DIP2A Functions as a FSTL1 Receptor

Abstract

FSTL1 is an extracellular glycoprotein whose functional significance in physiological and pathological processes is incompletely understood. Recently, we have shown that FSTL1 acts as a muscle-derived secreted factor that is up-regulated by Akt activation and ischemic stress and that FSTL1 exerts favorable actions on the heart and vasculature. Here, we sought to identify the receptor that mediates the cellular actions of FSTL1. We identified DIP2A as a novel FSTL1-binding partner from the membrane fraction of endothelial cells. Co-immunoprecipitation assays revealed a direct physical interaction between FSTL1 and DIP2A. DIP2A was present on the cell surface of endothelial cells, and knockdown of DIP2A by small interfering RNA reduced the binding of FSTL1 to cells. In cultured endothelial cells, knockdown of DIP2A by small interfering RNA diminished FSTL1-stimulated survival, migration, and differentiation into network structures and inhibited FSTL1-induced Akt phosphorylation. In cultured cardiac myocytes, ablation of DIP2A reduced the protective actions of FSTL1 on hypoxia/ reoxygenation-induced apoptosis and suppressed FSTL1-induced Akt phosphorylation. These data indicate that DIP2A functions as a novel receptor that mediates the cardiovascular protective effects of FSTL1.FSTL1 (follistatin-like 1; also referred to TSC36) is a secreted extracellular glycoprotein that was initially identified from a mouse osteoblast cell line as a transforming growth factor-␤ (TGF-␤) 2 -regulated gene (1). Prior studies have shown that FSTL1 can have antiproliferative effects on cells (2, 3) and that it can exert anti-inflammatory (4, 5) or pro-inflammatory (6) actions in animal models. Recently, we have found that FSTL1 is up-regulated in the myocardium in response to Akt1-induced hypertrophic growth and myocardial stress, including pressure overload, ischemia-reperfusion, and myocardial infarction (7). Systemic delivery of FSTL1 protects the heart from ischemiareperfusion injury in mice, which is accompanied by reduced myocyte apoptosis. More recently, we have shown that FSTL1 is up-regulated and secreted in skeletal muscle in response to Akt-mediated growth and ischemic injury (8). FSTL1 promotes endothelial cell function in vitro and accelerates ischemia-induced revascularization in vivo. Thus, we proposed that FSTL1 functions as an injury-induced secreted protein that protects against ischemic damage. However, the molecular mechanisms by which FSTL1 promotes cardiovascular cell protection and function are incompletely understood.FSTL1 possesses extracellular calcium-binding and follistatin-like domains and is categorized as the follistatin family member of proteins (9). Other members of the follistatin family act through their ability to function as extracellular binding partners of TGF-␤ superfamily proteins and antagonize the binding of these ligands to the receptors (10). However, it has never been documented that FSTL1 modulates cell function by binding members of the TGF-␤ superfamily, as do follistatin and FSTL3 (11...

Cardiac myocyte follistatin-like 1 functions to attenuate hypertrophy following pressure overload

“…The dose of recombinant Fstl1 protein required for half-maximal AMPK phosphorylation was 87 ng/mL, a level consistent with levels of circulating Fstl1 reported in healthy individuals (median = 7.18 ng/mL; range = 1.06-18.49 ng/mL) and in individuals with acute coronary syndrome (median = 13.50 ng/mL; range = 8.82-30.46 ng/mL) (22). Fstl1 can exert its actions via the candidate receptor Disco-interacting protein 2 homolog A (DIP2A) (15,16); however, preliminary analyses of whether DIP2A mediates Fstl1 actions on AMPK signaling in myocytes have been inconclusive. Consistent with observations of AMPK activation, an increase in myocardial eNOS phosphorylation at Ser1179 was observed in Fstl1-TG mice following TAC, whereas this phosphorylation was reduced in Fstl1-KO mice.…”

“…Although Fstl1 is categorized as a follistatin-like protein, it has relatively limited homology with other follistatin family proteins that classically act as binding partners of the TGF-β family proteins. In contrast to follistatin and Fstl3, Fstl1 can function as a ligand for a cell-surface receptor, and it does not appear to act as a high-affinity extracellular regulator of activin A-stimulated Smaand Mad-related protein (Smad) phosphorylation in cardiovascular cells (7,15,16). Previous studies have implicated a role for Fstl1 in inflammatory responses and in the control of tumor cell growth in vitro (17)(18)(19)(20).…”

Expression, characterization, and preliminary X-ray crystallographic analysis of recombinant murine Follistatin-like 1 expressed in Drosophila S2 cells

“…Recombinant forms of FSTL1 were also expressed in human cells and used to compare their structural and functional properties with those described for other members of the FST-SPARC protein family (23), but the biological activity assay for recombinant FSTL1 was not performed. Recently, Ouchi and colleagues, as well as our group, produced active recombinant FSTL1 protein in insect cell lines (Lepidopteran Sf9 or Drosophila S2 cells cells) respectively (34). In this study, we reported in detail the production of large amounts of highly purified and functional rFSTL1 using Drosophila S2 cells.…”

Chronic skeletal muscle ischemia preserves coronary flow in the ischemic rat heart

“…However, angiogenesis is regulated by complex molecular mechanisms involving the participation of multiple factors. Recent studies (25,26) on the interaction between skeletal muscle and the myocardium have implicated follistatin-like 1, an extracellular glycoprotein secreted by skeletal muscles in response to ischemic insult. Follistatin-like 1 was found to stimulate vascularization through its ability to activate Aktendothelial nitric oxide synthase signaling.…”