Disruption of astrocytic glutamine turnover by manganese is mediated by the protein kinase C pathway

Abstract

Manganese (Mn) is a trace element essential for normal human development and is required for the proper functioning of a variety of physiological processes. Chronic exposure to Mn can cause manganism, a neurodegenerative disorder resembling idiopathic Parkinson's disease (PD). Mn(II) neurotoxicity is characterized by astrocytic impairment both in the expression and activity of glutamine (Gln) transporters. Because protein kinase C (PKC) activation leads to the downregulation of a number of neurotransmitter transporters and Mn(II) increases PKC activity, we hypothesized that the PKC signaling pathway contributes to the Mn(II)-mediated disruption of Gln turnover. Our results have shown that Mn exposure increases the phosphorylation of both the PKCα and PKCδ isoforms. PKC activity was also shown to be increased in response to Mn(II) treatment. Corroborating our earlier observations, Mn(II) also caused a decrease in Gln uptake. This effect was blocked by PKC inhibitors. Notably, PKC activation caused a decrease in Gln uptake mediated by systems ASC and N, but had no effect on the activities of systems A and L. Exposure to α-phorbol 12-myristate 13-acetate significantly decreased SNAT3 (system N) and ASCT2 (system ASC) protein levels. Additionally, a co-immunoprecipitation study demonstrated the association of SNAT3 and ASCT2 with the PKCδ isoform, and Western blotting revealed the Mn(II)-mediated activation of PKCδ by proteolytic cleavage. PKC activation was also found to increase SNAT3 and ubiquitin ligase Nedd4-2 binding and to induce hyperubiquitination. Taken together, these findings demonstrate that the Mn(II)-induced dysregulation of Gln homeostasis in astrocytes involves PKCδ signaling accompanied by an increase in ubiquitin-mediated proteolysis.

Fig. 1.

Effect of Mn on astrocytic PKC isozymes mRNA and protein phosphorylation. Levels of PKCs mRNA and protein phosphorylation were determined in primary cultures of astrocytes treated with 100 μM, 500 μM, or 1 mM Mn(II). Expression of PKCα (A), PKCδ (C), and PKCβ (E) mRNA after 4, 8, or 24 h exposure to Mn(II) was measured by quantitative real-time PCR and normalized to levels of GAPDH mRNA. Levels of phosphorylated protein PKCα (B), PKCδ (D), and PKCβ (F) after 4, 8, and 24 h exposure to Mn(II) were determined by Western blotting and normalized to the levels of total protein PKCα, PKCβ, and PKCδ. Data represent the mean ± SD from three to four independent sets of cultures.

Fig. 2.

Effects of Mn on astrocytic PKC activity. PKC activity was determined by measuring phosphorylation of substrate peptide with radiolabeled ATP. Confluent cultures of astrocytes were treated with 100 μM, 500 μM, or 1 mM Mn(II) for 4, 8, and 24 h. Lysates containing 1000 μg of proteins were subjected for PKC activity measurement. Data represent the mean ± SD from three independent sets of cultures; *P < 0.05, **P < 0.01, and ***P < 0.001 control versus Mn(II)-exposed cells.

Fig. 3.

PKC downregulates astrocytic glutamine uptake. Total L-(G-³H)-Gln uptake was measured in control and 100 nM PMA treated for 1, 2, 4, 6, 8, and 24 h primary cultures of astrocytes. Data represent the mean ± SD from three independent sets of cultures each performed in triplicate; **P < 0.01, ***P < 0.001 control versus PMA-exposed cells (A). For competition analysis, cells were incubated for 4, 8, and 24 h with or without 100 nM PMA. Results are mean ± SD of three to four independent experiments each performed in triplicate; *P < 0.05 total versus total PMA-exposed cells, **P < 0.01 total versus total PMA-exposed cells, ^#P < 0.05 system N versus system N PMA-exposed cells, ^&P < 0.05 system ASC versus system ASC PMA-exposed cells, and ^&&P < 0.01 system ASC versus system ASC PMA-exposed cells following (B). Cells were treated with 500 μM and 1 mM Mn(II) in the presence or absence of 10 μM BIS II for 4, 8, and 24 h. Results are mean ± SD of three to four independent experiments each performed in triplicate; *P < 0.05 control versus Mn(II)-exposed cells, **P < 0.01 control versus Mn(II)-exposed cells, ^@P < 0.05 500 μM Mn(II) versus 500 μM Mn(II) and BIS II-exposed cells, and $P < 0.05 1 mM Mn(II) versus 1 mM Mn(II) and BIS II-exposed cells.

Fig. 4.

Effect of PKC activation on protein levels of Gln transporters in whole astrocyte lysates. Levels of LAT2 (A), SNAT2 (B), ASCT2 (C), and SNAT3 (D) in primary cultures of astrocytes treated with PMA (100 nM) for 4, 6, 8, and 24 h in whole cell lysates. Shown are results from typical Western blots and immunostaining images. For each lane, protein levels were adjusted to the levels of β-actin. Results are mean ± SD from three to five independent experiments each performed in triplicate; **P < 0.01 control and ***P < 0.01 control (0 h) versus PMA-exposed cells.

Fig. 5.

Effect of PKC activation on protein levels of astrocytic Gln transporters in plasma membranes. Levels of LAT2 (A), SNAT2 (B), ASCT2 (C), and SNAT3 (D) were determined upon PKC activation by 100 nM PMA for 4, 6, 8, and 24 h. All samples were adjusted to contain the same amount of proteins prior to loading on the gel. β-Actin was evaluated to assure the purity of cell surface proteins. Results are mean ± SD from three independent experiments each performed in triplicate; **P < 0.01 and ***P < 0.01 control (0 h) versus PMA-exposed cells.

Fig. 6.

PKCδ interacts with ASCT2 and SNAT3. Cells were exposed to 500 μM Mn(II) for 2, 4, 6, 8, and 24 h then lysed and subjected to immunoprecipitation with antibodies against ASCT2 (A), SNAT3 (B), LAT2 (C), or SNAT2 (D) and probed for PKCδ. In each experiment, an antibody against control IgG was additionally used for immunoprecipitation as the control for nonspecific binding. Similar results were obtained in three independent sets of cultures; *P < 0.05 and ***P < 0.001 control (0 h) versus Mn(II)-exposed.

Fig. 7.

Manganese mediates proteolytic cleavage of astrocytic PKCδ. Cells were exposed to 1 mM and 500 μM Mn(II) for 4, 8, and 24 h. Both native (72 kDa) and cleaved (40 kDa) PKCδ bands were determined by Western blotting. The blot is representative of three independent experiments; **P < 0.01 and ***P < 0.001 control (0 h) versus Mn(II)-exposed cells.

Fig. 8.

PKC stimulation influences astrocytic SNAT3 and Nedd4-2 interaction. Cells were exposure to 100 nM PMA for 1, 2, 6, 8, and 24 h and subjected to immunoprecipitation using an antibody against SNAT3 and were then probed for Nedd4-2. In each experiment, an antibody against control IgG was additionally used for immunoprecipitation as the control for nonspecific binding. Similar results were obtained in three to four independent sets of cultures; *P < 0.05, **P < 0.01, and ***P < 0.001 control (0 h) versus Mn(II)-exposed cells.

Fig. 9.

PMA exposure increases ubiquitination in astrocytes. Levels of protein ubiquitination after exposure to 100 nM PMA for 2, 4, 6, 8, and 24 h were determined by Western blotting and normalized to the levels of β-actin protein using an antibody that targets mono- and poly-ubiquitinated proteins. Data represent the mean ± SD from three independent sets of cultures each performed in triplicate; **P < 0.01 and ***P < 0.001 versus control (0 h) versus PMA-exposed cells.