Endocytic deficiency induced by intersectin-1s knockdown alters the Smad2/3-Erk1/2 signaling balance downstream of Alk5

Abstract

Recently, we demonstrated in cultured endothelial cells and in vivo that deficiency of an isoform of intersectin-1, ITSN-1s, impairs caveolae and clathrin-mediated endocytosis and functionally upregulates compensatory pathways and their morphological carriers (i.e. enlarged endocytic structures, membranous rings or tubules) that are normally underrepresented. We now show that these endocytic structures internalize the broadly expressed transforming growth factor β receptor I (TGFβ-RI or TGFBR1), also known as Alk5, leading to its ubiquitylation and degradation. Moreover, the apoptotic or activated vascular cells of the ITSN-1s-knockdown mice release Alk5-bearing microparticles to the systemic circulation. These interact with and transfer Alk5 to endocytosis-deficient endothelial cells, resulting in lung endothelial cell survival and phenotypic alteration towards proliferation through activation of Erk1 and Erk2 (also known as MAPK3 and MAPK1, respectively). We also show that non-productive assembly of the Alk5–Smad–SARA (Smad anchor for receptor activation, also known as ZFYVE9) signaling complex and preferential formation of the Alk5–mSos–Grb2 complex account for Erk1/2 activation downstream of Alk5 and proliferation of pulmonary endothelial cells. Taken together, our studies demonstrate a functional relationship between the intercellular transfer of Alk5 by microparticles and endothelial cell survival and proliferation, and define a novel molecular mechanism for TGFβ and Alk5-dependent Erk1/2MAPK signaling that is significant for proliferative signaling and abnormal growth.

Intersectin scaffold proteins and their role in cell signaling and endocytosis

“…Since ITSN and Grb2 compete for Sos [14], reduced levels of ITSN in cells may increase Sos availability for Grb2, favoring the Alk5-Sos1-Grb2 pathway over the Alk5-Smad-SARA route. Studies in knockdown ITSN1-S mouse lungs demonstrate a preference for the Alk5-Sos1-Grb2 pathway leading to persistent activation of ERK1/2 and the proliferation of pulmonary endothelial cells [55]. These findings suggest that ITSN attenuates Ras-ERK activation by the TGFβ pathway in contrast to previous work suggesting that ITSN promotes Ras activation [50, 59].…”

“…The impairment of these pathways leads to the upregulation of alternative pathways that include the presence of enlarged endocytic structures. These endocytic structures internalize the transforming growth factor β (TGFβ) receptor I, also named Alk5, resulting in its degradation [55, 56]. The effects of such degradation are important, since the TGFβ pathway is involved in cell proliferation, differentiation and apoptosis.…”

“…However, ITSN1 and EGFR cooperate to enhance transcriptional activation and this synergy requires ERK-MAPK activity [59]. As discussed above, ITSN1 attenuates Ras-ERK activation by TGFb in lung cell [55]. Thus, ITSN1 activates JNK and indirectly regulates ERK activation by receptors.…”

Plexiform Arteriopathy in Rodent Models of Pulmonary Arterial Hypertension

“…This interaction could rescue the aberrant phenotype, thereby improving the overall severity of the disease; this mechanism was previously demonstrated in other types of lung disease. 43,44 Although not tested, if true, administering PHD2-containing microparticles of hematopoietic cell origin may prove efficacious in treating idiopathic PAH patients. Regardless, this is the first study, to our knowledge, to generate a severe mouse model of PAH that recapitulates almost all key pathologic features exhibited in the human PAH phenotypes.…”

New insights into the functions of intersectin-1s

“…2 TGFb has been shown to induce Erk1/2 MAPK signaling in endothelial and epithelial cells, fibroblasts, breast and colorectal cancer to promote disassembly of adherens junctions, cell migration and proliferation. 3,4 Studies to understand the molecular mechanisms underlying TGFb-dependent Erk1/2 MAPK activation indicated that plasma membrane-bound activated Alk5/ TGFbRII heteromer recruits and phosphorylates the adaptor protein ShcA followed by the ShcA/Grb2/mSos complex assembly. 5 Localization of mSos/Grb2 at the plasma membrane level is the primary mechanism for Ras activation.…”