Biomedical subjects

Liliane Robillard

Publications and source records attributed to Liliane Robillard.

7 recordsLinked to original sources

PubMed2006

Urokinase receptors are required for alpha 5 beta 1 integrin-mediated signaling in tumor cells.

Up-regulation of urokinase receptors is common during tumor progression and thought to promote invasion and metastasis. Urokinase receptors bind urokinase and a set of beta1 integrins, but it remains unclear to what degree urokinase receptor/integrin binding is important to beta1 integrin signaling. Using site-directed mutagenesis, single amino acid mutants of the urokinase receptor were identified that fail to associate with either alpha3beta1 (D262A) or alpha5beta1 (H249A) but associate normally with urokinase. To study the effects of these mutations on beta1 integrin function, endogenous urokinase receptors were first stably silenced in tumor cell lines HT1080 and H1299, and then wild type or mutant receptors were expressed. Knockdown of urokinase receptors resulted in markedly reduced fibronectin and alpha5beta1-dependent ERK activation and metalloproteinase MMP-9 expression. Re-expression of wild type or D262A mutant receptors but not the alpha5beta1 binding-deficient H249A mutant reconstituted fibronectin responses. Because urokinase receptor.alpha5beta1 complexes bind in the fibronectin heparin-binding domain (Type III 12-14) whereas alpha5beta1 primarily binds in the RGD-containing domain (Type III 7-10), signaling pathways leading to ERK and MMP-9 responses were dissected. Binding to III 7-10 led to Src/focal adhesion kinase activation, whereas binding to III 7-14 caused Rac 1 activation. Tumor cells engaging fibronectin required both Type III 7-10- and 12-14-initiated signals to activate ERK and up-regulate MMP-9. Thus urokinase receptor binding to alpha5beta1 is required for maximal responses to fibronectin and tumor cell invasion, and this operates through an enhanced Src/Rac/ERK signaling pathway.

Cell Line, Tumor↗

PubMed2006

Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix.

Mechanisms leading to fibroblast accumulation during pulmonary fibrogenesis remain unclear. Although there is in vitro evidence of lung alveolar epithelial-to-mesenchymal transition (EMT), whether EMT occurs within the lung is currently unknown. Biopsies from fibrotic human lungs demonstrate epithelial cells with mesenchymal features, suggesting EMT. To more definitively test the capacity of alveolar epithelial cells for EMT, mice expressing beta-galactosidase (beta-gal) exclusively in lung epithelial cells were generated, and their fates were followed in an established model of pulmonary fibrosis, overexpression of active TGF-beta1. beta-gal-positive cells expressing mesenchymal markers accumulated within 3 weeks of in vivo TGF-beta1 expression. The increase in vimentin-positive cells within injured lungs was nearly all beta-gal-positive, indicating epithelial cells as the main source of mesenchymal expansion in this model. Ex vivo, primary alveolar epithelial cells cultured on provisional matrix components, fibronectin or fibrin, undergo robust EMT via integrin-dependent activation of endogenous latent TGF-beta1. In contrast, primary cells cultured on laminin/collagen mixtures do not activate the TGF-beta1 pathway and, if exposed to active TGF-beta1, undergo apoptosis rather than EMT. These data reveal alveolar epithelial cells as progenitors for fibroblasts in vivo and implicate the provisional extracellular matrix as a key regulator of epithelial transdifferentiation during fibrogenesis.

Animals↗

PubMed2006

Murine cathepsin F deficiency causes neuronal lipofuscinosis and late-onset neurological disease.

Cathepsin F (cat F) is a widely expressed lysosomal cysteine protease whose in vivo role is unknown. To address this issue, mice deficient in cat F were generated via homologous recombination. Although cat F-/- mice appeared healthy and reproduced normally, they developed progressive hind leg weakness and decline in motor coordination at 12 to 16 months of age, followed by significant weight loss and death within 6 months. cat F was found to be expressed throughout the central nervous system (CNS). cat F-/- neurons accumulated eosinophilic granules that had features typical of lysosomal lipofuscin by electron microscopy. Large amounts of autofluorescent lipofuscin, characteristic of the neurodegenerative disease neuronal ceroid lipofuscinosis (NCL), accumulated throughout the CNS but not in visceral organs, beginning as early as 6 weeks of age. Pronounced gliosis, an indicator of neuronal stress and neurodegeneration, was also apparent in older cat F-/- mice. cat F is the only cysteine cathepsin whose inactivation alone causes a lysosomal storage defect and progressive neurological features in mice. The late onset suggests that this gene may be a candidate for adult-onset NCL.

Age Factors↗

PubMed2005

Regulation of alpha5beta1 integrin conformation and function by urokinase receptor binding.

Urokinase-type plasminogen activator receptors (uPARs), up-regulated during tumor progression, associate with beta1 integrins, localizing urokinase to sites of cell attachment. Binding of uPAR to the beta-propeller of alpha3beta1 empowers vitronectin adhesion by this integrin. How uPAR modifies other beta1 integrins remains unknown. Using recombinant proteins, we found uPAR directly binds alpha5beta1 and rather than blocking, renders fibronectin (Fn) binding by alpha5beta1 Arg-Gly-Asp (RGD) resistant. This resulted from RGD-independent binding of alpha5beta1-uPAR to Fn type III repeats 12-15 in addition to type III repeats 9-11 bound by alpha5beta1. Suppression of endogenous uPAR by small interfering RNA in tumor cells promoted weaker, RGD-sensitive Fn adhesion and altered overall alpha5beta1 conformation. A beta1 peptide (res 224NLDSPEGGF232) that models near the known alpha-chain uPAR-binding region, or a beta1-chain Ser227Ala point mutation, abrogated effects of uPAR on alpha5beta1. Direct binding and regulation of alpha5beta1 by uPAR implies a modified "bent" integrin conformation can function in an alternative activation state with this and possibly other cis-acting membrane ligands.

Amino Acid Sequence↗

PubMed2002

Growth hormone-induced diacylglycerol and ceramide formation via Galpha i3 and Gbeta gamma in GH4 pituitary cells. Potentiation by dopamine-D2 receptor activation.

Growth hormone (GH) secretion is regulated by indirect negative feedback mechanisms. To address whether GH has direct actions on pituitary cells, lipid signaling in GH(4)ZR(7) somatomammotroph cells was examined. GH (EC(50) = 5 nm) stimulated diacylglycerol (DAG) and ceramide formation in parallel by over 10-fold within 15 min and persisting for >3 h. GH-induced DAG/ceramide formation was blocked by pertussis toxin (PTX) implicating G(i)/G(o) proteins and was potentiated 1.5-fold by activation of G(i)/G(o)-coupled dopamine-D2S receptors, which had no effect alone. Following PTX pretreatment, only PTX-resistant Galpha(i)3, not Galpha(o) or Galpha(i)2, rescued GH-induced DAG/ceramide signaling. GH-induced DAG/ceramide formation was also blocked in cells expressing Gbetagamma blocker GRK-ct. In GH(4)ZR(7) cells, GH induced phosphorylation of JAK2 and STAT5, which was blocked by PTX and mimicked by ceramide analogue C2-ceramide or sphingomyelinase treatment to increase endogenous ceramide. We conclude that in GH(4) pituitary cells, GH induces formation of DAG/ceramide via a novel Galpha(i)3/Gbetagamma-dependent pathway. This novel pathway suggests a mechanism for autocrine feedback regulation by GH of pituitary function.

Apomorphine↗

PubMed2002

G protein specificity: traffic direction required.

This review focuses on the coupling specificity of the Galpha and Gbetagamma subunits of pertussis toxin (PTX)-sensitive G(i/o) proteins that mediate diverse signaling pathways, including regulation of ion channels and other effectors. Several lines of evidence indicate that specific combinations of G protein alpha, beta and gamma subunits are required for different receptors or receptor-effector networks, and that a higher degree of specificity for Galpha and Gbetagamma is observed in intact systems than reported in vitro. The structural determinants of receptor-G protein specificity remain incompletely understood, and involve receptor-G protein interaction domains, and perhaps other scaffolding processes. By identifying G protein specificity for individual receptor signaling pathways, ligands targeted to disrupt individual pathways of a given receptor could be developed.

Adenylyl Cyclases↗

PubMed2001

Gbeta residues that do not interact with Galpha underlie agonist-independent activity of K+ channels.

Gbetagamma subunits interact directly and activate G protein-gated Inwardly Rectifying K(+) (GIRK) channels. Little is known about the identity of functionally important interactions between Gbetagamma and GIRK channels. We tested the effects of all mammalian Gbeta subunits on channel activity and showed that whereas Gbeta1-4 subunits activate heteromeric GIRK channels independently of receptor activation, Gbeta5 does not. Gbeta1 and Gbeta5 both bind the N and C termini of the GIRK1 and GIRK4 channel subunits. Chimeric analysis between the Gbeta1 and Gbeta5 proteins revealed a 90-amino acid stretch that spans blades two and three of the seven-propeller structure and is required for channel activation. Within this region, eight non-conserved amino acids were critical for the activity of Gbeta1, as mutation of each residue to its counterpart in Gbeta5 significantly reduced the ability of Gbeta1 to stimulate channel activity. In particular, mutation of residues Ser-67 and Thr-128 to the corresponding Gbeta5 residues completely abolished Gbeta1 stimulation of GIRK channel activity. Mapping these functionally important residues on the three-dimensional structure of Gbeta1 shows that Ser-67, Ser-98, and Thr-128 are the only surface accessible residues. Galpha(i)1 interacts with Ser-98 but not with Ser-67 and Thr-128 in the heterotrimeric Galphabetagamma structure. Further characterization of the three mutant proteins showed that they fold properly and interact with Ggamma2. Of the three identified functionally important residues, the Ser-67 and Thr-128 Gbeta mutants significantly inhibited basal currents of a channel point mutant that displays Gbetagamma-mediated basal but not agonist-induced currents. Our findings indicate that the presence of Gbeta residues that do not interact with Galpha are involved in Gbetagamma interactions in the absence of agonist stimulation.

Amino Acid Sequence↗