Abstract
Observations that Glioma-associated transcription factors Gli1 and Gli2 (Gli1/2), executers of the Sonic Hedgehog (Shh) signaling pathway and targets of the Transforming Growth Factor β (TGF-β) signaling axis, are involved in numerous developmental and pathological processes unveil them as attractive pharmaceutical targets. Unc-51-like serine/threonine kinase Ulk3 has been suggested to play kinase activity dependent and independent roles in the control of Gli proteins in the context of the Shh signaling pathway. This study aimed at investigating whether the mechanism of generation of Gli1/2 transcriptional activators has similarities regardless of the signaling cascade evoking their activation. We also elucidate further the role of Ulk3 kinase in regulation of Gli1/2 proteins and examine SU6668 as an inhibitor of Ulk3 catalytic activity and a compound targeting Gli1/2 proteins in different cell-based experimental models. Here we demonstrate that Ulk3 is required not only for maintenance of basal levels of Gli1/2 proteins but also for TGF-β or Shh dependent activation of endogenous Gli1/2 proteins in human adipose tissue derived multipotent stromal cells (ASCs) and mouse immortalized progenitor cells, respectively. We show that cultured ASCs possess the functional Shh signaling axis and differentiate towards osteoblasts in response to Shh. Also, we demonstrate that similarly to Ulk3 RNAi, SU6668 prevents de novo expression of Gli1/2 proteins and antagonizes the Gli-dependent activation of the gene expression programs induced by either Shh or TGF-β. Our data suggest SU6668 as an efficient inhibitor of Ulk3 kinase allowing manipulation of the Gli-dependent transcriptional outcome.
Highlights
Misregulation of cellular signaling pathways, that are important in embryonic development and maintaining adult homeostasis, leads to inherited as well as sporadic diseases
It has been previously shown that a tyrosine kinase inhibitor SU6668 can physically interact with ULK3 in a chromatographic pull-down assay [23]
Mechanistic differences specific for these intracellular signaling cascades are obvious: TGFβ/SMAD axis controls GLI2 transcription, whereas post-translational regulation of Gli2 protein activity is the central event in the Sonic Hedgehog (Shh) pathway
Summary
Misregulation of cellular signaling pathways, that are important in embryonic development and maintaining adult homeostasis, leads to inherited as well as sporadic diseases. One of such pathways, where a clear correlation between abnormal pathway activation and disease progression has been observed, is the Sonic Hedgehog (Shh) signaling pathway [1]. Disruption or misregulation of the Shh pathway results in various developmental abnormalities including holoprosencephaly, Pallister–Hall syndrome, Gorlin syndrome, Greig cephalopolysyndactyly, Abbreviations: AP, alkaline phosphatase; ASCs, adipose tissue derived stromal cells; GliACT, transcriptional activator form of Gli proteins; GliFL, full-length Gli proteins; Ptch, patched; RNAi, RNA interference; Shh, Sonic Hedgehog; Smo, Smoothened; shRNA, short hairpin RNA; siRNA, small interfering RNA; Sufu, Suppressor of Fused; TGF-β, Transforming Growth Factor β; Ulk, unc-51-like kinase 3; WB, Western blot; HSC, Hedgehog signaling complex; qRT-PCR, quantitative real-time PCR. Important is the TGF-β signaling pathway, with its role in various types of cancer, vascular diseases and fibrosis [2,3]
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