Upregulation of MMP-9 in MDCK epithelial cell line in response to expression of the Snail transcription factor

Abstract

Overexpression of the transcription factor Snail in epithelial MDCK cells promotes the epithelial-mesenchymal transition (EMT) and the acquisition of an invasive phenotype. We report here that the expression of Snail is associated with an increase in the promoter activity and expression of the matrix metalloproteinase MMP-9. The effect of Snail silencing on MMP-9 expression corroborates this finding. Induced transcription of MMP-9 by Snail is driven by a mechanism dependent on the MAPK and phosphoinositide 3-kinase (PI3K) signalling pathways. Although other regions of the promoter were required for a complete stimulation by Snail, a minimal fragment (nucleotides -97 to +114) produces a response following an increased phosphorylation of Sp-1 and either Sp-1 or Ets-1 binding to the GC-box elements contained in this region. The expression of a dominant negative form of MEK decreased these complexes. A moderate increase in the binding of the nuclear factor κB (NFκB) to the upstream region (nucleotide -562) of the MMP-9 promoter was also observed in Snail-expressing cells. Interestingly, oncogenic H-Ras (RasV12) synergistically co-operates with Snail in the induction of MMP-9 transcription and expression. Altogether, these results indicate that MMP-9 transcription is activated in response to Snail expression and that it might explain, at least in part, the invasive properties of the Snail-expressing cells.

Snail silencing effectively suppresses tumour growth and invasiveness

“…As shown in Figure 1a and Supplementary Figure S3A, either complete absence or very low levels (o10% over the corresponding control) of Snail mRNA was observed in all analysed independent clones, while control MDCK-Snail cells transfected with shEGFP showed levels of Snail transcript similar to those of parental MDCK-Snail and MDCK-Snail-EGFP cells ( Figure 1a and Supplementary Figure S3A, middle panels). Analysis of E-cadherin mRNA levels indicated robust expression of E-cadherin transcript in all independent clones with silenced Snail expression ( Figure 1a and Supplementary Figure S3A, upper panels), confirming and extending our recent results (Jorda et al, 2005). Re-expression of E-cadherin after Snail interference in the individual clones was further confirmed at the protein level, as assessed by Western blot (Figure 1b and Supplementary Figure S3B) and immunofluorescence analysis (Figure 2, E-CD panels).…”

“…We designed a 19-mer siRNA oligonucleotide directed to the N-terminal region of the first zing finger of mouse Snail mRNA (position from 573 to 591 in mouse cDNA; Jorda et al, 2005). Importantly, this specific nucleotide sequence is conserved between mouse and human Snail mRNA sequences (580-598 in human cDNA, accession number NM005985), but it is not present in Slug (also known as Snail2) mRNA of any species (Sefton et al, 1998;Manzanares et al, 2001).…”

“…Snail induces a full EMT when overexpressed in epithelial Madin Darby canine kidney (MDCK) cells leading to the acquisition of a motile/invasive phenotype (Cano et al, 2000;Peinado et al, 2004b). In agreement with this role, Snail (also known as Snail1) has also been found to downregulate the expression of additional epithelial genes, such as occluding, claudins or muc1 (Guaita et al, 2002;Ikenouchi et al, 2003;Ohkubo and Ozawa, 2004;Martinez-Estrada et al, 2005), or to induce the expression of mesenchymal and invasive genes, like fibronectin and metalloproteinase (MMP)-9 (Cano et al, 2000;Guaita et al, 2002;Jorda et al, 2005). Snail expression has been detected in increasing number of different human carcinoma and melanoma cell lines (reviewed in Peinado et al, 2004a).…”

“…These results indicate that additional mechanisms are required to maintain stable E-cadherin expression in in vitro culture; nevertheless, this situation can be overcome in in vivo tumour contexts (see below). We have recently reported that Snail silencing in MDCK-Snail cells efficiently suppressed the expression and secretion of MMP-9 (Jorda et al, 2005). Therefore, we analysed if stable Snail silencing in HaCa4 and CarB carcinoma cell lines was able to affect MMP-9 expression.…”

Akt2 interacts with Snail1 in the E-cadherin promoter

“…Repression of epithelial genes by Snail1 requires the binding of the C-terminal domain of this protein to 5 0 -CACCTG-3 0 elements present in these promoters and the interaction of the SNAG Snail1 sequence with co-repressors (Thiery et al, 2009;Garcı´a de Herreros et al, 2010). Conversely, activation of mesenchymal genes (fibronectin, vimentin) is thought to be a more indirect effect, partially dependent on Ecadherin downregulation, and requiring stimulation of several transduction pathways, such as those involving extracellular signal-regulated kinase-2 (ERK2), Sp1/Ets-1, nuclear factor-kB and b-catenin (Ohkubo and Ozawa 2003;Jorda`et al, 2005Jorda`et al, , 2007Solanas et al, 2008;Stemmer et al, 2008).…”