PARENT SESSION
Signal Transduction

WNT-2 SIGNALS THROUGH FRIZZLED-4 IN A RAT GRANULOSA CELL LINE (DC-3).

Finnson, Ken¹, Farookhi, Riaz ¹, ¹

ABSTRACT- Wnts are a family of secreted glycoproteins that regulate aspects of vertebrate embryonic and postnatal development. The wnt-canonical signaling pathway involves the interaction of wnt ligands with the 7-pass transmembrane frizzled (frz) receptor resulting in the inhibition of glycogen synthase kinase-3 (GSK-3) activity. In the absence of wnts, GSK-3 remains active and phosphorylates N-terminal serine-threonine residues of -catenin which targets -catenin for proteosomal degradation. Wnt signaling inhibits GSK-3 activity leading to an accumulation of unphosphorylated b-catenin which can enter the nucleus and interact with T-cell factor (TCF)/lymphocyte enhancer factor (LEF) transcription factors to modulate target gene expression. Wnts can also activate a non-canonical pathway which elevates intracellular calcium levels and increases activity of calcium dependent protein kinases including PKC. We have shown previously that both wnt-2 and frz-4 are expressed in rat granulosa cells suggesting that a wnt-2/frz-4 interaction may activate wnt signaling in these cells. To determine if wnt-2 can signal through frz-4 and if the wnt-2/frz-4 interaction activates the canonical pathway, an immortalized rat granulosa cell line (DC-3) which expresses frz-4 but not wnt-2 was transfected with either wnt-2 or a constitutively active form of -catenin (-cat-S37A). Activation of the wnt canonical pathway was assessed by a luciferase reporter construct containing tcf/lef binding sites upstream of a minimal promoter. Lithium, which mimics wnt signaling by inhibiting GSK-3, increased reporter activity in a dose-dependent manner indicating that the wnt-canonical pathway is functional in DC-3 cells. Neither wnt-2 nor -cat-S37A induced reporter activity. In the presence of lithium (20 mM) however, wnt-2 decreased reporter activity to ~20% of control levels while -cat-S37A increased reporter activity to ~120 % of control levels. These data indicate that wnt-2 can signal through frz-4 and that this signaling does not appear to directly involve the wnt canonical signaling pathway.

KEY WORDS: wnt-2, frizzled 4, granulosa cell line (DC3), -catenin