SCF/c-Kit-activated signaling and angiogenesis require Gαi1 and Gαi3

Abstract

The stem cell factor (SCF) binds to c-Kit in endothelial cells, thus activating downstream signaling and angiogenesis. Herein, we examined the role of G protein subunit alpha inhibitory (Gαi) proteins in this process. In MEFs and HUVECs, Gαi1/3 was associated with SCF-activated c-Kit, promoting c-Kit endocytosis, and binding of key adaptor proteins, subsequently transducing downstream signaling. SCF-induced Akt-mTOR and Erk activation was robustly attenuated by Gαi1/3 silencing or knockout (KO), or due to dominant negative mutations but was strengthened substantially following ectopic overexpression of Gαi1/3. SCF-induced HUVEC proliferation, migration, and capillary tube formation were suppressed after Gαi1/3 silencing or KO, or due to dominant negative mutations. In vivo, endothelial knockdown of Gαi1/3 by intravitreous injection of endothelial-specific shRNA adeno-associated virus (AAV) potently reduced SCF-induced signaling and retinal angiogenesis in mice. Moreover, mRNA and protein expressions of SCF increased significantly in the retinal tissues of streptozotocin-induced diabetic retinopathy (DR) mice. SCF silencing, through intravitreous injection of SCF shRNA AAV, inhibited pathological retinal angiogenesis and degeneration of retinal ganglion cells in DR mice. Finally, the expression of SCF and c-Kit increased in proliferative retinal tissues of human patients with proliferative DR. Taken together, Gαi1/3 mediate SCF/c-Kit-activated signaling and angiogenesis.

Figure 1

Double knockout of Gαi1 and Gαi3 in mouse embryonic fibroblasts (MEFs) abolishes SCF-induced signaling. The listed MEFs were stimulated with SCF at indicated concentrations and cultured for indicated periods, and the listed signaling protein levels were examined (A-F). “Ctrl” refers to PBS treatment. *P < 0.05 versus “WT MEFs”. ^#P < 0.05 (F). “N. S.” denotes P > 0.05.

Figure 2

Gαi1 and Gαi3 dominant negative (DN) mutants disrupt SCF-induced c-Kit internalization and binding of adaptor proteins and prevent downstream signaling activation. Stable WT MEFs, with the DN mutant-Gαi1 (murine) construct plus DN-Gαi3 construct (“DN-Gαi1/3”) or the vector control (“Vec”), were treated with SCF (50 ng/mL) for 5 min. The association of c-Kit, Grb2, Gab2, Shc, Gαi1, and Gαi3 was examined by co-immunoprecipitation (Co-IP) assays (A), and their expressions are shown in “Input” (A); The listed proteins in membrane fraction lysates and total cell lysates were examined (B-D). “Ctrl” refers to PBS treatment. *P < 0.05 versus “Vec”. ^#P < 0.05 (B).

Figure 3

Gαi1 and Gαi3 silencing prevents SCF-induced signaling and pro-angiogenic activity in endothelial cells. HUVECs were treated with SCF (50 ng/mL) for 5 min, and the association of c-Kit, Grb2, Gab2, Shc, Gαi1, and Gαi3 was examined by co-immunoprecipitation (Co-IP) assays (A and B). Their expressions are shown as “Input” (A and B). Stable HUVECs, with the lentiviral human Gαi1 shRNA and the lentiviral human Gαi3 shRNA (“shGαi1/3”) or scramble shRNA control (“shC”), were treated with SCF (50 ng/mL) for 15 min, and the listed proteins in membrane fraction lysates and total cell lysates were examined (C-E); HUVECs were further cultured, and cell proliferation (EdU nuclear incorporation, F), in vitro migration (G), and capillary tube formation (H) were assessed. “Veh” refers to vehicle control. * P< 0.05 versus “Veh” treatment in shC HUVECs. ^#P < 0.05. Scale bar = 100 μm.

Figure 4

SCF-induced signaling and pro-angiogenic activity are inhibited in endothelial cells with Gαi1 and Gαi3 mutations. HUVECs, with the dominant negative (DN) mutant-Gαi1 (human) construct plus the DN Gαi3 (human) (“DN-Gαi1/3”), construct or the vector control (“Vec”), were treated with SCF (50 ng/mL) for 15 min, and expression of listed proteins is shown (A and B). HUVECs were further cultured, and cell proliferation (C) and in vitro migration (D) were tested. * P< 0.05 versus “Vec” cells. “N. S.” denotes P > 0.05. Scale bar = 100 μm.

Figure 5

Gαi1 and Gαi3 overexpression strengthens SCF-induced signaling and pro-angiogenic activity in endothelial cells. HUVECs were transduced with the lentiviral human Gαi1-expressing construct plus the lentiviral human Gαi3-expressing vector, and two stable colonies, “oeGαi1/3-Slc1” and “oeGαi1/3-Slc2”, were obtained after selection. Control HUVECs were transduced with vector control (“Vec”). HUVECs were then treated with SCF (50 ng/mL) for 15 min and listed mRNA and protein levels were examined (A-C). HUVECs were further cultured, and cell proliferation (D), in vitro migration (E), and capillary tube formation (F) were tested. * P< 0.05 versus “Vec”. “N. S.” denotes P > 0.05.

Figure 6

Endothelial Gαi1/3 silencing prevents SCF-induced signaling and retinal angiogenesis in vivo. One-month-old C57B/6 adult mice with AAV5-TIE1-Gαi1 shRNA plus AAV5-TIE1-Gαi3 shRNA (“Gαi1/3-eKD”) or the AAV5-TIE1-scramble shRNA control (“Ct”) were injected intravitreously with SCF (0.5 ng in 0.2 μL). After 20 min, the retinal tissues were collected and expressions of listed mRNAs and proteins in fresh tissues are shown (A-D). Alternatively, the retinal vasculature was visualized via IB4 staining after 48 h (E). The expressions of listed mRNAs are shown (F-H). * P < 0.05 versus vehicle control (“Veh”, saline) # P < 0.05 vs. “Ct” group. “N. S.” denotes P > 0.05. Scale bar = 100 μm.

Figure 7

SCF shRNA inhibits pathological retinal angiogenesis in diabetic retinopathy (DR) mice. The retinal tissues of DR mice (90 days after the last STZ administration) and “mock” control mice (with citrate buffer administration) were separated, expressions of SCF mRNA and protein were tested, and the results were quantified (A-C). Day-30 after STZ administration, mice were injected intravitreously with AAV5-packed SCF shRNA (“shSCF-AAV5”, at 0.1 μL) or AAV5-packed scramble shRNA control (“shC-AAV5”, at 0.1 μL). After another 60 days, listed mRNAs and proteins in the retinal tissues were assessed (D-F, J-L). Alternatively, mice were infused with Evans blue (EB) for 2 h, and the percentage of EB leakage was quantified (G). IB4 staining was carried out to visualize the retinal vasculature (H, scale bar = 50 μm), and the average number of vascular branches were quantified (H). The retinal trypsin digestion assay was performed and the number of acellular capillaries per view were recorded (I). “Mock” refers to mice administered with citrate buffer. * P< 0.05.

Figure 8

SCF shRNA ameliorates degeneration of retinal ganglion cells (RGCs) in diabetic retinopathy (DR) mice. Day-30 after STZ administration, mice were injected intravitreously with AAV5-packed SCF shRNA (“shSCF-AAV5”, at 0.1 μL) or AAV5-packed scramble shRNA control (“shC-AAV5”, at 0.1 μL). After another 60 days, NeuN-positive RGCs in GCL were detected (A and B, scale bar = 50 μm). The listed human tissues were homogenized and mRNA and protein expressions of SCF and c-Kit were examined (C-E). “Mock” refers to mice administered with citrate buffer. “GCL” is the ganglion cell layer; “ONL” is the outer nuclear layer; “INL” is the inner nuclear layer. * P< 0.05 (A and B). * P< 0.05 vs. “Ctrl” tissues (C-E).