Tyrosines-740/751 of PDGFRβ contribute to the activation of Akt/Hif1α/TGFβ nexus to drive high glucose-induced glomerular mesangial cell hypertrophy

Abstract

Glomerular mesangial cell hypertrophy contributes to the complications of diabetic nephropathy. The mechanism by which high glucose induces mesangial cell hypertrophy is poorly understood. Here we explored the role of the platelet-derived growth factor receptor-β (PDGFRβ) tyrosine kinase in driving the high glucose-induced mesangial cell hypertrophy. We show that high glucose stimulates the association of the PDGFRβ with PI 3 kinase leading to tyrosine phosphorylation of the latter. High glucose-induced Akt kinase activation was also dependent upon PDGFRβ and its tyrosine phosphorylation at 740/751 residues. Inhibition of PDGFRβ activity, its downregulation and expression of its phospho-deficient (Y740/751F) mutant inhibited mesangial cell hypertrophy by high glucose. Interestingly, expression of constitutively active Akt reversed this inhibition, indicating a role of Akt kinase downstream of PDGFRβ phosphorylation in this process. The transcription factor Hif1α is a target of Akt kinase. siRNAs against Hif1α inhibited the high glucose-induced mesangial cell hypertrophy. In contrast, increased expression of Hif1α induced hypertrophy similar to high glucose. We found that inhibition of PDGFRβ and expression of PDGFRβ Y740/751F mutant significantly inhibited the high glucose-induced expression of Hif1α. Importantly, expression of Hif1α countered the inhibition of mesangial cell hypertrophy induced by siPDGFRβ or PDGFRβ Y740/751F mutant. Finally, we show that high glucose-stimulated PDGFRβ tyrosine phosphorylation at 740/751 residues and the tyrosine kinase activity of the receptor regulate the transforming growth factor-β (TGFβ) expression by Hif1α. Thus we define the cell surface PDGFRβ as a major link between high glucose and its effectors Hif1α and TGFβ for induction of diabetic mesangial cell hypertrophy.

Keywords: Diabetic nephropathy; Receptor tyrosine kinase; Renal hypertrophy.

Fig. 1

High glucose increases the association of PI 3 kinase with the PDGFRβ leading to its phosphorylation. (A, B and E) Mesangial cells were incubated with high glucose (HG, 25 mM glucose) for the indicated time periods. As control (0 h), 20 mM mannitol plus 5 mM glucose was used as described in the Materials and methods section. Equal amounts of cell lysates were immunoblotted with phospho-p85 (Tyr-458), p85, phospho-PDGFRβ (Tyr-857), phospho-PDGFRβ (Tyr-740), phospho-PDGFRβ (Tyr-751) and PDGFRβ antibodies as indicated. (C, F and G) Mesangial cells were treated with 0.1 μM JNJ prior to incubation with high glucose (HG) and normal glucose (NG) for 24 h. In panel C, the cell lysates were immunoblotted with the indicated antibodies. In panels F and G, the cell lysates were immunoprecipitated with PDGFRβ (panel F) or with p85 PI 3 kinase subunit (panel G) antibodies. The immunoprecipitates were immunoblotted with p85 (panel F) or PDGFRβ (panel G) antibodies, respectively. (D and H) Mesangial cells were transfected with scramble siRNA or siRNAs against PDGFRβ (panel D) or with vector or PDGFRβ (Y740/Y751F) mutant as described in the Materials and methods section. The transfected cells were incubated with high glucose (HG) or normal glucose (NG) as described above. The cell lysates were immunoblotted with the indicated antibodies.

Fig. 2

PDGFRβ regulates high glucose-stimulated Akt kinase activity. (A and B) Mesangial cells were treated with 0.1 μM JNJ for 1 h prior to incubation with high glucose (HG) or normal glucose (NG) for 24 h. Equal amounts of cell lysates were immunoblotted with the indicated antibodies. (C–F) Mesangial cells were transfected with siPDGFRβ (panels C and D) or with PDGFRβ Y740/751F (E and F) mutant. The cells were then incubated with high glucose (HG) or normal glucose (NG) for 24 h. The cell lysates were immunoblotted with indicated antibodies.

Fig. 3

PDGFRβ controls mesangial cell hypertrophy by high glucose. (A and B) Mesangial cells were treated 0.1 μM JNJ for an hour prior to incubation with high glucose (HG) for 24 h. Protein synthesis was measured by ³⁵S-methionine incorporation as described in the Materials and methods section (panel A). Mean ± SE of triplicate measurements is shown. *p < 0.01 vs NG; **p < 0.01 vs HG. Hypertrophy was determined as the ratio of total amount of protein to cell number as described. Mean ± SE of 6 measurements is shown. *p < 0.001 vs NG; **p < 0.05 vs HG [22,25]. (C–F) Mesangial cells were transfected with siPDGFRβ or scramble (panels C and D) or PDGFRβ mutant (Y740/751F) or vector (panels E and F). The transfected cells were incubated with normal glucose (NG) or high glucose (HG) for 24 h. The protein synthesis and hypertrophy were determined as described above. Mean ± SE of 3–6 measurements is shown. *p < 0.01 vs HG. For panels B and E, **p < 0.05 vs HG; for panels C, D and F, **p < 0.01 vs HG. The bottom panels show the expression of PDGFRβ and actin.

Fig. 4

Akt kinase regulates high glucose-induced PDGFRβ-mediated hypertrophy of mesangial cells. Mesangial cells were transfected with siPDGFRβ and Myr Akt (panels A and B) or PDGFRβ mutant (Y740/751F) and HA Myr Akt (panels C and D) as indicated. The transfected cells were incubated with normal glucose (NG) or high glucose (HG). The protein synthesis and hypertrophy were determined as described in the Materials and methods section [22,25]. Mean ± SE of 4 measurements is shown. *p < 0.01 vs NG; **p < 0.01 vs HG alone; ^@p < 0.01 vs HG + siPDGFRβ or PDGFRβ mutant; ^#p < 0.01 or 0.05 vs NG. The bottom panels show the expression of HA Myr Akt, PDGFRβ and actin.

Fig. 5

Akt-dependent Hif1α regulates mesangial cell hypertrophy. (A and G) Mesangial cells were treated with 0.1 μM JNJ (panel A) or 1 μM MK 2206 (panel G) for 1 h followed by incubation with normal glucose (NG) or high glucose (HG) for 24 h. The cell lysates were immunoblotted with Hif1α and actin antibodies. (B, H and I) Mesangial cells were transfected with dominant negative HA Akt K179M (panel B) or siPDGFRβ (panel H) or PDGFRβ mutant (Y740/751F) (panel I). The transfected cells were incubated with normal glucose or high glucose. The cell lysates were immunoblotted Hif1α, HA, PDGFRβ or actin antibodies as indicated. (C–F) Mesangial cells were transfected with siHif1α or scramble (panels C and D) or HA Hif1α expression vector (panels E and F). The protein synthesis and hypertrophy were determined as described in the Materials and methods section [22,25]. Mean ± SE of 3–6 measurements is shown. *p < 0.01 or 0.001 vs NG; **p < 0.01 vs HG. The bottom panels show the expression of Hif1α.

Fig. 6

Hif1α downstream of PDGFRβ controls high glucose-induced mesangial cell hypertrophy. Mesangial cells were transfected with siPDGFRβ or PDGFRβ mutant (Y740/751F) along with HA Hif1α. The transfected cells were incubated with normal glucose or high glucose. The protein synthesis and hypertrophy were determined as described in the Materials and methods section [22,25]. The bottom panels show the expression of HA Hif1α, PDGFRβ and actin. Mean ± SE of 3–6 measurement is shown. *p < 0. 01, 0.05 or 0.001 vs NG; **p < 0. 01, 0.05 or 0.001 vs HG; ^@p < 0.05, 0.01 or 0.001.

Fig. 7

PDGFRβ-stimulated Hif1α regulates high glucose-induced TGFβ expression. (A) Mesangial cells were treated with 0.1 μM JNJ for 1 h prior to incubation with high glucose (HG) or normal glucose (NG) for 24 h. The cell lysates were immunoblotted with TGFβ or actin antibodies. (B–G) Mesangial cells were transfected with siPDGFRβ (panel B) or PDGFRβ mutant (Y7f40/751F) (panel C) or siHIf1α (panel D) or HA Hif1α (panel E) or siPDGFRβ plus HA Hif1α (panel F) or PDGFRβ mutant (Y740/751F) plus HA Hif1α (panel G). The cell lysates were immunoblotted with TGFβ, PDGFRβ, HA and actin antibodies as indicated.