Catherine Monnot

Angiopoietin-Like 4 Is a Proangiogenic Factor Produced during Ischemia and in Conventional Renal Cell Carcinoma

Ischemic and solid tumor tissues are less well perfused than normal tissue, leading to metabolic changes and chronic hypoxia, which in turn promotes angiogenesis. We identified human angiopoietin-like 4 (angptl4) as a gene with hypoxia-induced expression in endothelial cells. We showed that the levels of both mRNA and protein for ANGPTL4 increased in response to hypoxia. When tested in the chicken chorioallantoic membrane assay, ANGPTL4 induced a strong proangiogenic response, independently of vascular endothelial growth factor. In human pathology, ANGPTL4 mRNA is produced in ischemic tissues, in conditions such as critical leg ischemia. In tumors, ANGPTL4 is produced in the hypoxic areas surrounding necrotic regions. We observed particularly high levels of ANGPTL4 mRNA in tumor cells of conventional renal cell carcinoma. Other benign and malignant renal tumor cells do not produce ANGPTL4 mRNA. This molecule therefore seems to be a marker of conventional renal cell carcinoma. ANGPTL4, originally identified as a peroxisome proliferator-activated receptor alpha and gamma target gene, has potential for use as a new diagnostic tool and a potential therapeutic target, modulating angiogenesis both in tumors and in ischemic tissues. This study also suggests that ANGPTL4 may provide a link between metabolic disorders and hypoxia-induced angiogenesis.

Polar Residues in the Transmembrane Domains of the Type 1 Angiotensin II Receptor Are Required for Binding and Coupling RECONSTITUTION OF THE BINDING SITE BY CO-EXPRESSION OF TWO DEFICIENT MUTANTS

Type 1 angiotensin receptors (AT) are G-protein coupled receptors, mediating the physiological actions of the vasoactive peptide angiotensin II. In this study, the roles of 7 amino acids of the rat AT receptor in ligand binding and signaling were investigated by performing functional assays of individual receptor mutants expressed in COS and Chinese hamster ovary cells. Substitutions of polar residues in the third transmembrane domain with Ala indicate that Ser, Ser, and Ser are not essential for maintenance of the angiotensin II binding site. Replacement of Asn or Ser does not alter the binding affinity for peptidic analogs, but modifies the ability of the receptor to interact with AT (DuP753)- or AT (CGP42112A)-specific ligands. These 2 residues are probably involved in determining the binding specificity for these analogs. The absence of G-protein coupling to the Ser mutant suggests that this residue, in addition to previously identified residues, Asp and Tyr, participates in the receptor activation mechanism. Finally, Lys (third helix) and Lys (fifth helix) mutants do not bind angiotensin II or different analogs. Co-expression of these two deficient receptors permitted the restoration of a normal binding site. This effect was not due to homologous recombination of the cDNAs but to protein trans-complementation.

Akt Is a Major Downstream Target of PI3-Kinase Involved in Angiotensin II–Induced Proliferation

Abstract—Different signal transduction cascades have been implicated in angiotensin II (Ang II)–mediated cell growth, such as the extracellular signal-regulated kinase 1/2 (ERK1/2) and the phosphatidylinositol 3-kinase (PI3K) pathways. To identify the downstream targets of PI3K involved in Ang II–induced proliferation, we used both rat aortic smooth muscle (RASM) cells and a CHO cell line stably expressing the rat AT1A receptor. The ERK1/2 and PI3K pathways are independently activated and implicated in Ang II–mediated DNA synthesis and cell number increase in these 2 cell lines. In addition, a specific inhibitor of Akt inhibited Ang II–induced Akt phosphorylation, DNA synthesis and proliferation in CHO-AT1A or RASM cells. A dominant-negative mutant of Akt was also found to selectively block Ang II–induced proliferation of CHO-AT1A cells. To further elucidate the signaling events leading to Akt activation, we used an AT1 receptor mutant (AT1AD74E), deficient for Gq protein coupling, and the intracellular calcium chelator BAPTA-AM. Although altered Akt and ERK1/2 activation was observed in the CHO-AT1AD74E cell line, blockade of intracellular calcium elevation did not affect phosphorylation of these kinases. These results provide the first evidence of a specific and necessary role of Akt in Ang II–induced proliferation through a Gq protein–dependent calcium-independent pathway.

Distribution of type 1 angiotensin II receptor subtype messenger RNAs in the rat fetus.

The localization of the two type 1 angiotensin II receptor subtype (AT1A and AT1B) messenger RNAs in the 19-day-old rat fetus was studied by in situ hybridization. AT1 receptor mRNAs were detected in target organs of the renin-angiotensin system such as the kidney, adrenal gland, liver, heart, large arteries, and pituitary gland. In addition, angiotensin II receptors were present in specialized mesenchymal cells surrounding the cartilage, in the pericardium, in the lung, and in the undifferentiated mesenchymal tissue. The AT1A subtype was predominant in all tissues and organs except the adrenal cortex and glomeruli in the kidney, which expressed both AT1A and AT1B mRNAs. The widespread distribution of AT1 receptors in tissues and organs involved in hydromineral equilibrium and blood pressure regulation shows that during fetal development angiotensin II may already act as a regulator of the cardiovascular system. An effect on cellular differentiation and/or proliferation via AT1 receptors is also suggested by their location in several mesenchymes.

A recombinant rat vascular AT1 receptor confers growth properties to angiotensin II in Chinese Hamster Ovary cells

A rat vascular AT1 receptor cDNA has been stably expressed into Chinese Hamster Ovary cells and the resulting recombinant AT1a receptor has been functionally characterized. This receptor binds 125I Sar1-angiotensin II with an affinity of 0.9 nM and the displacement of this ligand by a series of peptidic and nonpeptidic analogs is shown. Binding of angiotensin II to this receptor causes a rapid increase in inositol phosphate production, whereas this effect is not observed in nontransfected cells. Des-aspartyl1 angiotensin II and at a lesser extent angiotensin I are also able to produce an increase in inositol phosphates. More importantly, the actions of angiotensin II on cell division were clearly demonstrated in this model, since angiotensin II is able to stimulate DNA synthesis by 400% and double the cell population of the transfected cells in 36 hours in the absence of any other growth factor, whereas no effect is observed in nontransfected cells.

Knockdown of col22a1 gene in zebrafish induces a muscular dystrophy by disruption of the myotendinous junction

The myotendinous junction (MTJ) is the major site of force transfer in skeletal muscle, and defects in its structure correlate with a subset of muscular dystrophies. Col22a1 encodes the MTJ component collagen XXII, the function of which remains unknown. Here, we have cloned and characterized the zebrafish col22a1 gene and conducted morpholino-based loss-of-function studies in developing embryos. We showed that col22a1 transcripts localize at muscle ends when the MTJ forms and that COLXXII protein integrates the junctional extracellular matrix. Knockdown of COLXXII expression resulted in muscular dystrophy-like phenotype, including swimming impairment, curvature of embryo trunk/tail, strong reduction of twitch-contraction amplitude and contraction-induced muscle fiber detachment, and provoked significant activation of the survival factor Akt. Electron microscopy and immunofluorescence studies revealed that absence of COLXXII caused a strong reduction of MTJ folds and defects in myoseptal structure. These defects resulted in reduced contractile force and susceptibility of junctional extracellular matrix to rupture when subjected to repeated mechanical stress. Co-injection of sub-phenotypic doses of morpholinos against col22a1 and genes of the major muscle linkage systems showed a synergistic gene interaction between col22a1 and itga7 (α7β1 integrin) that was not observed with dag1 (dystroglycan). Finally, pertinent to a conserved role in humans, the dystrophic phenotype was rescued by microinjection of recombinant human COLXXII. Our findings indicate that COLXXII contributes to the stabilization of myotendinous junctions and strengthens skeletal muscle attachments during contractile activity.

Vascular smooth muscle cells efficiently activate a new proteinase cascade involving plasminogen and fibronectin

The plasminogen/plasmin system is involved in vascular wall remodeling after injury, through extracellular matrix (ECM) degradation and proteinase activation. Vascular smooth muscle cells (VSMCs) synthesize various components of the plasminogen/plasmin system. We investigated the conversion of plasminogen into plasmin in primary cultured rat VSMCs. VSMCs efficiently converted exogenous plasminogen into plasmin in a time‐ and dose‐dependent manner. We measured plasmin activity by monitoring the hydrolysis of Tosyl‐G‐P‐R‐Mca, a fluorogenic substrate of plasmin. Cell‐mediated plasmin activation was associated with the degradation of ECM, as revealed by fibronectin proteolysis. Plasmin also activated a proteinase able to hydrolyze Mca‐P‐L‐G‐L‐Dpa‐A‐R‐NH2, a fluorogenic substrate of matrix metalloproteinases (MMPs). However, this proteinase was not inhibited by an MMP inhibitor. Furthermore, this proteinase displayed similar biochemical and pharmacological properties to fibronectin‐proteinase, a recently identified zinc‐dependent metalloproteinase located in the gelatin‐binding domain of fibronectin. These results show that VSMCs convert exogenous plasminogen into plasmin in their pericellular environment. By hydrolyzing matrix protein plasmin activates a latent metalloproteinase that differs from MMP, fibronectin‐proteinase. This metalloproteinase may participate to vascular wall remodeling, in concert with other proteinases. J. Cell. Biochem. 88: 1188–1201, 2003.

Migration-stimulating factor displays HEXXH-dependent catalytic activity important for promoting tumor cell migration.

Like most extracellular matrix (ECM) components, fibronectin (Fn) is proteolyzed generating specific activities. Fibronectin proteinase (Fn‐proteinase) represents such a cryptic activity located in the gelatin‐binding domain (GBD) of Fn and displays a zinc metalloproteinase activity. The migration‐stimulating factor (MSF) is a truncated Fn isoform generated by alternative mRNA splicing and corresponds to the N‐terminal part of Fn that comprises the GBD. We show that several human mammary epithelial cells express MSF and constitutively produce Fn‐proteinase activity. Furthermore, recombinant MSF produced by HEK‐293 and MCF‐7 cells possesses a constitutive Fn‐proteinase activity. Mutating the putative zinc‐binding motif, HEXXH, of the protein abolishes its activity thereby demonstrating its specificity. Using PCR, we showed that MSF is barely expressed in normal breast tissues, whereas its expression is significantly increased in tumors. Furthermore, an association between MSF expression and invasive capacity is observed in various breast adenocarcinoma cell lines. Indeed, when stably transfected in non‐invasive MCF‐7 cells, MSF promotes cell migration in a mechanism mostly dependent on its Fn‐proteinase activity. In summary, our study shows that: (i) MSF displays constitutive Fn‐proteinase activity; (ii) MSF expression is induced in human breast cancer; and (iii) MSF confers pro‐migratory activity that depends mostly on its Fn‐proteinase activity. These results suggest that MSF may be involved in tumor progression.

Functional interactions of L-162,313 with angiotensin II receptor subtypes and mutants

A nonpeptide ligand, L-162,313 (5,7-dimethyl-2-ethyl-3-[[4-[2(n-butyloxycarbonylsulfonamido)-5-is obutyl-3-thienyl]phenyl]methyl]imidazo[4,5,6]pyridine) was characterized on the angiotensin II receptors. This compound displaced [125I][Sar1]angiotensin II from rat angiotensin AT1A, AT1B or AT2 receptor individually expressed in COS-7 cells (Ki = 207 nM, 226 nM and 276 nM, respectively). In monkey kidney cells expressing angiotensin AT1A or AT1B receptors, it stimulated inositol phosphate accumulation, but the maximal response was 34.9 and 23.3%, respectively, of those of angiotensin II. Furthermore, an antagonist effect of L-162.313 was observed in response to angiotensin II. Single-point substitutions in the second and third transmembrane domains of the rat angiotensin AT1A receptor, which impaired the binding of losartan (2-n-butyl-4-chloro-5-hydroxymethyl-1[(1H-tetrazol-5-yl)biphenyl-4 -yl)methyl]imidazole), also affected the binding of L-162,313. Losartan and L-162,313 require some common structural determinants for non-peptide recognition on the angiotensin AT1 receptor. Furthermore, some of these substitutions, which impaired the inositol phosphate accumulation in response to angiotensin II, also impaired the response to L-162,313. Angiotensin II and L-162,313 require common critical residues for angiotensin AT1 receptor activation.

The difficult challenge of cloning the angiotensin II receptor

The vasopressor peptide angiotensin II exerts its cellular effects through a membrane-bound receptor coupled to a G protein. Biochemical and pharmacological analyses of this receptor already identify two different membrane-bound receptors and one cytosoluble angiotensin-II-binding protein. Nevertheless, the purification of the membrane-bound form(s) appears to be difficult. In the absence of purified protein, two cloning strategies of the gene have been explored: (1) expression cloning, identifying the functions of the protein expressed from a cDNA library in COS cells or Xenopus oocytes, has been unsuccessful until now; (2) analogical cloning, trying to identify related members of the seven transmembrane segment receptor family, which could be related to angiotensin receptors, identifies the mas oncogene and two related genes. However, there are accumulating data to exclude their involvement in angiotensin binding.