Gene expression alterations during HGF-induced dedifferentiation of a renal tubular epithelial cell line (MDCK) using a novel canine DNA microarray

Balkovetz, Daniel F.; Gerrard, Edward R.; Li, Shixiong; Johnson, D. W.; Lee, James; Tobias, John W.; Rogers, Katherine K.; Snyder, Richard; Lipschutz, Joshua H.

doi:10.1152/ajprenal.00270.2003

Cited by 31 publications

(26 citation statements)

References 41 publications

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“…The fold changes for the conditions (HGF, HGF + PD, control, and PD alone) are shown in graph form. The fold change for fibronectin following stimulation with HGF for 24 hours as determined by realtime PCR was very similar to the 4.89-fold change obtained previously by real-time PCR [23]. Inhibition of upregulation was seen with the addition of PD or UO (not shown).…”

Section: Discussionsupporting

confidence: 86%

“…For a typical real-time PCR experiment, the calculated fold increase for fibronectin, normalized to the 18S ribosome (a control mRNA), was 4.14 +/− 0.69 for 24 h of HGF exposure compared to 0 h of HGF exposure (control) ( Figure 1). This was very similar to the fold increase in fibronectin mRNA by HGF as determined by microarray analysis and real-time PCR in our previous study [23]. The increase in fibronectin mRNA expression following 24 hours of exposure to HGF was prevented by the addition of the MEK inhibitors PD (Figure 1) and UO (not shown).…”

Section: Fibronectin Activation Via the Mapk/erk Pathway Using Real-tsupporting

confidence: 89%

“…Importantly, fibronectin, which we previously found in a microarray study to be induced by HGF [23], has been shown to be essential for kidney tubulogenesis [24,25]. Here we complete the pathway for tubulogenic fibronectin, by showing that it is activated by HGF via the intracellular ERK/MAPK signaling pathway.…”

Section: Introductionmentioning

confidence: 67%

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Intracellular signaling via ERK/MAPK completes the pathway for tubulogenic fibronectin in MDCK cells

Zhao

Greco

Hellman

et al. 2007

Biochemical and Biophysical Research Communications

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A classic in vitro model of branching morphogenesis utilizes the Madin-Darby canine kidney (MDCK) cell line. MDCK Strain II cells form hollow monoclonal cysts in a three-dimensional collagen matrix over the course of ten days and tubulate in response to hepatocyte growth factor (HGF). We and our colleagues previously showed that activation of the extracellular-signal regulated kinase (ERK, aka MAPK) pathway is necessary and sufficient to induce tubulogenesis in MDCK cells. We also showed in a microarray study that one of the genes upregulated by HGF was the known tubulogene fibronectin. Given that HGF activates a multitude of signaling pathways, including ERK/ MAPK, to test the intracellular regulatory pathway, we used two distinct inhibitors of ERK activation (U0126 and PD098059). Following induction of MDCK Type II cells with HGF, tubulogenic fibronectin mRNA was upregulated 4-fold by real time PCR, and minimal or no change in fibronectin expression was seen when HGF was added with either U0126 or PD098059. We confirmed these results using an MDCK cell line inducible for Raf, which is upstream of ERK. Following activation of Raf, fibronectin mRNA and protein expression were increased to a similar degree as was seen following HGF induction. Furthermore, MDCK Strain I cells, which originate from collecting ducts and have constitutively active ERK, spontaneously initiate tubulogenesis. We show here that MDCK Strain I cells have high levels of fibronectin mRNA and protein compared to MDCK Strain II cells. When U0126 and PD098059 were added to MDCK Strain I cells, fibronectin mRNA and protein levels were decreased to levels seen in MDCK Strain II cells. These data allow us to complete what we believe is the first description of a tubulogenic pathway from receptor/ligand (HGF/CMET), through an intracellular signaling pathway (ERK/MAPK), to transcription and, finally, secretion of a critical tubuloprotein (fibronectin).

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Section: Discussionsupporting

confidence: 86%

Section: Fibronectin Activation Via the Mapk/erk Pathway Using Real-tsupporting

confidence: 89%

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Intracellular signaling via ERK/MAPK completes the pathway for tubulogenic fibronectin in MDCK cells

Zhao

Greco

Hellman

et al. 2007

Biochemical and Biophysical Research Communications

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“…It was, therefore, not surprising to find that our dataset of differentially expressed genes was enriched in genes that encode proteins involved with cell adhesion and with the formation or remodeling of the ECM. In fact, microarray analysis of HGF-treated MDCK cells has shown that this treatment alters the expression of a large number of genes, particularly those involved in ECM associations (1,20). Additionally, an Affymetrix microarray analysis of HGF-stimulated MDCK cells revealed an upregulation of the expression of MMP13 and TIMP1 (14).…”

Section: Discussionmentioning

confidence: 99%

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

Wells

Yarborough

Lifton

et al. 2013

American Journal of Physiology-Cell Physiology

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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...

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“…Our first step was to establish an expression profile for known calcium channels in MDCK cells by reverse transcriptase PCR. Since c-met activation triggers dramatic changes in gene expression in MDCK cells (Balkovetz et al, 2004), calcium channel expression was determined before and after HGF treatment. We found detectable levels of expression of a number of Trp calcium channels, including TrpC 1, 4, 6 and 7, TrpV 1-4, TrpM 3-8 and PKD 1-2 (Fig.…”

Section: Identification Of Channels That Participate In Hgf Signalingmentioning

confidence: 99%

Plasma membrane ion fluxes and NFAT-dependent gene transcription contribute to c-met-induced epithelial scattering

Langford

Keyes

Hansen

2012

Journal of Cell Science

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SummaryHepatocyte growth factor (HGF) signaling drives epithelial cells to scatter by breaking cell-cell adhesions and causing them to migrate as solitary cells, a process that parallels epithelial-mesenchymal transition. HGF binds and activates the c-met receptor tyrosine kinase, but downstream signaling required for scattering remains poorly defined. We have applied a chemical biology approach to identify components of HGF signaling that are required for scattering in an in vitro model system. This approach yields a number of small molecules that block HGF-induced scattering, including a calcium channel blocker. We show that HGF stimulation results in sudden and transient increases in ion channel influxes at the plasma membrane. Although multiple channels occur in the membranes of our model system, we find that TrpC6 is specifically required for HGF-induced scattering. We further demonstrate that HGF-induced ion influxes through TrpC6 channels coincide with a transient increase in nuclear factor of activated T-cells (NFAT)-dependent gene transcription and that NFAT-dependent gene transcription is required for HGF-induced cell scattering.

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Gene expression alterations during HGF-induced dedifferentiation of a renal tubular epithelial cell line (MDCK) using a novel canine DNA microarray

Cited by 31 publications

References 41 publications

Intracellular signaling via ERK/MAPK completes the pathway for tubulogenic fibronectin in MDCK cells

Intracellular signaling via ERK/MAPK completes the pathway for tubulogenic fibronectin in MDCK cells

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

Plasma membrane ion fluxes and NFAT-dependent gene transcription contribute to c-met-induced epithelial scattering

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