Cyclic AMP and Calcium Regulate at a Transcriptional Level the ...

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CD7 is a 40-kDa cell surface glycoprotein expressed on T-cell precursors before their entry into the thymus during fetal development and whose functional role.

Vol. 267, No. 25, Issue of September 5 , pp. 18026-18031,1992 Printed in U.S.A.

THEJOURNALOF BIOLOGICAL CHEMISTRY Q 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Cyclic AMP and Calcium Regulate at a Transcriptional Level the Expression of the CD7 Leukocyte DifferentiationAntigen* (Received for publication, April 17, 1991)

Mercedes RinconS,Antonio Tugores, and MiguelLopez-Botet From the Seccidn de Znmunologia, Hospital de la Princesa, Universidad Autonoma de Madrid, Madrid 28006, Spain

CD7 is a 40-kDa cellsurface glycoprotein expressed on T-cell precursors before their entry into the thymus during fetal development and whose functional role remains uncertain. T-cell activationhas been shown to increase the expression of this surface molecule. In this report we describe the intracellular signals and the mechanisms involved in the regulation of CD7 antigen expression on human T lymphocytes. The elevation of intracellular calcium by using the A23187 ionophore increased the cell surface expression of CD7, whereas protein kinase C activation caused its down-regulation. Interestingly, the increase of intracellular cAMP with Bt,cAMP stimulated CD7 expression as well. Upregulation of CD7 on the cell surface following either BtzcAMP or calcium ionophore stimulation of T lymphocytes correlated with a raise of the steady-state levels of CD7-specific mRNA, without de nouo protein synthesis requirements. No differences between the half-life of basal CD7 mRNAand that induced by either BtzcAMP or calcium ionophore were detected. Run-on experiments showed that both stimuli enhanced the transcriptional rate of the CD7 gene. Our results provide the evidence for a positive regulatory effect mediated by cAMP on the expression of a leucocyte differentiation antigen.

The CD7 surface molecule is one of the earliest leukocyte differentiation antigens expressed during T-cellontogeny (1). CD7 antigen was formerly proposed to be a useful marker for the identification of T-cell acute lymphoblastic leukemia (2, 3). Nevertheless, recent reportshave described CD7’ nonlymphocytic leukemias, pointing out that the antigen maybe already expressed on pluripotential bone marrow progenitors

(4). Lazarovits andKarsh(5) have reported that the CD7specific mAb’ 7G5 partially inhibits theallogenic mixed lymphocyte reaction, whereas solid-phase-coupled anti-CD7 mAb has been shown to mediate comitogenic effects with anti-CD3 mAb on cultured T lymphocytes (6). So far, the biological *This workwas supported by INSALUD FISS Grant 89/0066. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $Recipient of a fellowship from the Ministerio de Educaci6n y Ciencia (Spain). To whom correspondence should be addressed Secci6n de Inmunologia, Hospital de la Princesa C/Diego de Le6n 62, Madrid 28006, Spain. The abbreviations used are: mAb, monoclonal antibody; Bt,cAMP, dibutyryl CAMP; CHX, cycloheximide; IL-2, interleukin 2; PBTL, peripheral blood T lymphocytes; PHA, phytohemagglutinin; PKA, CAMP-dependent protein kinase; PKC, proteinkinase C; PMA, phorbol 12-myristate 13-acetate; SCID, severe combined immunodeficiency disease; 8-Br-cAMP, 8-bromo-CAMP.

meaning of these findings is unknown, but the potential importance of the CD7 molecule has drawn special attention since Jung et al. (7) described a patient with severe combined immunodeficiency disease (SCID) whose only known phenotypic defect was an absence of CD7 expression on peripheral blood lymphocytes. cDNA cloning has showed that human CD7 bears significant homology with members of the immunoglobulin superfamily (8).Synthetic peptide technology and computer analysis of the primary aminoacid sequence have predicted a threedimensional structure of the N terminus of CD7, identifying putative binding regions based on Igk-chain homology. These analyses suggest similarities between the CD7 transmembrane region and thefusion sequences of both human immunodeficiency virus and respiratory syncitial virus (9). Since mitogenic activation of T-cells increases CD7cell surface expression (lo), we decided to examine the molecular mechanisms regulating CD7 expression on peripheral blood T-cells. Our results indicate that treatmentwith either a Ca2+ ionophore (A23187) or a cAMP analogue, dibutyryl cAMP (Bt,cAMP), stimulated CD7 expression on the surface of T lymphocytes, by increasing the steady-state-specific mRNA levels. The induced mRNA accumulation did not require de m u o proteinsynthesis and appeared secondary to anincreased gene transcription rather than to a modification of mRNA stability. EXPERIMENTALPROCEDURES

Materiak-The mAbs used in the present study were 3A1 antiCD7 ( l l ) , TS2/18 anti-CDZ (12), andSpV-T3b anti-CD3 (13). Phorbol 12-myristate 13-acetate (PMA), A23187 Ca2+ionophore, Bt,cAMP, prostaglandin E,, forskolin, cycloheximide (CHX), and actinomycin D were purchased from Sigma, phytohemagglutinin M (PHA) from Difco (Detroit, MI), [a-32P]dCTP and [a-”PIUTPfrom Du Pont-New England Nuclear Research, and RNase free-DNase I and Proteinase K from Boehringer Mannheim (Germany). Cell Cultures-Peripheral blood mononuclear cells were separated from heparinized venous blood by sedimentation of Ficoll-Hypaque (Pharmacia FineChemicals, Uppsala, Sweden) and cultured in RPMI 1640 (Flow, Irvine, Scotland) supplemented with 1% penicillin-streptomycin (Flow), 1% L-glutamine (Flow), and 5% fetalcalfserum (Flow). This is referred to as complete medium. Peripheral blood lymphocytes were purified from mononuclear cells by removal of adherent cells on plastic Petri dishes (1 h at 37 “C in 5% C o d . T lymphocyte-enriched populations (PBTL) were obtained by passing peripheral blood lymphocytes through a nylon wool column (14). Activation of PBTL was carried out by culturing 2 X lo6 cells/ml in a humidified atmosphere containing 5% CO, with ( a ) 0.5% (v/v) PHA, ( b ) anti-CD3 mAb directly bound to the plastic surface (0.5 Gg/well in the 24 well plates) as previously described (14), (c) A23187 calcium ionophore (1 CM), ( d ) PMA (5 ng/ml), and (e) BbcAMP (2 x 10-~ M). In several experiments parallel cultures were carried out in round-bottomed 96-well plates, and their proliferative response to the differentstimuli was determined by assessing [3H]thymidine incorporation as described (14). As previously described in detail (151, the early effect of T-cell mitogens on intracellular Ca‘+ was analyzed


Cyclic AMP and Calcium Induce by spectrofluorimetry. cAMP levels were measured as detailed elsewhere (14). T-cell blasts were obtained upon stimulation of PBTL for 4 days with PHA (0.5% v/v) a t 10' cells/ml in complete medium. Thereafter, cells were washed and grown for 4-5 days with recombinant interleukin-2 (IL-2) (10 units/ml), kindly provided by Hoffman-La Roche (Basel,Switzerland). T-cell lines CEM,Jurkat,andPEER, were grown in complete medium. Flow Cytometry Analysis-As previously described (14), cells were incubated with hybridoma supernatants containing mAb (100 m1/2 X 10' cells), washed, and further incubated with fluorescein isothyocyanate-labeled goat anti-mouse F(ab')* (Dakopatts, Copenhagen, Denmark). Samples were analyzed byflow cytometry(EPICS-C; Coulter Scientific, Harpenden, United Kingdom). RNA Isolation and Northern Blot Analysis-Total cellular95RNA was extracted as described (16) and quantified by absorbance at 260 nm. RNA (10 mg per lane) was denatured, electrophoresed through 2.2 M formaldehyde/l% agarose gels and blotted onto Biodyne nylon membranes (Pall Corp., Glen Cove, NY) as described (17). Filters were hybridized overnight, at 42 "C, under standard conditions (50% formamide, 5 X Denhardt's solution, 0.1% sodium dodecyl sulfate, 24 mM phosphate buffer, pH 7, 5 X SSC (1 X SSC = 0.15 M NaCl, 15 mM trisodium citrate), and250 pg/ml denatured salmon sperm DNA), with DNA probes radiolabeled by the random primer method (18). Autoradiographic bandintensities were quantifiedusinga 300-A Computing Densitometer (Molecular Dynamics, Sunnyvale, CAI. The CD7 probe was a full length cDNA cloned in rH3M vector, kindly provided by B. Seed (Boston, MA) (8).The 0-actinprobe was a HinfIIBamHI fragment (0.6 kb) from the 3'-untranslated region of the @actin cDNA (19). Run-on Transcription Assays-Nuclear run-on analyses were performedaccording to amethod previously described (20). Briefly, nuclei from 4 X IO7peripheral blood lymphocytes were incubated for 30 min at 30 "C with 100 pCi of [a-"P]UTP. After DNase I and proteinase K treatment, the reaction products were extracted with phenol/chloroform and precipitated. Samples were spun through a Sephadex G50 column to remove unincorporated nucleotides. The radiolabeled RNA (approximately 4 X 10' cpm) was hybridized to denatured double-stranded plasmid (10 pg) containing CD7 and pactin sequences which had beenslot-blotted onto a nitrocellulose filter; rH3M vector (10 pg) was used as a negative control; additionally, we used (2 pg) a fragment of the ribosomal DNA (pRA) cloned into pGEM4 (21). Hybridization was carried out for 36 h at 65 "C, and membranes were washed (20) and autoradiographed. RESULTSANDDISCUSSION

CD7 Gene Transcription


TABLEI CD7 surface expression after T-cell actiuation PBTL (2 X lo6 cells/ml) were incubated for 24 h in complete medium in the presence of the following reagents: (a) plastic-coupled anti-CD3 mAb, ( b ) PHA (0.5% v/v), ( c ) PMA (5 ng/ml), ( d ) Ca2+ , Bt,cAMP (2 X 10" M). After this ionophore A23187 (1 p ~ ) (e) incubation period, the expression of CD7 and CD2 antigens was analyzed by flow cytometry. CD7 Stimuli

76 Cells"

Medium Anti-CD3 92 mAb PHA PMA A23187 Bt2cAMP



% Cells


+54 +54 -21 +24 +24

85 88 91 88 87 82

+54 +41 +45 +21 -5

81 66 91 92

"The percentage of positive cells was calculated by substracting the fluorescence detected in the negative control (P3X63). bAfter incubation of the cells with different reagents, changes in linear mean fluorescence intensity (MFI) were calculated as follows: PMFI = MFI (stimulated cells) - MFI (untreated cells).

TABLE I1 Upregulation of CD7 expression in PBTL does not correlate with their proliferative response Stimuli

ACD7 A[ca*+l~a expression'

[3H]Thymidine incorporation'

nM MFI cpm x 10-3 132 +44 52.9 PHA (0.5%) 98 +20 7.1 Anti-CD3 mAb (2 pg/ml) 200 +47 5.3 A23187, 0.5 p M 192 +40 2.0 A23187, 0.1 p M 57 +11 1.2 A23187,0.05 p M Fura-2 AM-labeled PBTL (5 X 10' cell/ml) were stimulated with the different reagents and the early maximal increase of [Ca'+], was assessed by spectrofluorimetry (see "Experimental Procedures"). The data represent the increment of [Ca"], attained over the basal levels. See footnote Table I. e PBTL were incubatedeitheralone or in the presence of the different stimuli for 72 h in 96-well microtiter plates (1.5 X lo5 cells/ well) and labeled with ["]thymidine during the last 18 h. Data represent the mean of triplicate samples. [3H]Thymidine incorporation on unstimulated cultures was 1119 cpm. S.D. of triplicates was in every case 4 0 % of the mean.

CD7 Surface Expression Is Induced by Bt,cAMP and the A23187 Calcium Ionophore in Human T Lymphocytes-We analyzed by flow cytometry theexpression of the CD7 antigen T lymphocytes were purified A23187 that did not trigger a significant mitogenic response. during T-cell activation. Human from peripheral blood and incubated for 24 h in the presence Moreover, the CD7 up-regulation was detectable at concentrations of A23187 that induced an early increase of [Ca"], of different mitogenic stimuli. Both PHA and solid-phasecoupled anti-CD3 induced an up-regulation of CD7 cell sur- comparable to that mediatedby soluble anti-CD3 mAb. Simface expression (Table I) in agreement with previous reports ilarly, Ware et al. (25) haverecently shown that submitogenic concentrations of ionomycin up-regulate CD7 expression by (10). Activation of protein kinase C (PKC) associated with an increasing gene transcription. In contrast to the actionof Tincrease of intracytoplasmic Ca2+ concentration [Ca2+Ii,are cell mitogens, the ionophore mediated atransient effect which eventsknownto be physiologically triggered throughthe wascompletely inhibited by cyclosporin A, suggesting the CD3/T-cell receptor complex (CD3.TcR). As previously re- involvement of additional signals in mitogen-dependent upported (22, 23) stimulation of PBTL with soluble anti-CD3 regulation of CD7 expression. Since cAMP plays a role as a mAb raised the [Ca'+], up to 3-fold the basal levels as meas- second messengerin differentcellular systems, and,moreover, ured by spectrofluorimetry (not shown). To determine the we and others have previously shown that stimulation with nature of the intracellular signals involved in the regulation eitheranti-CD3 mAb (26, 27) orPHA (28)increases the of CD7 expression, we comparatively analyzed theeffects of intracellularCAMP,[CAMP],, in T-cells, we analyzed the either PKC activation or the increase of [Ca'+],. Treatment possible participation of the cyclic nucleotide in theregulation of T-cells for 24 h with the A23187 Ca2+ ionophoremarkedly of CD7. Interestingly, T-cell stimulation with Bt2cAMP also enhanced the surface expression of CD7 (Table I). In contrast, induced an overexpression of the molecule (Table I and Fig. PMA causeda down-regulation of CD7, as previously reported 11, comparable to that obtained with A23187. Adifferent by others (24).The mitogenic effect and the increaseof [Ca"] pattern of response to the same stimuli, observed for the CD2 triggered by different stimuliwhich enhanced CD7 expression molecule, is shown for comparison (Table I). As displayed in were compared. As shown in Table 11, the latter event apFig. 1, the increased expression of CD7 in the presence of peared unrelated to the proliferative response since it was A23187 or Bt,cAMP was detectable after 24 h, being more induced by either soluble anti-CD3 mAb or concentrationsof evident after 48 h of culture. Moreover, the combination of

Cyclic AMP Calcium Indzand

18028 2 4 h.

Ace CD7 Gene Transcription

48 h.







FIG. 1. Cell surface expression of CD7 in Bt2cAMP- and A23187-stimulated T lymphocytes. PBTL (2 X 10‘ cell/ml) were incubated in the presence of either medium (CONTROL.. . . . . .), Bt2cAMP (2 X lo-‘ M), Ca2+ ionophoreA23187 (1 p ~ or) BtZcAMP plus A23187. After treatment for 24 and 48 h cells were analyzed for CD7 expression by flow cytometry. P3X63 was used as a negative control (shaded area). TABLE111 Effect of Bt2cAMP and A23187 on CD7 expression on T blasts and cell lines T blasts were prepared as described under “Experimental Procedures.” T-cell lines were grown in complete medium. Each cell population was incubated (lo6cell/ml) for 24 h in the presence of medium, Bt2cAMP (2 X low4)or A23187 (1p M ) and assayed for CD7 expression by flow cytometry. Stimuli

T blasts %”


T-cell lines


Jurkat %






Medium 96 99 7743 84 75153 98 78 99 11143 86 98 157 BttcAMP 97 A2318783 123 98 43 98 78 97 158 “The percentage of positive cells was calculated by substracting the fluorescence detected inthe negative control (P3X63). * Linear mean fluorescence intensity (MFI) is expressed in arbitrary units. both stimuli produced an additive effect. We also analyzed the effect mediated bya differentcAMP analogue, 8-BrcAMP (loT3M), and by reagents able to activate adenylate cyclase increasing the [cAMPIi to 20-30 pmol/2 X loficells M) orto 12-20 pmol/2 X lo6 such as prostaglandinEz cells suchas forskolin (5 X M), as assessedby specific radioassay. Under such conditionswe observed a n increase of CD7 expression after treatment with 8-Br-CAMP (AMFI>14), prostaglandin EP (AMFI>13), or forskolin (AMFI>9) during 20 h, thus supporting that theeffect was due to the increase of CAMP]^. T o determine whether CD7 could be up-regulated on preactivated T-cells we analyzed the effect of either Bt2cAMP or calcium ionophore onT lymphoblasts. As shown in Table111, both stimuli were able to induce an overexpression of the antigeninnormalproliferating T-cells. Incontrast,no changes were detected in the different leukemia T-cell lines of both immature (CEM) or mature(JK, PEER) phenotypes which we examined (Table 111).Thus, the regulation of CD7 in T-cell lines appeared to be different from that of normal T-cells.

The elevation of[Ca’+Ii has alreadybeendescribed to induce the expression of certain surface antigens such 4F2, as a molecule associated with cell growth and activation (291, whereas the action mediated by Bt2cAMP was particularly striking. Although the elevation of [cAMPIi hasbeen shown to induce the expression of different genes, including human proenkephalin (30), vasoactive intestinal polypeptide (31), agonadotropine (32, 33), and rat somatostatin (34),CD7 constituted the first example of a T-cellmembrane molecule positively regulated by the cyclic nucleotide. So far, the increase of [cAMPIi induced by cAMP analogues or reagents thatactivateadenylate cyclase has beenfound toinhibit proliferation (14, 35, 36) as well as IL-2 production (37, 38) and IL-2 receptor (CD25) expression (14) in mitogen-stimulated human T-cells. This inhibition appeared tobe a consequence of a direct effect of CAMPon earlysignal transduction, during the activation process (14). Herein, we demonstrate that cAMP by itself can enhance the expression of certain cell surface antigens, and thus it might play a more complex regulatory role in T-cells. Our data are in line with similar observations preliminarily reportedby others (39, 40). Thehypothesisthat CAMP-dependentup-regulation of CD7 expression might be involved in the suppressive effect mediated by the cyclic nucleotide on T-cell function can be considered sincerecent studiesby Emara etal. (41,42) showed that cross-linking of cell-bound anti-CD7 mAbs could inhibit the mitogenic reponse in T-cells, and suggested a suppressive role for the putative naturalligand. Nevertheless, that possibility appears uncertain since both events do not correlate. In fact, mitogenic stimuli (PHA and anti-CD3 mAb) and Ca” ionophores up-regulate aswell CD7 expression but trigger Tcell proliferation. In addition, anti-CD7 mAb has been also reported to costimulate T-cell activation induced via CD3. TcR (6). Furthermore,we have previously shown that cAMP inhibits early stagesof T-cell receptor-mediatedsignal transduction (14) that precede de nouo gene expression. Whether the dual effects of cAMP may reflect the participation of different protein kinaseA isozymes is uncertain. Induced SurfaceExpression of the CD7 Molecule Correlates with Increased Specific mRNA Levels-Resting PBTL were treated for different times with either A23187 or Bt*cAMP, and total RNA was extracted from each sample. Northern blot hybridization studies were performed to compare the levels of CD7 mRNA under the different cultureconditions. As shown in Fig. 2, low levels of a specific 1.3-kb transcript were detected in resting T-cells and markedly increased upon stimulation with either BtzcAMP or A23187. CD7 mRNA levels detectably raised within the firsth of treatment, reaching a maximum after 12 h and persisting for at least 48 h. The calcium ionophore synergized with Bt2cAMP to yield a 10-fold induction as compared to the basal mRNA amount (data not shown). These data strongly suggested that both B.

A Time l h r l )




6 12 24







FIG. 2. Effect of BtzcAMP and A23187 on the levels of CD7 mRNA. PBTL (2 X 10“ cell/ml) were incubated for several time intervals in the presence of Bt,cAMP (2 X lo-’ M) ( A ) or Ca” ionophore A23187 (1 PM) ( B ) . Total RNA (10 pgllane) was isolated and analyzed by Northern blot, as described under “Experimental Procedures,” sequentially hybridizingwith radiolabeled CD7 and pactin cDNA probes.

Cyclic A M PCalcium andInduce CD7

Gene Transcription


reagents increasedCD7 expression by acting through different A complementary pathways. We analyzed as well the accumuAct D 2 4 8 " 2 8 8 -4 - 2 (hrsl lation of CD7-specific mRNAinthe leukemic T-cellline Stimuli - + + + " + + + + Jurkat that, as shown above, did not increase the surface expression of CD7 upon stimulation. The steady-statelevels .e * c .' of CD7 mRNA detectable in untreated cells were not significantly modified by A23187 and were minimally increased by 8t2 CAMP A23187 Bt,cAMP, as compared to the effectobserved on PBTL, further suggesting that CD7 is differently regulated in T-cell B lines (Fig. 3). CAMPand Calcium Activate CD7 Gene Transcription without Modification of the mRNA Stability-A number of distinct molecular mechanisms could account for the accumulationof CD7mRNAin T lymphocytes. T o analyzewhetherthe CAMP- and calcium-dependent induction mechanisms of CD7 gene expressionaremediated by changesinthe specific a mRNA stability, we comparatively estimated the half-life of the CD7 mRNA. Resting T-cells were incubated either with medium, Bt,cAMP, or A23187. After incubation for 12 h, the transcription inhibitor actinomycin D was added to the culture medium, and total RNA was isolated at different time intervals. Northern blot (Fig. and 4A) densitometric analysis (Fig. 4B) revealed that both Bt2cAMP- and Ca'+ ionophoreinduced CD7 mRNA levels displayed a similar half-life ( tlh= 0 ; ; i 6 8 5 h), shorter than thatobserved for unstimulated T-cells (tab = 7 h). The half-life of CD7 mRNA appears to be relatively T I M E (hrs) long as compared with that observed for oncogenes and lymFIG. 4. Stability of CD7mRNA in T-cells treated with phokines (tH= 30 min) (43), but fits into the average half-life either BtzcAMP or A23187. PBTL were incubated for 24 h either (t,,?= 8 h) reported for randomly chosen mammalianmh,VAs in medium alone, or with ( a ) Bt,cAMP (2 X 10" M ) and ( b ) A23187 (1 p ~ )At . the end ot the stimulation period, actinomycin D (Act D ) (44). cells were further incubated Since mRNA stabilizationwas not a mechanism leadingto (5 pg/ml) was added to the cultures, and for different times, followed by total RNA extractions. RNA samples the increased expressionof CD7, we attempted to determine (10 pg/lane) were analyzed by Northern blot hybridizing with a CD7 whether an enhancement in the transcriptional rate of the cDNA probe ( A ) . Autoradiograms were scanned and quantified as gene could be involved. For thispurpose, nuclei isolated from described under "Experimental Procedures" ( B ) . Symbols: 0, meT-cells incubated for 3 h in the presence of medium, Bt,cAMP, dium; 0, BtZcAMP; A, AA23187. or A23187 were subjected to run-on analysis. "'P-Labeled primary transcripswere hybridizedto filters containinga fulllength CD7 cDNA cloned into the rH3M plasmid. Nuclear run-on assays showed a low transcription rate of the CD7 gene in nonstimulated T-cells. Upon stimulation with either Bt2cAMP or A23187, the transcription rate of the CD7 gene 8-actin - 0 4 was induced 2- and %fold, respectively (Fig. 5). These results ."* CD7indicated that, at least, one of the mechanisms involved in hH3M " regulating CD7 expression is a n increase in the transcription "



1 2 3



FIG. 3. BtzcAMP and A23187 did not modify the levels of CD7 mRNA in the leukemic T-celllineJurkat. Cellswere incubated (0.5 X IOGcells/ml) for 12 h in the absence (lane I ) or in the presence of 0.2 mM Bt,cAMP ( h e 2 ) or 1 p~ A23187 (lane 3 ) in the culturemedium. Total RNA (IO pg) was isolated andsubjected to Northern hybridization by using specific CD7 and 8-actin cDNA probes.


FIG.5. Bt2cAMP- and A23187-stimulate CD7 gene transcription. Nuclear run-on transcription assays were performed on nuclei isolated from cellsthat were incubated for 3 h in medium alone . radiolabeled or with Bt,cAMP (2 X lo-" M ) or A23187 (1 p ~ ) The RNA washybridized to filters to which the following DNA were bound CD7 cDNA clonedinto nH3M; nH3M; 3'-untranslatedregion of 8-actin cloned into pBR327; and a fragment of ribosomal DNA cloned into pGEM4 ( p R A ) .

rate of the gene. Although we could not detect anydifferences between theCAMP-and calcium-dependent transcription rates, it is likely that different transcription factors are activated through eachpathway. Recently, agenomic clone of the CD7 gene has been isolatedand several cis regulatory elements such as a NF-kB site, a G-C box, a TRE, and an AP2 site have been proposed within the5' upstream region of the gene (45). Although NF-kB has been described to be activated by PKC through the release of a specific inhibitor in the cytoplasm (I-kB)(46),cDNA cloninghas shown a phosphorylation site for PKA thatmay enhance itsDNA binding avidity (47). Like phorbol esters and CAMP (48), free Ca" can also induce

Cyclic AMP and Calcium Induce


- "

- + + + + - -

CD7 Gene Transcription

quired for calcium-mediated transcriptional induction of the glucose-related genes P3C5 and P4A3 (57). Treatment with CHX alonefor 12 h induced an increase of - + - + - + CHX CD7 mRNA levels (Fig. 6). Previous reports have shown that protein synthesis inhibition by CHXcandramaticallyincrease thehalf-life of a variety of labile mRNAs (43) through CD7 several mechanisms that have been analyzed elsewhere (58). Particularly, the consensussequence A(U)nA, located at the 3"untranslated region of several mRNAs (59), appears tobe involved in their CHX-mediated stabilization. However, ArD-actln uffo and Seed (8) did not identify a 3' region containing this sequence in theCD7 cDNA,thus suggesting that theincrease levels in thepresence of CHX may involve FIG. 6. Effect of protein synthesis inhibition on Bt2cAMP- of the these mRNA and A23187-induced CD7 gene expression. PBTL were prein- other mechanisms unrelated to the presence of that sequence, cubated for 30 min in the presence or absence of CHX (10 pg/ml). as reported for CD2 regulation (60). Moreover, we cannot Thereafter, cells were treated with either medium alone, Bt2cAMP (2 exclude that CHX might play another role affecting gene X 10" M ) or A23187 (1 PM) for 12 h. Then, RNA was isolated and analyzed by Northern blot (10 pg/lane) by sequentially hybridizing expression. Recently, it has been proposed that CHX could account for the transcriptional inductionof nuclear protoonwith radiolabeled CD7 and &actin cDNA probes. cogenes c-fos and c-jun (61). Although the functional role of the CD7 molecule is a t c-fos expression, whose product is able toform heterodimers present unknown, Jung et al. (11) have described a case of with Jun/APl (49-51) and activate the transcription from SCID in which T-cells were shown to be selectively deficient promotersbearingtheTREconsensus sequence(52,53). for theexpression of the CD7 antigen. This abnormalitywas Additionally, AP2 has beenproposed to be regulated by both associated with defective T-cell proliferation and B cell difPKC and PKA (54). However, DNA binding and functional ferentiation, suggesting that CD7 could play an important assays are required to analyze the precise role of these cis role in thelymphoid ontogeny. The regulation of CD7 expreselements in the activation of CD7 gene transcription by Ca'+ sion during T-cell development from lymphoid progenitors and/or CAMP. and the role played by Ca'+ and cAMP need to be explored. Among the multiple mechanisms that may underlie lackthe In this report, we propose that the overexpression of CD7 of responsiveness of the leukemic cell lines to A23187 and uponactivation of T-cells is probably contributed by the Bt2cAMP, in terms of CD7 expression, the possibility that increase of [Ca'+], associated to thisprocess. Furthermore, an the DNA binding activity of transcription factors regulated increase of the cyclic nucleotide has been reported to occur by cAMP or Ca2+might be already induced has been considupon mitogenic stimulation via the T-cell receptor (26, 27) ered. Preliminary data2 indicate that the binding activityof and, thus, might also participate in the regulation of CD7 nuclear factor CREB, as analyzed by electrophoretic mobility expression, mediating a synergistic effect with Ca'+ as shown shiftassays,isconstitutivelyactivatedinJurkat cells as in the present study. Although the precise role of cAMP in compared to PBTL. T-cell activation is uncertain, the cyclic nucleotide could be CAMP-and Calcium-mediated Induction of CD7 Gene involved in the regulation of CD7 gene expression during Expression Do Not Require de Novo Protein Synthesis-To thymic and peripheral stages of development. better understand the regulationof the CD7 gene expression REFERENCES we investigated its protein synthesis requirements. To this end, PBTL were culturedalone or stimulated either with 1. Haynes, B. F., Denning, S. M., Singer, K. H., and Kurtzberg, J. (1989) Immunol. Today 10,87-91 Bt2cAMP orA23187 for 12 h. Parallel sampleswere preincu2. Link, M., Warnke, R., Finlay, J., Amylon, M., Miller, R., Dilley, J., and bated with CHX for 30 min, this protein synthesis inhibitor Levy, R. (1983)Blood 62,722-728 3. Vodinelich, L., Tax, W., Bai, Y., Pegran, S., Capel, P., and Greaves, M. F. being present in the culture medium during the stimulation (1983)Blood 62,1108-1113 period. As shown inFig. 6, CHXdid not abrogate the increase 4. Zutter. M. M.. Martin.. P. J... Hanke.. D... and Kidd. P. G. (1990)Leuk. Res. 14,'23-26 of CD7 mRNA induced by Bt2cAMP andcalcium ionophore. 5. Lazarovits, A. I., and Karsh, J. (1988)Trans lunt Proc 20,1253-1257 By contrast, a superinduction of the specific mRNA levels 6. Carrera. A. C.. Rinc6n. M.. SLnchez-MadriXF.. LoDez-Botet. M., and De L a n d h i , M. 0.(1988)j.Immunol. 141,1919-i924 was observed, indicating that denouo protein synthesis is not 7. Jung, L. K. L., Fu. S. M., Hara, T., Kappor, N., and Good, R. A. (1986)J. required to stimulate CD7 gene transcription and thus that Clin. Inuest. 77,940-946 8 . Aruffo, A., and Seed, B. (1987)EMBO J. 6,3313-3316 the putative regulatory factors involved are already present 9. Ware. R. F..Scearce. R. M.. Dietz. M. A.. Starmer. C. F.. Palker.. T.J... and in the cells. In this regard, it has been described that tranHaynei B.F. (1989)J. ImmuGl. 143,3632-3640 Y., Kobayashi. M., Yang, S. Y., Collins, N. H., Hoffmann, M. scription of certain genes induced by cAMP (31, 34) is inde- 10. Morishima, K., and Dupont, B. (1982)J. Immunol. 129, 1091-1098 pendent of protein synthesis. Thisprocess is mediatedby the 11. Haynes, B. F., Eisenbarth, G. S., and Fauci, A. S. (1979)Proc. Nutl. Acud. Sci. U. S. A. 76,5829-5833 CREB protein which is present in unstimulated cells as an 12. SHnchez-Madrid, F., Krensky, A. M., Ware, A. M., Robbins, E., Strominger, inactive form, being its transcription efficiency induced by J. L., Burakoff, S. J., and Springer, T.A. (1982)Proc. Nutl. Acud. S a . U. S. A . 79, 7489-7493 PKA-dependent phosphorylation (55). Although a CRE con- 13. Spits, H.,Yssel, H., Leeuwenberg, J., and De Vries, J. E. (1985)Eur. J. sensus has not been identified within the knownsequence of Immunol. 15,&3-91 M., Tugores, A,, L6pez-Rivas, A., Silva, A,,Alonso, M., de Landathe CD7 gene upstream region, the possibility that nuclear 14. RincBn, zuri, M. O., and bpez-Botet, M. 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Btp C A M P



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