Báo cáo Y học: Evidence for general stabilization of mRNAs in response to UV light pdf

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 inhi

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 kinase and affects a broad spectrum of mRNAs

Keywords: AU-rich element; MAPKAP kinase 2; mRNA stability; p38 MAP kinase; UV light

Higher organisms respond to an external insult by switching

on the expression of certain genes the products of which are

involved in the defense against pathogens and in tissue

repair Pathogen-derived material, direct cell stress and

endogenous mediators activate gene expression at multiple

levels, including transcriptional activation as well as

post-transcriptional mechanisms The importance of the latter

has been demonstrated in gene-targeted mice where

over-production of inflammatory proteins due to dysregulation

of mRNA degradation or translation caused severe disease

of the animals [1–5]

The molecular basis underlying the regulation of mRNA

translation and decay is not completely understood An

important type of regulatory mRNA element are AU-rich

elements (AREs), which are found in the 3¢-UTRs of many

rapidly inducible genes such as oncogenes and cytokine genes

[6,7] By imposing rapid degradation on the transcript, the

AREs limit basal expression and allow rapid reversion to

basal mRNA levels subsequent to gene induction Stability and translation of ARE-containing transcripts can be affected by signaling mechanisms activated by the damaging agents directly or by released inflammatory cytokines Cell stressors, infectious pathogens and inflammatory cytokines activate various signaling pathways simulta-neously Extensive overlap exists in the sets of pathways activated by the different agents Pathways activated include NF-jB and the mitogen-activated protein (MAP) kinase 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 Ca2+ 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,

E-mail: holtmann.helmut@mh-hannover.de

Abbreviations: ARE, AU-rich element; GFP, green fluorescent

pro-tein; GM-CSF, granulocyte–macrophage colony-stimulating factor;

IL, interleukin; LPS, lipopolysaccharide; MAP, mitogen-activated

protein; MK2, MAP kinase-activated protein kinase 2 (also named

MAPKAP kinase 2); MKK6, MAP kinase kinase 6.

*Note: these two authors contributed equally to this work.

(Received 5 July 2002, revised 2 October 2002,

accepted 8 October 2002)

limited to AU-rich transcripts, whereas UV light stabilizes a

broad spectrum of mRNAs either containing or lacking

AU-rich elements by a mechanism that is independent of the

p38/MK2 pathway

M A T E R I A L S A N D M E T H O D S

Cells and materials

HeLa cells constitutively expressing the tet transactivator

protein [25] (kindly provided by H Bujard, Center for

Molecular Biology, University of Heidelberg, Germany)

were cultured in Dulbecco’s modified Eagle’s medium

complemented with 5% fetal bovine serum Plasmids

ptet-BBB-I18972)1310 and ptet-BBB-GM-CSFARE encode the

rabbit b-globin mRNA with AU-rich regions of the Il-8

and GM-CSF transcripts respectively inserted into the

BglII site of the b-globin 3¢-UTR [16,26] The pUHC13-3

plasmid, kindly donated by H Bujard, contains the

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

(MKK62E), dominant negative p38, dominant negative

and constitutively active MK2 have been described [16]

To generate HeLa cells with inducible expression of

active MKK6, the MKK62E 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

New Jersey, Piscataway, NJ, USA Mouse monoclonal

antibodies 19F12 against HuR and 9H10 against hnRNP

A1 were kindly donated by H Furneaux, University of

Conneticut Health Center, Farmington, CT, USA, and

G Dreyfuss, University of Pennsylvania School of

Medicine, Philadelphia, PA, USA, respectively

Transfections and reporter assays for mRNA stability

Transient transfections by the calcium phosphate method

and RNA degradation kinetics were performed as

described [16] Briefly, cells (5· 106 seeded per

9-cm-diameter dish) were transfected with the indicated

plas-mids Amounts of plasmid DNA within each experiment

were kept constant by adding empty vector For each

kinetics of mRNA degradation assay, cells from one dish

were trypsinized and distributed into parallel cultures to

ensure equal transfection efficiency within the group of

samples The next day transcription from the

tetracycline-regulatable promoter was stopped by addition of

doxycycline (3 lgÆmL)1) At the indicated times thereaf-ter, total RNA was isolated, and Northern-blot analysis was performed using digoxigenin-labeled antisense RNA probes RNA half lives were determined as in [16], using

a video imaging system and the MOLECULAR ANALYST program (Bio-Rad)

Preparation of cytoplasmic extracts Cytoplasmic extracts were prepared as described by Wang

et al [22] All steps were carried out in the cold The cells (106per sample) were washed once with NaCl/Pi, harvested

by scraping, pelleted by centrifugation, and resuspended in

200 lL hypotonic buffer (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 1 lgÆmL)1 leupeptin, 1 lgÆmL)1 aprotinin and 0.5 mM 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 assays

Labeled RNA (typically 107)108c.p.m.Ælg)1) was synthes-ized by incubating 1–3 lg linearsynthes-ized plasmid DNA in a mixture of 50 lCi [a-32P]UTP (400 CiÆmmol)1, Hartmann Analytic, Braunschweig, Germany), 18 lMunlabeled UTP, 0.5 mMunlabeled ATP, CTP and GTP, 2 UÆlL)1T7 RNA polymerase or T3 RNA polymerase as required (both enzymes from Roche) and 2 UÆlL)1RNase inhibitor (MBI) for 1 h at 37C RNase-free DNase (Roche) was then added to a final concentration of 1 UÆlL)1 After incuba-tion for 15 min at 37C 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· 105c.p.m.) were incubated with cytoplasmic extracts (6 lg protein per sample) in 20 lL buffer containing 20 mM Hepes, pH 7.9, 100 mM KCl, 2 mM MgCl2, 3% (v/v) glycerol, 0.5 mM dithiothreitol, 0.5 mM phenyl-methanesulfonyl fluoride, 5 lgÆmL)1 pepstatin A and

200 lgÆmL)1tRNA for 10 min at 30C RNase T1 (30 units/sample) was then added, and incubation continued for

20 min at 37C Where indicated, antibodies were included for the last 10 min Samples were electrophoresed on a nondenaturing polyacrylamide gel (5% acrylamide in 0.25· Tris/borate/EDTA buffer) The gels were dried and autoradiographed

Western blot andin vitro kinase assay HeLa cells were lysed in 20 mMHepes, pH 7.5, containing

50 mMKCl, 2 mMMgCl2, 0.5 mMdithiothreitol, 0.5 mM phenylmethanesulfonyl fluoride, 5 lgÆmL)1 pepstatin,

5 lgÆmL)1leupeptin, 30 mM NaF, 15 mM b-glycerophos-phate and 0.2% Nonidet P-40 After 10 min on ice, lysates were centrifuged for 5 min at 10 000 g, and supernatants were saved (cytoplasm) Expression of GFP-MKK62Ewas 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-PTM; Milipore) After

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-MKK62E was detected by using the SuperSignal

chemiluminescence system (Pierce) For in vitro kinase

assays, 20 lg cytoplasmic proteins were diluted in kinase

buffer (20 mM Tris/HCl, pH 7.4, 5 mM MgCl2, 0.2 mM

dithiothreitol, 0.1 mM EDTA, 0.1 mM EGTA, 20 mM

b-glycerophosphate, 1 mMphenylmethanesulfonyl fluoride,

10 lMATP), and 4 lCi [c-32P]ATP and 1 lg recombinant

HSP27 (kindly provided by Matthias Gaestel, Medical

School Hannover, Germany) were added After 30 min at

30C, SDS/PAGE sample buffer was added, and samples

were boiled for 5 min and separated by SDS/PAGE

Phosphorylated HSP27 was detected by autoradiography

of the dried gel

R E S U L T S

UV light induces stabilization of AU-rich mRNAs

independently of the p38 MAP kinase/MK2 pathway

Mechanisms that affect mRNA turnover were studied in

HeLa-tTA cells expressing the tetracycline-sensitive

trans-activator [25] The decay of mRNAs expressed with the

tet-offsystem was followed subsequent to inhibition of their

transcription by the tetracycline analog doxycycline The

b-globin mRNA exhibits a long half-life under these

conditions (> 10 h [16], and additional data, not shown)

Decay of ARE-containing transcripts was investigated by

expressing b-globin reporter constructs containing the

regulatory region of the IL-8 3¢-UTR (BBB-IL-8972)1310)

and, to minimize the chance of detecting effects specific only

for that region, with the well characterized ARE of

GM-CSF (BBB-GMCSFARE) 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-8972)1310 and

BBB-GMCSFARE (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 (p38AGF) or MK2 (MK2K76R) were

coex-pressed (Fig 2A) As expected, the expression of each of

the mutants strongly interfered with the stabilization

induced by MKK62E, a selective activator of p38 MAP

kinase In contrast, neither the dominant-negative p38

MAP kinase mutant nor the dominant negative MK2

mutant affected stabilization induced by UV light

(Fig 2A) In support of this result, the pyridinyl imidazole

SB 203580, a selective p38 MAP kinase inhibitor, inhibited

mRNA stabilization induced by MKK62E, whereas it did

not have a significant effect on stabilization by UV light

(Fig 2B) The data indicate that stabilization in response to

UV light occurs independently of the p38 MAP kinase/

MK2 pathway

Fig 1 UV light induces stabilization of AU-rich mRNAs (A) Scheme

of mRNAs expressed b-Globin mRNAs with the ARE of GM-CSF (BBB-GMCSF ARE ) (Fig 1A) or with an ARE-containing region of IL-8 mRNA (BBB-IL-8 972 )1310 ) were expressed under the control of a tetracycline-regulatable promoter (for details see Materials and methods) (B) HeLa cells constitutively expressing the tet transactiva-tor protein were transfected with ptetBBB-IL-8972)1310and ptetBBB-GM-CSF 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-8972)1310 mRNA from an experiment performed as in (B), but with different doses of UV light.

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 ) or dominant negative MK2 (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-8972)1310 were quantified as described in Materials and Methods.

UV light but not activation of p38 MAP kinase increases

cytoplasmic HuR-binding activity

Analysis of protein–RNA binding showed that several

complexes were formed when GM-CSF ARE is incubated

with cytoplasmic extracts from control cells (Fig 3A, lane

1) Formation of two of the complexes increased with cytoplasmic extracts from UV-treated cells (Fig 3A, lanes 1–4) It has been reported recently that UV light induces translocation of the RNA-binding protein HuR to the cytoplasm, an effect that contributes to UV-induced stabi-lization of p21/WAF message [22] In accordance with that study, the two complexes modulated by UV light were supershifted by antibodies against HuR (Fig 3A, lanes 5–7) Antibodies against AUF1 induced a supershift of a different complex which was not influenced by UV treat-ment (lane 8), whereas antibodies against hnRNP-A1 did not affect any of the complexes (lane 9) The increase in formation of HuR-containing complexes on exposure to

UV light was also observed with the AU-rich IL-8 mRNA fragment (Fig 3A, lanes 10–12) Expression of Myc-tagged HuR increased the amounts of both UV-inducible com-plexes, and a further increase was observed in response to

UV light (Fig 3B, compare lanes 1 and 2 with lanes 3 and 4) On the other hand, active MKK6 did not result in any detectable change in the amounts of complexes containing coexpressed Myc-tagged HuR (Fig 3B, lanes 5 and 6), nor

in the pattern of other complexes formed with the RNA (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 MKK62E induced stabilization of the two ARE-containing mRNAs (BBB-GMCSFAREand BBB-IL8972-1310) but failed to exert any effect on the degradation rate of the GFP, luciferase and CAT-TIMP1 mRNAs (Fig 4A) In contrast, exposure

to UV light resulted in stabilization of all mRNAs investigated, including the short-lived CAT-TIMP1 and luciferase mRNAs as well as the long-lived GFP mRNA,

Fig 3 Effects of UV light and activation of p38/MK2 on complex

formation between AU-rich mRNAs and cytoplasmic proteins (A) HeLa

cells were left untreated or exposed to UV light (doses 1200 JÆm)2or as

indicated) Cytoplasmic extracts were prepared and incubated with

radiolabeled in vitro-transcribed RNAs consisting of the ARE of

GM-CSF or the AU-rich region of the IL-8 3¢-UTR (nucleotides

972–1310) Where indicated, antibodies specific for HuR (0.75 lg),

AUF1 (1 lL of a 1 : 10 dilution of serum) or hnRNP A1 (1 lL of a

1 : 10 dilution of ascites) were included Protein–RNA complexes were

separated on nondenaturing polyacrylamide gels and detected by

autoradiography Filled arrowheads indicate UV-induced complexes,

and open arrowheads indicate complexes supershifted by antibodies.

(B) HeLa cells transfected with expression vectors for Myc-tagged

HuR or MKK6 2E as indicated were left untreated or exposed to UV

light (1200 JÆm)2) Interaction of proteins from cytoplasmic extracts

with labeled GM-CSF ARE RNA was assayed as described in (A).

Fig 4 UV light and the p38/MK2 pathway induce mRNA stabilization with different transcript selectivities (A) Degradation of the indicated mRNAs, all expressed under the control of a tetracycline-regulatable promoter, was determined in HeLa cells expressing MKK6 2E or exposed to UV light as described in Fig 1 (B) Degradation of the indicated mRNAs was compared in cells transfected with an expres-sion vector for MEKK1D or with empty vector as control.

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 degradation of endogenous histone mRNA As shown in Fig 5A, UV light also induced stabilization of the endo-genous mRNA The histone transcripts do not contain an ARE in their 3¢-UTR, confirming the results in Fig 4 that this type of regulatory element is not essential for UV-induced stabilization Unlike most other mRNAs, the histone transcripts are not polyadenylated [32] Therefore this result indicates that the poly(A) tail is also dispensable

Fig 5 UV light but not activation of the p38/MK2 pathway induces stabilization of endogenous histone mRNA (A) Degradation of endogenous histone mRNA in untreated and UV-exposed cells was determined by Northern-blot analysis of total RNA isolated at the indicated times after inhibition of transcription with actinomycin D (5 lgÆmL)1) (B) HeLa cells stably transfected with a tetracycline-regulated expression vector for GFP-MKK6 2E were incubated for 24 h with tetracycline (100 ngÆmL)1) to suppress GFP-MKK6 2E expression, or without tetracycline to allow GFP-MKK6 2E expression, and with SB 203580 (2 l M ) as indicated Cytoplasmic lysates were assayed for GFP-MKK6 2E protein by Western blot, and for activation of the p38 MAP kinase/MK2 pathway by in vitro kinase assays with recombinant HSP27 as substrate (C) Stable HeLa cell transfectants were kept with (control, UV) or without (GFP-MKK6 2E ) 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).

for UV-induced stabilization The effect of the p38 MAP

kinase pathway on the stability of endogenous histone

mRNA was assayed in cells stably transfected with a

plasmid encoding constitutively active MKK62E 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-MKK62Edownstream of the tetracycline-regulatable

pro-moter Removal of tetracycline from the culture medium

resulted in GFP-MKK62Eexpression 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

of untransfected HeLa cells assayed in parallel (62 vs

68 min; not shown) Omission of tetracycline to induce

expression of GFP-MKK62Edid not alter histone mRNA

stability (Fig 5C), indicating that the p38 MAP kinase

pathway does not affect its degradation UV light induced

stabilization of the transcript, confirming the result in

untransfected cells shown in Fig 5A Importantly, the

addition of SB 203580, which inhibited p38 MAP kinase

activity (Fig 5B, lower panel), did not impair UV-induced

stabilization (Fig 5C) These results illustrate that UV light

can induce stabilization of a non-AU-rich endogenous

mRNA in a p38 MAP kinase-independent manner

The results in Figs 4 and 5 provide evidence for two

distinct modes of mRNA stabilization: selective,

ARE-dependent stabilization of mRNAs by activation of the p38

MAP kinase cascade, as opposed to a general mechanism of

stabilization triggered by UV light

D I S C U S S I O N

Extracellular stimuli affect gene expression in part by

modulating the degradation rate of mRNAs In the present

study we investigated the effect of UV light on the stability

of mRNAs expressed using the tet-off system which allows

the termination of only the synthesis of the mRNA under

study This enabled us to also investigate transcripts with

comparatively long half-lives The data in Fig 4 indicate

that UV light exerts a strong stabilizing effect on a range of

different mRNAs without apparent selectivity Most

no-tably, UV light stabilized mRNAs with a 3¢-UTR that did

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 was also insensitive to the inhibitor, as well as to coexpres-sion of inhibitory mutants of the p38 and MK2 kinases (Fig 2) Insensitivity of UV-induced stabilization to each of the two mutants alone would have left the possibility that p38 MAP kinase activates a different downstream compo-nent, or that MK2 is activated by a different upstream component The lackof inhibition by both kinase mutants together strongly indicates that the effect of UV light on mRNA stability is independent of the p38 MAP kinase/ MK2 pathway It cannot be excluded, however, that a p38/ MK2-dependent component of UV-induced mRNA stabil-ization may be detected if the predominant mechanism is inhibited

The mechanism of UV-induced stabilization remains unidentified The lackof stabilization of non-ARE tran-scripts by MEKK1 (Fig 4B), which in our cells is an effective activator of p38, JNK and NF-jB pathways, renders involvement of these pathways in this effect of UV light highly unlikely

Others have shown that UV-induced stabilization of p21 involves translocation of HuR to the cytoplasm [22] HuR can bind to AU-rich sequences [33], including those of the GM-CSF and IL-8 mRNAs (Fig 3) Although we also observed increased binding of HuR to these RNAs in the cytoplasm of cells exposed to UV light, the non-ARE transcripts did not interact with HuR in vitro (not shown) This may be due to technical limitations to detect such interactions in gel shifts, and/or to a more indirect association of HuR with those transcripts Alternatively, HuR may affect only the fate of a subset (i.e ARE-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

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

synthesis is impaired

In summary, the data presented here and in the literature

cited point to two different modes of mRNA stabilization

triggered by inflammatory stimuli and UV light The p38/

MK2 pathway, activated by inflammatory stimuli such as

IL-1 and LPS, induces stabilization of various

ARE-containing mRNAs Independently of this pathway, UV

light activates a more general mechanism of mRNA

stabilization, one that does not require an ARE or a

poly(A) tail as a cis-element

A C K N O W L E D G E M E N T S

This workwas supported by the Deutsche Forschungsgemeinschaft

(grants Ho1116/2, GK-GRK 99/2-98, SFB 566/A10 and Kr1143/2-3,

SFB 566/B06) We are grateful to Hermann Bujard, Matthias Gaestel

and Ann-Bin Shyu for providing plasmids and to Gary Brewer, Gideon

Dreyfuss and Henry Furneaux for providing antibodies.

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