Figure 4.

Shh-induced expression of Hk2 and concurrent activation of glycolysis. Shh and insulin/ IGF/ PI3K signaling pathways converge on Myc–Max effector complex to induce Hk2 expression and glycolysis. (A), (B) Isolated CGNPs were maintained in media with N2, Shh, neither or both. Media were changed after 24 hours in culture, after which 3 replicates per condition were maintained in normoxia for 24 hours, while 3 replicates per condition were concurrently subjected to hypoxia. Expression of Hk2, Hk1, IGFr, pIGFr, Akt, pAkt, and HP-Hif1α were demonstrated by Western blot (A), and the lactate concentration in media was quantified by enzymatic assay, presented as mean ± SEM, normalized for cell number (B). Addition of N2 alone increased Akt phosphorylation and mildly increased lactate production without inducing Hk2. Shh alone caused a modest increase in both Hk2 and lactate production. The combination of Shh and N2, however, markedly increased Hk2 expression and lactate production, indicating robust induction of glycolysis. Hypoxia alone induced near-maximal lactate production in the absence of Shh and N2, while also inducing moderate Hk2. Addition of Shh alone or N2 alone to hypoxic CGNPs did not further increase lactate, but the combination of Shh and N2 added to hypoxic CGNPs further increased both Hk2 and lactate. (C) Western blot analysis demonstrates that induction of Hk2 was modulated by Myc inhibitor 10058-F4 in isolated CGNPs maintained in Shh and N2. Reduced induction of Hk2 was dose dependent and paralleled the expression of Cyclin D2 and of Cip2a, a protein previously identified as down-regulated by 10058-F4.