Keratinocyte growth factor down-regulates intracellular ROS production induced by UVB

Background: Exposure to ultraviolet (UV) radiation causes a complex cellular response, mostly mediated by the production of reactive oxygen species (ROS), which can be counteracted by exogenous treatments and endogenous mechanisms with anti-oxidant and scavenger properties. Keratinocyte growth factor (KGF/FGF7), a member of the fibroblast growth factor family, promotes epithelial growth and differentiation and is involved in cell survival after oxidant injuries. Objective: We analyzed the role of KGF in the control of intracellular ROS production and oxidative stress after UVB exposure on KGF receptor (KGFR) transfected cells and human immortalized and primary keratinocytes. Methods: We assessed the intracellular ROS production measuring the intensity of the oxidation-sensitive fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA) by confocal microscopy, as well as the catalase activity by spectrophotometric assay. Moreover, morphological and biochemical analysis of actin cytoskeleton reorganization was evaluated as a further marker of oxidative damage. Results: Our data show that KGF significantly reduces intracellular ROS generation in response to UVB, preserves the decrease of catalase activity and prevents actin cytoskeleton rearrangement. Conclusion: Our results provide a further evidence that KGF may be crucial for an efficient skin photoprotection, demonstrating a direct role for KGF in the reduction of intracellular ROS content following UVB exposure. (C) 2009 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved

Lipid profile.

<p>(A) Total fatty acids in TG , (B) PL and (C) FFA fraction, (ng/mg dry weight-d.w.), in mycelia of WT and AfP33 strains grown in CD (25 mL) and incubated at 30°C from 24 to 168 hpi.</p

How Peroxisomes Affect Aflatoxin Biosynthesis in <i>Aspergillus Flavus</i>

<div><p>In filamentous fungi, peroxisomes are crucial for the primary metabolism and play a pivotal role in the formation of some secondary metabolites. Further, peroxisomes are important site for fatty acids β-oxidation, the formation of reactive oxygen species and for their scavenging through a complex of antioxidant activities. Oxidative stress is involved in different metabolic events in all organisms and it occurs during oxidative processes within the cell, including peroxisomal β-oxidation of fatty acids. In Aspergillus flavus, an unbalance towards an hyper-oxidant status into the cell is a prerequisite for the onset of aflatoxin biosynthesis. In our preliminary results, the use of bezafibrate, inducer of both peroxisomal β-oxidation and peroxisome proliferation in mammals, significantly enhanced the expression of pex11 and foxA and stimulated aflatoxin synthesis in A. flavus. This suggests the existence of a correlation among peroxisome proliferation, fatty acids β-oxidation and aflatoxin biosynthesis. To investigate this correlation, A. flavus was transformed with a vector containing P33, a gene from Cymbidium ringspot virus able to induce peroxisome proliferation, under the control of the promoter of the Cu,Zn-sod gene of A. flavus. This transcriptional control closely relates the onset of the antioxidant response to ROS increase, with the proliferation of peroxisomes in A. flavus. The AfP33 transformant strain show an up-regulation of lipid metabolism and an higher content of both intracellular ROS and some oxylipins. The combined presence of a higher amount of substrates (fatty acids-derived), an hyper-oxidant cell environment and of hormone-like signals (oxylipins) enhances the synthesis of aflatoxins in the AfP33 strain. The results obtained demonstrated a close link between peroxisome metabolism and aflatoxin synthesis.</p></div

Ultrastructural characterization.

<p>TEM examination of A. flavus mycelia. Expression of P33 in AfP33 cells (D, E, F, G) significantly increases number of peroxisomes in comparison with WT cells (A, B, C). Bar 0.5 μm in A, B, C, D; bar 0.25 μm in E; bar 0.5 μm in F, G. (P, Peroxisomes; M, mitochondria; Nu, nucleus).</p

Fungal growth and conidiogenesis.

<p>A) Fungal growth (mg dry weight-d.w/mL). B) conidiogenesis (n. conidia/mL).</p

Aflatoxin (µg/mL) synthesis, fungal growth (mg/mL d.w.), <i>pex11</i> and <i>foxA</i> mRNA relative expression in <i>A. flavus</i> 3357 (WT) grown in basal media (CD) for aflatoxin (AF) synthesis after 7 days at 30°C.

<p>Aflatoxin (µg/mL) synthesis, fungal growth (mg/mL d.w.), <i>pex11</i> and <i>foxA</i> mRNA relative expression in <i>A. flavus</i> 3357 (WT) grown in basal media (CD) for aflatoxin (AF) synthesis after 7 days at 30°C.</p

Expression of genes related to peroxisome proliferation and functionality.

<p>(A) Real Time PCR analysis of pex11 mRNA and (B) of foxA mRNA expression in AfP33 and WT strains grown in CD after different periods of incubation (24 up to 168 hpi) normalized to time 0 (inoculation).</p

Fluorescence analysis of AFDsRED and AfP33 strains.

<p>The fluorescence analysis was performed to compare the presence of peroxisomes (red) between (A, B) AfDsRED and (C, D) AfP33 strains. Cells not expressing p33 displayed a punctate pattern of fluorescence (A and detail in the boxed area) corresponding to scattered peroxisomes whereas cells expressing p33 showed the presence of red spots revealing an increased number of peroxisomes (C and detail in the boxed area). Nuclei are stained with DAPI. Scale bars =  10 mm (A–D); 5 mm (boxed areas in A and C).</p