tau phosphorylation status. In this assay, fusion proteins of Tau and Pin1 (peptidyl-prolyl cis-trans-isomerase 1) carrying complementary fragments of a luciferase protein serve as a sensor of altered protein-protein interaction between Tau and Pin1, a critical regulator of Tau dephosphorylation at several disease-associated proline-directed phosphorylation sites. Using this system, we identified several structurally distinct GABAA receptor modulators as novel regulators of Tau phosphorylation in a chemical library screen. GABAA receptor activation promoted specific phosphorylation of Tau at the AT8 epitope (Ser-199/Ser-202/Thr-205) in cultures of mature cortical neurons. Increased Tau phosphorylation by GABAA receptor activity was associated with reduced Tau binding to protein phosphatase 2A and was dependent on Cdk5 but not GSK3? kinase activity.

Nykanen, Niko-Petteri; Kysenius, Kai; Sakha, Prasanna; Tammela, Paivi; Huttunen, Henri J.

2012-01-01

339

[Screening of pregnane X receptor activation from ginsenosides].  

PubMed

In order to study effects of ginseng on the metabolism of drug belong to CYP3A4 substrate, screening of pregnane X receptor activation from ginsenosides was performed by reporter assay. Based on PXR-CYP3A stable translation cell lines, 13 ginsenosides were screened for pregnane X receptor activation by reporter assays, and RIF as the positive control. The effect of ginsenosides Rg1 onCYP3A4 mRNA expression was also investigated by RT-PCR. The PXR-CYP3A stable translation cell lines had good response to RIF, and the EC50 is 2.51 micro mol x L(-1). When the condition of final concentration was 10 micromol x L(-1), ginsenoside F2 and protopanaxatriol had moderate inductive effects on PXR. Panaxotriol, Rg2, pseudoginsenoside F11, Rg1, ginsenoside and Rb3 had inhibitory effects on PXR. Ginsenoside Rf1, Rg3, Rh2 and protopanaxdiol had no obvious effects on PXR. Rg1 down-regulated CYP3A4 mRNA expression in a concentration-dependent manner. Activation of pregnane X receptor by ginsenosides may influence the metabolism of drug belong to CYP3A4 substrate, and cause ginseng-drug interactions. PMID:23600156

Wang, Yu-Guang; Liu, Hao-Sheng; Zhang, Xian-Xie; Xiao, Yong; Lu, Bei-Bei; Ma, Zeng-Chun; Liang, Qian-De; Tang, Xiang-Lin; Xiao, Cheng-Rong; Tan, Hong-Ling; Zhang, Bo-Li; Gao, Yue

2013-01-01

340

Activation of RIG-I-like Receptor Signal Transduction  

PubMed Central

Mammalian cells have the ability to recognize virus infection and mount a powerful antiviral response. Pattern recognition receptor proteins detect molecular signatures of virus infection and activate antiviral signaling cascades. The RIG-I-like receptors are cytoplasmic DExD/H box proteins that can specifically recognize virus-derived RNA species as a molecular feature discriminating the pathogen from the host. The RIG-I-like receptor family is composed of three homologous proteins, RIG-I, MDA5, and LGP2. All of these proteins can bind double-stranded RNA species with varying affinities via their conserved DExD/H box RNA helicase domains and C-terminal regulatory domains. The recognition of foreign RNA by the RLRs activates enzymatic functions and initiates signal transduction pathways resulting in the production of antiviral cytokines and the establishment of a broadly effective cellular antiviral state that protects neighboring cells from infection and triggers innate and adaptive immune systems. The propagation of this signal via the interferon antiviral system has been studied extensively, while the precise roles for enzymatic activities of the RNA helicase domain in antiviral responses are only beginning to be elucidated. Here, current models for RLR ligand recognition and signaling are reviewed.

Bruns, Annie; Horvath, Curt M.

2011-01-01

341

The dynamic process of ?2-adrenergic receptor activation  

PubMed Central

G protein-coupled receptors (GPCRs) can modulate diverse signaling pathways, often in a ligand-specific manner. The full range of functionally relevant GPCR conformations is poorly understood. Here we use NMR spectroscopy to characterize the conformational dynamics of the transmembrane core of the ?2-adrenergic receptor (?2AR), a prototypical GPCR. We labeled ?2AR with 13CH3?-methionine and obtained HSQC spectra of unliganded receptor as well as receptor bound to an inverse agonist, an agonist, and a G protein-mimetic nanobody. These studies provide evidence for conformational states not observed in crystal structures, as well as substantial conformational heterogeneity in agonist- and inverse-agonist-bound preparations. They also show that for ?2AR, unlike rhodopsin, an agonist alone does not stabilize a fully active conformation, suggesting that the conformational link between the agonist-binding pocket and the G-protein-coupling surface is not rigid. The observed heterogeneity may be important for ?2AR’s ability to engage multiple signaling and regulatory proteins.

Nygaard, Rie; Zou, Yaozhong; Dror, Ron O.; Mildorf, Thomas J.; Arlow, Daniel H.; Manglik, Aashish; Pan, Albert C.; Liu, Corey W.; Fung, Juan Jose; Bokoch, Michael P.; Thian, Foon Sun; Kobilka, Tong Sun; Shaw, David E.; Mueller, Luciano; Prosser, R. Scott; Kobilka, Brian K.

2013-01-01

342

The dynamic process of ?(2)-adrenergic receptor activation.  

PubMed

G-protein-coupled receptors (GPCRs) can modulate diverse signaling pathways, often in a ligand-specific manner. The full range of functionally relevant GPCR conformations is poorly understood. Here, we use NMR spectroscopy to characterize the conformational dynamics of the transmembrane core of the ?(2)-adrenergic receptor (?(2)AR), a prototypical GPCR. We labeled ?(2)AR with (13)CH(3)?-methionine and obtained HSQC spectra of unliganded receptor as well as receptor bound to an inverse agonist, an agonist, and a G-protein-mimetic nanobody. These studies provide evidence for conformational states not observed in crystal structures, as well as substantial conformational heterogeneity in agonist- and inverse-agonist-bound preparations. They also show that for ?(2)AR, unlike rhodopsin, an agonist alone does not stabilize a fully active conformation, suggesting that the conformational link between the agonist-binding pocket and the G-protein-coupling surface is not rigid. The observed heterogeneity may be important for ?(2)AR's ability to engage multiple signaling and regulatory proteins. PMID:23374348

Nygaard, Rie; Zou, Yaozhong; Dror, Ron O; Mildorf, Thomas J; Arlow, Daniel H; Manglik, Aashish; Pan, Albert C; Liu, Corey W; Fung, Juan José; Bokoch, Michael P; Thian, Foon Sun; Kobilka, Tong Sun; Shaw, David E; Mueller, Luciano; Prosser, R Scott; Kobilka, Brian K

2013-01-31

343

?1- and ?5-containing Laminins Regulate the Development of Bile Ducts via ?1 Integrin Signals*  

PubMed Central

Signals derived from basal lamina components are important for developing three-dimensional architecture of epithelial tissues. Laminins consisting of ?, ?, and ? subunits in basal lamina play pivotal roles in the formation and maintenance of epithelial tissue structures. However, it remains unclear which laminin isoforms transmit signals and how epithelial cells receive them to regulate multiple developmental processes. In three-dimensional culture of a liver progenitor cell line, Hepatic Progenitor Cells Proliferating on Laminin (HPPL), the cells establish apicobasal polarity and form cysts with a central lumen. Neutralizing antibody against ?1 integrin blocked the formation and maintenance of the cyst structure, indicating that ?1 integrin signaling was necessary throughout the morphogenesis. Although the addition of ?1-containing laminin, a ligand of ?1 integrin, induced cyst formation, it was dispensable for the maintenance of the cyst, suggesting that HPPL produces another ligand for ?1 integrin to maintain the structure. Indeed, we found that HPPL produced ?5-containing laminin, and siRNA against laminin ?5 partially inhibited the lumen formation. In fetal liver, p75NTR+ periportal fibroblasts and bile duct epithelial cells, known as cholangiocytes, expressed ?1- and ?5-containing laminins, respectively. In laminin ?5 KO liver, cholangiocytes normally emerged, but the number of bile ducts was decreased. These results suggest that ?1-containing laminin is sufficient as a component of the basal lamina for the commitment of bipotential liver progenitors to cholangiocytes and the apicobasal polarization, whereas ?5-containing laminin is necessary for the formation of mature duct structures. Thus, ?1- and ?5-containing laminins differentially regulate the sequential events to form epithelial tissues via ?1 integrin signals.

Tanimizu, Naoki; Kikkawa, Yamato; Mitaka, Toshihiro; Miyajima, Atsushi

2012-01-01

344

Insect Repellents: Modulators of Mosquito Odorant Receptor Activity  

PubMed Central

Background DEET, 2-undecanone (2-U), IR3535 and Picaridin are widely used as insect repellents to prevent interactions between humans and many arthropods including mosquitoes. Their molecular action has only recently been studied, yielding seemingly contradictory theories including odorant-dependent inhibitory and odorant-independent excitatory activities on insect olfactory sensory neurons (OSNs) and odorant receptor proteins (ORs). Methodology/Principal Findings Here we characterize the action of these repellents on two Aedes aegypti ORs, AaOR2 and AaOR8, individually co-expressed with the common co-receptor AaOR7 in Xenopus oocytes; these ORs are respectively activated by the odors indole (AaOR2) and (R)-(?)-1-octen3-ol (AaOR8), odorants used to locate oviposition sites and host animals. In the absence of odorants, DEET activates AaOR2 but not AaOR8, while 2-U activates AaOR8 but not AaOR2; IR3535 and Picaridin do not activate these ORs. In the presence of odors, DEET strongly inhibits AaOR8 but not AaOR2, while 2-U strongly inhibits AaOR2 but not AaOR8; IR3535 and Picaridin strongly inhibit both ORs. Conclusions/Significance These data demonstrate that repellents can act as olfactory agonists or antagonists, thus modulating OR activity, bringing concordance to conflicting models.

Bohbot, Jonathan D.; Dickens, Joseph C.

2010-01-01

345

The human platelet ADP receptor activates Gi2 proteins.  

PubMed Central

We have previously shown that platelet ADP receptors are coupled to G-proteins by measuring the binding of [35S]guanosine-5'-[gamma-thio]triphosphate ([35S]GTP gamma S) to human platelet membranes stimulated with ADP. In order to identify the activated G-proteins, we used an approach which combines photolabelling of receptor-activated G-proteins with 4-azidoanilido-[alpha-32P]GTP and immunoprecipitation of the G-protein alpha-subunits with subtype-specific antibodies. Stimulation of human platelet membranes with ADP resulted in an increase in 4-azidoanilido-[alpha-32P]GTP incorporation into the immunoprecipitates of G alpha i but not of G alpha q proteins, whereas stimulation with the thromboxane analogue U46619 resulted in an increase in 4-azidoanilido-[alpha-32P]GTP incorporation into the immunoprecipitates of G alpha q but not of G alpha i proteins, and thrombin activated both G-proteins. This effect of ADP was concentration dependent and inhibited by the class P2 purinoceptor (P2T) antagonist ATP. Using specific antisera against subtypes of Gi proteins, we found that ADP stimulated labelling of the G alpha 12 immunoprecipitate, but not of the G alpha 13 precipitate. G alpha i1 was not detectable by immunoblotting of platelet membrane proteins. These data suggest that ADP inhibits cAMP formation by activation of G alpha 12 proteins and add evidence in support of the hypothesis that human platelet ADP receptors do not activate PLC through Gq activation. Images Figure 1 Figure 2 Figure 3 Figure 4

Ohlmann, P; Laugwitz, K L; Nurnberg, B; Spicher, K; Schultz, G; Cazenave, J P; Gachet, C

1995-01-01

346

Nuclear receptor corepressor-dependent repression of peroxisome-proliferator-activated receptor delta-mediated transactivation.  

PubMed Central

The nuclear receptor corepressor (NCoR) was isolated as a peroxisome-proliferator-activated receptor (PPAR) delta interacting protein using the yeast two-hybrid system. NCoR interacted strongly with the ligand-binding domain of PPAR delta, whereas interactions with the ligand-binding domains of PPAR gamma and PPAR alpha were significantly weaker. PPAR-NCoR interactions were antagonized by ligands in the two-hybrid system, but were ligand-insensitive in in vitro pull-down assays. Interaction between PPAR delta and NCoR was unaffected by coexpression of retinoid X receptor (RXR) alpha. The PPAR delta-RXR alpha heterodimer bound to an acyl-CoA oxidase (ACO)-type peroxisome-proliferator response element recruited a glutathione S-transferase-NCoR fusion protein in a ligand-independent manner. Contrasting with most other nuclear receptors, PPAR delta was found to interact equally well with interaction domains I and II of NCoR. In transient transfection experiments, NCoR and the related silencing mediator for retinoid and thyroid hormone receptor (SMRT) were shown to exert a marked dose-dependent repression of ligand-induced PPAR delta-mediated transactivation; in addition, transactivation induced by the cAMP-elevating agent forskolin was efficiently reduced to basal levels by NCoR as well as SMRT coexpression. Our results suggest that the transactivation potential of liganded PPAR delta can be fine-tuned by interaction with NCoR and SMRT in a manner determined by the expression levels of corepressors and coactivators.

Krogsdam, Anne-M; Nielsen, Curt A F; Neve, S?ren; Holst, Dorte; Helledie, Torben; Thomsen, Bo; Bendixen, Christian; Mandrup, Susanne; Kristiansen, Karsten

2002-01-01

347

[Studies on the synthesis of cholecystokinin A receptor antagonists. I. synthesis and cholecystokinin A receptor inhibitory activities of malonamide derivatives].  

PubMed

2-Substituted malonamide derivatives were synthesized and tested for cholecystokinin A (CCK-A) receptor inhibitory activity. Significant CCK-A receptor inhibitory activities were found in only three compounds (4g-i) which have carboxyl group. In order to study structure-activity relationships, carboxyethyl group was selected for 2-substituent and a number of N-substituted malonamides were prepared. After these compounds were tested for CCK-A receptor inhibitory activity, 4-(3,4-dichlorophenylcarbamoyl)-N,N-dipentylglutaramic acid (4h) was selected as the most preferred compound. PMID:8699319

Ajisawa, Y; Kitazawa, M; Uchida, M; Kobayashi, M

1996-01-01

348

Microvascular endothelial cells express a phosphatidylserine receptor: a functionally active receptor for phosphatidylserine-positive erythrocytes  

PubMed Central

Phosphatidylserine (PS)–positive erythrocytes adhere to endothelium and subendothelial matrix components. While thrombospondin mediates these inter-actions, it is unknown whether PS-associated erythrocyte-endothelial adhesion occurs in the absence of plasma ligands. Using ionophore-treated PS-expressing control HbAA erythrocytes, we demonstrate that PS-positive erythrocytes adhered to human lung microendothelial cells in the absence of plasma ligands, that this adhesion was enhanced following endothelial activation with IL-1?, TNF-?, LPS, hypoxia, and heme, and that this adhesive interaction was selective to erythrocyte PS. We next explored whether microendothelial cells express an adhesion receptor that recognizes cell surface–expressed PS (PSR) similar to that expressed on activated macrophages. We demonstrate constitutive expression of both PSR mRNA and protein that were up-regulated in a time-dependent manner following endothelial activation. While minimal PSR expression was noted on unstimulated cells, endothelial activation up-regulated PSR surface expression. In antibody-blocking studies, using PS-positive erythrocytes generated either artificially via ionophore treatment of control erythrocytes or from patients with sickle cell disease, we demonstrate that PSR was functional, supporting PS-mediated erythrocyte adhesion to activated endothelium. Our results demonstrate the existence of a novel functional adhesion receptor for PS on the microendothelium that is up-regulated by such pathologically relevant agonists as hypoxia, cytokines, and heme.

Betal, Suhita Gayen

2008-01-01

349

Detection of Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation  

PubMed Central

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of microbial (e.g., viruses, bacteria and parasite) infections, they also play an important role in the pathophysiology of infectious diseases, inflammatory diseases, and possibly in autoimmune diseases. Thus, the intensity and duration of TLR responses against infectious diseases must be tightly controlled. It follows that understanding the structural integrity of sensor receptors, their ligand interactions and signaling components is essential for subsequent immunological protection. It would also provide important opportunities for disease modification through sensor manipulation. Although the signaling pathways of TLR sensors are well characterized, the parameters controlling interactions between the sensors and their ligands still remain poorly defined. We have recently identified a novel mechanism of TLR activation by its natural ligand, which has not been previously observed 1,2. It suggests that ligand-induced TLR activation is tightly controlled by Neu1 sialidase activation. We have also reported that Neu1 tightly regulates neurotrophin receptors like TrkA and TrkB 3, which involve Neu1 and matrix metalloproteinase-9 (MMP-9) cross-talk in complex with the receptors 4. The sialidase assay has been initially use to find a novel ligand, thymoquinone, in the activation of Neu4 sialidase on the cell surface of macrophages, dendritic cells and fibroblast cells via GPCR G?i proteins and MMP-9 5. For TLR receptors, our data indicate that Neu1 sialidase is already in complex with TLR-2, -3 and -4 receptors, and is induced upon ligand binding to either receptor. Activated Neu1 sialidase hydrolyzes sialyl ?-2,3-linked ?-galactosyl residues distant from ligand binding to remove steric hinderance to TLR-4 dimerization, MyD88/TLR4 complex recruitment, NFkB activation and pro-inflammatory cell responses. In a collaborative report, Neu1 sialidase has been shown to regulate phagocytosis in macrophage cells 6. Taken together, the sialidase assay has provided us with powerful insights to the molecular mechanisms of ligand-induced receptor activation. Although the precise relationship between Neu1 sialidase and the activation of TLR, Trk receptors has yet to be fully elucidated, it would represent a new or pioneering approach to cell regulation pathways.

Abdulkhalek, Samar

2010-01-01

350

Liver X Receptor ? and Peroxisome Proliferator-Activated Receptor ? regulate cholesterol transport in cholangiocytes  

PubMed Central

Nuclear receptors (NRs) play crucial roles in regulation of hepatic cholesterol synthesis, metabolism and conversion to bile acids, but their actions in cholangiocytes have not been examined. In this study, we investigated the roles of NRs in cholangiocyte physiology and cholesterol metabolism and flux. We examined the expression of NRs and other genes involved in cholesterol homeostasis in freshly isolated and cultured rodent cholangiocytes and found that these cells express a specific subset of NRs which includes Liver X Receptor ? (LXR?) and Peroxisome Proliferator-Activated Receptor ? (PPAR?). Activation of LXR? and/or PPAR? in cholangiocytes induces ATP-binding cassette cholesterol transporter A1 (ABCA1) and increases cholesterol export at the basolateral compartment in polarized cultured cholangiocytes. In addition, PPAR? induces Niemann Pick C1 Like L1 (NPC1L1), which imports cholesterol into cholangiocytes and is expressed on the apical cholangiocyte membrane, via specific interaction with a PPRE within the NPC1L1 promoter. Based on these studies, we propose that (i) LXR? and PPAR? coordinate NPC1L1/ABCA1 dependent vectorial cholesterol flux from bile through cholangiocytes and (ii) manipulation of these processes may influence bile composition with important applications in cholestatic liver disease and gallstone disease, serious health concerns for humans.

Xia, Xuefeng; Jung, Dongju; Webb, Paul; Zhang, Aijun; Zhang, Bin; Li, Lifei; Ayers, Stephen D.; Gabbi, Chiara; Ueno, Yoshiyuki; Gustafsson, Jan-Ake; Alpini, Gianfranco; Moore, David D.; LeSage, Gene D.

2012-01-01

351

Structure and activity of OK-GC: a kidney receptor guanylate cyclase activated by guanylin peptides.  

PubMed

Uroguanylin, guanylin, and lymphoguanylin are small peptides that activate renal and intestinal receptor guanylate cyclases (GC). They are structurally similar to bacterial heat-stable enterotoxins (ST) that cause secretory diarrhea. Uroguanylin, guanylin, and ST elicit natriuresis, kaliuresis, and diuresis by direct actions on kidney GC receptors. A 3,762-bp cDNA characterizing a uroguanylin/guanylin/ST receptor was isolated from opossum kidney (OK) cell RNA/cDNA. This kidney cDNA (OK-GC) encodes a mature protein containing 1,049 residues sharing 72.4-75.8% identity with rat, human, and porcine forms of intestinal GC-C receptors. COS or HEK-293 cells expressing OK-GC receptor protein were activated by uroguanylin, guanylin, or ST13 peptides. The 3.8-kb OK-GC mRNA transcript is most abundant in the kidney cortex and intestinal mucosa, with lower mRNA levels observed in urinary bladder, adrenal gland, and myocardium and with no detectable transcripts in skin or stomach mucosa. We propose that OK-GC receptor GC participates in a renal mechanism of action for uroguanylin and/or guanylin in the physiological regulation of urinary sodium, potassium, and water excretion. This renal tubular receptor GC may be a target for circulating uroguanylin in an endocrine link between the intestine and kidney and/or participate in an intrarenal paracrine mechanism for regulation of kidney function via the intracellular second messenger, cGMP. PMID:10362777

London, R M; Eber, S L; Visweswariah, S S; Krause, W J; Forte, L R

1999-06-01

352

NK cell-activating receptors require PKC for sustained signaling, transcriptional activation, and IFN secretion  

Microsoft Academic Search

Natural killer (NK) cell sense virally in- fected cells and tumor cells through mul- tiple cell surface receptors. Many NK cell- activating receptors signal through immunoreceptor tyrosine-based activa- tion motif (ITAM)-containing adapters, which trigger both cytotoxicy and secre- tion of interferon-gamma (IFN-). Within the ITAM pathway, distinct signaling inter- mediates are variably involved in cytotox- icity and\\/or IFN- secretion. In

Ilaria Tassi; Marina Cella; Rachel Presti; Angela Colucci; Susan Gilfillan; Dan R. Littman; Marco Colonna

2008-01-01

353

Trace Amine Associated Receptor 1 Signaling in Activated Lymphocytes  

PubMed Central

Although most research to date on Trace Amine Associated Receptor 1 (TAAR1) has focused on its role in the brain, it has been recognized since its discovery in 2001 that TAAR1 mRNA is expressed in peripheral tissues as well, suggesting that this receptor may play a role in non-neurological pathways. This study reports TAAR1 expression, signaling and functionality in rhesus monkey lymphocytes. We detected a high level of TAAR1 protein in immortalized rhesus monkey B cell lines and a significant upregulation of TAAR1 protein expression in rhesus monkey lymphocytes following PHA treatment. Through screening a wide range of signaling pathways for their upregulation following TAAR1 activation by its potent agonist methamphetamine, we identified two transcription factors, CREB and NFAT, which are commonly associated with immune activation. Furthermore, we observed a TAAR1-dependent phosphorylation of PKA and PKC following treatment with methamphetamine in transfected HEK293 cells, immortalized rhesus monkey B cells and PHA-activated rhesus monkey lymphocytes. Accordingly, the high levels of TAAR1 that we observed on lymphocytes are inducible and fully functional, capable of transmitting a signal likely via PKA and PKC activation following ligand binding. More importantly, an increase in TAAR1 receptor expression is concomitant with lymphocyte immune activation, suggesting a possible role for TAAR1 in the generation or regulation of an immune response. TAAR1 is emerging as a potential therapeutic target, with regard to its ability to modulate brain monoamines. The current data raises the possibility that TAAR1-targeted drugs may also alter immune function.

Panas, Michael W.; Xie, Zhihua; Panas, Helen N.; Hoener, Marius C.; Vallender, Eric J.; Miller, Gregory M.

2013-01-01

354

Trace amine associated receptor 1 signaling in activated lymphocytes.  

PubMed

Although most research to date on Trace Amine Associated Receptor 1 (TAAR1) has focused on its role in the brain, it has been recognized since its discovery in 2001 that TAAR1 mRNA is expressed in peripheral tissues as well, suggesting that this receptor may play a role in non-neurological pathways. This study reports TAAR1 expression, signaling and functionality in rhesus monkey lymphocytes. We detected a high level of TAAR1 protein in immortalized rhesus monkey B cell lines and a significant upregulation of TAAR1 protein expression in rhesus monkey lymphocytes following PHA treatment. Through screening a wide range of signaling pathways for their upregulation following TAAR1 activation by its potent agonist methamphetamine, we identified two transcription factors, CREB and NFAT, which are commonly associated with immune activation. Furthermore, we observed a TAAR1-dependent phosphorylation of PKA and PKC following treatment with methamphetamine in transfected HEK293 cells, immortalized rhesus monkey B cells and PHA-activated rhesus monkey lymphocytes. Accordingly, the high levels of TAAR1 that we observed on lymphocytes are inducible and fully functional, capable of transmitting a signal likely via PKA and PKC activation following ligand binding. More importantly, an increase in TAAR1 receptor expression is concomitant with lymphocyte immune activation, suggesting a possible role for TAAR1 in the generation or regulation of an immune response. TAAR1 is emerging as a potential therapeutic target, with regard to its ability to modulate brain monoamines. The current data raises the possibility that TAAR1-targeted drugs may also alter immune function. PMID:22038157

Panas, Michael W; Xie, Zhihua; Panas, Helen N; Hoener, Marius C; Vallender, Eric J; Miller, Gregory M

2012-12-01

355

Purinergic receptors activating rapid intracellular Ca2+ increases in microglia  

PubMed Central

We provide both molecular and pharmacological evidence that the metabotropic, purinergic, P2Y6, P2Y12 and P2Y13 receptors and the ionotropic P2X4 receptor contribute strongly to the rapid calcium response caused by ATP and its analogues in mouse microglia. Real-time PCR demonstrates that the most prevalent P2 receptor in microglia is P2Y6 followed, in order, by P2X4, P2Y12, and P2X7 = P2Y13. Only very small quantities of mRNA for P2Y1, P2Y2, P2Y4, P2Y14, P2X3 and P2X5 were found. Dose-response curves of the rapid calcium response gave a potency order of: 2MeSADP>ADP=UDP=IDP=UTP>ATP>BzATP, whereas A2P4 had little effect. Pertussis toxin partially blocked responses to 2MeSADP, ADP and UDP. The P2X4 antagonist suramin, but not PPADS, significantly blocked responses to ATP. These data indicate that P2Y6, P2Y12, P2Y13 and P2X receptors mediate much of the rapid calcium responses and shape changes in microglia to low concentrations of ATP, presumably at least partly because ATP is rapidly hydrolyzed to ADP. Expression of P2Y6, P2Y12 and P2Y13 receptors appears to be largely glial in the brain, so that peripheral immune cells and CNS microglia share these receptors. Thus, purinergic, metabotropic, P2Y6, P2Y12, P2Y13 and P2X4 receptors might share a role in the activation and recruitment of microglia in the brain and spinal cord by widely varying stimuli that cause the release of ATP, including infection, injury and degeneration in the CNS, and peripheral tissue injury and inflammation which is signaled via nerve signaling to the spinal cord.

Light, Alan R.; Wu, Ying; Hughen, Ronald W.; Guthrie, Peter B.

2006-01-01

356

Liver X Receptors Regulate the Transcriptional Activity of the Glucocorticoid Receptor: Implications for the Carbohydrate Metabolism  

PubMed Central

GLUCOCORTICOIDS are steroid hormones that strongly influence intermediary carbohydrate metabolism by increasing the transcription rate of glucose-6-phosphatase (G6Pase), a key enzyme of gluconeogenesis, and suppress the immune system through the glucocorticoid receptor (GR). The liver X receptors (LXRs), on the other hand, bind to cholesterol metabolites, heterodimerize with the retinoid X receptor (RXR), and regulate the cholesterol turnover, the hepatic glucose metabolism by decreasing the expression of G6Pase, and repress a set of inflammatory genes in immune cells. Since the actions of these receptors overlap with each other, we evaluated the crosstalk between the GR- and LXR-mediated signaling systems. Transient transfection-based reporter assays and gene silencing methods using siRNAs for LXRs showed that overexpression/ligand (GW3965) activation of LXRs/RXRs repressed GR-stimulated transactivation of certain glucocorticoid response element (GRE)-driven promoters in a gene-specific fashion. Activation of LXRs by GW3965 attenuated dexamethasone-stimulated elevation of circulating glucose in rats. It also suppressed dexamethasone-induced mRNA expression of hepatic glucose-6-phosphatase (G6Pase) in rats, mice and human hepatoma HepG2 cells, whereas endogenous, unliganded LXRs were required for dexamethasone-induced mRNA expression of phosphoenolpyruvate carboxylase. In microarray transcriptomic analysis of rat liver, GW3965 differentially regulated glucocorticoid-induced transcriptional activity of about 15% of endogenous glucocorticoid-responsive genes. To examine the mechanism through which activated LXRs attenuated GR transcriptional activity, we examined LXR?/RXR? binding to GREs. Endogenous LXR?/RXR? bound GREs and inhibited GR binding to these DNA sequences both in in vitro and in vivo chromatin immunoprecipitation assays, while their recombinant proteins did so on classic or G6Pase GREs in gel mobility shift assays. We propose that administration of LXR agonists may be beneficial in glucocorticoid treatment- or stress-associated dysmetabolic states by directly and gene-specifically attenuating the transcriptional activity of the GR on glucose and/or lipid metabolism.

Nader, Nancy; Ng, Sinnie Sin Man; Wang, Yonghong; Abel, Brent S.; Chrousos, George P.; Kino, Tomoshige

2012-01-01

357

Depersonalization disorder may be related to glutamate receptor activation imbalance.  

PubMed

Low-dose ketamine administration mimics, both clinically and on gross neuroimaging, depersonalization disorder. The perceptual effects of ketamine may be due to secondary stimulation of glutamate release and lamotrigine, possibly by inhibited glutamate release, may reduce some of ketamine's so-called dissociative effects. However, lamotrigine does not seem to be useful in the treatment of depersonalization disorder. Glutamate release in prefrontal cortex is increased by subanaesthetic doses of ketamine, resulting in increased inhibition, possibly via intercalated GABAerg cells, of projections from amygdala, affecting structures critically involved in depersonalization. I speculate that, in depersonalization disorder, the increased glutamate activity in prefrontal cortex is due to intrinsic imbalance, resulting in long-term potentiation, at the postsynaptic glutamate receptors on the GABAerg interneurons while the same receptor abnormality at the synapses on the intercalated GABAerg cells of the amygdala result in long-term depression in the case of either normal or high glutamate release. PMID:21742442

Pikwer, Andreas

2011-10-01

358

Expression pattern of protease activated receptors in lymphoid cells.  

PubMed

Protease-activated receptors (PARs) are a subfamily of four G-protein-coupled receptors mediating multiple functions. PARs expression was studied in subpopulations of human lymphocytes. Our results indicate that natural killer cells expressed mRNA for PAR1, PAR2 and PAR3, CD4+ T cells expressed PAR1 and PAR2, while ?? and CD8+ T cells only expressed PAR1. PAR4 was absent at mRNA level and B cells did not express any PAR. Analyses of the cell surface PARs expression by flow cytometry were consistent with the mRNA data and also between different donors. PAR1 is the most abundant member of the PAR family present in lymphocytes. PMID:24637088

López, Mercedes L; Soriano-Sarabia, Natalia; Bruges, Gustavo; Marquez, María Elena; Preissner, Klaus T; Schmitz, M Lienhard; Hackstein, Holger

2014-01-01

359

Protease-activated receptor-3 (PAR3) regulates PAR1 signaling by receptor dimerization  

PubMed Central

Thrombin activates endothelial cell signaling by cleaving the protease-activated receptor-1 (PAR1). However, the function of the apparently nonsignaling receptor PAR3 also expressed in endothelial cells is unknown. We demonstrate here the crucial role of PAR3 in potentiating the responsiveness of PAR1 to thrombin. We tested the hypothesis that PAR1/PAR3 heterodimerization and its effect in modifying G protein selectivity was responsible for PAR3 regulation of PAR1 sensitivity. Using bioluminescent resonance energy transfer-2, we showed that PAR1 had comparable dimerization affinity for PAR3 as for itself. We observed increased G?13 coupling between the PAR1/3 heterodimer compared with the PAR1/1 homodimer. Moreover, knockdown of PAR3 moderated the PAR1-activated increase in endothelial permeability. These results demonstrate a role of PAR3 in allosterically regulating PAR1 signaling governing increased endothelial permeability. Because PAR3 is a critical determinant of PAR1 function, targeting of PAR3 may mitigate the effects of PAR1 in activating endothelial responses such as vascular inflammation.

McLaughlin, Joseph N.; Patterson, Myla M.; Malik, Asrar B.

2007-01-01

360

The Functions of Exogenous and Endogenous Laminin5 on Corneal Epithelial Cells  

Microsoft Academic Search

Recent evidence suggests that the basement membrane not only separates basal cells from Bowman's layer, but also has a crucial role in the proliferation, differentiation and migration of corneal epithelial cells. The basement membrane is composed of a mixture of matrix components including collagens, laminins and heparan sulfate proteoglycans. In these extracellular matrixes, laminin is a major component of the

Nobuyuki Ebihara; Hiroto Mizushima; Kaoru Miyazaki; Yasuo Watanabe; Seiichirou Ikawa; Kiyoo Nakayasu; Atsushi Kanai

2000-01-01

361

Laminin deposition is dispensable for vasculogenesis but regulates blood vessel diameter independent of flow  

Microsoft Academic Search

Basement membranes (BMs) consisting of laminins, collagens, and heparan sulfate proteogly- cans (HSPGs) are vital for proper endothelial cell function, but many aspects of their role in vascular development remain unknown. Here, we demonstrate that vascular structures within differentiating embryoid bodies are wrapped in a BM composed of 4- and 5-chain laminins, fibronectin, collagen IV, and HSPGs. In sprouting angiogenesis,

Lars Jakobsson; Anna Domogatskaya; Karl Tryggvason; David Edgar; Lena Claesson-Welsh

2007-01-01

362

Essential and overlapping roles for laminin alpha chains in notochord and blood vessel formation.  

PubMed

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 alpha, beta and gamma chain. We previously reported that two zebrafish loci, grumpy (gup) and sleepy (sly), encode laminin beta1 and gamma1, which are important both for notochord differentiation and for proper intersegmental blood vessel (ISV) formation. In this study we show that bashful (bal) encodes laminin alpha1 (lama1). Although the strongest allele, bal(m190), is fully penetrant, when compared to gup or sly mutant embryos, bal mutants are not as severely affected, as only anterior notochord fails to differentiate and ISVs are unaffected. This suggests that other alpha chains, and hence other isoforms, act redundantly to laminin 1 in posterior notochord and ISV development. We identified cDNA sequences for lama2, lama4 and lama5 and disrupted the expression of each alone or in mutant embryos also lacking laminin alpha1. When expression of laminin alpha4 and laminin alpha1 are simultaneously disrupted, notochord differentiation and ISVs are as severely affected as sly or gup mutants. Moreover, live imaging of transgenic embryos expressing enhanced green fluorescent protein in forming ISVs reveals that the vascular defects in these embryos are due to an inability of ISV sprouts to migrate correctly along the intersegmental, normally laminin-rich regions. PMID:16321372

Pollard, Steven M; Parsons, Michael J; Kamei, Makoto; Kettleborough, Ross N W; Thomas, Kevin A; Pham, Van N; Bae, Moon-Kyoung; Scott, Annabelle; Weinstein, Brant M; Stemple, Derek L

2006-01-01