A Phorbol Ester-Insensitive AP-1 Motif Mediates the Stimulatory Effect of Insulin on Rat Malic Enzyme Gene Transcription

Ryan S. Streeper, Stacey C. Chapman, Julio E. Ayala, Christina A. Svitek, Joshua K. Goldman, Alex Cave and Richard M. O’Brien

Department of Molecular Physiology and Biophysics Vanderbilt University Medical School Nashville, Tennessee 37232


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In liver, insulin stimulates the transcription of the gene encoding the cytosolic form of malic enzyme (ME) and modulates protein binding to two putative insulin response sequences (IRSs) in the ME promoter. One of these IRSs resembles that identified in the phosphoenolpyruvate carboxykinase (PEPCK) gene, whereas the other resembles that defined in the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. To assess the functional significance of these changes in protein binding, a series of truncated ME-chloramphenicol acetyltransferase (CAT) fusion genes were transiently transfected into rat H4IIE hepatoma cells. Deletion of the PEPCK-like IRS motif had no effect on the stimulation of CAT expression by insulin. Instead, the stimulatory effect of insulin was mediated through an AP-1 motif and an Egr-1 binding site that overlaps the GAPDH-like IRS motif. Both the ME AP-1 motif and the AP-1 motif identified in the collagenase-1 gene promoter were able to confer a stimulatory effect of insulin on the expression of a heterologous fusion gene, but surprisingly only the latter was able to confer a stimulatory effect of phorbol esters. Instead, the data suggest that AP-1 binds the ME AP-1 motif in an activated state such that phorbol ester treatment has no additional effect. The collagenase and ME AP-1 motifs were both shown to bind mainly Jun D and Fra-2, with similar affinities. However, the results of a proteolytic clipping bandshift assay suggest that these proteins bind the collagenase and ME AP-1 motifs in distinct conformations, which potentially explain the differences in phorbol ester signaling through these elements.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Malic enzyme (ME) catalyzes the oxidative decarboxylation of malate to pyruvate (1). Three isoforms of ME are found in mammalian tissues, each of which is encoded by a distinct gene: a cytosolic NADP+-linked enzyme, a mitochondrial NADP+-linked enzyme, and a mitochondrial enzyme that can utilize either NAD+ or NADP+ (2, 3). All three isoforms are widely expressed, although the relative amounts of each varies considerably between different tissues (2, 3). The cytosolic form of ME is most abundantly expressed in liver and adipose tissue where it plays a central role, in conjunction with the pentose phosphate pathway, in the generation of NADPH for fatty acid synthesis (see Ref. 1 for review). The activity of this enzyme is not regulated either allosterically or by posttranslational modification; however, the amount of the protein is regulated by a number of factors (1). For example, in rat liver, insulin stimulates the expression of the cytosolic form of ME by increasing both the transcription of the gene (4, 5) and stability of the mRNA (6).

cis-acting elements that mediate the action of insulin on gene transcription, referred to as insulin response sequences or elements (IRSs/IREs), have been identified in a number of genes but, unlike cAMP, which regulates gene transcription predominantly through one cis-acting element (7, 8), it is already apparent that a single consensus IRS does not exist (9). Three consensus IRSs have currently been identified. One of these has the sequence T(G/A)TTT(T/G) (G/T) and mediates the repression of PEPCK, insulin-like growth factor binding protein-1, tyrosine aminotransferase, apolipoprotein CIII, and glucose-6-phosphatase gene transcription by insulin (10, 11, 12, 13, 14). The other consensus IRSs are the serum response element (SRE; Refs. 9, 15) and the Ets motif (16), which mediate stimulatory effects of insulin on several genes. However, whereas the PEPCK-type IRS confers a selective effect of insulin (9) the serum response element and Ets motif can mediate the action of other hormones on gene transcription (see Refs. 17, 18 for review). Several additional IRSs have also been identified, but to date these elements appear to be unique to individual genes (9). Thus, this situation resembles that for phorbol esters, which can regulate gene transcription through at least eight distinct consensus sequences (19).

Data from a recent paper by Garcia-Jiménez et al. (5) suggested that the gene encoding the cytosolic form of ME may represent an exception to this complex paradigm. These investigators reported that sequences similar to the negative IRS identified in the PEPCK gene (10) and one of the two positive IRSs defined in the GAPDH gene, designated IRE-A (20), are both present in the ME promoter (5). Moreover, they demonstrated that nuclear extracts, isolated from the livers of diabetic rats, exhibited both increased protein binding to the PEPCK-like IRS motif and decreased binding to the GAPDH-like IRE-A motif in the ME promoter (5). Insulin treatment of the diabetic rats stimulated ME gene transcription and reversed these changes in protein binding back to the level seen in control animals (5). These observations raised the possibilities that 1) the negative PEPCK-like IRS motif mediates a positive effect of insulin on ME gene transcription and therefore represents a consensus IRS for both positive and negative effects of insulin and 2) that the GAPDH-like IRE-A motif represents a consensus IRE important for the insulin-stimulated transcription of genes other than GAPDH. To determine the functional significance of these observations, we have analyzed the effect of insulin on the expression of a series of truncated ME-CAT fusion genes. The results suggest that, in H4IIE hepatoma cells, an AP-1 motif and an Egr-1-binding site are required for the stimulatory effect of insulin on ME gene transcription. However, the ME AP-1 motif is atypical in that, in contrast to the well characterized AP-1 motif identified in the collagenase-1 promoter, it fails to confer a stimulatory effect of phorbol esters on the expression of a heterologous fusion gene. Instead, the data suggest that AP-1 binds the ME AP-1 motif in an activated state such that phorbol ester treatment has no additional effect. Based on the results from a series of experiments, we propose that the selective phorbol ester signaling through the collagenase and ME AP-1 motifs can be potentially explained by the observation that these elements bind AP-1 in distinct conformations.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Insulin Stimulates ME-CAT Fusion Gene Transcription
To begin to study the regulation of ME gene transcription by insulin, a ME-CAT fusion gene construct, containing ME promoter sequence from -882 to +39, relative to the major transcription start site at +1 (21), was transiently transfected into rat hepatoma H4IIE cells (Fig. 1Go). Insulin stimulated CAT expression directed by this construct ~3-fold under these conditions (Fig. 1Go), which is similar to the magnitude of insulin-stimulated ME gene transcription in rat liver (4, 5). This result suggests that an IRS is present in the ME promoter between -882 and +39. The maximal effect of insulin was seen at 10 nM; increasing the insulin concentration to 100 nM appeared to be toxic to these cells, based on their appearance, and significantly reduced CAT expression (data not shown). Insulin has a similar biphasic action on the expression of the genes encoding c-fos, c-myc, and JE (22).



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Figure 1. Insulin Stimulates ME-CAT Fusion Gene Transcription in H4IIE Cells

H4IIE cells were transiently transfected, as described in Materials and Methods, with a ME-CAT fusion gene containing promoter sequence from -882 to +39 (21 ). After transfection, cells were incubated for 20 h in serum-free medium, in the presence or absence of various concentrations of insulin. The cells were then harvested and CAT activity assayed as previously described (14 69 ). Results are presented as the ratio of CAT activity in insulin-treated vs. control cells (expressed as fold induction) and represent the mean of 3 experiments ± SEM.

 
To delineate the ME IRS, a series of 5'-deletion mutations of the ME promoter were constructed in the CAT reporter plasmid pCAT(An) (23), and the effect of insulin on CAT expression directed by these constructs was analyzed after transient transfection into H4IIE cells. Figure 2Go shows that insulin-stimulated ME-CAT gene transcription was reduced when the region of the promoter between -180 to -152 was deleted and was completely lost upon further deletion of the promoter sequence between -151 and -124.



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Figure 2. Deletion of the ME Promoter Sequence between -180 and -124 Abolishes the Stimulatory Effect of Insulin on ME-CAT Fusion Gene Transcription

H4IIE cells were transiently transfected, as described in Materials and Methods, with a series of ME-CAT fusion genes with 5' deletion end-points as shown on the abscissa. After transfection, cells were incubated for 20 h in serum-free medium, in the presence or absence of 10 nM insulin. The cells were then harvested and CAT activity assayed as previously described (14 69 ). Results are presented as the ratio of CAT activity in insulin-treated vs. control cells (expressed as fold induction) and represent the mean of 4–10 experiments ± SEM.

 
Insulin Stimulates Protein Binding to an Egr-1 and an AP-1 Motif in the ME Promoter
To determine whether the stimulation of ME gene transcription by insulin correlated with changes in protein binding to the ME promoter, nuclear extracts were prepared from control and insulin-treated H4IIE cells. Nuclear extract protein binding to three double-stranded oligonucleotides that span distinct regions of the ME promoter was assessed using the gel retardation assay (Fig. 3Go). When an oligonucleotide representing the ME promoter sequence between -181 and -145 was used as the labeled probe, an insulin-stimulated protein-DNA complex was detected (Fig. 3Go; see arrow). Similarly, when an oligonucleotide representing the ME promoter sequence between -161 and -123 was used as the labeled probe, two insulin-stimulated protein-DNA complexes were detected (Fig. 3Go; see arrows). In a comparison of nuclear extracts from control vs. insulin-treated cells, no consistent difference in protein binding was detected, when an oligonucleotide representing the ME PEPCK-like motif sequence between -699 and -664 was used as the labeled probe (Fig. 3Go).



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Figure 3. Insulin Stimulates Protein Binding to Oligonucleotides Representing the -181 to -145 and -161 to -123 Regions of the ME Promoter

Nuclear extracts were prepared from H4IIE cells incubated for 5 h in serum-free media (C) or serum-free media supplemented with 10 nM insulin (I). Protein binding to the three labeled oligonucleotide probes shown was analyzed using the gel retardation assay, as described in Materials and Methods, with three independent nuclear extract preparations (Preps. 1–3). In the representative autoradiograph shown only the retarded complexes are visible and not the free probe, which was present in excess. The insulin-stimulated protein-DNA complexes are indicated by the arrows.

 
A series of gel retardation experiments were performed to investigate the nature of the insulin-stimulated H4IIE cell protein-DNA complex detected with the -181/-145 oligonucleotide. Garcia-Jiménez et al. (5) reported that an insulin-stimulated liver protein-DNA complex could be detected when an oligonucleotide representing ME sequence from -175 to -156 was used as the labeled probe in a gel retardation assay. Since this sequence is included within the -181/-145 oligonucleotide, we hypothesized that the insulin-stimulated protein-DNA complex detected in H4IIE cells (Fig. 3Go) was the same as that detected by Garcia-Jiménez et al. in liver (5). Indeed, when an oligonucleotide (ME WT; Fig. 4AGo) representing the -175 to -156 ME sequence was used as the labeled probe in a gel retardation assay, an insulin-stimulated H4IIE cell protein-DNA complex was detected (Fig. 4BGo). In addition, a 100-fold molar excess of the unlabeled -181/-145 oligonucleotide competed effectively for protein binding to the -175/-156 labeled probe, indicating that the same insulin-stimulated protein-DNA complex binds both sequences (data not shown).



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Figure 4. The -175 to -156 Region of the ME Promoter Binds Egr-1

A, Panel A shows the sequence of oligonucleotides used in these studies. The core IRP-A and IRE-ABP binding motifs (20 24 ) in the GAPDH IRE-A sequence are boxed as are the IRP-A and Egr-1 binding motifs (25 26 ) in the wild-type (WT) ME -175/-156 sequence. The altered base pairs in the mutant (MUT) ME -175/-156 sequence are also boxed. B, The labeled ME WT oligonucleotide probe was incubated in the absence (-) or presence of a 100-fold molar excess of the unlabeled oligonucleotide competitors shown before addition of nuclear extract from control (C) or insulin-treated (I) H4IIE cells (Prep. 2 from Fig. 3Go). Protein binding was then analyzed using the gel retardation assay as described in Materials and Methods. In the representative autoradiograph shown only the retarded complexes are visible and not the free probe, which was present in excess. The insulin-stimulated protein-DNA complex, identified in Fig. 3Go, is indicated by the arrow. C, Nuclear extract from control (C) or insulin-treated (I) H4IIE cells (Prep. 2 from Fig. 3Go) was incubated with antibody dilution buffer (-) or various amounts of Egr-1 antiserum for 10 min at 4 C, before the addition of the labeled ME WT oligonucleotide probe and binding buffer and incubation for an additional 10 min at room temperature. Protein binding was then analyzed using the gel retardation assay as described in Materials and Methods. In the representative autoradiograph shown only the retarded complexes are visible and not the free probe, which was present in excess. The insulin-stimulated protein-DNA complex, identified in Fig. 3Go, is indicated by the arrow. 100 mM KCl was included in the binding reaction in panel B but not C which explains the differences in protein-DNA complex formation.

 
Two insulin response elements, designated IRE-A and IRE-B, have been identified in the GAPDH promoter (20). The IRE-A sequence has been shown to bind two proteins, designated IRP-A and IRE-ABP (Ref. 24 and Fig. 4AGo). The ME -175 to -156 sequence only contains the core-binding motif for IRP-A (Fig. 4AGo). To determine whether the insulin-stimulated protein-DNA complex detected with the ME -175/-156 oligonucleotide represents IRP-A, an oligonucleotide representing the GAPDH IRE-A sequence was synthesized (Fig. 4AGo). A 100-fold molar excess of this unlabeled IRE-A oligonucleotide did not compete for protein binding against the -175/-156 labeled probe, indicating that the insulin-stimulated protein-DNA complex binding this ME sequence is distinct from IRP-A (Fig. 4BGo). An oligonucleotide (ME MUT; Fig. 4AGo) representing the -175 to -156 ME sequence, but containing a mutation in the core IRP-A motif known to disrupt IRP-A binding to the GAPDH IRE-A (20), also failed to compete for protein binding against the wild-type -175/-156 labeled probe (Fig. 4BGo). This suggested that the insulin-stimulated protein-DNA complex contacts this sequence. Further inspection of the -175 to -156 ME sequence revealed the presence of a consensus Egr-1 binding motif (see Refs. 25, 26 for review) in addition to the IRP-A binding motif (Fig. 4AGo). The mutation present in the ME MUT oligonucleotide would be predicted to disrupt both IRP-A and Egr-1 binding, thus potentially explaining why this oligonucleotide does not compete for protein binding against the wild-type -175/-156 labeled probe (Fig. 4BGo). To confirm that the insulin-stimulated protein-DNA complex represents Egr-1, a supershift experiment was performed using a polyclonal antiserum raised against Egr-1 (Fig. 4CGo). The Egr-1 antiserum selectively supershifted the insulin-stimulated protein-DNA complex without affecting binding of the other protein-DNA complexes detected with the ME -175/-156 labeled probe (Fig. 4CGo).

Additional gel retardation experiments were also performed to investigate the nature of the two insulin-stimulated H4IIE cell protein-DNA complexes detected with the -161/-123 oligonucleotide (Fig. 3Go). A 100-fold molar excess of the unlabeled -161/-123 oligonucleotide competed effectively for protein binding against the -161/-123 labeled probe, indicating that the two insulin-stimulated protein-DNA complexes detected represent specific interactions (Fig. 5BGo; see arrows). By contrast, when a 100-fold molar excess of unlabeled oligonucleotides representing the ME Egr-1 motif, either the -181/-145 or -175/-156 sequence (Fig. 5AGo), were incubated with the -161/-123 labeled probe, no competition was seen (Fig. 5BGo). This suggests that neither of the insulin-stimulated protein-DNA complexes that are binding the -161 to -123 promoter sequence represents Egr-1.



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Figure 5. The -175 to -156 and -161 to -123 Regions of the ME Promoter Bind Distinct Insulin-stimulated Protein-DNA Complexes

A, Panel A shows a diagrammatic representation (drawn to scale) of the relative locations of the Egr-1 and AP-1 motifs in the oligonucleotides used in these studies. B, The labeled ME -161/-123 oligonucleotide probe was incubated in the absence (-) or presence of a 100-fold molar excess of the unlabeled oligonucleotide competitors shown (Table 1Go; Panel A) before addition of nuclear extract from insulin-treated H4IIE cells (Prep. 2 from Fig. 3Go). Protein binding was then analyzed using the gel retardation assay as described in Materials and Methods. In the representative autoradiograph shown only the retarded complexes are visible and not the free probe, which was present in excess. The insulin-stimulated protein-DNA complexes, identified in Fig. 3Go, are indicated by the arrows.

 
Inspection of the -161 to -123 sequence revealed the presence of a consensus AP-1 motif (see Ref. 27 for review) located between -132 and -126 (Table 1Go and Fig. 5AGo). Oligonucleotides representing the ME sequence between -138 and -123, and also the AP-1 motif sequence between -63 and -78 in the human collagenase-1 gene (28, 29, 30), were synthesized (Table 1Go) and used as competitors, at a 100-fold molar excess, in a gel retardation assay with the -161/-123 oligonucleotide as the labeled probe (Fig. 5BGo). Both oligonucleotides competed effectively against the labeled probe for binding of the two insulin-stimulated protein-DNA complexes (Fig. 5BGo; see arrows). Additional oligonucleotides were synthesized containing a mutated AP-1 motif within the context of the ME -161/-123, ME -138/-123, and collagenase -63/-78 sequences (Table 1Go). None of these oligonucleotides competed at a 100-fold molar excess against the wild-type ME -161/-123 labeled probe for binding of the two insulin-stimulated protein-DNA complexes (Fig. 5BGo), suggesting that both complexes recognize the AP-1 motif. The nature of the slower migrating insulin-stimulated protein-DNA complex is unknown, but supershift experiments identical to those described below with HeLa cell nuclear extracts, using antisera to the various isoforms of fos and jun, revealed that the major, faster migrating, insulin-stimulated protein-DNA complex contains members of the AP-1 family of transcription factors, primarily Fra-2 and Jun D (data not shown).


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Table 1. Sequence of Oligonucleotides used in These Studies

 
The ME AP-1 Motif Selectively Mediates Insulin and not Phorbol Ester Signaling
Figure 2Go shows that the effect of insulin on ME-CAT gene transcription is reduced when the region of the promoter between -180 to -152, which binds Egr-1 (Fig. 4Go), is deleted and is completely lost when the region of the promoter between -151 to -124, which binds AP-1 (Fig. 5Go), is deleted. To determine whether both the Egr-1 and AP-1 binding motifs can act as independent IRSs in H4IIE cells, these regions were analyzed for their ability to confer an insulin response in the context of a heterologous promoter. Oligonucleotides representing these motifs were synthesized and ligated into the polylinker of a heterologous thymidine kinase (TK) promoter-CAT fusion gene, containing TK promoter sequence from -105 to +51, and the resulting constructs were transiently transfected into H4IIE cells. Insulin had little effect on CAT expression directed by the basic TKCAT plasmid, but the ME AP-1 motif was able to confer a stimulatory effect of insulin on CAT expression when ligated to the TK promoter (Fig. 6Go). The effect of insulin was not enhanced by ligating multiple copies of the ME AP-1 motif to the TK promoter (Fig. 6Go). Importantly, when the mutated ME AP-1 motif (Table 1Go), which fails to bind either insulin-stimulated protein-DNA complex (Fig. 5BGo), was ligated into the TKCAT polylinker, it also failed to confer an effect of insulin on the expression of the fusion gene (Fig. 6Go). These results were expected since Kim and Kahn (31) had previously shown, in 3T3-F442A adipocytes, that insulin stimulates the expression of a heterologous fusion gene containing a multimerized collagenase AP-1 motif. The collagenase AP-1 motif was also able to confer a stimulatory effect of insulin on the expression of the TKCAT fusion gene in H4IIE cells (Fig. 6Go). In contrast to the ME AP-1 motif, when either single or multiple copies of an oligonucleotide representing the Egr-1 motif (ME WT; Fig. 4AGo) were ligated into the polylinker of the TKCAT fusion gene, they were unable to confer a stimulatory effect of insulin on CAT expression (data not shown).



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Figure 6. The ME Promoter Sequence between -161 and -123 Can Confer a Stimulatory Effect of Insulin on the Expression of a Heterologous Fusion Gene

H4IIE cells were transiently transfected, as described in Materials and Methods, with either the basic TKCAT vector, designated TK, or constructs, designated ME WT, ME MUT and Coll WT, in which oligonucleotides representing the wild-type (WT) or mutated (MUT) ME or collagenase promoter sequence from -161 to -123 and -63 to -78, respectively, as shown in Table 1Go, had been ligated into the BamHI site of the TK promoter either as a single copy, in the correct orientation relative to that in the ME gene promoter, or as multiple (3 4 ) copies. After transfection, cells were incubated for 20 h in serum-free medium in the presence (I) or absence (C) of 10 nM insulin. The cells were then harvested and CAT activity assayed as previously described (14 69 ). Results are presented as the ratio of CAT activity in insulin-treated vs. control cells (expressed as fold induction) and represent the mean of ± SEM of 3–11 experiments, in which each construct was assayed in duplicate.

 
While it is known that insulin treatment of various cell types stimulates the expression and phosphorylation of both c-fos and c-jun the specific protein kinases involved in these actions of insulin remain to be defined (see Discussion). By contrast, the action of phorbol esters on c-jun and c-fos expression and phosphorylation have been better characterized (see Ref. 27 for review). Given the long running controversy concerning the potential role of protein kinase C in insulin action (see Ref. 32 for review), it was of interest to determine whether phorbol esters could also stimulate ME gene transcription. To address this question the full-length -882/+39 ME-CAT fusion gene (21) was transiently transfected into HeLa cells, in which phorbol ester signaling through AP-1 motifs was initially studied (29, 30). There was almost no effect of phorbol esters on ME-CAT fusion gene transcription, whereas phorbol esters stimulated the expression of a collagenase-CAT fusion gene, containing human collagenase-1 promoter sequence from -518 to +64, more than 100-fold (Fig. 7AGo). In HeLa cells, insulin also stimulated collagenase-CAT gene transcription ~16-fold but had almost no effect on ME-CAT gene transcription (Fig. 7BGo). The stimulatory effects of both phorbol esters and insulin on collagenase gene transcription require an intact AP-1 motif in the collagenase promoter (29, 33). Therefore, while these observations were not informative regarding the role of protein kinase C in insulin action, they raised the question as to why, in HeLa cells, both insulin and phorbol esters markedly stimulate collagenase-CAT, but not ME-CAT, gene transcription even though both gene promoters contain AP-1 motifs.



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Figure 7. Insulin and Phorbol Esters Selectively Stimulate Collagenase, but not ME, Fusion Gene Transcription in HeLa Cells

A, HeLa cells were transiently transfected, as described in Materials and Methods, with either a ME-CAT fusion gene containing promoter sequence from -882 to +39 (21 ) or a collagenase-CAT fusion gene containing promoter sequence from -518 to +64. B, HeLa cells were transiently cotransfected with the same reporter gene constructs as above and an expression vector encoding the insulin receptor. After transfection, cells were incubated for 20 h in serum-free medium, either (A) in the presence or absence of various concentrations of PMA or (B) in the presence (I) or absence (C) of 10 nM insulin. The cells were then harvested and CAT activity assayed as previously described (14 69 ). Results are presented as the ratio of CAT activity in PMA or insulin-treated vs. control cells (expressed as fold induction) and represent the mean of 3–7 experiments ± SEM.

 
To determine whether these differences in the regulation of ME and collagenase gene transcription in HeLa cells reflected some inherent difference between the AP-1 motifs in each promoter, or whether the context in which the AP-1 motif is located is the relevant factor, the same heterologous ME-TKCAT and Coll-TKCAT constructs shown in Fig. 6Go were analyzed for their ability to mediate phorbol ester and insulin-stimulated CAT expression in HeLa cells. When these constructs were transiently transfected into HeLa cells, phorbol esters stimulated CAT expression directed by the basic TKCAT vector approximately 2-fold, but this effect was clearly enhanced by the presence of the collagenase, but not the ME AP-1 motif (Fig. 8AGo). In addition, this effect of phorbol esters was mediated only by the wild-type and not the mutated collagenase AP-1 motif (Fig. 8AGo and Table 1Go). This result suggests that the ME and collagenase AP-1 motifs are inherently functionally distinct with respect to mediating phorbol ester signaling in HeLa cells (Fig. 8AGo), although not with respect to insulin signaling in H4IIE cells (Fig. 6Go).



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Figure 8. Phorbol Esters Selectively Signal through the Collagenase but not the ME AP-1 Motif. The ME AP-1 Motif Markedly Enhances Basal TKCAT Gene Transcription

HeLa cells were transiently co-transfected, as described in Materials and Methods, with a ß-galactosidase expression vector and either the basic TKCAT vector, designated TK, or constructs, designated ME WT, ME MUT, Coll WT and Coll MUT, in which oligonucleotides representing either the wild-type or mutated ME or collagenase promoter sequence from -161 to -123 and -63 to -78, respectively, as shown in Table 1Go, had been ligated into the BamHI site of the TK promoter in multiple (3 4 ) copies. After transfection, cells were incubated for 20 h in serum-free medium in the presence (P) or absence (C) of 100 nM PMA. The cells were then harvested and both CAT and ß-galactosidase activity assayed as previously described (14 69 ). In panel A results are presented as the ratio of CAT activity, corrected for protein concentration in the cell lysate, in PMA-treated vs. control cells and are expressed as fold induction. In panels B and C results are presented as the ratio of CAT: ß-galactosidase activity in either control or PMA-treated cells, respectively, and are expressed as arbitary units. Results represent the mean of ± SEM of 5 experiments, in which each construct was assayed in duplicate.

 
Unfortunately, in HeLa cells, unlike H4IIE cells (Fig. 6Go), insulin markedly stimulates CAT expression directed by the control TKCAT fusion gene and ligation of multiple copies of the ME or collagenase AP-1 motif into the polylinker of the vector did not enhance the effect of insulin (data not shown). Therefore, it was not possible to determine whether insulin can also selectively activate gene transcription through the collagenase but not the ME AP-1 motif in HeLa cells. [This variable effect of insulin on TKCAT fusion gene transcription in H4IIE and HeLa cells is probably due in part to the presence of a cryptic IRS in the polylinker of this vector that is active in some cell lines but not others (9, 16).] The fact that there is almost no effect of insulin on the expression of the full-length ME-CAT fusion gene in HeLa cells (Fig. 7BGo), in contrast to H4IIE cells (Fig. 1Go), raises the possibility that there may be an inherent difference in insulin signaling through AP-1 motifs in the H4IIE and HeLa cell lines. Blackshear and colleagues have reported a similar situation with respect to insulin signaling through the c-fos SRE (15). Thus, in some cell lines the effect of insulin only required the core serum response factor binding site in the SRE, whereas in other cells the ternary complex factor-binding site in the SRE, adjacent to the serum response factor-binding site, was also required (15).

The ME and Collagenase AP-1 Motifs Bind AP-1 in Distinct Conformations
A number of parameters were investigated to determine why the ME AP-1 motif fails to mediate a phorbol ester-dependent increase in gene transcription when ligated to the heterologous TK promoter. An analysis of basal ME-TKCAT and Coll-TKCAT fusion gene transcription in HeLa cells revealed that ligation of the ME, but not the collagenase, AP-1 motif into the TKCAT polylinker markedly enhanced basal fusion gene transcription (Fig. 8BGo). This enhancement of basal fusion gene transcription was mediated only by the wild-type but not the mutated ME AP-1 motif (Fig. 8BGo and Table 1Go). This observation suggests that the reason why the ME AP-1 motif fails to mediate a phorbol ester-dependent increase in gene transcription, when ligated to the heterologous TK promoter, is that AP-1 is already bound in a fully activated state. Thus, phorbol ester treatment has no additional effect beyond that seen on the basic TKCAT vector alone (Fig. 8AGo). By contrast, ligation of the collagenase AP-1 motif into the TKCAT polylinker only modestly increases basal fusion gene transcription (Fig. 8BGo), but phorbol ester treatment enhances expression to a level similar to that obtained with the ME-TKCAT fusion gene (Fig. 8CGo). Based on these observations, the question as to why the ME AP-1 motif fails to mediate a phorbol ester-dependent increase in gene transcription when ligated to the heterologous TK promoter should be restated. The issue is why AP-1 binds the ME, but not the collagenase, AP-1 motif in an activated state.

Competition experiments revealed that a 100-fold molar excess of the unlabeled ME -138/-123 oligonucleotide (Table 1Go) competed effectively for specific protein binding when the oligonucleotide representing the -63/-78 collagenase AP-1 motif (Table 1Go) was used as the labeled probe in a gel retardation assay and vice versa (Fig. 9AGo). No difference was seen when a variable molar excess of the unlabeled ME -138/-123 and collagenase -63/-78 oligonucleotides were compared for their ability to compete for protein binding to the labeled ME -138/-123 probe, indicating that both oligonucleotides bind AP-1 with similar affinity (data not shown).



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Figure 9. The Collagenase and ME AP-1 Motifs Both Bind the Same Combination of fos and jun Transcription Factors

A, Protein binding to the two labeled oligonucleotide probes shown was analyzed using HeLa cell nuclear extracts and the gel retardation assay, as described in Materials and Methods. Where indicated, the labeled oligonucleotide probes were incubated in the absence (-) or presence of a 100-fold molar excess of the unlabeled ME -138/-123 or Coll -63/-78 oligonucleotides before the addition of nuclear extract. B, HeLa cell nuclear extract was incubated with antibody dilution buffer (None) or 1 µg of the indicated antisera for 10 min at 4 C, before the addition of the labeled ME -138/-123 (M) or Coll -63/-78 (C) oligonucleotide probes and binding buffer followed by incubation for an additional 10 min at room temperature. Protein binding was then analyzed using the gel retardation assay as described in Materials and Methods. In the representative autoradiographs shown only the retarded complexes are visible and not the free probe, which was present in excess. The AP-1 complex and a non-specific (NS) protein-DNA interaction are indicated by the arrows.

 
Since AP-1 is comprised of homodimers and heterodimers of various members of the fos and jun transcription factor families (34), we hypothesized that the ME and collagenase AP-1 motifs might selectively bind specific members of the fos/jun families. To test this hypothesis, polyclonal antisera to specific fos/jun family members were assayed for their ability to supershift the specific protein-DNA complex that binds the ME and collagenase AP-1 motifs (Fig. 9BGo). These experiments showed that the protein-DNA complex detected in the gel retardation assay contains mainly Fra-2 and Jun D with lesser amounts of c-fos and Jun B (Fig. 9BGo). However, no qualitative or quantitative difference was detected in AP-1 protein binding to the ME and collagenase probes (Fig. 9BGo). A faint protein-DNA interaction, which migrates slower than the AP-1 complex, was detected with the collagenase but not the ME AP-1 motif (Fig. 9BGo). However, it seems unlikely that this minor interaction could explain the selective enhancement of basal TKCAT gene transcription by the ME, but not the collagenase, AP-1 motif. Given the known action of phorbol esters on AP-1 expression and phosphorylation (see Discussion), the role of this minor interaction in phorbol ester signaling through the collagenase AP-1 motif is unclear.

The proteolytic band shift assay (35) was used to examine the possibility that the same transcription factors were binding both probes but in distinct conformations. To study the effect of partial protease digestion, HeLa nuclear extract was preincubated with either the labeled collagenase or ME oligonucleotides before the addition of various concentrations of chymotrypsin (Fig. 10Go). Distinct proteolytic products that selectively bind the collagenase, but not the ME oligonucleotide, probe were detected (Fig. 10Go). Several of these selectively bound products migrate faster than the nonspecific protein-DNA interaction (Fig. 10Go, lower arrow) so it is unclear as to whether these products are derived from proteolysis of this nonspecific protein-DNA interaction or the AP-1 complex. However, one of these selectively bound proteolytic products (Fig. 10Go, upper arrow) migrates slower than the nonspecific DNA-protein interaction and is therefore likely to be derived through proteolysis of the AP-1 complex. Thus, this experiment suggests that the proteins bound to the collagenase and ME AP-1 motifs have different surfaces exposed to proteolytic digestion indicative of a difference in binding conformation. If so, this difference in binding conformation could potentially explain the selective enhancement of basal TKCAT gene transcription by the ME AP-1 motif. It is possible that the faint protein-DNA interaction, which migrates slower than the AP-1 complex and selectively binds the collagenase but not the ME AP-1 motif, is the source of the proteolytic fragments that specifically bind the collagenase AP-1 motif (Fig. 10Go). However, this seems unlikely unless partial proteolysis of this unknown protein markedly increased its DNA-binding affinity.



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Figure 10. The Collagenase and ME AP-1 Motifs Bind AP-1 in Distinct Conformations

HeLa cell nuclear extract was incubated with the labeled ME -138/-123 (M) or Coll -63/-78 (C) oligonucleotide probes for 10 min at room temperature before the addition of various amounts of chymotrypsin and incubation for an additional 2 min at room temperature. Protein binding was then analyzed using the gel retardation assay as described in Materials and Methods. In the representative autoradiograph shown only the retarded complexes are visible and not the free probe, which was present in excess. A nonspecific (NS) protein-DNA interaction is indicated by the arrow as is the AP-1 complex and proteolytic fragments that specifically bind the Coll -63/-78 labeled probe.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In H4IIE cells the stimulation of ME gene transcription by insulin is mediated through two cis-acting elements in the promoter, an AP-1 motif and an Egr-1-binding site ( Figs. 2–6GoGoGoGoGo). The observation that insulin stimulates Egr-1 expression has been made in several cell types (36, 37, 38, 39, 40) but, to the best of our knowledge, ME is the first insulin-regulated gene identified in which this induction of Egr-1 gene expression has a specific functional consequence. Unlike the ME AP-1 motif (Fig. 6Go), this Egr-1 binding site does not mediate an insulin response in the context of the heterologous TK promoter (data not shown). This may indicate that this element only functions in the context of the native ME promoter. Alternatively, since Egr-1 motifs can mediate a stimulatory effect of serum when ligated to a minimal ß-globin promoter (41), it is possible that this element simply cannot mediate an effect of insulin in the context of the TK promoter. Such a scenario would be similar to the PEPCK IRS, which can mediate an insulin response in the context of the heterologous TKC-VI vector (10) but not TKCAT (42). The inverse is found with the ME AP-1 motif, which can mediate an insulin response in the context of the TKCAT (Fig. 6Go) but not TKC-VI vector (data not shown).

Various investigators have studied the signaling pathway through which insulin stimulates Egr-1 gene expression (38, 39, 40, 43). The phosphorylation of Shc (38) or insulin receptor substrate-1 (IRS-1) (39) by the receptors for insulin and IGF-I, respectively, both lead to the induction of Egr-1 gene expression. In fibroblasts, MAP kinase activation is not sufficient for IGF-I-stimulated Egr-1 gene expression (39), but it may be required since in 32D hematopoietic progenitor cells inhibition of MAP kinase blocks insulin-stimulated Egr-1 gene expression (38). By contrast, in experiments comparing the stimulation of Egr-1 gene expression by insulin in Rat 1 fibroblasts expressing either C-terminally truncated or normal insulin receptors, Olefsky and colleagues (40) found that insulin stimulated Egr-1 gene expression to the same extent in both cell lines, whereas MAP kinase activation was almost undetectable in cells expressing the C-terminally truncated insulin receptors (40). Interestingly, insulin-stimulated c-fos gene expression was also markedly reduced in the cells expressing C-terminally truncated insulin receptors, which suggests that distinct signaling pathways mediate the action of insulin on c-fos and Egr-1 gene expression (40). Since part of the effect of insulin on ME gene transcription may be mediated through an increase in c-fos gene expression (see below), this raises the possibility that the activation of multiple signal transduction pathways may be required for the effect of insulin on ME gene expression. The results of Olefsky and colleagues (40) were surprising because both the c-fos and Egr-1 promoters contain serum response elements (SREs) (25), and the effect of insulin on at least c-fos gene transcription is mediated through the SRE (see below). Moreover, using the same cell line expressing C- terminally truncated insulin receptors, Stumpo and Blackshear (43) observed the predicted result, that insulin-stimulated c-fos and Egr-1 gene expression were both reduced.

In contrast to the results described by Garcia-Jiménez et al. (5), no difference in protein binding was detected, comparing nuclear extracts from control and insulin-treated cells, when an oligonucleotide representing the ME PEPCK-like motif was used as the labeled probe in a gel retardation assay (Table 1Go and Fig. 3Go). The same result was obtained under the assay conditions described in Fig. 3Go and conditions identical to those described by Garcia-Jiménez et al. (data not shown). This discrepancy could reflect an inherent difference between extracts isolated from insulin-treated H4IIE cells (Fig. 3Go) and rat liver (5). However, in earlier studies, using liver nuclear extracts isolated from control, diabetic, and insulin-treated diabetic rats, we again detected no change in protein binding to the PEPCK IRS (44). Since these PEPCK IRS-binding proteins were later shown to represent members of the C/EBP family of transcription factors (45) and several recent publications report that insulin regulates expression of the genes encoding C/EBP{alpha}, ß, and {delta} (see Ref. 9 for review), the explanation for why we are unable to reproduce the result obtained by Garcia-Jiménez et al. (5) is unclear. Clearly though, the analysis of the effect of insulin on ME-CAT fusion gene transcription (Fig. 2Go) indicates that the ME PEPCK-like IRS motif has no apparent functional role with respect to insulin action, at least in H4IIE cells.

Insulin markedly induces the expression of several components of the AP-1 complex including c-jun (22, 37), Jun B (22, 37, 46), and c-fos (22, 47, 48) but only slightly stimulates Jun D expression (22, 37, 49). These effects of insulin are seen in multiple cell types. By contrast, insulin has no effect on Fos B mRNA levels (22, 37), while the stimulation of Fra-1 expression by insulin is seen in some (22, 50), although not in all cell types (37). The mechanism through which insulin induces the expression of these genes is unknown with the exception of c-fos. Blackshear and colleagues (51) have shown that the effect of insulin on c-fos gene transcription is mediated through the SRE in the c-fos promoter, although the precise trans-acting factor involved may vary between different cell types (15). Several studies suggest that the MAP kinase pathway is involved in insulin-stimulated c-fos gene transcription (52, 53, 54). For example, overexpression of wild-type p21ras enhances insulin-stimulated c-fos gene expression (52), whereas overexpression of dominant negative mutants of p21ras and Raf-1 block insulin-stimulated c-fos gene expression (53, 54).

The phosphorylation state of c-fos and c-jun is also stimulated by insulin (31, 55), and several insulin-regulated protein kinases are known to phosphorylate c-jun, with either stimulatory or inhibitory effects on c-jun DNA binding and/or transactivation potential, including MAP kinase, GSK-3, casein kinase II, and JNK (see Refs. 34, 56, 57, 58 for review). However, which of these kinases plays the central role in the regulation of c-jun phosphorylation by insulin in vivo is unclear. A further potential complication is that the action of insulin may be cell type specific. For example, while insulin has been shown to activate MAP kinase in multiple cell types (see Ref. 58 for review), the activation of JNK is seen in some cell types (59) but not others (33). Surprisingly, despite this wealth of information and the large number of insulin-regulated genes that have been studied in detail (see Ref. 9 for review), ME is only the second example, the first being collagenase (33), of an insulin-stimulated gene whose transcription is, at least in part, regulated through an AP-1 motif. Whether the stimulation of collagenase and ME gene transcription by insulin is principally mediated by an increase in the mass of the AP-1 complex or an alteration in the phosphorylation state of preexisting fos/jun proteins remains to be determined. In liver, however, inhibitors of protein synthesis block the induction of ME gene expression by insulin, suggesting that the former may be the more likely mechanism (4).

Based on the results of the experiments shown in Figs. 8–10GoGoGo, we suggest that a difference in AP-1 binding conformation could potentially explain the selective phorbol ester signaling through the collagenase AP-1 motif. This difference in AP-1 binding conformation is proposed to result in AP-1 binding the ME, but not the collagenase, AP-1 motif in an activated state such that phorbol ester treatment has no additional effect (Fig. 8BGo). Since the ME and collagenase AP-1 motifs share an identical core consensus sequence, TGACTCA, the difference in AP-1 binding conformation could be explained by the distinct flanking sequence on either side of the core AP-1 motif (Table 1Go). Indeed, Ryseck and Bravo (60) have shown that the flanking sequence on either side of a consensus AP-1 motif can influence the affinity of AP-1 binding. Moreover, this effect of the flanking sequence on binding affinity was not uniform for all fos/jun family members; thus, the composition of the AP-1 complex would be predicted to change depending on the precise flanking sequence (60). More recently, various biophysical approaches have demonstrated that the AP-1 flanking sequence can affect the conformation of fos/jun binding (61, 62). Our data suggests that, while the distinct flanking sequences affect the conformation of AP-1 binding (Fig. 10Go), the overall binding affinity of the AP-1 complex to the collagenase and ME AP-1 motifs are similar (Fig. 9AGo) and that the same fos/jun family members bind both motifs (Fig. 9BGo). Similar observations have been made in studies on the cAMP response element (CRE) in which the flanking sequence on either side of a consensus CRE motif can dramatically affect the magnitude of cAMP-induced gene transcription (63). Interestingly, insulin also stimulates the transcription of the human glutathione S-transferase P1–1 gene (GSTP1) through an undefined element located in the promoter sequence between -99 and +72 (64). An AP-1 motif, located between -65 to -59, has identical 5'- and 3'-flanking sequence to the ME AP-1 motif and, as with the ME gene, this AP-1 motif is unable to mediate a phorbol ester-dependent stimulation of GSTP1 gene transcription but is critical for high basal gene transcription (65, 66).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
[{alpha}-32P]dATP (>3000 Ci mmol-1) and [3H] acetic acid, sodium salt (>10 Ci mmol-1) were obtained from Amersham (Arlington Heights, IL) and ICN, respectively. Insulin was purchased from Collaborative Bioproducts. Phorbol 12-myristate 13-acetate (PMA) was obtained from Sigma Chemical Co. (St. Louis, MO). Specific antisera to c-fos (sc-52), Fra-1 (sc-183), Fra-2 (sc-604), c-jun (sc-45), Jun B (sc-46), Jun D (sc-74), and Egr-1 (sc-110) were all obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Plasmid Construction
DNA manipulations were accomplished by standard techniques (67). An ME-CAT plasmid containing rat ME promoter sequence from -882 to +39, relative to the major transcription start site, was obtained as a generous gift from Dr. Vera Nikodem (21). An XbaI-BamHI fragment of the rat ME promoter, spanning the promoter sequence from -775 to +38, was isolated from this plasmid and ligated, in the same orientation as that found in the endogenous ME gene, into the XbaI-BglII-digested polylinker of the pCAT(An) expression vector, a generous gift from Dr. Howard Towle (23). The pCAT(An) vector has polyadenylation signals located 5' of the polylinker to prevent read-through transcription (23). Control experiments demonstrated that there was no basal CAT expression and no effect of insulin when the empty vector, minus the ME promoter, was transiently transfected into H4IIE cells (data not shown). A series of truncated ME-CAT fusion genes was generated by restriction enzyme digestion using XbaI and the one of the following enzymes: EcoN I, Nae I, SfaN I and Eco47 III. The Klenow fragment of Escherichia coli DNA polymerase I was used to fill-in the non-compatible ends; after blunt-end ligation the resulting plasmids had calculated 5' end-points of -316, -180, -151, -123 which were confirmed by DNA sequencing using the USB Sequenase kit.

The plasmid pCol.Luc, containing the human collagenase-1 promoter ligated to the luciferase reporter gene, was a generous gift from Dr. Jeremy Tavaré (33). A BglII - HindIII fragment of the collagenase promoter, spanning the sequence from -518 to +64, was isolated from this plasmid and ligated, in the same orientation as that of the endogenous gene, into the BglII digested polylinker of the pCAT(An) expression vector. The non-compatible 3' HindIII - BglII junction was filled-in using the Klenow fragment of Escherichia coli DNA polymerase I before blunt-end ligation.

Plasmid TKCAT contains the herpes simplex virus thymidine kinase (TK) promoter sequence from -105 to +51 ligated to the CAT reporter gene and has a unique BamH I site in the polylinker at -105 (68). Various double-stranded complementary oligonucleotides, representing distinct regions of the ME or collagenase promoters (Table 1Go), were synthesized with BamHI compatible ends using a Perceptive Biosystems Nucleic Acid Synthesis System and were ligated into BamHI-cleaved TKCAT either as a single copy, in the correct orientation relative to that in the endogenous gene, or as multiple copies. The orientation and number of inserts was determined by restriction enzyme analysis and confirmed by DNA sequencing. All plasmid constructs were purified by centrifugation twice through cesium chloride gradients (67).

Cell Culture and Transient Transfection
A) Rat H4IIE hepatoma cells were grown to 40 to 70% confluence in T150 flasks in Dulbecco’s modified Eagle’s medium (DMEM) containing 2.5% (vol/vol) fetal calf serum and 2.5% (vol/vol) newborn calf serum and were then transiently transfected in solution using the calcium phosphate-DNA co-precipitation method as previously described (14, 69).

B) Human HeLa cervical carcinoma cells were grown to 90% confluence in T150 flasks in DMEM containing 10% (vol/vol) calf serum and were replated the day before use into 75 cm2 culture dishes. Attached cells were transfected by addition of 0.5 ml of a calcium phosphate-DNA co-precipitate (69), containing the reporter gene construct (15 µg) and an expression vector for ß-galactosidase (2.5 µg), to the 10 ml of culture medium. In some experiments (Fig. 7BGo) an expression vector encoding the insulin receptor (5 µg), courtesy of Dr. Jonathan Whittaker, was co-transfected with the reporter gene construct (15 µg) and the expression vector for ß-galactosidase (2.5 µg). After an overnight incubation the media was replaced with serum-free DMEM supplemented with or without various concentrations of PMA or 10 nM insulin as indicated in the FigureGo Legends. The cells were then incubated for a further 20 h prior to harvesting.

CAT and ß-Galactosidase Assays
Cells were harvested by trypsin digestion and sonicated in 300 µl of 250 mM Tris (pH 7.8) containing 2 mM PMSF. CAT and ß-galactosidase assays were performed exactly as previously described (14, 69). Since ß-galactosidase is very poorly expressed in H4IIE cells (14, 69), CAT activity was corrected for the protein concentration in the cell lysate, as measured by the Pierce BCA assay and each plasmid construct was analyzed in duplicate in multiple transfections, as specified in the FigureGo Legends. Because phorbol esters (and insulin) affect RSV-ß galactosidase expression in HeLa cells, CAT activity was either corrected for the protein concentration in the cell lysate (Fig. 7Go) or for ß-galactosidase activity with the data obtained from control and PMA-treated cells shown separately (Fig. 8Go).

Gel Retardation Assay
A) Labeled probes: double-stranded oligonucleotides representing various regions of the ME, collagenase, PEPCK or GAPDH promoters were synthesized with either BamHI (Table 1Go) or HindIII (Fig. 4Go) compatible ends, gel purified, annealed and then labeled with [{alpha}-32P]dATP using the Klenow fragment of Escherichia coli DNA polymerase I to a similar specific activity of approximately 2.5 µCi/pmol.

B) Nuclear extract preparation: H4IIE and HeLa nuclear extracts were prepared as previously described (69, 70) except that the nuclear pellet was extracted with 20 mM HEPES pH 7.8, 0.75 mM spermidine, 0.15 mM spermine, 0.2 mM EDTA, 2 mM EGTA, 2 mM DTT, 25% glycerol containing 200 mM NaCl, instead of 0.4 M ammonium sulfate, and the supernatant was used directly in gel retardation assays. The protein concentration of the nuclear extracts was determined using the Bio-Rad assay and was typically ~1 µg µl-1.

C) Standard binding assay: labeled oligonucleotide (7.5 fmol, ~30,000 cpm) was incubated with either H4IIE (5 µg) or HeLa (3 µg) nuclear extract, as indicated in the figure legends, in a final reaction volume of 20 µl containing 20 mM HEPES pH 7.8, 100 mM NaCl, 0.38 mM spermidine, 0.08 mM spermine, 0.1 mM EDTA, 1 mM EGTA, 2 mM DTT, 12.5% glycerol (vol/vol) and 1 µg of poly(dI-dC)·poly(dI-dC). When the ME -181/-145 (Fig. 3Go) or ME -175/-156 (Fig. 4Go) oligonucleotides were used as the labeled probes the HEPES concentration was increased to 40 mM and in some experiments (Fig. 3Go and 4BGo) 100 mM KCl was included in the binding reaction. After incubation for 10 min at room temperature, the reactants were loaded onto a 6% polyacrylamide gel and electrophoresed at room temperature for 90 min at 150 V in a buffer containing 25 mM Tris, 190 mM glycine and 1 mM EDTA. After electrophoresis the gels were dried, exposed to Kodak XAR5 film, and binding was analyzed by autoradiography.

D) Competition experiments: for competition experiments (Figs. 4Go, 5Go and 9A) or assessment of the relative affinity of binding (data not shown), unlabeled double-stranded oligonucleotide (1- to 100-fold molar excess) was mixed with the labeled oligomer before addition of nuclear extract. Binding was then analyzed by acrylamide gel electrophoresis as described above.

E) Gel supershift: gel supershift assays were carried out by incubating H4IIE (5 µg) or HeLa (3 µg) nuclear extract with varying amounts of antisera, for 10 min at 4 C, before the addition of the labeled oligonucleotide probe and binding buffer and incubation for an additional 10 min at room temperature. Where appropriate antisera were diluted in 10 mM HEPES pH 7.8 containing 1 µg µl-1 BSA.

F) Proteolytic cleavage: HeLa nuclear extract (3 µg) was pre-incubated for 10 min at room temperature with either the ME -138/-123 or Coll -63/-78 labeled probes, as described above, before addition of various amounts of chymotrypsin for an additional 2 min at room temperature. Proteolytic products were then analyzed by acrylamide gel electrophoresis as described above.


    ACKNOWLEDGMENTS
 
We thank Vera Nikodem and Jeremy Tavaré for the generous gifts of the rat ME and human collagenase-1 promoters, respectively, and Howard Towle and Jonathan Whittaker for the pCAT(An) and insulin receptor expression vector plasmids. We also thank Roland Stein and Rob Hall for helpful comments on the manuscript and Ron Wisdom, Lynn Matrisian, Howard Crawford and Linda Sealy for both advice and providing the AP-1 antibodies. The H4IIE and HeLa cell lines were kindly provided by Daryl Granner/Cathy Caldwell and Roland Stein/Eva Henderson, respectively. Data analysis was performed in part through the use of the VUMC Cell Imaging Resource (CA68485 and DK20593) and the research was supported by grants from the Mark Collie Foundation and NIH (RO1 DK52820 to R.O’B.).


    FOOTNOTES
 
Address requests for reprints to: Richard M. O’Brien, Department of Molecular Physiology and Biophysics, 761 MRB II, Vanderbilt University Medical School, Nashville, TN 37232-0615.

Received for publication March 3, 1998. Revision received July 22, 1998. Accepted for publication August 13, 1998.


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