Retinoic acid signaling is directly activated in cardiomyocytes and protects mouse hearts from apoptosis after myocardial infarction.

Retinoic acid (RA) is an essential signaling molecule for cardiac development and plays a protective role in the heart after myocardial infarction (MI). In both cases, the effect of RA signaling on cardiomyocytes, the principle cell type of the heart, has been reported to be indirect. Here we have developed an inducible murine transgenic RA-reporter line using CreERT2 technology that permits lineage tracing of RA-responsive cells and faithfully recapitulates endogenous RA activity in multiple organs during embryonic development. Strikingly, we have observed a direct RA response in cardiomyocytes during mid-late gestation and after MI. Ablation of RA signaling through deletion of the Aldh1a1/a2/a3 genes encoding RA-synthesizing enzymes leads to increased cardiomyocyte apoptosis in adults subjected to MI. RNA sequencing analysis reveals Tgm2 and Ace1, two genes with well-established links to cardiac repair, as potential targets of RA signaling in primary cardiomyocytes, thereby providing novel links between the RA pathway and heart disease.

Control of cell death/survival balance by the MET dependence receptor.

Control of cell death/survival balance is an important feature to maintain tissue homeostasis. Dependence receptors are able to induce either survival or cell death in presence or absence of their ligand, respectively. However, their precise mechanism of action and their physiological importance are still elusive for most of them including the MET receptor. We evidence that pro-apoptotic fragment generated by caspase cleavage of MET localizes to the mitochondria-associated membrane region. This fragment triggers a calcium transfer from endoplasmic reticulum to mitochondria, which is instrumental for the apoptotic action of the receptor. Knock-in mice bearing a mutation of MET caspase cleavage site highlighted that p40MET production is important for FAS-driven hepatocyte apoptosis, and demonstrate that MET acts as a dependence receptor in vivo. Our data shed light on new signaling mechanisms for dependence receptors' control of cell survival/death balance, which may offer new clues for the pathophysiology of epithelial structures.

Alternatively spliced isoforms of WT1 control podocyte-specific gene expression.

The Wilms' tumor suppressor WT1 is a key regulator of podocyte function that is mutated in Denys-Drash and Frasier syndromes. Here we have used an integrative approach employing ChIP, exon array, and genetic analyses in mice to address general and isoform-specific functions of WT1 in podocyte differentiation. Analysis of ChIP-Seq data showed that almost half of the podocyte-specific genes are direct targets of WT1. Bioinformatic analysis further identified coactivator FOXC1-binding sites in proximity to WT1-bound regions, thus supporting coordinated action of these transcription factors in regulating podocyte-specific genes. Transcriptional profiling of mice lacking the WT1 alternative splice isoform (+KTS) had a more restrictive set of genes whose expression depends on these alternatively spliced isoforms. One of these genes encodes the membrane-associated guanylate kinase MAGI2, a protein that localizes to the base of the slit diaphragm. Using functional analysis in mice, we further show that MAGI2α is essential for proper localization of nephrin and the assembly of the slit diaphragm complex. Finally, a dramatic reduction of MAGI2 was found in an LPS mouse model of glomerular injury and in genetic cases of human disease. Thus, our study highlights the central role of WT1 in podocyte differentiation, identifies that WT1 has a central role in podocyte differentiation, and identifies MAGI2α as the crucial isoform in slit diaphragm assembly, suggesting a causative role of this gene in the etiology of glomerular disorders.

The dependence receptor TrkC triggers mitochondria-dependent apoptosis upon Cobra-1 recruitment.

The neurotrophin receptor TrkC was recently identified as a dependence receptor, and, as such, it triggers apoptosis in the absence of its ligand, NT-3. The molecular mechanism for apoptotic engagement involves the double cleavage of the receptor's intracellular domain, leading to the formation of a proapoptotic "killer" fragment (TrkC KF). Here, we show that TrkC KF interacts with Cobra1, a putative cofactor of BRCA1, and that Cobra1 is required for TrkC-induced apoptosis. We also show that, in the developing chick neural tube, NT-3 silencing is associated with neuroepithelial cell death that is rescued by Cobra1 silencing. Cobra1 shuttles TrkC KF to the mitochondria, where it promotes Bax activation, cytochrome c release, and apoptosome-dependent apoptosis. Thus, we propose that, in the absence of NT-3, the proteolytic cleavage of TrkC leads to the release of a killer fragment that triggers mitochondria-dependent apoptosis via the recruitment of Cobra1.

Shedding-generated Met receptor fragments can be routed to either the proteasomal or the lysosomal degradation pathway.

The receptor tyrosine kinase Met and its ligand, the hepatocyte growth factor/scatter factor, are essential for embryonic development, whereas deregulation of Met signaling pathways is associated with tumorigenesis and metastasis. The presenilin-regulated intramembrane proteolysis (PS-RIP) is involved in ligand-independent downregulation of Met. This proteolytic process involves shedding of the Met extracellular domain followed by γ-secretase cleavage, generating labile intracellular fragments degraded by the proteasome. We demonstrate here that upon shedding both generated Met N- and C-terminal fragments are degraded directly in the lysosome, with C-terminal fragments escaping γ-secretase cleavage. PS-RIP and lysosomal degradation are complementary, because their simultaneous inhibition induces synergistic accumulation of fragments. Met N-terminal fragments associate with the high-affinity domain of HGF/SF, confirming its decoy activity which could be reduced through their routing to the lysosome at the expense of extracellular release. Finally, the DN30 monoclonal antibody inducing Met shedding promotes receptor degradation through induction of both PS-RIP and the lysosomal pathway. Thus, we demonstrate that Met shedding initiates a novel lysosomal degradation which participates to ligand-independent downregulation of the receptor.

The ganglioside G(D2) induces the constitutive activation of c-Met in MDA-MB-231 breast cancer cells expressing the G(D3) synthase.

We have recently established and characterized cellular clones deriving from MDA-MB-231 breast cancer cells that express the human G(D3) synthase (GD3S), the enzyme that controls the biosynthesis of b- and c-series gangliosides. The GD3S positive clones show a proliferative phenotype in the absence of serum or growth factors and an increased tumor growth in severe immunodeficient mice. This phenotype results from the constitutive activation of the receptor tyrosine kinase c-Met in spite of the absence of ligand and subsequent activation of mitogen-activated protein kinase/extracellular signal-regulated kinase and phosphoinositide 3-kinase/Akt pathways. Here, we show by mass spectrometry analysis of total glycosphingolipids that G(D3) and G(D2) are the main gangliosides expressed by the GD3S positive clones. Moreover, G(D2) colocalized with c-Met at the plasma membrane and small interfering RNA silencing of the G(M2)/G(D2) synthase efficiently reduced the expression of G(D2) as well as c-Met phosphorylation and reversed the proliferative phenotype. Competition assays using anti-G(D2) monoclonal antibodies also inhibit proliferation and c-Met phosphorylation of GD3S positive clones in serum-free conditions. Altogether, these results demonstrate the involvement of the disialoganglioside G(D2) in MDA-MB-231 cell proliferation via the constitutive activation of c-Met. The accumulation of G(D2) in c-Met expressing cells could therefore reinforce the tumorigenicity and aggressiveness of breast cancer tumors.

Met degradation: more than one stone to shoot a receptor down.

The receptor tyrosine kinase Met and its high-affinity ligand, the hepatocyte growth factor/scatter factor (HGF/SF), are essential to embryonic development. Deregulation of their signaling is associated with tumorigenesis and metastasis, notably through receptor overexpression. It is thus important to understand the mechanisms controlling Met expression. The ligand-dependent internalization of Met and its subsequent degradation in the lysosomal compartment are well described. This process is known to attenuate downstream Met signaling pathways. Yet internalized Met takes part directly in intracellular signaling by chaperoning signaling factors in the course of its trafficking. Furthermore, recent studies describe various new degradation mechanisms of membrane-anchored Met, involving proteolytic cleavages or association with novel partners. Although all these degradations are ligand-independent, they share, to different extents, some common features with canonical HGF/SF-dependent degradation. Interestingly, activated Met variants display resistance to degradation, suggesting defective degradation is involved in tumorigenesis. Conversely, forced degradation of Met through reinduction of one or more degradation pathways is a promising therapeutic strategy.

GD3 synthase expression enhances proliferation and tumor growth of MDA-MB-231 breast cancer cells through c-Met activation.

The disialoganglioside G(D3) is overexpressed in ∼50% of invasive ductal breast carcinoma, and the G(D3) synthase gene (ST8SIA1) displays higher expression among estrogen receptor-negative breast cancer tumors, associated with a decreased overall survival of breast cancer patients. However, no relationship between ganglioside expression and breast cancer development and aggressiveness has been reported. We have previously shown that overexpression of G(D3) synthase induces the accumulation of b- and c-series gangliosides (G(D3), G(D2), and G(T3)) at the cell surface of MDA-MB-231 breast cancer cells together with the acquisition of a proliferative phenotype in the absence of serum. Here, we show that phosphoinositide 3-kinase/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase pathways are constitutively activated in G(D3) synthase-expressing cells. Analysis of phosphorylation of tyrosine kinase receptors shows a specific c-Met constitutive activation in G(D3) synthase-expressing cells, in the absence of its ligand, hepatocyte growth factor/scatter factor. In addition, inhibition of c-Met or downstream signaling pathways reverses the proliferative phenotype. We also show that G(D3) synthase expression enhances tumor growth in severe combined immunodeficient mice. Finally, a higher expression of ST8SIA1 and MET in the basal subtype of human breast tumors are observed. Altogether, our results show that G(D3) synthase expression is sufficient to enhance the tumorigenicity of MDA-MB-231 breast cancer cells through a ganglioside-dependent activation of the c-Met receptor.