Electrical impedance spectroscopy and 3D cell cultures as emerging technologies for studying signal transduction pathways: the curious case of cathelicidins

This doctoral dissertation has two important parts. The first part covers experiments on elucidating the signal transduction pathways of recently discovered candidate bioactive peptides. The second part introduces 3D cell culture systems and expands on differential gene expression between 2D and 3D cell cultures using RNA-sequencing.Bioactive peptides perform countless functions in multicellular organisms as key players in cell-cell communication. They are involved in the regulation of various biochemical and biophysical processes such as cell growth, cell metabolism, reproduction, and homeostasis. Endogenous peptides can either signal over a short distance (auto- or paracrine signaling) or a long distance (endocrine signaling). Whatever their range of action, peptide signaling is usually accomplished through binding to a receptor protein expressed on target cells. Both the peptide as its cognate receptor(s) are potential targets for therapeutic interference, in case the peptide is implicated in a certain physiological or pathological process.Continuous improvement of peptide-extraction methods as well as mass spectrometry-based techniques enabled researchers to identify endogenous peptides in complex mixtures such as tissue extracts. By adapting peptidomics technologies previously used for invertebrates, the laboratory of prof. Schoofs systematically discovered more than 1000 new candidate bioactive peptides in various mouse and human tissues, from which approximately 700 were selected for synthesis and functional assessment.In this thesis project, candidate peptides were tested for activity on a collection of cultured cell lines representative for almost all cell types present in humans or mice. In a first stage, screening for activity was done using electrical impedance spectroscopy, a technique capable of detecting a cellular response regardless of the intracellular signaling cascade that is triggered. Once a response was detected upon exposure to a candidate peptide, further experiments using pharmacological agents were performed to identify its cognate receptor(s) and unravel its mechanism of action. Screening experiments indicated that the purity level of candidate peptides is important for adequate interpretation of results, as several effects evoked by crude (> 70% purity) peptides could not be reproduced with purified (> 95%) peptides. However, a significant number of pure peptides were shown to be biologically active, which opened ample perspectives to study their mode of action.We primarily focused on one peptide, codenamed P318. P318 is a novel fragment of the murine cathelicidin-related antimicrobial peptide (CRAMP). Cathelicidins play a crucial role in the immune system as they exhibit both immunomodulatory and direct antimicrobial activities. Probably the most widely studied member of the cathelidicin family is LL-37, the sole cathelicidin peptide found in humans. Various reports suggest that LL-37 exerts pleiotropic effects through interacting with various putative cell surface receptors and/or intracellular targets in a wide variety of cell lines. However, the molecular details by which LL-37 exerts its effect on host cells are often incompletely understood.Impedance measurements showed that LL-37 and P318 were able to activate a wide variety of cultured cell lines. Various cell lines exhibited unique impedance responses to LL-37 or P318, which may reflect different signaling events induced by the peptides. Subsequent experiments with pharmacological agents further indicated that the action of LL-37 or P318 is cell type-dependent. For example, LL-37 dose-dependently evoked calcium mobilization in HEK293T cells via the PLC-IP3R pathway. Strong indications were also found that LL-37 binds to the cell membrane rather than to specific ligand-binding sites of a receptor. However, in RAW264.7 macrophages as well as in A549 lung carcinoma cells (and not in HEK293T cells), the activation of a currently unknown Gai-coupled receptor(s) is involved in the signaling cascade induced by LL-37. Intriguingly, endocytic pathways also seem to play a role in A549 cells.Even though several parallel effects of P318 and LL-37 were discovered (e.g. in HEK293T cells), P318 exerted an effect on B16 melanoma cells through a different molecular mechanism than LL-37. In this cell line, P318 increased levels op cAMP and caused hyperpolarization across the cell membrane, after which the signal was transduced through an Akt-dependent pathway.In the final part of this doctoral dissertation, 3D cell culture systems were covered as they mimic in vivo cell and tissue biology more closely than 2D monolayer cultures. Multiple studies report differences in gene and protein expression between 3D spheroids and 2D cell cultures. These divergent gene expression profiles can lead to differences in receptor expression which in turn drive an altered response to candidate drugs. However, an easily accessible overview of differential receptor expression among cells cultured in 2D and 3D is currently not available. Therefore, we used RNA-sequencing to examine differentially expressed receptor genes between 2D and 3D cell cultures of HT-29 colorectal adenocarcinoma cells and identified 17 differentially expressed genes encoding GPCRs, as well as several clinically relevant RTKs and ion channels. In addition, we performed pathway enrichment analysis and showed that a number of pathways were altered among HT-29 spheroids and 2D cell cultures, including pathways associated with cell cycle progression and glucose metabolism.

Jaar van publicatie:2017