Ectopic Expression Of Pdx1 Research Articles

Pancreatic Lineage Specifier PDX1 Increases Adhesion and Decreases Motility of Cancer Cells.

Simple SummaryA possible way to inhibit tumor cells migration could be strengthening adhesion between tumor and stromal cells in primary solid tumors. Expanding on our previous findings, we have shown that ectopic expression of the pancreatic lineage specifier PDX1 reduces the migration potential of pancreatic cancer cells by increasing their adhesiveness and reducing the sensitivity to TGFβ1-induced epithelial-mesenchymal transition. We observed decreased expression levels of genes associated with promoting cell migration and increased expression of genes negatively affecting cell motility. Our study highlights a potential window for the PDX1 therapeutic application as an antimetastatic agent at the initial stages of cancer development.Intercellular interactions involving adhesion factors are key operators in cancer progression. In particular, these factors are responsible for facilitating cell migration and metastasis. Strengthening of adhesion between tumor cells and surrounding cells or extracellular matrix (ECM), may provide a way to inhibit tumor cell migration. Recently, we demonstrated that PDX1 ectopic expression results in the reduction of pancreatic cancer line PANC-1 cell motility in vitro and in vivo, and we now provide experimental data confirming the hypothesis that suppression of migration may be related to the effect of PDX1 on cell adhesion. Cell migration analyses demonstrated decreased motility of pancreatic Colo357 and PANC-1 cell lines expressing PDX1. We observed decreased expression levels of genes associated with promoting cell migration and increased expression of genes negatively affecting cell motility. Expression of the EMT regulator genes was only mildly induced in cells expressing PDX1 during the simulation of the epithelial-mesenchymal transition (EMT) by the addition of TGFβ1 to the medium. PDX1-expressing cancer cell lines showed increased cell adhesion to collagen type I, fibronectin, and poly-lysine. We conclude that ectopic expression of PDX1 reduces the migration potential of cancer cells, by increasing the adhesive properties of cells and reducing the sensitivity to TGFβ1-induced EMT.

PDX1, a key factor in pancreatic embryogenesis, can exhibit antimetastatic activity in pancreatic ductal adenocarcinoma.

Background: In cancer biology, metastasizing is one of the most poorly studied processes. Pancreatic ductal adenocarcinoma (PDAC) is characterized by early metastasis, which is the leading cause of death. The PDX1 protein is crucial for the development of cancer, and its low levels are characteristic of the most aggressive PDAC tumors. The PDX1 is a mediator of initiation and progression of PDAC. However, further studies are needed to elucidate the role of PDX1 in the cancer metastasis.Purpose: To confirm the hypothesis that PDX1 in PDAC plays suppressor role of epithelial–mesenchymal transition (EMT), and to study its possible ability to inhibit metastasis.Methods: A PDX1-overexpressing PDAC cell line was obtained by lentiviral transduction of PANC-1 cells. PDX1 overexpression was confirmed by RT-PCR and Western blotting. Effects of PDX1 ectopic expression on cell proliferation and motility were determined in PANC-1 cells using MTS, cell cycle analysis, transwell and wound-healing assay. EMT genes expression was analyzed in PDX1-overexpressing and Control PANC-1. Finally, the migration potential of pancreatic cancer cells expressing PDX1 was evaluated using a zebrafish embryo model.Results: The motility of human PDAC cells PANC-1 considerably decreased at ectopic expression of PDX1. The decreased expression of ZEB1, the key factor of EMT, and almost unchanged expression of the genes that characterize the epithelial state suggest a decrease in the EMT ability. Suppression of PDX1 expression by siRNA knockdown restored the PANC1 motility.Conclusion: The results obtained suggest a possible therapeutic use of PDX1 delivery into PDAC patients with a reduced or absent expression of PDX1 in the most aggressive tumors.

Combinations of Activin A or Nicotinamide with the Pancreatic Transcription Factor PDX1 Support Differentiation of Human Amnion Epithelial Cells Toward a Pancreatic Lineage.

The differentiation of multipotent stem cells toward a pancreatic lineage provides us with an alternative cell-based therapeutic approach to type 1 diabetes and enables us to study pancreas development. The current study aims to study the effect of growth factors such as activin A or nicotinamide, alone and in combinations with the transcription factor, PDX1 (pancreatic and duodenal homeobox-1), on human amnion epithelial cells (hAECs) toward a pancreatic lineage. Ectopic expression of Pdx1 followed by treatment of hAECs with nicotinamide for 4 days resulted in strong induction of pancreatic endoderm and pancreatic progenitor genes, including NKX6.1 and NEUROD1. Pancreatic lineage cells expressing PDX1, SOX17, and RFX6 are derived from Pdx1-transduced hAECs treated with activin A or nicotinamide, but not cells treated with activin A or nicotinamide alone. Our study provides a novel culture protocol for generating pancreas-committed cells from hAECs and reveals an interplay between Pdx1 and activin A/nicotinamide signaling in early pancreatic fate determination.

The combined effect of PDX1, epidermal growth factor and poly-L-ornithine on human amnion epithelial cells' differentiation.

BackgroundIt has been suggested that the ectopic expression of PDX1, a dominant pancreatic transcription factor, plays a critical role in the developmental programming of the pancreas even from cells of unrelated tissues such as keratinocytes and amniotic fluid stem cells. In this study we have chosen to drive pancreatic development in human amnion epithelial cells by inducing endogenous PDX1 expression. Further, we have investigated the role of Epidermal Growth Factor (EGF) and Poly-L-Ornithine (PLO) on this differentiation process.ResultsHuman amnion epithelial cells expressed high levels of endogenous PDX1 upon transduction with an adenoviral vector expressing murine Pdx1. Other markers of various stages of pancreatic differentiation such as NKX6.1, SOX17, RFX6, FOXA2, CFTR, NEUROD1, PAX4 and PPY were also expressed upon Pdx1 transduction.Although initial expression of pancreatic progenitor markers was higher in culture conditions lacking EGF, for a sustained and increased expression EGF was required. Culture on PLO further increased the positive impact of EGF.ConclusionPancreatic marker expression subsequent to mPdx1 transduction suggests that this approach may facilitate the in vitro differentiation of hAECs into cells of the endocrine pancreas. This result may have important implications in diabetes therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-016-0108-y) contains supplementary material, which is available to authorized users.

The Temporal and Hierarchical Control of Transcription Factors-Induced Liver to Pancreas Transdifferentiation

Lineage-specific transcription factors (TFs) display instructive roles in directly reprogramming adult cells into alternate developmental fates, in a process known as transdifferentiation. The present study analyses the hypothesis that despite being fast, transdifferentiation does not occur in one step but is rather a consecutive and hierarchical process. Using ectopic expression of Pdx1 in human liver cells, we demonstrate that while glugacon and somatostatin expression initiates within a day, insulin gene expression becomes evident only 2–3 days later. To both increase transdifferentiation efficiency and analyze whether the process indeed display consecutive and hierarchical characteristics, adult human liver cells were treated by three pancreatic transcription factors, Pdx1, Pax4 and Mafa (3pTFs) that control distinct hierarchical stages of pancreatic development in the embryo. Ectopic expression of the 3pTFs in human liver cells, increased the transdifferentiation yield, manifested by 300% increase in the number of insulin positive cells, compared to each of the ectopic factors alone. However, only when the 3pTFs were sequentially supplemented one day apart from each other in a direct hierarchical manner, the transdifferentiated cells displayed increased mature β-cell-like characteristics. Ectopic expression of Pdx1 followed by Pax4 on the 2nd day and concluded by Mafa on the 3rd day resulted in increased yield of transdifferentiation that was associated by increased glucose regulated c-peptide secretion. By contrast, concerted or sequential administration of the ectopic 3pTFs in an indirect hierarchical mode resulted in the generation of insulin and somatostatin co-producing cells and diminished glucose regulated processed insulin secretion. In conclusion transcription factors induced liver to pancreas transdifferentiation is a progressive and hierarchical process. It is reasonable to assume that this characteristic is general to wide ranges of tissues. Therefore, our findings could facilitate the development of cell replacement therapy modalities for many degenerative diseases including diabetes.

Reprogramming of mice primary hepatocytes into insulin-producing cells by transfection with multicistronic vectors.

The neogenesis of insulin-producing cells (IPCs) from non-beta-cells has emerged as a potential method for treating diabetes mellitus (DM). Many groups have documented that activation of pancreatic transcription factor(s) in hepatocytes can improve the hyperglycemia in diabetic mice. In the present study, we explored a novel protocol that reprogrammed primary hepatocytes into functional IPCs by using multicistronic vectors carrying pancreatic and duodenal homeobox-1 (Pdx1), neurogenin 3 (Ngn3), and v-musculoaponeurotic fibrosarcoma oncogene homolog A (MafA). These triple-transfected cells activated multiple beta-cell genes, synthesized and stored considerable amounts of insulin, and released the hormone in a glucose-regulated manner in vitro. Furthermore, when transplanted into streptozotocin-induced diabetic mice, the cells markedly ameliorated glucose tolerance. Our results indicated that ectopic expression of Pdx1, Ngn3, and MafA facilitated hepatocytes-to-IPCs reprogramming. This approach may offer opportunities for treatment of DM.

Control of Cell Identity in Pancreas Development and Regeneration

The endocrine and exocrine cells in the adult pancreas are not static, but can change their differentiation state in response to injury or stress. This concept of cells in flux means that there may be ways to generate certain types of cells (such as insulin-producing β-cells) and prevent formation of others (such as transformed neoplastic cells). We review different aspects of cell identity in the pancreas, discussing how cells achieve their identity during embryonic development and maturation, and how this identity remains plastic, even in the adult pancreas.

Pdx1and controlled culture conditions induced differentiation of human amniotic fluid-derived stem cells to insulin-producing clusters

This study investigated the differentiation of human amniotic fluid-derived stem cells (hAFSCs) into insulin-producing clusters in vitro. Adenovirally-delivered mouse Pdx1 (Ad-Pdx1) induced human Pdx1 expression in hAFSCs and enhanced the coordinated expression of downstream β-cell markers. When Ad-Pdx1-transduced hAFSCs were sequentially treated with activin A, bFGF and nicotinamide and the culture plate surface coated with poly-l-ornithine, the expression of islet-associated human mRNAs for Pdx1, Pax6, Ngn3 and insulin was increased. C-peptide ELISA confirmed that Ad-Pdx1-transduced hAFSCs processed and secreted insulin in a manner consistent with that pathway in pancreatic β-cells. To sustain the β-cell-like phenotype and investigate the effect of three-dimensional (3D) conformation on the differentiation of hAFSCs, Pdx1-transduced cells were encapsulated in alginate and cultured long-term under serum-free conditions. Over 2 weeks, partially differentiated hAFSC clusters increased in size and increased insulin secretion. Taken together, these data demonstrate that ectopic Pdx1 expression initiates pancreatic differentiation in hAFSCs and that a β-cell-like phenotype can be augmented by culture conditions that mimic the stromal components and 3D geometry associated with pancreatic islets.

Ectopic PDX-1 Expression Directly Reprograms Human Keratinocytes along Pancreatic Insulin-Producing Cells Fate

BackgroundCellular differentiation and lineage commitment have previously been considered irreversible processes. However, recent studies have indicated that differentiated adult cells can be reprogrammed to pluripotency and, in some cases, directly into alternate committed lineages. However, although pluripotent cells can be induced in numerous somatic cell sources, it was thought that inducing alternate committed lineages is primarily only possible in cells of developmentally related tissues. Here, we challenge this view and analyze whether direct adult cell reprogramming to alternate committed lineages can cross the boundaries of distinct developmental germ layers.Methodology/Principal FindingsWe ectopically expressed non-integrating pancreatic differentiation factors in ectoderm-derived human keratinocytes to determine whether these factors could directly induce endoderm-derived pancreatic lineage and β-cell-like function. We found that PDX-1 and to a lesser extent other pancreatic transcription factors, could rapidly and specifically activate pancreatic lineage and β-cell-like functional characteristics in ectoderm-derived human keratinocytes. Human keratinocytes transdifferentiated along the β cell lineage produced processed and secreted insulin in response to elevated glucose concentrations. Using irreversible lineage tracing for KRT-5 promoter activity, we present supporting evidence that insulin-positive cells induced by ectopic PDX-1 expression are generated in ectoderm derived keratinocytes.Conclusions/SignificanceThese findings constitute the first demonstration of human ectoderm cells to endoderm derived pancreatic cells transdifferentiation. The study represents a proof of concept which suggests that transcription factors induced reprogramming is wider and more general developmental process than initially considered. These results expanded the arsenal of adult cells that can be used as a cell source for generating functional endocrine pancreatic cells. Directly reprogramming somatic cells into alternate desired tissues has important implications in developing patient-specific, regenerative medicine approaches.

Endoderm and mesoderm reciprocal signaling mediated by CXCL12 and CXCR4 regulates the migration of angioblasts and establishes the pancreatic fate

We have discovered that angioblasts trigger an early inductive event in pancreatic differentiation. This event occurs soon after gastrulation, before the formation of blood vessels. Morphological studies revealed that Lmo2-expressing angioblasts reside in proximity to the somitic mesoderm and the gut endoderm from which pancreatic progenitors arise. The chemokine ligand CXCL12 expressed in the gut endoderm functions to attract the angioblasts that express its receptor CXCR4. Angioblasts then signal back to the gut endoderm to induce Pdx1 expression. Gain-of-function and loss-of-function experiments for CXCL12 and CXCR4 were performed to test their function in blood vessel formation and pancreatic differentiation. The ectopic expression of Cxcl12 in the endoderm attracted the angioblasts and induced ectopic Pdx1 expression, resulting in an expanded pancreatic bud and an increased area of insulin-expressing cells. By contrast, in chick embryos treated with beads soaked in AMD3100, an inhibitor of CXCR4, the migration of angioblasts towards the Cxcl12-expressing gut endoderm was arrested, causing a malformation of blood vessels. This led to the generation of a smaller pancreatic bud and a reduced area of insulin-expressing cells. Taken together, these results indicate that the gut endoderm and angioblasts attract each other through reciprocal CXCL12 and CXCR4 signaling. This has a pivotal role in the fate establishment of the pancreatic progenitor cells and in the potentiation of further differentiation into endocrine β-cells.

NKX6.1 Promotes PDX-1-Induced Liver to Pancreatic β-Cells Reprogramming

Reprogramming adult mammalian cells is an attractive approach for generating cell-based therapies for degenerative diseases, such as diabetes. Adult human liver cells exhibit a high level of developmental plasticity and have been suggested as a potential source of pancreatic progenitor tissue. An instructive role for dominant pancreatic transcription factors in altering the hepatic developmental fate along the pancreatic lineage and function has been demonstrated. Here we analyze whether transcription factors expressed in mature pancreatic β-cells preferentially activate β-cell lineage differentiation in liver. NKX6.1 is a transcription factor uniquely expressed in β-cells of the adult pancreas, its potential role in reprogramming liver cells to pancreatic lineages has never been analyzed. Our results suggest that NKX6.1 activates immature pancreatic markers such as NGN-3 and ISL-1 but not pancreatic hormones gene expression in human liver cells. We hypothesized that its restricted capacity to activate a wide pancreatic repertoire in liver could be related to its incapacity to activate endogenous PDX-1 expression in liver cells. Indeed, the complementation of NKX6.1 by ectopic PDX-1 expression substantially and specifically promoted insulin expression and glucose regulated processed hormone secretion to a higher extent than that of PDX-1 alone, without increasing the reprogrammed cells. This may suggest a potential role for NKX6.1 in promoting PDX-1 reprogrammed cells maturation along the β-cell-like lineage. By contrast, NKX6.1 repressed PDX-1 induced proglucagon gene expression. The individual and concerted effects of pancreatic transcription factors in adult extra-pancreatic cells, is expected to facilitate developing regenerative medicine approaches for cell replacement therapy in diabetics.

Overexpression of the Pdx-1 Homeodomain Transcription Factor Impairs Glucose Metabolism in Cultured Rat Hepatocytes

The Pdx-1 transcription factor plays crucial functions both during pancreas development and in the adult β cells. Previous studies have indicated that ectopic Pdx-1 expression in liver or intestinal primary and immortalized cells is sufficient to promote activation of insulin gene expression. This work is focused on the molecular and physiological consequences of Pdx-1 overexpression in liver cells. We present evidence that Pdx-1 affects the level of expression of one of the four mammalian hexokinase isozymes. These are glucose phosphorylating enzymes involved in essential cellular functions such as glucose sensing, metabolic energy production and apoptosis. Specifically, our data show that over-expression of Pdx-1 in cultured hepatocytes is able to repress the expression of hexokinase 2 (Hxk 2) and the phenomenon is mediated via binding of Pdx-1 to a specific sequence on the Hxk 2 gene promoter. As a consequence, liver cells over-expressing Pdx-1 present interesting alterations concerning glucose metabolism.

Ectopic PDX-1 expression in liver ameliorates type 1 diabetes

Type 1 diabetes mellitus (T1DM) results from a specific autoimmune mediated destruction of the pancreatic β-cells. PDX-1 induced developmentally redirected liver cells were suggested to restore the ablated pancreatic function in chemically induced diabetes. However, developmentally redirected liver cells, may have acquired along with the desired β-cell characteristics and functions, also undesired sensitivity to autoimmune attack and therefore may be inefficient in ameliorating T1DM. This study analyzes whether subjects with β-cell autoimmunity could benefit from Ad-CMV-PDX-1 gene therapy. Using the model of cyclophosphamide-accelerated diabetes in non-obese diabetic (CAD-NOD) mice, we report that recombinant adenovirus mediated PDX-1 gene therapy, ameliorates hyperglycemia in CAD-NOD mice. Our data demonstrate that 43% of the overtly diabetic CAD-NOD mice treated with Ad-CMV-PDX-1 became normoglycemic and maintained a stable body weight. Ectopic PDX-1 expression induced pancreatic gene expression and insulin production in the mice livers. The amelioration of hyperglycemia, in PDX-1 treated diabetic mice was associated with an immune modulation manifested by Th1 to Th2 shift in the autoimmune T-cell response to antigens associated with NOD diabetes. Thus, liver-to-pancreas transdifferentiation ameliorates T1DM in a process which is associated with a concomitant modulation of the autoimmune attack. Our findings suggest a beneficial therapeutic effect of the PDX-1 gene therapy for treating autoimmune type 1 diabetes mellitus (T1DM).

Forced expression of PDX-1 induces insulin production in intestinal epithelia

In the developmental stage, pancreas derives from the endodermal cells where the transcription factor, pancreatic duodenal homeobox gene-1 (pdx-1) is expressed. In adulthood, pdx-1 expression is localized to pancreatic beta cells, which is necessary for maintenance of beta cell function. Recently, ectopic expression of pdx-1 in the liver successfully induced insulin production and ameliorated hyperglycemia. Our study was designed to investigate the effects of forced expression of pdx-1 in ileal epithelia by adenovirus-mediated gene transfer. The recombinant, replication-deficient adenovirus carrying the pdx-1 gene was constructed using the COS-TPC method. ICR mice were treated with intraperitoneal injection of 220 mg/kg streptozotocin (STZ). After determining the hyperglycemia, a loop of ileum was constructed and the adenovirus solutions (Ad-pdx-1 and Ad-lacZ 1 x 10(8) PFU/body) were injected into the lumen of the ileal loop. In this model, immunohistochemical or fluorescent analyses of PDX-1 and insulin in the adenovirus-infected ileal epithelia were carried out. Reverse transcription polymerase chain reaction of pdx-1 and other pancreatic markers were investigated. Blood glucose concentrations were measured by drawing blood from ocular veins. Immunoreactive insulin extracted from the adenovirus-infected ileum was measured. Ad-pdx-1 induced ectopic PDX-1 expression in the ileum. The PDX-1 positive cells in the ileal epithelia were positive for insulin; mRNA of insulin-1, insulin-2 and pdx-1 were expressed in mice infected with Ad-pdx-1. Hyperglycemia was improved in STZ-treated mice infected with Ad-pdx-1. Immunoreactive insulin in the ileum extract was increased significantly in mice with Ad-pdx-1. Gene transfer of PDX-1 in intestinal epithelia could be a promising strategy for diabetes mellitus by inducing ectopic insulin producing cells.

Combined ectopic expression of Pdx1 and Ptf1a/p48 results in the stable conversion of posterior endoderm into endocrine and exocrine pancreatic tissue

Patterning of the embryonic endoderm into distinct sets of precursor cells involves the precisely regulated activities of key transcription regulators. Ectopic, pan-endodermal activation of XPtf1a/p48 during pancreas precursor cell stages of Xenopus embryogenesis results in an expansion of the pancreatic territory, precisely within the borders of XlHbox8 expression. A combination of both activities is sufficient to expand the pancreatic precursor cell population also into more posterior portions of the endoderm. Both treatments result in the formation of a giant pancreas that persists up to late tadpole stages of development and carries both supernumerary endocrine and exocrine cells. A combination of XPtf1a/p48 and XlHbox8 is thus sufficient to convert nonpancreatic endodermal cells into pancreatic precursor cells.

Ectopically expressed PDX-1 in liver initiates endocrine and exocrine pancreas differentiation but causes dysmorphogenesis

To date, the potency of pancreatic and duodenal homeobox gene 1 (PDX-1) in inducing differentiation into insulin-producing cells has been demonstrated in some cells and tissues. In order to carry out efficient screening of somatic tissues and cells that can transdifferentiate into β-cell-like cells in response to PDX-1, we generated CAG-CAT-PDX1 transgenic mice carrying a transgene cassette composed of the chicken β-actin gene (CAG) promoter and a floxed stuffer DNA sequence (CAT) linked to PDX-1 cDNA. When the mice were crossed with Alb-Cre mice, which express the Cre recombinase driven by the rat albumin gene promoter, PDX-1 was expressed in more than 50% of hepatocytes and cholangiocytes. The PDX-1 (+) livers expressed a variety of endocrine hormone genes such as insulin, glucagon, somatostatin, and pancreatic polypeptide. In addition, they expressed exocrine genes such as elastase-1 and chymotrypsinogen 1B. However, the mice exhibited marked jaundice due to conjugated hyperbilirubinemia, and the liver tissue displayed abnormal lobe structures and multiple cystic lesions. Thus, the in vivo ectopic expression of PDX-1 in albumin-producing cells was able to initiate but not complete the differentiation of liver cells into pancreatic cells. The conditional PDX-1 transgenic mouse system developed in this study appeared to be useful for efficient screening of PDX-1 responsive somatic tissues and cells.

Beta-cell neogenesis induced by adenovirus-mediated gene delivery of transcription factor pdx-1 into mouse pancreas.

beta-cell neogenesis is expected to provide a new therapy for diabetes. Numerous studies have demonstrated that transcriptional regulation involving pdx-1 is essential for endocrine neogenesis in vivo and in vitro. Therefore, it is possible that ectopic expression of pdx-1 in the pancreas could induce endocrine neogenesis. To test this possibility, we performed safe and efficient gene delivery of the pdx-1 gene into the mouse pancreas through the common bile duct using adenoviral vectors, and examined the effects of the ectopic expression of pdx-1. Here we show that adenovirus-mediated expression of pdx-1 can activate the endogenous pdx-1 gene, leading to beta-cell neogenesis and ductal proliferation. This technique is similar to the endoscopic retrograde cholangiopancreatography, which has been already established as a safe procedure for humans. Thus, beta-cell neogenesis induced by adenovirus-mediated expression of pdx-1 provides a novel strategy for gene therapy for a cure for diabetes mellitus.

Key events of pancreas formation are triggered in gut endoderm by ectopic expression of pancreatic regulatory genes.

The mechanisms by which the epithelium of the digestive tract and its associated glands are specified are largely unknown. One clue is that several transcription factors are expressed in specific regions of the endoderm prior to and during organogenesis. Pdx-1, for example, is expressed in the duodenum and pancreas and Pdx-1 inactivation results in an arrest of pancreatic development after buds formation. Similarly, ngn3 is transiently expressed in the developing pancreas and a knockout results in the absence of endocrine cells. This paper focuses on the question of whether these and other transcription factors, known to be necessary for pancreatic development, are also sufficient to drive a program of pancreatic organogenesis. Using in ovo electroporation of chick embryos, we show that ectopic expression of Pdx-1 or ngn3 causes cells to bud out of the epithelium like pancreatic progenitors. The Pdx-1-expressing cells extinguish markers for other nonpancreatic regions of the endoderm and initiate, but do not complete, pancreatic cytodifferentiation. Ectopic expression of ngn3 is sufficient to turn endodermal cells of any region into endocrine cells that form islets expressing glucagon and somatostatin in the mesenchyme. The results suggest that simple gene combinations could be used in stem cells to achieve specific endodermal tissue differentiation.