Intersectin-1s Regulates the Mitochondrial Apoptotic Pathway in Endothelial Cells

Abstract

Intersectins (ITSNs) are multidomain adaptor proteins implicated in endocytosis, regulation of actin polymerization, and Ras/MAPK signaling. We have previously shown that ITSN-1s is required for caveolae fission and internalization in endothelial cells (ECs). In the present study, using small interfering RNA to knock down ITSN-1s protein expression, we demonstrate a novel role of ITSN-1s as a key antiapoptotic protein. Knockdown of ITSN-1s in ECs activated the mitochondrial pathway of apoptosis as determined by genomic DNA fragmentation, extensive mitochondrial fission, activation of the proapoptotic proteins BAK and BAX, and cytochrome c efflux from mitochondria. ITSN-1 knockdown acts as a proapoptotic signal that causes mitochondrial outer membrane permeabilization, dissipation of the mitochondrial membrane potential, and generation of reactive oxygen species. These effects were secondary to decreased activation of Erk1/2 and its direct activator MEK. Bcl-X L overexpression prevented BAX activation and the apoptotic ECs death induced by suppression of ITSN-1s. Our findings demonstrate a novel role of ITSN-1s as a negative regulator of the mitochondrial pathway-dependent apoptosis secondary to activation of the Erk1/2 survival signaling pathway.ITSN-1 is a multidomain adaptor protein, which binds to and scaffolds the endocytic machinery of clathrin-and caveolaemediated endocytic pathways (1-3). Two major ITSN-1 transcripts have been described in mammals, the ubiquitously expressed ITSN-1s and the neuron specific long isoform, 4,5). A highly similar human gene, ITSN-2, generates by alternative splicing two ITSN-2 isoforms widely expressed in human tissues (lung endothelium included) and showing a high degree of similarity to ITSN-1 proteins (2, 6). ITSN-1s, the only ITSN-1 isoform present in the ECs lining the blood vessels wall (3), comprises two Eps15 homology domains, a central coiled-coil domain, and five consecutive SH3 2 domains (SH3A to -E) (1, 7). Although ITSN-1 is best known for its role in endocytosis, recent studies also implicate ITSNs as mediators of the MAPK signaling pathway (8). ITSNs interact via the SH3A domain with the mammalian Son-of-sevenless, a guanine-nucleotide exchange factor for Ras (9). The association of ITSN with mammalian Son-of-sevenless leads to increased levels of RasGTP with functional consequences in signaling Erk1/2 activation (9). Activated Ras initiates the phosphorylation cascade, leading to phosphorylation and activation of p42/ p44 MAPKs (Erk1 and Erk2). In both neurons and nonneuronal cell lines, overexpression of the SH3A to -E, presumed to function in a dominant negative manner (11), attenuated the activation of EGF-mediated Ras/MAPK pathway (8). The concept of ITSNs as mediators of MAPK signaling is further supported by studies showing that full-length ITSN overexpression in HEK cells generated increased Ras-GTP levels associated with cytoplasmic vesicles, resulting in the c-Jun NH 2 -terminal kinase (JNK) activation (12). More recent work (13) in NIH 3T3 fibroblast...

Molecular determinants of endothelial transcytosis and their role in endothelial permeability

“…Once released from the plasma membrane, caveolae form free transport vesicles that reach the opposite front of the cell where they discharge their contents by fusion with the plasma membrane. Moreover, siRNA-mediated downregulation of intersectin-1s protein expression in cultured ECs caused a significant decrease in caveolae number, consistent with intersectin-1s requirement for recruiting and organizing caveolae endocytic and transcytotic machinery (157).…”

Regulation of Neuron Survival through an Intersectin-Phosphoinositide 3′-Kinase C2β-AKT Pathway

“…Indeed, loss-of-function alleles of Drosophila ITSN resulted in larval lethality (14,17), consistent with this possibility. In addition, a recent study by Predescu and colleagues (22) demonstrated that transient silencing of ITSN in human microvascular endothelial cells activated a mitochondrial apoptotic pathway, leading to death of the ITSN-silenced cells. However, the mechanism by which ITSN protects cells from apoptosis was not determined.…”

Modulation of Intersectin-1s Lung Expression Induces Obliterative Remodeling and Severe Plexiform Arteriopathy in the Murine Pulmonary Vascular Bed

“…47,48 Recently, we have shown that chronic ITSN deficiency up-regulated extracellular signal regulated kinase (Erk) 1/2 MAPK activity, suppressed the proapoptotic Bad, and altered Smads signaling downstream of the transforming growth factor-b receptor-1, resulting in a proliferative apoptotic-resistant EC phenotype. 12 In a PAH environment, ITSN deficiency and concurrent expression of granzyme B cleavage products of ITSN, able to regulate the p38 to Erk1/2 activity ratio, appear to be critical for initiation of the aberrant EC proliferation.…”