PRAS40 acts as a nodal regulator of high glucose-induced TORC1 activation in glomerular mesangial cell hypertrophy

Abstract

Diabetic nephropathy manifests aberrant activation of TORC1, which senses key signals to modulate protein synthesis and renal hypertrophy. PRAS40 has recently been identified as a raptor-interacting protein and is a component and a constitutive inhibitor of TORC1. The mechanism by which high glucose stimulates TORC1 activity is not known. PRAS40 was identified in the mesangial cells in renal glomeruli and in tubulointerstitium of rat kidney. Streptozotocin-induced diabetic renal hypertrophy was associated with phosphorylation of PRAS40 in the cortex and glomeruli. In vitro, high glucose concentration increased PRAS40 phosphorylation in a PI 3 kinase- and Akt-dependent manner, resulting in dissociation of raptor-PRAS40 complex in mesangial cells. High glucose augmented the inactivating and activating phosphorylation of 4EBP-1 and S6 kinase, respectively, with concomitant induction of protein synthesis and hypertrophy. Expression of TORC1-nonphosphorylatable mutant of 4EBP-1 and dominant-negative S6 kinase significantly inhibited high glucose-induced protein synthesis and hypertrophy. PRAS40 knockdown mimicked the effect of high glucose on phosphorylation of 4EBP-1 and S6 kinase, protein synthesis, and hypertrophy. To elucidate the role of PRAS40 phosphorylation, we used phosphorylation-deficient mutant of PRAS40, which in contrast to PRAS40 knockdown inhibited phosphorylation of 4EBP-1 and S6 kinase, leading to reduced mesangial cell hypertrophy. Thus, our data identify high glucose-induced phosphorylation and inactivation of PRAS40 as a central node for mesangial cell hypertrophy in diabetic nephropathy.

Figure 1

Hyperglycemia induces kidney hypertrophy and phosphorylation of PRAS40 in rats. Diabetes was induced by injecting streptozotocin into rats and kidneys were harvested as described in Materials and Methods. Three groups of animals were used, control, diabetes and diabetes with insulin treatment. (A and B) Renal hypertrophy is expressed as a ratio of kidney weight to total body weight at sacrifice (panel A) and prior to streptozotocin injection (panel B). Panel C shows the kidney weight of the animals at sacrifice. C, control: n= 5; D, diabetes; n = 6; DI, diabetes with insulin; n = 6. In panel A, *p < 0.001 vs. control, **p < 0.001 vs diabetes obtained by ANOVA. In panel B, *p < 0.05 vs. control, **p < 0.05 vs diabetes. In panel C, *p < 0.05 vs. control, **p < 0.05 vs diabetes. (D) Localization of PRAS40 in rat kidney. Acetone-fixed renal sections were stained with nonimmune rabbit IgG or PRAS40 antibody as described in the Materials and Methods. Panel a, IgG control; Panel b, PRAS40 staining. (E) Kidney cortical tissues were lysed in RIPA buffer and lysates were immunoblotted with phospho-PRAS40 (upper panel) and PRAS40 antibodies (lower panel), respectively. C, control animal; D, diabetic animal; DI, diabetic animal treated with insulin. (F) Quantification of the data presented in Fig. 1E. n = 3 animals *p < 0.001 vs. control; **p < 0.01 vs diabetes, obtained by ANOVA. (G) Glomeruli from these rats were isolated as described in the Materials and Methods. Glomerular lysates were immunoblotted with phospho-PRAS40 (upper panel) and PRAS40 antibodies (lower panel), respectively. C, control; D, diabetic. DI, diabetic animal treated with insulin. (H) Quantification of the data presented in 1G. n = 3 animals *p < 0.001 vs. control; **p < 0.001 vs diabetic, obtained by ANOVA.

Figure 2

Phosphorylation of PRAS40 coincides with activation of PI 3 kinase and Akt. (A – C) Mesangial cells were incubated with 25 mM glucose for indicated periods of time. 5 mM glucose + 20 mM mannitol was used as the osmotic control (lane 1). Lysates were immunoblotted with phospho-PRAS40 (panel A), phospho-Akt (panel B) and phospho-p85 (panel C) antibodies (top panels). Middle panels show immunoblotting of the same samples with PRAS40, Akt and p85 antibodies and bottom panels show immunoblotting with actin antibody. Representative blots from four independent experiments are shown in panels A and B. Representative of two independent experiments is shown in panels C and D. Quantification of these data is shown in Supplemental Fig. S2A-S2C. (D) High glucose increases PI 3 kinase activity in mesangial cells. Anti-phospho-tyrosine immunoprecipitates of lysates of cells harvested at indicated time-points were assayed for PI 3 kinase activity as described in the Materials and Methods. Quantification of these data is shown in Supplemental Fig. S2D.

Figure 3

PI 3 kinase/Akt signaling regulates phosphorylation of PRAS40. (A) Mesangial cells were incubated with 25 μM of Ly294002 for 1 hour prior to incubation with high glucose (HG) for 15 minutes. (B – D) Mesangial cells were infected with 50 moi of AdΔ p85 (panel B), Ad PTEN (panel C) and Ad DN Akt (panel D) for 48 hours prior to incubation with high glucose for 15 minutes (HG). As control, infection with Ad GFP was used. Also 5 mM glucose plus 20 mM mannitol was used as control (LG). Equal amounts of cell lysates were immunoblotted with indicated antibodies. Representative of three independent experiments is shown for each panel. Quantification of the data is presented in Supplementary Fig. S3.

Figure 4

High glucose induces dissociation of PRAS40-Raptor complex and phosphorylation of 4EBP-1 and S6 kinase. (A and B) Mesangial cells were incubated with high glucose (HG, 25 mM glucose) for 15 min. LG, low glucose (5 mM glucose plus 20 mM mannitol). The cell lysates were immunoprecipitated with Raptor (panel A) and PRAS40 (panel B) antibodies respectively. The immunoprecipitates were immunoblotted with PRAS40 and Raptor antibodies as indicated (top two panels). Cell lysates were immunoblotted with PRAS40 and Raptor antibodies as indicated (bottom two panels). (C and D) Mesangial cells were incubated with high glucose as described in the Fig. 2 legends. Cell lysates were immunoblotted with phospho-4EBP-1 (p4EBP-1), 4EBP-1, phospho-S6 kinase (pS6K), S6 kinase (S6K) and actin antibodies, as indicated. Representative of four (panel A and B) and three (panels C and D) independent experiments is shown. Quantification of the data presented in each panel is shown in Supplementary Fig. S4A – S4D.

Figure 5

High glucose mediated phosphorylation of 4EBP-1 and S6 kinase is PI 3 Kinase- and Akt-dependent. (A) Mesangial cells were incubated with 25 μM of Ly294002 for 1 hour prior to incubation with HG for15 minutes as described in the Fig. 3A legend. (B – D) Mesangial cells were infected with 50 moi of Ad Δ p85 (panel B), Ad PTEN (panel C) and Ad DN Akt (panel D) as described in the legends of Fig. 3B-3D. Cell lysates were immunoblotted with phospho-4EBP-1 (p-4EBP-1), 4EBP-1, and other indicated antibodies (left panels). In the right panels, the cell lysates were immunoblotted with phospho-S6 kinase (p-S6K), S6 kinase (S6K) and indicated antibodies. Representative of three independent experiments is shown. Quantification of these data is shown in Supplementary Fig. S5A – S5D.

Figure 6

4EBP-1 and S6 kinase regulate high glucose-induced protein synthesis and hypertrophy. Mesangial cells were infected with 50 moi of Ad 4EBP-1μ (panels A, B and C) and transfected with dominant negative S6 kinase (D and E) as described in the Materials and Methods. Cells were treated with 25 mM glucose (HG) for 24 hours. Control cells were treated with 5 mM glucose + 20 mM mannitol (LG) for 24 hours. (A and D) Protein synthesis was determined by ³⁵S Methionine incorporation as described in Materials and Methods (Mahimainathan et al., 2006; Senthil et al., 2002). Mean ± SE of three measurements is shown. *p < 0.01 vs. control; **p < 0.05 vs. HG in panel A; *p < 0.05 vs. control; **p < 0.05 vs. HG in panel D. (C) Treated cells were fixed and the surface area of a minimum of 20 cells were determined using a polygonal measurement tool as described in the Materials and Methods. *p < 0.001 vs. control; **p < 0.001 vs. HG. Bottom panels show immunoblotting of cell lysates with 4EBP-1, HA and actin antibodies to show 4EBP-1 and dominant negative S6 kinase expression levels. (B and E) Cellular hypertrophy was determined by measurement of total protein per cell as described in Materials and Methods (Mahimainathan et al., 2006). Mean ± SE of three measurements is shown. *p < 0.05 vs. control; **p < 0.01 vs. HG in panel B; *p < 0.01 vs. control; **p < 0.05 vs. HG in panel E. Bottom panels show immunoblotting of cell lysates with 4EBP-1, HA and actin antibodies to show 4EBP-1 and dominant negative S6 kinase expression levels.

Figure 7

shRNA-mediated downregulation of PRAS40 increases phosphorylation of 4EBP-1 and S6 kinase. (A) Mesangial cells were transfected with a plasmid expressing PRAS40-specific shRNA (shPRAS40) or plasmid expressing scrambled RNA. Cell lysates were immunoblotted with PRAS40 and actin antibodies as indicated. (B and C) Mesangial cells were transfected with shPRAS40 or scrambled RNA expression plasmids. Transfected cells were incubated with high glucose for 15 minutes as described in the legends to the Fig. 3A and 3B. Mannitol was used as the osmotic control. Lysates were immunoblotted with phospho-4EBP-1 (p-4EBP-1), PRAS40, 4EBP-1, phospho-S6 kinase (p-S6K), S6 kinase (S6K) and actin antibodies as indicated. Representative of three independent experiments for panels B and C is shown. Quantification of these data is shown in Supplemental Fig. S7A and S7B.

Figure 8

shRNA-targeted downregulation of PRAS40 increases protein synthesis and hypertrophy. Mesangial cells were transfected with shPRAS40 and scrambled RNA as described in the Fig. 7B and 7C. The transfected cells were incubated with high glucose (HG) for 24 hours as described in the legend of Fig. 6. Mannitol was used as the osmotic control as described in Fig. 6. (A) Protein synthesis was determined by ³⁵S Methionine incorporation as described in Materials and Methods. Mean ± SE of three measurements is shown. *p < 0.05 vs. control; (B) Cellular hypertrophy was determined by measurement of total protein per cell as described in the Materials and Methods (Mahimainathan et al., 2006). Mean ± SE of three measurements is shown. *p < 0.01 vs. control. Bottom panels show immunoblotting of cell lysates with PRAS40 and actin antibodies as indicated to show expression levels.

Figure 9

Phosphorylation of PRAS40 at Thr246 is critical for subsequent phosphorylation of 4EBP-1 (panel A), S6 kinase (panel B), and mTOR (panel C). Mesangial cells were transfected with plasmid expressing HA-tagged phosphorylation defective mutant of PRAS40 (PRAS40T246A) or vector. The transfected cells were treated with high glucose (HG) for 15 minutes as described in the legends of Fig. 3A and 3B. Mannitol was used as the osmotic control. Lysates were immunoblotted with phospho-4EBP-1 (p-4EBP-1), HA (to detect PRAS40T246A expression), 4EBP-1, phospho-S6 kinase (p-S6K), S6 kinase (S6K), phospho-mTOR (p-mTOR), mTOR and actin antibodies as indicated in each panel. Representative of three independent experiments is shown for each panel. Quantification of these data is presented in Supplementary Fig. S8A – S8C.

Figure 10

Thr246 phosphorylation of PRAS40 is critical for protein synthesis and hypertrophy in mesangial cells in the absence of apoptosis. Mesangial cells were transfected with the PRAS40T246A mutant plasmid or vector plasmid. Transfected cells were treated with high glucose (HG) for 24 hours as described in the legends to the Fig. 6. (A) Protein synthesis was determined by ³⁵S-Methionine incorporation as described in Materials and Methods. Mean ± SE of three measurements is shown. *p < 0.01 vs. control; **p < 0.01 vs. high glucose-treated. (B) Cellular hypertrophy was determined by measurement of total protein per cell as described in Materials and Methods (Mahimainathan et al., 2006). Mean ± SE of three measurements is shown. *p < 0.05 vs. control; **p < 0.05 vs. high glucose treated. Bottom panels show immunoblotting of cell lysates with HA and actin antibodies as indicated to show expression levels. (C) Apoptosis of mesangial cells was determined using Annexin V method as described in the Materials and Methods (Das et al., 2007; Ghosh Choudhury et al., 2003; Venkatesan et al., 2008). The numbers in the bottom right quadrants show percentage of mesangial cells apoptosis.