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. Author manuscript; available in PMC: 2008 Feb 16.
Published in final edited form as: Biochem Biophys Res Commun. 2006 Dec 22;353(3):793–798. doi: 10.1016/j.bbrc.2006.12.106

Intracellular Signaling via ERK/MAPK Completes the Pathway for Tubulogenic Fibronectin in MDCK Cells

Zhao Liu 1, Andres J Greco 1, Nathan E Hellman 1, June Spector 1, Jonathan Robinson 1, Oliver T Tang 1, Joshua H Lipschutz 2
PMCID: PMC1839983  NIHMSID: NIHMS16683  PMID: 17196167

Abstract

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).

Keywords: ERK, MDCK, Tubulogenesis, Fibronectin

Introduction

Epithelial organs such as the kidney, lung, and salivary gland develop from a process termed branching morphogenesis [1]. Though branching morphogenesis is incompletely understood, genes involved in this process are conserved in different organs, and indeed throughout evolution. Of the mammalian organs, kidney development is probably best understood [2] and the kidney is particularly well suited for studying the two basic building blocks of branching morphogenesis, cysts and tubules [3].

Due to the complexity of organogenesis (the human kidney is composed of more than twenty cell types and one million nephrons [3, 4]) and the transitory nature of cyst and tubule formation, it is difficult to study these processes in vivo. Relatively little, therefore, was known about the initiation of cyst and tubule formation prior to the development of an in vitro assay. The MDCK cell lines were derived from the kidney tubules of a normal cocker spaniel in 1958 [5, 6] and have been one of the most widely used reagents for studying important and fundamental issues in epithelial cell biology [7]. When MDCK cells are seeded singly within a three-dimensional collagen matrix, they form monoclonal cysts over ten days [8, 9]. Exposure of preformed MDCK cysts to HGF causes the cysts to develop branching tubules [10] in a process that resembles renal branching morphogenesis in vivo [4].

The vast majority of studies examining cyst and tubule formation using MDCK cells were performed with Strain II cells [1, 11, 12]. MDCK Strain I cells, derived from an early passage of the cell population, and MDCK Strain II cells, which predominate in later passages, originated from separate nephron segments [13, 14]. MDCK Strain I cells were determined to be of cortical collecting duct cell origin based on their high electrical resistance, their responsiveness to vasopressin and the absence of more proximal marker enzymes, such as alkaline phosphatase and γ-glutamyl transferase. MDCK Strain II cells resemble more proximal renal tubular epithelial cells [14]. Another major difference between MDCK Strain I and Strain II cells, is the presence of high levels of active ERK in MDCK Strain I, compared to Strain II, cells [15].

Detailed studies using MDCK Strain II cells grown in a collagen matrix until the cyst stage and induced with HGF showed that tubulogenesis consists of two morphologically-defined stages: an initiation phase termed the partial epithelial-mesenchymal transition (p-EMT) that occurs in the first 24 hours following HGF induction and subsequent redifferentiation [3, 11, 16]. In morphologic terms, the p-EMT phase involves formation of actin extensions and chains of cells, which have lost their polarity, extending off the basolateral surface of the cysts [16]. HGF (aka scatter factor) is mitogenic, motogenic, and morphogenic and binding of HGF to its CMET tyrosine kinase receptor, which is located on the basolateral surface of MDCK cells [17], activates a multitude of signaling pathways including: phosphoinositide 3-kinase, phospholipase C, protein tyrosine phosphatase 2, cytosolic phospholipase A2, and ERK/MAPK to name a few (as reviewed in [18]).

Recently, we and our colleagues showed that the mitogen-activated protein (MAP) kinase pathway of Raf-MEK-ERK is necessary and sufficient to initiate the p-EMT stage of tubulogenesis in the MDCK/HGF system [11, 19]. The ERK/MAPK pathway, which is downstream of receptor tyrosine kinases, leads to phosphorylation, and hence activation, of ERK and has been shown to be important in branching morphogenesis in several systems, from Drosophila to mammals [20, 21]. ERK/MAPK has also been shown to be necessary for branching morphogenesis of the ureteric bud, the collecting duct progenitor, in the embryonic kidney [22].

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.

Materials and Methods

MDCK culture, HGF treatment, and RNA Isolation

Low passage MDCK Strain I (obtained from Karl Matlin, University of Cincinnati) and Strain II (obtained from Keith Mostov, UCSF) cells were used. Cells were cultured in modified Eagle's minimum essential medium supplemented with 5% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. MDCK cells were seeded at confluency on 24-mm Transwell filter units coated with collagen (Costar, Cambridge, MA). Cell monolayers were used for experiments after six days of culture with daily changes in medium. Recombinant HGF at 100 ng/ml was added to the basolateral compartment of MDCK cell monolayers following a 1 h pretreatment either with or without U0126 (Promega) or PD098059 (Sigma), both inhibitors of MEK, at 10 μM and 50μM respectively. HGF at 100 ng/ml has been shown to induce tubulogenesis in MDCK cells [1, 19]. Recombinant human HGF was generously provided by the late R. Schwall (Genentech, South San Francisco).

MDCK Strain II cells containing inducible Raf fused to the estrogen receptor [26] were grown in a similar manner to that described above, except on plastic. In these cells, the kinase activity of Raf was induced using an estrogen analog, 4-hydroxytamoxifen (4-HT) (Sigma) at a concentration of 1 μM as described previously [26].

Total RNA was obtained from MDCK cell monolayers using the Rneasy Protocol (Qiagen) for the isolation of total RNA from animal cells. RNA concentration and purity was measured by determining the 260 nm/ 280 nm ratio. All ratios were >1.8.

Real-time PCR

Fifteen ug of total RNA, collected from MDCK cells grown for six days on Transwell filters and exposed to zero, or twenty-fours of HGF +/− UO126 or PD09859, were converted to first-strand cDNA. cDNA and the TaqMan primer/probe system, individualized for each mRNA, were used in conjunction with the 7700 PRISM Sequence Detection Instrument (both Applied Biosystems) as described in the Applied Biosystems Technical Manual. The values were normalized to a control mRNA, the 18S ribosome.

Western Blotting

Western blot analysis was performed as described previously [27]. For total ERK analysis, filters were probed with goat-anti-ERK1/2 polyclonal IgG (1:1000) (Santa Cruz Biotechnology). Filters were then probed with horseradish peroxidase-labeled donkey anti-goat at 1:15,000 dilution. For active ERK/MAPK analysis, filters were probed with Phospho-p44/42(ERK 1/2) MAPK Antibody (1:1000) (Cell Signaling Technologies). Filters were also probed with antibodies against claudin-2 (Zymed, rabbit polyclonal IgG, 1:250) and fibronectin (Biogenesis Ltd, rabbit polyclonal IgG, 1:750). Filters were then probed with horseradish peroxidase-labeled goat anti-rabbit at 1:15,000 dilution. Filters were developed using SuperSignal West Femto maximum sensitivity substrate kit (Pierce) and visualized on Kodak X-OMAT film (Eastman Kodak, Rochester, NY).

Statistics

To determine the p values for the real-time PCR experiments, the data for all experiments were combined and an ANOVA analysis was performed with pairwise comparisons of conditions.

Results

Fibronectin Activation Via the MAPK/ERK Pathway Using Real-Time PCR

Fibronectin has been shown to be essential for the initiation of tubulogenesis [24, 25]. To test if fibronectin mRNA expression was upregulated by HGF via the ERK/MAPK pathway, we used real-time PCR. Sequence information for canine fibronectin (NCBI, GenBank No. U16207) was used to design the primers and probes. 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). Similar results were seen at the protein level (data not shown).

Figure 1. Fibronectin mRNA Expression Levels in MDCK Strain II Cells by Real-time PCR.

Figure 1

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RNA was harvested from MDCK Strain II cells grown on filters for six days and exposed for 24 hours to HGF, HGF + PD, control medium, or PD alone. The RNA was converted to cDNA and amplified using the TaqMan primer/probe fluorescence assay on a 7700 PRISM instrument. The primer and probe were designed to specifically recognize fibronectin cDNA. When the reaction product amplification exceeds the threshold value, the corresponding cycle number is termed CT. Fold change between conditions was calculated through an exponential function of the observed difference in CT as previously described [33]. Fold changes for the control samples (no HGF exposure) were normalized and are listed as 1. 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 real-time 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). The real-time PCR data were normalized to a control mRNA, the 18S ribosome, and were performed three times with triplicate samples. The p values in were determined by combining all the experimental data and performing ANOVA with pairwise comparisons.

Confirmation of the Real Time PCR Results Using MDCK Cells Expressing Inducible Raf

To confirm that fibronectin expression was upregulated via the MAPK/ERK pathway, we used an MDCK Strain II cell line in which ERK activation is conditionally regulated [26]. These cells (MDCK Raf:ER) stably express an inducible form of Raf-1 kinase, due to transfection with a plasmid construct containing a fusion of the Raf-1 kinase domain with the estrogen receptor ligand binding domain [28]. Binding of the estrogen analog 4-hydroxytomoxifen (4-HT) to the estrogen receptor moiety activates the Raf-1 domain, which then leads to phosphorylation and activation of downstream MEK and ERK [11, 26].

MDCK Raf:ER cells were plated at confluency and grown for six days, prior to stimulation for 24 hours with 4-HT (1 μM), 4-HT plus UO, 4-HT plus PD, or control medium. Cells stimulated with 4-HT had obvious changes in cell morphology, and these morphologic changes were blocked by the addition of UO or PD (Figure 2A). Following stimulation with 4-HT the MDCK Raf:ER cells expressed high levels of active (phosphorylated) ERK and activation of ERK was inhibited by UO or PD. The amount of total ERK (active plus inactive) was the same in all conditions (Figure 2B). Following 24 hours of exposure to 4-HT, 4-HT plus UO, 4-HT plus PD, or control medium, mRNA was harvested and real-time PCR was performed using primers for claudin-2 and fibronectin. Claudin-2 mRNA expression, which we and others previously showed to be regulated via the ERK/MAPK pathway [15, 29] was dramatically downregulated by activation of Raf (Figure 2C), while fibronectin was significantly upregulated by activation of Raf (Figure 2D). The Raf-induced regulation of claudin-2 and fibronectin mRNA expression was completely blocked by the addition of UO or PD (Figure 2C,D). Similar results were seen at the protein level for claudin-2 and fibronectin expression (Figure 2E,F).

Figure 2. Raf Activation Differentially Regulates Expression of Claudin-2 and Fibronectin.

Figure 2

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MDCK Raf:ER cells were plated at confluency and grown for six days, prior to stimulation for 24 hours with 4-hydroxy tamoxifen (4-HT) at 1 μM, 4-HT plus UO, 4-HT plus PD, or control medium. A) Cells stimulated with 4-HT had obvious changes in cell morphology under light microscopy, and these morphologic changes were blocked by the addition of UO or PD (not shown). Following the addition of 4-HT, cells had a fibroblastic appearance along with the loss of the classic MDCK “cobblestone” pattern and “domes” (circular structures representing sections of the cell monolayer that have lifted off, or “domed”, as a result of secretion). B) Following stimulation with 4-HT the MDCK Raf:ER cells expressed high levels of active (phosphorylated) ERK. Activation of ERK was prevented when UO or PD were included with 4-HT. The amount of total ERK was similar in all the conditions. C,D) Following 24 hours of exposure to 4-HT, 4-HT plus UO, 4-HT plus PD, or control medium, mRNA was harvested and real-time PCR was performed using primers for claudin-2 (C) and fibronectin (D). Claudin-2 mRNA was dramatically downregulated by activation of Raf and fibronectin mRNA was significantly upregulated by activation of Raf. Changes in claudin-2 and fibronectin mRNA expression by 4-HT were prevented by the addition of UO or PD. E,F) Following 24 hours of exposure to 4-HT, 4-HT plus UO, 4-HT plus PD, or control medium, the cells were lysed with SDS and protein was collected for Western blot. Equal amounts of protein per BCA were loaded in each lane. Claudin-2 protein expression was dramatically downregulated by activation of Raf (E) and fibronectin was significantly upregulated by activation of Raf (F). Changes in claudin-2 and fibronectin protein expression following the addition of 4-HT were prevented by adding UO or PD. The p values in (C,D) were determined by combining all the experimental data and performing ANOVA with pairwise comparisons.

Fibronectin is Constitutively Expressed in MDCK Strain I Cells in an ERK Dependent Manner

MDCK Strain I cells, typical of other collecting duct cells, have high levels of active ERK and we previously showed that MDCK Strain I cells undergo spontaneous tubulogenesis when placed in a collagen matrix [19]. We examined fibronectin mRNA (Figure 3A) and protein levels (Figure 3B) and found both to be elevated in MDCK Strain I cells compared to MDCK Strain II cells. Furthermore, treatment with UO or PD decreased fibronectin mRNA and protein expression in MDCK Strain I cells to levels seen in MDCK Strain II cells (Figure 3A,B).

Figure 3. mRNA and Protein Expression Levels for Fibronectin in MDCK Strain I Versus Strain II Cells by Real-time PCR.

Figure 3

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A) RNA was harvested from MDCK Strain I and Strain II cells grown on filters for six days. The RNA was converted to cDNA and amplified using the TaqMan system and a primer/probe specific for fibronectin cDNA. The fold changes for MDCK Strain I, MDCK Strain I + PD09859, and MDCK Strain II cells are shown in graph form. The presence of PD or UO (not shown) decreased fibronectin mRNA expression in MDCK Strain I cells to levels seen in MDCK Strain II cells. The real-time PCR data were normalized to a control mRNA, the 18S ribosome. B) Fibronectin protein expression in MDCK Strain I, MDCK Strain I + PD09859, MDCK Strain II, and MDCK Strain II + PD09859 cells is shown by Western blot. The presence of PD or UO (not shown) decreased fibronectin protein expression in MDCK Strain I cells to levels seen in MDCK Strain II cells.

Discussion

The data we report here completes what we believe is the first description of an entire tubulogenic pathway from receptor/ligand interaction (HGF/CMET), through an intracellular signaling pathway (ERK/MAPK), to transcription and, finally, secretion of a critical protein (fibronectin) (Figure 4).

Figure 4. The Completed Tubulogenic Signaling Pathway for Fibronectin.

Figure 4

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During tubulogenesis in the MDCK cells, HGF binds to the CMET receptor tyrosine kinase receptor and activates the ERK/MAPK pathway, which leads to transcription and secretion of tubulogenic fibronectin.

Fibronectin has previously been shown to be essential for tubulogenesis using both the MDCK/HGF system [24] and organ culture models of developing submandibular salivary gland, lung, and kidney [25]. Fibronectin is an extracellular adhesion molecule that acts as an extracellular matrix linker by binding to integrin receptors [30]. Jiang and colleagues used stable transfectants expressing fibronectin antisense RNA and arginine-glycine-aspartate (RGD) peptides to disturb interactions between fibronectin and cell surface integrins, which prevented the initiation of tubulogenesis following HGF induction of MDCK cells cysts grown in a collagen matrix [24]. Sakai and colleagues showed that decreasing the fibronectin concentration using small interfering RNA or adding anti-fibronectin or anti-integrin antibodies blocked cleft formation between cells and, therefore, initiation of branching morphogenesis. Conversely, exogenous fibronectin accelerated cleft formation and tubulogenesis. Fibronectin expression is essential for cleft formation due to the need for conversion of cell-cell adhesions to cell-matrix adhesions in cells undergoing p-EMT and tubulogenesis [25].

There is also support in the literature for a link between fibronectin expression and regulation via the ERK/MAPK signaling pathway. Fibronectin expression was shown to increase in an ERK/MAPK dependent manner in endothelial cells following stimulation with high levels of glucose [31].

Branching morphogenesis of tubular epithelium is a common and important feature of vertebrate organogenesis [1, 22, 32]. The MAPK/ERK pathway, which is downstream of receptor tyrosine kinases, leads to the phosphorylation, and hence activation, of ERK and has been shown to be important in branching morphogenesis in several systems, including the Drosophila trachea [20] and murine salivary gland [21]. Importantly, it was shown that active ERK regulates branching morphogenesis in the developing kidney and that PD09859 reversibly inhibits branching in a dose dependent manner [22].

We previously showed in MDCK cells grown in a collagen matrix and stimulated with HGF, that active ERK is necessary and sufficient to initiate tubulogenesis [11, 19]. MDCK Strain I cells have high levels of active ERK and spontaneously initiate tubulogenesis [19]. We show here that MDCK Strain I cells also have high levels of fibronectin, which is regulatable via the ERK/MAPK pathway. While a number of different intracellular signaling cassettes have been implicated in HGF-induced tubulogenesis in MDCK cells [18], our data clearly demonstrate that tubulogenic fibronectin is regulated via the ERK/MAPK pathway in response to HGF.

Acknowledgments

This work was supported by NIH grants DK069909 and DK070980 to J.H.L. The P30 Center Grant (DK50306) is gratefully acknowledged for microscopy services.

Footnotes

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