(Received for publication, December 6, 1995; and in revised form, February 5, 1996)
From the
Cloning of the 5`-flanking region of the rat pp120 gene has indicated that it is a housekeeping gene: it lacks a functional TATA box and contains several Sp1 binding sites and multiple transcription initiation sites at nucleotides -101, -71, -41, and -27 spread over a GC-rich area. A fragment between nucleotides -21 and -1609 exhibited promoter activity when ligated in a sense orientation into a promoterless luciferase reporter plasmid and transiently transfected into rat H4-II-E hepatoma cells. 5` progressive deletion and block substitution analyses revealed that the three proximal Sp1 boxes (boxes 3, 5, and 6) are required for basal transcription of the pp120 gene. Promoter activity was stimulated 2-3-fold in response to insulin, dexamethasone, insulin plus dexamethasone, and cAMP. Although unaltered by phorbol esters alone, promoter activity was stimulated 4-5-fold in response to phorbol esters plus cAMP. Several motifs resembling response elements for insulin (in the rat phosphoenolpyruvate carboxykinase gene), glucocorticoids, cAMP, and phorbol esters as well as a number of putative binding sites for activating proteins-1 (Jun/Fos) and -2, and liver-specific factors were detected. The role of these sites in tissue-specific expression of pp120 remains to be investigated.
pp120/ecto-ATPase, ()a substrate of the insulin
receptor tyrosine kinase, is an integral liver plasma membrane
glycoprotein(1, 2, 3, 4) . It is
identical to HA4, a liver bile canalicular domain glycoprotein (5) and to a rat Ca
/Mg
ecto-ATPase(6) , which has been identified as a cell
adhesion molecule (cell-CAM(
)-105) (7, 8) and a bile acid transporter(9) .
Moreover, pp120 shares sequence homology with human biliary
glycoprotein and other members of the carcinoembryonic antigen (CEA)
immunoglobulin superfamily(10) .
Molecular cloning revealed
that the rat pp120 gene consists of nine exons, the 7th of which (53
bp) is alternatively spliced during mRNA processing, generating two
alternatively spliced variants that differ in the intracellular
cytoplasmic domain(11) . The truncated isoform lacks 61 of the
71 amino acid cytoplasmic domain, including basal (Ser)
and insulin-stimulated (Tyr
) phosphorylation
sites(4) .
The function of pp120 remains unclear. We have observed that the rate of insulin-stimulated internalization and degradation of the ligand-receptor complex is 2-3-fold higher in NIH 3T3 cells coexpressing insulin receptors and pp120 than in cells expressing insulin receptors alone, suggesting a role for pp120 in the hepatic clearance of insulin from circulation(12) . Since pp120 may play a physiological role in insulin metabolism, it has become essential to understand the mechanism of its in vivo expression, both basal and regulated.
Glucocorticoid treatment of rats increased pp120 protein level 2-3-fold in liver plasma membrane(1) . To understand the molecular basis for the regulation by glucocorticoids, we have cloned the 5`-flanking region and demonstrated a functional promoter activity of the pp120 gene. This region lacks a TATA box and contains multiple transcription initiation sites as well as potential binding sites for basal and regulatory transcriptional elements. Additionally, we have observed that Sp1 plays an important role in determining basal promoter activity of the gene.
To obtain intron 1 sequence,
plasmid clone p-2(12) was used as template and -100, T3, and T7
pBluescript II KS
-specific oligonucleotides as initial
primers.
An aliquot of amplified DNA (50 ng) was
ligated into pCRII plasmid (Invitrogen, San Diego). Constructs
containing the genomic DNA insert in either sense or reverse
orientation were treated with XhoI and HindIII, and
the DNA was analyzed on 1% agarose gel and electroeluted in 0.2
TBE (Tris-borate-EDTA) buffer at 60 V for 3 h. Purified genomic DNA was
then ligated at the XhoI and HindIII sites of
pGL3-BASIC promoterless firefly luciferase reporter plasmid (Promega).
The same PCR technique was employed to synthesize the 5` deletion
products using the same (-21) antisense primer and different sense
21-mer primers whose 5` ends were at nt -439, -249,
-194, -147, -131, -124, and -112,
respectively. Amplified genomic DNA was subsequently subcloned at the XhoI and HindIII sites of pGL3-BASIC plasmid.
Scanning mutants between nt -209 and -128 were created in the -249/-21 promoter fragment by replacing 20 bp of the native sequence with 20 bp of a heterologous sequence that includes an EcoRI restriction site (5`-CTCgaattcGATGGCGGTAT-3`) in a PCR-based reaction. Mutant promoters were prepared by EcoRI restriction and ligation of the appropriate pairs of the 5` and 3` fragments. The mutant promoter fragments were cloned into the XhoI/HindIII sites of pGL3-BASIC plasmid. Using this procedure, three mutant constructs were synthesized and designated as -249Mut3pLuc, -249Mut5pLuc, and -249Mut6pLuc containing block substitution mutation from nt -209 to -189, -169 to -149, and -148 to -128, respectively.
Figure 1:
Map of the 5`-flanking region and
intron 1 of the pp120 gene. Genomic DNA of -2, a bacteriophage
containing the 5`-flanking region of the pp120 gene(11) , was
cleaved at the intronic XhoI sites (X) to yield
different DNA fragments that were subcloned into the pBluescript II
KS
plasmid vector. p-2(12) subclone contains exons 2
and most of the intervening introns 1 and 2 sequences, whereas p-2(7)
contains exon 1 and greater than 10 kb of the 5`-flanking region of the
gene. p-2(7) was further cleaved with either HindIII (H) or EcoRI (E) and self-religated to yield
p-2(7-HindIII) and p-2(7-EcoRI),
respectively.
Figure 2:
Nucleotide sequence of the 5`-flanking
region of the rat pp120 gene. Numbers on the right are nucleotide number relative to +1 at the
ATG translation initiation codon (indicated by an arrow). Nucleotides upstream of this codon are labeled with negative numbers. Potential regulatory cis-elements
are indicated either by boxes or underlining. They
include binding sites for HNF-1 and HNF-5, CAAT enhancer-binding
protein (C/EBP), liver factor-A1 (LF-A1-RS),
activating proteins-1 and -2 (AP-1 and AP-2),
glucocorticoid response element (GRE), insulin response
element (IRE), and Sp1. Sp1 boxes are in bold and are
designated Sp1 box 1-6.
The
nucleotide sequence of intron 1 (769 bp) was similarly determined using
plasmid clone p-2(12) as template (data not shown) and beginning with
-100, an exon 2-specific primer, and plasmid-specific T3 and T7
primers.
Sequence analysis by MacVector 4.5 program (IBI) revealed
the presence of several putative DNA-binding elements that may play a
role in basal and regulated pp120 transcriptional activity. These
include multiple putative Sp1 binding sites in the proximal promoter (Table 2) and a number of motifs conforming to the consensus
binding site for HNF-1/HP-1 liver-specific transcription factor (nt
-1624 to -1612) and for LF-A1-RS (Table 3), a trans-acting factor that is required for the expression of
several genes such as human apolipoprotein A1, haptoglobin-related
genes, and human -antitrypsin in
hepatocytes(17) . Overlapping sequences matching perfectly the
consensus binding sequence for HNF-5 (nt -1359 to -1373), a
liver-specific factor in the rat tyrosine aminotransferase
gene(18) , were characteristically found in proximity to other
liver-specific factor binding sites such as CAAT enhancer-binding
protein (nt -1376 to -1346) binding sites in the
liver-specific rat tyrosine aminotransferase gene (TTTGTTTT, (18) ).
Potential sites for activating proteins AP-1 (Jun/Fos) and AP-2 were also found (Table 4). Nucleotide sequences matching 9 out of 10 bp in the insulin response element of the rat phosphoenolpyruvate carboxykinase gene (PEPCK, (19) ), 12 out of 15 bp in the loose consensus sequence of glucocorticoid response element (20) , 8 out of 8 bp in TPA response element(21) , and 6 out of 6 bp in cAMP response element (22) were also identified (Table 4).
Figure 3:
Identification of the transcription start
sites for the rat pp120 gene. The TAP reverse ligation PCR method was
applied to total rat livers. RNA was sequentially treated with DNase I,
CIP, and TAP before ligation to an RNA linker (Table 1) as
described under ``Experimental Procedures.'' Reactions were
conducted in the absence(-) or presence (+) of TAP and avian
myeloblastosis virus reverse transcriptase as indicated. 100 ng of
ligated RNA was reverse transcribed using exon 1-specific -45
antisense primer and amplified with nested exon 1-specific
-38 and
-19 antisense primers (Table 1). The PCR products were
analyzed on a 6% polyacrylamide-urea sequencing gel. The size of the
PCR products (bands A-D) was determined by comparison
with a sequencing ladder obtained by the dideoxy sequencing of
p-2(7-HindIII) plasmid using
-19
primer.
Figure 4:
The
5`-flanking region of the rat pp120 gene contains functional promoter
activity in H4-II-E rat hepatoma cells. A 1.6-kb fragment (nt
-1609 to -21) from the 5`-flanking region was subcloned
into a promoterless luciferase reporter plasmid in both sense and
reverse orientation. These constructs were transiently transfected into
H4-II-E rat hepatoma cells, and the luciferase activity was determined
in cell lysates 48 h after transfection. A hGH reporter plasmid (pXGH5)
was cotransfected to monitor transfection efficiency. For comparison,
luciferase activity in lysates from untransfected cells was included (Untrsft). Results were normalized for GH secretion and
expressed as mean ± S.D. of triplicate transfections in relative
light units. The graph represents typical results from four separate
experiments.
Figure 5: Effect of progressive 5` deletions on basal promoter activity in H4-II-E cells. A series of promoter fragments with different 5` ends (nt -1609, -439, -249, -194, -147, -131, -124, -112) and a common 3` end (nt -21) was ligated into the pGL3 promoterless plasmid and transiently transfected into H4-II-E hepatoma cells. Deletion constructs are schematically shown on the left. The relative location of the six potential Sp1 boxes in the -439/-21 promoter fragment are schematically shown below. Transfections were performed in triplicate, and the luciferase activity in the cell lysates was measured 48 h after transfection. Luciferase activity (mean ± S.D. in relative light units) of each construct is graphically shown on the right panel and has been normalized for equal hGH levels in the media. For comparison, luciferase activity in cell lysates of untransfected cells was included. The graph represents typical results of four separate experiments.
In addition to the 5` deletion analysis described above, a 20-bp nucleotide block substitution (which selectively mutated boxes 2, 3, 4/5) in the -249/-21 promoter fragment was also made to assess the functional significance of these respective Sp1 boxes in basal promoter activity (Fig. 6). Mutation of Sp1 box 2 did not alter promoter activity of the -249/-21 promoter fragment (-249Mut3pLuc 5,965.5 ± 381.69 versus -249pLuc 4,422.4 ± 971.42), confirming the previous observation that box 2 did not play a significant role in the overall pp120 promoter activity. However, mutation of the region spanning Sp1 box 3 (-249Mut5) or that spanning Sp1 box 4 and box 5 together (-249Mut6) in the -249/-21 promoter fragment led to a significant decrease (p < 0.05) in luciferase activity relative to the intact -249pLuc construct (-249Mut5pLuc 1,501.9 ± 579.37 and -249Mut6pLuc 1,173.1 ± 293.07 versus -249pLuc 4,422.4 ± 971.42). Thus, selective block mutation of either box 3 or boxes 4/5 led to a comparable reduction in the overall activity of the pp120 promoter, indicating that these Sp1 binding sites are equally important. It is unclear whether boxes 4 and 5 are both essential for activity, since mutations in -249Mut6pLuc did target both of these boxes. However, 5` deletion analysis suggests that box 4 did not contribute to basal activity since deletion of box 4 did not further decrease basal activity of the pp120 promoter (-147pLuc versus -131pLuc). Taken together, 5` deletion and block mutation analyses suggest that the collective presence of Sp1 boxes 3, 5, and 6 is needed for full basal activity of the pp120 promoter in H4-II-E cells.
Figure 6: Effect of block substitution of specific Sp1 boxes in the -249/-21 promoter fragment on basal promoter activity in H4-II-E cells. A series of 20-bp (5`-CTCGAATTCGATGGCGGTAT-3`) block mutations was prepared in the -249/-21 promoter fragment (-249pLuc), which overlapped and mutated specific potential Sp1 boxes. -249Mut3pLuc, -249Mut5pLuc, and -249Mut6pLuc constructs containing block mutations spanning Sp1 box 2 in nt -209 to -189, Sp1 box 3 in nt -169 to -149, and Sp1 boxes 4/5 in nt -148 to -128, respectively, were thus obtained. Luciferase activity in lysates derived from cells transfected with these constructs is graphically shown on the right. Luciferase activity was normalized for GH secreted in the media and calculated as mean ± S.D. in relative light units of triplicate transfections. The graph represents typical results of three different experiments.
Figure 7:
Regulation of the pp120 promoter activity
in H4-II-E cells by insulin, dexamethasone, cAMP, and phorbol esters. A
1.6-kb fragment (nt -1609 to -21) of the 5`-flanking
region was subcloned into pGL3 and transfected into H4-II-E cells using
DEAE-dextran as described in the legend to Fig. 4. Transfected
cells were incubated overnight in serum-containing medium followed by a
24-h incubation in serum-free medium. Insulin (Ins, 1 mg/ml),
dexamethasone (Dex, 1 mM), cAMP (1 mM), and
PMA (1 mM) were added and the incubation continued for an
additional 24 h. Untreated transfected cells (plain) were
included as control. Luciferase activity was measured in the cell
lysates, normalized for hGH secretion, and calculated as mean ±
S.D. in relative light units of triplicate transfections. The graph
represents typical results of four different
experiments.
pp120, a 120-kDa rat liver plasma membrane glycoprotein, is
an endogenous substrate of the insulin receptor
kinase(1, 3, 4) . It is identical to a
Ca/Mg
ecto-ATPase (cell-CAM-105),
possessing two ATP binding sites and three immunoglobulin-like loops in
its extracellular domain(6, 10) . In this report, we
have cloned the 5`-flanking region of the rat pp120 gene and showed
that it contains functional promoter activity when transfected into
H4-II-E rat hepatoma cells.
The 5`-flanking region of the pp120 gene
shares 60% homology with that of the human tumor marker CEA (Fig. 8), a member of the immunoglobulin superfamily
gene(25) . Since CEA has been implicated in neoplasia and cell
adhesion (26) and pp120 has been proposed to play a role in
insulin clearance from blood(12) , identification of basal and
regulatory DNA elements may therefore provide important insight into
the molecular mechanisms underlying regulation of expression of these
gene products.
Figure 8: Comparison of the nucleotide sequence of the 5`-flanking region of the rat liver pp120 and the human CEA genes. Approximately 1 kb of the 5`-flanking region of each of the rat pp120 (GenBank U27207) and the human CEA (GenBank HUMCEA01) genes was compared using the GeneWorks software program (Intelligenetics). Sequence homologies are shown as boxes.
Similar to immunoglobulin genes such as neural cell adhesion molecule and Thy-1 (27, 28) and to many genes that are involved in signal transduction(29, 30, 31, 32, 33, 34, 35, 36, 37) , the rat pp120 appears to belong to the GC-rich promoter class of TATA-less housekeeping genes. This class usually contains several transcription start sites in close proximity to clustered potential Sp1 binding sites spread over a GC-rich area(24, 38, 39, 40, 41) . The region between nt -165 and -25 in the proximal pp120 promoter is GC-rich (62%), and it contains four transcription start sites (nt -101, -71, -41, and -27) as well as four potential binding sites for Sp1 ( Table 2and Fig. 5) which are clustered in a 72% GC-rich domain (nt -165 to -114). Although further studies such as specific binding of purified Sp1 to Sp1 boxes in DNase I footprinting and protection assays are required to determine whether Sp1 binding to boxes 3, 5, and 6 is essential for efficient transcription of the TATA-less pp120 gene, our observations suggest that these boxes are essential for basal functional promoter activity of the gene. At least two of these boxes (boxes 3 and 5) may act independently as was reported for the Sp1 boxes of the TATA-less gene of the rat insulin-like growth factor-binding protein-2(42) . It is interesting that box 3 contains elements that fully match the consensus sequence for Sp1 binding site, and boxes 6 and 5 contain either a single C to A mutation at position 5 (box 6) or in addition to another mutation at position 1 of the Sp1 canonical motif (box 5) (Table 2). The minimal effect exhibited by these mutations on the pp120 promoter activity is in agreement with the notion that Sp1 binding sites containing the C to A mutation at position 5 are fully functional and bind Sp1 with high affinity in many natural promoters (43, 44, 45) and that mutation at position 1 is the least important(43) . In addition to the C to A mutation at position 5, box 4 contains mutation at position 8. Although this mutation may be responsible for the apparent minimal role of box 4, we cannot exclude in these studies the possibility of a functional role for box 4 in the transcription of the pp120 gene. Individual block mutation of boxes 4 and 5 is required to address this question in more detail. The apparent nonfunctionality of Sp1 boxes 1 and 2 is not unexpected since they are spatially separated from box 3 by 250 and 42 nt, respectively. Additionally, these boxes are located outside the GC-rich area (nt -25 to -165) of proximal pp120 promoter.
Sp1-activating proteins acting either alone (46, 47) or in conjunction with a transcriptional initiator located downstream from the Sp1 binding site (48, 49) attract the transcription factor IID complex to direct proper transcription. pp120 proximal promoter sequence contains a 7-bp fragment (CCAAATC, nt -45 to -38) that includes the transcription start site at nt -41, thus conforming to the loose consensus sequence for a transcriptional initiator (PyPyA*N T/A PyPy) at the start site (*)(49) . In the pp120 gene, this putative initiator is part of an AGCCAAAT sequence (nt -47 to -39) that matches 6 out of 8 bp in the consensus octamer binding site found in immunoglobulin genes (ATGCAAAT, (28) ). Whether this potential initiator-octamer binding site complex participates in the transcriptional activation of the pp120 gene remains to be investigated.
A number of motifs for liver-specific transcription factors are present in the 5`-flanking region of the pp120 gene. Some of these binding sites, such as those for HNF-5, are characteristically located in proximity to other liver-specific factors, such as the potential CAAT enhancer-binding protein binding sites in the pp120 gene (nt -1373 to -1359). Although many of these factors are neither restricted to nor sufficient to confer hepatocyte specificity, they are commonly classified as liver-specific transcription factors(50) . Nonetheless, they have been implicated in polarized epithelium expression(50) . Hence, it is of future interest to investigate whether these potential liver-specific factors play a role in pp120 expression on the bile canalicular domain of hepatocytes.
Nucleotide sequences matching response elements for cAMP and TPA were found in the 5`-flanking region of the pp120 gene. TPA and cAMP may exert their effects by binding either to their respective DNA binding sites (TPA response element and cAMP response element, respectively) or through the activation of AP-1 (TPA, (51) ) or AP-2 (TPA and cAMP, (52) ). pp120 promoter activity was not affected by PMA alone, whereas it was elevated 4-5-fold in response to both cAMP and PMA. This suggests a synergistic effect of PMA and cAMP on the promoter activity of pp120. cAMP and PMA may coordinate their concerted effect by binding either to the same site, such as AP-2, or to different sites where the binding of one is required to mediate regulation by the other. Although nucleoside monophosphates are not substrates of the enzymatic activity that is believed to be associated with the protein gene product(53) , our observations of the up-regulatory effect of cAMP on pp120 promoter activity warrant further investigations.
Dexamethasone treatment of rats increased pp120 protein expression 2-3-fold in hepatocytes(1) . Hence, the 2-fold increase in pp120 promoter activity by glucocorticoids suggests that the up-regulatory effect of glucocorticoids on the pp120 protein occurs at the transcriptional level. These observations are in marked contrast to the post-transcriptional down-regulatory effect of glucocorticoids on the insulin receptor substrate-1 mRNA levels, the occurrence of which is not accompanied by any effect on the insulin receptor substrate-1 promoter activity in transfected 3T3-F442A adipocytes(37) .
The up-regulatory effect of insulin on the pp120 promoter activity suggests that insulin may act at the transcriptional level to regulate pp120 expression, an insulin receptor substrate. In view of the proposed role of pp120 to regulate hepatic insulin clearance from blood (12) , modulation of pp120 gene expression may represent an important feedback loop in insulin action in liver. The promoter activity of insulin receptor substrate-1 was not altered by insulin treatment of transfected 3T3-F442A adipocytes(37) . Thus, pp120 constitutes a first example of an insulin receptor substrate whose promoter activity is up-regulated by insulin. The cis-regulatory elements required to regulate transcription of the pp120 gene by insulin have not been determined. Insulin may act through nuclear factors binding to the sequence that matches the insulin response element in the PEPCK gene (Table 4) or to a novel site. Insulin down-regulates the transcriptional rate and counteracts the up-regulatory effect of glucocorticoids on PEPCK, the enzyme that catalyzes the rate-limiting step in gluconeogenesis(19) . Unlike the PEPCK gene, there is no apparent interaction between insulin and glucocorticoids to regulate transcription of the pp120 gene.
In the present report, we have identified the presence of potential cis-elements that constitute the binding sites of various trans-acting factors on the pp120 promoter. Further studies of the interaction between these putative binding sites with specific trans-acting nuclear factors will provide important insight into the elements required for tissue-specific expression of the rat pp120 gene.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U27207 [GenBank]and U27208 [GenBank]for the 5`-flanking region and intron 1 sequences of the pp120/ecto-ATPase gene, respectively, and HUMCEA01 for the 5`-flanking region of the human CEA gene(16) .