Mmp17b Is Essential for Proper Neural Crest Cell Migration In Vivo

The extracellular matrix plays a critical role in neural crest (NC) cell migration. In this study, we characterize the contribution of the novel GPI-linked matrix metalloproteinase (MMP) zebrafish mmp17b. Mmp17b is expressed post-gastrulation in the developing NC. Morpholino inactivation of mmp17b function, or chemical inhibition of MMP activity results in aberrant NC cell migration with minimal change in NC proliferation or apoptosis. Intriguingly, a GPI anchored protein with metalloproteinase inhibitor properties, Reversion-inducing-Cysteine-rich protein with Kazal motifs (RECK), which has previously been implicated in NC development, is expressed in close apposition to NC cells expressing mmp17b, raising the possibility that these two gene products interact. Consistent with this possibility, embryos silenced for mmp17b show defective development of the dorsal root ganglia (DRG), a crest-derived structure affected in RECK mutant fish sensory deprived (sdp). Taken together, this study has identified the first pair of MMP, and their putative MMP inhibitor RECK that functions together in NC cell migration.     

miR-200b regulates cell migration via Zeb family during mouse palate development

Palate development requires coordinating proper cellular and molecular events in palatogenesis, including the epithelial–mesenchymal transition (EMT), apoptosis, cell proliferation, and cell migration. Zeb1 and Zeb2 regulate epithelial cadherin (E-cadherin) and EMT during organogenesis. While microRNA 200b (miR-200b) is known to be a negative regulator of Zeb1 and Zeb2 in cancer progression, its regulatory effects on Zeb1 and Zeb2 in palatogenesis have not yet been clarified. The aim of this study is to investigate the relationship between the regulators of palatal development, specifically, miR-200b and the Zeb family. Expression of both Zeb1 and Zeb2 was detected in the mesenchyme of the mouse palate, while miR-200b was expressed in the medial edge epithelium. After contact with the palatal shelves, miR-200b was expressed in the palatal epithelial lining and epithelial island around the fusion region but not in the palatal mesenchyme. The function of miR-200b was examined by overexpression via a lentiviral vector in the palatal shelves. Ectopic expression of miR-200b resulted in suppression of the Zeb family, upregulation of E-cadherin, and changes in cell migration and palatal fusion. These results suggest that miR-200b plays crucial roles in cell migration and palatal fusion by regulating Zeb1 and Zeb2 as a noncoding RNA during palate development.     

Snai1 regulates cell lineage allocation and stem cell maintenance in the mouse intestinal epithelium

Snail family members regulate epithelial‐to‐mesenchymal transition (EMT) during invasion of intestinal tumours, but their role in normal intestinal homeostasis is unknown. Studies in breast and skin epithelia indicate that Snail proteins promote an undifferentiated state. Here, we demonstrate that conditional knockout of Snai1 in the intestinal epithelium results in apoptotic loss of crypt base columnar stem cells and bias towards differentiation of secretory lineages. In vitro organoid cultures derived from Snai1 conditional knockout mice also undergo apoptosis when Snai1 is deleted. Conversely, ectopic expression of Snai1 in the intestinal epithelium in vivo results in the expansion of the crypt base columnar cell pool and a decrease in secretory enteroendocrine and Paneth cells. Following conditional deletion of Snai1, the intestinal epithelium fails to produce a proliferative response following radiation‐induced damage indicating a fundamental requirement for Snai1 in epithelial regeneration. These results demonstrate that Snai1 is required for regulation of lineage choice, maintenance of CBC stem cells and regeneration of the intestinal epithelium following damage.     

Pim protein kinase-3 is regulated by TNF-α and promotes endothelial cell sprouting

Tumor necrosis factor-α (TNF-α) plays an important role in pathological angiogenesis associated with inflammatory response. Pim-3 kinase belonging to serine/threonine protein kinases is a potent suppressor of myc-induced apoptosis. We have recently demonstrated that Pim-3 plays an essential role in endothelial cell (EC) spreading and migration. In this study, we showed that TNF-α transiently increased Pim-3 mRNA expression, and this was mediated through Tumor necrosis factor-α receptor-1 (TNFR1) pathway in ECs. TNF-α could promote stabilization of Pim- 3 mRNA in ECs. Small-interfering RNA (siRNA)-mediated gene knockdown of Pim-3 significantly impaired TNF-α-induced formation of EC membrane protrusions in vitro. Furthermore, Pim-3 silencing inhibited EC sprouting in subcutaneous Matrigel in vivo. eNOS mRNA abundance was lower in Pim-3 siRNA transfected ECs compared with the control ECs. These observations suggest that Pim-3 plays a role in TNF-α-induced angiogenesis.     

Dioxin Receptor Expression Inhibits Basal and Transforming Growth Factor β-induced Epithelial-to-mesenchymal Transition

Recent studies have emphasized the role of the dioxin receptor (AhR) in maintaining cell morphology, adhesion, and migration. These novel AhR functions depend on the cell phenotype, and although AhR expression maintains mesenchymal fibroblasts migration, it inhibits keratinocytes motility. These observations prompted us to investigate whether AhR modulates the epithelial-to-mesenchymal transition (EMT). For this, we have used primary AhR^+/+ and AhR^−/− keratinocytes and NMuMG cells engineered to knock down AhR levels (sh-AhR) or to express a constitutively active receptor (CA-AhR). Both AhR^−/− keratinocytes and sh-AhR NMuMG cells had increased migration, reduced levels of epithelial markers E-cadherin and β-catenin, and increased expression of mesenchymal markers Snail, Slug/Snai2, vimentin, fibronectin, and α-smooth muscle actin. Consistently, AhR^+/+ and CA-AhR NMuMG cells had reduced migration and enhanced expression of epithelial markers. AhR activation by the agonist FICZ (6-formylindolo[3,2-b]carbazole) inhibited NMuMG migration, whereas the antagonist α-naphthoflavone induced migration as did AhR knockdown. Exogenous TGFβ exacerbated the promigratory mesenchymal phenotype in both AhR-expressing and AhR-depleted cells, although the effects on the latter were more pronounced. Rescuing AhR expression in sh-AhR cells reduced Snail and Slug/Snai2 levels and cell migration and restored E-cadherin levels. Interference of AhR in human HaCaT cells further supported its role in EMT. Interestingly, co-immunoprecipitation and immunofluorescence assays showed that AhR associates in common protein complexes with E-cadherin and β-catenin, suggesting the implication of AhR in cell-cell adhesion. Thus, basal or TGFβ-induced AhR down-modulation could be relevant in the acquisition of a motile EMT phenotype in both normal and transformed epithelial cells.     

Expression analysis of zebrafish membrane type-2 matrix metalloproteinases during embryonic development

Membrane tethered matrix metalloproteinases (MMPs) cleave a variety of extracellular matrix (ECM) and non-ECM targets and play important roles during embryonic development and tumor progression. Membrane tethered MMPs in particular are important regulators of both tissue invasion and morphogenesis. Much attention has been given to understanding the function of human and mouse MMP14 (also called membrane type-1 MMP, MT1-MMP) and our own data have linked zebrafish Mmp14 to the regulation of gastrulation cell movements. However, less is known regarding the expression and function of other membrane tethered MMPs. We report the cloning and gene expression analysis of zebrafish mmp15a and mmp15b (MT2-MMP) during early embryonic and larval development. Our data show that mmp15a exhibits limited expression prior to segmentation stages and is first detected in the tectum and posterior tailbud. At 24hours post-fertilization (hpf) mmp15a localizes to the caudal hematopoietic tissue, pectoral fin buds, and mandibular arch. By contrast, mmp15b is strongly expressed during gastrula stages before becoming restricted to the polster and anterior neural plate. From 24 to 48hpf, mmp15b expression is detected in the pharyngeal arches, fin buds, otic vesicle, pronephric ducts, proctodeum, tail epidermis, posterior lateral line primordia, and caudal notochord. During the larval period beginning at 72hpf, mmp15b expression becomes restricted to the brain ventricular zone, pharyngeal arches, pectoral fins, and the proctodeum. Many of the mmp15-expressing tissues have been shown to express genes encoding components of the ECM including collagens, fibronectin, and laminins. Our data thus provide a foundation for uncovering the role of Mmp15-dependent pericellular proteolysis during zebrafish embryonic development.     

Increased activity of mesenchymal ALK2‐BMP signaling causes posteriorly truncated microglossia and disorganization of lingual tissues

Proper development of taste organs including the tongue and taste papillae requires interactions with the underlying mesenchyme through multiple molecular signaling pathways. The effects of bone morphogenetic proteins (BMPs) and antagonists are profound, however, the tissue‐specific roles of distinct receptors are largely unknown. Here, we report that constitutive activation (ca) of ALK2‐BMP signaling in the tongue mesenchyme (marked by Wnt1‐Cre) caused microglossia—a dramatically smaller and misshapen tongue with a progressively severe reduction in size along the anteroposterior axis and absence of a pharyngeal region. At E10.5, the tongue primordia (branchial arches 1–4) formed in Wnt1‐Cre/caAlk2 mutants while each branchial arch responded to elevated BMP signaling distinctly in gene expression of BMP targets (Id1, Snai1, Snai2, and Runx2), proliferation (Cyclin‐D1) and apoptosis (p53). Moreover, elevated ALK2‐BMP signaling in the mesenchyme resulted in apparent defects of lingual epithelium, muscles, and nerves. In Wnt1‐Cre/caAlk2 mutants, a circumvallate papilla was missing and further development of formed fungiform papillae was arrested in late embryos. Our data collectively demonstrate that ALK2‐BMP signaling in the mesenchyme plays essential roles in orchestrating various tissues for proper development of the tongue and its appendages in a region‐specific manner.     

Membrane-type 1 matrix metalloproteinase regulates cell migration during zebrafish gastrulation: evidence for an interaction with non-canonical Wnt signaling

Key to invasiveness is the ability of tumor cells to modify the extracellular matrix, become motile, and engage in directed migration towards the vasculature. One significant protein associated with metastatic progression is membrane-type 1 matrix metalloproteinase (MT1-MMP/MMP14). How MMP14 activity is coordinated with other signaling pathways to regulate cell migration in vivo is largely unknown. Here we have used zebrafish embryogenesis as a model to understand the potential relationship between MMP14-dependent pericellular proteolysis, cell polarity, and motility. Knockdown of zebrafish Mmp14 function disrupted gastrulation convergence and extension cell movements and craniofacial morphogenesis. Using time-lapse imaging and morphometric analyses, we show that Mmp14 is required for proper cell polarity underlying the directed migration of mesodermal cells during gastrulation. We have identified a genetic interaction between mmp14 and non-canonical Wnt signaling, a pathway that also regulates cell polarity in embryonic tissues and is increasingly being linked with tumor cell migration. Finally, we demonstrate that Van Gogh-like 2, a key regulator of the non-canonical Wnt pathway, co-localizes with MMP14 and becomes redistributed towards the leading edge of polarized human cancer cells. Together, our results support the notion that pathways regulating pericellular proteolysis and cell polarity converge to promote efficient cell migration.     

Genes involved in the immediate early response and epithelial-mesenchymal transition are regulated by adipocytokines in the female reproductive tract

Obesity increases the risk of female reproductive tract cancers, but the underlying mechanistic link between the two is ill‐defined. Thus, the objective of the current study was to identify obesity‐dependent changes in the expression of immediate early (IE) genes that contribute to cell proliferation and differentiation, and epithelial–mesenchymal transition (EMT) genes that promote cell migration. When HeLa cells were treated for 0–48 hr with IGF‐1, leptin, TNFα, or IL‐6, each individual adipocytokine altered the abundance of IE (cJUN, cFOS, and cMYC) and EMT (SNAI1, SNAI2, and TWIST1) mRNA abundance. For example, IGF‐1 increased cJUN and cFOS and decreased cMYC; leptin increased cFOS; IL‐6 increased cFOS and cMYC; and TNFα increased cJUN and cFOS mRNA abundance. Likewise, EMT gene expression was altered by IGF‐1, TNFα, and IL‐6. SNAI1 was increased by IGF‐1 and IL‐6; SNAI2 was increased by IGF‐1 and TNFα; and TWIST1 was increased by TNFα and IL‐6. Chronic exposure to adipocytokines also altered EMT gene expression in the whole uterus of obese compared to normal‐weight mice. Specifically, there was no difference in cJun, cFos, or cMyc mRNA abundance between normal‐weight and obese animals. Snai1, Snai2, and Twist1 mRNA abundance, however, was increased in the uterus of obese females and correlated with increased circulating IGF‐1 levels. These data indicate that obesity‐dependent alterations in adipocytokine levels regulate the expression of genes associated with cell proliferation and migration, and therefore may provide a plausible mechanism for obesity‐dependent increases in cancers of the female reproductive tract. Mol. Reprod. Dev. 79:128–137, 2012. © 2011 Wiley Periodicals, Inc.     

MMP2/3 promote the growth and migration of laryngeal squamous cell carcinoma via PI3K/Akt-NF-κB-mediated epithelial–mesenchymal transformation

Laryngeal squamous cell carcinoma (LSCC) is an aggressive malignancy with metastatic potential and high mortality worldwide. Matrix metalloproteinases (MMPs) are a group of extracellular proteolytic enzymes. Although MMPs have been proved to be essential in the process of tumor migration and metastasis in most types of carcinomas, the expression and function of MMP2/3 in LSCC remain unknown. Here, we investigated the roles of MMP2/3 in LSCC and elucidated the underlying mechanism. We found that levels of MMP2/3 were significantly higher in clinical LSCC samples than in paired normal sample examined by real-time polymerase chain reaction and western blot assays. Moreover, overexpression of MMP2/3 promoted the proliferation and migration of LSCC cells by Cell Counting Kit-8, transwell, and scratch wound-healing assays in vitro. Furthermore, levels of epithelial cell markers (E-cadherin and occludin) were decreased following MMP2/3 overexpression, with increased levels of mesenchymal cell markers (N-cadherin and vimentin), suggesting the involvement of epithelial–mesenchymal transformation (EMT) process in MMP2/3-mediated LSCC cell migration. By using short hairpin RNA, knockdown of MMP2/3 expression inhibited the proliferation, migration, and EMT process in LSCC cells in vitro. More importantly, we confirmed that MMP2/3 promoted LSCC in the PI3K/Akt-NF-κB-dependent manner. This study provides insight into MMP2/3-mediated LSCC development and lays a foundation for potential pharmacological targets to LSCC.     

Elevated glucose inhibits VEGF-A-mediated endocardial cushion formation: modulation by PECAM-1 and MMP-2

Atrioventricular (AV) septal defects resulting from aberrant endocardial cushion (EC) formation are observed at increased rates in infants of diabetic mothers. EC formation occurs via an epithelial-mesenchymal transformation (EMT), involving transformation of endocardial cells into mesenchymal cells, migration, and invasion into extracellular matrix. Here, we report that elevated glucose inhibits EMT by reducing myocardial vascular endothelial growth factor A (VEGF-A). This effect is reversed with exogenous recombinant mouse VEGF-A165, whereas addition of soluble VEGF receptor-1 blocks EMT. We show that disruption of EMT is associated with persistence of platelet endothelial cell adhesion molecule-1 (PECAM-1) and decreased matrix metalloproteinase-2 (MMP-2) expression. These findings correlate with retention of a nontransformed endocardial sheet and lack of invasion. The MMP inhibitor GM6001 blocks invasion, whereas explants from PECAM-1 deficient mice exhibit MMP-2 induction and normal EMT in high glucose. PECAM-1-negative endothelial cells are highly motile and express more MMP-2 than do PECAM-1-positive endothelial cells. During EMT, loss of PECAM-1 similarly promotes single cell motility and MMP-2 expression. Our findings suggest that high glucose-induced inhibition of AV cushion morphogenesis results from decreased myocardial VEGF-A expression and is, in part, mediated by persistent endocardial cell PECAM-1 expression and failure to up-regulate MMP-2 expression.     

Vasoprotective Effects of Urocortin 1 against Atherosclerosis In Vitro and In Vivo

Aim

Atherosclerosis is the complex lesion that consists of endothelial inflammation, macrophage foam cell formation, vascular smooth muscle cell (VSMC) migration and proliferation, and extracellular matrix production. Human urocortin 1 (Ucn1), a 40-amino acid peptide member of the corticotrophin-releasing factor/urotensin I family, has potent cardiovascular protective effects. This peptide induces potent and long-lasting hypotension and coronary vasodilation. However, the relationship of Ucn1 with atherosclerosis remains unclear. The present study was performed to clarify the effects of Ucn1 on atherosclerosis.

Methods

We assessed the effects of Ucn1 on the inflammatory response and proliferation of human endothelial cells (ECs), human macrophage foam cell formation, migration and proliferation of human VSMCs, extracellular matrix expression in VSMCs, and the development of atherosclerosis in apolipoprotein E-deficient (Apoe ^−/−) mice.

Results

Ucn1 significantly suppressed cell proliferation without inducing apoptosis, and lipopolysaccharide-induced up-regulation of monocyte chemoattractant protein-1 and intercellular adhesion molecule-1 in human ECs. Ucn1 significantly reduced oxidized low-density lipoprotein-induced foam cell formation with a significant down-regulation of CD36 and acyl-CoA:cholesterol acyltransferase 1 in human monocyte-derived macrophages. Ucn1 significantly suppressed the migration and proliferation of human VSMCs and increased the activities of matrix metalloproteinase-2 (MMP2) and MMP9 in human VSMCs. Intraperitoneal injection of Ucn1 into Apoe ^−/− mice for 4 weeks significantly retarded the development of aortic atherosclerotic lesions.

Conclusions

This study provided the first evidence that Ucn1 prevents the development of atherosclerosis by suppressing EC inflammatory response and proliferation, macrophage foam cell formation, and VSMC migration and proliferation. Thus, Ucn1 could serve as a novel therapeutic target for atherosclerotic cardiovascular diseases.     

A sea urchin in vivo model to evaluate Epithelial‐Mesenchymal Transition

Epithelial‐mesenchymal transition (EMT) is an evolutionarily conserved cellular program, which is a prerequisite for the metastatic cascade in carcinoma progression. Here, we evaluate the EMT process using the sea urchin Paracentrotus lividus embryo. In sea urchin embryos, the earliest EMT event is related to the acquisition of a mesenchymal phenotype by the spiculogenetic primary mesenchyme cells (PMCs) and their migration into the blastocoel. We investigated the effect of inhibiting the epidermal growth factor (EGF) signaling pathway on this process, and we observed that mesenchyme cell differentiation was blocked. In order to extend and validate our studies, we investigated the migratory capability and the level of potential epidermal growth factor receptor (EGFr) targets in a breast cancer cell line after EGF modulation. Altogether, our data highlight the sensitivity of the sea urchin embryo to anti‐EMT drugs and pinpoint the sea urchin embryo as a valuable in vivo model system for studying EMT and the screening of anti‐EMT candidates.     

Effects of morphological patterning on endothelial cell migration

The migration of vascular endothelial cells (ECs) plays an important role in vascular remodeling. Here we studied the effects of cell morphology on the migration of bovine aortic ECs by culturing cells on micropatterned strips of collagen matrix (60-, 30-, and 15-microm wide). The spreading areas of the cells on 15- and 30-microm wide strips were 30% lower than those on 60-microm wide strips and unpatterned collagen. The cells on 15-microm wide strips completely aligned in the direction of the strip, and had significantly lower shape index than those in all other groups. On strips of all widths, ECs tended to migrate in the direction of strips. ECs on 15-microm wide strips had highest speed, particularly in the direction of the strip. Vinculin staining showed that the leading edge of ECs on 15-microm wide strips had focal adhesions that were oriented with their lamellipodial protrusion and the direction of cell migration; this arrangement of the focal adhesions may promote EC migration. The present study provides direct evidence on the role of cell morphology in EC migration, and will help us to understand the mechanisms of EC migration during angiogenesis and wound healing.     

Transdifferentiation-inducing HCCR-1 oncogene

Background

Cell transdifferentiation is characterized by loss of some phenotypes along with acquisition of new phenotypes in differentiated cells. The differentiated state of a given cell is not irreversible. It depends on the up- and downregulation exerted by specific molecules.

Results

We report here that HCCR-1, previously shown to play an oncogenic role in human cancers, induces epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) in human and mouse, respectively. The stem cell factor receptor CD117/c-Kit was induced in this transdifferentiated (EMT) sarcoma tissues. This MET occurring in HCCR-1 transfected cells is reminiscent of the transdifferentiation process during nephrogenesis. Indeed, expression of HCCR-1 was observed during the embryonic development of the kidney. This suggests that HCCR-1 might be involved in the transdifferentiation process of cancer stem cell.

Conclusions

Therefore, we propose that HCCR-1 may be a regulatory factor that stimulates morphogenesis of epithelia or mesenchyme during neoplastic transformation.     

Opposing actions of Notch1 and VEGF in post-natal cardiac valve endothelial cells

The endothelium of the cardiac valves is unique compared the rest of the vasculature in its ability to undergo an endothelial-to-mesenchymal transformation (EMT) in vitro in response to transforming growth factor-β (TGF-β). EMT is a critical event during embryonic valve development, and both VEGF-A and Notch1 have been shown to function in this process. Here we investigate the effects of VEGF-A and Notch1 on EMT in clonal endothelial cell (EC) populations isolated from adult aortic valve leaflets. VEGF-A inhibited TGF-β-induced EMT. Endothelial growth, however, was not affected by VEGF-A or TGF-β. A positive role for Notch1 was revealed in three experiments: (1) TGF-β induced Notch1 mRNA in valve ECs, (2) a γ-secretase inhibitor of Notch1 signaling blocked EMT, and (3) overexpression of a ligand-independent form of Notch1 induced EMT. These results demonstrate, for the first time, that VEGF-A and Notch1 play opposing roles in regulating EMT in post-natal valve endothelium.