Maarten F. Bijlsma

Hedgehog-Stimulated Chemotaxis Is Mediated by Smoothened Located Outside the Primary Cilium

Hedgehog stimulates a chemotactic or transcriptional response depending on the intracellular trafficking of the co-receptor Smoothened. Signaling Move from Outside the Cilium Hedgehog (Hh) can trigger transcriptional responses that control differentiation or it can stimulate chemotaxis or axonal pathfinding. The transcriptional response to Hh requires trafficking of the receptor Smoothened (Smo) to the primary cilium. Bijlsma et al. showed that cells expressing mutants of Smo that could not traffic to the primary cilium or cells lacking components needed for maintenance of the primary cilium exhibited a robust Smo-mediated chemotactic response, but failed to exhibit a transcriptional response. Cells expressing mutants that could not traffic to the primary cilium showed an enhanced chemotactic response. The intracellular trafficking of Smo was altered in the absence of the primary cilium or for Smo mutants without the ability to target to the cilium. Experiments with pharmacological inhibitors indicated that pertussis toxin–sensitive G proteins and autocrine leukotriene signaling contributed to the chemotactic response. Thus, Hh can stimulate a chemotactic or transcriptional response, which depends on the intracellular trafficking of Smo and involves distinct signaling pathways. Regulation of the Hedgehog (Hh) pathway relies on an interaction of two receptors. In the absence of Hh, Patched1 (Ptch1) inhibits the pathway. Binding of the ligand Hh to Ptch1 stimulates the localization of the activating receptor Smoothened (Smo) to the primary cilium, which is required for the transcriptional Hh response. Hh can also induce chemotaxis through a nontranscriptional pathway. We assessed the effects of defective ciliary localization of Smo on its subcellular trafficking and ability to mediate chemotactic signaling. Cells expressing mutants of Smo that could not localize to the primary cilium or cells lacking the primary cilium showed altered intracellular trafficking of Smo and, in response to Hh or Smo agonists, decreased transcriptional signaling and enhanced chemotactic responsiveness. Thus, the ciliary localization machinery appears to transport Smo to subcellular sites where it can mediate transcriptional signaling and away from locations where it can mediate chemotactic signaling. The subcellular localization of Smo is thus a crucial determinant of its signaling characteristics and implies the existence of a pool of Smo dedicated to chemotaxis.

Human plasma very low density lipoprotein carries Indian hedgehog.

Hedgehog is one of the major morphogens and fulfils critical functions in both the development and maintenance of the vasculature. Hedgehog is highly hydrophobic and its diffusion toward target tissues remains only partly understood. In Drosophila, hedgehog transport via lipophorins is relevant for development, but neither the presence nor a function for a mammalian Hedgehog carried by human plasma lipoproteins has been established. We investigated the presence of Hedgehog on lipoprotein particles and determined its importance for maintaining the endothelium. LTQ-Orbitrap XL analysis of defined plasma lipoproteins revealed that Indian Hedgehog (Ihh) is present in the human very low density lipoprotein (VLDL) fraction but not in other plasma lipoprotein fractions (low density lipoprotein (LDL) and high density lipoprotein (HDL)). Using the same approach, neither Sonic Hedgehog nor Desert Hedgehog could be detected in plasma lipoprotein fractions. Most likely, primary white adipocytes are the source of Ihh loading on VLDL as both transcriptome as well as immunofluorescence analysis showed high expression of Ihh in these cells. Additionally, we show that the endothelial compartment is most likely to be affected by the presence of Ihh on VLDL. Indeed, VLDL increased survival of primary endothelial cells, suggesting that Ihh transport by VLDL is important for maintaining the human endothelium. In conclusion, our study shows that VLDL carries Ihh throughout the body in mammals and Hedgehog signaling by human plasma VLDL particles may affect blood vessel pathophysiology. A combination of three state-of-the-art technologies, proteomics, genomics, and confocal microscopy, appeared to be a powerful tool for analyzing plasma lipoprotein-associated proteins.

Assessment of the stromal contribution to Sonic Hedgehog-dependent pancreatic adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. It is typically detected at an advanced stage, at which the therapeutic options are very limited. One remarkable feature of PDAC that contributes to its resilience to treatment is the extreme stromal activation seen in these tumors. Often, the vast majority of tumor bulk consists of non‐tumor cells that together provide a tumor‐promoting environment. One of the signals that maintains and activates the stroma is the developmental protein Sonic Hedgehog (SHH). As the disease progresses, tumor cells produce increasing amounts of SHH, which activates the surrounding stroma to aid in tumor progression. To better understand this response and identify targets for inhibition, we aimed to elucidate the proteins that mediate the SHH‐driven stromal response in PDAC. For this a novel mixed‐species coculture model was set up in which the cancer cells are human, and the stroma is modeled by mouse fibroblasts. In conjunction with next‐generation sequencing we were able to use the sequence difference between these species to genetically distinguish between the epithelial and stromal responses to SHH. The stromal SHH‐dependent genes from this analysis were validated and their relevance for human disease was subsequently determined in two independent patient cohorts. In non‐microdissected tissue from PDAC patients, in which a large amount of stroma is present, the targets were confirmed to associate with tumor stroma versus normal pancreatic tissue. Patient survival analysis and immunohistochemistry identified CDA, EDIL3, ITGB4, PLAUR and SPOCK1 as SHH‐dependent stromal factors that are associated with poor prognosis in PDAC patients. Summarizing, the presented data provide insight into the role of the activated stroma in PDAC, and how SHH acts to mediate this response. In addition, the study has yielded several candidates that are interesting therapeutic targets for a disease for which treatment options are still inadequate.

Non-cell-autonomous signaling by Shh in tumors: challenges and opportunities for therapeutic targets.

Importance of the field: The Hedgehog (Hh) pathway is required during many developmental events; in adults the Hedgehog pathway is involved in the maintenance of several stem cell niches. It is therefore not surprising that aberrantly regulated Hh pathway activity can cause birth defects in the developing organism, as well as neoplastic disease later in life. Areas covered in this review: As a consequence of the involvement in pathogenesis, the Hh pathway components are subject to an intense scrutiny as potential targets for therapeutic agents. We aim to provide an overview of the biology of the Hh proteins and the cellular response, in conjunction with potential therapeutic interventions. What the reader will gain: Specifically, we focus on the recently discovered non-cell-autonomous Shh signaling used by tumors and the implications of this for the design of treatment strategies. This should provide the reader with up-to-date knowledge on the role of the Hh pathway in tumor progression and the options to treat these malignancies. Take home message: An important concept that we advocate in this review is the need to recognize the need to target both the stromal and the tumor compartment in malignancies that rely on paracrine Shh signaling.

The Hedgehog morphogen in myocardial ischemia-reperfusion injury.

The developmental Hedgehog (Hh) protein family is known to be pivotal in many embryonic patterning events and the number of processes in which Hh plays an essential role is expanding persistently. Recently, it has become clear that the Hh pathway is not only active in the developing embryo but also in the adult organism. For example, Hh has been suggested to salvage ischemia-induced tissue damage although endogenous Hh might be deleterious during the early phase of myocardial ischemia–reperfusion. The current review provides an overview of the history of Hh biology and discusses some novel insights on Hh cell biology. Hh function in pathophysiology as well as recent findings concerning Hh signaling in ischemia models, especially in light of cardiovascular disease, is discussed in more detail and future perspectives are proposed.