Role of keratinocyte growth factor receptor (KGFR/FGFR2b) expression and signaling in the control of human keratinocyte differentiation

The fibroblast growth factor receptors (FGFRs) trigger divergent responses, such as proliferation and differentiation, and the cell type as well as the context-dependent signaling are crucial for the functional outcome. The FGFR2b/KGFR is expressed exclusively on epithelial cells and plays a key role in skin homeostasis. In the first part of this work I analyzed in vitro the role of KGFR in the early differentiation of keratinocytes modulating its expression by KGFR cDNA transient transfection or KGFR siRNA microinjection and inducing a synchronous wave of differentiation in pre-confluent cells. Immunofluorescence, biochemical and molecular approaches demonstrated that KGFR overexpression increased the early differentiation marker keratin 1 at both transcriptional and translational levels, while receptor depletion reduced it. Ligand-dependent receptor activation and signaling were required for this differentiative effect. Overexpression of kinase negative KGFR mutant or Tyr769 KGFR signaling mutant, which is not able to recruit and activate PLCγ showed that the receptor kinase activity, but not its PLCγ-mediated signaling, is required for differentiation. Reduction of K1 expression, obtained by AKT inhibition, demonstrated that the PI3K/Akt signaling pathway is involved in the control of KGFR-mediated keratinocyte differentiation. This in vitro experimental model indicates that KGFR expression represents a key event regulating keratinocyte early differentiation during the switch from undifferentiated to differentiating cells. Since membrane and actin cytoskeleton dynamics during phagocytosis can be triggered and amplified by the signal transduction of receptor tyrosine kinases and the epidermal keratinocytes appear to use the phagocytic mechanism of uptake to ingest melanosomes released by the melanocytes playing a pivotal role in the transfer process, in the second part of this work I investigate the contribution of KGFR expression, activation and signaling in regulating the phagocytic process. We have previously demonstrated that the keratinocyte growth factor KGF/FGF7 promotes the melanosome uptake through activation of its receptor tyrosine kinase KGFR. Phagocytosis was analyzed in vitro using fluorescent latex beads on human keratinocytes induced to differentiate. KGFR depletion by siRNA microinjection and overexpression by transfection of KGFR wild type or defective mutants previously described were performed to demonstrate the direct effect of the receptor on phagocytosis. Colocalization of the phagocytosed beads with the internalized receptors in phagolysosomes was analyzed by optical sectioning and 3D reconstruction. KGFR ligands triggered phagocytosis in differentiated keratinocytes and receptor kinase activity and signaling were required for these effects, suggesting that KGFR expression/activity and PLCγ signaling pathway play crucial roles in phagocytosis

Extravasation injury of balanced electrolyte solution simulates the clinical condition of necrotizing fasciitis: A case report

Extravasation injury (EI) is an iatrogenic condition that occurs preferentially in neonatal and pediatric patients when the injection of fluid substances by intravenous access is required and it accidentally leaks into the adjacent tissues or in spaces outside of vascular compartment. Different types and amount of substances once undergoing extravasation can affect the EI differently [1]. In some instances immediate measures such as saline washout, local antidotes, enzymatic debridement and surgical interventions can be required in order to prevent the occurrence of a growing injury avoiding the progression of the EI to a medical emergency [6]. Here we report an unusual case of a preterm 2-month-old male patient in which the extravasation of balanced electrolyte solution on the upper right arm resulted in the development of full-thickness skin necrosis appearing as the clinical condition of necrotizing fasciitis. The management of necrotic tissue was performed using escharectomy as well as autograft skin under conditions of general anesthesia

The Collection of Adipose Derived Stem Cells using Water-Jet Assisted Lipoplasty for their Use in Plastic and Reconstructive Surgery: A Preliminary Study

The graft of autologous fat for the augmentation of soft tissue is a common practice frequently used in the field of plastic and reconstructive surgery. In addition, the presence of adipose derived stem cells (ASCs) in adipose tissue stimulates the regeneration of tissue in which it is applied after the autologous fat grafting improving the final clinical results. Due to these characteristics, there is an increasing interest in the use of ASCs for the treatment of several clinical conditions. As a consequence, the use of clean room environment is required for the production of cell-based therapies. The present study is aimed to describe the biological properties of adipose tissue and cells derived from it cultured in vitro in clean room environment according to current regulation. The collection of adipose tissue was performed using the water-jet assisted liposuction in order to preserve an high cell viability increasing their chances of future use for different clinical application in the field of plastic and reconstructive surgery

The Use of Quercetin to Improve the Antioxidant and Regenerative Properties of Frozen or Cryopreserved Human Amniotic Membrane

none7noPurpura, Valeria; Benedetti, Serena; Bondioli, Elena; Scarpellini, Francesca; Giacometti, Agnese; Albertini, Maria Cristina; Melandri, DavidePurpura, Valeria; Benedetti, Serena; Bondioli, Elena; Scarpellini, Francesca; Giacometti, Agnese; Albertini, Maria Cristina; Melandri, David

The Use of Quercetin to Improve the Antioxidant and Regenerative Properties of Frozen or Cryopreserved Human Amniotic Membrane

The biological properties of the human amniotic membrane (HAM) and its characteristic ability to be a reservoir of growth factors promoting wound healing make it an ideal biological dressing for the treatment of different clinical conditions, such as burns and non-healing wounds. However, the application of a preservation method on the HAM is required during banking to maintain biological tissue properties and to ensure the release overtime of protein content for its final clinical effectiveness after application on the wound bed. Although cryopreservation and freezing are methods widely used to maintain tissue properties, reactive oxygen species (ROS) are produced within tissue cellular components during their switching from frozen to thawed state. Consequently, these methods can lead to oxidative stress-induced cell injury, affecting tissue regenerative properties and its final clinical effectiveness. Taking advantage of the antioxidant activity of the natural compound quercetin, we used it to improve the antioxidant and regenerative properties of frozen or cryopreserved HAM tissues. In particular, we evaluated the oxidative damage (lipid peroxidation, malondialdehyde) as well as the regenerative/biological properties (bFGF growth factor release, wound healing closure, structure, and viability) of HAM tissue after its application. We identified the effectiveness of quercetin on both preservation methods to reduce oxidative damage, as well as its ability to enhance regenerative properties, while maintaining the unaltered structure and viability of HAM tissue. The use of quercetin described in this study appears able to counteract the side effects of cryopreservation and freezing methods related to oxidative stress, enhancing the regenerative properties of HAM. However, further investigations will need to be performed, starting from these promising results, to identify its beneficial effect when applied on burns or non-healing wounds

The receptor tyrosine kinase FGFR2b/KGFR controls early differentiation of human keratinocytes.

The FGFRs trigger divergent responses, such as proliferation and differentiation, and the cell type as well as the context-dependent signaling are crucial for the functional outcome. The FGFR2b/KGFR is expressed exclusively on epithelial cells and plays a key role in skin homeostasis. Here we analyzed in vitro the role of KGFR in the early differentiation of keratinocytes modulating its expression by KGFR cDNA transient transfection or KGFR siRNA microinjection and inducing a synchronous wave of differentiation in pre-confluent cells. Immunofluorescence, biochemical and molecular approaches demonstrated that KGFR overexpression increased the early differentiation marker keratin 1 at both transcriptional and translational levels, while receptor depletion reduced it. Ligand-dependent receptor activation and signaling were required for this differentiative effect. Overexpression of kinase negative KGFR mutant or Tyr769 KGFR signaling mutant, which is not able to recruit and activate PLC-γ, showed that the receptor kinase activity, but not its PLCγ-mediated signaling, is required for differentiation. Reduction of K1 expression, obtained by AKT inhibition, demonstrated that the PI3K/Akt signaling pathway is involved in the control of KGFR-mediated keratinocyte differentiation. This in vitro experimental model indicates that FGFR2b/KGFR expression represents a key event regulating keratinocyte early differentiation during the switch from undifferentiated to differentiating cells

HPV16 E5 and KGFR/FGFR2b interplay in differentiating epithelial cells

The E5 oncogenic protein of the human papillomavirus type 16 (HPV16 E5) cooperates in epithelial transformation perturbing the behaviour of differentiating suprabasal cells. Among the receptor tyrosine kinases deregulated by 16E5 expression, the key paracrine mediator of epithelial homeostasis keratinocyte growth factor receptor (KGFR/FGFR2b) is altered in its signaling and endocytic traffic in undifferentiated keratinocytes expressing 16E5 and it would represent a major target of the viral protein in differentiated cells. With the aim to specifically address the possible interplay of 16E5 with KGFR/FGFR2b in cells already committed to differentiation, we took advantage of an in vitro model for forced overexpression or depletion of KGFR in E5 expressing human keratinocytes under synchronous waves of differentiation. Quantitative RT-PCR, biochemical and immunofluorescence analysis showed that KGFR down-modulation is responsible for a E5-mediated decrease of the early differentiation marker K1 and that the receptor re-expression as well as triggering of its kinase activity and signaling are able to efficiently counteract the impairment of differentiation, providing a further demonstration of the tumor-suppressive role of KGFR in the new unexplored context of HPV16 E5-mediated carcinogenesis. In addition, KGFR induced a ligand-dependent decrease of p63 through a miR-203 independent mechanism and this effect was blocked by inhibition of the PI3K/Akt signaling, which is the main pathway involved in KGFR-dependent keratinocyte differentiation, suggesting that alterations of the KGFR/p63 crosstalk are responsible for the impairment of keratinocyte differentiation induced by 16E5 and that the opposite tumor-suppressive action of KGFR and oncogenic role of E5 might both involve p63

FGF7/KGF regulates autophagy in keratinocytes: A novel dual role in the induction of both assembly and turnover of autophagosomes

Autophagy is a degradative pathway through which cells overcome stressful conditions and rapidly change their phenotype during differentiation. Despite its protective role, when exacerbated, autophagy may lead to cell death. Several growth factors involved in cell survival and in preventing differentiation are able to inhibit autophagy. Here we investigated the autophagic role of FGF7/KGF, an important player in epithelial cell protection and differentiation. Biochemical and quantitative fluorescence approaches showed that FGF7 and its signaling induce autophagy in human keratinocytes and the use of specific inhibitors indicated that this effect is independent of the PI3K-AKT-MTOR pathway. The selective block of autophagosome-to-lysosome fusion clarified that FGF7 induces autophagy stimulating autophagosome formation. However, quantitative fluorescence approaches also indicated that, upon a prolonged autophagic stimulus, FGF7 is able to accelerate autophagosome turnover. Moreover, in differentiating keratinocytes, the use of the autophagic inhibitor 3-MA as well as the depletion of BECN1 and ATG5, 2 essential regulators of the process, counteracted the FGF7-induced increase of the differentiation marker KRT1/K1, suggesting that autophagy is required for the FGF7-mediated early differentiation. These results provide the first evidence of a role of FGF7 in the regulation of sequential steps of the autophagic process and strengthen the hypothesis of a direct interplay between autophagy and differentiation. On the other hand, the ability of FGF7 to accelerate autophagosome turnover, preventing their dangerous accumulation, is consistent with the well-established protective role played by the growth factor in epithelial cells

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