Local simvastatin and inflammation during periodontal mini-flap wound healing: Exploratory results.

BACKGROUND: The objective of this exploratory study was to evaluate inflammatory markers in periodontal maintenance patients from a randomized, double-masked, parallel intervention clinical trial comparing local simvastatin (SIM) to carrier alone following mini-flap access. METHODS: Fifty patients with a 6-9-mm inflamed pocket during periodontal maintenance therapy (PMT) were treated with papilla reflection (PR)/root planing and placement of 2.2-mg simvastatin in methylcellulose (SIM/MCL) or methylcellulose alone (MCL). A small piece of interproximal soft tissue was harvested at baseline and 2 weeks postoperatively, gingival crevicular fluid (GCF) obtained at baseline, 2 weeks and 12 months, and bleeding on probing (BOP) and clinical attachment level (CAL) were measured at baseline and 12 months. Pro-inflammatory interleukin (IL)-6 and anti-inflammatory IL-10 gene activation were determined by reverse transcriptase polymerase chain reaction (rt-PCR). GCF IL-1ß, IL-6, IL-10, and vascular endothelial growth factor (VEGF-A) were measured with multiplex technology. Comparisons between groups and over time used logistic regression and general estimating equations. Associations between inflammatory markers and 12-month outcomes used Wilcoxon rank sum tests or Pearson correlations. RESULTS: Patients in the SIM group had 4.17 greater odds (p = 0.047) of improved BOP at 12 months. Median IL-6 and VEGF were significantly increased for all patients after 2 weeks of healing (p < 0.0001 and p = 0.03, respectively), while median IL-10 gene activation was increased after 2 weeks in SIM/MCL (NS). Overall, elevated GCF IL-10 at 2 weeks was significantly correlated with improved CAL at 12 months (r = -0.32, p = 0.03). CONCLUSIONS: Local SIM/MCL may have anti-inflammatory effects that potentially are associated with improved long-term CAL outcomes.

Standardized Rat Model Testing Effects of Inflammation and Grafting on Extraction Healing.

BACKGROUND: Loss of alveolar ridge width and height after tooth extraction is well documented, but models to evaluate ridge preservation are neither standardized nor cost-effective. This rat model characterizes the pattern of bone turnover and inflammation after extraction and bone grafting with or without local simvastatin (SIM). METHODS: Fifty retired-breeder rats underwent extraction of the maxillary right first molar and standard surgical defect creation under inhalation/local anesthesia. The left side of each animal served as unmanipulated control. Untreated groups (n = 8 to 9 per group) were compared (analysis of variance, t test) at days 0, 7, 14, and 28 for alveolar ridge height and width and for markers of inflammation and bone turnover by microcomputed tomography, histology, and enzyme-linked immunosorbent assay. Seventeen additional specimens had defects grafted with either bone mineralized matrix (BMM) or a BMM+SIM conjugate. RESULTS: Extraction-induced bone loss (BL) was noted on buccal, palatal, and interproximal height (P <0.05) and ridge width (P <0.01). Week 1 inflammation positively correlated with ridge height; thereafter, a more intense inflammatory reaction corresponded to reduction in alveolar bone height and density (r = 0.74; P <0.05; Spearman). BMM+SIM preserved the most interproximal bone height (P <0.01), increased ridge width and bone density (P <0.01), enhanced 7-day prostaglandin E2 (P <0.01), and reduced 28-day inflammation density (P <0.05). CONCLUSIONS: The standard defect used in the current study paralleled human postextraction alveolar BL. Defect grafting, especially BMM+SIM, reduced inflammation and preserved bone.

Transforming growth factor-ß1 activates ΔNp63/c-Myc to promote oral squamous cell carcinoma.

OBJECTIVE: During the development of oral squamous cell carcinoma (OSCC), the transformed epithelial cells undergo increased proliferation resulting in tumor growth and invasion. Interestingly, throughout all phases of differentiation and progression to OSCC, transforming growth factor-ß1 (TGF)-ß1 induces cell cycle arrest or apoptosis; however, the role of TGF-ß1 in promoting cancer cell proliferation has not been explored in detail. The purpose of this study was to identify the effect of TGF-ß1 on OSCC cell proliferation. STUDY DESIGN: Using both human OSCC samples and cell lines (UMSCC38 and UMSCC11B), we assessed protein, mRNA, gene expression, and protein-DNA interactions during OSCC progression. RESULTS: Our results showed that TGF-ß1 increased OSCC cell proliferation by upregulating the expression of ΔNp63 and c-Myc oncogenes. Although the basal OSCC cell proliferation is sustained by activating ΔNp63, increased induction of c-Myc causes unregulated OSCC cell proliferation. Following induction of the cell cycle by ΔNp63 and c-Myc, cancer cells that halt c-Myc activity undergo epithelial mesenchymal transition or invasion while those that continue to express ΔNp63/c-Myc undergo unlimited progression through the cell cycle. CONCLUSIONS: OSCC proliferation is manifested by the induction of c-Myc in response to TGF-ß1 signaling, which is essential for OSCC growth. Our data highlight the potential role of TGF-ß1 in the induction of cancer progression and invasion of OSCC.

Nicotine Exposure During Pregnancy Results in Persistent Midline Epithelial Seam With Improper Palatal Fusion.

INTRODUCTION: Nonsyndromic cleft palate is a common birth defect (1:700) with a complex etiology involving both genetic and environmental risk factors. Nicotine, a major teratogen present in tobacco products, was shown to cause alterations and delays in the developing fetus. METHODS: To demonstrate the postpartum effects of nicotine on palatal development, we delivered three different doses of nicotine (1.5, 3.0, and 4.5mg/kg/d) and sterile saline (control) into pregnant BALB/c mice throughout their entire pregnancy using subcutaneous micro-osmotic pump. Dams were allowed to deliver (~day 21 of pregnancy) and neonatal assessments (weight, length, nicotine levels) were conducted, and palatal tissues were harvested for morphological and molecular analyses, as well as transcriptional profiling using microarrays. RESULTS: Consistent administration of nicotine caused developmental retardation, still birth, low birth weight, and significant palatal size and shape abnormality and persistent midline epithelial seam in the pups. Through microarray analysis, we detected that 6232 genes were up-regulated and 6310 genes were down-regulated in nicotine-treated groups compared to the control. Moreover, 46% of the cleft palate-causing genes were found to be affected by nicotine exposure. Alterations of a subset of differentially expressed genes were illustrated with hierarchal clustering and a series of formal pathway analyses were performed using the bioinformatics tools. CONCLUSIONS: We concluded that nicotine exposure during pregnancy interferes with normal growth and development of the fetus, as well results in persistent midline epithelial seam with type B and C patterns of palatal fusion. IMPLICATIONS: Although there are several studies analyzing the genetic and environmental causes of palatal deformities, this study primarily shows the morphological and large-scale genomic outcomes of gestational nicotine exposure in neonatal mice palate.The previous version was incorrect. New authors Ali Nawshad, Hasan Otu, Janki Sharma, and Elizabeth Sheldon have been included in this version; the funding and acknowledgement sections have been updated accordingly; the article title, some text, and one supplementary data file have been edited; and the corresponding author has been changed. The original corresponding author regrets these earlier errors.

Ephrin reverse signaling mediates palatal fusion and epithelial-to-mesenchymal transition independently of Tgfß3.

The mammalian secondary palate forms from shelves of epithelia-covered mesenchyme that meet at midline and fuse. The midline epithelial seam (MES) is thought to degrade by apoptosis, epithelial-to-mesenchymal transition (EMT), or both. Failure to degrade the MES blocks fusion and causes cleft palate. It was previously thought that transforming growth factor ß3 (Tgfß3) is required to initiate fusion. Members of the Eph tyrosine kinase receptor family and their membrane-bound ephrin ligands are expressed on the MES. We demonstrated that treatment of mouse palates with recombinant EphB2/Fc to activate ephrin reverse signaling (where the ephrin acts as a receptor and transduces signals from its cytodomain) was sufficient to cause mouse palatal fusion when Tgfß3 signaling was blocked by an antibody against Tgfß3 or by an inhibitor of the TgfßrI serine/threonine receptor kinase. Cultured palatal epithelial cells traded their expression of epithelial cell markers for that of mesenchymal cells and became motile after treatment with EphB2/Fc. They concurrently increased their expression of the EMT-associated transcription factors Snail, Sip1, and Twist1. EphB2/Fc did not cause apoptosis in these cells. These data reveal that ephrin reverse signaling directs palatal fusion in mammals through a mechanism that involves EMT but not apoptosis and activates a gene expression program not previously associated with ephrin reverse signaling.

TGFß3 regulates periderm removal through ΔNp63 in the developing palate.

The periderm is a flat layer of epithelium created during embryonic development. During palatogenesis, the periderm forms a protective layer against premature adhesion of the oral epithelia, including the palate. However, the periderm must be removed in order for the medial edge epithelia (MEE) to properly adhere and form a palatal seam. Improper periderm removal results in a cleft palate. Although the timing of transforming growth factor ß3 (TGFß3) expression in the MEE coincides with periderm degeneration, its role in periderm desquamation is not known. Interestingly, murine models of knockout (-/-) TGFß3, interferon regulatory factor 6 (IRF6) (-/-), and truncated p63 (ΔNp63) (-/-) are born with palatal clefts because of failure of the palatal shelves to adhere, suggesting that these genes regulate palatal epithelial differentiation. However, despite having similar phenotypes in null mouse models, no studies have analyzed the possible association between the TGFß3 signaling cascade and the IRF6/ΔNp63 genes during palate development. Recent studies indicate that regulation of ΔNp63, which depends on IRF6, facilitates epithelial differentiation. We performed biochemical analysis, gene activity and protein expression assays with palatal sections of TGFß3 (-/-), ΔNp63 (-/-), and wild-type (WT) embryos, and primary MEE cells from WT palates to analyze the association between TGFß3 and IRF6/ΔNp63. Our results suggest that periderm degeneration depends on functional TGFß3 signaling to repress ΔNp63, thereby coordinating periderm desquamation. Cleft palate occurs in TGFß3 (-/-) because of inadequate periderm removal that impedes palatal seam formation, while cleft palate occurs in ΔNp63 (-/-) palates because of premature fusion.

Systematic analysis of palatal transcriptome to identify cleft palate genes within TGFß3-knockout mice alleles: RNA-Seq analysis of TGFß3 Mice.

BACKGROUND: In humans, cleft palate (CP) accounts for one of the largest number of birth defects with a complex genetic and environmental etiology. TGFß3 has been established as an important regulator of palatal fusion in mice and it has been shown that TGFß3-null mice exhibit CP without any other major deformities. However, the genes that regulate cellular decisions and molecular mechanisms maintained by the TGFß3 pathway throughout palatogenesis are predominantly unexplored. Our objective in this study was to analyze global transcriptome changes within the palate during different gestational ages within TGFß3 knockout mice to identify TGFß3-associated genes previously unknown to be associated with the development of cleft palate. We used deep sequencing technology, RNA-Seq, to analyze the transcriptome of TGFß3 knockout mice at crucial stages of palatogenesis, including palatal growth (E14.5), adhesion (E15.5), and fusion (E16.5). RESULTS: The overall transcriptome analysis of TGFß3 wildtype mice (C57BL/6) reveals that almost 6000 genes were upregulated during the transition from E14.5 to E15.5 and more than 2000 were downregulated from E15.5 to E16.5. Using bioinformatics tools and databases, we identified the most comprehensive list of CP genes (n = 322) in which mutations cause CP either in humans or mice, and analyzed their expression patterns. The expression motifs of CP genes between TGFß3+/- and TGFß3-/- were not significantly different from each other, and the expression of the majority of CP genes remained unchanged from E14.5 to E16.5. Using these patterns, we identified 8 unique genes within TGFß3-/- mice (Chrng, Foxc2, H19, Kcnj13, Lhx8, Meox2, Shh, and Six3), which may function as the primary contributors to the development of cleft palate in TGFß3-/- mice. When the significantly altered CP genes were overlaid with TGFß signaling, all of these genes followed the Smad-dependent pathway. CONCLUSIONS: Our study represents the first analysis of the palatal transcriptome of the mouse, as well as TGFß3 knockout mice, using deep sequencing methods. In this study, we characterized the critical regulation of palatal transcripts that may play key regulatory roles through crucial stages of palatal development. We identified potential causative CP genes in a TGFß3 knockout model, which may lead to a better understanding of the genetic mechanisms of palatogenesis and provide novel potential targets for gene therapy approaches to treat cleft palate.

Induction of palate epithelial mesenchymal transition by transforming growth factor ß3 signaling.

Transforming growth factor (TGFß)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 TGFß3 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 TGFß3 facilitates palatal seam disintegration. Here, we demonstrate that TGFß3 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 TGFß3-activated Snail and SIP1, combined with Smad4, bind to the E-Cadherin promoter to repress its transcription in response to TGFß3 signaling. These results suggest that TGFß3 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 TGFß3 during palate development may have a significant therapeutic potential.

Transforming growth factor-ß activates c-Myc to promote palatal growth.

During palatogenesis, the palatal mesenchyme undergoes increased cell proliferation resulting in palatal growth, elevation and fusion of the two palatal shelves. Interestingly, the palatal mesenchyme expresses all three transforming growth factor (TGF) ß isoforms (1, 2, and 3) throughout these steps of palatogenesis. However, the role of TGFß in promoting proliferation of palatal mesenchymal cells has never been explored. The purpose of this study was to identify the effect of TGFß on human embryonic palatal mesenchymal (HEPM) cell proliferation. Our results showed that all isoforms of TGFß, especially TGFß3, increased HEPM cell proliferation by up-regulating the expression of cyclins and cyclin-dependent kinases as well as c-Myc oncogene. TGFß activated both Smad-dependent and Smad-independent pathways to induce c-Myc gene expression. Furthermore, TBE1 is the only functional Smad binding element (SBE) in the c-Myc promoter and Smad4, activated by TGFß, binds to the TBE1 to induce c-Myc gene activity. We conclude that HEPM proliferation is manifested by the induction of c-Myc in response to TGFß signaling, which is essential for complete palatal confluency. Our data highlights the potential role of TGFß as a therapeutic molecule to correct cleft palate by promoting growth.

Implications of TGFß on Transcriptome and Cellular Biofunctions of Palatal Mesenchyme.

Development of the palate comprises sequential stages of growth, elevation, and fusion of the palatal shelves. The mesenchymal component of palates plays a major role in early phases of palatogenesis, such as growth and elevation. Failure in these steps may result in cleft palate, the second most common birth defect in the world. These early stages of palatogenesis require precise and chronological orchestration of key physiological processes, such as growth, proliferation, differentiation, migration, and apoptosis. There is compelling evidence for the vital role of TGFß-mediated regulation of palate development. We hypothesized that the isoforms of TGFß regulate different cellular biofunctions of the palatal mesenchyme to various extents. Human embryonic palatal mesenchyme (HEPM) cells were treated with TGFß1, ß2, and ß3 for microarray-based gene expression studies in order to identify the roles of TGFß in the transcriptome of the palatal mesenchyme. Following normalization and modeling of 28,869 human genes, 566 transcripts were detected as differentially expressed in TGFß-treated HEPM cells. Out of these altered transcripts, 234 of them were clustered in cellular biofunctions, including growth and proliferation, development, morphology, movement, cell cycle, and apoptosis. Biological interpretation and network analysis of the genes active in cellular biofunctions were performed using IPA. Among the differentially expressed genes, 11 of them are known to be crucial for palatogenesis (EDN1, INHBA, LHX8, PDGFC, PIGA, RUNX1, SNAI1, SMAD3, TGFß1, TGFß2, and TGFßR1). These genes were used for a merged interaction network with cellular behaviors. Overall, we have determined that more than 2% of human transcripts were differentially expressed in response to TGFß treatment in HEPM cells. Our results suggest that both TGFß1 and TGFß2 orchestrate major cellular biofunctions within the palatal mesenchyme in vitro by regulating expression of 234 genes.

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-ß1), 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-ß1. 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.

Role of prostaglandin pathway and alendronate-based carriers to enhance statin-induced bone.

This study investigated the role of the prostaglandin (PG) pathway in locally applied, simvastatin-induced oral bone growth. The possibility of enhancing long-term bone augmentation with an alendronate-based carrier was initiated. Mandibles of 44 mature female rats were treated bilaterally with the following combinations: 2 mg of simvastatin in ethanol (SIM-EtOH), EtOH, 2 mg of simvastatin acid complexed with alendronate-beta-cyclodextrin conjugate (SIM/ALN-CD), ALN-CD, or ALN. Bone wash technology (injection of PBS and re-collection by suction) was used to sample injection sites at baseline (day 0), and 3, 7, 14, and 21 days post-treatment. After 21-24 or 48 days, histomorphometric analysis was done. The amount of PGE(2) in bone wash fluid was measured by ELISA, normalized by total protein, and compared between high and low bone growth groups (ANOVA) and correlated with subsequent bone histology at 21 days (Spearman). SIM-stimulated PGE(2) synthase and EP4 receptor mRNA in murine osteoblast and fibroblast cell lines were evaluated with real-time PCR. Single injections of 2 mg of SIM-EtOH induced significantly more new bone than control side after 21 days. PGE(2)/protein ratios peaked at day 7 and were correlated with the subsequent 21-day new bone width. The correlations at day 14 between PGE(2) and new bone width changed to a negative relationship in the test group. SIM-stimulated osteoblasts expressed increased mRNA levels of PGE receptor EP4, while SIM activated PGE synthesis in fibroblasts. SIM/ALN-CD tended to preserve bone long-term. Findings suggest that PGE pathway activation and higher levels of PGE(2) during the first week following SIM-induced bone growth are desirable, and alendronate-beta-cyclodextrin conjugates not only act as tissue-specific carriers, but preserve new bone.

Mechanisms of transforming growth factor ß induced cell cycle arrest in palate development.

Immaculate and complete palatal seam disintegration, which takes place at the last phase of palate development, is essential for normal palate development. And in absence of palatal midline epithelial seam (MES) disintegration, cleft palate may arise. It has been established that transforming growth factor (TGF) ß induces both epithelial mesenchymal transition (EMT) and/or apoptosis during MES disintegration. It is likely that MES might cease cell cycle to facilitate cellular changes prior to undergoing transformation or apoptosis, which has never been studied before. This study was designed to explore whether TGFß, which is crucial for palatal MES disintegration, is capable of inducing cell cycle arrest. We studied the effects of TGFß1 and TGFß3, potent negative regulators of the cell cycle, on p15ink4b activity in MES cells. We surprisingly found that TGFß1, but not TGFß3, plays a major role in activation of the p15ink4b gene. In contrast, following successful cell cycle arrest by TGFß1, it is TGFß3 but not TGFß1 that causes later cellular morphogenesis, such as EMT and apoptosis. Since TGFß signaling activates Smads, we analyzed the roles of three Smad binding elements (SBEs) on the p15ink4b mouse promoter by site specific mutagenesis and found that these binding sites are functional. The ChIP assay demonstrated that TGFß1, not TGFß3, promotes Smad4 binding to two 5' terminal SBEs but not the 3' terminal site. Thus, TGFß1 and TGFß3 play separate yet complimentary roles in achieving cell cycle arrest and EMT/apoptosis and cell cycle arrest is a prerequisite for later cellular changes.

Type I collagen promotes epithelial-mesenchymal transition through ILK-dependent activation of NF-kappaB 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.

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.

Mechanisms of palatal epithelial seam disintegration by transforming growth factor (TGF) beta3.

TGFbeta3 signaling initiates and completes sequential phases of cellular differentiation that is required for complete disintegration of the palatal medial edge seam, that progresses between 14 and 17 embryonic days in the murine system, which is necessary in establishing confluence of the palatal stroma. Understanding the cellular mechanism of palatal MES disintegration in response to TGFbeta3 signaling will result in new approaches to defining the causes of cleft palate and other facial clefts that may result from failure of seam disintegration. We have isolated MES primary cells to study the details of MES disintegration mechanism by TGFbeta3 during palate development using several biochemical and genetic approaches. Our results demonstrate a novel mechanism of MES disintegration where MES, independently yet sequentially, undergoes cell cycle arrest, cell migration and apoptosis to generate immaculate palatal confluency during palatogenesis in response to robust TGFbeta3 signaling. The results contribute to a missing fundamental element to our base knowledge of the diverse roles of TGFbeta3 in functional and morphological changes that MES undergo during palatal seam disintegration. We believe that our findings will lead to more effective treatment of facial clefting.

Complexity in interpretation of embryonic epithelial-mesenchymal transition in response to transforming growth factor-beta 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-beta) is a potent inducer of EMT in various developmental and tumor systems. The analysis of TGF-beta 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-beta 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-beta 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-beta signaling in vivo to better understand the controlled, yet delicate mechanism of embryonic EMT.

TGFbeta3 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 TGFbeta3 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 beta-catenin), binds with the promoter of the E-cadherin gene to repress its transcription in response to TGFbeta3 signaling. Furthermore, we found that TGFbeta3 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 beta-catenin. We proved that TGFbeta3 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-beta/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.

Transforming growth factor-beta 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 beta-catenin. However, more recent studies have implicated a significant role of the transforming growth factor-beta (TGF-beta) in causing EMT in both development and pathology. The ability of TGF-beta 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-beta and their potential cross-interaction with traditional Wnt signaling molecules to bring about EMT during embryogenesis and tumor metastasis.