Adult and fetal human mesangial cells interact with specific laminin domains

1991 ◽  
Vol 261 (4) ◽  
pp. F688-F695
Author(s):  
B. S. Weeks ◽  
J. B. Kopp ◽  
S. Horikoshi ◽  
F. B. Cannon ◽  
M. Garrett ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Mesangial Cell ◽  
Mesangial Cells ◽  
Cell Attachment ◽  
Binding Protein ◽  
Laminin Binding Protein ◽  
Laminin Binding

Mesangial cells are centrally located pericytes in the renal glomerulus. They are surrounded by an extracellular matrix and directly contact the glomerular basement membrane in vivo. Because these interactions are critical for renal development and function, we have studied human mesangial cell interactions with laminin, a major adhesive component of basement membranes present in the extracellular matrix of the mesangium. Human fetal and adult mesangial cell attachment was stimulated by both laminin and the laminin-derived synthetic peptides YIGSR-NH2, CQAGTFALRGDNPQG-NH2, and CIKVAVS-NH2. Furthermore, mesangial cells spread on laminin as well as on both the RGD-containing and CIKVAVS peptides. When added in solution, all three peptides inhibited mesangial cell attachment to laminin, and the latter two peptides inhibited mesangial cell spreading on laminin. Laminin affinity column chromatography demonstrated several low-molecular-mass laminin-binding proteins ranging from between 35 and 42 kDa, which predominated in fetal mesangial cells, whereas a higher molecular mass laminin-binding protein of 65 kDa was predominant in adult mesangial cells. Western blot analysis with an anti-32-kDa laminin-binding protein antibody showed increased expression of both 31- and 42-kDa proteins in fetal mesangial cells when compared with the adult. The antisera to the 32-kDa laminin-binding protein also inhibited fetal mesangial spreading on the CIKVAVS peptide. Western blot analysis with an anti-67-kDa laminin-binding protein antibody revealed a 110-kDa protein in adult mesangial cells that was not present in fetal mesangial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals The ErbB2/Neu/HER2 receptor is a new calmodulin-binding protein

2004 ◽  
Vol 381 (1) ◽  
pp. 257-266 ◽  
Author(s):  
Hongbing LI ◽  
Juan SÁNCHEZ-TORRES ◽  
Alan del CARPIO ◽  
Valentina SALAS ◽  
Antonio VILLALOBO
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Tyrosine Kinases ◽  
Western Blot Analysis ◽  
Binding Protein ◽  
Signalling Pathways ◽  
Breast Adenocarcinoma ◽  
Dependent Manner ◽  
Her2 Receptor

We have demonstrated previously that the EGFR (epidermal growth factor receptor) is a calmodulin (CaM)-binding protein. To establish whether or not the related receptor ErbB2/Neu/HER2 also binds CaM, we used human breast adenocarcinoma SK-BR-3 cells, because these cells overexpress this receptor thus facilitating the detection of this interaction. In the present paper, we show that ErbB2 could be pulled-down using CaM–agarose beads in a Ca2+-dependent manner, as detected by Western blot analysis using an anti-ErbB2 antibody. ErbB2 was also isolated by Ca2+-dependent CaM-affinity chromatography. We also demonstrate using an overlay technique with biotinylated CaM that CaM binds directly to the immunoprecipitated ErbB2. The binding of biotinylated CaM to ErbB2 depends strictly on the presence of Ca2+, since it was prevented by the presence of EGTA. Moreover, the addition of an excess of free CaM prevents the binding of its biotinylated form, demonstrating that this was a specific process. We excluded any interference with the EGFR, as SK-BR-3 cells express considerably lower levels of this receptor, and no detectable EGFR signal was observed by Western blot analysis in the immunoprecipitated ErbB2 preparations used to perform the overlay assays with biotinylated CaM. We also demonstrate that treating living cells with W7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide], a cell-permeant CaM antagonist, down-regulates ErbB2 phosphorylation, and show that W7 does not interfere non-specifically with the activity of ErbB tyrosine kinases. We also show that W7 inhibits the phosphorylation (activation) of both ERK1/2 (extracellular-signal-regulated kinases 1 and 2) and Akt/PKB (protein kinase B), in accordance with the inhibition observed in ErbB2 phosphorylation. In contrast, W7 treatment increased the phosphorylation (activation) of CREB (cAMP-response-element-binding protein) and ATF1 (activating transcription factor-1), two Ca2+-sensitive transcription factors that operate downstream of these ErbB2 signalling pathways, most likely because of the absence of calcineurin activity. We conclude that ErbB2 is a new CaM-binding protein, and that CaM plays a role in the regulation of this receptor and its downstream signalling pathways in vivo.

scholarly journals Isolation and characterization of a laminin-binding protein from rat and chick muscle.

1988 ◽  
Vol 107 (2) ◽  
pp. 687-697 ◽  
Author(s):  
D E Hall ◽  
K A Frazer ◽  
B C Hann ◽  
L F Reichardt
Keyword(s):  
Skeletal Muscle ◽  
Extracellular Matrix ◽  
Amino Acid ◽  
Binding Protein ◽  
Light And Electron Microscopy ◽  
Muscle Membrane ◽  
Total Amino Acid ◽  
Isolation And Characterization ◽  
Laminin Binding Protein ◽  
Laminin Binding

A major laminin-binding protein (LBP), distinct from previously described LBPs, has been isolated from chick and rat skeletal muscle (Mr 56,000 and 66,000, respectively). The purified LBPs from the two species were shown to be related antigenically and to have similar NH2-terminal amino acid sequences and total amino acid compositions. Protein blots using laminin and laminin fragments provided evidence that this LBP interacts with the major heparin-binding domain, E3, of laminin. Studies on the association of this LBP with muscle membrane fractions and reconstituted lipid vesicles indicate that this protein can interact with lipid bilayers and has properties of a peripheral, not an integral membrane protein. These properties are consistent with its amino acid sequence, determined from cDNAs (Clegg et al., 1988). Examination by light and electron microscopy of the LBP antigen distribution in skeletal muscle indicated that the protein is localized primarily extracellularly, near the extracellular matrix and myotube plasmalemma. While a form of this LBP has been identified in heart muscle, it is present at low or undetectable levels in other tissues examined by immunocytochemistry indicating that it is probably a muscle-specific protein. As this protein is localized extracellularly and can bind to both membranes and laminin, it may mediate myotube interactions with the extracellular matrix.

Plasmin Proteolysis of Endothelial Cell and Vessel Wall Associated Tissue Factor Pathway Inhibitor

2001 ◽  
Vol 86 (09) ◽  
pp. 923-928 ◽  
Author(s):  
Paul Stalboerger ◽  
Carmelo Panetta ◽  
Robert Simari ◽  
Noel Caplice
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Cell Surface ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Vessel Wall ◽  
Anticoagulant Activity ◽  
Vascular Cells ◽  
Normal Vessel

SummaryPlasmin is an important protease that mediates clot fibrinolysis and vessel wall extracellular matrix proteolysis. Recently, in vitro studies have suggested that plasmin can cleave and inactivate recombinant TFPI, a major inhibitor of TF-mediated coagulation. We hypothesized that such an interaction may occur in vascular cells expressing TFPI, or in the vessel wall, with implications for thrombolysis. In a series of experiments, we examined the effects of plasmin on cell surface and extracellular matrix (ECM) associated TFPI in endothelial cells (EC) in culture and on EC and smooth muscle cells (SMC) in the vessel wall. Plasmin (0.2 μM) decreased cell surface and matrix associated TFPI activity in cultured endothelial cells by 77 ± 5 % and 69 ± 6% respectively (p < 0.01). Plasminogen, the proenzyme form of plasmin had no such effect on cell surface TFPI or matrix TFPI. Cell surface TFPI antigen measured by fluorescence activated cell sorter (FACS) was also significantly reduced by plasmin. Proteolysis of conditioned medium TFPI was suggested by loss of a ~45kD TFPI on Western Blot analysis following plasmin treatment. Plasmin also proteolysed a ~45kD TFPI protein in the intact ECM of EC, an effect which was inhibited by preincubation with aprotinin, a plasmin inhibitor. Incubation of similar concentrations of plasmin, with homogenates of normal vessel decreased a ~45kD TFPI immunoreactive band on Western blot analysis. Plasmin also decreased surface TFPI activity on frozen sections of normal vessel as measured by an amidolytic assay. Finally, plasmin treatment of atherosclerotic plaque sections caused complete removal of TFPI immunoreactivity associated with luminal EC and intimal SMC, when compared to control treated plaque (n = 3). Together these data suggest that plasmin proteolyses the majority of EC-associated (surface and matrix) TFPI and may remove TFPI from the luminal surface and intima of the vessel wall. TFPI proteolysis in cultured EC was associated with significant reduction in TFPI anticoagulant activity. These data provide evidence that plasmin degradation of TFPI occurs in vascular cells and in the vessel wall and may have implications for rethrombosis following thrombolysis in vivo.

Effect of 32/67 kDa laminin-binding protein antibody on mouse embryo implantation

Reproduction ◽  
2000 ◽  
pp. 137-142 ◽  
Author(s):  
C Zhang ◽  
E Duan ◽  
Y Cao ◽  
G Jiang ◽  
G Zeng
Keyword(s):  
Extracellular Matrix ◽  
Mouse Embryo ◽  
Binding Protein ◽  
Embryo Implantation ◽  
Continuous Section ◽  
Rabbit Igg ◽  
Laminin Binding Protein ◽  
Ectoplacental Cone ◽  
Laminin Binding

Mouse embryo implantation depends on the complex interaction between the embryo trophoblast cells and the uterine environment, which deposits an extracellular matrix with abundant amounts of laminin. Intrauterine injection and blastocyst or ectoplacental cone culture models were used to study the effect of 32/67 kDa laminin-binding protein antibody on mouse embryo implantation in vivo and in vitro. Intrauterine injection of 32/67 kDa laminin-binding protein antibody (0.4 mg in 1 ml Ham's F-10 medium, 5 microl per mouse) into the left uterine horns of mice (n = 22) on day 3 of pregnancy inhibited embryo implantation significantly (P < 0.001) compared with the contralateral horns that had been injected with normal rabbit IgG. A continuous section study on day 5 after injection showed that the embryos in the control uteri implanted normally and developed healthily, but there were no embryos or the remaining embryos had disintegrated in the uteri injected with 32/67 kDa laminin-binding protein antibody. Blastocysts or ectoplacental cones were cultured in media containing 32/67 kDa laminin-binding protein antibody (0.2 mg ml(-1)) on laminin-coated dishes with normal rabbit IgG at the same concentration as in the controls. The 32/67 kDa laminin-binding protein had no effect on blastocyst or ectoplacental cone attachment, but prohibited the blastocyst or ectoplacental cone outgrowth and primary or secondary trophoblast giant cell migration. These results indicate that 32/67 kDa laminin-binding protein antibody blocked mouse embryo implantation by preventing embryo trophoblast cell invasion and migration through the uterine decidual basement membrane-like extracellular matrix which has a high laminin content.

scholarly journals Ribonucleic acid-binding protein CPSF6 promotes glycolysis and suppresses apoptosis in hepatocellular carcinoma cells by inhibiting the BTG2 expression

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Yang Liu ◽  
Hongbo Zou ◽  
Qichao Xie ◽  
Lan Zou ◽  
Rui Kong ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Rna Binding ◽  
Binding Protein ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Glucose Assay ◽  
Normal Hepatocyte ◽  
Hcc Cells

AbstractHepatocellular carcinoma (HCC) is currently the sixth most common malignancy and the second major cause of tumor-related deaths in the world. This study aimed to investigate the role of cleavage and polyadenylation factor-6 (CPSF6) and B-cell translocation gene 2 (BTG2) in regulating the glycolysis and apoptosis in HCC cells. The RNA and protein expression of CPSF6 and BTG2 in normal hepatocyte and HCC were, respectively, detected by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis and Western blot analysis. The viability and apoptosis of transfected Huh-7 cells were, respectively, analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay. The expression of apoptosis-related proteins and HK-2 in transfected Huh-7 cells was also detected by Western blot analysis. The levels of glucose and lactate in the culture supernatant of transfected Huh-7 cells were, respectively, detected with the glucose assay kit and lactate assay kit. The interaction of CPSF6 and BTG2 was confirmed by RNA binding protein immunoprecipitation (RIP) assay. As a result, CPSF6 expression was increased while BTG2 expression was decreased in Huh-7 cells. Interference with CPSF6 suppressed the viability and glycolysis, and promoted the apoptosis of Huh-7 cells. Furthermore, CPSF6 interacted with BTG2 and interference with CPSF6 upregulated the BTG2 expression and inhibited the protein kinase B (AKT)/extracellular signal-regulated kinase (ERK)/nuclear factor (NF)-κB pathway. Interference with BTG2 could partially reverse the above cell changes caused by interference with CPSF6. In conclusion, CPSF6 inhibited the BTG2 expression to promote glycolysis and suppress apoptosis in HCC cells by activating AKT/ERK/NF-κB pathway.

Alendronate promotes the gene expression of extracellular matrix mediated by SP-1/SOX-9

2021 ◽  
pp. 096032712098887
Author(s):  
L Wang ◽  
B Mi ◽  
Y Zhang ◽  
H Yan ◽  
H Zhu
Keyword(s):  
Gene Expression ◽  
Extracellular Matrix ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Specific Inhibitor ◽  
Expression Level ◽  
Dependent Manner ◽  
Qrt Pcr ◽  
Tnf Α

Background and purpose: Osteoarthritis (OA) is a disease with significant degenerative changes of articular cartilage, which is reported to be closely related to the integrity of chondrocytes extracellular matrix (ECM). Alendronate belongs to the family of bisphosphonates with promising cartilage repair function. In the present study, the effects of Alendronate on the gene expression of chondrocytes ECM and the potential mechanism will be investigated to explore the potential therapeutic property of Alendronate on OA. Methods: Human SW1353 chondrocytes were stimulated with 1 and 2 μM Alendronate for 12 h. The gene expression of Col2α1, COL9α2, and Acan in the treated chondrocytes was determined by qRT-PCR. QRT-PCR and western blot analysis were used to evaluate the expression level of SOX-9 in the treated chondrocytes. The expression level of SP-1 was checked by qRT-PCR and immunostaining. SiRNA against SP-1 was transfected into chondrocytes to knockdown the expression of SP-1. The levels of p-ERK1/2 and total ERK1/2 were examined using western blot analysis. TNF-α was used to induce an OA-like in vitro model in the chondrocytes for therapeutic evaluations. Results: Treatment with Alendronate increased the levels of ECM related genes ( Col2α1, COL9α2, and Acan) in a dose-dependent manner through increasing the expression of SOX-9, a central regulator of ECM genes. Additionally, our findings demonstrate that the effects of Alendronate in the expression of SOX-9 are mediated by SP-1 as silencing of SP-1 abolished these effects. Notably, Alendronate increased the phosphorylation of ERK1/2 and inhibition of ERK1/2 using its specific inhibitor U0126 blocked the expression of SP-1. Finally, we found that treatment with Alendronate could rescue TNF-α-induced reduction of Col2α1, COL9α2, Acan and SOX-9. Conclusion: Our data indicated that Alendronate might promote the gene expression of extracellular matrix through SOX-9 mediated by the ERK1/2/SP1 signaling pathway.

scholarly journals Cystathionine β-synthase and cystathionine γ-lyase double gene transfer ameliorate homocysteine-mediated mesangial inflammation through hydrogen sulfide generation

2011 ◽  
Vol 300 (1) ◽  
pp. C155-C163 ◽  
Author(s):  
Utpal Sen ◽  
Srikanth Givvimani ◽  
Oluwasegun A. Abe ◽  
Eleanor D. Lederer ◽  
Suresh C. Tyagi
Keyword(s):  
Hydrogen Sulfide ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Mesangial Cells ◽  
Regulatory Role ◽  
Glomerular Mesangial Cells ◽  
Endogenous Production ◽  
Inflammatory Molecules

Elevated level of homocysteine (Hcy) induces chronic inflammation in vascular bed, including glomerulus, and promotes glomerulosclerosis. In this study we investigated in vitro mechanism of Hcy-mediated monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) induction and determined the regulatory role of hydrogen sulfide (H2S) to ameliorate inflammation. Mouse glomerular mesangial cells (MCs) were incubated with Hcy (75 μM) and supplemented with vehicle or with H2S (30 μM, in the form of NaHS). Inflammatory molecules MCP-1 and MIP-2 were measured by ELISA. Cellular capability to generate H2S was measured by colorimetric chemical method. To enhance endogenous production of H2S and better clearance of Hcy, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) genes were delivered to the cells. Oxidative NAD(P)H p47 phox was measured by Western blot analysis and immunostaining. Phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun NH2-terminal kinase (JNK1/2) were measured by Western blot analysis. Our results demonstrated that Hcy upregulated inflammatory molecules MCP-1 and MIP-2, whereas endogenous production of H2S was attenuated. H2S treatment as well as CBS and CSE doubly cDNA overexpression markedly reduced Hcy-induced upregulation of MCP-1 and MIP-2. Hcy-induced upregulation of oxidative p47 phox was attenuated by H2S supplementation and CBS/CSE overexpression as well. In addition to that we also detected Hcy-induced MCP-1 and MIP-2 induction was through phosphorylation of ERK1/2 and JNK1/2. Either H2S supplementation or CBS and CSE doubly cDNA overexpression attenuated Hcy-induced phosphorylation of these two signaling molecules and diminished MCP-1 and MIP-2 expressions. Similar results were obtained by inhibition of ERK1/2 and JNK1/2 using pharmacological and small interferring RNA (siRNA) blockers. We conclude that H2S plays a regulatory role in Hcy-induced mesangial inflammation and that ERK1/2 and JNK1/2 are two signaling pathways involved this process.

scholarly journals Amino acid sequence and distribution of mRNA encoding a major skeletal muscle laminin binding protein: an extracellular matrix-associated protein with an unusual COOH-terminal polyaspartate domain.

1988 ◽  
Vol 107 (2) ◽  
pp. 699-705 ◽  
Author(s):  
D O Clegg ◽  
J C Helder ◽  
B C Hann ◽  
D E Hall ◽  
L F Reichardt
Keyword(s):  
Extracellular Matrix ◽  
Amino Acid ◽  
Membrane Protein ◽  
Amino Acid Sequence ◽  
Biochemical Characterization ◽  
Muscle Protein ◽  
Binding Protein ◽  
Single Gene ◽  
Laminin Binding Protein ◽  
Laminin Binding

Two cDNAs encoding an abundant chicken muscle extracellular matrix (ECM)-associated laminin-binding protein (LBP) have been isolated and sequenced. The predicted primary amino acid sequence includes a probable signal peptide and a site for N-linked glycosylation, but lacks a hydrophobic segment long enough to span the membrane. The COOH terminus consists of an unusual repeat of 33 consecutive aspartate residues. Comparison with other sequences indicates that this protein is different from previously described LBPs and ECM receptors. RNA blot analysis of LBP gene expression showed that LBP mRNA was abundant in skeletal and heart muscle, but barely detectable in other tissues. Blots of chicken genomic DNA suggest that a single gene encodes this LBP. The amino acid sequence and mRNA distribution are consistent with the biochemical characterization described by Hall and co-workers (Hall, D. E., K. A. Frazer, B. C. Hahn, and L. F. Reichardt. 1988. J. Cell Biol. 107:687-697). These analyses indicate that LBP is an abundant ECM-associated muscle protein with an unusually high negative charge that interacts with both membranes and laminin, and has properties of a peripheral, not integral membrane protein. Taken together, our studies show that muscle LBP is a secreted, peripheral membrane protein with an unusual polyaspartate domain. Its laminin and membrane binding properties suggest that it may help mediate muscle cell interactions with the extracellular matrix. We propose the name "aspartactin" for this LBP.

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