Ali Nawshad

Transforming Growth Factor-β Signaling during Epithelial-Mesenchymal Transformation: Implications for Embryogenesis and Tumor Metastasis

The molecular mechanisms of epithelial-mesenchymal transformation (EMT) have long been studied to gain a greater understanding of this distinct change in cellular morphology. Early studies of the developing embryo have designated the involvement of Wnt signaling in EMT, through an activated complex of the lymphoid-enhancing factor-1 (LEF-1) transcription factor and the cell adhesion molecule β-catenin. However, more recent studies have implicated a significant role of the transforming growth factor-β (TGF-β) in causing EMT in both development and pathology. The ability of TGF-β isoforms to signal through a variety of molecules such as Smads, phosphatidylinositol 3-kinase (PI3K), and mitogen-activated protein kinase (MAPK) creates an incredible complexity as to their role in this transition. Here we assess the biochemical signaling pathways of TGF-β and their potential cross-interaction with traditional Wnt signaling molecules to bring about EMT during embryogenesis and tumor metastasis.

TGFβ3 signaling activates transcription of the LEF1 gene to induce epithelial mesenchymal transformation during mouse palate development

Epithelial mesenchymal transformation (EMT) of the medial edge epithelial (MEE) seam creates palatal confluence. This work aims to elucidate the molecular mechanisms by which TGFβ3 brings about palatal seam EMT. We collected mRNA for PCR analysis from individual transforming MEE cells by laser microdissection techniques and demonstrated that TGFβ3 stimulates lymphoid-enhancing factor 1 (LEF1) mRNA synthesis in MEE cells. We show with antisense β-catenin oligonucleotides that up-regulated LEF1 is not activated by β-catenin in palate EMT. We ruled out other TGFβ3 targets, such as RhoA and MEK1/2 pathways, and we present evidence using dominant-negative Smad4 and dominant-negative LEF1 showing that TGFβ3 uses Smads both to up-regulate synthesis of LEF1 and to activate LEF1 transcription during induction of palatal EMT. When phospho-Smad2 and Smad4 are present in the nucleus, LEF1 is activated without β-catenin. Our paper is the first to show that the Smad2,4/LEF1 complex replaces β-catenin/LEF1 during activation of EMT in vivo by TGFβ3.

TGFβ3 inhibits E-cadherin gene expression in palate medial-edge epithelial cells through a Smad2-Smad4-LEF1 transcription complex

Dissociation of medial-edge epithelium (MEE) during palate development is essential for mediating correct craniofacial morphogenesis. This phenomenon is initiated by TGFβ3 upon adherence of opposing palatal shelves, because loss of E-cadherin causes the MEE seam to break into small epithelial islands. To investigate the molecular mechanisms that cause this E-cadherin loss, we isolated and cultured murine embryonic primary MEE cells from adhered or non-adhered palates. Here, we provide the first evidence that lymphoid enhancer factor 1 (LEF1), when functionally activated by phosphorylated Smad2 (Smad2-P) and Smad4 (rather than β-catenin), binds with the promoter of the E-cadherin gene to repress its transcription in response to TGFβ3 signaling. Furthermore, we found that TGFβ3 signaling stimulates epithelial-mesenchymal transformation (EMT) and cell migration in these cells. LEF1 and Smad4 were found to be necessary for up-regulation of the mesenchymal markers vimentin and fibronectin, independently of β-catenin. We proved that TGFβ3 signaling induces EMT in MEE cells by forming activated transcription complexes of Smad2-P, Smad4 and LEF1 that directly inhibit E-cadherin gene expression.

Microarray analysis of gene expression during epithelial-mesenchymal transformation

One of the most fundamental biological processes in development, as well as a primary mechanism for tumor metastasis, is epithelial–mesenchymal transformation (EMT). To gain a greater understanding of this transition, we have obtained a genomic profile of the critical stages before and during this rapid change in morphology in the developing mouse palate. By isolating the medial edge epithelium of each palatal shelf, we were able to obtain pure gene expression data without contamination from surrounding mesenchymal cells. Our results support the important role of the TGF‐β/Smad signal transduction pathway in the stimulation of EMT by means of up‐regulation of the EMT‐inducing gene, LEF‐1. We document changes in gene expression profiles during palatal adherence and subsequent transformation of the medial edge epithelial seam that suggests a high number of LEF‐1 target genes promote cellular transformation to mesenchyme. These include genes involved in cell adhesion, polarity, cytoskeletal dynamics, migration, and intracellular signaling. This knowledge of the changes in gene expression levels during palatogenesis should lead to a better understanding of the mechanisms of EMT. Developmental Dynamics 234:132–142, 2005.

Palatal Seam Disintegration: To Die or Not to Die? That Is No Longer the Question

Formation of the medial epithelial seam (MES) by palatal shelf fusion is a crucial step of palate development. Complete disintegration of the MES is the final essential phase of palatal confluency with surrounding mesenchymal cells. In general, the mechanisms of palatal seam disintegration are not overwhelmingly complex, but given the large number of interacting constituents; their complicated circuitry involving feedforward, feedback, and crosstalk; and the fact that the kinetics of interaction matter, this otherwise simple mechanism can be quite difficult to interpret. As a result of this complexity, apparently simple but highly important questions remain unanswered. One such question pertains to the fate of the palatal seam. Such questions may be answered by detailed and extensive quantitative experimentation of basic biological studies (cellular, structural) and the newest molecular biological determinants (genetic/dye cell lineage, gene activity, kinase/enzyme activity), as well as animal model (knockouts, transgenic) approaches. System biology and cellular kinetics play a crucial role in cellular MES function; omissions of such critical contributors may lead to inaccurate understanding of the fate of MES. Excellent progress has been made relevant to elucidation of the mechanism(s) of palatal seam disintegration. Current understanding of palatal seam disintegration suggests epithelial–mesenchymal transition and/or programmed cell death as two most common mechanisms of MES disintegration. In this review, I discuss those two mechanisms and the differences between them. Developmental Dynamics 237:2643–2656, 2008.

Type I collagen promotes epithelial-mesenchymal transition through ILK-dependent activation of NF-κB and LEF-1

Collagen I has been shown to promote epithelial-mesenchymal transition (EMT), a critical process of embryonic development and disease progression. However, little is known about the signaling mechanisms by which collagen I induces this cellular transformation. Here we show that collagen I causes ILK-dependent phosphorylation of IkappaB and subsequent nuclear translocation of active NF-kappaB, which in turn promotes increased expression of the Snail and LEF-1 transcription factors. ILK also causes inhibitory phosphorylation of GSK-3beta, a kinase that prevents functional activation of both Snail and LEF-1. These transcription factors alter expression of epithelial and mesenchymal markers to initiate EMT and stimulate cell migration. These data provide a foundation for understanding the mechanisms by which collagen I stimulates EMT and identify potential therapeutic targets for suppressing this transition in pathological conditions.

Discoidin domain receptor 2 is a critical regulator of epithelial-mesenchymal transition.

Discoidin domain receptor 2 (DDR2) is a collagen receptor that is expressed during epithelial-mesenchymal transition (EMT), a cellular transformation that mediates many stages of embryonic development and disease. However, the functional significance of this receptor in EMT is unknown. Here we show that Transforming Growth Factor-beta1 (TGF-β1), a common stimulator of EMT, promotes increased expression of type I collagen and DDR2. Inhibiting expression of COL1A1 or DDR2 with siRNA is sufficient to perturb activity of the NF-κB and LEF-1 transcription factors and to inhibit EMT and cell migration induced by TGF-β1. Furthermore, knockdown of DDR2 expression with siRNA inhibits EMT directly induced by type I collagen. These data establish a critical role for type I collagen-dependent DDR2 signaling in the regulation of EMT.

Complexity in interpretation of embryonic epithelial-mesenchymal transition in response to transforming growth factor-β signaling

Epithelial-mesenchymal transition (EMT) is a highly conserved and fundamental process that governs morphogenesis in development and may also contribute to cancer metastasis. Transforming growth factor (TGF-β) is a potent inducer of EMT in various developmental and tumor systems. The analysis of TGF-β signal transduction pathways is now considered a critically important area of biology, since many defects occur in these pathways in embryonic development. The complexity of TGF-β signal transduction networks is overwhelming due to the large numbers of interacting constituents, complicated feedforward, feedback and crosstalk circuitry mechanisms that they involve in addition to the cellular kinetics and enzymatics that contribute to cell signaling. As a result of this complexity, apparently simple but highly important questions remain unanswered, that is, how do epithelial cells respond to such TGF-β signals? System biology and cellular kinetics play a crucial role in cellular function; omissions of such a critical contributor may lead to inaccurate understanding of embryonic EMT. In this review, we identify and explain why certain conditions need to be considered for a true representation of TGF-β signaling in vivo to better understand the controlled, yet delicate mechanism of embryonic EMT.

Laser capture microdissection (LCM) for analysis of gene expression in specific tissues during embryonic epithelial-mesenchymal transformation

The analysis of gene expression in developing organs is a valuable tool for the assessment of genetic fingerprints during the various stages of tissue differentiation and epithelial–mesenchymal transformation (EMT). However, the variety of differentiating cells and the close association of epithelial and mesenchymal cells makes it difficult to extract protein and mRNA from specific cells and tissue and, thus, to assign expressed genes to specific cell populations. We report here the analysis of LEF1 mRNA in epithelial and mesenchymal cells isolated by LCM from different stages of EMT during development of the mouse palate and describe our techniques in detail. By applying a laser capture microdissection (LCM) technique and real‐time polymerase chain reaction, we were able to determine mRNA levels that accurately reflect changes in gene expression in specific cells. The sensitivity of the technique is remarkable. Indeed, the mRNAs can be detected for many proteins too low in abundance to stain with antibodies. These techniques will enable embryologists to collect homogeneous groups of cells from heterogeneous populations in developing organs, which otherwise would not be available for gene analysis. Developmental Dynamics 230:529–534, 2004.

Induction of palate epithelial mesenchymal transition by transforming growth factor β3 signaling

Transforming growth factor (TGFβ)3 is essential for palate development, particularly during the late phase of palatogenesis when the disintegration of the palatal medial edge seam (MES) occurs resulting in mesenchymal confluence. The MES is composed of medial‐edge epithelium (MEE) of opposite palatal shelves; its complete disintegration is essential for mediating correct craniofacial morphogenesis. This phenomenon is initiated by TGFβ3 upon adherence of opposing palatal shelves, and subsequently epithelial–mesenchymal transition (EMT) instigates the loss of E‐Cadherin, causing the MES to break into small epithelial islands forming confluent palatal mesenchyme; however, apoptosis and cell migration or in combination of all are other established mechanisms of seam disintegration. To investigate the molecular mechanisms that cause this E‐Cadherin loss, we isolated and cultured murine embryonic primary MES cells from adhered palates and employed several biological approaches to explore the mechanism by which TGFβ3 facilitates palatal seam disintegration. Here, we demonstrate that TGFβ3 signals by activating both Smad‐dependent and Smad‐independent pathways. However, activation of the two most common EMT related transcription factors, Snail and SIP, was facilitated by Smad‐independent pathways, contrary to the commonly accepted Smad‐dependent pathway. Finally, we provide the first evidence that TGFβ3‐activated Snail and SIP1, combined with Smad4, bind to the E‐Cadherin promoter to repress its transcription in response to TGFβ3 signaling. These results suggest that TGFβ3 uses multiple pathways to activate Snail and SIP1 and these transcription factors repress the cell–cell adhesion protein, E‐Cadherin, to induce palatal epithelial seam EMT. Manipulation and intervention of the pathways stimulated by TGFβ3 during palate development may have a significant therapeutic potential.