G protein α12 (Gα12) is a negative regulator of kidney injury molecule-1-mediated efferocytosis

Abstract

Kidney injury molecule-1 (KIM-1) is a receptor for the "eat me" signal, phosphatidylserine, on apoptotic cells. The specific upregulation of KIM-1 by injured tubular epithelial cells (TECs) enables them to clear apoptotic cells (also known as efferocytosis), thereby protecting from acute kidney injury. Recently, we uncovered that KIM-1 binds directly to the α-subunit of heterotrimeric G₁₂ protein (Gα₁₂) and inhibits its activation by reactive oxygen species during renal ischemia-reperfusion injury (Ismail OZ, Zhang X, Wei J, Haig A, Denker BM, Suri RS, Sener A, Gunaratnam L. Am J Pathol 185: 1207-1215, 2015). Here, we investigated the role that Gα₁₂ plays in KIM-1-mediated efferocytosis by TECs. We showed that KIM-1 remains bound to Gα₁₂ and suppresses its activity during phagocytosis. When we silenced Gα₁₂ expression using small interefering RNA, KIM-1-mediated engulfment of apoptotic cells was increased significantly; in contrast overexpression of constitutively active Gα₁₂ (QLGα₁₂) resulted in inhibition of efferocytosis. Inhibition of RhoA, a key effector of Gα₁₂, using a chemical inhibitor or expression of dominant-negative RhoA, had the same effect as inhibition of Gα₁₂ on efferocytosis. Consistent with this, silencing Gα₁₂ suppressed active RhoA in KIM-1-expressing cells. Finally, using primary TECs from Kim-1^+/+ and Kim-1^-/- mice, we confirmed that engulfment of apoptotic cells requires KIM-1 expression and that silencing Gα₁₂ enhanced efferocytosis by primary TECs. Our data reveal a previously unknown role for Gα₁₂ in regulating efferocytosis and that renal TECs require KIM-1 to mediate this process. These results may have therapeutic implications given the known harmful role of Gα₁₂ in acute kidney injury.

Keywords: G protein; Gα12; kidney; kidney injury molecule-1 (KIM-1); phagocytosis.

Fig. 1.

Kidney injury molecule-1 (KIM-1) mediates the uptake of apoptotic cells (AC). A: various cell lines were screened for level of KIM-1 and Gα₁₂ expression via Western blotting. These cells included human embryonic kidney-293 (HEK-293), porcine proximal tubular epithelial cells (LLC-PK₁), and human renal adenocarcinoma cell lines (769P). HEK-293 transfected with human KIM-1 and Gα₁₂ served as a positive control. Lysates were obtained and run on Western blots and immunoblotted (IB) for KIM-1, Gα₁₂, and actin. B: HEK-293 stably expressing either a control vector (pcDNA-GFP) or KIM-1-green fluorescent protein (GFP) were fed pHrodo-labeled (Red) AC for various time points (in hours) as indicated. The percentage of phagocytosis was determined by flow cytometry measurement of GFP- and high pHrodo Red double-positive cells (n = 3). *P < 0.05, **P < 0.005, ***P < 0.01 by unpaired t-test. C: HEK-293 cells stably expressing KIM-1-GFP were fed pHrodo-labeled AC (red) for various time points. Cells were fixed and stained with rhodamine-phalloidin to visualize F-actin (red) and 4,6-diamidino-2-phenylindole (DAPI) to visualize the nucleus and apoptotic cells (blue; ×600; bars = 5 μm). AC labeled with both pHrodo Red and DAPI (blue) appear as purple/pink in the merged image. Arrows indicates the AC in the vicinity of KIM-1. D: HEK-293 stably expressing KIM-1-GFP were treated with vehicle (DMSO) or the actin polymerization inhibitor cytochalasin D (1 μM) for 30 min and were then fed fluorescently labeled AC for 90 min. The percentage of phagocytosis represents the percentage of KIM-1-GFP-positive cells with high fluorescence of pHrodo Red of engulfed AC as measured by flow cytometry (n = 3). **P < 0.005 by unpaired t-test.

Fig. 2.

KIM-1 interacts with Gα₁₂ during efferocytosis. A: HEK-293 cells stably expressing human KIM-1-GFP with a high level of endogenous Gα₁₂ were stimulated with AC for indicated time points and used for coimmunoprecipitation (IP) using antibody against Gα₁₂. Densitometric ratio of the coimmunoprecipitated KIM-1 during AC stimulation compared with unstimulated sample was quantified (n = 3). *P < 0.05 1-way ANOVA. B: glutathione-S-transferase (GST) and GST-Gα₁₂ pull down was performed on HEK-293-KIM-1-GFP cells that were left unstimulated or stimulated with AC for 90 min. All samples were analyzed by SDS-PAGE followed by IB with antibodies against KIM-1, Gα₁₂, and actin. The input lane represents 5% of the lysate. The data represent 3 independent experiments. C: HEK-293 cells were transfected with KIM-1-wild-type (WT) and a GFP-tagged Gα₁₂ construct and fed pHrodo Red fluorescently labeled AC (blue/red) for 30 min before fixation, and detection of surface KIM-1 using anti-KIM-1 antibody against mucin domain (AKG) and Alexa 555-conjugated secondary antibody (×600; bars = 40 μm). Arrows indicate where phagocytic cup formed. D: images of colocalization of both KIM-1 and Gα₁₂ were analyzed using the Van Steensel approach, where the cross correlation function (CCFs) was calculated with a pixel shift ± 20. The results shown are representative of 4 images of 3 independent experiments (1-way ANOVA).

Fig. 3.

KIM-1 downregulates Gα₁₂ activity during KIM-1-mediated efferocytosis. HEK-293 stably expressing KIM-1-GFP (A) or pcDNA (control vector; B) were either left untreated (none) or fed AC for various time points (15, 30, 60, and 90 min). Cell lysate were subjected to a GST-tetratricopeptide repeat (TPR) pull-down assay to measure active Gα₁₂ as described in experimental procedures. Samples were analyzed by SDS-PAGE followed by IB with antibodies against Gα₁₂ and actin. The total Gα₁₂ lanes represent 5% of the lysate were used for the GST-TPR pull down. Densitometric analysis of the relative ratio of the active Gα₁₂ to total Gα₁₂ compared with nontreated samples is shown as numbers below blots. The average of the ratio of the active to total Gα₁₂ compared with no treatment was blotted as a representation of 3 independent experiments (n = 3). *P < 0.05 by unpaired t-test.

Fig. 4.

Gα₁₂ negatively regulates KIM-1-mediated efferocytosis. A: HEK-293 cells stably expressing wild-type KIM-1 were transfected with either a control vector encoding GFP (pcDNA-GFP), wild-type GFP-tagged Gα₁₂ (pcDNA-WTGα₁₂-GFP), or constitutively active GFP-tagged Gα₁₂ (pcDNA-QLGα₁₂-GFP). B: LLC-PK₁ cells stably expressing KIM-1 were transfected with the same constructs as in A. Cells in A and B were fed AC that are fluorescently labeled with pHrodo Red for 90 min. The percentage of uptake of AC was determined by flow cytometry as described in experimental procedures. The ratio of phagocytosis compared with the control vector was determined by flow cytometry after gating on GFP-positive cells (n = 3). *P < 0.05, ***P < 0.001 by unpaired t-test. C: HEK-293 cells stably expressing wild-type KIM-1 were grown on coverslips and transfected with either pcDNA-GFP or pcDNA-QLGα₁₂-GFP and fed fluorescently labeled (pHrodo Red) AC for 90 min. Confocal microscopy images were taken to quantify the number of AC bound to the phagocytic cells. The results were plotted as a representation of 8 sections from 4 independent experiments (n = 4; ×400; bars = 5 μm). *P < 0.05. D: HEK-293 cells stably expressing KIM-1 were transfected with 50 pmol of control small interfering (si) RNA or siRNA against Gα₁₂ for 24 h, and level of Gα₁₂, KIM-1, and actin was determined by IB with indicated antibodies. A graph of the densitometric ratio of the Gα₁₂ in siRNA-treated samples to nontransfected samples represents 4 independent experiments (n = 4). *P < 0.05 by unpaired t-test. E: HEK-293-KIM-1-GFP-expressing cells transfected with either control or Gα₁₂ siRNA were fed fluorescently labeled (pHrodo Red) AC to measure phagocytosis as in A and B. F: cells in E were visualized with confocal microscopy to count the number of AC. The number of AC bound to the cells that were seen in each field was plotted based on analysis of 8 representative sections from each of 4 independent experiments with ∼120 GFP-positive (KIM-1-GFP) cells counted for each section (n = 4). *P < 0.05 by unpaired t-test.

Fig. 5.

RhoA is a downstream mediator of Gα₁₂ in KIM-1-mediated phagocytosis. A: HEK-293 cells stably expressing KIM-1 were treated for 4 h with vehicle (DMSO), Rho inhibitor (cell-permeable C3 transferase; 0.5 μg/ml), or Rho kinase (ROCK) inhibitor Y27632 (10 μM). B: cells were transfected with cyan fluorescent protein (CFP)-tagged control vector, CFP-tagged wild-type RhoA, CFP-tagged dominant negative RhoA (T19N), or CFP-tagged constitutively active RhoA (Q62L). These cells (A and B) were incubated with pHrodo Red-labeled AC for 90 min, and the percentage of phagocytosis by CFP-positive cells was measured by flow cytometry (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired t-test. C: HEK-293 cells stably expressing KIM-1 were transfected with either control siRNA or siRNA against Gα₁₂ for 24 h and then stimulated with AC for 30 or 90 min. D: HEK-293-KIM-1 cells were transfected with a control vector or constitutively active Gα₁₂ (QLGα₁₂). Levels of active RhoA (in C and D) were measured using a G-LISA RhoA activation assay kit. Total RhoA levels in lysates were measured using an RhoA ELISA. The levels of normalized active RhoA were calculated and graphed (n = 3). *P < 0.05, ***P < 0.001 by unpaired t-test.

Fig. 6.

RhoA activity is increased during the course of KIM-1-mediated efferocytosis. HEK-293 stably expressing KIM-1-GFP (A) or pcDNA (control vector; B) were fed AC for various time points (15, 30, 60, and 90 min). Cell lysate were loaded with nonhydrolyzable GTP (GTPyS) as a positive control or GDP as a negative control. The level of active RhoA was measured using an active RhoA (RhoA-GTP) pull-down assay as described in experimental procedures. Samples were analyzed by SDS-PAGE followed by IB with antibodies against RhoA and actin. Densitometric analysis of the relative ratio of active RhoA to the respective total fractions compared with the untreated sample is shown as numbers below blots. The average ratio of active to total RhoA compared with no treatment was graphed as a representation of 3 independent experiments (n = 3). *P < 0.05 by unpaired t-test.

Fig. 7.

Rac1 is involved in KIM-1-mediated efferocytosis. A: HEK-293 cells were cotransfected with a construct encoding wild-type KIM-1 and either a CFP-tagged control vector (control) or CFP-tagged dominant negative Rac1 (Rac1-T17N). B: HEK-293-KIM-1 cells were treated for 4 h with vehicle (DMSO) or a Rac1 inhibitor (NSC23766; 50 μM). Cells in A and B were fed to pHrodo-labeled AC for 90 min, and the percentage of uptake was measured by flow cytometry. In A, the percentage of pHrodo Red high fluorescent cells was determined for CFP-positive cells (n = 3). **P < 0.01, ***P < 0.001 by unpaired t-test.

Fig. 8.

KIM-1 is a major phagocytic receptor in proximal tubular epithelial cells (TECs), and Gα₁₂ is a negative regulator. A: proximal TECs isolated from C57BL/6 wild-type (Kim-1^+/+) and Kim-1 knockout (Kim-1^−/−) mice were cultured, and the level of mouse Kim-1 expressed was measured by Western blotting. B: surface expression of mouse Kim-1 on TECs was determined by flow cytometry using PE-conjugated anti-mouse Tim-1/Kim-1 antibody (R&D Systems; n = 3). *P < 0.05 by unpaired t-test. C: TECs were fed pHrodo red-labeled AC for various time points (in hour), and the percentage of phagocytosis of fluorescently labeled AC was measured by flow cytometry (n = 3). *P < 0.05 by 1-way ANOVA. D: TECs were cultured on glass coverslips and fed green-labeled AC for 3 h. Cells were fixed and surface stained for mouse Kim-1 using an anti-mouse Kim-1 antibody and Cy5-conjugated secondary antibody (×600; bars = 10 μm). E: TECs isolated from wild-type C57BL/6 mice were left untreated (live), subjected to UV for 3 min, followed by incubation overnight (to stimulate apoptosis), or subjected to heat shock at 65°C for 15 min (to stimulate necrosis). The different type of cell death was confirmed using PI and annexin V staining followed by flow cytometry. Data represent 3 independent experiments. F: live, apoptotic, or necrotic cells labeled with pHrodo Red were fed to either Kim-1^+/+ or Kim-1^−/− TECs, and the percentage of phagocytosis was measured using flow cytometry (n = 3) **P < 0.005 by unpaired t-test. G: TECs isolated from wild-type C57BL/6 mice were transfected with 50 pmol of control siRNA or siRNA against mouse Gα₁₂ for 24 h, and the level of Gα₁₂, mouse Kim-1, and actin was determined by Western blotting. H: Gα₁₂ siRNA-treated cells were subjected to phagocytosis assay, and the percentage of phagocytosis of pHrodo-labeled AC was measured by flow cytometry (n = 4). *P < 0.5 by unpaired t-test.