The role of keratin intermediate filaments in the colon epithelial cells

Keratins (K) are cytoskeletal proteins mainly expressed in the epithelium and constitute the largest subgroup of intermediate filaments (IFs). Simple epithelial keratins (SEKs) K7-K8 and K18-K20 are the major IF elements in the colon. SEK mutations are known to cause around 30 human diseases, mainly affecting liver and skin. However, so far no strong associations between K8 mutations and the development of human colitis have been found. The keratin contribution to colonic health comes from the K8 knock-out (K8-/-) mouse model, which develops an early chronic inflammation and hyperproliferation in the colon. The aim of this thesis was to investigate how keratins contribute to intestinal health and disease mainly by the experimental analysis using the K8-/- mouse colon and cell culture models. The work described here is divided into three studies. The first study revealed involvement of keratins in Notch1 signaling, which is the master regulator of cell fate in the colon. Immunoprecipitation and immunostaining, both in vitro and in vivo showed that K8 binds and co-localizes with Notch1. Interestingly, overexpression of keratins enhanced Notch1 levels and stabilized Notch intracellular domain (NICD), leading to higher activity of Notch signaling. The dramatic decrease in Notch activity in the K8-/- colon resulted in a differentiation shift towards goblet and enteroendocrine cells. The second study focused on the involvement of keratins in colitis-associated cancer (CAC). Although, the K8-/- inflamed colon did not develop colorectal cancer (CRC) spontaneously, it was dramatically more susceptible to induced CRC in two CRC models: azoxymethane (AOM) and multiple intestinal neoplasia (ApcMin/+). To understand how the loss of K8 contributes to CAC, the epithelial inflammasome signaling pathway was analyzed. The released component of active inflammasome, cleaved caspase-1 and its downstream protein, interleukin (IL)-18, were significantly increased in K8-/- and K8-/-ApcMin/+ colons. The inflammasome pathway has recently been suggested to control the levels of IL-22 binding protein (IL-22BP), which is a negative regulator of IL-22 activity. Interestingly, the activated inflammasome correlated with an upregulation of IL-22 and a complete loss of IL-22BP in the K8-null colons. The activation of IL-22 was confirmed by increased levels of downstream signaling, which is phosphorylated signal transducer and activator of transcription 3 (P-STAT3), a transcription factor promoting proliferation and tissue regeneration in the colon. The objective of the third study, was to examine the role of keratins in colon energy metabolism. A proteomic analysis identified mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) as the major ownregulated protein in the K8-/- colonocytes. HMGCS2 is the rate-limiting enzyme in ketogenesis, where energy from bacterially produced short chain fatty acids (SCFAs), mainly butyrate, is converted into ketone bodies in colonic epithelium. Lower levels and activity of HMGCS2 in the K8-/- colon resulted in a blunted ketogenesis. The studies upstream from HMGCS2, identified decreased levels of the SCFA-transporter monocarboxylate transporter 1 (MCT1), which led to increased SCFA content in the stool suggesting impaired butyrate transport through the colonic epithelium. Taken together, the results of the herein thesis indicate that keratins are essential regulators of colon homeostasis, in particular epithelial differentiation, tumorigenesis and energy metabolism.

Imaging techniques applied for detection of secretion, transgene delivery and G proteincoupled receptors in reproductive and endocrine cells in vivo and in vitro

Post-testicular sperm maturation occurs in the epididymis. The ion concentration and proteins secreted into the epididymal lumen, together with testicular factors, are believed to be responsible for the maturation of spermatozoa. Disruption of the maturation of spermatozoa in the epididymis provides a promising strategy for generating a male contraceptive. However, little is known about the proteins involved. For drug development, it is also essential to have tools to study the function of these proteins in vitro. One approach for screening novel targets is to study the secretory products of the epididymis or the G protein-coupled receptors (GPCRs) that are involved in the maturation process of the spermatozoa. The modified Ca2+ imaging technique to monitor release from PC12 pheochromocytoma cells can also be applied to monitor secretory products involved in the maturational processes of spermatozoa. PC12 pheochromocytoma cells were chosen for evaluation of this technique as they release catecholamines from their cell body, thus behaving like endocrine secretory cells. The results of the study demonstrate that depolarisation of nerve growth factor -differentiated PC12 cells releases factors which activate nearby randomly distributed HEL erythroleukemia cells. Thus, during the release process, the ligands reach concentrations high enough to activate receptors even in cells some distance from the release site. This suggests that communication between randomly dispersed cells is possible even if the actual quantities of transmitter released are extremely small. The development of a novel method to analyse GPCR-dependent Ca2+ signalling in living slices of mouse caput epididymis is an additional tool for screening for drug targets. By this technique it was possible to analyse functional GPCRs in the epithelial cells of the ductus epididymis. The results revealed that, both P2X- and P2Y-type purinergic receptors are responsible for the rapid and transient Ca2+ signal detected in the epithelial cells of caput epididymides. Immunohistochemical and reverse transcriptase-polymerase chain reaction (RTPCR) analyses showed the expression of at least P2X1, P2X2, P2X4 and P2X7, and P2Y1 and P2Y2 receptors in the epididymis. Searching for epididymis-specific promoters for transgene delivery into the epididymis is of key importance for the development of specific models for drug development. We used EGFP as the reporter gene to identify proper promoters to deliver transgenes into the epithelial cells of the mouse epididymis in vivo. Our results revealed that the 5.0 kb murine Glutathione peroxidase 5 (GPX5) promoter can be used to target transgene expression into the epididymis while the 3.8 kb Cysteine-rich secretory protein-1 (CRISP-1) promoter can be used to target transgene expression into the testis. Although the visualisation of EGFP in living cells in culture usually poses few problems, the detection of EGFP in tissue sections can be more difficult because soluble EGFP molecules can be lost if the cell membrane is damaged by freezing, sectioning, or permeabilisation. Furthermore, the fluorescence of EGFP is dependent on its conformation. Therefore, fixation protocols that immobilise EGFP may also destroy its usefulness as a fluorescent reporter. We therefore developed a novel tissue preparation and preservation techniques for EGFP. In addition, fluorescence spectrophotometry with epididymal epithelial cells in suspension revealed the expression of functional purinergic, adrenergic, cholinergic and bradykinin receptors in these cell lines (mE-Cap27 and mE-Cap28). In conclusion, we developed new tools for studying the role of the epididymis in sperm maturation. We developed a new technique to analyse GPCR dependent Ca2+ signalling in living slices of mouse caput epididymis. In addition, we improved the method of detecting reporter gene expression. Furthermore, we characterised two epididymis-specific gene promoters, analysed the expression of GPCRs in epididymal epithelial cells and developed a novel technique for measurement of secretion from cells.

From epithelial to mesenchymal: regulation of invasive cancer cell motility

Metastasis is the main cause of death among cancer patients. In order to initiate the metastatic cascade cancer cells have to undergo epithelial-to-mesenchymal transition (EMT). In EMT epithelial cells lose their cell-cell and cell-extracellular matrix (ECM) contacts and become more motile. The expression of the transcription factor Slug and of the mesenchymal intermediate filament vimentin is induced during EMT. Vimentin is often overexpressed in malignant epithelial cancers but the functional role of vimentin remains incompletely understood. In addition, kinases such as AKT and ERK are known to be involved in the regulation of EMT and cancer cell motility but the mechanisms underlining their functions are often unclear. Integrins are heterodimeric receptors that attach cells to the surrounding tissue and participate in regulating cell migration and invasion. Changes in integrin activity are linked to increased cell motility and further cancer metastasis. The aim for my PhD studies was to investigate the role of cellular signalling pathways and vimentin in the regulation of cancer cell motility and EMT. Our results revealed that in prostate cancer the downregulation of AKT1 and AKT2, but not AKT3, induces activation of cell surface 1-integrins leading to enhanced cell adhesion, migration and invasion. In addition, our findings demonstrated a reciprocal regulatory interaction between vimentin and ERK2 facilitating ERK-mediated phosphorylation of Slug at serine-87 (S87) in breast cancer. Surprisingly, Slug S87 phosphorylation is dispensable for E-cadherin repression but essential for the induction of vimentin and Axl expression in early onset of EMT. Our findings reveal previously unknown mechanistic information of how prostate and breast cancer cell motility and disease progression is regulated