Dictyostelium Differentiation-inducing Factor-3 Activates Glycogen Synthase Kinase-3β and Degrades Cyclin D1 in Mammalian Cells

Abstract

In search of chemical substances applicable for the treatment of cancer and other proliferative disorders, we studied the signal transduction of Dictyostelium differentiation-inducing factors (DIFs) in mammalian cells mainly using HeLa cells. Although DIF-1 and DIF-3 both strongly inhibited cell proliferation by inducing G 0 /G 1 arrest, DIF-3 was more effective than DIF-1. DIF-3 suppressed cyclin D1 expression at both mRNA and protein levels, whereas the overexpression of cyclin D1 overrode DIF-3-induced cell cycle arrest. The DIF-3-induced decrease in the amount of cyclin D1 protein preceded the reduction in the level of cyclin D1 mRNA. The decrease in cyclin D1 protein seemed to be caused by accelerated proteolysis, since it was abrogated by N-acetyl-Leu-Leu-norleucinal, a proteasome inhibitor. DIF-3-induced degradation of cyclin D1 was also prevented by treatment with lithium chloride, an inhibitor of glycogen synthase kinase-3␤ (GSK-3␤), suggesting that DIF-3 induced cyclin D1 proteolysis through the activation of GSK-3␤. Indeed, DIF-3 dephosphorylated Ser 9 and phosphorylated tyrosine on GSK-3␤, and it stimulated GSK-3␤ activity in an in vitro kinase assay. Moreover, DIF-3 was revealed to induce the nuclear translocation of GSK-3␤ by immunofluorescent microscopy and immunoblotting of subcellular protein fractions. These results suggested that DIF-3 activates GSK-3␤ to accelerate the proteolysis of cyclin D1 and that this mechanism is involved in the DIF-3-induced G 0 /G 1 arrest in mammalian cells.Differentiation-inducing factors (DIFs) 1 were identified in Dictyostelium discoideum as the morphogens required for stalk cell differentiation of Dictyostelium (1). In the DIF family, DIF-1 (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)-1-hexanone) was the first to be identified, and DIF-3, the monochlorinated analogue of DIF-1, is a natural metabolite of DIF-1 in Dictyostelium (2). However, the actions of DIFs are not limited to Dictyostelium. They also have strong effects on mammalian cells. DIF-1 and/or DIF-3 strongly inhibit proliferation and induce differentiation in several leukemia cells, such as the murine erythroleukemia cell line B8, human leukemia cell line K562, and human myeloid leukemia cell line HL-60 (3, 4). DIF-3 has been reported to have the most potent antiproliferative effect on mammalian leukemia cells among DIF analogues examined to date (5). Recently, we found that DIF-1 strongly inhibits proliferation and induces differentiation in human vascular smooth muscle cells, indicating that cells sensitive to DIFs are not limited to transformed cells (6).However, the target molecule (receptor) of DIFs is unknown, and it is not clear even in Dictyostelium how DIFs induce an antiproliferative effect and cell differentiation. DIFs are small hydrophobic molecules and are therefore expected to be able to cross cell membranes without requiring channels or carriers. Also, the rapidity with which DIFs induce prestalk cell-specific gene expression suggests that they directly regulate gene expression. The...

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“…This result suggests that DIFs inhibited cyclin D1 mRNA expression via the inhibition of β-catenin / TCF-dependent transcription activity. On the other hand, we also found that the activated GSK-3β translocated to the nucleus and phosphorylated cyclin D1 on Thr 286 to trigger the degradation of cyclin D1 by an ubiquitin-dependent mechanism (28,30,31). Correlated with the above observations, DIFs induced G0 / G1 cell cycle arrest, which was rescued by the overexpression of cyclin D1 (28), suggesting that DIFs were likely to induce cell cycle arrest by reducing the expression of cyclin D1.…”

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“…Therefore, activation of GSK-3β is expected to cause a reduction in both protein and mRNA levels of cyclin D1 through independent pathways. Indeed, DIFs induced phosphorylation of the Thr 286 residue of cyclin D1 and β-catenin degradation, resulting in the reduction of cyclin D1 in the protein and mRNA levels (82,84,85). We also found that DIFs reduced the activity of a TCF reporter plasmid and a reporter gene driven by the human cyclin D1 promoter (83).…”

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“…Differentiation-inducing factors (DIF) are effector molecules that have a phenol unit and can alter cell chemistry. DIF-1 and DIF-3 have been reported to inhibit b-catenin by activating GSK3-b [10].…”