engleski

Interaction of Gli1, SuFu and GSK3b in centrosomes of HEK293 cells

Background The Hedgehog (Hh) signaling pathway is a developmental pathway, mostly inactivated in adult tissues. Aberrant activation has been found in various tumor types, such as lung, breast, ovarian and colon. The pathway is activated by the ligand Hedgehog that causes its receptor Pathed to release its repression over the coreceptor Smoothened. This triggers a cascade of events in the cytoplasm leading to activation of transcription factor Gli1. The interactions between the transcription factor Gli1 and its regulators Suppressor of Fused (SuFu) and GSK3 or the role of GSK3 in activated cells are not fully understood yet. Recently the primary cilium was shown to play an important role in signal transduction. The human embryonic kidney cells, HEK293 show an interesting pattern of Gli1, SuFu and GSK3 accumulation in the centrosome, which gives rise to the basal body of the primary cilium. Since the primary cilia of these cells were undetectable we wanted to investigate if it is possible for these proteins to interact in the centrosome in the absence of a primary cilium. Materials and Methods HEK293 cells were cultured in MEM containing 10% FBS and treated with Shh protein (3ng/l) for 48h and with lithium chloride (LiCl) (20 mM) for 24h. For immunofluorescent staining cells were paraformaldehyde fixed and permeabilized with methanol. Primary antibodies against Gli1, SuFu, GSK3, -tubulin, Gli2 and Gli3 (Santa Cruz Biotechnology, USA) and the corresponding secondary FITC or Texas Red conjugated antibodies (Santa Cruz Biotechnology, USA) were used. mRNA expression levels were analyzed by real-time PCR and expressed as fold change relative to Arp as the housekeeping gene. Dynabeads Co-immunoprecipitation kit (Invitrogen, USA) was used for co-immunoprecipitation. Results and Discussion Exogenous Shh protein treatment causes a shift in protein localization, Gli1 translocates to the nucleus and SuFu remains in the cytoplasm. The amount of cells with visible accumulations of these proteins in the centrosome decreases from 80% to 19%. This suggests that the pathway is fully active and functioning properly. Preliminary results reveal that Gli1 and SuFu form a complex in these cells, suggesting that their interaction is independent of the primary cilium. Gli2 is undetectable in these cells, while Gli3 localizes to vesicles in the cytoplasm. Therefore it is likely that Gli1 is the main mediator of signal transduction. To examine the effect of GSK3inhibition on protein localization and interactions, we treated the cells with a GSK3 inhibitor (LiCl). Treatment elevates the pathway activity, increases expression of GLI1 and PTCH1 and also causes a shift in protein localization consistent with pathway activation. Conclusion Our results propose that HEK293 cells have an active Hh signaling pathway, with the regulatory processes between Gli1, SuFu and GSK3 taking place in the centrosome.

Hedgehog signaling

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