Insulin-like growth factor binding protein-3 mediates cytokine-induced mesangial cell apoptosis

Abstract

Mesangial cells are critical for glomerular filtration. Mesangial cell dysfunction, the hallmark of diabetic nephropathy, results from disordered mesangial growth induced by cytokines, abnormal hemodynamic influence, and metabolic factors associated with chronic hyperglycemia. Insulin-like growth factors (IGFs) and their high affinity binding proteins (IGFBPs) exert major actions on mesangial cell survival, but their underlying mechanisms remain unclear. In light of emerging IGF-independent roles for IGFBP-3, we investigated IGFBP-3 actions during mesangial cell apoptosis induced by cytokine or high glucose concentration. Quantified by DNA fragmentation ELISA and Annexin V flow cytometry, apoptosis occurred in rat mesangial cells (RMC) exposed to 2 microg/mL IGFBP-3 for 24 h under high ambient or standard glucose. Anti-sense IGFBP-3 oligo at 10 microg/mL significantly inhibited apoptosis induced by 100 ng/mL TNF-alpha, serum-free conditions, or high (25 mM) glucose. Increased IGFBP-3 release associated with high ambient glucose or TNF-alpha was inhibited by pre-treatment with anti-sense oligo. Under serum-free conditions, recombinant human IGFBP-3 blocked Akt phosphorylation at threonine 308 (pThr308), whereas anti-sense oligo treatment was associated with enhanced pThr308 activity. In summary, these data support a novel mechanism for TNF-alpha-induced mesangial cell apoptosis mediated by IGFBP-3 and present regulation of pThr308 activity as a novel mechanism underlying IGFBP-3 action.

Fig. 1

RhIGFBP-3 induced apoptosis in RMC. FACS analysis: The cells were treated with or without 2 μg/mL IGFBP-3 in SFM containing 5 mM glucose for 2 h (a) or 24 h (b). Black lines represent rhIGFBP-3 treatment, and blue lines represent SF control. Annexin (upper panel) reflects apoptosis, and propidium iodide stain (lower panel) depicts necrosis. DNA fragmentation ELISA: (c) cells were treated with or without 2 μg/mL rhIGFBP-3 under 5 or 25 mM glucose-containing media for 24 h. Data were represented as means ± SE (n = 8; *p < 0.05 vs. SFM control by ANOVA).

Fig. 2

Anti-sense IGFBP-3 oligo inhibited SFM-induced apoptosis by DNA fragmentation ELISA. Cells were incubated with dose ranges of oligos for 24 h under SFM containing 5 mM glucose. Data are presented as percentage of SFM control. Histogram depicts means ± SE (n = 8, *p < 0.05, **p < 0.0001 vs. SF control by ANOVA).

Fig. 3

Anti-sense IGFBP-3 oligo inhibited TNF-α-induced apoptosis by FACS. (a) Cells were incubated with 100 ng/mL TNF-α (black lines) or SFM control (blue lines) containing 5 mM glucose for 24 h. Annexin (upper panel) reflects apoptosis, and PI (lower panel) reflects necrosis. Cells pre-treated with or without sense or anti-sense oligo for 30 min, then 100 ng/mL TNF-α for 2 h (b) or 24 h (c). Data presented in the histograms are expressed as percentage of SF control with means ± SE (n = 3; *p < 0.05 vs. SF control; ^#p < 0.05 vs. TNF-α). (d) DNA fragmentation ELISA: cells pre-treated with or without oligo before 24 h of TNF-α under SFM with 5 or 25 mM glucose. Data represented percentage of SF control, with histogram depicting means ± SE (n = 8; *p < 0.05 vs. SF control; ^#p < 0.05 vs. TNF-α by ANOVA).

Fig. 4

DNA fragmentation ELISA. Data represent means ± SE (n = 8; **p < 0.01 vs. 2 h under identical glucose; ^##p < 0.01 vs. 24 h SFM under identical glucose; ^©p < 0.01 vs. 24 h SFM under 5 mM glucose, by ANOVA).

Fig. 5

Anti-sense oligo inhibit TNF-α-induced IGFBP-3 synthesis by Western blot of cell lysates. Cells were pre-treated with or without oligo (10 μg/mL) for 30 min, before incubated with TNF-α under 5 mM or 25 mM glucose for 24 h. Representative blot from three independent experiments.

Fig. 6

Thr-308 Akt phosphorylation ELISA. Data represent percentage of SF control expressed as mean ± SE (n = 7; **p < 0.01 vs. SF control by ANOVA).