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

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).

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

“…It has previously been shown that fibronectin plays an essential role in the partial EMT that occurs early in the process of HGF-stimulated tubule formation. In addition, it has been previously shown that disrupting fibronectin expression impairs cyst growth and prevents the initiation of tubulogenesis in MDCK cells grown in 3D culture (16,26,47,50). Taken together, these findings suggest that proteins involved in remodeling the ECM play an important role in the development and expansion of multicellular epithelial structures in 3D culture.…”

Macrophage Stimulating Protein May Promote Tubular Regeneration after Acute Injury

“…42 Blockade of the Mek-dependent pathway of ERK activation with PD98059 significantly reduced the biologic effects of MSP on TEC, suggesting the involvement of this pathway in MSP/RON signal transduction as previously shown for tubulogenesis induced by MET receptor activation. 27,[43][44][45] Apart from the inhibition of apoptosis, MSP preserved the expression of several molecules involved in physiologic tubular functions. Indeed, in cisplatin-treated TEC, MSP maintained the ability to internalize albumin and the expression of the endocytic receptor megalin that is essential for protein reabsorption.…”

Matrix Metalloproteinase 13 (MMP13) and Tissue Inhibitor of Matrix Metalloproteinase 1 (TIMP1), Regulated by the MAPK Pathway, Are Both Necessary for Madin-Darby Canine Kidney Tubulogenesis

“…In these cells, the kinase activity of Raf was induced using an estrogen analog, 4-hydroxytamoxifen (4-HT) (Sigma), at a concentration of 1 M as described previously (25,26).…”