Pyruvate kinase isoform expression alters nucleotide synthesis to impact cell proliferation

Abstract

Metabolic regulation influences cell proliferation. The influence of pyruvate kinase isoforms on tumor cells has been extensively studied, but whether PKM2 is required for normal cell proliferation is unknown. We examine how PKM2 deletion affects proliferation and metabolism in nontransformed, nonimmortalized PKM2-expressing primary cells. We find that deletion of PKM2 in primary cells results in PKM1 expression and proliferation arrest. PKM1 expression, rather than PKM2 loss, is responsible for this effect, and proliferation arrest cannot be explained by cell differentiation, senescence, death, changes in gene expression, or prevention of cell growth. Instead, PKM1 expression impairs nucleotide production and the ability to synthesize DNA and progress through the cell cycle. Nucleotide biosynthesis is limiting, as proliferation arrest is characterized by severe thymidine depletion, and supplying exogenous thymine rescues both nucleotide levels and cell proliferation. Thus, PKM1 expression promotes a metabolic state that is unable to support DNA synthesis.

Figure 1. PKM2 deletion with PKM1 expression results in proliferation arrest

(A) The effect of 4-OHT treatment on pyruvate kinase isoform expression in PKM2^fl/fl Cre-ER MEFs was assessed by Western blot analysis. PKM2^fl/fl Cre-ER MEFs were treated with 4-OHT (PKM2^Δ/Δ Cre-ER) or vehicle (PKM2^fl/fl Cre-ER) for the indicated number of days. Muscle or H1299 cancer cell lysate were included as control samples that express only PKM1 or PKM2, respectively. (B) Proliferation of PKM2^fl/fl Cre-ER MEFs following vehicle (PKM2^fl/fl Cre-ER) or 4-OHT (PKM2^Δ/Δ Cre-ER) treatment was assessed by counting cell numbers as shown. (C) Proliferation of Cre negative PKM2^fl/fl MEFs treated with vehicle or 4-OHT was assessed by counting cell numbers as shown. (D) Western blot analysis of MEFs generated from PKM2^fl/+ Cre-ER mice. 4-OHT treatment results in PKM2^fl/Δ MEFs that express both PKM2 and PKM1 protein. Western blot analysis of 100 ng of recombinant PKM1 or PKM2 (rPKM1, rPKM2) are included as controls. (E) Proliferation of PKM2^fl/+ Cre-ER MEFs treated with vehicle (PKM2^fl/+) or with 4-OHT (PKM2^Δ/+) was assessed by counting cell numbers as shown. (F) Proliferation of wildtype MEFs treated with vehicle or 1 μM of PKM2 activator TEPP-46 was assessed by counting cell numbers over time as shown. All data are displayed as means ± SEM, n=3. See also Figure S1.

Figure 2. PKM2^Δ/Δ cells undergo cell cycle arrest

(A) Quantification of cell viability by propidium iodide staining after treatment of PKM2^fl/fl MEFs with vehicle (PKM2^fl/fl) or 4-OHT (PKM2^Δ/Δ). Data are displayed as means ± SEM, n=3. (B) Classical cell senescence was assayed by acid stable β-galactosidase activity. The top panels (labeled Early passage) assess acid stable β-galactosidase activity in PKM2^fl/fl and PKM2^Δ/Δ MEFs 9 days after vehicle or 4-OHT treatment, respectively. Bottom panels (labeled Late passage) assess acid stable β-galactosidase activity inPKM2^fl/fl and PKM2^Δ/Δ MEFs after 12 days in culture. The PKM2^fl/fl MEFs had undergone proliferation arrest at this time point. (C) Population doublings over 12 days for myoblasts isolated from three different PKM2^fl/fl Cre-ER mice that were treated with vehicle (PKM2^fl/fl) or 4-OHT (PKM2^Δ/Δ). Data are displayed as means ± SEM, n=3. (D) Images of myoblasts generated from PKM2^fl/fl Cre-ER mice 9 days after treatment with vehicle (PKM2^fl/fl) or 4-OHT (PKM2^Δ/Δ) are shown. PKM2^fl/fl myoblasts induced to differentiate by culture in low-serum for 9 days are also presented (right panel). (E) EdU uptake of PKM2^fl/fl and PKM2^Δ/Δ MEFs was measured in PKM2^fl/fl Cre-ER MEFs 7 days after vehicle or 4-OHT treatment. Data are displayed as means ± SEM, n=3. (F) DNA content was assessed using propidium iodide staining and flow cytometry in PKM2^fl/fl and PKM2^Δ/Δ MEFs 7 days after PKM2^fl/fl Cre-ER MEFs were treated with vehicle or 4-OHT. (G) DNA content was assessed following dual staining with FxCycle Violet and EdU using flow cytometry in PKM2^fl/fl and PKM2^Δ/Δ MEFs 7 days after PKM2^fl/fl Cre-ER MEFs were treated with vehicle or 4-OHT. See also Figure S2.

Figure 3. PKM2^Δ/Δ cells remain metabolically active and are larger than PKM2^fl/fl cells

(A) Glucose uptake rates; (B) lactate excretion rates; (C) the relative production of ¹⁴C-labeled CO₂ from uniformly ¹⁴C-labeled glucose; (D) average cell volumes; (E) total protein content; and (F) single cell dry mass of PKM2^fl/fl and PKM2^Δ/Δ MEFs 7 days after treatment of PKM2^fl/fl MEFs with vehicle or 4-OHT. All data are displayed as means ± SEM, n=3 (*p < 0.0001). See also Figure S3.

Figure 4. Pyruvate kinase isoform expression affects the use of specific metabolic pathways

(A) Labeling of intracellular metabolites with [U-¹³C]glucose or (B) [U-¹³C]glutamine was measured using mass spectrometry in PKM2^fl/fl and PKM2^Δ/Δ MEFs 7 days after PKM2^fl/fl Cre-ER MEFs were treated with vehicle or 4-OHT. Complete isotopomer distributions used to calculate these values are included in Table S1. The y-axis for all graphs in (A) is the calculated percent total glucose contribution (Fendt et al., 2013a). See also Figure S4.

Figure 5. Nucleotide deficiency is limiting for proliferation of PKM2^Δ/Δ MEFs

(A) Pool sizes of intracellular nucleotides in PKM2^fl/fl and PKM2^Δ/Δ MEFs were measured 7 days after PKM2^fl/fl Cre-ER MEFs were treated with vehicle or 4-OHT. Pool sizes of intracellular nucleotides in PKM2^Δ/Δ MEFs supplemented with 250 μM thymine were also measured 7 days after 4-OHT treatment. Data are displayed as means ± SEM, n=4. (B) Dynamic labeling of intracellular metabolites with [U-¹³C]glucose and [U-¹³C]glutamine was measured using mass spectrometry in PKM2^fl/fl and PKM2^Δ/Δ MEFs 7 days after PKM2^fl/fl Cre-ER MEFs were treated with vehicle or 4-OHT. The complete isotopomer distributions for all measurements are included in Table S2. (C) Metabolic fluxes of central carbon metabolism in PKM2^fl/fl and PKM2^Δ/Δ MEFs. Fluxes were modeled based on dynamic and steady state [U-¹³C]glucose and [U-¹³C]glutamine labeling data. With the exception of NTP and lactate, biomass and extraction fluxes are not depicted (see supplement for complete flux model). Significant differences (>40%, based on 95% confidence interval) between fluxes of PKM2^fl/fl and PKM2^Δ/Δ MEFs are highlighted in grey. See also Figure S5, Supplemental Experimental Procedures, and ¹³C-Metabolic flux analysis model.

Figure 6. Genome-scale gene expression analysis reveals no correlation between gene expression and proliferation arrest of PKM2^Δ/Δ cells

(A) Unsupervised hierarchical clustering of PKM2^fl/fl and PKM2^Δ/Δ MEF expression profiles. RNA was isolated from PKM2^fl/fl and PKM2^Δ/Δ MEFs 7 days after PKM2^fl/fl Cre-ER MEFs were treated with vehicle or 4-OHT. MEFs were generated from two independent PKM2^fl/fl Cre-ER MEFs. RNA was analyzed using an Affymetrix GeneChip Mouse Gene 1.0 ST array. (B) Unsupervised hierarchical clustering of PKM2^fl/fl and PKM2^Δ/Δ MEF expression profiles for genes involved in nucleotide metabolism. See also Figure S6.

Figure 7. Thymine supplementation restores proliferation of PKM2^Δ/Δ MEFs

(A) Population doublings over 10 days of PKM2^fl/fl and PKM2^Δ/Δ MEFs were measured with or without the indicated metabolites supplemented in the media at a concentration of 250 μM. Data are displayed as means ± SEM, n=3. *Population doubling is statistically significant (p < 0.05) when compared to the population doubling of PKM2^Δ/Δ MEFs without any supplements. (B) Three representative images of DNA strands incorporating CldU (red) and IdU (green) from PKM2^fl/fl and PKM2^Δ/Δ MEFs five days after PKM2^fl/fl Cre-ER MEFs were treated with vehicle or 4-OHT are shown; scale bar = 15 μm. (C) Quantitation of the percentage of DNA strands incorporating both CldU and IdU is shown. For this analysis 237 strands from PKM2^fl/fl MEFs and 54 strands from PKM2^Δ/Δ MEFs prepared as described in (B) were evaluated. See also Figure S7.