Adiponectin stimulates proliferation of adult hippocampal neural stem/progenitor cells through activation of p38 mitogen-activated protein kinase (p38MAPK)/glycogen synthase kinase 3β (GSK-3β)/β-catenin signaling cascade - PubMed (original) (raw)

Adiponectin stimulates proliferation of adult hippocampal neural stem/progenitor cells through activation of p38 mitogen-activated protein kinase (p38MAPK)/glycogen synthase kinase 3β (GSK-3β)/β-catenin signaling cascade

Di Zhang et al. J Biol Chem. 2011.

Abstract

Adiponectin is the most abundant adipokine secreted from adipocytes. Accumulating evidence suggests that the physiological roles of adiponectin go beyond its metabolic effects. In the present study, we demonstrate that adiponectin receptors 1 and 2 (AdipoR1 and AdipoR2) are expressed in adult hippocampal neural stem/progenitor cells (hNSCs). Adiponectin treatment increases proliferation of cultured adult hNSCs in a dose- and time-dependent manner, whereas apoptosis and differentiation of adult hNSCs into neuronal or glial lineage were not affected. Adiponectin activates AMP-activated protein kinase and p38 mitogen-activated protein kinase (p38MAPK) signaling pathways in adult hNSCs. Pretreatment with the p38MAPK inhibitor SB203580, but not the AMP-activated protein kinase inhibitor Compound C, attenuates adiponectin-induced cell proliferation. Moreover, adiponectin induces phosphorylation of Ser-389, a key inhibitory site of glycogen synthase kinase 3β (GSK-3β), and this effect can be blocked by inhibition of p38MAPK with SB203580. Levels of total and nuclear β-catenin, the primary substrate of GSK-3β, were increased by adiponectin treatment. These results indicate that adiponectin stimulates proliferation of adult hNSCs, via acting on GSK-3β to promote nuclear accumulation of β-catenin. Thus, our studies uncover a novel role for adiponectin signaling in regulating proliferation of adult neural stem cells.

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Figures

FIGURE 1.

FIGURE 1.

Expression of AdipoR1 and AdipoR2 in cultured adult hippocampal neural stem/progenitor cells. A, Western blot demonstrating the presence of AdipoR1 (46 kDa, top) and AdipoR2 (37 kDa, bottom) in cultured adult hippocampal neural stem/progenitor cells. B, immunocytochemical staining showing the colocalization of AdipoR1 (green, top) and AdipoR2 (green, bottom) with nestin (red), a neural stem cell marker. DAPI (blue) reveals nuclear counterstaining. Scale bar, 10 μm.

FIGURE 2.

FIGURE 2.

Effects of adiponectin on proliferation of adult hippocampal neural stem/progenitor cells. A, immunocytochemistry showing that cells are positive for the neural stem cell marker nestin (red). DAPI (blue) reveals nuclei. Scale bar, 20 μm. B and C, cells were treated with various doses of globular adiponectin (B) or full-length adiponectin (C) for 48 h. D, cells were treated with 3 μg/ml globular adiponectin (gAd) for 24, 48, or 72 h. Cell proliferation was determined by the MTT assay. Data are expressed as mean ± S.E. (n = 6 per group for B and n = 3 per group for C and D). *, p < 0.01; **, p < 0.001, ***, p < 0.0001, compared with vehicle controls. Error bars, S.E.

FIGURE 3.

FIGURE 3.

Effect of adiponectin on PARP cleavage in cultured adult hippocampal neural stem/progenitor cells. Adult hippocaampal neural stem/progenitor cells were incubated with various doses of globular adiponectin (gAd) for 48 h. Cleavage of PARP precursor protein was determined by Western blot analysis. A, representative immunoblots of full-length and cleaved PARP. B, quantitative data showing the effect of adiponectin on cleaved PARP. C, quantitative data showing the effect of adiponectin on full-length PARP. Data are expressed as mean ± S.E. (error bars), n = 4 per group.

FIGURE 4.

FIGURE 4.

Effect of adiponectin on adult hippocampal neural stem/progenitor cell differentiation. A, cells were incubated with globular adiponectin (gAd; 3 μg/ml) and treated with 1 μ

m

retinoic acid and 0.5% FBS in culture medium for 6 days. The differentiation into neuronal or glial lineage was assessed by examining Tuj1, a neuronal marker, or GFAP, an astrocyte marker. Data are expressed as mean ± S.E. (error bars), n = 3 per group. B, representative images showing immunocytochemical staining for Tuj1 and GFAP and DAPI nuclear counterstain. Scale bar, 40 μm.

FIGURE 5.

FIGURE 5.

Effect of inhibition of AMPK and p38MAPK on adiponectin-induced proliferation of adult hippocampal neural stem/progenitor cells. A, effect of adiponectin on phosphorylation of AMPK (left) and p38MAPK (right) in adult hippocampal neural stem/progenitor cells at different time points (0, 15, and 30 min). Data are expressed as mean ± S.E. (error bars), n = 4 per group for AMPK, n = 3 per group for p38MAPK. *, p < 0.05; **, p < 0.01 compared with control. B, effect of inhibition of AMPK on adiponectin-induced cell proliferation. Cells were incubated with various concentrations of Compound C (0.02–2.0 μ

m

) for 2 h, followed by treatment with globular adiponectin (gAd; 3 μg/ml) for 48 h. Cell proliferation was assessed by MTT assay. C, effect of inhibition of p38MAPK on adiponectin-induced cell proliferation. Cells were incubated with various concentrations of SB203580 (1–10 μ

m

) for 2 h followed by treatment with globular adiponectin (3 μg/ml) for 48 h. Data in B and C are expressed as mean ± S.E., n = 6 per group. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with vehicle-vehicle control; ##, p < 0.01 compared with the vehicle plus 1.0 μ

m

SB203580 treatment; ++, p = 0.01 compared with the adiponectin-vehicle treatment.

FIGURE 6.

FIGURE 6.

Effect of adiponectin on Ser-389 phosphorylation of GSK-3β in cultured adult hippocampal neural stem/progenitor cells. A, cells were incubated with 3 μg/ml globular adiponectin (gAd) for different time periods followed by immunoblotting with anti-phosphorylated Ser-389 (S389) of GSK-3β and anti-GSK-3β antibodies. Data are expressed as mean ± S.E. (error bars), n = 3 per group. *, p < 0.05 compared with control. B, cells were pretreated with a 3.0 μ

m

concentration of the p38MAPK inhibitor SB203580 at 2 h prior to adiponectin treatment (3.0 μg/ml) for 30 min. Representative immunoblots showing inhibition of phosphorylation of p38MAPK and Ser-389 phosphorylation of GSK-3β by SB203580. Similar results were obtained from two independent experiments. C, levels of β-catenin following adiponectin treatment for 48 h. Left, whole cell level of β-catenin. Right, nuclear level of β-catenin. Data are expressed as mean ± S.E., n = 3–4 per group. *, p < 0.05; **, p < 0.01 compared with vehicle control.

FIGURE 7.

FIGURE 7.

Signaling pathways possibly involved in adiponectin-induced proliferation of adult hippocampal neural stem/progenitor cells. Adiponectin activates p38MAPK, which phosphorylates GSK-3β on Ser-389, leading to inhibition of GSK-3β activity. This effect in turn results in reduced degradation of its substrate β-catenin and causes an accumulation of β-catenin in the nucleus, where it interacts with members of the lymphoid enhancer factor/T-cell factor (LEF/TCF) family of transcription factors and stimulates transcription of target genes, promoting neural stem/progenitor cell proliferation.

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