Epithelial morphogenesis of MDCK cells in three-dimensional collagen culture is modulated by interleukin-8

Abstract

Under these conditions, MDCK cells spontaneously formed either hollow spherical cysts or flat monolayer sheets, respectively. Microarray analysis of gene expression revealed a twofold or greater expression difference in 732 gene sets from MDCK cysts compared with monolayers (false discovery rate or FDR-adjusted P values Ͻ0.05). Interleukin-8 (IL-8) was reproducibly found to be among the genes whose expression was most dramatically upregulated, and this behavior was verified through real-time PCR analysis. The level of IL-8 protein expression was significantly increased in 3D MDCK cultures compared with that detected in cells in 2D culture. Hepatocyte growth factor (HGF) induces MDCK cells in 3D culture to form linear tubule-like structures. We found that HGF stimulation caused MDCK cells in 3D culture to decrease the expression of IL-8 at both the mRNA and protein levels. Furthermore, the addition of recombinant IL-8 to HGF-stimulated 3D MDCK cultures was sufficient to partially reverse the tubulogenic effects of HGF, resulting in the formation of cystic structures. These data suggest that IL-8 participates in the formation of cystic structures by MDCK cells in 3D culture and that HGF may stimulate tubulogenesis through the suppression of IL-8. renal epithelial cells; interleukin-8; hepatocyte growth factor; morphogenesis; Madin-Darby canine kidney IN TISSUES SUCH AS the lungs, intestines, and kidneys, epithelial cells form barriers between an organism and its external milieu. The surface membranes of these epithelial cells are exposed to disparate environments and are divided into discrete apical and basolateral domains (4,8,37,39). These membrane domains are established and maintained through the directed trafficking and selective retention of cellular components. In addition to this capacity to generate polarized plasma membrane domains with distinct biochemical and physiological properties, epithelial cells must also be able to organize themselves into complex multicellular architectures. The formation and maintenance of these structures is dependent both upon intercellular interactions between epithelial cells and upon cell-matrix interactions.Interactions between an individual cell and its surrounding environment are essential in the activation of signaling networks that subsequently regulate polarity and the determination of epithelial structural morphology (6,7,9,40,48). As epithelial cells establish polarity and interactions with their environment they can begin to function together to generate organized multicellular structures. This property is exemplified by comparing the behaviors of epithelial cell lines, such as Madin-Darby canine kidney (MDCK) cells, grown in twodimensional (2D) vs. three-dimensional (3D) culture conditions. MDCK cells form flat monolayer sheets when grown atop tissue culture plastic, permeable filter supports, and various extracellular matrix (ECM) substrates in traditional 2D culture conditions. When grown in 3D culture, in which cells are embedded within an ECM substrate, MDCK...

Novel MAPK-dependent and -independent tubulogenes identified via microarray analysis of 3D-cultured Madin-Darby canine kidney cells

“…Given that macrophage infiltration (often mediated by chemokines) has been implicated recently in abnormal cyst formation in ADPKD (17) and that an increase in levels of CCL-2 has been correlated with acute kidney injury (31), our results point to biologically significant targets, previously not identified using 2D culture. Supporting this idea is the fact that the gene most highly downregulated, exclusively in 3D, in response to HGF, was IL-8 (#1 in Table 1), which was recently shown to participate in the formation of cysts by MDCK cells in 3D culture, with HGF stimulating tubulogenesis through the suppression of IL-8 (45).…”

Identifying ultrasensitive HGF dose-response functions in a 3D mammalian system for synthetic morphogenesis

“…Because of this, the transcriptome profile that it induces has been previously explored in canine MDCK cells grown in both 2D cell culture and 3D cell culture48495051. These transcriptome studies identified HGF-responsive genes, such as matrix metalloproteinase 1 (MMP1), but used single high doses of HGF (e.g.…”

“…Overall, we identified ~12,000 genes (out of 25,000 annotated genes) that were differentially-expressed between the presence or absence of HGF. These included genes that were previously reported to be activated by HGF, including MMP1, TIMP1 and IL8495051. By collecting gene expression data for various doses of HGF we were able to go one step further and characterise more detailed dose-response functions.…”

The influence of GAP‐43 on orientation of cell division through G proteins

“…In order to verify the assumption, a three-dimensional (3D) epithelial cyst culture of Madin-Darby Canine Kidney (MDCK) cells transfected with GAP-43, were used in the present study, to induce the polarized epithelia within 3-4 days, since MDCK cells in 3D conditions can orientate a single, central lumen, which makes the orientation of cell division more accurately (Wells et al, 2013). This can mimic formation of the neural tube (which is a pseudo stratified epithelium with the apical cell surface oriented toward a single, central lumen) in vivo and to reconstitute efficient and faithful division of NPCs.…”