The PI3K/Akt signaling pathway is involved in KGFR-mediated early differentiation.

<p>(A) HaCaT KGFR and HaCaT cells were serum starved for 4 h and treated with 100 ng/ml KGF for 10′ at 37°C. Western blot analysis using anti-Bek and anti-phospho-Akt polyclonal antibodies shows that Akt protein phosphorylation is evident following KGF treatment and it is specifically blocked by the Akt inhibitor in HaCaT control cells. In KGFR transfected cells, the Akt phosphorylation is more intense and clearly detectable also in serum-free untreated cells. The specific band corresponding to the receptor is well visible only in KGFR transfected cells. The equal loading was assessed with anti-actin antibody. For the densitometric analysis of band corresponding to phospho-Akt protein band the values from three independent experiments were normalized, expressed as fold increase and reported in graph as mean values +/− SE. Student's t test was performed and significance level has been defined as *p<0.001 vs the corresponding KGF-untreated cells, **p<0.005 vs the corresponding Akt inhibitor-untreated cells, ***p<0.001 vs the corresponding untransfected cells and ****p<0.005 vs the corresponding Akt inhibitor-untreated cells. (B) HaCaT KGFR and HaCaT cells were serum starved for 12 h and then stimulated with 20 ng/ml KGF for the last 24 h at 37°C in presence or not of the specific Akt inhibitor. Quantitative immunofluorescence analysis shows that the Akt inhibitor drastically blocks the increase of K1 positive cells observed upon KGFR overexpression and KGF stimulation. The Akt inhibitor decreases the KGF-mediated differentiation also in cells expressing endogenous levels of the receptor. Results are expressed as mean values ± SE. Student's t test was performed and significance level has been defined as *p<0.001 and **p<0.001 vs the corresponding, KGF-treated cells. Bar, 10 µm.</p

KGF-induced KGFR activation is required for the induction of early differentiation.

<p>HaCaT KGFR cells were treated with TG as above, serum starved for 12 h and stimulated the last 24 h at 37°C with 20 ng/ml KGF. Quantitative immunofluorescence analysis performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024194#pone-0024194-g002" target="_blank">figure 2D</a> shows that KGF stimulation increases the percentage of K1 positive cells. This KGF-induced increase is higher in cells overexpressing KGFR compared to cells expressing endogenous receptor levels and is particularly evident in undifferentiated cells, while the TG-treated cells are almost all K1 positive independently on KGF addition. No significant increase in the percentage of K1 positive cells is induced by KGFR overexpression in KGF untreated cells as a consequence of serum deprivation. Results are expressed as mean values ± SE. Student's t test was performed and significance level has been defined as *: NS vs the corresponding surrounding cells that do not display KGFR overexpression, **p<0.01 and ***p<0.001 vs the corresponding unstimulated cells. Bar, 10 µm.</p

KGFR expression triggers the early differentiation in undifferentiated keratinocytes.

<p>(A) HaCaT cells were transfected with the pCI-neo expression vector containing human KGFR cDNA (HaCaT KGFR). After 24 h from transfection, the KGFR and K1 mRNA transcript levels were quantitated by real-time RT-PCR: a clear fold increase in both K1 mRNA (right panel) and KGFR mRNA (left panel) expression is observed in HaCaT KGFR cells compared to control ones. (B) Western blot analysis on HaCaT KGFR and HaCaT cells using anti-K1 monoclonal antibody and with anti-Bek polyclonal antibodies, which recognize the KGFR/FGFR2b protein, shows that the band of K1 is increased by KGFR overexpression in HaCaT KGFR compared to control cells. The specific band corresponding to KGFR protein is well visible only upon KGFR transfection. The equal loading was assessed with anti-actin antibody. For densitometric analysis of the band corresponding to K1 protein the values from three independent experiments were normalized, expressed as fold increase and reported in graph as mean values +/− standard deviation (SD). Student' t test was performed and significance level has been defined as *p<0.005 vs the corresponding untransfected cells.</p

KGFR-mediated early differentiation is independent on PLC-γ recruitment and activation.

<p>(A) HaCaT cells transiently transfected with KGFR WT, with KGFR Y769F signaling mutant or with KGFR Y656F/Y657F kinase dead mutant were treated with TG as above. Quantitative immunofluorescence analysis performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024194#pone-0024194-g002" target="_blank">figure 2D</a> shows that, similarly to KGFR WT, KGFR Y769F overexpression significantly increases the percentage of cells expressing K1 either in TG-treated and in the control cells, while the KGFR Y656F/Y657F overexpression does not affect it. Results are expressed as mean values ± SE. Student's t test was performed and significance level has been defined as *p<0.001, **p<0.01, ***p<0.001 and ****p<0.05 vs the corresponding surrounding cells that do not display KGFR overexpression. Bar, 10 µm. (B) HaCaT cells were transfected and treated with TG and KGF as above. KGFR mRNA (left panel) and K1 mRNA (right panel) transcript levels were quantitated by real-time RT-PCR: a ligand-dependent increase in K1 mRNA expression is observed in cells transfected with KGFR WT and with KGFR Y769F but not in cells transfected with KGFR Y656F/Y657F. A ligand-dependent down-modulation in KGFR mRNA expression is observed in HaCaT KGFR WT cells and in HaCaT KGFR Y769F cells but not in KGFR Y656F/Y657F cells. Student's t test was performed and significance level has been defined as *p<0.001, **p<0.05, ***p<0.005 and ****p<0.001 vs the corresponding unstimulated cells.</p

KGFR/FGFR2b depletion inhibits early differentiation in keratinocytes.

<p>(A) HaCaT cells were coinjected with KGFR siRNA to obtain KGFR silencing and mouse IgG to identify the microinjected cells. Control cells were injected with an unrelated siRNA. After injection, cells were treated with TG as above. Quantitative immunofluorescence analysis using anti-K1 polyclonal antibodies shows that the percentage of K1 positive cells in KGFR-depleted and TG-treated cells is significantly decreased if compared either to uninjected cells in the same microscopic field or to cells injected with control siRNA. The quantitative analysis was assessed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024194#pone-0024194-g002" target="_blank">figure 2D</a>. Results are expressed as mean values ± SE. Student's t test was performed and significance level has been defined as *p<0.001 vs the corresponding uninjected cells. Bar, 10 µm. (B) Western blot analysis with anti-Bek polyclonal antibodies in HaCaT cells transfected with KGFR siRNA or with the control unrelated siRNA and treated with TG as above. The KGFR protein expression is down-regulated in KGFR siRNA-transfected cells. The equal loading was assessed with anti-actin antibody. For densitometric analysis of the band corresponding to KGFR protein, the values from three independent experiments were normalized, expressed as fold increase and reported in graph as mean values +/− SE. Student's t test was performed and significance level has been defined as *p<0.01 vs the corresponding uninjected cells.</p

Thapsigargin-induced differentiation is associated with increased KGFR expression.

<p>(A) HaCaT cells were treated with TG 0.5 µM for 1 h at 37°C following by incubation at 37°C for 48 h. Cells treated with equal amount of DMSO solvent were used as a control. Phase contrast microscopy analysis shows that the TG-treated cells appear detached each other and elongated, while control cells are closely packed and polygonal (left panels). Quantitative immunofluorescence analysis using anti-Ki67 and anti-K1 antibodies shows that TG treatment significantly decreases the percentage of cells expressing the proliferative marker Ki67 and increases that of cells positive for the early differentiation marker (right panels). Cell nuclei were visualized by DAPI. The quantitative analysis was assessed by counting for each sample a total of 50 cells, randomly observed in 10 microscopic fields from three different experiments. Cut-off of the K1 signal intensity was determined for TG-treated and control samples as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024194#s4" target="_blank">materials and methods</a>. Results are expressed as mean values ± standard errors (SE). Student's t test was performed and significance level has been defined as *p<0.001 and **p<0.05 vs the corresponding untreated cells. (B) Quantitative real-time RT-PCR of KGFR and K1 mRNA transcript levels on TG-treated and control HaCaT cells shows an evident increase in both KGFR mRNA (left panel) and K1 mRNA (right panel) expression upon TG-treatment. Student's t test was performed and significance level has been defined as *p<0.005 and **p<0.001 vs the corresponding untreated cells. (C) Western blot analysis shows that the KGFR band, which is hardly detectable in control cells, becomes well visible in TG-treated cells. Enhancement of K1 protein expression is also evident upon TG treatment. The equal loading was assessed with anti-actin antibody. For densitometric analysis of the bands, the values from three independent experiments were normalized, expressed as fold increase and reported in graphs as mean values +/− SD. Student's t test was performed and significance level has been defined as *p<0.001 and **p<0.001 vs the corresponding untreated cells.</p

The KGFR polarization is dependent on receptor endocytosis.

<p>HaCaT KGFR cells were incubated at 4°C with the anti-Bek polyclonal antibodies to selectively stain the plasma membrane receptors and exclusively follow them during endocytosis, and then stimulated with KGF or with FGF10 for 5, 10 and 30 minutes at 37°C. The plasma membrane was decorated with the plasma membrane marker WGA-FITC. KGFR staining appears continuous and uniformly distributed on the cell surface of untreated cells, discontinuous on the plasma membrane and in some intracellular dots underlying the cell surface upon 5 minutes of KGF stimulation, and polarized at both the plasma membrane and in intracellular dots at the leading edge of migrating cells upon 10 and 30 minutes of KGF stimulation. After FGF10 stimulation the polarization appears delayed and evident only upon 30 minutes of treatment. The staining of the marker WGA appears uniformly distributed along the plasma membrane at all time points. Bars: 10 µm.</p

KGFR expression and polarization are involved in cell motility.

<p>A) HaCaT KGFR and HaCaT cells were seeded on coverslip and grown until confluence. A cell-free area was introduced in a monolayer of cells using a steril tip and then cells were immediately fixed (T0) or allowed to migrate from the edge of the scratch for 20 h at 37°C in the presence or not of KGF or FGF10. The cell-free area, evident in samples at time 0 (T0) from the scratch, is only partially repopulated in untreated cells. KGF stimulation induces a more intense cell migration if compared to FGF10 in HaCaT cells; KGFR overexpression induces a significant increase of cell migration upon both growth factors stimulation. Bar: 80 µm. Immunofluorescence analysis using anti-Bek antibodies in HaCaT KGFR cells shows some cells that clearly overexpresses KGFR. The receptor staining is uniformly distributed on the plasma membrane in unstimulated cells or in cells fixed at time 0 (T0) from the scratch, but is polarized at the leading edge of migrating cells that repopulate the scratch area upon KGF of FGF10 stimulation. Bar: 10 µm. B) Cells migration was quantified measuring the mean gap distance between the edges of the scratch area as reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029159#s4" target="_blank">Materials and Methods</a>. Student’s T test was performed and significance level has been defined as above. * NS vs the corresponding untransfected cells; **p< 0,0001 vs the corresponding untransfected cells; ***p<0,0001 vs the corresponding untransfected cells.</p

Src activity is required for KGFR and cortactin colocalization.

<p>A) HaCaT KGFR cells, treated or not with the growth factors, were incubated at 4°C with the anti-Bek polyclonal antibodies before cell fixation, as above, to selectively stain the plasma membrane KGFR, or with an anti-Bek monoclonal antibody after cell fixation and permeabilization, to visualize simultaneously the intracellular and plasma membrane KGFRs. Double immunofluorescence analysis, using anti-cortactin monoclonal antibody or anti-cortactin polyclonal antibodies, shows that in untreated cells the KGFR signal is localized along the entire surface of the cell plasma membrane, as well as in intracellular dots, and the cells do not show migratory features. The cortactin staining appears mainly localized on small intracellular dots dispersed throughout the cytoplasm, and only partially overlapping with KGFR positive dots. After treatment with either KGF or FGF10, cortactin and KGFR colocalization is significantly increased, evident not only in intracellular endocytic dots (arrows), but also at the level of the plasma membrane, where cortactin is translocated (arrowheads). Upon ligand treatment HaCaT KGFR cells located on the periphery of the colonies show a typical migratory phenotype, with a clearly defined leading edge, where the intracellular yellow dots stained for both cortactin and KGFR appear to be concentrated. Treatment with SU6656 reduces the colocalization between KGFR and cortactin, at a level comparable to that observed in untreated cells, and abolishes the migratory features. Images shown were obtained by 3D reconstruction of a selection of three out of the total number of the serial optical sections as reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029159#pone-0029159-g002" target="_blank">figure 2</a>. Bars: 10 µm. B) Quantitative analysis of the percentage of colocalization of KFGR and cortactin was performed by serial optical sectioning and 3D reconstruction, as reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029159#s4" target="_blank">Materials and Methods</a>. Results are expressed as mean values +/- SE (standard errors): the percentage of colocalization was calculated analyzing a minimum of 50 cells for each treatment randomly taken from three independent experiments. Student's T test was performed and significance levels have been defined. *p<0,001 vs the corresponding untreated cells; **p<0,001 vs the corresponding untreated cells; ***p<0,001 vs the corresponding cells –SU6656; ****p<0,001 vs the corresponding cells –SU6656. C) Double immunofluorescence analysis, using anti-cortactin polyclonal antibodies and anti-early endosome antigene 1 (EEA1) monoclonal antibody, shows that the colocalization between cortactin and EEA1, already visible in untreated HaCaT cells, is increased upon KGF or FGF10 treatment and appears mostly relocalized in dots at the leading edge of migrating cells (arrows). The treatment with SU6656 significantly reduces the cortactin/EEA1 colocalization, as well as the migratory phenotype, upon either KGF or FGF10 stimulation. Images shown were obtained by 3D reconstruction of a selection of three out of the total number of the serial optical sections as reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029159#pone-0029159-g002" target="_blank">figure 2</a>. Bars: 10 µm. D) Quantitative analysis of the percentage of colocalization of cortactin with EEA1 was performed by serial optical sectioning and 3D reconstruction as above. Results are expressed as mean values +/- SE; the percentage of colocalization was calculated analyzing a minimum of 50 cells for each treatment randomly taken from three independent experiments. Student's T test was performed and significance levels have been defined. *p<0,005 vs the corresponding untreated cells; **p<0,005 vs the corresponding untreated cells; ***p<0,001 vs the corresponding cells –SU6656; ****p<0,005 vs the corresponding cells –SU6656.</p

Src signaling is required for KGFR endocytosis.

<p>A) HaCaT KGFR cells were incubated at 4°C with the anti-Bek polyclonal antibodies, to selectively stain the plasma membrane receptors, and then treated with KGF or FGF10 to induce receptor internalization from the plasma membrane. Double immunofluorescence analysis, using anti-cortactin monoclonal antibody, shows that in untreated cells the KGFR signal appears uniformly distributed on the cell surface, while the cortactin signal is evident in dots dispersed throughout the cytoplasm, which correspond to sorting endosomes. Virtually no colocalization is observed between the two proteins. After KGF or FGF10 stimulation, HaCaT KGFR cells show a migratory phenotype and the internalized KGFR appear in endocytic dots polarized at the leading edge of migrating cells, in which the receptor significantly colocalizes with cortactin (arrows). Treatment with SU6656 is able to block the ligand-induced KGFR internalization, and consequently its colocalization with cortactin in endocytic dots: the receptor staining is uniformly distributed on the plasma membrane, while the cortactin labeling remains dispersed throughout the cytosol, as observed in untreated cells. Images shown were obtained by 3D reconstruction of a selection of three out of the total number of the serial optical sections, as reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029159#pone-0029159-g002" target="_blank">figure 2</a>. Bars: 10 µm. B) Quantitative analysis of percentage of HaCaT KGFR cells showing internalized KGFR was performed by counting 100 cells that overexpress KGFR for each condition, randomly taken from 10 microscopic fields in three different experiments, and values are expressed as the mean value ± standard errors (SE). C) Quantitative analysis of the percentage of colocalization of KGFR with cortactin was performed as described above. The percentage of colocalization was calculated analyzing a minimum of 50 cells for each treatment randomly taken from three independent experiments. Results are expressed as mean values +/- SE. Student's T test was performed and significance levels have been defined. Student's T test was performed and significance level has been defined as above. *p<0,005 vs the corresponding untreated cells; **p<0,005 vs the corresponding untreated cells; ***p<0,001 vs the corresponding cells –SU6656; ****p<0,005 vs the corresponding cells –SU6656.</p