Supplementary MaterialsNIHMS954252-supplement-supplement_1. process. The transcription factor Hif1 is a target of Akt kinase. siRNAs against Hif1 inhibited the high glucose-induced mesangial cell hypertrophy. In contrast, increased expression of Hif1 induced hypertrophy similar to high glucose. We found that inhibition of PDGFR and expression of PDGFR Y740/751F mutant significantly inhibited the high glucose-induced expression of Hif1. Importantly, expression of Hif1 AMD3100 reversible enzyme inhibition countered the inhibition of mesangial cell hypertrophy induced by siPDGFR or PDGFR Y740/751F mutant. Finally, we show that high glucose-stimulated PDGFR tyrosine phosphorylation at 740/751 residues and the tyrosine kinase activity of the receptor regulate the transforming growth factor- (TGF) expression by Hif1. Thus we define the cell surface PDGFR as a major link between high glucose and its effectors Hif1 and TGF for induction of diabetic mesangial cell hypertrophy. for 20 min at 4 C. The supernatant was collected. After determining the protein concentration, equal amounts of proteins were separated by SDS polyacrylamide gel electrophoresis. The separated proteins were transferred to PVDF membrane. The membrane containing the proteins was immunoblotted with indicated antibodies. The AMD3100 reversible enzyme inhibition protein bands were developed with HRP-conjugated secondary antibody using ECL reagent as described previously [4,22,23]. For immunoprecipitation, equal amounts of proteins were incubated with the indicated antibody on ice for 30 min. Protein G agarose conjugated beads were then added. The mixture was rotated at 4 C for overnight. The immune beads were washed with RIPA buffer and resuspended in SDS sample buffer [21]. The denatured proteins were separated by electrophoresis. The separated proteins were then immunoblotted with the indicated antibody as described above. 2.4. Transfection The mesangial cells were seeded at 80% confluency. Next day, the cells were transfected with the plasmid vectors or siRNAs against PDGFR using FuGENE HD according to vendors protocol as described previously [4,22]. After 24 h of transfection, the cells were starved in serum free medium and treated with high glucose as described above. 2.5. Protein synthesis After incubation with high glucose, the mesangial cells were incubated with 35S-methionine and protein synthesis was determined as [35S]-methionine incorporation as described previously [4,5,22]. 2.6. Measurement of cellular hypertrophy At the end of the incubation period, the mesangial cells were trypsinized. The cells were counted in a hemocytometer. After counting, the cells were centrifuged at 4000 at 4 C. The cell pellet was washed with PBS and lysed in RIPA buffer as described above. The protein content in the cells was determined. Hypertrophy was expressed as an increase in the ratio of total cellular protein content to the cell number as described previously [4,22]. 2.7. Statistics The mean SE of indicated measurements is shown. The significance of the results was determined using the Graph Pad Prism software. Analysis of variance followed by Students-Newman-Keuls analysis was used as described previously [24,25]. A p value of 0.05 was considered significant. 3. Results 3.1. Tyrosine phosphorylation of PDGFR is necessary for high glucose-induced PI 3 kinase phosphorylation Recent work demonstrated that the regulatory subunit of PI RBX1 3 AMD3100 reversible enzyme inhibition kinase, the p85 protein, is tyrosine phosphorylated when the PI 3 kinase is activated [26]. We examined the AMD3100 reversible enzyme inhibition tyrosine phosphorylation of p85 by high glucose. Incubation of mesangial cells with high glucose increased the phosphorylation of p85 at Tyr-458 in a time-dependent manner (Fig. 1A). Since PI 3 kinase is activated by growth factor receptors and recent report showed increased expression of PDGFR in diabetic renal glomeruli [18], we tested the status of tyrosine phosphorylation of this receptor in mesangial cells. High glucose time-dependently enhanced the autophosphorylation of PDGFR at Tyr-857 (Fig. 1B). To determine the requirement of the tyrosine kinase activity of PDGFR for phosphorylation of p85 subunit of PI 3 kinase, we used JNJ-10198409 (JNJ), a PDGFR inhibitor [27]. JNJ inhibited the tyrosine phosphorylation of p85 concomitant with attenuation of the autophosphorylation of the PDGFR (Fig. 1C). Furthermore, the requirement of PDGFR for p85 phosphorylation was also confirmed by siRNAs against this tyrosine kinase (Fig. 1D). Open in a separate window Fig. 1 High glucose increases the association of PI 3 kinase with the PDGFR 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-PDGFR (Tyr-857), phospho-PDGFR (Tyr-740), phospho-PDGFR (Tyr-751) and PDGFR antibodies as indicated. (C, F and AMD3100 reversible enzyme inhibition G) Mesangial cells were treated with 0.1 M JNJ prior to incubation with high.