Page 96 - Helicobacter pylori and Gastric Cancer: From Tumor microenvironment to Immunotherapy

observe that purmorphamine results in inhibition of PKA enzymatic activity, an effect which is also seen following stimulation of fibroblasts with Hedgehog and is thus dominant over the Smoothened-independent inhibition of its enzymatic activity. Intriguingly, purmorphamine results in a downregulation of Rho-associated protein kinase (ROCK). ROCK is important for a variety of cellular processes, but in particular for cytoskeletal reorganization[52]. It was earlier established that Smoothened is a powerful mediator of chemotactic responses, but only so when not located at the primary cilium[34]. At the primary cilium, Smoothened loses its capacity to stimulate chemotaxis. The apparent downregulation of ROCK activity following purmorphamine stimulation is thus best explained by a purmorphamine- dependent recruitment of Smoothened to the primary cilium. The strong canonical responses to purmorphamine stimulation observed by others would agree with this notion, as would the marked downregulation of PKA activity in our profiles. In some aspects, the rapid Smoothened-independent effects and rapid Smoothened- dependent effects on cellular kinase activities studied in our experimental set up, are similar, as both provoke mTOR activation and, in our model system, activation of Wnt signaling. In this sense, non-canonical signaling downstream of Patched and Smoothened may converge to produce the final phenotype. It is important to stress that our set up does not allow studying the effects of canonical Hedgehog signaling, which requires transcriptional responses. Generally speaking canonical signaling and non-canonical signaling by morphogens counteract each other and the effects observed in this study partially substantiate that notion for Hedgehog signaling as well. Not seen downstream of specific Smoothened stimulation were strong pro- inflammatory responses, which therefore seem mainly Patched-dependent. Generally speaking, Patched-specific signaling events (i.e. the effects of Hedgehog stimulation on Smoothened-/- fibroblasts) were less pronounced as those provoked by purmorphamine stimulation as also evident from the number of peptides that became significantly phosphorylated (see experimental procedures), in casu 180 peptides in Hedgehog-stimulated Smoothened-/- fibroblasts and 134 in purmorphamine-stimulated wild type fibroblasts. It thus appears that the major branch of non-canonical Hedgehog signaling is downstream of Patched but not of Smoothened.
Chapter 4
Chapter 4
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