Limits...
Live imaging and gene expression analysis in zebrafish identifies a link between neutrophils and epithelial to mesenchymal transition.

Freisinger CM, Huttenlocher A - PLoS ONE (2014)

Bottom Line: Chronic inflammation is associated with epithelial to mesenchymal transition (EMT) and cancer progression however the relationship between inflammation and EMT remains unclear.Live imaging revealed that expression of HRas(V12) in the epidermis results in EMT and chronic neutrophil and macrophage infiltration.Surprisingly, we also found a cell autonomous role for Cxcr2 signaling in transformed cells for both neutrophil recruitment and EMT related gene expression associated with Ras transformation.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pediatrics and Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.

ABSTRACT
Chronic inflammation is associated with epithelial to mesenchymal transition (EMT) and cancer progression however the relationship between inflammation and EMT remains unclear. Here, we have exploited zebrafish to visualize and quantify the earliest events during epithelial cell transformation induced by oncogenic HRas(V12). Live imaging revealed that expression of HRas(V12) in the epidermis results in EMT and chronic neutrophil and macrophage infiltration. We have developed an in vivo system to probe and quantify gene expression changes specifically in transformed cells from chimeric zebrafish expressing oncogenic HRas(V12) using translating ribosomal affinity purification (TRAP). We found that the expression of genes associated with EMT, including slug, vimentin and mmp9, are enriched in HRas(V12) transformed epithelial cells and that this enrichment requires the presence of neutrophils. An early signal induced by HRas(V12) in epithelial cells is the expression of il-8 (cxcl8) and we found that the chemokine receptor, Cxcr2, mediates neutrophil but not macrophage recruitment to the transformed cells. Surprisingly, we also found a cell autonomous role for Cxcr2 signaling in transformed cells for both neutrophil recruitment and EMT related gene expression associated with Ras transformation. Taken together, these findings implicate both autocrine and paracrine signaling through Cxcr2 in the regulation of inflammation and gene expression in transformed epithelial cells.

Show MeSH

Related in: MedlinePlus

Cxcr2 signaling in HRasV12 transformed epithelial cells is required for neutrophil recruitment and EMT related gene expression.(A) For analysis of tissue specific Cxcr2 expression TRAP was performed on 3.5 dpf transgenic krt4-EGFP-L10a and mpx-EGFP-L10a larvae and one-step RT-PCR was performed. cxcr2 expression is observed in the epidermis and in neutrophils. mpx expression is only observed in neutrophils supporting that there is not neutrophil contamination in the epidermal samples. (B) Schematic diagram to illustrate the cell transplantation used to generate chimeric HRasV12 expressing larvae in which the transformed cells express either control MO or Cxcr2 MO. (C–D) Fluorescent Z stack projections of live 3.5 dpf of transgenic mpx:GFP (green neutrophils) larvae with control MO in the HRasV12 expressing cells (C) or with cxcr2 MO within the HRasV12 expressing cells (D). (E) Quantification of C–D (as a ratio of neutrophils per transformed cell) shows a statistically significant decrease in neutrophil recruitment to HRasV12 expressing cells that have cxcr2 MO compared to HRasV12 expressing cells that have control MO. (F) Quantitative RT-PCR (one representative graph shown n = 3) indicates a statistically significant decrease in slug, vimentin and mmp9 transcripts in HRasV12 expressing cells that have cxcr2 MO compared to HRasV12 expressing cells with control MO. (G) Schematic illustrating the requirement for Cxcr2 in neutrophils for initial neutrophil recruitment to transformed cells as well as a cell autonomous function of Cxcr2 in transformed cells to mediate changes associated with EMT. * = P<.05, ** = P<.01, *** = P<.001. Scale bar = 20 microns.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4221567&req=5

pone-0112183-g006: Cxcr2 signaling in HRasV12 transformed epithelial cells is required for neutrophil recruitment and EMT related gene expression.(A) For analysis of tissue specific Cxcr2 expression TRAP was performed on 3.5 dpf transgenic krt4-EGFP-L10a and mpx-EGFP-L10a larvae and one-step RT-PCR was performed. cxcr2 expression is observed in the epidermis and in neutrophils. mpx expression is only observed in neutrophils supporting that there is not neutrophil contamination in the epidermal samples. (B) Schematic diagram to illustrate the cell transplantation used to generate chimeric HRasV12 expressing larvae in which the transformed cells express either control MO or Cxcr2 MO. (C–D) Fluorescent Z stack projections of live 3.5 dpf of transgenic mpx:GFP (green neutrophils) larvae with control MO in the HRasV12 expressing cells (C) or with cxcr2 MO within the HRasV12 expressing cells (D). (E) Quantification of C–D (as a ratio of neutrophils per transformed cell) shows a statistically significant decrease in neutrophil recruitment to HRasV12 expressing cells that have cxcr2 MO compared to HRasV12 expressing cells that have control MO. (F) Quantitative RT-PCR (one representative graph shown n = 3) indicates a statistically significant decrease in slug, vimentin and mmp9 transcripts in HRasV12 expressing cells that have cxcr2 MO compared to HRasV12 expressing cells with control MO. (G) Schematic illustrating the requirement for Cxcr2 in neutrophils for initial neutrophil recruitment to transformed cells as well as a cell autonomous function of Cxcr2 in transformed cells to mediate changes associated with EMT. * = P<.05, ** = P<.01, *** = P<.001. Scale bar = 20 microns.

Mentions: Previous studies have shown that Cxcr2 expression is necessary for neutrophil recruitment to exogenous Cxcl8, indicating that neutrophil Cxcr2 mediates chemotaxis to Cxcl8 in vivo. It is possible that Cxcr2 mediates EMT gene expression in transformed epithelial cells indirectly through its effects on neutrophil recruitment. However, Cxcr2 is also expressed in tumor cells, suggesting that Cxcr2 may play cell autonomous roles in epithelial cells. Indeed, we found that zebrafish epithelial cells express cxcr2 (Figure 6A), suggesting that Cxcr2 in epithelial cells may affect gene expression changes induced by cell transformation independent of the effects of Cxcr2 in neutrophils. To determine if there is a cell autonomous role for Cxcr2 in transformed epithelial cells we utilized a cell transplantation strategy to deplete Cxcr2 specifically in transformed cells without altering Cxcr2 signaling in neutrophils (Figure 6B). Cells from embryos expressing RFP-HRasV12 in cxcr2 morphants or control morphants were transplanted into Tg(mpx:GFP) embryos. Interestingly, we found a significant decrease in neutrophil recruitment toward Cxcr2-deficient HRasV12 expressing cells even though the neutrophils expressed Cxcr2 (Figure 6D and E), suggesting that Cxcr2 signaling in transformed cells is necessary for neutrophil recruitment. To determine if Cxcr2-deficient epithelial cells that express HRasV12 induce EMT associated genes, we tested the expression of vimentin, mmp9 and slug in the Cxcr2-deficient and control transformed epithelial cells. Surprisingly, expression of vimentin, slug and mmp9 were reduced in Cxcr2-deficient transformed epithelial cells compared to control (Figure 6F); indicating a cell autonomous role for Cxcr2 signaling in epithelial cells in the induction of EMT related gene expression.


Live imaging and gene expression analysis in zebrafish identifies a link between neutrophils and epithelial to mesenchymal transition.

Freisinger CM, Huttenlocher A - PLoS ONE (2014)

Cxcr2 signaling in HRasV12 transformed epithelial cells is required for neutrophil recruitment and EMT related gene expression.(A) For analysis of tissue specific Cxcr2 expression TRAP was performed on 3.5 dpf transgenic krt4-EGFP-L10a and mpx-EGFP-L10a larvae and one-step RT-PCR was performed. cxcr2 expression is observed in the epidermis and in neutrophils. mpx expression is only observed in neutrophils supporting that there is not neutrophil contamination in the epidermal samples. (B) Schematic diagram to illustrate the cell transplantation used to generate chimeric HRasV12 expressing larvae in which the transformed cells express either control MO or Cxcr2 MO. (C–D) Fluorescent Z stack projections of live 3.5 dpf of transgenic mpx:GFP (green neutrophils) larvae with control MO in the HRasV12 expressing cells (C) or with cxcr2 MO within the HRasV12 expressing cells (D). (E) Quantification of C–D (as a ratio of neutrophils per transformed cell) shows a statistically significant decrease in neutrophil recruitment to HRasV12 expressing cells that have cxcr2 MO compared to HRasV12 expressing cells that have control MO. (F) Quantitative RT-PCR (one representative graph shown n = 3) indicates a statistically significant decrease in slug, vimentin and mmp9 transcripts in HRasV12 expressing cells that have cxcr2 MO compared to HRasV12 expressing cells with control MO. (G) Schematic illustrating the requirement for Cxcr2 in neutrophils for initial neutrophil recruitment to transformed cells as well as a cell autonomous function of Cxcr2 in transformed cells to mediate changes associated with EMT. * = P<.05, ** = P<.01, *** = P<.001. Scale bar = 20 microns.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4221567&req=5

pone-0112183-g006: Cxcr2 signaling in HRasV12 transformed epithelial cells is required for neutrophil recruitment and EMT related gene expression.(A) For analysis of tissue specific Cxcr2 expression TRAP was performed on 3.5 dpf transgenic krt4-EGFP-L10a and mpx-EGFP-L10a larvae and one-step RT-PCR was performed. cxcr2 expression is observed in the epidermis and in neutrophils. mpx expression is only observed in neutrophils supporting that there is not neutrophil contamination in the epidermal samples. (B) Schematic diagram to illustrate the cell transplantation used to generate chimeric HRasV12 expressing larvae in which the transformed cells express either control MO or Cxcr2 MO. (C–D) Fluorescent Z stack projections of live 3.5 dpf of transgenic mpx:GFP (green neutrophils) larvae with control MO in the HRasV12 expressing cells (C) or with cxcr2 MO within the HRasV12 expressing cells (D). (E) Quantification of C–D (as a ratio of neutrophils per transformed cell) shows a statistically significant decrease in neutrophil recruitment to HRasV12 expressing cells that have cxcr2 MO compared to HRasV12 expressing cells that have control MO. (F) Quantitative RT-PCR (one representative graph shown n = 3) indicates a statistically significant decrease in slug, vimentin and mmp9 transcripts in HRasV12 expressing cells that have cxcr2 MO compared to HRasV12 expressing cells with control MO. (G) Schematic illustrating the requirement for Cxcr2 in neutrophils for initial neutrophil recruitment to transformed cells as well as a cell autonomous function of Cxcr2 in transformed cells to mediate changes associated with EMT. * = P<.05, ** = P<.01, *** = P<.001. Scale bar = 20 microns.
Mentions: Previous studies have shown that Cxcr2 expression is necessary for neutrophil recruitment to exogenous Cxcl8, indicating that neutrophil Cxcr2 mediates chemotaxis to Cxcl8 in vivo. It is possible that Cxcr2 mediates EMT gene expression in transformed epithelial cells indirectly through its effects on neutrophil recruitment. However, Cxcr2 is also expressed in tumor cells, suggesting that Cxcr2 may play cell autonomous roles in epithelial cells. Indeed, we found that zebrafish epithelial cells express cxcr2 (Figure 6A), suggesting that Cxcr2 in epithelial cells may affect gene expression changes induced by cell transformation independent of the effects of Cxcr2 in neutrophils. To determine if there is a cell autonomous role for Cxcr2 in transformed epithelial cells we utilized a cell transplantation strategy to deplete Cxcr2 specifically in transformed cells without altering Cxcr2 signaling in neutrophils (Figure 6B). Cells from embryos expressing RFP-HRasV12 in cxcr2 morphants or control morphants were transplanted into Tg(mpx:GFP) embryos. Interestingly, we found a significant decrease in neutrophil recruitment toward Cxcr2-deficient HRasV12 expressing cells even though the neutrophils expressed Cxcr2 (Figure 6D and E), suggesting that Cxcr2 signaling in transformed cells is necessary for neutrophil recruitment. To determine if Cxcr2-deficient epithelial cells that express HRasV12 induce EMT associated genes, we tested the expression of vimentin, mmp9 and slug in the Cxcr2-deficient and control transformed epithelial cells. Surprisingly, expression of vimentin, slug and mmp9 were reduced in Cxcr2-deficient transformed epithelial cells compared to control (Figure 6F); indicating a cell autonomous role for Cxcr2 signaling in epithelial cells in the induction of EMT related gene expression.

Bottom Line: Chronic inflammation is associated with epithelial to mesenchymal transition (EMT) and cancer progression however the relationship between inflammation and EMT remains unclear.Live imaging revealed that expression of HRas(V12) in the epidermis results in EMT and chronic neutrophil and macrophage infiltration.Surprisingly, we also found a cell autonomous role for Cxcr2 signaling in transformed cells for both neutrophil recruitment and EMT related gene expression associated with Ras transformation.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pediatrics and Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.

ABSTRACT
Chronic inflammation is associated with epithelial to mesenchymal transition (EMT) and cancer progression however the relationship between inflammation and EMT remains unclear. Here, we have exploited zebrafish to visualize and quantify the earliest events during epithelial cell transformation induced by oncogenic HRas(V12). Live imaging revealed that expression of HRas(V12) in the epidermis results in EMT and chronic neutrophil and macrophage infiltration. We have developed an in vivo system to probe and quantify gene expression changes specifically in transformed cells from chimeric zebrafish expressing oncogenic HRas(V12) using translating ribosomal affinity purification (TRAP). We found that the expression of genes associated with EMT, including slug, vimentin and mmp9, are enriched in HRas(V12) transformed epithelial cells and that this enrichment requires the presence of neutrophils. An early signal induced by HRas(V12) in epithelial cells is the expression of il-8 (cxcl8) and we found that the chemokine receptor, Cxcr2, mediates neutrophil but not macrophage recruitment to the transformed cells. Surprisingly, we also found a cell autonomous role for Cxcr2 signaling in transformed cells for both neutrophil recruitment and EMT related gene expression associated with Ras transformation. Taken together, these findings implicate both autocrine and paracrine signaling through Cxcr2 in the regulation of inflammation and gene expression in transformed epithelial cells.

Show MeSH
Related in: MedlinePlus