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.

FIGURE 1.

Interaction of FSTL1 with a novel binding protein, DIP2A. A, detection of FSTL1-binding protein in membrane fractions of HUVECs. Membrane fractions were incubated in the presence or absence of FLAG-FSTL1 protein (2 μg/ml) and then immunoprecipitated with anti-FLAG affinity gel, followed by SDS-PAGE. Proteins were stained with carrier-complexed silver. The arrow indicates possible binding protein partners of FSTL1. B, DIP2A is immunoprecipitated by FLAG-FSTL1 from HUVEC membranes. Immunoprecipitated material was subjected to SDS-PAGE, followed by Western blot (WB) analysis with anti-DIP2A and anti-FSTL1 antibodies. C, FSTL1 is immunoprecipitated with nickel resin when DIP2A-His is present in the cell lysates. COS-7 cells were transfected with DIP2A-His or mock-transfected. Cell lysates were incubated in the presence or absence of recombinant FSTL1 protein (rFstl1; 400 ng) for 1 h and then subjected to immunoprecipitation with nickel resin. Immunoprecipitated material was subjected to SDS-PAGE, and Western blot analysis was performed with anti-DIP2A and anti-FSTL1 antibodies.

FIGURE 2.

Involvement of DIP2A in FSTL1 binding to endothelial cells. A, detection of DIP2A on the cell surface of HUVECs. HUVECs were incubated with anti-DIP2A antibody (red; mouse IgG, 5 μg/ml) or control mouse IgG (black) for 60 min. Cells were stained with Alexa Fluor® 488-conjugated secondary antibody and analyzed using a FACScan. B, immunocytochemical analysis of HUVECs with anti-DIP2A antibody. HUVECs were incubated with anti-DIP2A antibody, followed by staining with Alexa Fluor® 594-conjugated secondary antibody (red). Nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). Representative pictures are shown. C, reduction of DIP2A mRNA expression in HUVECs following transfection with siRNA against DIP2A. At 48 h after transfection of HUVECs with siRNA against DIP2A or control siRNA, DIP2A mRNA levels were determined by quantitative real-time PCR analysis and expressed relative to 36B4 levels (n = 3). D, effect of knockdown of DIP2A on the binding of FSTL1 to HUVECs. HUVECs were transfected with siRNA targeting DIP2A (○) or unrelated siRNA (●), incubated with increasing concentrations of biotinylated recombinant FSTL1 for 60 min, and treated with streptavidin-conjugated horseradish peroxidase, followed by incubation with QuantaBlu fluorogenic peroxidase substrate. The binding was assessed with a fluorescent microplate reader (n = 6–7). The data were analyzed with Microsoft Excel to generate a logarithmic trend line.

FIGURE 3.

Contribution of DIP2A to FSTL1-induced endothelial cell survival and function. HUVECs were transduced with siRNA against DIP2A or control siRNA. A and B, effect of DIP2A deletion on FSTL1-induced inhibition of HUVEC death. A, HUVECs transduced with siRNA were treated with recombinant FSTL1 protein (rFstl1; 100 ng/ml) or vehicle in serum-free medium for 48 h. B, after transfection with siRNAs, HUVECs were transduced with Ad-FSTL1 or Ad-β-galactosidase (Ad-βgal) for 8 h, followed by incubation in serum-free medium for 48 h. HUVEC death was assessed by a quantitative methanethiosulfonate-based assay. C, knockdown of DIP2A blocks the inhibitory actions of FSTL1 on HUVEC apoptosis caused by serum deprivation. HUVECs were cultured as described for B. HUVEC apoptosis was assessed by the degree of nucleosome fragmentation. D and E, role of DIP2A in endothelial cell network formation in response to FSTL1. D, after siRNA transfection, HUVECs were deprived of serum for 16 h and seeded on Matrigel-coated culture dishes in the presence of recombinant FSTL1 (100 ng/ml) or vehicle. Representative cultures are shown (upper panels). Quantitative analyses of network formation are shown (lower panels). E, following siRNA transfection, HUVECs were transduced with Ad-FSTL1 and Ad-β-galactosidase for 8 h and incubated in serum-free medium for 16 h, followed by subjection to Matrigel matrix. Quantitative analyses of network formation are shown. F, DIP2A is involved in FSTL1-stimulated endothelial cell migration. HUVECs were transduced with Ad-FSTL1 and Ad-β-galactosidase for 8 h. After 24 h of serum starvation, a modified Boyden chamber assay was performed. Results are shown as means ± S.E. (n = 4–7). Results are expressed relative to the values compared with control siRNA with vehicle or Ad-β-galactosidase.

FIGURE 4.

Involvement of DIP2A in FSTL1-mediated Akt signaling. HUVECs were transfected with siRNA against DIP2A or control siRNA. A and B, DIP2A mediates the stimulatory actions of FSTL1 on Akt phosphorylation in HUVECs. A, after 16 h of incubation in serum-free medium, siRNA-transfected HUVECs were treated with recombinant FSTL1 (rFstl1; 100 ng/ml) or vehicle for 30 min. B, after transfection with siRNA, HUVECs were transduced with Ad-FSTL1 and Ad-β-galactosidase for 8 h and incubated in serum-free medium for 24 h. Akt phosphorylation (P-Akt) levels were determined by Western blot analysis. Representative blots are shown from one of three independent experiments.

FIGURE 5.

Contribution of DIP2A to FSTL1 actions on cardiac myocytes. NRVMs were transfected with siRNA targeting rat DIP2A or control siRNA, followed by transduction with Ad-FSTL1 and Ad-β-galactosidase (Ad-βgal). A, reduction of DIP2A mRNA expression in NRVMs transfected with siRNA against DIP2A. At 48 h after transfection of NRVMs with siRNA against DIP2A or control siRNA, DIP2A mRNA levels were measured by quantitative real-time PCR analysis and expressed relative to glyceraldehyde-3-phosphate dehydrogenase levels (n = 3). B, involvement of DIP2A in the protective effects of FSTL1 against hypoxia/reoxygenation (H/R)-induced NRVM apoptosis. After transfection with adenovirus, NRVMs were subjected to 12 h of hypoxia, followed by 24 h of reoxygenation. Apoptotic activity was assessed by the degree of nucleosome fragmentation. Results are shown as means ± S.E. The scale from 0 to 1.0 is minimized to highlight differences in nucleosome fragmentation values. C, DIP2A mediates FSTL1-induced Akt signaling in cardiac myocytes. After transduction with adenovirus, NRVMs were cultured in serum-free medium for 24 h. Akt phosphorylation (P-Akt) levels were determined by Western blot analysis. Representative blots are shown from one of three independent experiments.