Fumihiko Ishimaru, Department of Medicine, University of Okayama, 2-5-1 Shikatacho, Okayama 700-8558, Japan. E-mail: email@example.com
Summary. The cell surface zinc metalloproteinase CD10/neutral endopeptidase 24.11 (NEP) is expressed on normal and malignant lymphoid progenitors, granulocytes and a variety of epithelial cells. Because CD10/NEP functions as part of a regulatory loop that controls local concentrations of peptide substrates and associated peptide-mediated signal transduction, its role in each tissue is different depending on the availability of substrate. To characterize further how this widely distributed molecule is regulated differentially in each tissue, we analysed the major type 2 CD10/NEP promoter and found three functionally important transcription factor binding sites, one of which was identical to CCAAT-binding transcription factor/nuclear transcription factor Y. In this report, we analyse the type 1 CD10/NEP promoter and found a functionally important transcription factor binding site in the 5′-untranslated region. The results of the competition and supershift experiments demonstrated that the functionally important transcription factor was identical to Sp1. Our results suggest that ubiquitously expressed Sp1 may play an important role in differentiation stage-specific regulation of CD10/NEP expression in lymphoid lineage.
CD10/neutral endopeptidase 24.11 (NEP) is a member of a family of membrane-bound metallopeptidases that are expressed in a highly restricted fashion in normal and malignant haematopoietic cells, but expressed widely in a variety of epithelial cells including bronchial epithelial cells, bone marrow stromal cells, renal proximal tubular cells and certain tumour cell lines (LeTarte et al, 1988; Shipp et al, 1988; LeBien & McCormack, 1989; Shipp & Look, 1993). CD10/NEP cleaves small peptides on the amino-terminal side of hydrophobic amino acids, hydrolysing substrates including enkephalin, atrial natriuretic factor, substance P and bombesin-like peptides (Erdos & Skidgel, 1989; Shipp et al, 1991). In all the cell types in which CD10/NEP function has been studied, the enzyme downregulates peptide-mediated signal transduction by reducing the local concentrations of a specific peptide substrate. Because CD10/NEP functions as part of a regulatory loop that controls the local concentrations of peptide substrates and associated peptide-mediated signal transduction, its role in each tissue is different depending on the availability of substrate. For example, CD10/NEP reduces enkephalin-mediated analgesia and accumulation of amyloid beta peptide in the nervous system (Erdos & Skidgel, 1989; Iwata et al, 2001), atrial natriuretic factor-mediated diuresis in kidney (Erdos & Skidgel, 1989), peptide-mediated inflammatory responses (Martins et al, 1990) and cellular proliferation of lung (Shipp et al, 1991) and prostate (Papandreou et al, 1998).
In earlier studies, we demonstrated that the CD10/NEP gene contains two different 5′ exons (exons 1 and 2a/b) that splice into a common exon (exon 3) that contains the translation initiation codon (D'Adamio et al, 1989). Three types of CD10/NEP transcripts result from alternative splicing of these specific 5′-untranslated regions (5′-UTRs). Alternative promoters have been linked to tissue- and/or developmental stage-specific gene expression (Schibler & Sierra, 1987). As CD10/NEP expression is tissue specific and differentiation stage specific in the lymphoid lineage (Shipp & Look, 1993), analyses of these promoters are of particular interest. These type 1 and 2 CD10/NEP regulatory regions are both characterized by the presence of multiple transcription initiation sites and the absence of classic TATA boxes and consensus initiator elements (Ishimaru & Shipp, 1995). In the majority of tissues examined to date, type 2 CD10/NEP transcripts were more abundant; the abundance of type 1 transcripts was more variable, with the highest type 1 levels in fetal thymus and pre-B-lymphoblastic leukaemia cell lines, suggesting that type 1 transcripts may play an important role in lymphoid differentiation (Ishimaru & Shipp, 1995). To characterize further how this widely distributed molecule is regulated differentially in each tissue, we analysed the major type 2 CD10/NEP promoter and found three functionally important transcription factor binding sites (Ishimaru et al, 1997). One of them was identical to CCAAT-binding transcription factor/nuclear transcription factor Y (CBF/NFY), and our results suggested that ubiquitously expressed CBF/NFY might mediate tissue-specific expression of CD10/NEP by alternative splicing (Ishimaru et al, 1997). In this report, we focused on the type 1 CD10/NEP promoter to examine the differentiation stage-specific regulation of CD10/NEP expression in the lymphoid lineage. We found a functionally important transcription factor binding site in the 5′-UTR, which was identical to the ubiquitously expressed transcription factor, Sp1. Sp1 was found to be involved in tissue-specific gene expression and the control of transcription following a number of different stimuli, in response to oncogenes, antimetabolites, growth stimulation and differentiation, demonstrating that Sp1-dependent transcription can be highly regulated (Black et al, 2001).
Materials and methods
Cell lines. Cell lines used to assess CD10/NEP-driven luciferase activity included the human acute lymphoblastic leukaemia cell lines, Nalm6 (pre-B cell) and Raji (mature B cell). Nalm6 and Raji were grown in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal calf serum.
Determination of CD10/NEP promoter activity by luciferase activity. A series of CD10/NEP genomic fragments containing overlapping segments of 5′ CD10/NEP sequence was subcloned into the promoterless luciferase vector, pXP2. CD10/NEP genomic fragments were obtained from previously characterized CD10/NEP constructs (Haouas et al, 1995; Ishimaru & Shipp, 1995) by restriction endonuclease digestion. Transient transfections were performed as described previously (Ishimaru et al, 1997). Briefly, 5·0 × 107 Nalm6 and Raji were electroporated with 20 µg of pXP2 alone, cytomegalovirus (CMV)-pXP2 or CD10/NEP-pXP2 construct and 2 µg of CMV human growth hormone (GH) plasmid in RPMI medium at 300 V, 960 µF. Luciferase activity was measured in relative light units (RLU) at 8 h after transfection using a luminometer (Analytical Luminescence Laboratory, San Diego, CA, USA), and GH level was measured by radioimmunoassay (Nichols Institute Diagnostics, San Juan Capistrano, CA, USA). Thereafter, the RLUs from individual transfections were normalized for transfection efficiency by standardizing RLU for GH production driven by the CMV-GH internal control.
DNase I footprinting. Nuclear extracts for DNase I footprinting were prepared as described previously (Dignam et al, 1983; Ishimaru et al, 1997). The 602-bp fragment, extending from bp −46 to +556 of the type 1 CD10/NEP promoter, was 32P labelled as described previously (Ishimaru et al, 1997). DNA-binding reactions were carried out in a 20-µl volume with 1 × 105 c.p.m. of labelled DNA fragment, 4 µg of the synthetic polymer ds-poly(dI–dC) (Pharmacia Biotech, Piscataway, NJ, USA) and 100 µg of nuclear protein in a final buffer concentration of 10 mmol/l Hepes, pH 8·0, 30 mmol/l KCl, 5 mmol/l MgCl2, 1 mmol/l EDTA, 12% glycerol, 0·5 mmol/l dithiothreitol and 0·2 mmol/l phenylmethylsulphonyl fluoride. Reactions were incubated for 60 min on ice followed by 60 s digestion at room temperature with 6 µl of freshly diluted (10 µg/ml) DNase I (Worthington Biochemical, Freehold, NJ, USA). The DNase I digestion reactions were stopped by the addition of 100 µl of stop mixture (20 mmol/l Tris, pH 7·5, 20 mmol/l EDTA, 0·5% sodium dodecyl sulphate and 100 µg/ml proteinase K). The samples were incubated at 37°C for 30 min, extracted with phenol–chloroform–isoamyl alcohol, precipitated with ethanol and analysed on a 6% polyacrylamide sequencing gel.
Mobility shift assay. Mobility shift assay was performed as described previously (Buratowski & Chodosh, 1996; Ishimaru et al, 1997). In brief, double-stranded oligonucleotides were 32P labelled using polynucleotide kinase. DNA-binding reactions were set up with 1 × 104 c.p.m. of probe, 2 µg of ds-poly(dI–dC) and 5 µg of nuclear protein in the same buffer as for DNase I footprinting. Reactions were incubated on ice for 15 min and electrophoresed on a 5% polyacrylamide gel in 0·5× Tris–borate–EDTA (TBE) at 4°C. For competition experiments, 100-fold molar excess of double-stranded unlabelled oligonucleotides was added to the binding reaction mixture together with the other components. The oligonucleotides of consensus sequence were Sp1: 5′-CCAGGAAAGGAAGGGGGCTGGCTCGGAGG-3′ (Zhang et al, 1994) and Oct-1: 5′-TGTCGAATGCAAATCACTAGAA-3′ (Santa Cruz Biotechnology, Santa Cruz, CA, USA). For supershift experiments, 1 µl of anti-Sp1 and anti-Bcl-6 antibody (Santa Cruz Biotechnology) was added to the binding reaction mixture 15 min before the addition of the probe.
Site-directed mutagenesis. Mutated CD10/NEP promoter fragments were generated by polymerase chain reaction (PCR) as described previously (Ishimaru et al, 1997) with CD10/NEP(−511/+556)pBluescript (Stratagene, La Jolla, CA, USA) plasmid as a template. The sequences of the mutated constructs were confirmed by sequence analysis.
The deletion of the 5′-UTR (+121/+556) decreased promoter activity in pre-B-cell line Nalm6
Because the abundance of CD10/NEP mRNA, especially type 1 transcripts, was higher in pre-B-cell line Nalm6 than in mature B-cell line Raji (Ishimaru & Shipp, 1995), we speculated that there should be a differentiation stage-specific regulatory mechanism of CD10/NEP expression in the lymphoid lineage. To examine the differentiation stage-specific regulation of CD10/NEP expression further, we generated a series of deletion constructs of the type 1 regulatory region and tested their promoter activity by transient transfection using the luciferase assay. As reported previously (Ishimaru & Shipp, 1995), using the CD10/NEP(−511/+556)-pXP2 construct, we could detect promoter activity in the pre-B-cell line Nalm6. However, this minimal type 1 promoter was inactive in the mature B-cell line Raji (data not shown). With the deletion of the 5′-UTR (+121/+556), the promoter activity dropped dramatically to baseline levels in Nalm6 (Fig 1). The inclusion of the upstream region up to −1854 yielded no additional activity. These results demonstrated that the region +121/+556 plays an important role in the regulation of CD10/NEP expression in the pre-B-cell line Nalm6.
Three regions of the 5′-UTR preferentially interacted with nuclear proteins from Nalm6
We speculated that pre-B-cell-specific transcription factors might bind the 5′-UTR. To analyse the transcription factors responsible for the differentiation stage-specific regulation of CD10/NEP expression, we performed DNase I footprinting with a DNA fragment containing the 5′-UTR −46/+556 and nuclear proteins from Nalm6 and Raji. Three regions of the 5′-UTR (I: +93/+132; II: +323/+352; III: +432/+461) interacted preferentially with nuclear proteins from the pre-B-cell line, Nalm6, rather than from the mature B-cell line, Raji (Fig 2A–C), which indicated that the transcription factors responsible for region I, II and III appeared to be pre-B cell specific.
To confirm further the results of DNase I footprinting, we performed a mobility shift assay using double-stranded oligonucleotides specific for regions I, II and III (oligos I, II and III respectively) (Fig 3). Using oligo I as a probe, one major shifted band was detected with nuclear protein from Nalm6; however, two major shifted bands were detected with nuclear protein from Raji (Fig 4A). These bands showed competition when a 100-fold molar excess of self-oligonucleotides was used, showing that these bindings were specific. Using oligos II and III as a probe, one major shifted band was detected with nuclear protein from Nalm6 (Fig 4B and C respectively). However, these bands were also detected with nuclear protein from Raji, although less abundantly, which suggested that these transcription factors were relatively pre-B cell specific.
Mutation of the region +323/+352 significantly decreased the promoter activity in Nalm6
To study further the interaction of nuclear protein with each region, the KpnI restriction site was introduced into each region (Fig 5) and tested for the ability to compete for the binding activity. Mutated oligonucleotides could not compete with the nuclear protein binding to wild-type oligonucleotides or bind to nuclear proteins (data not shown).
To analyse the function of the protein–DNA interaction in each region, the same mutation was generated in each region of the CD10/NEP(−511/+556)-pXP2 construct. Transient transfection experiments comparing the wild-type construct (WT) and the mutated construct demonstrated that region II was critical for CD10/NEP promoter activity (Fig 6). Mutation of region II (II-M) significantly decreased promoter activity down to 54·3% in Nalm6 (P < 0·001). On the other hand, mutation of regions I (I-M) and III (III-M) did not affect promoter activity.
The transcription factor that binds to the region +323/+352 was identical to Sp1
Because we found the GC-rich sequence in region II, a competition experiment was performed using nuclear protein from Nalm6 to determine whether Sp1 was responsible for the protein–DNA interaction in region II. The shifted band was competed efficiently with both oligo II and Sp1 oligo (Zhang et al, 1994) but not with Oct-1 oligo (Fig 7). To confirm the result of the competition experiment, anti-Sp1 antibody was used in the supershift experiment. We detected the supershifted band with anti-Sp1 antibody but not with anti-Bcl-6 antibody, and the supershifted band was also detected with nuclear protein from Raji (Fig 7). These results suggested that the transcription factor responsible for region II was identical to Sp1. Although the mutation of region I (I-M) did not affect promoter activity, we found that the transcription factors responsible for region I were members of the Oct family (data not shown).
We found a functionally important transcription factor binding site in the 5′-UTR of the type 1 CD10/NEP promoter, which was identical to Sp1. As the abundance of CD10/NEP mRNA, especially for type 1 transcripts, was higher in the pre-B-cell line than in the mature B-cell line, our results suggested that ubiquitously expressed Sp1 might play an important role in differentiation of the stage-specific regulation of CD10/NEP expression in the lymphoid lineage. Recent reports have suggested that hypermethylation of the 5′-UTR of CD10/NEP is associated with a loss of CD10/NEP expression (Usmani et al, 2000), and an androgen response element is located in the 3′-UTR of CD10/NEP mRNA (Shen et al, 2000).
The characteristics of the type 1 CD10/NEP promoter resemble those of promoters for housekeeping genes (Ishimaru & Shipp, 1995), and Sp1 has been implicated in the transcription of TATA-less promoters of housekeeping genes (Pugh & Tjian, 1991) and the protection of CpG islands (Gardiner-Garden & Frommer, 1987) from methylation (Brandeis et al, 1994). They share some common features: they have wide tissue distribution, a low constitutive level of expression and generally multiple transcription initiation sites. However, these hallmarks are not restricted to promoters for housekeeping genes. Promoters of several oncogenes and several growth factor receptor genes also belong to this category. An unusually long (> 200 nucleotides) GC-rich 5′-UTR of the type 1 CD10/NEP promoter is also a common feature of most mRNAs coding for proto-oncogenes and other factors related to cell proliferation (Kozak, 1991). Previous studies have suggested that hairpin loops in the 5′-UTR affect the translation efficiency of the mRNA (Kozak, 1989), and premature termination is sometimes associated with secondary structure (Spencer & Groudine, 1990). Our results suggested that the 5′-UTR could also regulate gene expression through the transcriptional mechanism. There are several reports that support transcriptional regulation by the 5′-UTR (Amrolia et al, 1995; Valhmu et al, 1998).
Although Sp1 is expressed ubiquitously, substantial variations in expression level at different stages of development and differentiation have been reported (Saffer et al, 1991). Well-developed, fully differentiated cells with specialized functions primarily exhibited the lowest amounts of Sp1. Several lines of evidence also suggest the important role of Sp1 in the differentiation process. Marked downregulation of Sp1 expression was demonstrated during myogenesis (Vinals et al, 1997), and reduced DNA binding of Sp1 in differentiated chondrocytes has been reported in comparison with fibroblast-like dedifferentiated chondrocytes (Dharmavaram et al, 1997). However gene-targeting studies of Sp1 have shown recently that it is dispensable for growth and differentiation of primitive cells (Marin et al, 1997). Another unique feature of Sp1 as a transcription factor is its synergistic activation and interaction with other transcription factors (Li et al, 1991). Sp1 has been shown to interact directly with the TATA-box protein (Gill et al, 1994). When bound to distant sites, Sp1 can interact with it, thus looping out the intervening DNA (Su et al, 1991). Sp1 may establish interactions between promoters and distant regulatory elements through such a looping mechanism. In our study, the promoter activity of the type 1 CD10/NEP regulatory region (−511/+556) was minimal and, including the upstream region up to −1854, yielded no additional activity. We can speculate that an enhancer or another transcription factor, which might interact with Sp1, could also play an important role in the regulation of CD10/NEP expression.
We gratefully acknowledge Margaret A. Shipp (Dana-Farber Cancer Institute) for her helpful comments throughout this project.