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. 2012 Sep;132(9):2148-57.
doi: 10.1038/jid.2012.78. Epub 2012 May 17.

Heparin-binding EGF-like growth factor promotes epithelial-mesenchymal transition in human keratinocytes

Stefan W Stoll  1 , Laure Rittié, Jessica L Johnson, James T Elder
Affiliations

Heparin-binding EGF-like growth factor promotes epithelial-mesenchymal transition in human keratinocytes

Stefan W Stoll et al. J Invest Dermatol. 2012 Sep.

Abstract

We have shown that autocrine proliferation of human keratinocytes (KCs) is strongly dependent upon amphiregulin (AREG), whereas blockade of heparin-binding EGF-like growth factor (HB-EGF) inhibits KC migration in scratch wound assays. Here we demonstrate that expression of soluble HB-EGF (sHB-EGF) or full-length transmembrane HB-EGF (proHB-EGF), but not proAREG, results in profound increases in KC migration and invasiveness in monolayer culture. Coincident with these changes, HB-EGF significantly decreases mRNA expression of several epithelial markers including keratins 1, 5, 10, and 14 while increasing expression of markers of cellular motility including SNAI1, ZEB1, COX-2, and MMP1. Immunostaining revealed HB-EGF-induced expression of the mesenchymal protein vimentin and decreased expression of E-cadherin, as well as nuclear translocation of β-catenin. Suggestive of a trade-off between KC motility and proliferation, overexpression of HB-EGF also reduced KC growth by >90%. We also show that HB-EGF is strongly induced in regenerating epidermis after partial-thickness wounding of human skin. Taken together, our data suggest that expression of HB-EGF in human KCs triggers a migratory and invasive phenotype with many features of epithelial-mesenchymal transition (EMT), which may be beneficial in the context of cutaneous wound healing.

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Conflict of interest statement

CONFLICT OF INTEREST:

The authors state no conflict of interest.

Figures

Figure 1
Figure 1
HB-EGF overexpression strongly changes KC morphology. (A) N/TERT with inducible expression of HB-EGF were grown to approx. 40% confluence and incubated for 60 h +/− TET. Normalized HB-EGF mRNA levels (* =p< 0.05, n= 3–6) and protein shedding (* =p< 0.0005, n= 3) into CM were analyzed by QRT-PCR and ELISA, respectively. (B) N/TERT with and w/o constitutive expression of proHB-EGF or proAREG. Treatment with rhHB-EGF or EGF was for 72 h. (C) N/TERT were exposed to CM from N/TERT or cells expressing His-tagged sHB-EGF for 72h with and without prior depletion of sHB-EGF using nickel affinity chromatography. (D) N/TERT with inducible expression of sHB-EGF or proHB-EGF were incubated +/− TET with and w/o 10 µg/ml HB-EGF Abs, 1 µM PD158780 or 40 µM GM6001 for 1–3 days.
Figure 1
Figure 1
HB-EGF overexpression strongly changes KC morphology. (A) N/TERT with inducible expression of HB-EGF were grown to approx. 40% confluence and incubated for 60 h +/− TET. Normalized HB-EGF mRNA levels (* =p< 0.05, n= 3–6) and protein shedding (* =p< 0.0005, n= 3) into CM were analyzed by QRT-PCR and ELISA, respectively. (B) N/TERT with and w/o constitutive expression of proHB-EGF or proAREG. Treatment with rhHB-EGF or EGF was for 72 h. (C) N/TERT were exposed to CM from N/TERT or cells expressing His-tagged sHB-EGF for 72h with and without prior depletion of sHB-EGF using nickel affinity chromatography. (D) N/TERT with inducible expression of sHB-EGF or proHB-EGF were incubated +/− TET with and w/o 10 µg/ml HB-EGF Abs, 1 µM PD158780 or 40 µM GM6001 for 1–3 days.
Figure 1
Figure 1
HB-EGF overexpression strongly changes KC morphology. (A) N/TERT with inducible expression of HB-EGF were grown to approx. 40% confluence and incubated for 60 h +/− TET. Normalized HB-EGF mRNA levels (* =p< 0.05, n= 3–6) and protein shedding (* =p< 0.0005, n= 3) into CM were analyzed by QRT-PCR and ELISA, respectively. (B) N/TERT with and w/o constitutive expression of proHB-EGF or proAREG. Treatment with rhHB-EGF or EGF was for 72 h. (C) N/TERT were exposed to CM from N/TERT or cells expressing His-tagged sHB-EGF for 72h with and without prior depletion of sHB-EGF using nickel affinity chromatography. (D) N/TERT with inducible expression of sHB-EGF or proHB-EGF were incubated +/− TET with and w/o 10 µg/ml HB-EGF Abs, 1 µM PD158780 or 40 µM GM6001 for 1–3 days.
Figure 1
Figure 1
HB-EGF overexpression strongly changes KC morphology. (A) N/TERT with inducible expression of HB-EGF were grown to approx. 40% confluence and incubated for 60 h +/− TET. Normalized HB-EGF mRNA levels (* =p< 0.05, n= 3–6) and protein shedding (* =p< 0.0005, n= 3) into CM were analyzed by QRT-PCR and ELISA, respectively. (B) N/TERT with and w/o constitutive expression of proHB-EGF or proAREG. Treatment with rhHB-EGF or EGF was for 72 h. (C) N/TERT were exposed to CM from N/TERT or cells expressing His-tagged sHB-EGF for 72h with and without prior depletion of sHB-EGF using nickel affinity chromatography. (D) N/TERT with inducible expression of sHB-EGF or proHB-EGF were incubated +/− TET with and w/o 10 µg/ml HB-EGF Abs, 1 µM PD158780 or 40 µM GM6001 for 1–3 days.
Figure 2
Figure 2
HB-EGF expression strongly reduces KC cell numbers in vitro. N/TERT with inducible expression HB-EGF were plated and incubated +/− TET. (A, B): Time course of KC growth with and without HB-EGF expression (+/− TET). Day 0 denotes the time of TET treatment. Cells were fixed and stained with crystal violet (A) and absorption at 590 nm was measured after extraction of crystal violet with 10% acetic acid (B), mean +/−SEM, n=4 except day 0 and day 6, n=2, * =p<0.05 and ** =p<0.00001 (two-tailed Student’s t-test). (C) N/TERT-KC were incubated +/− TET for 6 days and after trypsinization, cell number was determined by hemacytometer counting. Data are expressed as percent of control, mean +/− SEM, n=6 for sHBEGF (p<0.0001, two-tailed Student’s t-test) and n=2 for proHBEGF. Representative photos at day 6 are shown below the graph.
Figure 2
Figure 2
HB-EGF expression strongly reduces KC cell numbers in vitro. N/TERT with inducible expression HB-EGF were plated and incubated +/− TET. (A, B): Time course of KC growth with and without HB-EGF expression (+/− TET). Day 0 denotes the time of TET treatment. Cells were fixed and stained with crystal violet (A) and absorption at 590 nm was measured after extraction of crystal violet with 10% acetic acid (B), mean +/−SEM, n=4 except day 0 and day 6, n=2, * =p<0.05 and ** =p<0.00001 (two-tailed Student’s t-test). (C) N/TERT-KC were incubated +/− TET for 6 days and after trypsinization, cell number was determined by hemacytometer counting. Data are expressed as percent of control, mean +/− SEM, n=6 for sHBEGF (p<0.0001, two-tailed Student’s t-test) and n=2 for proHBEGF. Representative photos at day 6 are shown below the graph.
Figure 3
Figure 3
A. HB-EGF induces vimentin protein expression in KC. N/TERT KC with constitutive expression of AREG or HB-EGF were treated with and without EGF or PD158780 for 24 h and vimentin immunoreactivity was detected by fluorescence microscopy. Nuclei were counterstained with DAPI. B. HB-EGF expression in KC leads to nuclear accumulation of β-catenin. KC were cultured in the presence or absence of EGF or PD158780. KC were stained with β-catenin Abs and visualized by immunofluorescence microscopy. Arrows indicate positive nuclear staining. The western blot demonstrates increased nuclear accumulation of β-catenin in HB-EGF-expressing cells. C. Immunofluorescence microscopy of E-cadherin staining in N/TERT KC. Please note the membranous E-cadherin staining in control cells that is strongly decreased in EGF-treated or HB-EGF expressing cells.
Figure 3
Figure 3
A. HB-EGF induces vimentin protein expression in KC. N/TERT KC with constitutive expression of AREG or HB-EGF were treated with and without EGF or PD158780 for 24 h and vimentin immunoreactivity was detected by fluorescence microscopy. Nuclei were counterstained with DAPI. B. HB-EGF expression in KC leads to nuclear accumulation of β-catenin. KC were cultured in the presence or absence of EGF or PD158780. KC were stained with β-catenin Abs and visualized by immunofluorescence microscopy. Arrows indicate positive nuclear staining. The western blot demonstrates increased nuclear accumulation of β-catenin in HB-EGF-expressing cells. C. Immunofluorescence microscopy of E-cadherin staining in N/TERT KC. Please note the membranous E-cadherin staining in control cells that is strongly decreased in EGF-treated or HB-EGF expressing cells.
Figure 3
Figure 3
A. HB-EGF induces vimentin protein expression in KC. N/TERT KC with constitutive expression of AREG or HB-EGF were treated with and without EGF or PD158780 for 24 h and vimentin immunoreactivity was detected by fluorescence microscopy. Nuclei were counterstained with DAPI. B. HB-EGF expression in KC leads to nuclear accumulation of β-catenin. KC were cultured in the presence or absence of EGF or PD158780. KC were stained with β-catenin Abs and visualized by immunofluorescence microscopy. Arrows indicate positive nuclear staining. The western blot demonstrates increased nuclear accumulation of β-catenin in HB-EGF-expressing cells. C. Immunofluorescence microscopy of E-cadherin staining in N/TERT KC. Please note the membranous E-cadherin staining in control cells that is strongly decreased in EGF-treated or HB-EGF expressing cells.
Figure 4
Figure 4
HB-EGF overexpression strongly alters keratin and EMT/invasion-related gene expression in human KC. N/TERT KC were incubated in the presence or absence of TET for 60 h and gene expression was analyzed by QRT-PCR. (A) Gene expression was analyzed with TaqMan assays specific for human keratins as indicated. Data are expressed as percent of control (− TET), mean +/− SEM, n = 3–6. The reduction was significant for all data points with p<0.005 vs controls except for the sHB-EGF-induced reduction of K14 mRNA expression (p<0.022) as assessed by two-tailed, one-sample t-test. (B) HB-EGF increases invasion-related gene expression. Data are expressed as relative mRNA levels, mean +/− SEM, n= 3–6, * = p< 0.05, ** = p<0.005 vs control (− TET).
Figure 4
Figure 4
HB-EGF overexpression strongly alters keratin and EMT/invasion-related gene expression in human KC. N/TERT KC were incubated in the presence or absence of TET for 60 h and gene expression was analyzed by QRT-PCR. (A) Gene expression was analyzed with TaqMan assays specific for human keratins as indicated. Data are expressed as percent of control (− TET), mean +/− SEM, n = 3–6. The reduction was significant for all data points with p<0.005 vs controls except for the sHB-EGF-induced reduction of K14 mRNA expression (p<0.022) as assessed by two-tailed, one-sample t-test. (B) HB-EGF increases invasion-related gene expression. Data are expressed as relative mRNA levels, mean +/− SEM, n= 3–6, * = p< 0.05, ** = p<0.005 vs control (− TET).
Figure 5
Figure 5
Hypermotility and invasiveness as a function of HB-EGF overexpression in human KC. N/TERT KC and HT1080 cells were seeded in serum-free medium on growth factor reduced matrigel and incubated for 36 h with serum-containing media as a chemoattractant as described in Material and Methods. Invading cells were stained with crystal violet and photographed. Photos show representative fields, invasion assays with HT1080 and MDA-MB231 cells are shown for comparison. Results shown are representative of 3 independent experiments, similar results as shown here for sHB-EGF were also obtained with proHB-EGF (not shown). The typical cell morphology of the various lines is shown in the panels above the invasion assay photos.
Figure 6
Figure 6
HB-EGF expression in human skin wounds. HB-EGF mRNA expression was analyzed by QRT-PCR in regenerating interfollicular epidermis isolated by laser capture microdissection from skin biopsies taken one to four week after CO2 laser-generated partial thickness wounds. Data are normalized to the control gene RPLP0 (36B4) and are expressed as fold-change (log-scale) relative to unwounded skin, n=6.
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