C/EBPbeta contributes to cAMP-activated transcription of phosphoenolpyruvate carboxykinase in LLC-PK1-F+ cells

Xiangdong Liu, Quynh-Thu Wall, Lynn Taylor, and Norman P. Curthoys

Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523-1870


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Phosphoenolpyruvate carboxykinase (PEPCK) is a key regulatory enzyme in renal gluconeogenesis. Activation of various PEPCK-2300Luc reporter constructs in LLC-PK1-F+ cells, a gluconeogenic line of porcine renal proximal tubule-like cells, by protein kinase A (PKA) is mediated, in part, through the cAMP-response element (CRE)-1 of the PEPCK promoter. Incubation of a CRE-1 containing oligonucleotide with nuclear extracts from LLC-PK1-F+ cells produced multiple bands, all of which were blocked by antibodies that are specific for C/EBPbeta but not for C/EBPalpha or C/EBPdelta . Treatment of cells with cAMP did not affect the expression of C/EBPbeta , but the observed binding activity was increased nearly threefold. Mutation of CRE-1 to a Gal-4 binding site reduced the PKA-dependent activation of PEPCK-2300Luc to 40% of that observed with the wild-type construct. Coexpression of a chimeric protein containing a Gal-4 binding domain and the transactivation domain of C/EBPbeta , but not of C/EBPalpha or CRE binding protein (CREB), restored full activation by PKA. A deletion construct that lacks the activation domain of C/EBPbeta functions as a dominant negative inhibitor. Thus the binding of C/EBPbeta to the CRE-1 may contribute to the cAMP-dependent activation of the PEPCK promoter in kidney cells.

renal gluconeogenesis; adenosine-3',5'-cyclic monophosphate-response element-1; protein kinase A


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE CYTOSOLIC FORM OF phosphoenolpyruvate carboxykinase (PEPCK) is encoded by a single copy gene that is developmentally regulated and is expressed in a tissue- and cell-specific manner (12). It is expressed predominantly in liver, kidney, and adipose tissues. Transcription of PEPCK is suppressed during fetal development but is dramatically induced at birth. Transcription of the PEPCK gene in the postnatal liver is stimulated by glucagon via cAMP, thyroid hormone, and glucocorticoids, whereas insulin and phorbol esters inhibit its expression. Expression of a chimeric PEPCK-bovine growth hormone gene in transgenic mice revealed that a relatively small region within the PEPCK promoter (-460 to +73 bp) contains most of the information required for conferring the appropriate pattern of developmental, tissue-specific, hormonal, and dietary regulation of the PEPCK gene (18, 19). However, this region is highly complex. DNase I footprinting analysis of this segment with rat liver nuclear extracts identified at least eight protein binding domains, termed cAMP response element (CRE)-1, CRE-2, and P1 through P6 (30).

The initial cAMP-response element (CRE-1) within the PEPCK promoter can bind a number of transcription factors, including CRE binding protein (CREB) (23), c-Fos/c-Jun (11), and C/EBP (23). The CRE-1 element was able to confer cAMP responsiveness to a neutral promoter when transfected into human choriocarcinoma cells (1). However, the extent of cAMP induction mediated by CRE-1 alone was much less than that observed with the intact PEPCK promoter, suggesting that other elements are also required for full cAMP responsiveness. Transient transfection assays performed in HepG2 liver cells using a series of deletions and block mutations of a PEPCK-490-chloroamphenicol acetyltyransferase (CAT) construct established that both the CRE-1 and P3(I) elements are important for cAMP induction (15). Mutation of both the CRE-1 and P3(I) sequences resulted in the complete loss of induction by either 8-bromo-cAMP or by the catalytic subunit of protein kinase A (PKA).

Use of dominant negative constructs established that both CREB and C/EBP are involved in mediating the cAMP-dependent activation of PEPCK gene expression in liver cells (27, 28). Experiments using Gal-4 fusion proteins (28) indicated that CREB preferentially binds to the CRE-1 element and interacts with upstream transcription factors to activate PEPCK transcription in HepG2 cells. Similar experiments have shown that the synergistic effect of the P3(I) and P4 elements can be mediated by C/EBPalpha (27) and to a lesser extent by C/EBPbeta (24). However, more recent data obtained by constitutive expression of specific antisense RNAs indicate that C/EBPalpha , and not C/EBPbeta , participates in cAMP-dependent activation of PEPCK transcription in H4IIE cells (8). The latter conclusion is supported by studies using mice homozygous for deletions in the genes that encode either C/EBPalpha or C/EBPbeta (7). C/EBPalpha was found to be required for the cAMP activation of PEPCK transcription in the neonatal liver. In contrast, C/EBPbeta was normally not essential, but it could compensate for the loss of C/EBPalpha if induced sufficiently. P3(II), which contains an activator protein (AP)-1-like element, also participates in this process, since the activity of the liver-specific region cannot be mimicked by multiple copies of other well-characterized C/EBP binding sites (29). Furthermore, supershift analysis suggests that a c-Fos/c-Jun protein from HepG2 cells binds to an oligonucleotide containing the P3(II) site. Thus a c-Fos/c-Jun protein also appears to be involved in cAMP activation of the PEPCK gene in liver cells (29).

While hepatic gluconeogenesis is essential for maintaining blood glucose levels, in kidney this process is coupled to ammoniagenesis and the maintenance of acid-base balance. The renal PEPCK is expressed solely within the proximal tubular segment of the nephron (2). Various hormones such as angiotensin II and parathyroid hormone are known to affect cAMP levels within the renal proximal tubule. The two hormones primarily regulate renal sodium, bicarbonate, and phosphate reabsorption. However, angiotensin II also stimulates renal ammoniagenesis (5), and parathyroid hormone stimulates renal gluconeogenesis (25). Previous experiments demonstrated that there are marked differences in the footprinting patterns observed with nuclear extracts prepared from rat liver and kidney (30), suggesting that kidney and liver may utilize different trans-acting factors to regulate the PEPCK gene. Thus it is physiological relevant to determine the specific cis/trans interactions that mediate the cAMP-dependent activation of the PEPCK gene in renal proximal tubule cells.

The cAMP-dependent activation of the PEPCK gene in LLC-PK1-F+ cells, a porcine gluconeogenic proximal tubule-like line of cells (10), mapped to only the CRE-1 and P3(II) elements (17). Mutation of the P3(I) and P4 elements had no effect on cAMP stimulation of the PEPCK-490CAT construct when transfected into the kidney cells. By using dominant negative constructs, it was shown that the renal response of the PEPCK gene to cAMP may be mediated, in part, by an isoform of C/EBP but not by CREB (17). In the current study, the endogenous protein in LLC-PK1-F+ cells that binds to the CRE-1 element of the PEPCK promoter was characterized. Of the various C/EBP isoforms, only antibodies against C/EBPbeta were able to block the specific binding of a CRE-1 oligonucleotide to nuclear protein(s) from LLC-PK1-F+ cells. Furthermore, luciferase assays using various Gal-4 constructs indicated that the transactivation domain of C/EBPbeta , but not that of C/EBPalpha or CREB, restores full activation by PKA. Thus the binding of C/EBPbeta to the CRE-1 element may contribute to the cAMP-dependent activation of the PEPCK promoter in kidney cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. [14C]chloramphenicol (specific activity, 56 mCi/mmol) and T4 polynucleotide kinase were obtained from Amersham. [gamma -32P]ATP (specific activity, 5,600 Ci/mmol) was obtained from ICN. 8-(4-Chlorophenylthio)-cAMP (CPT-cAMP) was purchased from Boehringer Mannheim. The Dual-Luciferase Reporter Assay Kit was purchased from Promega. Oligonucleotides containing consensus sequences for CRE and AP-1 elements were purchased from Santa Cruz Biotechnology. Antibodies specific for the alpha -, beta -, and delta -isoforms of C/EBP used in the supershift assays were obtained from Dr. Steven L. McKnight (Tularik). The expression vector for C/EBPbeta was provided by Dr. Richard Hanson (Case Western Reserve Univ., Cleveland, OH). GST-C/EBPalpha , GST-C/EBPbeta , and GST-C/EBPdelta fusion constructs were from Dr. Wen-Hwa Lee (Univ. of Texas, San Antonio, TX). The C/EBPbeta Delta Spl construct was obtained from Dr. Akira (Hyogo College of Medicine, Hyogo, Japan). The expression vectors for Gal-4-CREB and Gal-4-C/EBPalpha (Galpha 2) were obtained from Dr. William Roesler (Univ. of Saskatchewan, Saskatoon, SK, Canada), whereas the Gal-4-C/EBPbeta -(1---138) construct was obtained from Dr. Edwards Park (Univ. of Tennessee Health Sciences Center, Memphis, TN). Other biochemicals were purchased from Sigma or Fluka.

Cell cultures. LLC-PK1-F+ cells were originally isolated by Gstraunthaler and Handler (10) and were obtained from Dr. Gerhard Gstraunthaler (Univ. of Innsbruck, Innsbruck, Austria). The cells were cultured on 10-cm plates in a 50:50 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with 10% fetal bovine serum. The medium contained 5 mM glucose and 25 mM NaHCO3 and was adjusted to pH 7.4.

Nuclear extract isolation and electrophoretic mobility shift assays. Nuclear extracts were isolated from LLC-PK1-F+ cells as described by Lee et al. (14) except that Tris · HCl (pH 8.0) was replaced with HEPES (pH 7.9), and 1 mM dithiothreitol, 10 µM antipain, 10 µg/ml aprotinin, and 1 µM pepstatin were added to both the lysis and extraction buffers. Double-stranded deoxyribonucleotide probes were synthesized by Macromolecular Resources (Ft. Collins, CO) as complementary oligonucleotides containing 5'-BamHI overhangs. They were annealed in 50 mM NaCl, 66 mM Tris · HCl, and 6.6 mM MgCl2, pH 7.5, by heating to 85°C and cooling to 25°C. The various oligonucleotide probes and their position in the PEPCK promoter include the following: CRE-1, bases -99 to -77; the CRE-1 block mutation (mCRE-1), bases -99 to -77 but containing the same 5-bp mutation as found in the CRE-1 block mutation of the PEPCK-490CAT construct (16); and P3(II), bases -266 to -246. The sequences of the sense strands are as follows: CRE-1, 5'-GATCCGGCCCCTTACGTCAGAGGCGAG-3'; mCRE-1, 5'-GATCCGGCCCCTGCATGCAGAGGCGAG-3'; and P3(II), 5'-GATCTCAAAGTTTAGTCAATCAAAC-3'; where the sequences derived from the PEPCK promoter are underlined, the mutated bases are italicized, and the CRE-1 or AP-1 elements are in bold. The sequence of the consensus CRE and AP-1 oligonucleotides with the binding elements underlined are 5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3' and 5'-CGCTTGATGACTCAGCCGGAA-3', respectively.

The double-stranded oligonucleotides were 5'-end labeled with [gamma -32P]ATP using T4 polynucleotide kinase (31). The indicated amount of nuclear extract was incubated for 10 min on ice with 400 ng poly[dI-dC], 15 ng pUC19, and 20 fmol of labeled oligonucleotide before being applied to a 5% polyacrylamide gel. In the competition experiments, the double-stranded unlabeled competitor oligonucleotide was preincubated with the other reagents for 10 min on ice before addition of the labeled oligonucleotide and incubation for another 10 min. Alternatively, the indicated amount of antibody was preincubated with 400 ng poly[dI-dC], 15 ng pUC19, and nuclear extract for 40 min on ice, and then the labeled probe was added and incubated for another 20 min on ice before being applied to a 5% polyacrylamide gel. In all cases, the electrophoresis was performed at 170 V for 1.5 h at 4°C.

CAT assay. LLC-PK1-F+ cells were split and replated on 10-cm plates at ~30% confluence. The cells were grown for 20 h in culture and transfected by calcium phosphate precipitation of DNA (4). The precipitated DNA contained 10 µg of the PEPCK-490CAT construct, 2 µg of cytomegalo virus (CMV)-beta -galactosidase, and, where indicated, 5 µg of a C/EBPbeta expression vector. The total DNA in each transfection was maintained at 40 µg by addition of salmon sperm DNA. At 20 h posttransfection, the cells were homogenized and assayed for beta -galactosidase activity. Samples of homogenate (20-100 µl) containing equivalent units of beta -galactosidase activity were used to measure CAT activity (9). The acetylated products and the unreacted substrate were separated by thin-layer chromatography, and the percent conversion was quantitated using a PhosphorImager (Molecular Dynamics).

Synthesis of luciferase reporter constructs. The various luciferase constructs were assembled in pGL2-Basic (Promega), which contains the firefly luciferase gene preceded by a large multicloning site. pGL2-Basic was linearized with NheI, blunted with Klenow, and then restricted with BglII. To obtain the -2,300-bp promoter segment of the PEPCK gene, pPEPCK-2300CAT was partially digested with XbaI, and the linear plasmid was isolated on an agarose gel and purified using GeneClean (BIO 101). The XbaI overhangs were blunted with Klenow, and the DNA was subsequently restricted with BglII. The 2.4-kb fragment was isolated and ligated into the restricted pGL2-Basic plasmid to produce pPEPCK-2300Luc. A 428-bp NdeI and BglII restriction fragment was removed from pPEPCK-2300Luc. The same restriction enzymes were used to digest the PEPCK-490CAT constructs containing the individual block mutations in the CRE-1, P2, and P3(II) sites (16). The isolated fragments containing the mutations were ligated into the linearized PEPCK-2300Luc plasmid to yield pPEPCKmCRE-1Luc, pPEPCKmP2Luc, and pPEPCKmP3IILuc, respectively. A 1,566-bp FseI and ClaI fragment from pPEPCKmCRE-1Luc was then cloned into pPEPCKmP3IILuc to produce a construct containing the double mutation. The 428-bp NdeI/BglII fragment containing the mCRE-1 element was also subcloned into pUC19. The mCRE-1 sequence contains a unique SphI site (16). Complimentary oligonucleotides, containing a Gal-4 binding site with SphI overhangs, were annealed and ligated into pUC19/mCRE-1 that had been linearized with SphI. The sequence of the sense strand is 5'-CGGGAGTACTGTCCTCCGCATG-3', where the Gal-4 binding site is underlined. The resulting plasmid was restricted with NdeI and BglII, and the promoter fragment containing the Gal-4 binding site was cloned into pPEPCK-2300Luc to yield pPEPCKGal4Luc. For each plasmid, all of the created mutations and ligated junctions were confirmed by dideoxynucleotide sequencing.

Luciferase assay. At 3 days postsplitting into 6-well plates, the LLC-PK1-F+ cells were transfected by calcium phosphate precipitation (4). Each sample contained 0.1 µg of pRL-null (Promega), 0.6 µg of a pPEPCK-2300Luc plasmid, and, where indicated, 2 µg of a chimeric Gal-4 and/or 1 µg of PKA expression vectors. Sufficient salmon sperm DNA was added so that all samples contained 5 µg of DNA. Approximately 24 h later, the transfection media was removed and fresh media were added. The cells were cultured for an additional 24 h and washed with 2 ml of phosphate-buffered saline, and cell extracts were prepared using 250 µl of passive lysis buffer (Promega). The extracts were assayed with a Turner Design 20/20 Luminometer using the reagents contained in the Dual-Luciferase Reporter Assay System (Promega). The firefly luciferase activities obtained from the various pPEPCK-2300Luc plasmids were standardized vs. the corresponding Renilla luciferase activities to correct for differences in transfection efficiency. In a single experiment, each transfection was performed in triplicate.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previous studies using a series of deletions and block mutations of a PEPCK-490CAT construct identified the CRE-1 and P3(II) sites as the primary elements within the PEPCK promoter (Fig. 1) that are essential for cAMP-dependent activation of the PEPCK gene in LLC-PK1-F+ cells (17). This analysis was repeated using a more sensitive and more responsive set of luciferase constructs (Fig. 2). Cotransfection of an expression vector that encoded the catalytic subunit of PKA produced a 30-fold activation of the PEPCK-2300Luc construct. Mutation of the CRE-1 element reduced basal activity by 20%. However, PKA activation of PEPCKmCRE-1Luc was reduced to one-third of the wild-type construct (11-fold). In contrast, mutation of the P2 element reduced basal expression to one-fifth of the basal expression of PEPCK-2300Luc, whereas PKA activation of PEPCKmP2Luc was at least as great (64-fold) as was observed with PEPCK-2300Luc. Mutation of the P3(II) element also had a significant effect on basal expression without affecting PKA activation. Basal expression of PEPCKmP3IILuc was reduced to 65% of the wild-type vector, but coexpression of PKA again caused a 30-fold increase from the reduced basal activity. The double mutant, PEPCKmCRE-1/mP3IILuc, exhibited properties equal to the sum of the two individual mutants. Basal expression was equivalent to that observed with PEPCKmP3IILuc (65%), whereas PKA activation was equivalent to that of the PEPCKmCRE-1Luc construct (9-fold).


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Fig. 1.   The proximal promoter region of the cytosolic phosphoenolpyruvate carboxykinase (PEPCK) gene. The locations of cis-acting elements within the PEPCK promoter are drawn to scale. Only the elements shaded in black were footprinted with proteins contained in rat renal nuclear extracts (30). The sequence of the cAMP-response element (CRE)-1 and P3(II) elements and the transcription factors known to bind to these sites are also shown. CREB, CRE binding protein.



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Fig. 2.   Protein kinase A (PKA)-dependent activation of various PEPCK-2300Luc constructs in LLC-PK1-F+ cells. The PEPCK-2300Luc and pRL-null plasmids were cotransfected into subconfluent LLC-PK1-F+ cells either without (solid bars) or with (shaded bars) a plasmid that encodes the catalytic subunit of PKA. The resulting firefly and Renilla luciferase activities were measured with the Dual-Luciferase Reporter Assay Kit. The reported data are means ± SD of 3 separate experiments (n = 9). wt, Wild type.

The potential role of a C/EBP protein in cAMP-dependent activation of PEPCK transcription in LLC-PK1-F+ cells was previously demonstrated by the finding that coexpression of either of two dominant negative forms of C/EBP blocks the cAMP-dependent stimulation of PEPCK-490CAT activity (17). Electrophoretic mobility shift assays were used to further characterize the protein that binds to the CRE-1 element (Fig. 3). Nuclear extracts from subconfluent LLC-PK1-F+ cells produced multiple bands when incubated with the CRE-1 probe (Fig. 3, lane 2). The specificity of the apparent binding was determined from competition experiments in which a 50- or 500-fold excess of various unlabeled competitors was preincubated with the nuclear extract. A 500-fold excess of unlabeled CRE-1 or an oligonucleotide containing a consensus CRE site completely inhibited the observed binding. In contrast, unlabeled mCRE-1, in which the CRE-1 sequence contains the same 5-bp mutation as the CRE-1 block mutant of PEPCK-490CAT, did not compete. Similarly, an oligonucleotide containing the P3(II) site of the PEPCK promoter failed to inhibit the binding to the CRE-1 probe, indicating that different proteins bind to the CRE-1 and P3(II) elements. The oligonucleotide containing a consensus AP-1 site exhibited only a slight competition, suggesting that c-Fos/c-Jun proteins are not primarily responsible for the observed binding. The cumulative data strongly suggest that the observed binding to the CRE-1 oligonucleotide is specific and represents interactions characteristic of the CRE-1 element.


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Fig. 3.   Specificity of binding to the CRE-1 oligonucleotide. The binding reaction was performed using 5 µg of a nuclear extract from LLC-PK1-F+ cells and 20 fmol of the 32P-labeled CRE-1 oligonucleotide (lane 2). A 50- or 500-fold excess of various competitors was included in the binding reactions shown in lanes 3-12. Lane 1 contains a control sample in which no nuclear extract was added. The gel was dried and imaged with a Molecular Dynamics PhosphorImager. B, bound probe; N, nonspecific binding; mCRE-1, CRE-1 block mutation; AP-1, activator protein-1.

The possibility that the protein in the nuclear extract from subconfluent LLC-PK1-F+ cells that binds to the CRE-1 element is an isoform of C/EBP was examined by supershift or immunoblocking analysis (Fig. 4). A polyclonal antibody against C/EBPbeta was able to supershift the binding of a GST-C/EBPbeta fusion protein in a concentration-dependent manner (lanes 2-4). Preincubation with increasing concentrations of anti-C/EBPbeta antibody blocked the binding of all proteins in the nuclear extract from LLC-PK1-F+ cells to the CRE-1 probe, suggesting that differentially modified forms of C/EBPbeta or heterodimers formed between C/EBPbeta and different partners are responsible for the multiple bands. The anti-C/EBPbeta antibody did not cross-react with the GST-C/EBPalpha or GST-C/EBPdelta proteins (data not shown). Furthermore, the above experiment was repeated using polyclonal antibodies specific for C/EBPalpha and C/EBPdelta (Fig. 5). The two antibodies supershifted the respective GST-C/EBPalpha and GST-C/EBPdelta fusion proteins but neither supershifted nor blocked the binding of nuclear proteins from LLC-PK1-F+ cells to the CRE-1 oligonucleotide.


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Fig. 4.   Immunoblocking assay using C/EBPbeta -specific antibody. Binding reactions were performed using 5 µl of a 1:10 dilution of GST-C/EBPbeta (lanes 2-5) or 2 µg of nuclear extract from LLC-PK1-F+ cells (lanes 6-9), 20 fmol of the 32P-CRE-1 oligonucleotide, and the indicated volumes of the C/EBPbeta antibody. Lane 1 contains a control sample in which no nuclear extract was added.



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Fig. 5.   Immunoblocking assay using C/EBPalpha (A)- and C/EBPdelta (B)-specific antibodies. Binding reactions were performed using GST-C/EBPalpha or GST-C/EBPdelta (lanes 2-5) or 2 µg of nuclear extract from LLC-PK1-F+ cells (lanes 6-9), 20 fmol of the 32P-CRE-1 oligonucleotide, and the indicated volumes of the specific antibody. Lane 1 contains a control sample in which no nuclear extract was added.

Various Gal-4 constructs were used to determine the ability of different transcription factors to restore PKA-dependent activation of PEPCK-2300Luc in LLC-PK1-F+ cells (Fig. 6). The firefly luciferase activity measured in LLC-PK1-F+ cells transiently transfected with PEPCK-2300Luc is strongly activated by cotransfection with an expression vector that encodes the catalytic subunit of PKA. When the CRE-1 site of the PEPCK-2300Luc construct was mutated, the fold activation by PKA was reduced to 38% of that observed with the wild-type construct. This result is similar to that observed with the PEPCKmCRE-1Luc construct. Conversion of the mutated CRE-1 site to a Gal-4 binding site had little effect on the fold activation by PKA. However, cotransfection of this reporter construct with an expression vector that encoded a chimeric protein containing a Gal-4 binding domain and the transactivation domain of C/EBPbeta restored the level of PKA activation to that observed with the wild-type construct. In contrast, coexpression of Gal-4-C/EBPalpha or Gal-4-CREB proteins had either no effect or further reduced PKA activation, respectively.


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Fig. 6.   Reconstitution of PKA activation of PEPCK-2300Luc activity using various Gal-4 chimeric proteins. A: various Gal-4 chimeric proteins contain the DNA binding domain (amino acids 1-147) of the Gal-4 transcription factor (hatched bar) and the indicated amino acids of the transactivation domain of C/EBPbeta , C/EBPalpha , or CREB (open bar). B: LLC-PK1-F+ cells were transfected with the wild-type (wt) PEPCK-2300Luc plasmid or the PEPCK-2300Luc plasmids that contained either mCRE-1 or the Gal-4 binding site (Gal4) in the presence or absence of the expression vector that encodes the catalytic subunit of PKA. Where indicated, the expression vector that encodes the chimeric Gal-4-C/EBPbeta , Gal-4-C/EBPalpha , or Gal-4-CREB protein was included in the transfection. The data represent means ± SE from 3 separate experiments.

Western blot analysis indicated that the level of C/EBPbeta expressed in LLC-PK1-F+ cells is not affected by treatment with CPT-cAMP or forskolin (Fig. 7A). In addition, the pattern of specific binding to the CRE-1 probe was not altered. However, the apparent binding activity was increased significantly in nuclear extracts prepared from cells treated with CPT-cAMP (Fig. 7B). The increase in apparent binding was specific to the CRE-1 probe, since the apparent binding observed with an Sp-1 containing oligonucleotide was unaffected by using nuclear extracts obtained from cAMP-treated and untreated cells (data not shown). When normalized to the Sp-1 controls, the binding activity was increased 2.7-fold within 2 h after treatment with cAMP, and the observed increase was sustained for 24 h.


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Fig. 7.   Effect of cAMP treatment on the level of C/EBPbeta and the CRE-1 binding activity in nuclear extracts of LLC-PK1-F+ cells. A: samples containing 5 µg of nuclear protein derived from LLC-PK1-F+ cells that were treated for 2 h in the absence (-) or presence (+) of 0.5 mM 8-(4-chlorophenylthio) (CPT)-cAMP (cAMP) or 10 µM forskolin (for) were separated by SDS-PAGE and blotted with antibodies specific for C/EBPbeta . B: binding reaction was performed using 20 fmol of 32P-CRE-1 oligonucleotide and 5 µg of nuclear extracts from cells that had been treated for 2, 8, or 24 h either in standard medium (lanes 2, 4, and 6) or in medium containing 100 µM CPT-cAMP (lanes 3, 5, and 7). Lane 1 contains a control sample in which no nuclear extract was added.

The importance of the activation domain of C/EBPbeta in mediating cAMP activation of PEPCK transcription was investigated by comparing the activation produced by coexpression of C/EBPbeta or of a deletion construct (Fig. 8). Coexpression of C/EBPbeta produced a sixfold stimulation of PEPCK-490CAT activity, whereas coexpression of Delta Spl significantly reduced basal activity. Delta Spl is a C/EBPbeta construct in which amino acids 41-205 are deleted, and thus it lacks the transactivation domain (22). Expression of the catalytic subunit of PKA produced a fourfold activation of PEPCK-490CAT that was further enhanced (2-fold) by the coexpression of C/EBPbeta . However, coexpression of Delta Spl completely abolished the activation caused by the catalytic subunit of PKA. Thus deletion of the activation domain of C/EBPbeta generates a dominant negative effector.


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Fig. 8.   Effect of expression of full-length or a deleted form of C/EBPbeta on the basal and PKA-activated transcription of PEPCK-490CAT in LLC-PK1-F+ cells. A: expression vectors encode full-length C/EBPbeta or the Delta Spl deletion that lacks amino acids 41-205. B: effect of the C/EBPbeta constructs on PEPCK-490CAT activity was determined in the absence (-) or presence (+) of the catalytic subunit of PKA. Data are means of duplicate transfections ± one-half of the range.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CREB was originally purified by DNA affinity chromatography using the CRE from the somatostatin gene as the ligand (21). On phosphorylation by PKA, CREB recruits a CREB binding protein (CBP) that acts as an essential coactivator (13). This sequence of events has become the paradigm to explain how cAMP-dependent activation of transcription is mediated in a variety of systems. Roesler et al. (28) demonstrated that cotransfection of HepG2 cells with KCREB, a dominant negative form of CREB that contains a point mutation in the DNA binding domain (32), blocked the PKA-dependent activation of PEPCK-490CAT. Subsequent experiments clearly document that CREB or C/EBPalpha , but not C/EBPbeta , can interact with CRE-1 to mediate the cAMP-dependent induction of PEPCK mRNA in various hepatoma cells (26).

Similar experiments performed in LLC-PK1-F+ kidney cells produced very different results (17). Cotransfection of KCREB had little effect on basal or PKA-stimulated expression of PEPCK-490CAT. However, cotransfection of CREB inhibited the basal and PKA-stimulated activities of PEPCK-490CAT by 70%, and these effects were reversed by coexpression of KCREB. A similar effect was observed in the reconstitution experiments using the Gal-4-CREB expression vector (Fig. 6). This construct resulted in further inhibition of PKA-stimulated luciferase activity of the pPEPCKGal4Luc plasmid. However, Western blot analysis indicates that the LLC-PK1-F+ cells express CREB at a level comparable with that observed in various hepatoma cells (data not shown). CREB-specific antibodies were also effective in supershifting the complex formed between the CRE-1 oligonucleotide and recombinant CREB. However, the same antibody failed to supershift or block the formation of any of the bands observed when the CRE-1 probe was incubated with the nuclear extracts of LLC-PK1-F+ cells (data not shown). These observations suggest that the endogenous CREB-like protein in LLC-PK1-F+ cells may be a unique isoform. Alternatively, the binding of CREB to CRE-1 may be inhibited by the binding of other transcription factors. For example, NF1-C, which binds effectively to the adjacent P1 site and is highly expressed in kidney, abrogates both CREB and PKA stimulation of PEPCK-490CAT activity when expressed in HepG2 cells (6). The presence of a unique coactivator could also affect CREB interactions with the CRE-1. The coexpression of CBP partially reversed the CREB-dependent inhibition of the PKA-stimulated PEPCK-490CAT activity in LLC-PK1-F+ cells (17). This finding was previously interpreted to indicate that the LLC-PK1-F+ cells lack CBP and express an alternative coactivator. Western blot analysis also indicated that LLC-PK1-F+ cells express very little, if any, CBP (data not shown). Thus cAMP activation of the PEPCK gene in kidney cells must differ significantly from the mechanism characterized in liver cells.

PEPCK-2300Luc, when expressed in subconfluent LLC-PK1-F+ cells, is stimulated 30-fold by cotransfection of the catalytic subunit of PKA. The observed activation was significantly greater than previously reported for the PEPCK-490CAT construct (17). Part of the greater response was due to the incorporation of the longer segment of promoter sequence, since the corresponding PEPCK-490Luc construct is activated only 15-fold by PKA (data not shown). As observed with the previous CAT assays (17), mutation of the CRE-1 element had a slight effect on basal activity (decreased by 20%) but a greater effect on PKA activation. In both sets of experiments, mutation of the CRE-1 reduced the fold activation by PKA of the reporter activities to about one-third of the wild type. The data obtained with the CAT assays suggested that the remaining activation may be due to AP-1 binding to the P3(II) element (17). However, assays with the more responsive luciferase constructs suggest that the P3(II) site contributes to basal expression and does not mediate the effect of PKA. Thus some element in addition to the CRE-1 must also contribute to the PKA-dependent activation of the PEPCK promoter in LLC-PK1-F+ cells.

Nuclear extracts of HepG2 cells contain at least two proteins that form specific complexes with the CRE-1 oligonucleotide (28). Formation of one of the observed complexes was blocked by preincubation with antibodies to CREB, the other by antibodies to C/EBP. Neither antibody formed a supershift of the respective complex. In contrast, all of the specific binding interactions observed with the nuclear proteins from LLC-PK1-F+ cells were blocked with antibodies specific for C/EBPbeta (Figs. 4 and 5). However, this polyclonal antibody supershifted the complex formed between the GST-C/EBPbeta fusion protein and the CRE-1 element. Thus the added domain may either block or alter the conformation of an epitope that is adjacent to the DNA binding domain of C/EBPbeta . The binding of an antibody to this epitope could block DNA binding of the wild-type C/EBPbeta . Alternatively, the binding of the antibodies to C/EBPbeta could produce conformational changes that reduce its affinity for the CRE-1 element, and the additional domain of the GST-C/EBPbeta fusion protein may restrict the conformation of the C/EBPbeta DNA binding domain sufficiently to prevent such changes. Curiously, two other C/EBPbeta -specific antibodies obtained from Santa Cruz Biotechnology also blocked binding of the nuclear protein from LLC-PK1-F+ cells without forming a supershift (data not shown). Thus the current experiments identify C/EBPbeta as one isoform of C/EBP that is expressed in LLC-PK1-F+ cells. This conclusion was confirmed by Western blot analysis (Fig. 7A). Furthermore, the Gal-4 experiments indicate that C/EBPbeta can contribute to the cAMP-dependent activation of transcription in the LLC-PK1-F+ cells by binding to the CRE-1 region of the PEPCK promoter. Only the Gal-4-C/EBPbeta chimera was able to restore full PKA activation to the PEPCKGal4Luc construct (Fig. 6). In this experiment, Gal-4-C/EBPalpha or Gal-4-CREB chimeras had little effect or were inhibitory, respectively. These results are the complete opposite of data previously reported with HepG2 cells (27, 28). To verify this difference, the plasmids used in the current experiments were transfected into HepG2 cells and shown to produce the previously reported results (unpublished data of M. Mallozzi, Q.-T. Wall, and N. P. Curthoys).

Electrophoretic mobility shift assays using nuclear extracts from LLC-PK1-F+ cells (Fig. 3) produced multiple bands that may represent homodimeric and heterodimeric forms (3) of C/EBPbeta . For example, the upper band may contain a heterodimer of C/EBPbeta and activating transcription factor (ATF)-2, since it is selectively supershifted with antibodies specific for ATF-2 (unpublished data of A. Tang, L. Taylor, and N. P. Curthoys). C/EBPbeta was previously shown to be an in vitro substrate for PKA (20). Furthermore, on phosphorylation by PKA, C/EBPbeta was translocated into the nucleus where it then activated transcription of c-Fos (20). Nuclear extracts prepared from cAMP-treated LLC-PK1-F+ cells exhibited about a threefold higher binding activity than those obtained from untreated cells. The kinetics of the observed increase in binding activity and the absence of a corresponding increase in the level of C/EBPbeta protein suggest that phosphorylation of C/EBPbeta may contribute to the observed response. In addition, deletion of the activation domain of C/EBPbeta produced a dominant negative inhibitor that reduced both the basal and the PKA-stimulated activity of PEPCK-490CAT (Fig. 8). All of these observations are consistent with the conclusion that C/EBPbeta contributes to the cAMP-dependent activation of PEPCK transcription in LLC-PK1-F+ cells.

Mutation of the CRE-1 element produces a slight, but highly reproducible, decrease (20%) in either CAT (17) or luciferase basal reporter activities. In contrast, addition of the dominant negative Delta Spl construct causes a fourfold reduction in basal CAT activity (Fig. 8). Identical results were obtained with the PEPCK-2300Luc reporter construct (data not shown). Therefore, the binding of C/EBPbeta to the CRE-1 element and to a second site may also contribute to the basal activity of the PEPCK promoter in LLC-PK1-F+ cells. Furthermore, PKA activation is only partially inhibited by mutation of the CRE-1 element, but it is completely reversed by addition of the dominant negative Delta Spl construct. Thus the postulated second binding site for C/EBPbeta may also contribute to the PKA-dependent activation in LLC-PK1-F+ cells. The identification and characterization of this putative site will require further analysis.

Tissue-specific regulation of a gene may be achieved by utilizing different combinations of cis-elements and trans-acting factors. In the case of cAMP activation of the PEPCK gene, liver cells use the CRE-1 element and a "liver-specific region" containing the P3(I), P4, and P3(II) elements. The individual elements apparently bind CREB, a C/EBP protein, and c-Fos/c-Jun, respectively (26). In contrast, in kidney cells, binding of C/EBPbeta to the CRE-1 element may contribute to the cAMP-dependent activation of PEPCK transcription.


    ACKNOWLEDGEMENTS

The assistance of Dr. Richard W. Hanson, Dr. William Roesler, Dr. Edwards Park, Dr. Steven McKnight, Dr. Wen-Hwa Lee, and Dr. Shizuo Akira, who provided various plasmids or antibodies used in this study, is greatly appreciated.


    FOOTNOTES

This research was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-43704 awarded to N. P. Curthoys.

Address for reprint requests and other correspondence: N. P. Curthoys, Dept. of Biochemistry and Molecular Biology, Colorado State Univ., Ft. Collins, CO 80523-1870 (E-mail: NCurth{at}lamar.ColoState.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 18 July 2000; accepted in final form 21 May 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Renal Fluid Electrolyte Physiol 281(4):F649-F657
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