Site home page Conference home page Discussion
The Influence of UVA and UVB on the Tyrosine Kinase Profile of Normal Human Keratinocytes

Gabriele Klosner, Roland Varecka,* and Franz Trautinger

Division of Special and Environmental Dermatology, Dept.Dermatol., Univ.Vienna; *Boehringer-Ingelheim Austria GmbH;Vienna, Austria
 
 

INTRODUCTION

Protein tyrosine kinases (PTKs) play a significant role in signalling pathways that regulate cell proliferation, differentiation, transformation and immune responses.

After exposure to stress PTKs have been described to be involved in the induction of growth arrest and apoptosis. Exposure of human skin to UV induces major changes in the genetic program of the exposed cells leading to immediate and long term skin changes.

Although it can be assumed that UV-induced modifications of signal transduction involving PTKs regulate these processes, details as to the specific changes in PTK expression after UV exposure are unknown.

METHODS

To investigate PTK expression in normal human keratinocytes (HNK) we employed a reversed transcriptase-PCR approach using degenerate primers derived from the conserved catalytic domain of PTKs (table 1).

PCR products were cloned and PTKs from randomly picked colonies (up to n = 87 per screen) were identified by sequence analysis. PTK profiles of sham-irradiated, UVA (filtered metal halide lamp, 60 J/cm²), and UVB (filtered metal halide lamp, 256 mJ/cm²) treated HNK were analyzed 7 h after exposure.

Results were analyzed by chi-square test for statistical significance.

RESULTS

We identified 14 PTKs including 3 receptor kinases (axl, cak, fgfr2) and 11 non-receptor kinases (abl1, abl2, lck, map4k2, fyn, yes, src, cnk, ptk6, mstr1, jak1). The PTK profile of HNK was characterized by a predominance of abl2.

Differential screening revealed a further induction of abl2 expression by UVA. UVB had no influence on abl2 but predominantly induced the expression of the receptor kinases of the axl-family. Both treatments lead to a downregulation of src-family kinases (src, fyn, yes) (table 2, table 3).

Overall evaluation revealed that the effect of UVA and UVB on receptor vs. non-receptor kinases is reciprocal: UVA stimulates expression of non-receptor kinases and inhibits receptor PTKs, while UVB had the opposite effect (table 2, table 3).

The differences reached statistical significance at p<0.0001.

DISCUSSION

These results for the first time provide information on the PTK expression profile of HNK and its modification by UV.

The observed UV effects are wavelength dependent and affect PTKs which are involved in the regulation of gene transcription, cell death, and proliferation.

We conclude that regulation of PTK expression is part of genetic program that mediates late effects of UVA and UVB through specific alterations in phosphorylation dependent signalling cascades.
 
 

Table 1: Primer strategy and kinase domains of PTKs amplified from HNK. Conserved amino acids are indicated in bold letters.

Degenerate primers used for amplification of PTKs
5 primer
Protein     K       (V/I)   (A/S/T)   D       F       G
DNA         AA(A/G) (A/G)TN (A/G/T)CN GA(T/C) TT(T/C) GG
3 primer
Protein     G        (F/Y)   (S/A)   W   V      D
DNA       NCC (A/C)(A/T)A  NG(A/C) CCA NAC AG(T/C)

Receptor TK                                                                              name
dfg msrnlyagdyyrvqgravlpirwmawecilmgkfttas   dvw  cak
dfg lardinnidyykkttngrlpvkwmapealfdrvythqs   dvw  fgfr2
dfg lsrkiysgdyyrqgcasklpvkwlalesladnlytvhs   dvw  tyro3
Non-Receptor TK
dfg  lsrlmtgdtytahagakfpikwtapeslaynkfsiks   dvw  abl1
dfg  lsrlmtgdtytahagakfpikwtapeslayntfsiks   dvw  abl2
dfgltkaietdkeyytvkddrdspvfwyapeclmqskfyias   dvw  jak1
dfg  larliedneytaregakfpikwtapeainygtftiks   dvw  lck
dfg   vsgeltasvakrrsfigtpywmapevaaverkggynelcdvw  map4k2
dfg   larlikedvylshdhnipykwtapealsrghystks   dvw  ptk6
dfg  larliedneytarqgakfpikwtapeaalygrftiks   dvw  src
dfg  larliedneytarqgakfpikwtapeaalygrftiks   dvw  yes
dfg  larliedneytarqgakfpikwtapeaalygrftiks   dvw  fyn
dfg   laarleppeqrkkticgtpnyvapevllrqghgpea   dvw  cnk
 
 

Table 2: Frequency of PTKs in untreated HNK and after exposure to UVA and UVB. 14 individual kinases could be identified; numbers denote their absolute frequency in a randomly picked sample of clones.
 
 
con
UVA
UVB
cak*
6
4
12
fgfr2*
4
2
1
tyro3/axl*
10
7
26
abl1
10
9
10
abl2
23
56
17
cnk
0
0
2
fyn
7
0
3
jak1
0
0
1
lck
6
1
10
map4k2
2
0
1
mstr1
0
0
1
ptk6
0
0
2
src
2
0
0
yes
2
4
1
total
72
83
87

* receptor tyrosine kinase
 
 
con
UVA
UVB
receptor 
tyrosine kinase
20
13
39
non-receptor 
tyrosine kinase
52
70
48
total
72
83
87

Table 3: Relative frequency of PTKs in untreated HNK and after exposure to UVA and UVB. Numbers denote the percentage of each individual kinase calculated from table 1.
 
con
UVA
UVB
cak*
8 %
5 %
14 %
fgfr2*
6 %
2 %
1 %
tyro3/axl*
14 %
8 %
30 %
abl1
14 %
11 %
11 %
abl2
32 %
67 %
20 %
cnk
0 %
0 %
2 %
fyn
10 %
0 %
3 %
jak1
0 %
0 %
1 %
lck
8 %
1 %
11 %
map4k2
3 %
0 %
1 %
mstr1
0 %
0 %
1 %
ptk6
0 %
0 %
2 %
src
3 %
0 %
0 %
yes
3 %
5 %
1 %

* receptor tyrosine kinase
 
 
con
UVA
UVB
receptor 
tyrosine kinase
28 %
16 %
45 %
non-receptor 
tyrosine kinase
72 %
84 %
55 %