Ellen Van Obberghen-Schilling

cDNA cloning and expression of a hamster α‐thrombin receptor coupled to Ca2+ mobilization

The serine protease α‐thrombin (thrombin) potently stimulates G‐protein‐coupled signaling pathways and DNA synthesis in CCL39 hamster lung fibroblasts. To clone a thrombin receptor cDNA, selective amplification of mRNA sequences displaying homology to the transmembrane domains of G‐protein‐coupled receptor genes was performed by polymerase chain reaction. Using reverse transcribed poly(A)+ RNA from CCL39 cells and degenerate primers corresponding to conserved regions of several phospholipase C‐coupled receptors, three novel putative receptor sequences were identified. One corresponds to an mRNA transcript of 3.4 kb in CCL39 cells and a relatively abundant cDNA. Microinjection of RNA transcribed in vitro from this cDNA in Xenopus oocytes leads to the expression of a functional thrombin receptor. The hamster thrombin receptor consists of 427 amino acid residues with 8 hydrophobic domains, including one at the extreme N‐terminus that is likely to represent a signal peptide. A thrombin consensus cleavage site is present in the N‐terminal extracellular region of the receptor sequence followed by a negatively charged cluster of residues present in a number of proteins that interact with the anion‐binding exosite of thrombin.

Transforming growth factor beta: biochemistry and roles in embryogenesis, tissue repair and remodeling, and carcinogenesis.

Transforming growth factor (TGF)- β plays essential roles in embryogenesis, particularly during periods of morphogenesis. Some of the same embryological mechanisms are reiterated in the adult during the normal processes of tissue remodeling and repair and aberrantly in various pathological processes, including carcinogenesis. This chapter highlights the new advances in the understanding of the complex biology of TGF- β and discusses the chemistry of TGF- β . The broad range of biological activities of TGF- β makes it highly likely that other peptide activities—purified by presumably novel and specific assays—will result from TGF- β once their amino acid sequence is determined. TGF- β 1 and 2 are two homologous forms of a homodimeric peptide with molecular weight of 25,000. Every chain of the peptide contains 112 amino acids of which nine are cysteine residues. The chapter reviews the structure of TGF- β 1 and 2 and TGF- β gene family. The biological activities of the members of the TGF- β family are described in the chapter. The chapter further reviews the regulation of gene activity by TGF- β, antibodies to TGF- β , and role of TGF- β in embryogenesis, tissue repair and remodeling, and carcinogenesis and other proliferative diseases.

Transforming Growth Factor β Induces Rosettes of Podosomes in Primary Aortic Endothelial Cells

ABSTRACT Cytoskeletal rearrangements are central to endothelial cell physiology and are controlled by soluble factors, matrix proteins, cell-cell interactions, and mechanical forces. We previously reported that aortic endothelial cells can rearrange their cytoskeletons into complex actin-based structures called podosomes when a constitutively active mutant of Cdc42 is expressed. We now report that transforming growth factor beta (TGF-β) promotes podosome formation in primary aortic endothelial cells. TGF-β-induced podosomes assembled together into large ring- or crescent-shaped structures. Their formation was dependent on protein synthesis and required functional Src, phosphatidylinositide 3-kinase, Cdc42, RhoA, and Smad signaling. MT1-MMP and metalloprotease 9 (MMP9), both upregulated by TGF-β, were detected at sites of podosome formation, and MT1-MMP was found to be involved in the local degradation of extracellular matrix proteins beneath the podosomes and required for the invasion of collagen gels by endothelial cells. We propose that TGF-β plays an important role in endothelial cell physiology by inducing the formation of podosomal structures endowed with metalloprotease activity that may contribute to arterial remodeling.

ILK is required for the assembly of matrix-forming adhesions and capillary morphogenesis in endothelial cells

Integrins play a key role in regulating endothelial cell survival, migration and differentiated function during angiogenic blood-vessel remodeling. Integrin-linked kinase (ILK) is a multidomain protein that interacts with the cytoplasmic tail of integrin β subunits and is thought to participate in integrin-mediated signal transduction. We report here that attenuation of ILK expression in cultured bovine aortic endothelial cells by RNA interference had marked effects on surface distribution of α5β1 integrin and the organization of cell-matrix adhesions characterized by the disappearance of fibrillar (3D-like) adhesions that are rich in α5β1 and paxillin, and associated fibrillar fibronectin matrix. This defect was not caused by a decrease in fibronectin mRNA levels or by intracellular retention of the protein. Adhesion to surface-adsorbed matrix proteins based on β1 and β3 integrin was enhanced following ILK depletion, whereas cell spreading, migration and multilayer alignment into capillary-like structures on Matrigel were impaired. We conclude that ILK is an important regulator of the endothelial phenotype and vascular network formation by directing the assembly and/or maturation of α5β1-competent matrix-forming adhesions.

Fibronectin and tenascin-C: accomplices in vascular morphogenesis during development and tumor growth

In addition to soluble factors, the extracellular matrix (ECM) also plays a vital role in normal vasculogenesis and in the pathological angiogenesis of many disease states. Here we will review what is known about the role of the ECM molecules fibronectin and tenascin-C in the vasculature and highlight a potential collaborative interplay between these molecules in developmental and tumorigenic angiogenesis. We will address the evolution of these modular proteins, their cellular interactions and how they become assembled into an insoluble matrix that impacts the assembly of other ECM proteins and the bioavailability of pro-angiogenic factors. The role of fibronectin and tenascin-C networks in tumor angiogenesis and metastasis will be described. We will elaborate on lessons learned about their role in vessel function from the functional ablation or the ectopic expression of both molecules. We will also elaborate on potential mechanisms of how fibronectin and tenascin-C affect cell adhesion and signaling that are relevant to angiogenesis.

Regulation of cell-matrix adhesion dynamics and Rac-1 by integrin linked kinase

Extracellular matrix (ECM) receptors of the integrin family initiate changes in cell shape and motility by recruiting signaling components that coordinate these events. Integrin‐linked kinase (ILK) is one such partner of β1 integrins that participates in dynamic rearrangement of cell‐matrix adhesions and cell spreading by mechanisms that are not well understood. To further elucidate the role of ILK in these events, we engineered a chimeric molecule comprising ILK fused to a membrane‐targeted green fluorescent protein (ILK‐GFP‐F). ILK‐GFP‐F is highly enriched in cell‐matrix adhesions, and its expression in fibroblasts leads to an accumulation of focal adhesions (2–5 µm) and elongated adhesions (>5 µm). ILK‐GFP‐F enhances cell spreading on fibronectin and induces a constitutive increase in the levels of GTP‐bound Rac‐1. Conversely, ILK knock‐down by siRNA transfection decreases active Rac‐1. Endogenous ILK was found to associate with PKL (paxillin kinase linker) and the Rac/Cdc42 guanine nucleotide exchange factor βPIX. Further, expression of a dominant negative βPIX mutant reversed the increase in active Rac‐1 levels of ILK‐GFP‐F‐expressing cells, thus placing βPIX in the pathway leading from ILK to Rac‐1 activation. However, expression of constitutively active Rac only partially restores the spreading defects of ILK‐depleted cells, suggesting that an additional ILK‐dependent signal is required for cell spreading.—Boulter, E., Grall, D., Cagnol, S., and Van Obberghen‐Schilling, E. Regulation of cell‐matrix adhesion dynamics and Rac‐1 by integrin linked kinase. FASEB J. 20, E640‐E651 (2006)

An anchorage-dependent signal distinct from p42/44 MAP kinase activation is required for cell cycle progression

Most normal cells require both mitogens and integrin-mediated attachment for growth. It is generally accepted that the p42/p44 MAP kinase module, which can be activated by both growth factors and adhesion, plays a critical role in G0 to S phase progression of quiescent cells. Studies on various cultured fibroblasts have shown that removal of anchorage leads to cell cycle arrest in G1 and it has been proposed that adhesion-dependent G1 progression requires the joint regulation of p42/p44 MAP kinase by integrins and growth factors. In quiescent CCL39 lung fibroblasts, MAP kinase activation in response to serum becomes compromised when cells are placed in suspension. Under these conditions, serum-stimulated cells arrest their growth in mid-G1 with reduced cyclin D1 expression and increased p21Cip/Waf1 expression, as compared to their attached counterparts. To determine whether a casual link exists between sub-optimal activation of MAP kinase in non-adherent cells and the observed G1 block, we used a variant of CCL39 stably expressing an estrogen-inducible activated-Raf-1 construct (ΔRaf-1:ER). We found that even strong and sustained activation of MAP kinase with estradiol, in addition to serum, is not able to boost cyclin D1 expression levels or stimulate hyperphosphorylation of pRb in suspended CCL39-ΔRaf-1:ER cells. These results indicate that p42/p44 MAP kinase activation is not a limiting factor for G1 to S phase transit in absence of anchorage. Thus, at least one adhesion-mediated signalling event, distinct from MAP kinase activation is required for maximal cyclin D1 induction and hyperphosphorylation of pRb.

Mitogen-potentiating action and binding characteristics of insulin and insulin-like growth factors in Chinese hamster fibroblasts.

alpha-Thrombin alone is able to stimulate DNA synthesis reinitiation of G0-arrested Chinese hamster lung fibroblasts (CC139) as well as continued growth of these cells in serum-free medium. Although insulin at high concentrations (1-10 micrograms/ml) is not intrinsically mitogenic for these cells, it potently enhances the growth-promoting action of thrombin. The generation time of CC139 cells in the defined medium, transferrin, alpha-thrombin, insulin, is around 15 h. To determine whether this effect of insulin is mediated via putative receptors for the insulin-like growth factors (IGFs) on these cells, we examined the abilities of two IGFs, Multiplication-Stimulating Activity (MSA) and IGF-I, to potentiate the thrombin-induced reinitiation of DNA synthesis. Both IGFs were found to be as effective as insulin for this biological effect; however, much lower concentrations were required to elicit half-maximal response, 100 ng/ml of MSA and 30 ng/ml of IGF-I. Detailed binding studies using 125I-labelled insulin, MSA, and IGF-I revealed that CC139 cells specifically bind all three polypeptides with IC50 values for the corresponding ligands of 1-2 ng/ml, 80-100 ng/ml, and 30-40 ng/ml, respectively. 125I-MSA binding was insulin-insensitive, whereas insulin did compete with 125I-IGF-I for binding to CC139 cells. These results indicate that CC139 cells possess at least two types of IGF receptors, an insulin-insensitive IGF receptor with high affinity for MSA which apparently mediates its biological effect, and an insulin-sensitive IGF-I receptor. Insulin appears to exert its mitogen-potentiating activity in CC139 fibroblasts by interacting with the IGF-I receptor.

Autocrine fibronectin directs matrix assembly and crosstalk between cell−matrix and cell−cell adhesion in vascular endothelial cells

Cellular fibronectin (cFN) variants harboring extra FN type 3 repeats, namely extra domains B and A, are major constituents of the extracellular matrix around newly forming blood vessels during development and angiogenesis. Their expression is induced by angiogenic stimuli and their assembly into fibrillar arrays is driven by cell-generated tension at α5β1 integrin-based adhesions. Here, we examined the role and functional redundancy of cFN variants in cultured endothelial cells by isoform-selective RNA interference. We show that FN fibrillogenesis is a cell-autonomous process whereby basally directed secretion and assembly of cellular FN are tightly coupled events that play an important role not only in signaling at cell–matrix adhesions but also at cell–cell contacts. Silencing of cFN variants differentially affects integrin usage, cell spreading, motility and capillary morphogenesis in vitro. cFN-deficient cells undergo a switch from α5β1- to αvβ3-based adhesion, accompanied by a Src-regulated disruption of adherens junctions. These studies identify a crucial role for autocrine FN in subendothelial matrix assembly and junctional integrity that provides spatially and temporally restricted control of endothelial plasticity during angiogenic blood vessel remodeling.

RhoA inhibition is a key step in pituicyte stellation induced by A1-type adenosine receptor activation

Pituicyte stellation in vitro represents a useful model with which to study morphological changes that occur in vivo in these cells during times of high neurohypophysial hormone output. This model has helped us establish the hypothesis of a purinergic regulation of pituicyte morphological plasticity. We first show that ATP induces stellation in 37% of pituicytes, an effect that is secondary to the metabolism of ATP to adenosine. Adenosine‐induced stellation of pituicytes appears to be mediated by A1‐type receptors. The effect is independent of intracellular calcium and does not involve the mitogen‐activated protein kinase pathway. The basal (nonstellate) state of pituicytes depends on tonic activation of a Rho GTPase because both C3 transferase (a Rho inhibitor) and Y‐27632 (an inhibitor of p160Rho kinase) can induce stellation. Lysophosphatidic acid, a Rho activator, blocks the morphogenic effect of adenosine dose‐dependently. Using a specific RhoA pull‐down assay, we also show that downregulation of activated RhoA is the key event coupling A1 receptor activation to pituicyte stellation, via F‐actin depolymerization and microtubule reorganization. Finally, both vasopressin and oxytocin can prevent or reverse adenosine‐induced stellation. The effects of vasopressin, and those of high concentrations of oxytocin, are mediated through V1a receptors. Placed within the context of the relevant literature, our data suggest the possibility of a purinergic regulation of pituicyte morphological plasticity and subsequent modulation of hormone release, with these hormones providing a negative feedback mechanism. GLIA 38:351–362, 2002.