JOURNAL OF INTERFERON AND CYTOKINE RESEARCH 17:747-755 (1997)Mary Ann Liebert, Inc.

Stabilization of Invariant Chain mRNA by12-0-Tetradecanoylphorbol-13-Acetate Is Blocked by IFN-

in a Murine Lymphoma Cell Line

NENG-YAO SHIH, IRENE SOESILO, and GEORGIA FLOYD-SMITH

ABSTRACT

Activation of protein kinase C (PKC) by 12-0-tetradecanoylphorbol-13-acetate (TPA) increased steady-statelevels of mRNA encoding the major histocompatibility complex (MHC) class II antigen I-A^ and the class IIantigen-associated invariant chain (Ii, CD74) in A20 lymphoma cells and in normal mouse cells. The in¬crease in Ii mRNA levels appeared to be due to a slight increase in the rate of gene transcription and an in¬crease in the stability of Ii mRNA. The half-life of Ii mRNA increased from 12 h to >24 h following treat¬ment with TPA, as determined by Northern blot analysis following actinomycin D treatment or by the[3H]-uridine pulse-chase method. Interferon-y (IFN-y), which has been well characterized as a cytokine thatinduces class II antigens and the Ii, increased Ii expression sjightly in A20 cells. However, cotreatment of cellswith TPA and IFN-y resulted in a block in the TPA-induced increase in Ii expression. Transcription of theli gene was minimally affected following treatment with IFN-y alone, and cells treated with both TPA andIFN-y had the same transcription rate as the control cells. IFN-y did, however, block stabilization of Ii mRNAby TPA. Activation of PKC by TPA, which was previously shown to lead to membrane translocation anddownregulation, was not inhibited by IFN-y. Therefore, IFN-y appeared to block a downstream signal trans¬duction pathway activated by PKC that controls stability of Ii mRNA.

INTRODUCTION

The protein kinase C (PKC) FAMILY of enzymes can mod¬ulate cellular physiology by controlling the expression of

regulated target genes.(1'2' Isoforms of PKC have been classi¬fied into three groups: (1) the conventional isoforms, PKC-a,PKC-jSI, PKC-0II, and PKC- , which require Ca2+, diacyl-glycerol (DAG), and phosphatidylserine for activation, the new

isoforms, PKC- , PKC-e, PKC-r, (L), PKC-0, and PKC-µ,which require DAG and phosphatidylserine but not Ca2+ foractivation, and the atypical isoforms, which may be activatedby phosphatidylserine, free fatty acids, and other lipid secondmessengers but not DAG or Ca2+.(2'3) The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), is a potent activatorof conventional PKCs and novel PKCs but not atypical PKCs.Activators of PKC can induce or increase expression of the ma¬

jor histocompatibility complex (MHC) class IT antigens*4,5* andan associated invariant chain (Ii),*6' which is essential for anti¬gen processing and presentation by MHC class II antigens.<7·8)TPA treatment increases Ii mRNA levels 5-8-fold in A20cells,(6) a cell line that expresses high levels of Ii constitu¬

tively.<6·9·10' DAG, the natural activator of PKC, also increasesIi mRNA levels in A20 cells, and specific PKC inhibitors can

block this increase.Expression of the MHC class II and Ii genes, with few ex¬

ceptions,*11·12' is coregulated.*13' Both MHC class II antigensand Ii are constitutively expressed in lymphocytes, dendriticcells, and macrophages, and both can be induced in other celltypes by interferon-y (LFN-y). IFN-yhas been extensively char¬acterized as a cytokine that activates a signal transducer and ac¬

tivator of transcription (Stat) protein, Static*, on binding to itsreceptor.*14' Stati dimerizes and then binds to a transcriptionalregulatory element called the gamma-TFN activation sequence(GAS), leading to rapid induction of a set of genes containingthis element. The promotor regions of MHC class II antigensand Ii do not, however, contain GAS elements but do containconserved sequences designated the H, X, and Y boxes thatfunction as constitutive and IFN-y-inducible enhancers.*13'

PKC activators, such as TPA, have been well characterizedas agents that induce gene transcription.*15*16> However, it isunclear how these agents might modulate expression of MHCclass II or Ii genes. As TPA can also selectively stabilize*17·18'

Molecular and Cellular Biology Program and Department of Biology, Arizona State University, Tempe, Arizona.

747

748 SHIH ET AL.

or destabilize niRNA,*19~21' the mechanism by which TPA in¬creases Ii mRNA levels was investigated. In this report, induc¬tion of the Ii by TPA is shown to be primarily due to increasedstability of Ii mRNA. Addition of IFN-y to TPA-treated cellsblocks stabilization of Ii mRNA. This is not because of down-regulation or inactivation of PKC-a or PKC- but rather ap¬pears to be the result of an TFN-y-mediated block in a PKC-mediated signaling pathway that controls Ii mRNA stability.

tion of goat antimouse immunoglobulin conjugated to horse¬radish peroxidase (Gibco-BRL) was used as a secondary anti¬body. A l:750-fold dilution of polyclonal antiscrum against theC-terminus of PKC- (amino acid 662-673; R&D Systems,Minneapolis, MN) was used to detect PKC-r?6' and the boundantibodies detected by addition of a 1:1000-fold dilution of goatantirabbit immunoglobulin (Gibco-BRL) conjugated to horse¬radish peroxidase. Horseradish peroxidase activity was detectedusing a chemiluminescent substrate and autoradiography.

MATERIALS AND METHODS

Cells

A20 cells were obtained from American Type Culture Col¬lection (Rockville, MD) and maintained as previously de¬scribed.*6' Before treatment with TPA, LFN-y, or equivalent vol¬ume of the solvent controls, DMSO and PBS, the cells were

transferred to tissue culture dishes in fresh medium at a celldensity of 2-3 X 106/ml.

Whole spleens were obtained from 3^1-month-old Balb/cmice. Nonadherent cells were extruded from punctured spleensby injection of RPMI-1640 medium. The cells were purifiedas described elsewhere'22' and further purified by centrifuga¬tion on Ficoll-Paque as recommended by the manufacturer(Pharmacia Biotech, Piscataway, NJ). The cells were resus¬

pended in RPMI-1640 plus 10% FBS and incubated at 37°Cfor 2 h before treatment.

Immunodetection of li, PKC-a, and PKC-h

For detection of Ii, homogenates were prepared from 5 X107 cells/treatment in 500 /xl of ice-cold homogenization buffer[20 mM Tris-HCl, pH 7.4, 5 mM EGTA, 1 mM phenyl-methylsulfonyl fluoride (PMSF), 5 /¿g/ml leupeptin, 5 /Ag/mlaprotinin, 1% Triton X-100] by 30 strokes of a tightly fittingDounce homogenizer as described elsewhere*21' and analyzedon Western blots using 1 µg/ml of the monoclonal antibody(mAb), In-1 (J. Miller, University of Chicago, Chicago, IL), ap¬plied to the membranes at 4°C overnight. Goat antirat antisera,conjugated with horseradish peroxidase (Gibco-BRL, Gaithers¬burg, MD) was subsequently added at a l:3000-fold dilution.Bound antibody was detected using a chemiluminescent sub¬strate (LumiGLO, Kirkegaard and Perry Laboratories, Gaithers¬burg, MD) and autoradiography. Western blots were quantitatedby densitometry (Bioimage, Millipore, Bedford, MA).

For detection of PKC-a and PKC- , cell homogenates were

prepared and fractionated into the cytoplasmic and membranefractions as done previously.*21' PKC-a was detected using a 3/ig/ml dilution of monoclonal anticatalytic domain (UpstateBiotechnology Incorporated, Lake Placid, NY). A 1:1250 dilu-

Northern blot analysisTotal cellular RNA was extracted from control and treated

cells using the method of Chomczynski and Sacchi*23' withmodifications described by Puissant and Houdebine.*24' Twentymicrogram samples were resolved on 1.2% agarose gels andtransferred to Magna Graph nylon membranes as recommendedby the manufacturer (MSI, Wcstboro, MA). Synthetic oligonu-cleotide probes complementary to Ii, I- ß, or GAPDH mRNA(Table 1) were labeled at their 3'-termini using terminal trans-ferase and |a-32P]dATP (specific activity 3000 Ci/mmol), as

described by Deng and Wu.'25' Labeled oligonucleotide probes(5 X 106 cpm/ml) were hybridized to the Northern blots as de¬scribed previously.*6,10' Labeled mRNAs were visualized by au¬

toradiography and quantitated by densitometry (Bioimage, Mil¬lipore, Bedford, MA). Autoradiogram exposures were withinthe linear response range for the film as determined using a cal¬ibrated standard. Significance was determined as described pre¬viously.'6,21'Nuclear runoff analysis

Cells were lysed in NP-40 lysis buffer (10 mM Tris-Cl, pH7.4, 10 mM NaCl, 5 mM MgCl2, 0.5% NP-40) and centrifugedat 500g for 5 min at 4°C. Nuclei were quick-frozen in liquidnitrogen and stored in 30% glycerol storage buffer (50 mM Tris-Cl, pH 8.0, 2 mM MgCl2, 0.1 mM EDNA, 30% glycerol) at-70°C. Transcription elongation reactions (400 µ ) were per¬formed in buffer (5 mM Tris-HCl, pH 8.0, 150 mM KC1, 2.5mM MgCl2, 2.5 mM DTT, 0.5 mM ATP, 0.5 mM CTP, 0.5mM GTP) containing 100 pJCi of [a-32P]UTP (3000 Ci/mmol,Dupont NEM, Boston, MA) at 26°C for 30 min.*21' The labelednuclear transcripts were purified and counted in a liquid scin¬tillation analyzer (Packard Instrument Co., Meriden, CT).

Full-length li and GAPDH cDNA inserts were purified frompcEXV3-mIi31 *26' and pB7,*27' respectively, and 2-3 µg was

immobilized on nylon transfer membranes. Labeled transcripts(6 X 106 cpm) were hybridized to the immobilized, denaturedIi and GAPDH cDNAs for 3 days at 42°C, then washed. Boundradioactivity was detected by autoradiography and quantifiedby densitometry.

mRNA

Table 1. Antisense Oligonucleotides Used to Detect Ii, FAß and GAPDH mRNA

Location Oligonucleotide sequence

IiFAßGAPDHc-fos

1874-1879262-300131-170

2448-2483

5'-CAGAAGATAGGTCTTCCATGTCCAGTGGCTCTTTAG5'-CAGGACCTGGAAGTTCCAGTCGCCATTCCTAATA5'-CCATGTAGTTGAGGTCAATGAAGGGGTCGTTGATGGCAAC5 ' -CTGTGCAGAGGCTCCCAGTCTGCTGCATAGAAGGA

INVARIANT CHAIN mRNA STABILITY 741

IFN- TPA

li

+ + +

Time (h) 12 24 48 48 48 48 48

FIG. 1. Expression of li in 20 cells treated with TPA, IFN-y, or a combination of TPA and IFN- . Cells were incubatedwith 100 ng/ml TPA for 12, 24, or 48 h (first three lanes). Cellswere incubated with the solvent control (0.01% DMSO) withor without 500 U/ml IFN- or 100 ng/ml TPA for 48 h as in¬dicated. Ii was immunodetected using the In-1 mAb on a West¬ern blot containing 40 µg protein per treatment.

Pulse-chase analysis of mRNA half-lifeMeasurement of the turnover rate of Ti mRNA was performed

as described by Zhu et al.,*20' with some modifications. Cells(2-3 X 107) were pulsed with 100 /xCi/ml of [5,6-3H]uridinefor 48 h and chased for 2 h with medium containing unlabeled10 mM uridine and 10 mM cytidine. Two hours before treat¬

ment, the medium was exchanged, and the concentration of uri¬dine and cytidine was reduced to 1 mM. Following treatments,total RNA was purified and counted in a liquid scintillation an¬

alyzer. The specific activity of incorporation of [5,6-3H]uridinewas 6.8-7.5 X 104 cpm/Mg RNA. Labeled RNA was detectedby hybridization of 150 µg of total RNA (5 X 106 cpm/ml) to2 µg Ii or GAPDH cDNAs immobilized on a nylon transfermembrane. The bound transcripts were eluted in 1 NaOH andcounted in a liquid scintillation analyzer. Background was theamount of radioactive RNA bound to vector DNA. An aliquotequal to one-tenth the RNA hybridized to cDNA probes was

blotted onto a nylon transfer membrane and hybridized to 32P-labeled antisense GAPDH oligonucleotide to confirm that eachsample was equally applied.

RESULTS

Induction of Ii by TPA

Although Ii is abundantly expressed in A20 cells, Ii mRNAhas been shown to increase 5-8-fold over a 48 h time course

when PKC is activated by TPA or DAG.'6' The increase in IimRNA levels seen at 24 h and 48 h is also reflected by an in¬crease in the amount of Ii delected on immunoblots, which showa significant (p < 0.001) increase of 2-3-fold between 24 h and48 h (Fig. 1). Addition of IFN-y to TPA-treated cells blocksthe TPA-induced increase in Ii mRNA (Fig. 2) and the amountof Ii detected on immunoblots at 24 h and 48 h (Fig. 1). Treat¬ment of A20 cells with IFN- alone increases Ii by about 30%when compared with the control cells.

Induction of I-Af¡ by TPA

Because the Ii and the MHC class II antigens are generallycoregulated,'13' the effect of TPA treatment on expression of

I-A^ in A20 cells was tested. As shown in Figure 3, TPA treat¬ment does not increase I-A^ mRNA at 24 h but does signifi¬cantly increase I-A^ mRNA about 3-fold at 48 h. As Ii mRNAincreases 2-^l-fold within 24 h and 5-8-fold at 48 h after TPAtreatment, Ii and I-Ap appear to be partially coregulated. How¬ever, they may differ in the kinetics and magnitude of the in¬duction.

Induction of I-A$ and li by TPA in mouse spleen cells

To determine if upregulation of I-A^ and li mRNAs occursin physiologically normal cells as well as the A20 tumor-de¬rived cell line,'9' expression of FAß and Ti mRNAs in spleen cells given control or TPA treatment was compared. As shown

? ^^ .

.c

Invariant chain

GAPDH-

ControlTPAIFN- TPA/IFN-

Time (h)FIG. 2. Regulation of Ii mRNA levels by TPA, IFN-y, or

TPA plus IFN-y. (A) Cells were incubated with 100 ng/ml TPA,500 U/ml IFN-y, or 100 ng/ml TPA plus 500 U/ml IFN-y for24 h or 48 h. Northern blots containing 20 µg total cellularRNA for each sample were analyzed for expression of Ii andGAPDH mRNA. (B) The ratio of Ii/GAPDH was expressed as

percent of control. Each value represents the mean and SD oftwo or three independent experiments.

750 SHIH ET AL.

in Figure 4, treatment of cells with TPA causes a significantincrease in both Ii and I-Ap mRNA, with Ii mRNA appearingto show a larger increase. Regulation of Ii and I-Ap mRNA in cells is, therefore, similar to that observed in A20 cells, sug¬gesting that TPA treatment induces a mechanism that functionsin physiologically normal cells as well as A20 cells.

Transcription of the li geneTo determine if Ii is transcriptionally induced by TPA and

if cotreatment with IFN-y alters transcriptional regulation ofthe li gene, nuclear runoff assays were done on nuclei isolatedfrom control cells, TPA-treated cells, cells treated with IFN-y,and cells treated with both TPA and IFN-y. Transcription ofthe Ii is not increased 2-24 h after treatment with TPA (datanot shown). At 24 h, transcription of the Ii appeared to increase10%-20%. However, this was not significant (p < 0.1), and at48 h, transcription rates for all four treatments were indistin-

C TPA

guishable (Fig. 5). As expected, IFN-y alone slightly increasesli gene transcription at 24 h but not at 48 h. The transcriptionrate for cells treated with both TPA and IFN-y is indistin¬guishable from the controls at both 24 h and 48 h.

// mRNA stabilization by TPA

As treatment of cells with TPA does not lead to a largechange in Ii transcription, the effect of TPA on Ii mRNA sta¬bility was determined in cells treated for 24 h with TPA or thesolvent control following addition of the transcription inhibitor,

C TPATime (h) [ 48 I 24 48

I-Ar—

Time (h) 148 I 24 48l

GAPDH

300-

coo

enco

Q.

FIG. 3. Induction of I-Aß by TPA in A20 cells. (A) A20 lymphoma cells were incubated with 100 ng/ml TPA or an equalvolume of solvent for 24 h or 48 h. Northern blots containing20 µg RNA per sample were analyzed for expression of I-Apand GAPDH. (B) The ratio of I-A^/GAPDH was determinedand is expressed as a percent of control. Each value shows themean and SD of three independent experiments.

FIG. 4. Regulation of I-A^ and Ii mRNA in mouse spleen cells. (A) Mouse spleen cells were purified and incubated as

described in Materials and Methods. Cells were incubated with100 ng/ml TPA or the solvent control for 24 h or 48 h. Analy¬sis of 10 µg total RNA for expression of I-A^, Ii, and GAPDHmRNA was done by Northern blotting. (B) The ratios ofI-Aß/GAPDH and Ii/GAPDH were determined and are ex¬

pressed as the percent of control. Each value shows the mean

and SD of two or three independent experiments.

INVARIANT CHAIN mRNA STABILITY 751

actinomycin D. Residual li and GAPDH mRNAs were detectedon Northern blots (Fig. 6A), and the ratio of Ii/GAPDH was

determined (Fig. 6B), as TPA previously was shown to havelittle effect on stability of GAPDH mRNA.'20,2" In controlcells, the ratio of Ii/GAPDH remained nearly constant, whereasaddition of TPA increased the ratio of Ii/GAPDH mRNA (Fig.6A). The time course for decay of Ii mRNA in A20 cells treatedwith TPA, IFN-y, or TPA plus IFN-y is shown in Figure 6B.Treatment with TPA increases stability of the mRNA above

A ^ «*A ^ c/GAPDH -—.</—» —

that seen in the control, whereas treatment with IFN-y alonedoes not alter the stability of Ii mRNA. Addition of IFN-y toTPA-treated cells blocks the TPA-induced increase in Ii mRNA.

The stability of Ii mRNA was also assayed in the absence ofa transcriptional blocker, by the |3H]uridine pulse-chasemethod.*20,21' Ii mRNA is relatively stable, with a half-life ofabout 12 h in control cells (Fig. 7A), which is similar to thehalf-life of GAPDH mRNA.'20' TPA treatment prolongs thehalf-life of Ii mRNA from about 12 h to >24 h, whereas, thereis little difference in the half-life of GAPDH mRNA, which isabout 12 h in both TPA-treated and control cells (Fig. 7B). Thehalf-life of Ii mRNA in cells treated with IFN-y alone is 12-13h, not significantly different from the control. The half-life ofIi mRNA is 14-15 h in cells treated with both TPA and IFN-

Control TPA

GAPDH

Time(h) 0 12 24 30 36 0 12 24 30 36

oO

CO2

Q.

ControlVZZ TPA^S IFN-y^ TPA/IFN-

Time (h)FIG. 5. Transcription of the li gene is unaffected by TPA,IFN-y, or a combination of TPA and IFN-y. Cells (1-2 X 108were treated with 100 ng/ml TPA, 500 U/ml IFN-y, or 100ng/ml TPA plus 500 U/ml IFN-y, or solvent control, DMSOand PBS, for 24 h (A) or 48 h ( ). The transcription rates of Iiand GAPDH genes were determined by nuclear runoff assaysas described in Materials and Methods. (C) The relative amountsof radiolabeled transcripts detected in nuclear runoff assayswere quantified by densitometry. The ratio of Ii/GAPDH was

expressed as percent of control and is the mean and SD of twoor three replicate experiments.

rr<

Time (h)FIG. 6. The effects of TPA, IFN- , or TPA plus IFN-y onthe stability of Ii mRNA. (A) A20 cells were incubated with100 ng/ml TPA or with solvent controls for 24 h. Transcriptionwas terminated by addition of 10 /¿g/ml actinomycin D, and to¬tal RNA was extracted at the indicated times. Residual mRNAfor Ii and GAPDH was detected on the Northern blot at the in¬dicated times. ( ) A20 cells were incubated with 100 ng/mlTPA ( ), 500 U/ml IFN-y (A), 100 ng/ml TPA plus 500 U/mlIFN-y ( ). or solvent controls (·) for 24 h. Transcription wasterminated by addition of 10 µg/ml actinomycin D, and totalRNA was extracted. Residual mRNA for Ii and GAPDH wasdetected on Northern blots and quantified by densitometry. Therelative stability of Ii mRNA normalized to GAPDH (tm = 12h) was obtained from three independent experiments.

752 SHIH ET AL.

y, which is considerably reduced when compared with cellstreated with TPA alone. As IFN- does not appear to directlydestabilize li mRNA compared wilh controls, these data sug¬gest that IFN-y blocks activation of an mRNA stabilizationmechanism by TPA.

Activation of PKC-a or PKC-hWestern blot analysis using eight antibodies specific for

PKC isoforms detected two PKC isoforms in A20 cells: PKC- and PKC- .*6' Because IFN- blocks stabilization of IimRNA by PKC activators in A20 cells, the effect of IFN-yon activation of PKC-a and PKC- was determined. Two as¬

says were used to detect activation of PKCs in vivo: (1)translocation of activated PKC-a and PKCS from the cytosolto the membrane fraction*1,28' and (2) transcriptional induc¬tion of the c-fos oncogene.'1,6' As shown in Figure 8A, PKC- is translocated from the cytosol to the membrane fractionfollowing treatment with TPA. IFN- alone does not alter thedistribution of PKC-a, and addition of IFN-y to TPA-treatedcells does not block translocation. Likewise, treatment withTPA induces translocation of PKC- from the cytosol to themembrane fraction (Fig. 8B), whereas the effect of treatmentwith IFN-yis indistinguishable from that of the control. PKC- translocates to the membrane fraction within 20 min afterTPA treatment, and translocation is not blocked by additionof IFN-y. Activation of PKC- is also associated with in¬creased phosphorylation of PKC- .'29' IFN-y alone did not al¬ter phosphorylation of PKC- when compared with the con¬

trol, nor did IFN-y block TPA-induced phosphorylation ofPKC- (data not shown). PKC activation has long been knowto initiate a protein kinase signaling cascade leading to in¬duction of the c-fos oncogene.'1,2' As shown in Figure 8C,IFN-y does not induce c-fos, nor does IFN-y block c-fos geneinduction by TPA.*6' These data suggest that the mechanismfor repression of the TPA-induced increase in Ii mRNA byIFN-y is noi due to a functional block in PKC-a or PKC- activation, nor was there an apparent block in signaling events

leading to transcriptional activation of the c-fos gene. Rather,IFN-y appeared to block another signaling pathway down¬stream of PKC activation.

DISCUSSION

PKC activation not only increases expression of li in A20cells but also increases levels of the MHC class II mRNA,I-Ap, consistent with a general coregulation of MHC class IIand li gene expression. The magnitude of this increase differsbetween li and MHC class II mRNAs, suggesting that specificmechanisms may differ.

The PKC-activated signaling pathway upregulating Ii and

I-Ap mRNA appears to be functional in normal cells as wellas A20 cells. However, the time course for induction of thesemRNAs in cells appears to be more prolonged when com¬

pared with that of A20 cells. Likewise, in normal cells, themagnitude of the increase in MHC class II mRNA appears tobe less than that seen for Ii mRNA. The mechanism upregu¬lating these mRNAs in A20 cells is, therefore, likely to be a

normal physiologic response to activation of PKC.

In A20 cells, Ii expression is constitutively high and insen¬sitive to IFN-y treatment. Cotreatment with IFN- and TPAdocs, however, abrogate the TPA-induced increase in Ii mRNA,which does not appear to involve a TPA-induced increase ingene transcription. The Ii promoter does contain a functionalNF-xB binding site.*10,30' However, the absence of transcrip¬tional induction by TPA suggests that TPA is not involved in

-r—15

Time (h)

Time (h)FIG. 7. Pulse-chase analysis of Ii mRNA stability in TPA-treated, IFN-y-treated, or TPA plus IFN-y-treated A20 cells.Cells labeled with [5,6-3H|uridine were incubated with 100ng/ml TPA D), 500 U/ml IFN-y (A), 100 ng/ml TPA plus500 U/ml IFN-y (T), or solvent controls (·, O) for the desig¬nated times. RNA hybridizing to Ii (A) and GAPDH (B) cDNAwas quantitated as described in Materials and Methods. Eachvalue is expressed as the relative percentage of radioactivemRNA at the time of treatment. Each value is the mean of twoindependent experiments.

INVARIANT CHAIN mRNA STABILITY 753

activating - in A20 cells. This is probably because of thepresence of fully active NF- in A20 cells,*10' which is notbound to IkB.

Increased expression of Ii in TPA-treated cells appears tobe due to an increase in Ii mRNA stability. Ii mRNA is rel¬atively stable, with a half-life of about 12 h, which is simi¬lar to that observed for four MHC class II genes in WEHI-3 and A20 cells.'31' Long-lived mRNAs are frequentlyexpressed constitutively,*20' although some regulatory mech¬anisms have been identified that either stabilize or destabi-

CytoplasmPKC-a- §

MembranePKC-a

-

i

CytoplasmPKC-

MembranePKC-

4*It

^

¥#^c-fos

~~

#«

GAPDH-

'm

FIG. 8. Effects of TPA, IFN-y, or a combination of TPA andIFN-y on PKC-a and PKC- activation. (A and B) Cells wereincubated with 100 ng/ml TPA, 500 U/ml IFN-y, a combina¬tion of 100 ng/ml TPA and 500 U/ml IFN-y, or solvent con¬

trols for 20 min. Cell extracts were fractionated into the cyto-plasmic and membrane fractions by centrifugation as describedin Materials and Methods. The PKC-a (A) and two phosphor¬ylation variants of PKC- (B) were detected using peptide-spe-cific antibodies and visualized Western blot analysis. (C) Ex¬pression of c-fos mRNA in cells treated for 30 min with TPA,IFN-y, a combination of TPA and IFN-y, or solvent controls(PBS, DMSO) was detemtined by Northern blot analysis of 20/ug total cellular RNA per treatment.

lize long-lived mRNAs. Labile proteins have been identifiedthat appear to stabilize human MHC class II mRNAs by a

factor of 8-10-fold in cells.'32' In 70Z/3 cells, Aa mRNAis induced greater than 10-fold by IL-4 after 65 h by a mech¬anism that involves primarily mRNA stabilization, with lit¬tle effect on transcription.*33' The relatively long time course

for increased expression of the Ii after TPA treatment isconsistent with stabilization of an mRNA with a half-life of12 h.

PKC activators have very specific effects on the stabilityof mRNAs. Most mRNAs that are stabilized by TPA haverelatively shorl half-lives.*17' However, TPA has been foundto stabilize some long-lived mRNAs, including those ofapolipoprotein E'18' and the heat shock proteins, hsp70 andhsp90.'34' Elements within the 3'-untranslated region, suchas the AUUUA sequences, are known to control stability ofunstable mRNAs.*17' Less is known about stabilization mech¬anisms for long-lived mRNAs, although these mechanismsmay involve mRNA binding proteins.'32' It is not knownwhich regions of the Ii mRNA are important for its stability.However, this mRNA does not contain an AUUUA motif inthe 3'-untranslated region'35' consistent with its relativelyhigh stability.

Cotreatment with IFN-y blocks the TPA-induced stabi¬lization of Ii mRNA by an apparently novel mechanism thatdoes not involve either blocking activation of PKC or inter¬action with signaling components leading to activation of c-

fus. Although IFN-y has been previously shown to increasethe stability of some mRNAs, including c-/ím\*36' this is thefirst report demonstrating a system in which IFN- blocksstabilization of an mRNA by another agent. As treatment withIFN-y alone does not cause direct destabilization of li mRNAand does not directly affect transcription, these data suggestthat IFN-y blocks a step in one of the signal transductionpathway involving PKC that leads to mRNA stability.

IFN- does not block activation of the either PKC-a or

PKC- in A20 cells. Both PKC-a and PKC- are translocatedto the membrane fraction within 20 min of TPA treatmentwith or without IFN- . In addition, transcriptional activationof the c-fos gene by TPA is unaffected by IFN- , althoughIFN-y does increase c-fos mRNA by stabilizing its mRNA.'36'These data suggest that IFN- blocks a PKC-dependent sig¬nal transduction pathway downstream from PKC activation.This is in contrast to the observations of others,'37' who showthat IFN-y blocks TPA-induced activation of Raf-1 by block¬ing PKC- activation. Signal transduction pathways activatedby IFN-y and TPA would thus appear to intersect in diverseways that may show cell type specificity.

Control of gene expression is now widely recognized to in¬volve both transcriptional and posttranscriptional processes. Be¬cause the Ii and MHC class II antigens are usually coregulated,it is of interest to determine if PKC activation alters the stabil¬ity of MHC class II mRNAs. In A20 cells, TPA treatment in¬creases the steady-state levels of both Ii and FAß mRNAs, al¬though I-Aß mRNA is increased only 3-fold over a longer timecourse, suggesting that the Ii and MHC class II mRNAs are not

tightly coregulated at the level of mRNA stability. Differentialregulation of mRNA stability might account for some situationsin which Ii and MHC class II genes show different regula-tion.*11,12'

754 SHIH ET AL.

ACKNOWLEDGMENTS

We thank Drs. J.F. Mushinski and D.E. Sabath for kindlysending the recombinant plasmids, pL- and pB7, respectively.We also thank Dr. J. Miller for the monoclonal antibody, In-1,and the recombinant plasmid, pcEXV3-mK31. This researchwas supported by grant AI24915 from the National Institutesof Health and by the Arizone State University Faculty Grant-In-Aid program.

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Address reprint requests to:Dr. Georgia Floyd-Smith

Department of ZoologyArizona State University

Tempe, AZ 85287-1501

Received 5 May 1997/Accepted 13 August 1997