TGF-β regulation of gene expression at early and late stages of HPV16-mediated transformation of human keratinocytes

Abstract

In our in vitro model for HPV16-mediated transformation, HPV16-immortalized human keratinocytes (HKc/HPV16) give rise to differentiation resistant, premalignant cells (HKc/DR). HKc/DR, but not HKc/HPV16, are resistant to growth inhibition by transforming growth factor beta (TGF-β), due to a partial loss of TGF-β receptor type I. We show that TGF-β activates a Smad-responsive reporter construct in HKc/DR to about 50% of the maximum levels of activation observed in HKc/HPV16. To investigate the functional significance of residual TGF-β signaling in HKc/DR, we compared gene expression profiles elicited by TGF-β treatment of HKc/HPV16 and HKc/DR on Agilent 44k human whole genome microarrays. TGF-β altered the expression of cell cycle and MAP kinase pathway genes in HKc/HPV16, but not in HKc/DR. However, epithelial-mesenchymal transition (EMT) responses to TGF-β were comparable in HKc/HPV16 and HKc/DR, indicating that the signaling pathways through which TGF-β elicits growth inhibition diverge from those that induce EMT in HPV16-transformed cells.

Keywords: EMT; HPV; Human keratinocytes; Ski; Smad; TGF-beta.

Figure 1. TGF-β activation of a Smad-responsive luciferase reporter construct in HKc/HPV16 and HKc/DR

Four HKc/HPV16 and their corresponding HKc/DR lines (d-1, d-2, d-4 and d-5) were transiently transfected in triplicate wells per experimental condition with the p6SBE-Luc reporter construct and pRL-SV40. Cells were treated without or with the indicated concentrations of TGF-β1 24 h after transfection, and harvested for dual luciferase assay after 22 h of TGF-β treatment. Firefly luciferase values, measured in Relative Light Units (RLU), were normalized against Renilla luciferase values, and the results expressed as fold induction over control (without TGF-β).

Figure 2. Experimental design and initial analysis of microarray experiments

(A) Experimental design: Four separate HKc/HPV16 lines and their respective HKc/DR counterparts were treated without or with 40 pM TGF-β for 48 h before harvesting. Labeled RNA samples, using a dye-swap design, were hybridized to Agilent 4x44k whole human genome microarrays for each of the HKc/HPV16 and HKc/DR lines, resulting in eight microarrays per transformation stage of the model system. RNA amplification, hybridization, washing and scanning for each individual donor (HKc/HPV16 and HKc/DR) were performed at the same time on separate dates. (B) Scatter plot of gene expression levels detected by microarray analysis described in A. Red: genes up-regulated by ≥ 2- fold; green: genes down-regulated by ≥ 2- fold; gray: genes exhibiting no statistically significant change, or < 2-fold change in expression between TGF-β treated and control cells. (C) Numbers of genes significantly changed in HKc/HPV16 and HKc/DR as a consequence of TGF-β treatment. (D) Venn diagrams showing how many genes are up- or down-regulated (≥ 2 fold) in all four HKc/HPV16 and HKc/DR lines after TGF-β treatment, and how many of the TGF-β regulated genes are common between HKc/HPV16 and HKc/DR. red: ≥ 2-fold up-regulated, green ≥ 2-fold down-regulated.

Figure 2. Experimental design and initial analysis of microarray experiments

(A) Experimental design: Four separate HKc/HPV16 lines and their respective HKc/DR counterparts were treated without or with 40 pM TGF-β for 48 h before harvesting. Labeled RNA samples, using a dye-swap design, were hybridized to Agilent 4x44k whole human genome microarrays for each of the HKc/HPV16 and HKc/DR lines, resulting in eight microarrays per transformation stage of the model system. RNA amplification, hybridization, washing and scanning for each individual donor (HKc/HPV16 and HKc/DR) were performed at the same time on separate dates. (B) Scatter plot of gene expression levels detected by microarray analysis described in A. Red: genes up-regulated by ≥ 2- fold; green: genes down-regulated by ≥ 2- fold; gray: genes exhibiting no statistically significant change, or < 2-fold change in expression between TGF-β treated and control cells. (C) Numbers of genes significantly changed in HKc/HPV16 and HKc/DR as a consequence of TGF-β treatment. (D) Venn diagrams showing how many genes are up- or down-regulated (≥ 2 fold) in all four HKc/HPV16 and HKc/DR lines after TGF-β treatment, and how many of the TGF-β regulated genes are common between HKc/HPV16 and HKc/DR. red: ≥ 2-fold up-regulated, green ≥ 2-fold down-regulated.

Figure 2. Experimental design and initial analysis of microarray experiments

(A) Experimental design: Four separate HKc/HPV16 lines and their respective HKc/DR counterparts were treated without or with 40 pM TGF-β for 48 h before harvesting. Labeled RNA samples, using a dye-swap design, were hybridized to Agilent 4x44k whole human genome microarrays for each of the HKc/HPV16 and HKc/DR lines, resulting in eight microarrays per transformation stage of the model system. RNA amplification, hybridization, washing and scanning for each individual donor (HKc/HPV16 and HKc/DR) were performed at the same time on separate dates. (B) Scatter plot of gene expression levels detected by microarray analysis described in A. Red: genes up-regulated by ≥ 2- fold; green: genes down-regulated by ≥ 2- fold; gray: genes exhibiting no statistically significant change, or < 2-fold change in expression between TGF-β treated and control cells. (C) Numbers of genes significantly changed in HKc/HPV16 and HKc/DR as a consequence of TGF-β treatment. (D) Venn diagrams showing how many genes are up- or down-regulated (≥ 2 fold) in all four HKc/HPV16 and HKc/DR lines after TGF-β treatment, and how many of the TGF-β regulated genes are common between HKc/HPV16 and HKc/DR. red: ≥ 2-fold up-regulated, green ≥ 2-fold down-regulated.

Figure 3. RT/PCR validation of microarray results

Fold-change induced by TGF-β of the expression of a panel of genes, determined by microarray analysis (open bars) and RT-PCR (solid bars) in HKc/HPV16 (A) and HKc/DR (B).

Figure 3. RT/PCR validation of microarray results

Fold-change induced by TGF-β of the expression of a panel of genes, determined by microarray analysis (open bars) and RT-PCR (solid bars) in HKc/HPV16 (A) and HKc/DR (B).

Figure 4. KEGG pathway analysis of differentially expressed genes

(A) Summary of KEGG analysis results for genes belonging to the cell cycle, MAPK, focal adhesion/ECM-receptor interaction and cell communication pathways found changed by TGF-β treatment in HKc/HPV16 (upper panel) and HKc/DR (lower panel). Red: ≥ 2-fold up-regulated; green β 2-fold down-regulated by TGF-β. (B, C) Gene ontology analysis of differentially expressed genes as determined using the GeneSifter software. The y-axis represents the percentage of genes changed up or down, within each group of genes (in each cellular process) that changed in response to 48 h of TGF-β treatment (40 pM) in HKc/HPV16 (B) and HKc/DR (C) by β 2-fold. Data are grouped into different ontology groups (x-axis) and each is further separated into increased (red bars) and decreased (green bars) expression.

Figure 4. KEGG pathway analysis of differentially expressed genes

(A) Summary of KEGG analysis results for genes belonging to the cell cycle, MAPK, focal adhesion/ECM-receptor interaction and cell communication pathways found changed by TGF-β treatment in HKc/HPV16 (upper panel) and HKc/DR (lower panel). Red: ≥ 2-fold up-regulated; green β 2-fold down-regulated by TGF-β. (B, C) Gene ontology analysis of differentially expressed genes as determined using the GeneSifter software. The y-axis represents the percentage of genes changed up or down, within each group of genes (in each cellular process) that changed in response to 48 h of TGF-β treatment (40 pM) in HKc/HPV16 (B) and HKc/DR (C) by β 2-fold. Data are grouped into different ontology groups (x-axis) and each is further separated into increased (red bars) and decreased (green bars) expression.

Figure 5. TGF-β decreases E-cadherin and increases fibronectin protein levels in HKc/HPV16 and HKc/DR

Whole-protein lysates were prepared from HKc/HPV16 and HKc/DR treated without or with TGF-β (40 pM) for 48 h (A) or for the indicated times (B) and Western blot analysis was performed for E-cadherin (A), fibronectin (B) or tubulin (A, B) as a loading control. (C) Control and TGF-β treated (40 pM for 96 h) HKc/HPV16 and HKc/DR were fixed in 4% paraformaldehyde. Slides were stained with DAPI to visualize nuclei (blue; panels a, d, g, and j) and with anti-fibronectin antibody (FN1) (red; panels b, e, h, and k). Merged images are presented in panels c, f, i, and l.

Figure 5. TGF-β decreases E-cadherin and increases fibronectin protein levels in HKc/HPV16 and HKc/DR

Whole-protein lysates were prepared from HKc/HPV16 and HKc/DR treated without or with TGF-β (40 pM) for 48 h (A) or for the indicated times (B) and Western blot analysis was performed for E-cadherin (A), fibronectin (B) or tubulin (A, B) as a loading control. (C) Control and TGF-β treated (40 pM for 96 h) HKc/HPV16 and HKc/DR were fixed in 4% paraformaldehyde. Slides were stained with DAPI to visualize nuclei (blue; panels a, d, g, and j) and with anti-fibronectin antibody (FN1) (red; panels b, e, h, and k). Merged images are presented in panels c, f, i, and l.

Figure 6. TGF-β induces morphological changes, re-organizes actin cytoskeleton, and enhances cell migration in HKc/HPV16 and HKc/DR

(A) Cells were treated with or without TGF-β (40 pM for 4 days) and examined under phase contrast using a Zeiss Axiovert 200 inverted microscope. White arrows show projections protruding out from the cell surface of HKc/HPV16 and HKc/DR. (B) F-actin was stained with Alexa fluor-488 labeled Phalloidin and nuclei with DAPI in HKc/HPV16 and HKc/DR, which were then visualized under a confocal microscope. F-actin, green: panels a, d, g and j; nuclei, blue: panels b, e, h and k. Merged images are presented in panels c, f, i, and l. (C) Quantification of scratch assay. HKc/HPV16 and HKc/DR were grown to confluence and treated with or without 40 pM TGF-β1 for 12 h. Wounds were then incised by scratching the cell monolayer with a pipet tip, and incubation with or without TGF-β1 continued for 36 h. Images were captured under phase-contrast microscopy at 6 h intervals after the incision and quantified with ImageJ. At least ten measurements of the width of the gap were assessed for each time point, starting at the 6 h time point and continuing until 36 h after the scratch.

Figure 6. TGF-β induces morphological changes, re-organizes actin cytoskeleton, and enhances cell migration in HKc/HPV16 and HKc/DR

(A) Cells were treated with or without TGF-β (40 pM for 4 days) and examined under phase contrast using a Zeiss Axiovert 200 inverted microscope. White arrows show projections protruding out from the cell surface of HKc/HPV16 and HKc/DR. (B) F-actin was stained with Alexa fluor-488 labeled Phalloidin and nuclei with DAPI in HKc/HPV16 and HKc/DR, which were then visualized under a confocal microscope. F-actin, green: panels a, d, g and j; nuclei, blue: panels b, e, h and k. Merged images are presented in panels c, f, i, and l. (C) Quantification of scratch assay. HKc/HPV16 and HKc/DR were grown to confluence and treated with or without 40 pM TGF-β1 for 12 h. Wounds were then incised by scratching the cell monolayer with a pipet tip, and incubation with or without TGF-β1 continued for 36 h. Images were captured under phase-contrast microscopy at 6 h intervals after the incision and quantified with ImageJ. At least ten measurements of the width of the gap were assessed for each time point, starting at the 6 h time point and continuing until 36 h after the scratch.

Figure 6. TGF-β induces morphological changes, re-organizes actin cytoskeleton, and enhances cell migration in HKc/HPV16 and HKc/DR

(A) Cells were treated with or without TGF-β (40 pM for 4 days) and examined under phase contrast using a Zeiss Axiovert 200 inverted microscope. White arrows show projections protruding out from the cell surface of HKc/HPV16 and HKc/DR. (B) F-actin was stained with Alexa fluor-488 labeled Phalloidin and nuclei with DAPI in HKc/HPV16 and HKc/DR, which were then visualized under a confocal microscope. F-actin, green: panels a, d, g and j; nuclei, blue: panels b, e, h and k. Merged images are presented in panels c, f, i, and l. (C) Quantification of scratch assay. HKc/HPV16 and HKc/DR were grown to confluence and treated with or without 40 pM TGF-β1 for 12 h. Wounds were then incised by scratching the cell monolayer with a pipet tip, and incubation with or without TGF-β1 continued for 36 h. Images were captured under phase-contrast microscopy at 6 h intervals after the incision and quantified with ImageJ. At least ten measurements of the width of the gap were assessed for each time point, starting at the 6 h time point and continuing until 36 h after the scratch.