Evidence for general stabilization of mRNAs in response to UV light
Frank Bollig*, Reinhard Winzen*, Michael Kracht, Beniam Ghebremedhin, Birgit Ritter, Arno Wilhelm,
Klaus Resch and Helmut Holtmann
Institute of Pharmacology, Medical School Hannover, Germany
mRNA stabilization plays an important role in the changes
in protein expression initiated by inducers of inflammation
or direct cell stress such as UV light. This study provides
evidence that stabilization in response to UV light differs
from that induced by proinflammatory stimuli such as
bacterial lipopolysaccharide or interleukin (IL)-1. Firstly,
UV-induced stabilization is independent of the p38 MAP
kinase pathway, which has previously been shown to medi-
ate stabilization induced by IL-1 or lipopolysaccharide.
UV-induced mRNA stabilization was insensitive to the
dominant negative forms of p38 MAP kinase and its sub-
strate MAP kinase-activated protein kinase 2 (MK2), or to
the p38 MAP kinase inhibitor SB 203580, demonstrating
that it occurs through a different signaling mechanism.
Secondly, UV-induced stabilization exhibits a different
transcript selectivity. Activation of the p38 MAP kinase
pathway, by expressing active MAP kinase kinase 6, induced
stabilization only of transcripts containing AU-rich
elements. UV light also induced stabilization of transcripts
lacking AU-rich elements. This effect could not be mimicked
by expressing MEKK1, an upstream activator of the p38,
JNK, ERK and NF-jB pathways. UV light also stabilized
endogenous histone mRNA, which lacks AU-rich elements
and a poly(A) tail. This effect was not mimicked by active
MAP kinase kinase 6 and not sensitive to a p38 MAP kinase
inhibitor. This suggests that UV light induces stabilization
through a mechanism that is independent of p38 MAP

cascades. The JNK pathway has been reported to stabilize
the short-lived interleukin (IL)-2 mRNA on activation of the
T-cell line Jurkat [8] and the IL-3 mRNA in the murine mast
cell line PB-3c [9]. Several groups have shown an mRNA-
stabilizing effect of protein kinase C activation and/or
increased intracellular Ca
2+
concentrations [6,8–12]. The
results of others, including our own, show that stabilization
of several ARE-containing mRNAs, triggered by IL-1 or
bacterial lipopolysaccharide (LPS), involves activation of
p38 MAP kinase [13–17] and its substrate MAP kinase-
activated protein kinase 2 (MK2) [16–18]. Consistent with
these findings, MK2-deficient mice exhibit reduced synthesis
of several cytokines in response to LPS [19].
Similarly to LPS and IL-1, UV light is a potent inducer of
inflammation and induces expression of numerous genes
including cytokines and oncogenes [20,21], which is in part
due to the stabilization of mRNAs [22,23]. UV light
strongly activates stress signaling pathways, including the
p38/MK2 pathway [24]. However, the signaling mecha-
nisms involved in mRNA stabilization in response to UV
light have not been identified, nor has the transcript
selectivity of UV-induced stabilization been defined.
In this study, we show that, in HeLa cells, mRNA
stabilization induced through the p38/MK2 pathway is
Correspondence to H. Holtmann, Institute of Pharmacology,
Medical School Hannover, Carl-Neuberg Strasse-1,
D-30625 Hannover, Germany.
Fax: + 49 511 5324081, Tel.: + 49 511 5322800,

Photinus pyralis luciferase cDNA downstream of a
tetracycline-regulated promoter [25]. pUHD10-CAT-
TIMP1 was generated by excising the IL-8 fragment of
pUHD10-CAT-IL-8 [16] with BamHI and inserting a
fragment of human TIMP1 (nucleotide 19–782, accession
no. NM_003254) generated by RT-PCR with primers
containing BamH1 sites. To obtain pUHD10-GFP a
fragment of pEGFP-C1 (Clontech) including the green
fluorescent protein (GFP) cDNA and 3¢ adjacent restric-
tion sites was amplified with XbaI-flanked primers and
inserted into the XbaI site of pUHD10.3 [25]. Expression
plasmids for constitutively active MAP kinase kinase 6
(MKK6
2E
), dominant negative p38, dominant negative
and constitutively active MK2 have been described [16].
To generate HeLa cells with inducible expression of
active MKK6, the MKK6
2E
cDNA was placed in-frame
downstream of the GFP cDNA in pUHD10-GFP. HeLa
cells were cotransfected with this plasmid and a plasmid
for puromycin resistance, and stable transfectants selected
by culture in 1 lgÆmL
)1
puromycin. Myc-tagged HuR
was expressed with the plasmid pTet-Myc-over-HuR [27]
(a gift from A B. Shyu, University of Texas, Houston,
TX, USA). Rabbit antiserum against AUF1 was a gift
from G. Brewer, University of Medicine and Dentistry of

Cytoplasmic extracts were prepared as described by Wang
et al. [22]. All steps were carried out in the cold. The cells
(10
6
per sample) were washed once with NaCl/P
i
,harvested
by scraping, pelleted by centrifugation, and resuspended in
200 lL hypotonic buffer (10 m
M
Hepes, pH 7.9, 10 m
M
KCl, 1.5 m
M
MgCl
2
,1lgÆmL
)1
leupeptin, 1 lgÆmL
)1
aprotinin and 0.5 m
M
phenylmethanesulfonyl fluoride).
Then 25 lL of the same buffer including 2.5% (v/v)
Nonidet P-40 was added. After centrifugation at 1000 g
for 4 min, the supernatants were removed, freeze–thawed
five times, and cleared by centrifugation. Aliquots were
frozen at )70 °C.
In vitro
transcription and electrophoretic mobility-shift

tion for 15 min at 37 °C the RNA was passed through a
NucTrap push column (Stratagene) to remove free nucle-
otides, and stored at )80 °C. Radiolabeled RNA probes
(1.5 · 10
5
c.p.m.) were incubated with cytoplasmic extracts
(6 lg protein per sample) in 20 lL buffer containing 20 m
M
Hepes, pH 7.9, 100 m
M
KCl, 2 m
M
MgCl
2
, 3% (v/v)
glycerol, 0.5 m
M
dithiothreitol, 0.5 m
M
phenyl-
methanesulfonyl fluoride, 5 lgÆmL
)1
pepstatin A and
200 lgÆmL
)1
tRNA for 10 min at 30 °C. RNase T1 (30
units/sample) was then added, and incubation continued for
20 min at 37 °C. Where indicated, antibodies were included
for the last 10 min. Samples were electrophoresed on a
nondenaturing polyacrylamide gel (5% acrylamide in

were saved (cytoplasm). Expression of GFP-MKK6
2E
was
analyzed by Western blotting as described elsewhere [16].
Briefly, cytoplasmic proteins were separated by SDS/PAGE
and electrophoretically transferred to poly(vinylidene
difluoride) membranes (Immobilon-P
TM
;Milipore).After
Ó FEBS 2002 General mRNA stabilization by UV light (Eur. J. Biochem. 269) 5831
blocking with 5% dried milk in Tris-buffered saline, the
membranes were incubated with monoclonal antibodies
against GFP (Roche Diagnostics) for 16 h, washed and
incubated with peroxidase-coupled second antibody.
GFP-MKK6
2E
was detected by using the SuperSignalÒ
chemiluminescence system (Pierce). For in vitro kinase
assays, 20 lg cytoplasmic proteins were diluted in kinase
buffer (20 m
M
Tris/HCl, pH 7.4, 5 m
M
MgCl
2
,0.2m
M
dithiothreitol, 0.1 m
M
EDTA, 0.1 m

972)1310
)
and, to minimize the chance of detecting effects specific only
for that region, with the well characterized ARE of
GM-CSF (BBB-GMCSF
ARE
). In agreement with previous
studies [6,16,26], the mRNAs derived from both constructs
were rapidly degraded in unstimulated cells (Fig. 1B).
Exposure to UV light (UV-B) induced marked and dose-
dependent stabilization of both hybrid mRNAs (Fig. 1B,C).
According to kinetic studies, the increase in stability
persisted for about 14 h after exposure to UV light and
gradually disappeared thereafter (not shown).
As reported previously [16], activators of the p38
MAP kinase/MK2 pathway induce stabilization of
AU-rich mRNAs, including BBB-IL-8
972)1310
and BBB-
GMCSF
ARE
(Fig. 1A). To determine whether mRNA
stabilization induced by UV light also involved the p38
MAP kinase pathway, dominant-negative mutants of p38
MAP kinase (p38
AGF
) or MK2 (MK2
K76R
)werecoex-
pressed (Fig. 2A). As expected, the expression of each of

ARE
. At 2 h after UV-B exposure (1200 JÆm
)2
), doxycycline
(3 lgÆmL
)1
) was added. Total RNA was isolated at the indicated times
and analyzed by Northern blotting. Ethidium bromide staining of 28S
rRNA is shown to allow comparison of RNA amounts loaded. (C)
Quantification of results for BBB-IL-8
972)1310
mRNA from an
experiment performed as in (B), but with different doses of UV light.
5832 F. Bollig et al.(Eur. J. Biochem. 269) Ó FEBS 2002
Fig. 2. mRNA stabilization by UV light is independent of the p38/MK2 pathway. Degradation of BBB-IL-8
972)1310
and BBB-GM-CSF
ARE
tran-
scripts was determined as in Fig. 1 in untreated or UV-exposed HeLa cells cotransfected with empty vector or expression vectors for constitutively
active MKK6 (MKK6
2E
). (A) Plasmids encoding dominant negative p38 MAP kinase (p38
AGF
)ordominantnegativeMK2(MK2
K76R
)were
cotransfected as indicated. (B) Cells received SB 203580 (2 l
M
) or vehicle 3 h before the assay of RNA stability. Half-lives for BBB-IL-8

(additional results, not shown).
UV light but not p38 MAP kinase activation induces
stabilization of non-AU-rich mRNAs
The observation that stabilization of mRNAs on exposure
to UV light is independent of the p38 MAP kinase pathway
suggested that the mechanism of stabilization itself may be
different. To elucidate this, transcript selectivity of the two
ways of inducing stabilization was compared. For this
purpose, additional mRNAs lacking AU-rich sequences
were included in the experiments (Fig. 4). The GFP mRNA
contains a short 3¢-UTR that consists of vector-derived
sequences. The luciferase mRNA derived from the plasmid
pUHC13-3 [25] harbors a long 3¢-UTR with regions of high
A + U content, but with no overlapping AUUUA motifs
nor UUAUUUA U/A U/A motif suggested to confer
regulation of stability [28–30]. The TIMP1 cDNA was
cloned downstream of a 196-nucleotide CAT fragment to
express a CAT-TIMP1 hybrid RNA that can be distin-
guished from endogenous TIMP1 transcript. The 3¢-UTR
of TIMP1 is short (96 nucleotides) and devoid of AU-rich
regions. The basal half-life of the GFP transcript is long
(5 h), whereas that of luciferase and CAT-TIMP1 tran-
scripts is rather short (Fig. 4A). Expression of MKK6
2E
induced stabilization of the two ARE-containing mRNAs
(BBB-GMCSF
ARE
and BBB-IL8
972-1310
) but failed to exert

promoter, was determined in HeLa cells expressing MKK6
2E
or
exposed to UV light as described in Fig. 1. (B) Degradation of the
indicatedmRNAswascomparedincellstransfectedwithanexpres-
sion vector for MEKK1D or with empty vector as control.
5834 F. Bollig et al.(Eur. J. Biochem. 269) Ó FEBS 2002
Ó FEBS 2002 General mRNA stabilization by UV light (Eur. J. Biochem. 269) 5835
the half-life of which was reproducibly found to be extended
(Fig. 4A). This indicates that UV light can induce stabi-
lization of a much broader spectrum of mRNAs including
species containing and lacking AREs in their 3¢-UTR. Of
note, the mRNAs devoid of AU-rich sequences did not
interact with HuR in electrophoretic mobility-shift assays
(data not shown).
Expression of a truncated mutant of the MAP triple
kinase MEKK1 (MEKK1-D) is known to activate the p38
MAP kinase and also the JNK, ERK and NF-jB signaling
pathways ([16,31] and references cited therein). Expression
of MEKK1-D, in agreement with previously published
results [16], induced stabilization of the two AU-rich
mRNAs. However, it did not induce stabilization of the
non-AU-rich mRNAs (Fig. 4B). This indicates that
the pathways activated by MEKK1-D are not sufficient
to induce the UV-activated mechanism of mRNA
stabilization.
UV light but not p38 MAP kinase activation induces
stabilization of endogenous histone mRNA
To ensure that the results are not only applicable to mRNAs
expressed from transfected plasmids, we investigated the

) tetracycline to suppress or to allow GFP-MKK6
2E
expression, respectively,
as described for (B). SB 203580 (2 l
M
) was added where indicated. Exposure to UV light (UV) was performed 2 h before addition of actinomycin
D. Degradation of histone mRNA was determined as in (A).
5836 F. Bollig et al.(Eur. J. Biochem. 269) Ó FEBS 2002
for UV-induced stabilization. The effect of the p38 MAP
kinase pathway on the stability of endogenous histone
mRNAwasassayedincellsstablytransfectedwitha
plasmid encoding constitutively active MKK6
2E
.This
procedure allowed the expression of the active kinase in
all cells as opposed to transient transfection which affects
only part of the cells. The stable transfectants express GFP-
MKK6
2E
downstream of the tetracycline-regulatable pro-
moter. Removal of tetracycline from the culture medium
resulted in GFP-MKK6
2E
expression as observed by green
fluorescence (not shown) and Western blot (Fig. 5B, upper
panel). It also resulted in activation of the p38 MAP kinase
pathway, as determined by phosphorylation of recombinant
HSP27 in in vitro kinase assays with cytoplasmic lysates
(Fig. 5B, lower panel). The half-life of endogenous histone
mRNA in the cells kept with tetracycline was similar to that

not contain an ARE, a type of cis-element involved in the
control of many cytokine and oncogene mRNAs (e.g.
[6,7,16]).
Results corresponding to mRNAs derived from trans-
fected tet-off plasmids were obtained with endogenous
histone mRNA (Fig. 5). Although lacking an ARE, histone
mRNA was also markedly stabilized on treatment of cells
with UV light. Furthermore, as histone mRNA does not
carry a poly(A) tail at its 3¢ end [32], this structural element
common to most other mRNAs is apparently also dispen-
sable for UV-induced stabilization.
The inflammatory stimuli LPS and IL-1 induce stabi-
lization of several AU-rich mRNAs by activating the p38
MAP kinase/MK2 pathway [16,18]. However, p38 MAP
kinase activation did not affect the degradation of non-ARE-
containing transcripts, as shown for the plasmid-derived
mRNAs (Fig. 4) as well as for the endogenous histone
mRNA (Fig. 5). Thus with respect to selectivity of the
mRNAs affected, the effect of UV light differs from
stabilization induced by the p38 MAP kinase/MK2 pathway.
Although UV light activates p38 MAP kinase and MK2
[24], stabilization of mRNAs in response to UV light occurs
mainly through a mechanism independent of these kinases.
This is suggested by the different transcript selectivity of
stabilization by UV light as opposed to activation of the
p38/MK2 pathway (discussed above), as well as by the
results obtained when this pathway is inhibited. The p38
MAP kinase inhibitor SB 203580 failed to inhibit UV-
induced stabilization of endogenous histone mRNA
(Fig. 5). Stabilization of ARE-containing reporter mRNAs

containing) of mRNAs in response to UV light. Of note,
activation of the p38 pathway did not seem to affect HuR
binding to the GM-CSF ARE. This is in agreement with a
recent report by Dean and colleagues [34] who did not
observe a change in levels of HuR in p100 or nuclear
fractions of a macrophage-like cell line in response to LPS.
UV light, unlike IL-1, damages cells directly. It induces
activation of caspases and cell death. Under the condi-
tions applied in this study, about 30% of the cells
undergo apoptosis within 24 h of UV exposure. Caspases
have been shown to cleave translation initiation factors
[35]. This effect is expected to alter mRNA metabolism
and could also be involved in the stabilization observed.
However, addition of the caspase inhibitor Z-VAD.fmk
effectively reduced apoptosis in response to UV light, but
did not interfere with the UV-induced mRNA stabiliza-
tion (not shown). This argues against the involvement of
caspase activation.
One of the direct effects of UV light is the site-specific
damage of 28S rRNA [36]. This has been suggested to be
Ó FEBS 2002 General mRNA stabilization by UV light (Eur. J. Biochem. 269) 5837
involved in triggering of the ribotoxic stress response which
includes translational inhibition and activation of stress
kinase pathways and gene expression. It is possible that
ribosomal damage or its consequences leads to inhibition of
mRNA decay mechanisms. As it is known that UV light
also targets the transcriptional machinery [37,38], stabiliza-
tion of mRNAs may represent an element of the cellular
response to direct damage. This may serve to prevent
mRNAs from being degraded in conditions where new

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