Extracellular Matrix Proteins Laminin Research Articles

Cell Surface Proteoglycans Syndecan-1 and -4 Bind Overlapping but Distinct Sites in Laminin α3 LG45 Protein Domain

Keratinocyte migration during epidermal repair depends on interactions between cellular heparan sulfate proteoglycan receptors, syndecan-1 and -4, and the C-terminal globular domains (LG45) of the extracellular matrix protein laminin 332. This study investigates the molecular basis of the binding specificity of the syndecan-1 and -4 receptors expressed by human keratinocytes. We used site-directed mutagenesis to alter a recombinant LG45 protein by substituting the most critical basic residues with glutamine. All proteins were expressed in mammalian cells, purified, and characterized biochemically. We used in vitro binding assays, including surface plasmon resonance, to examine interactions between mutated LG45 and heparan sulfates, syndecan-1 and -4. We identify a major heparin binding domain on the outer edge of a β-strand of LG45 surrounded by a track of converging low affinity residues. This domain harbors distinctive syndecan-1 and -4 binding-specific sequences. This is the first study to demonstrate a binding specificity of two proteoglycans produced by a single cell type. In addition, we found that although syndecan-1 interacts exclusively through its glycosaminoglycan chains, syndecan-4 binding relies on both its core protein and its heparan sulfate chains. These results suggest that LG45 may trigger different signals toward keratinocytes depending on its interaction with syndecan-1 or -4.

Matrix Metalloproteinase-9 Regulates Survival of Neurons in Newborn Hippocampus

The number of neurons in the adult rodent brain is strongly influenced by events in early postnatal life that eliminate approximately half of the neurons. Recently, we reported that neurotrophins induced survival of neonatal rat hippocampal neurons by promoting neural activity and activation of the Ser/Thr kinase, Akt. The survival of neurons also depended on integrin signaling, but a role for the extracellular matrix (ECM) in this mechanism was yet to be explored. Here, we show that levels of the matrix metalloproteinase-9 (MMP9) decrease, and the level of the ECM protein laminin increases in rat hippocampus during the period of neuronal death. Hippocampi from MMP9 null mice showed higher levels of laminin expression than wild type at P1 and no further increase at P10. In vitro, the matrix metalloproteinase inhibitor FN-439 promoted survival of neurons in a laminin-integrin β1-dependent manner. Blocking laminin signaling attenuated activation of Akt by depolarization. In vivo, injecting FN-439 into the neonatal hippocampus increased the level of laminin and promoted neuronal survival through an integrin-dependent mechanism. These results show signals from the ECM are not simply permissive but rather actively regulated, and they interact with neuronal activity to control the number of hippocampal neurons. This work is the first to report a role for MMP9 in regulating neuronal survival through the developmental process that establishes the functional brain.

Transforming Growth Factor Beta-Induced (TGFBI) Is an Anti-Adhesive Protein Regulating the Invasive Growth of Melanoma Cells

Melanoma is a malignancy characterized by high invasive/metastatic potential, with no efficient therapy after metastasis. Understanding the molecular mechanisms underlying the invasive/metastatic tendency is therefore important. Our genome-wide gene expression analyses revealed that human melanoma cell lines WM793 and especially WM239 (vertical growth phase and metastatic cells, respectively) overexpress the extracellular matrix (ECM) protein transforming growth factor β induced (TGFBI). In adhesion assays, recombinant TGFBI was strongly anti-adhesive for both melanoma cells and skin fibroblasts. TGFBI further impaired the adhesion of melanoma cells to the adhesive ECM proteins fibronectin, collagen-I, and laminin, known to interact with it. Unexpectedly, WM239 cells migrated/invaded more effectively in three-dimensional collagen-I and Matrigel cultures after knockdown of TGFBI by shRNA expression. However, in the physiological subcutaneous microenvironment in nude mice, after TGFBI knockdown, these cells showed markedly impaired tumor growth and invasive capability; the initially formed small tumors later underwent myxoid degeneration and completely regressed. By contrast, the expanding control tumors showed intense TGFBI staining at the tumor edges, co-localizing with the fibrillar fibronectin/tenascin-C/periostin structures that characteristically surround melanoma cells at invasion fronts. Furthermore, TGFBI was found in similar fibrillar structures in clinical human melanoma metastases as well, co-localizing with fibronectin. These data imply an important role for TGFBI in the ECM deposition and invasive growth of melanoma cells, rendering TGFBI a potential target for therapeutic interventions.

Transdifferentiation of hepatic oval cells into pancreatic islet b-cells

Insulin production by beta-cells derived from hepatic oval cells is a promising new approach for the treatment of diabetes. Hepatic oval cells can be redirected to the beta-cell linage by an appropriate combination of high extracellular glucose, specific extracellular matrix proteins (laminin and fibronectin), cytokines (activin A), and the expression of several differentiation-related transcription factors (Pdx-1, Ngn-3, MafA). We explore the process of hepatic oval cell transdifferentiation into pancreatic islet beta-cells and the cellular signaling pathways involved.

Engineering pro-angiogenic peptides using stable, disulfide-rich cyclic scaffolds

AbstractFragments from the extracellular matrix proteins laminin and osteopontin and a sequence from VEGF have potent proangiogenic activity despite their small size (< 10 residues). However, these linear peptides have limited potential as drug candidates for therapeutic angiogenesis because of their poor stability. In the present study, we show that the therapeutic potential of these peptides can be significantly improved by “grafting” them into cyclic peptide scaffolds. Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II) and sunflower trypsin inhibitor-1 (SFTI-1), naturally occurring, plant-derived cyclic peptides of 34 and 14 residues, respectively, were used as scaffolds in this study. Using this approach, we have designed a peptide that, in contrast to the small peptide fragments, is stable in human serum and at nanomolar concentration induces angiogenesis in vivo. This is the first report of using these scaffolds to improve the activity and stability of angiogenic peptide sequences and is a promising approach for promoting angiogenesis for therapeutic uses.

Biocompatibility and Structural Stability of a Laminin Biopolymer

Polylaminin (polyLM) is a polymerized form of the extracellular matrix protein laminin obtained upon pH acidification. Here microscopy and spectroscopic tools are used to study the cell compatibility and the structural stability of polyLM, aiming at establishing its robustness as a biopolymer for therapeutic use. PolyLM is cell compatible as judged by the efficiency of attachment and neuritogenesis. It is resistant to low temperature. Addition of urea or an increase in hydrostatic pressure leads to polymer disassembly. PolyLM biofilms remain stable for 48 h in contact with cell culture medium. The sedimented polymer recovered after centrifugation and adsorbed on a glass coverslip preserved its original structure and its biological properties.

A novel method for obtaining semi-thin cross sections of the Drosophila heart and their labeling with multiple antibodies

The Drosophila heart has become an exciting model for elucidating the molecular basis for cardiac function in higher organisms. To complement the genetic approaches that have recently identified an array of genes essential for cardiac function, we developed a method to obtain optimal semi-thin cross sections of embryonic, larval, and adult fly hearts in a desired orientation. A procedure for fluorescent labeling of these sections with multiple markers has also been developed, allowing the detection of proteins at high subcellular resolution. Sections obtained by our method reveal changes in cell shape between embryonic heart and aorta cardioblasts and elucidate the morphology of the adult heart. Analysis of the adult heart reveals the precise cardiac tube morphology, differential distribution of the extracellular matrix protein Laminin within the cardiac tube, as well as individual hand-positive, and Held Out Wings (HOW)-positive luminal cells that might represent blood cells.In summary, our method enables visualization of cross sections of the embryonic and adult hearts at high resolution while maintaining the ability to co-label the sections with multiple markers, thereby facilitating the analysis of cardiac tube formation and maintenance at different developmental stages.

IgA1 immune complexes from pediatric patients with IgA nephropathy activate cultured human mesangial cells

Circulating immune complexes (CIC) containing galactose (Gal)-deficient IgA1 from adults with IgA nephropathy (IgAN) induce proliferation of cultured mesangial cells, but activities of CIC from pediatric patients with the disease have not been studied. CIC of different sizes were isolated from sera of pediatric and adult IgAN patients and their effects on cultured human mesangial cells (MC) were assessed by measuring cellular proliferation, expression of IL-6 and IL-8 and laminin and phosphotyrosine signaling. Large CIC from pediatric IgAN patients (>800 kDa) containing Gal-deficient IgA1 stimulated cellular proliferation, whereas in some patients, smaller CIC were inhibitory. Addition of stimulatory and inhibitory CIC to MC differentially altered phosphorylation patterns of three major tyrosine-phosphorylated proteins of molecular mass 37, 60 and 115 kDa. The stimulatory CIC transiently increased tyrosine-phosphorylation of the 37-kDa protein and decreased phosphorylation of the other two proteins, whereas the inhibitory CIC increased phosphorylation of all three proteins. Furthermore, we investigated the influence of IgA1-containing CIC from sera of children with IgAN with clinically active disease (i.e., abnormal urinalysis and/or serum creatinine concentration) or inactive disease (i.e., normal urinalysis and serum creatinine concentration) on the expression of IL-6 and IL-8 genes by mesangial cells. Real-time reverse transcription-polymerase chain reaction results showed that the CIC from a patient with active disease stimulated MC to express the two cytokine genes at higher levels than did the CIC from a patient with inactive disease. Moreover, stimulatory CIC increased production of the extracellular matrix protein laminin. These data indicate that sera of pediatric IgAN patients contain biologically active CIC with Gal-deficient IgA1.

The N‐terminal part of Als1 protein from Candida albicans specifically binds fucose‐containing glycans

The opportunistic pathogen Candida albicans expresses on its surface Als (Agglutinin like sequence) proteins, which play an important role in the adhesion to host cells and in the development of candidiasis. The binding specificity of these proteins is broad, as they can bind to various mammalian proteins, such as extracellular matrix proteins, and N- and E-cadherins. The N-terminal part of Als proteins constitutes the substrate-specific binding domain and is responsible for attachment to epithelial and endothelial cells. We have used glycan array screening to identify possible glycan receptors for the binding domain of Als1p-N. Under those conditions, Als1p-N binds specifically to fucose-containing glycans, which adds a lectin function to the functional diversity of the Als1 protein. The binding between Als1p-N and BSA-fucose glycoconjugate was quantitatively characterized using surface plasmon resonance, which demonstrated a weak millimolar affinity between Als1p-N and fucose. Furthermore, we have also quantified the affinity of Als1p-N to the extracellular matrix proteins proteins fibronectin and laminin, which is situated in the micromolar range. Surface plasmon resonance characterization of Als1p-N-Als1p-N interaction was in the micromolar affinity range.

Mutant p53 Disrupts MCF-10A Cell Polarity in Three-dimensional Culture via Epithelial-to-mesenchymal Transitions

Mutant p53 is not only deficient in tumor suppression but also acquires additional activity, called gain of function. Mutant p53 gain of function is recapitulated in knock-in mice that carry one null allele and one mutant allele of the p53 gene. These knock-in mice develop aggressive tumors compared with p53-null mice. Recently, we and others showed that tumor cells carrying a mutant p53 are addicted to the mutant for cell survival and resistance to DNA damage. To further define mutant p53 gain of function, we used the MCF-10A three-dimensional model of mammary morphogenesis. MCF-10A cells in three-dimensional culture undergo a series of morphological changes and form polarized and growth-arrested spheroids with hollow lumen, which resembles normal glandular architectures in vivo. Here, we found that endogenous wild-type p53 in MCF-10A cells was not required for acinus formation, but knockdown of endogenous wild-type p53 (p53-KD) led to partial clearance of cells in the lumen due to decreased apoptosis. Consistent with this, p53-KD altered expression patterns of the cell adhesion molecule E-cadherin, the cytoskeletal marker β-catenin, and the extracellular matrix protein laminin V. We also found that ectopic expression of the mutant G245S led to a phenotype similar to p53-KD, whereas a combination of ectopic expression of siRNA-resistant G245S with p53-KD led to a less cleared lumen. In contrast, ectopic expression of mutant R248W, R175H, and R273H disrupted normal acinus architectures with filled lumen and led to formation of irregular and multiacinus structures regardless of p53-KD. In addition, these mutants altered normal expression patterns and/or levels of E-cadherin, β-catenin, laminin V, and tight junction marker ZO-1. Furthermore, epithelial-to-mesenchymal transitions (EMT) markers, Snail, Slug, and Twist, were highly induced by mutant p53 and/or p53-KD. Together, we postulate that EMT represents a mutant p53 gain of function and mutant p53 alters cell polarity via EMT.

The in vivo performance of an enzyme-assisted self-assembled peptide/protein hydrogel

We demonstrate the distribution of the important extracellular matrix protein laminin in a novel biomaterial consisting of a hydrogel underpinned by nanofibrillar networks. These are formed by the immobilised enzyme mediated self-assembly of fmoc-L 3 (9-fluorenylmethoxycarbonyl-tri-leucine). The peptide assembly yields nanofibrils formed of β-sheets that are locked together via π-stacking interactions. This ordering allows the localisation of the peptide sidechains on the surface, creating a hydrophobic environment. This induces the formation of bundles of these nanofibrils producing a clear hydrogel. This mechanism enables the three dimensional distribution of laminin throughout the network via supramolecular interactions. These forces favour the formation and improve the order of the network itself, as observed by spectroscopic and mechanical testing. In order to test the stability and suitability of this class of material for in vivo applications, we utilise microinjection to deliver the biomaterial under fine spatial control into a dystrophic zebrafish model organism, which lacks laminin as a result of a genetic mutation. Using confocal and transmission electron microscopy, we confirm that the biomaterial remains stable structurally, and is confined spatially to the site of injection.

Abstract 1527: Hepatocyte growth factor modulates adhesion of ovarian cancer cells to the peritoneum mesothelium through p70 S6 kinase

Abstract Ovarian cancer has the highest mortality of all gynecological cancers. These deaths are mainly due to the highly metastatic characteristic with already widespread peritoneal dissemination and malignant ascites at the time of diagnosis. However the molecular mechanisms that regulate ovarian cancer progression are poorly understood. A key step in this process is the adhesion of ovarian cancer cells to the peritoneal mesothelium. Hepatocyte growth factor (HGF) is present at high concentrations in ovarian cancer ascitic fluid. We show here a role for HGF signaling in the regulation of the peritoneal adhesion of ovarian cancer cells. HGF increased binding of ovarian cancer cells to peritoneal extracellular matrix (ECM) proteins fibronectin and laminin and their receptors α5, α6, and β1 integrin expression. Blockade of HGF resulted in reduced attachment of ovarian cancer cells to these ECM proteins, with a concomitant reduction of α5, α6, and β1 integrin expression, confirming that the effect was HGF specific. Similarly, the peritoneal ECM adhesion could be reversed by neutralizing antibodies to α5, α6, and β1 integrin. The adhesive phenotype and integrin expression activated by HGF could be blocked by specific inhibition of p70 S6 kinase (p70S6K) using short hairpin RNA or small molecule inhibitor, suggesting that HGF transmits the signal through p70S6K. Importantly, it also inhibited adhesion to primary human peritoneal mesothelial cells. These results identify a potential novel mechanism for HGF-induced peritoneal adhesion, which is mediated through p70S6K through regulation of α5, α6, and β1 integrin (This work is supported by the Hong Kong Research Grants Council Grant HKU7599/05M to A.S.T.W.). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1527. doi:10.1158/1538-7445.AM2011-1527

Molecular and Functional Characterization of Mouse S5D-SRCRB: A New Group B Member of the Scavenger Receptor Cysteine-Rich Superfamily

The scavenger receptor cysteine-rich superfamily (SRCR-SF) members are transmembrane and/or secreted receptors exhibiting one or several repeats of a cysteine-rich protein module of ∼100 aa, named scavenger receptor cysteine-rich (SRCR). Two types of SRCR domains (A or B) have been reported, which differ in the number of coding exons and intradomain cysteines. Although no unifying function has been reported for SRCR-SF members, recognition of pathogen-associated molecular patterns (PAMPs) was recently shown for some of them. In this article, we report the structural and functional characterization of mouse S5D-SRCRB, a new group B member of the SRCR-SF. The s5d-srcrb gene maps at mouse chromosome 7 and encompasses 14 exons extending over 15 kb. The longest cDNA sequence found is 4286 bp in length and encodes a mature protein of 1371 aa, with a predicted M(r) of 144.6 kDa. Using an episomal mammalian-expression system, a glycosylated soluble recombinant form >200 kDa was obtained and used as immunogen for the generation of specific rat mAbs. Subsequent immunohistochemical and real-time PCR analysis showed significant S5D-SRCRB expression in murine genitourinary and digestive tracts. S5D-SRCRB was shown to bind endogenous extracellular matrix proteins (laminin and galectin-1), as well as PAMPs present on Gram-positive and Gram-negative bacteria and fungi. PAMP binding by S5D-SRCRB induced microbial aggregation and subsequent inhibition of PAMP-induced cytokine release. These abilities suggest that S5D-SRCRB might play a role in the innate defense and homeostasis of certain specialized epithelial surfaces.

Two Distinct Filopodia Populations at the Growth Cone Allow to Sense Nanotopographical Extracellular Matrix Cues to Guide Neurite Outgrowth

BackgroundThe process of neurite outgrowth is the initial step in producing the neuronal processes that wire the brain. Current models about neurite outgrowth have been derived from classic two-dimensional (2D) cell culture systems, which do not recapitulate the topographical cues that are present in the extracellular matrix (ECM) in vivo. Here, we explore how ECM nanotopography influences neurite outgrowth.Methodology/Principal FindingsWe show that, when the ECM protein laminin is presented on a line pattern with nanometric size features, it leads to orientation of neurite outgrowth along the line pattern. This is also coupled with a robust increase in neurite length. The sensing mechanism that allows neurite orientation occurs through a highly stereotypical growth cone behavior involving two filopodia populations. Non-aligned filopodia on the distal part of the growth cone scan the pattern in a lateral back and forth motion and are highly unstable. Filopodia at the growth cone tip align with the line substrate, are stabilized by an F-actin rich cytoskeleton and enable steady neurite extension. This stabilization event most likely occurs by integration of signals emanating from non-aligned and aligned filopodia which sense different extent of adhesion surface on the line pattern. In contrast, on the 2D substrate only unstable filopodia are observed at the growth cone, leading to frequent neurite collapse events and less efficient outgrowth.Conclusions/SignificanceWe propose that a constant crosstalk between both filopodia populations allows stochastic sensing of nanotopographical ECM cues, leading to oriented and steady neurite outgrowth. Our work provides insight in how neuronal growth cones can sense geometric ECM cues. This has not been accessible previously using routine 2D culture systems.

Surface-modified nanofibrous biomaterial bridge for the enhancement and control of neurite outgrowth

Biomaterial bridges constructed from electrospun fibers offer a promising alternative to traditional nerve tissue regeneration substrates. Aligned and unaligned polycaprolactone (PCL) electrospun fibers were prepared and functionalized with the extracellular matrix proteins collagen and laminin using covalent and physical adsorption attachment chemistries. The effect of the protein modified and native PCL nanofiber scaffolds on cell proliferation, neurite outgrowth rate, and orientation was examined with neuronlike PC12 cells. All protein modified scaffolds showed enhanced cellular adhesion and neurite outgrowth compared to unmodified PCL scaffolds. Neurite orientation was found to be in near perfect alignment with the fiber axis for cells grown on aligned fibers, with difference angles of less than 7° from the fiber axis, regardless of the surface chemistry. The bioavailability of PCL fibers with covalently attached laminin was found to be identical to that of PCL fibers with physically adsorbed laminin, indicating that the covalent chemistry did not change the protein conformation into a less active form and the covalent attachment of protein is a suitable method for enhancing the biocompatibility of tissue engineering scaffolds.

Dystroglycan modulates the ability of insulin‐like growth factor‐1 to promote oligodendrocyte differentiation

The adhesion receptor dystroglycan positively regulates terminal differentiation of oligodendrocytes, but the mechanism by which this occurs remains unclear. Using primary oligodendrocyte cultures, we identified and examined a connection between dystroglycan and the ability of insulin-like growth factor-1 (IGF-1) to promote oligodendrocyte differentiation. Consistent with previous reports, treatment with exogenous IGF-1 caused an increase in MBP protein that was preceded by activation of PI3K (AKT) and MAPK (ERK) signaling pathways. The extracellular matrix protein laminin was further shown to potentiate the effect of IGF-1 on oligodendrocyte differentiation. Depletion of the laminin receptor dystroglycan using siRNA, however, blocked the ability of IGF-1 to promote oligodendrocyte differentiation of cells grown on laminin, suggesting a role for dystroglycan in IGF-1-mediated differentiation. Indeed, loss of dystroglycan led to a reduction in the ability of IGF-1 to activate MAPK, but not PI3K, signaling pathways. Pharmacological inhibition of MAPK signaling also prevented IGF-1-induced increases in myelin basic protein (MBP), indicating that MAPK signaling was necessary to drive IGF-1-mediated enhancement of oligodendrocyte differentiation. Using immunoprecipitation, we found that dystroglycan, the adaptor protein Grb2, and insulin receptor substrate-1 (IRS-1), were associated in a protein complex. Taken together, our results suggest that the positive regulatory effect of laminin on oligodendrocyte differentiation may be attributed, at least in part, to dystroglycan's ability to promote IGF-1-induced differentiation.

Changes of glycoprotein and collagen immunolocalization in the uterine artery wall of postmenopausal women with and without pelvic organ prolapse

Pelvic organ prolapse (POP) is accompanied by an altered composition of the extracellular matrix (ECM). However, it is unclear whether the changed ECM is the cause or the consequence of POP, as stretching of the tissue may have an effect on the composition of the ECM. To address this question, we analyzed the connective tissues of the uterine artery wall of postmenopausal women with and without POP. The uterine artery wall is stretched in patients with POP, but this stretching is unlikely to cause the POP. Twenty-one women (13 with POP and 8 without POP) hospitalized for hysterectomy were included in this study. Tissue samples from the uterine artery were analyzed for collagen (types I, III, IV, V and VI) and other ECM proteins (fibronectin, laminin, tenascin, vitronectin and elastin) using immunofluorescence microscopy. Results revealed that uterine artery samples of women with prolapse showed a significantly weaker immunoreactivity to type VI collagen, vitronectin and elastin and a stronger immunostaining for type III collagen and tenascin as compared to control samples.Our results suggest that the ECM may be altered in response to mechanical stretch. Changes in the ECM composition as observed in POP may not necessarily be the reason for the development of pelvic floor relaxation in postmenopausal women.

Dip‐pen nanolithography on SiOx and tissue‐derived substrates: comparison with multiple biological inks

There has been extensive interest in the micro and nanoscale manipulation of various substrates in the past few decades. One promising technique is dip-pen nanolithography which has shown the capability to pattern substrates of all forms including, tissue-derived substrates. Patterning of tissue-derived substrates is of particular interest, as it would facilitate studies into controlling cell morphology and cell-substrate interaction. To expand the field into this area both peptides and bioactive collagen-binding peptide-linked biomolecules were patterned to the inner collagenous zone of the Bruch's membrane (BM). Collagen-binding peptide, and extra cellular matrix (ECM) proteins laminin and fibronectin were patterned on the BM and SiO(x). The lithographic protocol was facilitated by Triton X-100 which was used to clean the tissue-derived construct after harvesting. This produced a collagen-exposed BM which was more hydrophilic (contact angle 67 degrees +/-8.49 degrees) surface compared with other cleaning methods but it maintained similar surface roughness (root-mean-square) 80+/-18 nm and collagen exposure. This type of surface can be readily patterned with the chosen inks under lower humidity conditions.

Laminin enhances β2‐adrenergic receptor stimulation of L‐type Ca2+ current via cytosolic phospholipase A2 signalling in cat atrial myocytes

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.

Effect of thyroid hormone depletion on cultured murine cerebral cortex astrocytes

Astrocytes play a crucial role in several steps of brain development, such as the proliferation of neural precursors, neuronal migration and differentiation, axonal growth, and synaptogenesis. Astrocyte generation and maturation is dramatically modulated by thyroid hormones (THs). Here, we propose a modified model for studying THs action on astroglial cells, in vitro. We investigated the effect of depletion of THs from fetal bovine serum (FBS) on the expression of the astrocyte maturation markers, GFAP (glial fibrillary acidic protein) and S100β, and the extracellular matrix (ECM) proteins laminin and fibronectin in cultured astrocytes. To accomplish this, murine cortical astrocytes were cultured in medium supplemented with THs-depleted serum, in contrast to the traditional techniques that use normal FBS which contains considerable amounts of THs. Immunostaining revealed that depletion of THs from FBS did not affect astrocyte proliferation, as observed by the number of astrocytes labeled for the proliferation antigen, Ki67. Surprisingly, western blot and RT-PCR assays revealed decreased levels of GFAP and S100β in astrocytes cultured with depleted serum. These events were reversed by addition of THs to the medium. Immunostaining and western blot assays did not reveal any difference in the organization and synthesis of the ECM protein, laminin; whereas the levels of fibronectin were decreased by 50% in THs-depleted serum. The fact that decreased expression of GFAP and fibronectin is associated with hypothyroidism in vivo suggests that our work might provide a useful model to assess in vitro the molecular mechanism underlying astrocytic maturation under conditions of THs deficiency.