Keratinocyte growth factor can increase the expression of the EDA
+fibronectin
Krisztina Vas1, Bernadett Kormos2, Attila Bebes1, Anikó Göblös1, Márta Széll2, Lajos Kemény1,2, Zsuzsanna Bata-Csörgő1,2
1) University of Szeged, Department of Dermatology and Allergology, Szeged, Hungary
2) University of Szeged, Dermatological Research Group of the Hungarian Academy of Sciences, Szeged, Hungary
Introduction:
Psoriasis is considered to be a multigenic inflammatory skin disease with hyperproliferation and abnormal differentiation of the epidermal keratinocytes. In our previous work we showed that the fibronectin splice variant EDA+FN (oncofetal fibronectin), its receptor the α5- integrin, the keratinocyte growth factor (KGF), and its receptor (KGFR) are overexpressed in psoriatic uninvolved skin, compared to normal skin. EDA+FN and KGF both stimulate keratinocyte proliferation, moreover KGF is also known to induce α5-integrin expression.
Aim:
To measure the effect of exogenous KGF on the EDA+FN production of fibroblasts, keratinocytes and HaCaT cells.
Results:
Materials and Methods:
Cell cultures: Fibroblasts(5th passage)Keratinocytes (3rd passage) andHaCaT cells were seeded into 6 well plates at density of 0.3 x 106 cells per cm2 in the appropriate media and were maintained in humidified atmosphere containing 5% CO2. These cells were incubated with different concentrations of human recombinant KGF (10 ng/ml, 25 ng/ml, 50 ng/ml, 100 ng/ml). We measured the EDA+FN gene and protein levels 24 hours after exogenous KGF treatment.
Real-time RT-PCR:
18S rRNA expression served as internal control. Results are expressed as mean
± SEM. Relative expressions were calculated using the∆∆CT method.
Immunocytochemistry on cultured human fibroblasts, keratinocytes and HaCaT cells: primary antibody: mouse MoAb for EDA+FN (IST-9),DAPI was used for nuclear staining.
secondary antibody: goat anti-mouse IgG-Alexa 546 Flow cytometry:
primary antibodies: mouse MoAb for EDA+FN (IST-9), mouse MoAB IgG1 (isotype control), secondary antibody: goat anti-mouse IgG-Alexa 647
Conclusion:
Our results suggest that KGF can promote the production of EDA
+FN, therefore it may contribute to
the altered homeostasis in the uninvolved skin of psoriatic patients.
Funding: OTKA NK 77434, OTKA K 83277, TÁMOP 4.2.2/B-10/1- 2010-0012,TÁMOP 4.2.1/B-09/1/KONV-2010-0005E-mail: krisztina.vas@mail.derma.szote.u-szeged.hu
0 1 2 3 4 5 6
0 1 2 3 4 5 6
0 1 2 3 4 5 6 In fibroblasts 24 hours after exogen KGF (25ng/ml)
treatment a 2.5- fold increase in EDA+FN mRNA expression was observed
No changes were detected 24 hours after KGF treatment in EDA+FN mRNA expression in HaCaT cells
Relativeexpressionof EDA+FNmRNA
Relativeexpressionof EDA+FNmRNA Relativeexpressionof EDA+FNmRNA
After 24 hours KGF treatment EDA+FN mRNA expression did not change in keratinocytes
n=2 n=3 n=3
0ng / ml KGF 10 ng / ml KGF 25ng / ml KGF
Normal human fibroblasts Normal human keratinocytes HaCaT cells
0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6
0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6
0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 In normal human keratinocytes the EDA+FN protein
levels significantly decreased at higher concentrations of KGF
In HaCaT keratinocytes exogen KGF treatment did not significantly alter the EDA+FN protein levels
0 ng/ml 10 ng/ml 25ng/ml 50 ng/ml 100 ng/ml 0 ng/ml 10 ng/ml 25ng/ml 50 ng/ml 100 ng/ml
0 ng/ml 10 ng/ml 25ng/ml 50 ng/ml 100 ng/mlKGF KGF KGF
EDA+FN protein expression significantly increased in fibroblasts 24 hours after exogen KGF treatment
n=4 n=4 n=4
* p<0.01
* p<0.01
Protein expressionof EDA+FN byflowcytometry Protein expressionof EDA+FN byflowcytometry
Protein expressionof EDA+FN byflowcytometry
blue: DAPI red: EDA+FN
EDA+FN immunostainingonhuman fibroblasts 24 hours after exogen KGF treatment EDA+FN immunostaining onhuman keratinocytes 24 hours after exogen KGF treatment EDA+FN immunostaining onHaCaT cells 24 hours after exogen KGF treatment
blue: DAPI red: EDA+FN
blue: DAPI red: EDA+FN
0 ng/ml KGF
10 ng/ml KGF
25 ng/ml KGF
0 ng/ml KGF
10 ng/ml KGF
25 ng/ml KGF
0 ng/ml KGF
10 ng/ml KGF
25 ng/ml KGF 0ng / ml KGF 10 ng / ml KGF 25ng / ml KGF 0ng / ml KGF 10 ng / ml KGF 25ng / ml KGF
