Substitution for mesenchyme by basement-membrane-like substratum and epidermal growth factor in inducing branching morphogenesis of mouse salivary epithelium

Mouse salivary epithelium cannot undergo branching morphogenesis in the absence of the surrounding mesenchyme. To clarify the nature of the mesenchymal influence on the epithelium, we have investigated the culture conditions in which the epithelium could normally branch in the absence of mesenchymal cells. Combination of basement-membrane-like substratum (Matrigel) and epidermal growth factor (EGF) could substitute for the mesenchyme, the epithelium showing typical branching morphogenesis. Transforming growth factor alpha had the same effect as EGF. Matrigel plus basic fibroblast growth factor or transforming growth factor beta 1 and collagen gel plus EGF were not sufficient to support the branching of the epithelium. These results clearly reveal that the role of mesenchyme in salivary morphogenesis is both to provide the epithelium with an appropriate substratum and to accelerate growth of the epithelium.     

Ingression of primary mesenchyme cells of the sea urchin embryo: a precisely timed epithelial mesenchymal transition

Epithelial-mesenchyme transitions (EMTs) are familiar to all scholars of development. Each animal system utilizes an EMT to produce mesenchyme cells. In vertebrates, for example, there are a number of EMTs that shape the embryo. Early, entry of epiblast cells into the primitive streak is followed by the emergence of mesoderm via an EMT process. The departure of neural crest cells from the margin of the neural folds is an EMT process, and the delamination of cells from the endomesoderm to form the supporting mesenchyme of the lung, liver, and pancreas are EMTs. EMTs are observed in Drosophila following invagination of the ventral furrow, and even in Cnidarians, which have only two germ layers, yet mesoglial and stem cells delaminate from the epithelia and occupy the matrix between the ectoderm and endoderm. This review will focus on a classic example of an EMT, which occurs in the sea urchin embryo. The primary mesenchyme cells (PMCs) ingress from the vegetal plate of this embryo precociously and in advance of archenteron invagination. Because ingression is precisely timed, the PMC lineage precisely known, and the embryo easily observed and manipulated, much has been learned about how the ingression of PMCs works in the sea urchin. Though the focus of this review is the sea urchin PMCs, there is evidence that all EMTs share many common features at both cellular and molecular levels, and many of these mechanisms are also shown to be involved in tumor progression, especially metastasizing carcinomas.     

Identification and characterization of PlAlix,the Alix homologue from the Mediterranean sea urchin Paracentrotus lividus

The sea urchin provides a relatively simple and tractable system for analyzing the early stages of embryo development. Here, we use the sea urchin species, Paracentrotus lividus, to investigate the role of Alix in key stages of embryogenesis, namely the egg fertilization and the first cleavage division. Alix is a multifunctional protein involved in different cellular processes including endocytic membrane trafficking, filamentous (F)‐actin remodeling, and cytokinesis. Alix homologues have been identified in different metazoans; in these organisms, Alix is involved in oogenesis and in determination/differentiation events during embryo development. Herein, we describe the identification of the sea urchin homologue of Alix, PlAlix. The deduced amino acid sequence shows that Alix is highly conserved in sea urchins. Accordingly, we detect the PlAlix protein cross‐reacting with monoclonal Alix antibodies in extracts from P. lividus, at different developmental stages. Focusing on the role of PlAlix during early embryogenesis we found that PlAlix is a maternal protein that is expressed at increasingly higher levels from fertilization to the 2‐cell stage embryo. In sea urchin eggs, PlAlix localizes throughout the cytoplasm with a punctuated pattern and, soon after fertilization, accumulates in larger puncta in the cytosol, and in microvilli‐like protrusions. Together our data show that PlAlix is structurally conserved from sea urchin to mammals and may open new lines of inquiry into the role of Alix during the early stages of embryo development.     

Melatonin protects ADSCs from ROS and enhances their therapeutic potency in a rat model of myocardial infarction

Myocardial infarction (MI) is a major cause of death and disability worldwide. In the last decade, mesenchymal stem cells (MSCs) based cell therapy has emerged as a promising therapeutic strategy. Although great advance have been made using MSCs to treat MI, the low viability of transplanted MSCs severely limits the efficiency of MSCs therapy. Here, we show evidence that ex vivo pre‐treatment with melatonin, an endogenous hormone with newly found anti‐oxidative activity, could improve survival and function of adipose tissue derived MSCs (ADSCs) in vitro as well as in vivo. ADSCs with 5 μM melatonin pre‐treatment for 24 hrs showed increased expression of the antioxidant enzyme catalase and Cu/Zn superoxide dismutase (SOD‐1), as well as pro‐angiogenic and mitogenic factors like insulin‐like growth factor 1, basic fibroblast growth factor, hepatocyte growth factor (HGF), epidermal growth factor. Furthermore, melatonin pre‐treatment protected MSCs from reactive oxygen species (ROS) induced apoptosis both directly by promoting anti‐apoptosis kinases like p‐Akt as well as blocking caspase cascade, and indirectly by restoring the ROS impaired cell adhesion. Using a rat model of MI, we found that melatonin pre‐treatment enhanced the viability of engrafted ADSCs, and promoted their therapeutic potency. Hopefully, our results may shed light on the design of more effective therapeutic strategies treating MI by MSCs in clinic.     

Development and growth of mouse embryonic kidney in organ culture and modulation of development by soluble growth factor

Differentiation of the metanephrogenic mesenchyme is triggered by an inductive tissue interaction between an inducer tissue and the mesenchyme. It is generally believed that the epithelial ureter bud acts as an inducer during in vivo development. In response to the inductive stimulus most of the mesenchymal cells convert into epithelial cells, while a small fraction differentiates into stromal cells. In vitro, differentiation of isolated mesenchyme to epithelium can be induced by a variety of embryonic tissues, but nothing is known about the molecular nature of the inducing stimulus. In recent years, large numbers of polypeptide growth factors have been described, which in addition to proliferative effects were shown to exert effects on a variety of biological phenomena such as chemotaxis, inflammation, tissue repair, or induction of embryonic development. We therefore analyzed whether growth factors in the absence of inducer tissue can induce isolated kidney mesenchyme to differentiate into epithelium or interstitium. As expected, both growth and differentiation into epithelium were stimulated by an inducer tissue, the spinal cord. We found that none of the various growth factors tested (including epidermal growth factor, transforming growth factors alpha and beta, insulin-like growth factors I and II, fibroblast growth factor, platelet-derived growth factor, and retinoic acid) could mimick the effect of an inducer tissue, although we tested the factors over a wide concentration range. One of the tested factors, epidermal growth factor (EGF) stimulated the mesenchymal cells to become stromal cells, although it could not stimulate development into epithelium. EGF could stimulate stromal development both when the mesenchyme was cultured in isolation and when the mesenchyme was stimulated by an inducer tissue to become epithelium. The expansion of the stromal compartment in response to EGF treatment occurred at the expense of the epithelial cells, but EGF could not completely suppress the formation of epithelium. These data suggest the presence of EGF receptors in the developing kidney, but since application of soluble EGF leads to abnormal development, soluble EGF cannot be the natural ligand. We suggest that locally produced mitogens with an EGF-like structure may regulate the relative amounts of stroma (interstitium) and epithelium in the developing kidney.     

3D spheroid culture enhances survival and therapeutic capacities of MSCs injected into ischemic kidney

Three‐dimensional (3D) cell culture has been reported to increase the therapeutic potentials of mesenchymal stem cells (MSCs). In this study, we aimed to investigate the therapeutic effects of 3D spheroids of human adipose‐derived MSCs for acute kidney injury (AKI). In vitro studies indicated that 3D spheroids of MSCs produced higher levels of extracellular matrix proteins (including collagen I, fibronectin and laminin), and exhibited stronger anti‐apoptotic and anti‐oxidative capacities than two‐dimensional (2D) cultured cells. Furthermore, 3D culture increased the paracrine secretion of cytokines by MSCs, including angiogenic factors (VEGF and basic fibroblast growth factor), anti‐apoptotic factors (epidermal growth factor and hepatocyte growth factor), the anti‐oxidative factor insulin‐like growth factor and the anti‐inflammatory protein tumour necrosis factor‐alpha stimulated gene/protein 6. Consistent with in vitro experiments, 3D spheroids of MSCs showed enhanced survival and paracrine effects in vivo. More importantly, when injected into the kidney of model rats with ischemia‐reperfusion (I/R)‐induced AKI, 3D spheroids were more beneficial in protecting the I/R kidney against apoptosis, reducing tissue damage, promoting vascularization and ameliorating renal function compared with 2D cultured cells. Therefore, the 3D culture strategy improved the therapeutic effects of MSCs, and might be promising for AKI treatment.     

Human umbilical cord mesenchymal stem cells facilitate the up‐regulation of miR‐153‐3p,whereby attenuating MGO‐induced peritoneal fibrosis in rats

MiRNAs contribute greatly to epithelial to mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs), which is a crucial step in peritoneal fibrosis (PF). In this study, we tried to profile whether miRNA expression differences exist after human umbilical cord mesenchymal stem cells (hUCMSCs) treatment in PF rats and investigate the possible role of miR‐153‐3p involved in anti‐EMT process. We randomly assigned 34 rats into three groups: control group (Group Control), MGO‐induced PF rats (Group MGO) and hUCMSCs‐treated rats (Group MGO + hUCMSCs). MiRNA microarrays and real‐time PCR analyses were conducted in three groups. α‐SMA, Snail1 and E‐cadherin expression were detected by Western blot. Luciferase reporter assays were used to detect the effects of miR‐153‐3p overexpression on Snai1 in rat peritoneal mesothelial cells (RPMCs). We identified differentially expressed miRNAs related to EMT, in which miR‐153‐3p demonstrated the greatest increase in Group MGO + hUCMSCs. Transient cotransfection of miR‐153‐3p mimics with luciferase expression plasmids resulted in a significant repression of Snai1 3′‐untranslated region luciferase activity in RPMCs. These studies suggest that miR‐153‐3p is a critical molecule in anti‐EMT effects of hUCMSCs in MGO‐induced PF rats. MiR‐153‐3p might exert its beneficial effect through directly targeting Snai1.     

Localization and quantitation of 125I-epidermal growth factor binding in mouse embryonic tooth and other embryonic tissues at different developmental stages

We have shown earlier that epidermal growth factor (EGF) inhibits morphogenesis and cell differentiation in mouse embryonic teeth in organ culture. This inhibition depends on the stage of tooth development so that only teeth at early developmental stages respond to EGF (A-M. Partanen, P. Ekblom, and I. Thesleff (1985) Dev. Biol. 111, 84-94). We have now studied the quantity and pattern of EGF binding in teeth at various stages of development by incubating the dissected tooth germs with 125I-labeled EGF. Although the quantity of 125I-EGF binding per microgram DNA stays at the same level, localization of 125I-EGF binding by autoradiography reveals that the distribution of binding sites changes dramatically. In bud stage the epithelial tooth bud that is intruding into the underlying mesenchyme has binding sites for EGF, but the condensation of dental mesenchymal cells around the bud does not bind EGF. At the cap stage of development the dental mesenchyme binds EGF, but the dental epithelium shows no binding. This indicates that the dental mesenchyme is the primary target tissue for the inhibitory effect of EGF on tooth morphogenesis during early cap stage. During advanced morphogenesis the binding sites of EGF disappear also from the dental papilla mesenchyme, but the dental follicle which consists of condensed mesenchymal cells surrounding the tooth germ, binds EGF abundantly. We have also studied EGF binding during the development of other embryonic organs, kidney, salivary gland, lung, and skin, which are all formed by mesenchymal and epithelial components. The patterns of EGF binding in various tissues suggest that EGF may have a role in the organogenesis of epitheliomesenchymal organs as a stimulator of epithelial proliferation during initial epithelial bud formation and branching morphogenesis. The results of this study indicate that EGF stimulates or maintains proliferation of undifferentiated cells during embryonic development and that the expression of EGF receptors in different organs is not related to the age of the embryo, but is specific to the developmental stage of each organ.     

Heterochronic activation of VEGF signaling and the evolution of the skeleton in echinoderm pluteus larvae

The evolution of the echinoderm larval skeleton was examined from the aspect of interactions between skeletogenic mesenchyme cells and surrounding epithelium. We focused on vascular endothelial growth factor (VEGF) signaling, which was reported to be essential for skeletogenesis in sea urchin larvae. Here, we examined the expression patterns of vegf and vegfr in starfish and brittle stars. During starfish embryogenesis, no expression of either vegfr or vegf was detected, which contrast with previous reports on the expression of starfish homologs of sea urchin skeletogenic genes, including Ets, Tbr, and Dri. In later stages, when adult skeletogenesis commenced, vegfr and vegf expression were upregulated in skeletogenic cells and in the adjacent epidermis, respectively. These expression patterns suggest that heterochronic activation of VEGF signaling is one of the key molecular evolutionary steps in the evolution of the larval skeleton. The absence of vegf or vegfr expression during early embryogenesis in starfish suggests that the evolution of the larval skeleton requires distinct evolutionary changes, both in mesoderm cells (activation of vegfr expression) and in epidermal cells (activation of vegf expression). In brittle stars, which have well‐organized skeletons like the sea urchin, vegfr and vegf were expressed in the skeletogenic mesenchyme and the overlying epidermis, respectively, in the same manner as in sea urchins. Therefore, the distinct activation of vegfr and vegf may have occurred in two lineages, sea urchins and brittle stars.     

Reciprocal interactions between epithelium, mesenchyme, and epidermal growth factor (EGF) in the regulation of mandibular mitotic activity in the embryonic chick

Mandibular epithelia and mesenchyme from chick embryos of Hamburger and Hamilton (H.H.) stage 18-25 were cultured intact, in isolation, or in recombinations in the presence or absence of 5-40 ng/ml epidermal growth factor (EGF). 3H-thymidine labelling demonstrated that mesenchyme influenced epithelial mitotic activity and vice versa. EGF can substitute for the epithelial effect. The stimulation of mesenchymal proliferation by H.H. 18 and 22 epithelia correlated with high levels of epithelial proliferation. Epithelial proliferation was low at H.H. 25 and unaffected by mesenchyme or by EGF. Epithelial stimulation of mesenchymal proliferation began earlier (H.H. 18) than did mesenchymal stimulation of epithelial proliferation (H.H. 22); i.e., within the ages tested, the epithelium initiated these reciprocal mitogenic interactions. That epithelial dependence on mesenchyme coincided with epithelial bone-evoking properties, suggested a) that mesenchyme promotes or maintains epithelial bone-promoting activity and b) that the critical differentiative influence of epithelium on mesenchyme is a mitogenic one. The temporal correlation between a sharp decline in mesenchymal proliferation and termination of the osteogenic epithelial-mesenchymal interaction at H.H. 25 further supports a connection between epithelial maintenance of mesenchymal proliferation and epithelial evocation of osteogenesis.     

Epidermal growth factor inhibits morphogenesis and cell differentiation in cultured mouse embryonic teeth

Although local epithelial-mesenchymal tissue interactions which are presumably mediated by extracellular matrix molecules are important regulators of tooth morphogenesis and differentiation, our studies have indicated that these developmental processes also depend on circulating molecules. The iron-carrying serum protein transferrin is necessary for the early morphogenesis of mouse tooth in organ culture (A-M. Partanen, I. Thesleff, and P. Ekblom, 1984, Differentiation 27, 59-66). In the present study we have examined the effects of other growth factors on mouse tooth germs grown in a chemically defined medium containing transferrin. Fibroblast growth factor and platelet derived growth factor had no detectable effects but epidermal growth factor (EGF) inhibited dramatically the morphogenesis of teeth, and prevented odontoblast and ameloblast cell differentiation. EGF stimulated cell proliferation in the explants measured as [3H]thymidine incorporation in DNA. However, when the distribution of dividing cells was visualized in autoradiographs, it was observed that cell proliferation was stimulated in the dental epithelium but was inhibited in the dental mesenchyme. The inhibition of cell proliferation in the dental mesenchyme apparently caused the inhibition of morphogenesis. We do not know whether the dental epithelium or mesenchyme was the primary target for the action of EGF in the inhibition of morphogenesis. It is, however, apparent that the response of the dental mesenchymal cells to EGF (inhibition of proliferation) is regulated by their local environment, since EGF enhanced proliferation when these cells were disaggregated and cultured as monolayers. This indicates that the organ culture system where the various embryonic cell lineages are maintained in their original environment corresponds better to the in vivo situation when the roles of exogenous growth factors during development are examined.     

miR‐1‐3p and miR‐206 sensitizes HGF‐induced gefitinib‐resistant human lung cancer cells through inhibition of c‐Met signalling and EMT

Hepatocyte growth factor (HGF) overexpression is an important mechanism in acquired epidermal growth factor receptor (EGFR) kinase inhibitor gefitinib resistance in lung cancers with EGFR activating mutations. MiR‐1‐3p and miR‐206 act as suppressors in lung cancer proliferation and metastasis. However, whether miR‐1‐3p and miR‐206 can overcome HGF‐induced gefitinib resistance in EGFR mutant lung cancer is not clear. In this study, we showed that miR‐1‐3p and miR‐206 restored the sensitivities of lung cancer cells PC‐9 and HCC‐827 to gefitinib in present of HGF. For the mechanisms, we demonstrated that both miR‐1‐3p and miR‐206 directly target HGF receptor c‐Met in lung cancer. Knockdown of c‐Met mimicked the effects of miR‐1‐3p and miR‐206 transfections Meanwhile, c‐Met overexpression attenuated the effects of miR‐1‐3p and miR‐206 in HGF‐induced gefitinib resistance of lung cancers. Furthermore, we showed that miR‐1‐3p and miR‐206 inhibited c‐Met downstream Akt and Erk pathway and blocked HGF‐induced epithelial‐mesenchymal transition (EMT). Finally, we demonstrated that miR‐1‐3p and miR‐206 can increase gefitinib sensitivity in xenograft mouse models in vivo. Our study for the first time indicated the new function of miR‐1‐3p and miR‐206 in overcoming HGF‐induced gefitinib resistance in EGFR mutant lung cancer cell.     

Astragaloside IV modulates TGF‐β1‐dependent epithelial‐mesenchymal transition in bleomycin‐induced pulmonary fibrosis

Epithelial‐mesenchymal transition (EMT) plays an important role in idiopathic pulmonary fibrosis (IPF). Astragaloside IV (ASV), a natural saponin from astragalus membranaceus, has shown anti‐fibrotic property in bleomycin (BLM)‐induced pulmonary fibrosis. The current study was undertaken to determine whether EMT was involved in the beneficial of ASV against BLM‐induced pulmonary fibrosis and to elucidate its potential mechanism. As expected, in BLM‐induced IPF, ASV exerted protective effects on pulmonary fibrosis and ASV significantly reversed BLM‐induced EMT. Intriguing, transforming growth factor‐β1 (TGF‐β1) was found to be up‐regulated, whereas Forkhead box O3a (FOXO3a) was hyperphosphorylated and less expressed. However, ASV treatment inhibited increased TGF‐β1 and activated FOXO3a in lung tissues. TGF‐β1 was administered to alveolar epithelial cells A549 to induce EMT in vitro. Meanwhile, stimulation with TGF‐β1‐activated phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) pathway and induced FOXO3a hyperphosphorylated and down‐regulated. It was found that overexpression of FOXO3a leading to the suppression of TGF‐β1‐induced EMT. Moreover, ASV treatment, similar with the TGF‐β1 or PI3K/Akt inhibitor, reverted these cellular changes and inhibited EMT in A549 cells. Collectively, the results suggested that ASV significantly inhibited TGF‐β1/PI3K/Akt‐induced FOXO3a hyperphosphorylation and down‐regulation to reverse EMT during the progression of fibrosis.     

Disulfiram combined with copper inhibits metastasis and epithelial–mesenchymal transition in hepatocellular carcinoma through the NF‐κB and TGF‐β pathways

Late‐stage hepatocellular carcinoma (HCC) usually has a low survival rate because of the high risk of metastases and the lack of an effective cure. Disulfiram (DSF) has copper (Cu)‐dependent anticancer properties in vitro and in vivo. The present work aims to explore the anti‐metastasis effects and molecular mechanisms of DSF/Cu on HCC cells both in vitro and in vivo. The results showed that DSF inhibited the proliferation, migration and invasion of HCC cells. Cu improved the anti‐metastatic activity of DSF, while Cu alone had no effect. Furthermore, DSF/Cu inhibited both NF‐κB and TGF‐β signalling, including the nuclear translocation of NF‐κB subunits and the expression of Smad4, leading to down‐regulation of Snail and Slug, which contributed to phenotype epithelial–mesenchymal transition (EMT). Finally, DSF/Cu inhibited the lung metastasis of Hep3B cells not only in a subcutaneous tumour model but also in an orthotopic liver metastasis assay. These results indicated that DSF/Cu suppressed the metastasis and EMT of hepatic carcinoma through NF‐κB and TGF‐β signalling. Our study indicates the potential of DSF/Cu for therapeutic use.