6-OHDA treatment activates HIF-1α and HIF-2α in primary cultured astrocytes, but not in VM neurons
To investigate if HIFs are specifically activated in astrocytes treated with 6-OHDA, we used Western blots to quantify HIF-1α and HIF-2α expression in primary cultured astrocytes and VM neurons treated with 6-OHDA. Increased expressions of HIF-1α and HIF-2α was observed in primary cultured astrocytes treated with 10 μM 6-OHDA for 24 h compared to control (Figure. 1a, P < 0.01; Figure. 1b, P < 0.05). However, protein levels of HIF-1α and HIF-2α remained unchanged in VM neurons after application of 6-OHDA compared to control (Figure. 1c and Figure. 1d; P > 0.05). These results indicate that 6-OHDA treatment activates HIF-1α and HIF-2α in primary cultured astrocytes, but not in VM neurons.
6-OHDA-induced overexpressions of DMT1 and FPN1 in cultured astrocytes do not require HIF-1a upregulation
We next tested the effects of BAY 87-2243, a potent and selective HIF-1α inhibitor, on primary cultured astrocytes. Cells were pretreated with 10 μM BAY 87-2243 for 48 h, and then co-treated with 10 μM BAY 87-2243 and 10 μM 6-OHDA for 24 h. Expression of HIF-1α increased in primary cultured astrocytes treated with 10 μM 6-OHDA for 24 h, compared with the control group (Figure. 2a; P < 0.05). In contrast, the expression of HIF-1α decreased in primary cultured astrocytes treated with 10 μM BAY 87-2243, compared with the control group (Figure. 2a; P < 0.05). Furthermore, BAY 87-2243 pretreatment decreased the expression of HIF-1α in 6-OHDA-treated cells, compared with the 6-OHDA group (Figure. 2a; P < 0.05). To further investigate whether HIF-1α modulates the expressions of DMT1 and FPN1 induced by 6-OHDA in primary cultured astrocytes, measured the expressions of DMT1 and FPN1 after inhibiting HIF-1α with BAY 87-2243 (Figure. 2b and Figure. 2c). We observed that DMT1 and FPN1 expression increased in primary cultured astrocytes treated with 10 μM 6-OHDA for 24 h, compared with the control group (Figure. 2b, P < 0.01; Figure. 2c, P < 0.05). However, the expressions of DMT1 and FPN1 remained unchanged in astrocytes treated with 10 μM BAY 87-2243 alone, compared with the control group (Figure. 2b; Figure. 2c; P > 0.05). Expressions of DMT1 and FPN1 did not change in the BAY 87-2243 pretreatment group, compared to the 6-OHDA group (Figure. 2B; Figure. 2C; P > 0.05). Overall, these results demonstrate that BAY 87-2243 suppressed expression of HIF-1α and inhibited the overexpression of HIF-1α in primary cultured astrocytes treated with 6-OHDA. However, BAY 87-2243 did not inhibit the up-regulation of DMT1 and FPN1 by 6-OHDA, suggesting that HIF-1α does not contribute to these processes.
HIF-2α upregulation is required for 6-OHDA induced overexpressions of DMT1 and FPN1
We next investigated the effects of a potent and selective inhibitor of HIF-2α, which acts specifically on the subunits of HIF-2α. Primary cultured astrocytes were pretreated with 10 μM HIF-2α inhibitor for 24 h, and then co-treated with 10 μM HIF-2α inhibitor and 10 μM 6-OHDA for 24 h. Expression of HIF-2α increased in primary cultured astrocytes treated with 10 μM 6-OHDA for 24 h, compared with the control group (Figure. 3a; P < 0.05). In contrast, treatment with 10 μM HIF-2α inhibitor decreased the expression of HIF-2α in primary cultured astrocytes, compared with the control group (Figure. 3a; P < 0.05). Furthermore, HIF-2α inhibitor pretreatment decreased the expression of HIF-2α in 6-OHDA-treated cells, compared with the 6-OHDA group (Figure. 3a; P < 0.01). To obtain direct evidence about whether HIF-2α contributes to modulating the expression of DMT1 and FPN1 in activated primary cultured astrocytes, we measured expression levels of DMT1 and FPN1 in primary cultured astrocytes treated with HIF-2α inhibitor. Expressions of DMT1 and FPN1 increased in primary cultured astrocytes treated with 10 μM 6-OHDA for 24 h, compared with the control group (Figure. 3b, P < 0.05; Figure. 3c, P < 0.01). However, DMT1 and FPN1 expression decreased in primary cultured astrocytes treated with 10 μM HIF-2α inhibitor, compared to the control group (Figure. 3b, P < 0.01; Figure. 3c, P < 0.05 ). Similarly, HIF-2α inhibitor pretreatment decreased the expressions of DMT1 and FPN1 in 6-OHDA-treated cells, compared with the 6-OHDA group (Figure. 3b, P < 0.05; Figure. 3c, P < 0.01). In summary, the HIF-2α inhibitor suppressed expression of HIF-2α, as expected, and prevented the overexpression of HIF-2α in primary cultured astrocytes by 6-OHDA. These results suggest that HIF-2α modulates the influence of 6-OHDA on DMT1 and FPN1 expression.
HIF-2α was involved in the increased ferrous iron influx and efflux in 6-OHDA-treated primary cultured astrocytes
The fluorescent dye calcein was used to monitor ferrous iron influx into primary astrocytes during a 1 mM ferrous iron perfusion. The fluorescence intensity declined gradually inside cells, indicating a transmembrane ferrous iron influx (Figure. 4). We investigated the roles of astrocytes in regulating iron homeostasis under oxidative conditions. In agreement with our previous data for primary astrocytes11, cells treated with 10 μM 6-OHDA showed a more rapid fluorescence quenching and a decrease in fluorescence intensity compared with controls. However, pretreatment with 10 μM HIF-2α inhibitor for 24 h fully blocked this process and produced a fluorescence intensity similar to control, indicating that the HIF-2α inhibitor suppresses the increased ferrous iron influx caused by 6-OHDA.
For the iron efflux assay, we used DFO, a membrane-impermeable, potent, specific iron chelator. When cells were perfused with 1 mM DFO, a marked increase in fluorescence intensity was observed in the 10 μM 6-OHDA-treated group. These results indicate that 6-OHDA enhances iron transportation rate in primary cultured astrocytes. However, pretreatment with 10 μM HIF-2α inhibitor for 24 h fully blocked this process and yielded a fluorescence intensity similar to control levels, indicating that the HIF-2α inhibitor suppresses the ferrous iron efflux caused by 6-OHDA.
PKC pathway was involved in the 6-OHDA induced activation of HIF-2α in primary cultured astrocytes.
To investigate if PKC pathway is involved in the activation of HIF-2α in astrocytes, we quantify HIF-2α expression in primary cultured astrocytes treated with PMA or 6-OHDA. Primary cultured astrocytes were pretreated with 0.4 μM Bisl for 24 h, and then co-treated with 0.4 μM Bisl and 0.2 μM PMA or 10 μM 6-OHDA for 24 h. Expression of HIF-2α increased after treatment of primary astrocytes with 0.2 μM PMA or 10 μM 6-OHDA for 24 h, compared with the control group (Figure. 5a; Figure. 5b; P < 0.05). However, HIF-2α expression did not change when astrocytes were treated with 0.4 μM Bisl for 24 h, compared to the control group (Figure. 5a; Figure. 5b; P > 0.05). Next, primary cultured astrocytes were pretreated with 0.4 μM Bisl for 24 h, and then co-treated with 0.4 μM Bisl and 0.2 μM PMA or 10 μM 6-OHDA for 24 h. Pretreatment with Bisl decreased HIF-2α expression in PMA-treated cells or 6-OHDA-treated cells, compared with the PMA or 6-OHDA group (Figure. 5a; Figure. 5b; P < 0.05), but expression of HIF-2α did not differ from the control (Figure. 5a; Figure. 5b; P > 0.05).
We next examined whether expression levels of DMT1 and FPN1 increase following HIF-2α activation due to HIF/HRE binding. To confirm that the activated HIF-2α is functional, DMT1 and FPN1 expression were investigated. The expression of DMT1 increased in primary cultured astrocytes treated with 0.2 μM PMA or 10 μM 6-OHDA for 24 h, compared with the control group (Figure. 5c; Figure. 5e; P < 0.05). In contrast, the expression of DMT1 did not change in primary cultured astrocytes after 0.4 μM Bisl treatment, compared with the control group (Figure. 5c; Figure. 5e; P > 0.05). However, Bisl pretreatment decreased the expression of DMT1 in PMA-treated cells or 6-OHDA-treated cells, compared with the PMA or 6-OHDA group (Figure. 5c; Figure. 5e; P < 0.05), but expression of DMT1 did not differ from control (Figure. 5c; Figure. 5e; P > 0.05).
The expression of FPN1 increased in primary cultured astrocytes treated with 0.2 μM PMA or 10 μM 6-OHDA for 24 h, compared with the control group (Figure. 5d; Figure. 5f; P < 0.05). However, the expression of FPN1 did not change in astrocytes treated with 0.4 μM Bisl, compared to the control group (Figure. 5d; Figure. 5f; P > 0.05). In contrast, pretreatment with Bisl decreased the expression of FPN1 in PMA-treated cells or 6-OHDA-treated cells, compared with the PMA or 6-OHDA group(Figure. 5d; Figure. 5f; P < 0.05), but expression of FPN1 did not differ from control (Figure. 5d; Figure. 5f; P > 0.05). These results indicate that in primary cultured astrocytes, 6-OHDA upregulates HIF-2α expression via PKC activation and that the activated HIF-2α is functional.
ROS and NO were involved in the 6-OHDA induced activation of HIF-2α in primary cultured astrocytes.
Since both oxidative stress and NO activate HIFs, we assumed that upregulation of HIF-2α may be initiated by intracellular oxidative stress alone or in combination with NO production induced by pro-inflammatory cytokines. To test this hypothesis, we investigated the expression of HIF-2α in 6-OHDA-treated primary cultured astrocytes after pretreatment with the radical scavenger NAC and the inducible NO synthase (iNOS) inhibitor L-NAME. Expression of HIF-2α significantly increased after primary cultured astrocytes were treated with 10 μM 6-OHDA for 24 h, compared with the control group (Figure. 6a; P < 0.01). However, pretreatment with 1 mM NAC or 1 mM L-NAME decreased the expression of HIF-2α in 6-OHDA-treated primary cultured astrocytes, compared with 6-OHDA group (Figure. 6a; P < 0.05). However, expression of HIF-2α remained unchanged in both the NAC pretreatment group and the L-NAME pretreatment group compared with the control group (Figure. 6a; P > 0.05).
To confirm that the HIF-2α expressed is functional, DMT1 and FPN1 expression were quantified. We found that the expressions of DMT1 and FPN1 increased in 10 μM 6-OHDA treated primary cultured astrocytes, compared with the control group (Figure. 6b, P < 0.05; Figure. 6c, P < 0.05). However, pretreatment with either NAC (Figure. 6b, P < 0.05; Figure. 6c, P < 0.05) or L-NAME (Figure. 6b, P < 0.01; Figure. 6c, P < 0.05) decreased the expression of DMT1 and FPN1 in 6-OHDA-treated cells, compared with the 6-OHDA group. Expression of DMT1 and FPN1 remained unchanged when cells were pretreated with either NAC or L-NAME, compared with the control group (Figure. 6b, P > 0.05; Figure. 6c, P > 0.05). These results indicate that in primary cultured astrocytes, ROS and NO upregulate HIF-2α expression and that the HIF-2α expressed is functional.
ROS and NO were involved in the activation of PKCδ phosphorylation by 6-OHDA in primary cultured astrocytes.
To further study the mechanism about the activation of HIF-2α by 6-OHDA in primary cultured astrocytes. Used western blots, we observed the level of PKCδ phosphorylation in 6-OHDA-treated primary cultured astrocytes after pretreatment with NAC and L-NAME. PKC δ phosphorylation increased after primary cultured astrocytes were treated with 10 μM 6-OHDA for 24 h, compared with the control group (P< 0.05). Pretreatment with NAC decreased PKC δ phosphorylation in 6-OHDA-treated primary cultured astrocytes, compared with the 6-OHDA group (P< 0.05). Pretreatment with 1 mM L-NAME decreased PKC δ phosphorylation in 6-OHDA-treated primary cultured astrocytes, compared with the 6-OHDA group (P< 0.05, Fig.7). These results indicated that ROS and NO were involved in the activation of PKCδ phosphorylation by 6-OHDA in primary cultured astrocytes.