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The activation-dependent induction of APN-(CD13) in T-cells is controlled at different levels of gene expression

Recently, it was shown that aminopeptidase N (E.C. 3.4.11.2, CD13) is up-regulated during mitogenic stimulation of peripheral T-cells. In this study, we demonstrate that the half-life of APN mRNA was considerably prolonged in these cells leading to a 2.7-fold increase of APN transcript level. The apparent half-life time of the APN transcript was investigated by the RNA synthesis inhibitor-chase method using actinomycin D. The steady-state APN mRNA levels was determined by a competitive RT-PCR. The half-lives estimated in resting T-cells, natural killer cells and permanently growing tumour cells varied between 3.5 and 6 h. Finally, nuclear run-on assays revealed that the APN gene expression of stimulated T-cells is controlled by increased promoter activity as well. These studies suggest a control of APN gene expression at the post-transcriptional level in addition to promoter-mediated regulation.


Introduction
The cell-surface antigen CD 13, also known as aminopeptidase N (APN, E.C. 3.4.11.2), is a 150 kDa glycoprotein [1,2]. It is a stalked integral membrane peptidase, mainly expressed in the small intestinal and kidney brush borders but also found in brain, lung, liver and in the hematopoietic system [3][4][5]. Studies aimed at the functional role of APN revealed that this enzyme is involved in different processes such as hydrolysis of nutrients, inactivation of bioactive peptides, binding of corona viruses, mediating CMV infection, regulation of tumour-cell invasion, and degradation of the extracellular matrix and antigen presentation [3,[6][7][8][9][10][11][12].
The aim of this study was to investigate APN gene regulation in mitogen-stimulated T-cells. Here, we present data suggesting that APN gene expression of these cells is controlled at both the transcriptional and post-transcriptional level.
Peripheral T-cells were enriched from the MNC population by nylon adherence as described by others [17]. FACS analyses indicated a purity of CD3+ cells between 85 and 95%. Natural killer (NK) cells were enriched by depletion of CD3 + and CD19+ cells using the MACS system (Miltenyi Biotec) as previously described [18]. In general, NK cells consist of more than 90% CD16 + /CD56 + cells, whereas CD3 + cells were less than 5%.

Enzymatic assay
Ala-pNA hydrolysing activity of cells or their lysates, respectively, was determined in triplicate by measuring the hydrolysis of the chromogenic substrate Ala-pNA (2.5 mM) as described previously [16].

Competitive PCR
RNA samples of resting or stimulated cells were obtained using the 'RNeasy' kit (Qiagen) according to the manufacturer's instructions. Total RNA (250 ng-1 ug) was mixed with an aliquot of a standard APN mRNA fragment and transcribed into cDNA by 50 U of MMLV reverse transcriptase (USB) using an APN-specific primer (5'-gcggtagaagcccgccaggtca).
An aliquot of the cDNA mixture was used directly for enzymatic amplifications which were performed in a 40 (xl reaction volume containing 0.5 U 'Gold-Star' polymerase (Eurogentec), 0.25 mM dNTP, 2.5 mM MgCl 2 , 0.2 pmol of primers (forward: 5'-gccgtgtgcacaatcatcgcact; reverse: 5'-caccagggagcccttgaggtg) and IX reaction buffer (Eurogentec) in the thermocycler Autogene II (CLF, Emmersacker, Germany). Initial denaturation at 94°C for 3 min was followed by 30 cycles with denaturation at 96.5°C for 0.4 min, annealing at 54°C for 0.6 min and elongation at 72°C for 0.8 min. The final extension step was 72°C for 10 min. An aliquot of the PCR reaction was electrophoretically separated in a 1.6% agarose gel (Biozym, Germany), documented by a video image system (GelPrint 2000i, MWG Biotech, Germany) and densitometrically analysed using RFLP 3.0 software (Scanalytics, USA).

Nuclear run-on assay
Nuclear run-on assays were performed from isolated nuclei derived from resting and stimulated T-cells as described by others with some modifications [20], Briefly, 1-2 XlO 7 cells were harvested, incubated on ice for 10 min, washed twice in ice-cold PBS (pH 7.4) and lysed in NP-40 buffer (10 mM Tris-HCl, pH 7.4; 10 mM NaCl; 3 mM MgCl 2 ; 0.5% NP-40). After an incubation on ice for 5 min, nuclei were isolated by centrifugation (500 Xg, 4°C, 5 min), resuspended in NP-40 wt sf <-587bp <-489bp < 320bp Fig. 1. Determination of the APN mRNA content of restin (A) or stimulated (B) T-cells after RNA synthesis inhibition by actinomycin D. Cells were cultured in the presence of 5 ug/ml actinomycin D for 6 h, then their APN-transcript levels were estimated as described in Section 2. The figure represents three independent experiments using the same T-cells. Equal amounts of RNA and standard fragment were used in the both samples of each experiment (0 h, 6 h). Summarizing data from six T-cell populations, resting or stimulated T-cells contained 2.9 ± 1.6 or 7.8 ± 3.9 pg APN mRNA/ug RNA, respectively. buffer again and centrifugation was repeated. The nuclear pellets were resuspended in 100 ju. 1 of storage buffer containing 50 mM Tris-HCl (pH 8.3), 40% glycerol, 5 mM MgCl 2 , 0.1 mM EDTA and stored at -70°C until use.
An aliquot of this solution was used to determine the specific activity of the nuclear lysate using the counter SMG 20046 (RFT, Germany). DNA slot-blots were prepared using the plasmid pSKAPN containing the full-length APN cDNA (kind gift from A.T. Look [4]).

Half-life time of APN transcripts in hematopoietic cells
The rate of decay of A P N m R N A in different hematopoietic cells was investigated using the R N A synthesis inhibitor actinomycin D . Cells were cultured in the presence of 5 |J.g/ml actinomycin D for 1-6 h. At different times, their A P N m R N A content was quantified using a competitive R T -P C R approach. The inhibition of newly transcribed messenger R N A by actinomycin D leads to a continued decrease of m R N A species, the time course of which is depended on their rates of decay. The reduction of A P N R N A level is  3.5 ±0.7 (n = 6) 6 ±1.9 (» = 3) 3.5 ±1.9 (« = 3) 4.5 ± 0.9 (n = 3) yes yes yes not determined Half-life times were determined by competitive RT-PCR as demonstrated in Fig. 1. Data represent means of at least three experiments («). No noticeable decrease of steady-state APN mRNA level was observed in stimulated T-, NK and H9 cells until 6 h co-culture with actinomycin D as shown for stimulated T-cells in Fig. 2. measured as the time-dependent decrease of the ratio between the APN wild-type and the APN standard fragment in RT-PCR. Fig. 1A represents three independent quantification experiments using resting T-cells from one donor. Summarizing the data obtained with resting T-cells from six healthy donors, the half-life time of APN transcript in resting T-cells was determined to be approximately 3.5 h (Fig. 2). The apparent half-life of APN transcript in resting NK cells was estimated as about 6 hours, however, NK-cell data showed more variance than those of T-cells ( Table 1). The APN mRNA halflives of permanently growing tumour cells such as the T-cell line H9 or the histiocytic cell line U937 were estimated to be 3.5 or 4.5 h, respectively (Table 1).

Stabilization of APN transcript and increase of APN gene promoter activity in stimulated T-cells
Purified resting T-cells were stimulated for 3 days as described in Section 2. The induction of APN expression was confirmed by measuring APN-derived enzymatic activity. For instance, stimulated T-cells contained 15-30 pkat/10 6 cells Ala-pNA hydrolysing activity compared to 4-7 pkat/10 6 cells determined in resting T-cells. The steady-state level of APN mRNA was increased 2.7 ± 1.4-fold in these cells. Interestingly, the half-lives of the APN transcript was considerably prolonged in stimulated cells compared to their unstimulated counterparts. The APN mRNA level of stimulated T-cells remained unchanged up to 6 h of cultivation in the presence of actinomycin D as shown in Figs. IB and 2. Furthermore, a stabilization of APN transcript was also observed in stimulated NK and H9 cells (data not shown).
To investigate whether the promoter activity of the APN gene additionally contributes to the increased steady-state APN transcript levels in stimulated T-cells, nuclear run-on assays using nuclei derived from these cells were performed. Slot-blots were hybridized with equal amounts of labelled nuclear lysates. Densitometrical analysis of hybridization signals revealed an increase of promoter activity up to 172.5 ±38.9% (« = 3) in stimulated T-cells compared to those of resting T-cells (data not shown).

Discussion
During the last years it has been shown that within the hematopoietic system CD 13 expression is not restricted to myelomonocytic cells. It has been longly known that this antigen is frequently expressed on lymphoid tumour clones. The frequencies of such CD13-positive clones vary between 5 and 30% [13,21]. Recently, it has been proven that other immune cells such as resting lymphocytes and NK cells express membrane-bound APN as well. In contrast to myelomonocytic cells, these cells express low amounts of APN; however, their CD 13 surface expression is increased by different mitogenic stimuli or after co-cultivation with synoviocytes or other CD13-expressing cells [16,[22][23][24]. The functional relevance of this induction is unclear as yet, but it has been proposed that APN like other membrane-bound peptidases such as neutral endopeptidase (E.C. 3.4.24.11,CD10), aminopeptidase A (E.C. 3.4.11.7) or angiotensin-converting enzyme (E.C. 3.4.15.1,CD 143) may function as a regulatory molecule that degrades bioactive peptides [5]. The induction of CD 13 on Tcells is generally accompanied by an increase of the corresponding enzymatic activity and APN mRNA level [16,24]. To gain insight into the mechanism underlying the CD 13 induction on T-lymphocytes, we investigated the transcriptional regulation of the APN gene. Actinomycin D inhibits nearly all RNA synthesis by intercalation into DNA. Furthermore, it also prevents the binding of ribosomes to transcripts and, therefore, subsequent protein synthesis [25]. Using this approach, the half-lives of the APN transcript were determined in different immune cells. The average half-life span of human transcripts is about 10 h, ranging from less than 15 min to more than 24 h [26]. Talking into account these data, the APN half-lives of 3.5-6 h could be considered as medium. Since no data on APN half-life time in any tissue are available, the data obtained in this study can only be compared to those of other glycoproteins. Comparable half-life times were described for annexin VII (3-6 h), lactoferrin (8-9 h) and myeloperoxidase (4-5 h) [27,28].
The transcriptional regulation of APN gene expression was revealed by two excellent studies. Both Shapiro and co-workers as well as Olsen and co-workers identified separate promoters that initiate transcription in intestinal epithelial and myeloid cells. In epithelial cells, transcription is controlled by a classical promoter containing a TATA box immediately upstream to the first codon of APN, whereas an alternative GC-rich promoter located about 8 kb upstream from coding region is active in myeloid cells as well as in fibroblasts [29,30]. Both promoter regions considerably differ with respect to transcription factor-binding sites. The transcription factors LF-B1 and Sp-1 represent the major regulators in epithelial and liver cells, whereas myb and Ets-2 both transactivate CD13 expression in myeloid cells [29,31]. The importance of promoter-mediated regulation is emphasised by reports showing that modulation of APN surface expression on endometrial cells and leukaemic B-cells is directly linked to the activity of the tissue-specific APN gene promoter [32,33].
The transcription of the APN gene in hematopoietic cells is controlled by the alternative promoter [31]. RNA synthesis inhibition leads to a linear decrease of steady-state APN mRNA levels with half-life times of 3.5-6 h in different hem-atopoietic cells. Therefore, it could be assumed that factors involved in APN mRNA degradation seem to be more stable than transcription factors which control the constitutive APN gene expression in resting T-cells. The activation-dependent induction of APN gene expression in T-cells may be due to the action of new transcription factors, an assumption which is supported by the increase of APN-promoter activity detected in stimulated T-cells. Shapiro and co-workers identified putative Sp-1, PU-AP-2, myb-and ETF-binding sites in the alternative promoter which probably contributes to the transcriptional activation of the APN gene [30]. However, the prolonged half-life time of APN-transcript in stimulated Tcells as well as the discrepancy between the 2.7-fold increase of APN transcript level and the 1.7-fold rise of transcription rate in these cells suggest the appearance or disappearance of factors directly involved in the degradation of APN transcript. Post-transcriptional regulation of mRNA decay after stimulation is a common regulatory mechanism described for other transcripts such as the CD 11 a, the mMCP-2 (chymase-homolog protein) [34,35].
Taken together, the results of this study clearly show that the APN gene expression of stimulated T-cells is controlled at both the transcriptional and post-transcriptional level. Since stabilization of APN mRNA was observed in other stimulated cells such as H9 and NK cells, as well, it may be concluded that this process represents a general mechanism of APN induction in hematopoietic cells.