Inositol synthesis regulates the activation of GSK-3α in neuronal cells

Abstract

The synthesis of inositol provides precursors of inositol lipids and inositol phosphates that are pivotal for cell signaling. Mood stabilizers lithium and valproic acid, used for treating bipolar disorder, cause cellular inositol depletion, which has been proposed as a therapeutic mechanism of action of both drugs. Despite the importance of inositol, the requirement for inositol synthesis in neuronal cells is not well understood. Here, we examined inositol effects on proliferation of SK-N-SH neuroblastoma cells. The essential role of inositol synthesis in proliferation is underscored by the findings that exogenous inositol was dispensable for proliferation, and inhibition of inositol synthesis decreased proliferation. Interestingly, the inhibition of inositol synthesis by knocking down INO1, which encodes inositol-3-phosphate synthase, the rate-limiting enzyme of inositol synthesis, led to the inactivation of GSK-3α by increasing the inhibitory phosphorylation of this kinase. Similarly, the mood stabilizer valproic acid effected transient decreases in intracellular inositol, leading to inactivation of GSK-3α. As GSK-3 inhibition has been proposed as a likely therapeutic mechanism of action, the finding that inhibition of inositol synthesis results in the inactivation of GSK-3α suggests a unifying hypothesis for mechanism of mood-stabilizing drugs. Inositol is an essential metabolite that serves as a precursor for inositol lipids and inositol phosphates. We report that inhibition of the rate-limiting enzyme of inositol synthesis leads to the inactivation of glycogen synthase kinase (GSK) 3α by increasing inhibitory phosphorylation of this kinase. These findings have implications for the therapeutic mechanisms of mood stabilizers and suggest that inositol synthesis and GSK 3α activity are intrinsically related.

Keywords: Inositol; bipolar disorder; glycogen synthesis kinase; inositol depletion; myo-inositol-3-phosphate synthase; valproic acid.

Fig. 1. Exogenous inositol is not essential for cell proliferation or maintaining inositol homeostasis in SK-N-SH cells

(A) SK-N-SH cells were inoculated at a concentration of 5,000 cells per well in 96-well plates at day 0, and cell numbers were estimated by the proliferation assay described under “Materials and Methods.” (B) Intracellular inositol levels were assayed in cells cultured in inositol-deficient media without (control) or with inositol supplement (0.5, 1, 5, 10 mM). The data shown in A and B are the average of at least three experiments ± S.D, n≥3.

Fig. 2. Inositol biosynthesis is essential for cell proliferation

(A) Western blot analysis of Ino1 protein levels (left) and quantitative analysis of INO1 knockdown efficiency (right). (B) Inositol levels were measured in control and INO1 knockdown cells cultured in DMEM supplemented with 10% serum. (C) Cell proliferation was assayed as described in Fig. 1A, and cell numbers were estimated 4 days after inoculation. (D) Control and INO1 knockdown cells (about 1×10⁶) were plated in 100-mm dishes and photographed at day 2 using a microscope at 200 X magnification. The data in A, B, and C are presented as the mean ± S.D, n=3, and **p< 0.01.

Fig. 3. Exogenous inositol does not regulate transcription of the genes for inositol biosynthesis or uptake

(A) Inositol levels were measured in control or INO1 knockdown cells in inositol-rich (I+) and inositol-deficient (I-) media. Cells were cultured in DMEM with 10% serum to reach 70% confluence, washed twice with PBS, and replenished with media as indicated. The data are presented as the mean ± S.D, n=3, and **p<0.01. (B) mRNA levels of INO1 were measured in SK-N-SH cells incubated in the presence of inositol (0, 0.1, 1, 10 mM) for the indicated times (1, 5, and 10 hours) after growth in inositol-deficient media. Values were normalized to the internal control SDHA (succinate dehydrogenase complex, subunit A). INO1 mRNA levels normalized to SDHA were represented as fold change relative to cells exposed to 0 mM inositol for 1 hour. (C) Western blot analysis of Ino1 protein levels. SK-N-SH cells were cultured to reach about 70% confluence and then were incubated with either serum or inositol for indicated times. Cells were harvested and lysed for Western blot analysis as described under “Materials and Methods.” Actin was used as the loading control. The figure is representative of experiments in triplicate. (D) mRNA levels of Na+/inositol transporter SMIT1 and (E) H+/inositol transporter HMIT were measured as described above. The data in B, D, and E are presented as the mean ± S.D, n≥3.

Fig. 4. INO1 knockdown and VPA treatment lead to inactivation of GSK3α

(A) Western blot analysis and quantification of Ino1 and inhibitory phosphorylation levels of GSK-3α (Ser21) and GSK-3β (Ser9). Actin was used as the loading control. Scrambled control and Ino1 knockdown (shRNA_INO1_1 and shRNA_INO1_2) SK-N-SH cells were cultured to about 70% confluence, and cells were refreshed with media containing 10% serum (+serum) or no serum (-serum) for 4 hours. Cells were harvested and lysed for Western blot analysis as described under “Materials and Methods.” The figure is representative of experiments in triplicate. The quantification data are presented as the mean ± S.D, n=3, and *p< 0.05. (B) Intracellular inositol levels were measured in SK-N-SH cells after exposure to VPA for the indicated times. The data are presented as the mean ± S.D, n=3, and **p< 0.01. (C) Western blot analysis and quantification of the protein levels of GSK-3α (Ser21) and GSK-3β (Ser9) and total protein levels of GSK-3α and GSK-3β. Actin was used as the loading control. The figure is representative of at least three independent experiments. The quantification data are presented as the mean ± S.D, n≥4, and *p< 0.05, **p<0.01.