©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Novel Nerve Growth Factor-responsive Element in the Stromelysin-1 (Transin) Gene That Is Necessary and Sufficient for Gene Expression in PC12 Cells (*)

Sunita deSouza (1), Janis Lochner (2), Cynthia M. Machida (1), Lynn M. Matrisian (3), Gary Ciment (1)(§)

From the (1) Department of Cell Biology & Anatomy, School of Medicine, Oregon Health Sciences University, Portland, Oregon 97201, the (2) Department of Chemistry, Lewis & Clark College, Portland, Oregon 97219, and the (3) Department of Cell Biology, School of Medicine, Vanderbilt University, Nashville, Tennessee 37232

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Stromelysin-1 (ST-1) is an extracellular matrix metalloproteinase whose expression is transcriptionally regulated by nerve growth factor (NGF) in the PC12 rat pheochromocytoma cell line. In this paper, we define sequences in the proximal ST-1 promoter that contain a novel NGF-responsive element(s). We show that this cis-acting promoter element can bind nuclear proteins from both untreated and NGF-treated PC12 cells in a specific and saturable manner and is sufficient to confer NGF-inducibility to a heterologous promoter. At least a portion of this NGF-responsive element lies within a 12-base pair region between positions -241 and -229 of the ST-1 promoter and bears no sequence homology to other known transcriptional elements. In contrast to what has been reported for fibroblasts, an AP1 site centered around position -68 does not seem to be involved in the growth factor regulation of ST-1 in PC12 cells. These results suggest that the NGF regulation of ST-1 gene expression involves different promoter elements, and possibly different transcription factors, from that described for ST-1 induction by other growth factors.


INTRODUCTION

Nerve growth factor (NGF)() was the first neurotrophic factor discovered in a class of molecules responsible for the development, differentiation, and growth of the nervous system. NGF supports the survival and maintenance of sympathetic and sensory neurons of the peripheral nervous system and promotes the differentiation of selected cholinergic and adrenergic neurons of the central nervous system (1, 2) . NGF can also induce neural crest-derived adrenal medullary cells to transdifferentiate into sympathetic neurons (3) , a phenomenon recapitulated in vitro by the PC12 rat pheochromocytoma cell line (4) . Following several days of exposure to NGF, PC12 cells undergo transcriptionally dependent transformation into a neuronal phenotype characterized by the extension of neurites, the development of electrical excitability, and the expression of genes encoding neuronal cell-specific proteins (4, 5) .

Unlike NGF, which triggers neuronal differentiation, epidermal growth factor (EGF) stimulates proliferation of PC12 cells without differentiation. Both NGF and EGF are ligands for receptor tyrosine kinases, which can activate similar intracellular signaling molecules such as phospholipase C-, phosphoinositol 3-kinase, and ras (6, 7, 8, 9, 10, 11, 12, 13, 14) . Within minutes, both NGF and EGF induce several immediate-early genes encoding transcriptional regulatory proteins, including c- fos, c- jun, and NGF-IA/ egr-1 in PC12 cells (15, 16, 17, 18, 19, 20) . Despite similarities in the signaling pathways, these growth factors have very different effects on the physiology and morphology of PC12 cells. Neuronal differentiation by NGF is accompanied by expression of a subset of genes which encodes proteins that are important for the differentiated phenotype. Most of these genes are transcriptionally active hours after NGF addition, including those encoding tyrosine hydroxylase (21) , neuropeptide Y (22) , peripherin (23) and other neurofilament components (24) , brain type II Nachannels (25) , SCG10 (26) , VGF (27) , and stromelysin (also known as transin) (28) . None of these late genes are activated by EGF, suggesting that some critical aspects of these two signaling pathways differ. In order to better understand the mechanisms underlying the development and expression of the neuronal phenotype in PC12 cells, several groups, including ours, have examined the transcriptional regulation of NGF-induced genes (29, 30, 31, 32) . In this study, we utilize the stromelysin gene as a model for NGF-induced regulation of gene expression.

Stromelysin-1 (ST-1) is a member of the matrix metalloproteinase gene family which includes interstitial collagenase and the gelatinases (33, 34) . ST-1 itself is known to degrade various components of the extracellular matrix associated with basal laminae (35) and has been implicated in tissue remodelling events associated with embryonic development, tumor metastasis, and axonal invasiveness (36, 37, 38) . In fibroblasts, ST-1 is transcriptionally induced by EGF, platelet-derived growth factor (PDGF) and the phorbol ester 12- O-tetradecanoylphorbol-13-acetate (39, 40, 41) . This induction can be transcriptionally inhibited by transforming growth factor -1 through a 10-bp sequence in the ST-1 proximal promoter region known as the transforming growth factor -1 inhibitory element (42, 43, 44) . Recently, PDGF has been shown to induce ST-1 gene expression in NIH 3T3 cells via a 6-bp palindromic sequence in the distal ST-1 promoter (45) . In PC12 cells, however, we found that NGF, but not EGF nor PDGF, increased the levels of ST-1 mRNA (28) . This increase in ST-1 mRNA is at least a hundredfold over initially undetectable levels, making ST-1 one of the most highly induced NGF-responsive late gene products described (21) .

In this study, we utilize the ST-1 promoter to determine the mechanisms responsible for the NGF induction of ST-1 gene expression in PC12 cells. It was previously shown that a 750-bp fragment of the proximal ST-1 promoter contained sequences which were sufficient for the NGF induction of this gene (28) . Here we identify a 12-bp region of the ST-1 promoter which contains at least a portion of a novel NGF-responsive element. We show that the promoter region containing this element binds nuclear proteins from both untreated and NGF-treated PC12 cells and is sufficient to confer NGF responsiveness to a heterologous promoter. This characterization of cis-acting sequences mediating the effects of NGF may provide insights into the mechanisms underlying the growth factor-specific expression of late genes during neuronal development.


MATERIALS AND METHODS

Oligonucleotides and Plasmids

5` promoter deletion mutants of the p750TRCAT plasmid were generated using an Erase-a-Basekit (Promega), as described (44) . Deletions were analyzed by DNA sequencing with Sequenase (Version 2.0, U. S. Biochemical Corp.). The pCATbasicTK vectors (pCBTK) was the gift of Dr. Bruce Magun (Oregon Health Sciences University) (46) . In some experiments, a modified form of the pCBTK vector was used in which an AP1 site was inserted upstream of the thymidine kinase (TK) promoter. This vector is referred to as ``pAS.'' The NRR-ABC fragment was obtained by polymerase chain reaction (PCR) amplification of the p715TRCAT vector using appropriate oligonucleotides with BglII and ClaI restriction sites placed at the termini. These oligonucleotides were: 5`-GCACAGATCTCTTCTGGAAGTTCTTTGTAC-3` (upstream) and 5`-GCACATCGATAAATGCTTCCTGCCTTAG-3` (downstream). The PCR product was then subcloned into the BglII and ClaI sites of the pCBTK vector. The NRR-A, NRR-B, and NRR-C fragments were obtained by restriction digestion of the NRR-ABC fragment using MaeIII and HaeIII, followed by gel isolation. The NRR-C fragment was treated with Klenow polymerase to fill in the overhangs and then blunt-end-ligated into the SalI site of the pAS vector.

Cell Culture and Transient Transfection Assays

Stock cultures of PC12 cells were maintained in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum and 5% horse serum at 37 °C in a 95% air, 5% COatmosphere. The original PC12 cell line (subclone GR-5) was obtained from Dr. Rae Nishi (Oregon Health Sciences University). For transient transfections, PC12 cells were plated at an initial density of 2 10cells per 10-cm Primariaplate (Falcon Plastics) 2 days before transfections. Calcium phosphate-DNA precipitates containing 15 µg of DNA were added to the cultures for 4 h (47) . The cells were then ``shocked'' for 2.0 min with 15% glycerol in HEPES-buffered (25 m M; pH 7.0) saline solution. The cultures were washed twice with phosphate-buffered saline, allowed to recover overnight in serum-containing medium, and then cultured for 24 h in ``N2-supplemented'' medium (48) . The next morning, NGF (50 ng/ml) or EGF (5 ng/ml) was added directly to the cultures for an additional 24 h prior to harvest. Protein levels were assayed (49) and 50-µg aliquots were used in a kinetic chloramphenicol acetyltransferase (CAT) assay using [H]acetyl coenzyme A, as described (50) .

Gel Mobility Shift Assays

Nuclei from PC12 cells were prepared using a modified version of the method of Hagenbüchle and Wellauer (51) in which intact nuclei are used instead of extracts. PC12 cells were first dissociated by treatment with PBS, pelleted by centrifugation, and then resuspended in 0.3 M sucrose in Buffer A (60 m M KCl, 15 m M NaCl, 0.15 m M spermine, 0.5 m M spermidine, 15 m M HEPES, pH 7.8, 14 m M mercaptoethanol, 0.5 m M phenylmethylsulfonyl fluoride and 8 µl/ml aprotinin). Nonidet P-40 was then added to this nuclear preparation to a final concentration of 0.1% (v/v). Cells were lysed in a Dounce homogenizer using 30 strokes with a type B pestle, and then the suspension was centrifuged atop a 0.9 M sucrose cushion in buffer A. The nuclei pellet was resuspended and treated again to Dounce homogenization in buffer A (without Nonidet P-40) with 5 strokes with the pestle. After the second centrifugation, the pellet was resuspended in a small volume of buffer B (75 m M NaCl, 0.5 m M EDTA, 20 m M Tris, pH 7.9, 0.8 m M dithiothreitol, 0.1 m M PMSF, and 50% glycerol) and the number of nuclei in each extraction was determined. Nuclei from untreated and NGF-treated PC12 cells were then adjusted to a final concentration of 10/ml, and 50-µl aliquots were stored at -80 °C.

DNA binding reactions involved first preincubating the nuclei in 10 µl containing 20 µg of bovine serum albumin, 15 µg of poly(dI-dC), and unlabeled competitor DNAs for 30 min at room temperature. End-labeled probes (10,000 cpm; specific activity = 10cpm/µg) were added to the nuclei in a final buffer concentration of 15 m M HEPES (PH 7.5), 60 M M KCL, 5 M M MGCL, 2 M M EDTA, AND 12% GLYCEROL. BINDING REACTIONS WERE ALLOWED TO INCUBATE FOR 30 MIN ON ICE. SAMPLES WERE THEN SUBJECTED TO ELECTROPHORESIS IN 6% SDS-POLYACRYLAMIDE GELS (ACRYLAMIDE:BIS RATIO OF 37:1) AT 4 °C IN 0.5 TBE (50 M M TRIS-HCL, PH 8.3, 41 M M BORIC ACID, 0.5 M M EDTA). GELS WERE DRIED, AND RADIOLABELED BANDS WERE VISUALIZED BY AUTORADIOGRAPHY.

Site-directed Mutagenesis of the NGF-responsive Region

Five versions of the ST-1 promoter were generated using the Altered Sitesin vitro mutagenesis system (Promega). These 5 plasmids contained contiguous 6-bp nested mutations spanning the 30-bp NRR-C` region (see Fig. 6 A). The EcoRI fragment of p715TRCAT (containing the promoter) was subcloned into pAlter-1 in the sense orientation and used as a template for the mutagenesis reaction with the following oligonucleotides: mutant 1 (m1), 5`-CAGCTTCTGAAGGATATAGTACTTTTCCAAAGTAG-3`; mutant 2 (m2), 5`-GAAGGATAGTTACAAGACTGAAAGTAGAAAAAAATGCC-3`; mutant 3 (m3), 5`-GATAGTTACATTTTCCG-TATCTGAAAAAAATGCCCC-3`; mutant 4 (m4), 5`-CATTTTCCAAAGTATTACTGAATGCCCCAGTTTTC-3`; and mutant 5 (m5), 5`-CCAA-AGTAGAAAAAGCCTATGCAGTTTTCTCTTTTGC-3`. Annealing, extension, and screening of mutations were carried out using protocols supplied by the manufacturer. Mutated forms of the promoter were then reintroduced into the pCBTK vector by PCR amplification of the mutant NRR-ABC region and subsequently subcloned into the BglII and ClaI sites of pCBTK.


Figure 6: Competition gel mobility shift assay with DNA containing nested mutations, showing that the 3`-end of the 30-bp sequence is necessary for nuclear protein binding. A, DNA sequences of the wild type and 5 mutated forms of the 30-bp NRR-C` region which were used as competitors in the experiment represented in B. B, P-labeled NRR-C was incubated with nuclei from NGF-treated PC12 cells and subjected to electrophoresis. Competitor unlabeled NRR-ABC fragments from either wild type ( wt) or mutant plasmids were included in the binding reactions at 25- or 100-fold molar excesses over labeled probe, as indicated by the ramps at the top of the gel. The arrow on the right points to the shifted proteinDNA complex. Note that wild type and m1, m2, and m3 were able to compete for nuclear protein binding, but that m4 and m5 were not.




RESULTS

Transient Transfection Studies Using a Mutated AP1 Site in the ST-1 Promoter

In previous work, Kerr et al. (41) showed that the AP1 site at position -65 of the ST-1 promoter mediated the EGF induction of ST-1 gene expression in NIH 3T3 cells. To determine whether this AP1 site was necessary for NGF induction of ST-1, PC12 cells were transiently transfected with either the parental p750TRCAT vector containing a 753-base pair region of the ST-1 promoter, or a site-directed mutant of this vector in which two base substitutions were introduced into the AP1 site, rendering it functionally inactive (41) . Fig. 1shows that PC12 cells transfected with the wild type p750TRCAT plasmid showed a 5- to 7-fold NGF-induced increase in CAT activity as compared with untreated control cells. Basal levels of CAT activity were significantly lower in cells transfected with the plasmid containing the mutated AP1 site as compared with those transfected with the wild type AP1 site ( p < 0.001), whereas NGF still caused a significant increase in CAT levels ( p < 0.050). In neither case was there a statistically significant change in CAT activities following exposure to EGF. Since the ability of the mutated plasmid to respond to NGF was not lost, we infer that the AP1 site is not directly responsible for the NGF responsiveness of the ST-1 promoter. This experiment was performed three times with qualitatively similar results.


Figure 1: The AP1 site is not directly involved in the NGF responsiveness of the ST-1 promoter. PC12 cells were transiently transfected with a plasmid containing 750 bp of the ST-1 promoter driving expression of the reporter gene, chloramphenicol acetyltransferase (CAT) containing the native AP1 site or with a plasmid in which two base changes were made in the AP1 site (indicated by asterisks). CAT enzymatic activity was assayed in PC12 cells that were either untreated ( C; open bars), treated with 50 ng/ml NGF ( N; black bars), or treated with 5 ng/ml EGF ( E; stippled bars). Results from Student's t test analysis between bracketed samples is indicated. N.S., not statistically significant.



5`-Deletion Analysis of the ST-1 Promoter

The observation that a mutated AP1 site failed to abolish the NGF induction of CAT indicates that a different element must confer NGF responsiveness to the ST-1 promoter. To localize this element, 5`-deletion mutants of the parental p750TRCAT plasmid were generated (see Fig. 2 A; the arrows indicate the various deletion mutants). PC12 cells were then transiently transfected with these plasmids and treated for 24 h with either control culture medium or medium supplemented with NGF or EGF. As a control for transfection efficiencies, all of the cultures were co-transfected with a second plasmid containing the luciferase reporter gene driven by the Rous sarcoma virus constitutive promoter. Fig. 2 B shows that removal of the terminal 38 bases from the 5` end of the parental plasmid resulted in an increase in NGF-responsive CAT expression from approximately 5-fold to 11-fold above basal levels, indicating the possible presence of a negative regulatory element in this region. In contrast, there was no statistically significant change in basal or EGF-induced levels of CAT expression. Deletion of the subsequent 400 base pairs from the 5` end of the ST-1 promoter, however, produced no consistent or statistically significant changes in the levels of NGF responsiveness. Although basal levels of CAT expression remained unchanged following deletions of this region, the levels of EGF-induced gene expression were slightly higher with the p573TRCAT and p578TRCAT plasmids. This observation, however, was not pursued further.


Figure 2: The region between positions -247 and -315 in the ST-1 promoter is necessary for NGF responsiveness. A, sequence of the ST-1 promoter (39) numbered with respect to the transcription start site. Putative regulatory elements are bracketed and include a transforming growth factor -1 inhibitory element, NGF-IA (also known as zif 268, Tis 8), CAAT box, TATA box, and AP1 site. The narrow underlined sequence corresponds to a 9-bp palindromic sequence. The thick underlined sequence corresponds to the 12-bp sequence that contains at least a portion of the NGF-responsive element. The arrows and numbers below the sequence refer to the various 5`-deletions analyzed for promoter activity. B, analysis of ST-1 promoter activity in transient transfection assays. PC12 cells were incubated with medium in the absence (control; open squares) or presence of either NGF (50 ng/ml; closed circles) or EGF (5 ng/ml; closed squares) for 24 h. All cultures were co-transfected with a plasmid containing the luciferase gene driven by the Rous sarcoma virus promoter. The results presented here have been normalized against luciferase levels. Note that there is a significant decrease in NGF-induced CAT activities between positions -315 and -247. This experiment has been repeated three times with qualitatively similar results.



The largest change in NGF responsiveness occurred following the deletion of a 68-base pair region between positions -315 and -247, in which the fold NGF induction fell from approximately 8-fold to about 1.5-fold (averaging the results of three experiments). In contrast, this deletion produced no significant differences in either basal levels or EGF-induced levels of CAT expression. Further deletions in the ST-1 promoter had only a slight effect on NGF responsiveness. However, removal of the region between -247 and -228 resulted in a small but significant increase in EGF-responsive CAT expression, indicating the possible presence of a growth factor-specific silencer region. This experiment was performed three times with qualitatively similar results. These results indicate that the region between -315 and -247 bp upstream of the ST-1 transcription start site contains sequences that are required for NGF-responsiveness.

Gel Mobility Shift Experiments Using Various Regions of the ST-1 Promoter

DNA sequences which function as regulatory elements are likely to bind nuclear proteins (52) . To determine whether the region between positions -247 and -315 would bind such a protein, gel mobility shift assays were performed. As a probe for these experiments, we first generated a polymerase chain reaction (PCR) fragment which included the region between -247 and -315 plus significant portions of 5`- and 3`-flanking DNA (labeled in Fig. 3 A, NRR-ABC for ``NGF-responsive region'') to ensure that this probe would contain the entire protein binding site. PC12 cells were then treated for 2 h in the presence or absence of NGF, and then nuclei were isolated and incubated with P-end-labeled probe, allowing proteinDNA complexes to form. Fig. 3 B is an autoradiograph of a nondenaturing gel loaded with these binding reactions showing that, in the absence of nuclear proteins, no shifted complexes were present ( lane 1). When nuclei from either control or NGF-treated PC12 cells were used, several shifted bands were visible ( lanes 2 and 3). The arrow at the left indicates the most heavily labeled band, which was subsequently found to be the only one which displayed specific and saturable DNA-protein binding.


Figure 3: Nuclear protein(s) bind to a 60-bp NRR-C region of the ST-1 promoter. A, schematic of the NRR-ABC fragment containing the region between positions -315 and -247 of the ST-1 promoter, plus flanking DNA. Also diagrammed are the 45-bp NRR-A, 43-bp NRR-B, and 60-bp NRR-C restriction fragments. B, gel mobility shift assay using different regions of the ST-1 promoter. DNA fragments were first end-labeled with P, incubated with nuclei from either untreated ( C) or 2-h NGF-treated ( N) PC12 cells, and then subjected to electrophoresis in a 6% acrylamide gel under nondenaturing conditions, as described under ``Materials and Methods.'' Note that, in the absence of nuclei, all of the label is present at a single band ( asterisks), but in the presence of nuclei from either untreated or NGF-treated cells, the NRR-ABC ( lanes 1-3) and NRR-C fragments ( lanes 10-12) produce shifted complexes containing multiple bands ( arrows).



To dissect this protein binding region further, the NRR-ABC fragment was digested with HaeIII and MaeIII restriction enzymes, which cut the fragment into 3 similarly sized pieces (termed NRR-A, NRR-B, and NRR-C; see Fig. 3A). Each of these pieces was then end-labeled and used in gel mobility shift assays, as described above. Lanes 4-9 of Fig. 3 B show that neither the NRR-A nor NRR-B fragments produced shifted proteinDNA complexes, whereas the NRR-C fragment did with nuclei from either control or NGF-treated PC12 cells (Fig. 3 B, lanes 10-12). This experiment was performed three times with qualitatively similar results. These results suggest, therefore, that a region within the NRR-C fragment binds DNA-binding protein(s), and that this protein(s) is present constitutively.

To determine whether these DNAprotein interactions were specific and saturable, competition gel shift assays were performed. In these experiments, molar excesses of unlabeled DNA corresponding either to the full-length NRR-ABC or each of the three fragments was included in the incubation mixture along with radiolabeled probe. Figs. 4 A and 4 B show competition mobility shift assays using the full-length NRR-ABC with nuclei isolated from untreated and NGF-treated cells. In either case, only the full-length NRR-ABC and the NRR-C could compete for the shifted complex, and this competition was apparent when a molar excess of as little as 25 unlabeled-to-labeled DNA was used ( lanes 3-6 and 15-18). Fig. 4, C and D, shows the converse experiment and that the NRR-C radiolabeled probe was effectively competed for binding with both NRR-ABC and NRR-C unlabeled DNA. Again, this competition was seen when using nuclei from either NGF-treated or control PC12 cell cultures. This experiment has been performed three times with qualitatively similar results. Several other nonspecific oligonucleotides were also used as competitors, and none were able to disrupt binding of nuclear protein(s) to the NRR-ABC (data not shown). These results are consistent, therefore, with the notion that a specific and saturable protein binding element can be found within region NRR-C, and that the protein recognizing this element is present in the nuclei of both NGF-treated and untreated PC12 cells. Interestingly, the NRR-B fragment contains a 9-base pair palindromic sequence centered around position -268 (Fig. 2 A, single underlined sequence). When an oligomer corresponding to this region was used in competition gel mobility shift assays, however, it was also unable to compete with the NRR-ABC probe for binding (data not shown), consistent with the notion that the NRR-B fragment does not contain this regulatory element.


Figure 4: Binding of nuclear protein(s) from both untreated and NGF-treated PC12 cells to the NRR-C is specific and saturable.P-labeled DNA fragments corresponding to different regions of the ST-1 promoter were incubated with nuclei from either NGF-treated or untreated PC12 cells and electrophoresed as described under ``Materials and Methods.'' Competitor nonlabeled DNA ( NRR-ABC, NRR-A, NRR-B, and NRR-C) was included in the binding reactions at increasing amounts of 25-, 50-, 100-, and 200-fold molar excess over labeled probe (represented by the ramps at the top of each gel). The arrows at the right identify the shifted complexes which showed specific and saturable DNA-protein binding. A, the probe used was the NRR-ABC fragment (see Fig. 3 A), and nuclei were from untreated PC12 cells. B, the probe used was the NRR-ABC fragment, and nuclei were from NGF-treated (50 ng/ml) cells. C, the probe used was the NRR-C fragment (see Fig. 3 A), and nuclei were from untreated cells. D, the probe used was the NRR-C fragment, and nuclei were from NGF-treated cells. Essentially identical results were obtained in two other experiments.



Since the NRR-C contains a large segment of DNA flanking the original deletion at position -247 (see Fig. 2A), it seems likely that the protein binding element would be found near this position in the 5`-half of the NRR-C fragment. To test this assumption, additional competition mobility shift assays were performed using a 30-bp synthetic oligonucleotide that corresponded to the first 30 bp of NRR-C. Fig. 5 A shows that this oligonucleotide (termed NRR-C`) competed with radiolabeled NRR-C for binding to nuclear proteins in both control ( lanes 3-7) and NGF-treated ( lanes 9- 13) PC12 cells. The arrow points to the shifted complex that was specifically competed by NRR-C`, and the asterisk indicates another complex which is not competed with unlabeled DNA. Fig. 5B shows that the NRR-C` oligonucleotide, when used as a labeled probe, could also bind nuclear proteins in both NGF-treated and untreated PC12 cells, indicating that this 30-bp region of the ST-1 promoter contains the binding site(s) for nuclear proteins from PC12 cells. These experiments were performed three times with qualitatively similar results.


Figure 5: The region between -229 and -259 of the ST-1 promoter contains the nuclear protein binding site. A, P-labeled NRR-C was incubated with nuclei from either untreated ( C; lanes 2-7) or NGF-treated ( N; lanes 8-13) PC12 cells and electrophoresed, as described under ``Materials and Methods.'' Competitor unlabeled synthetic oligonucleotides corresponding to the NRR-C` region of the ST-1 promoter was included in the binding reactions at increasing amounts of 25-, 50-, 100-, 200-, and 400-fold molar excess over labeled probe as indicated by the ramps on the top of the gel. The arrow on the right points to the shifted protein-DNA complex that is specifically competed out by the NRR-C`. The asterisk (*) indicates a shifted complex which is not competed out by NRR-C`. B, P-labeled NRR-C ( lanes 1-3) or radiolabeled NRR-C` ( lanes 4-6) was mixed with nuclei from either untreated ( C) or NGF-treated ( N) PC12 cells and electrophoresed as described under ``Materials and Methods.''



To further localize the NGF-responsive element within the 30-bp region, we performed site-directed mutagenesis. For these studies, 5 mutant plasmids were first generated using the Altered Sitessystem (Promega) containing contiguous 6-bp mutations spanning the NRR-C` region (see Fig. 6A). The NRR-ABC region of each of these mutant plasmids was then isolated and used in gel mobility shift assays as competitors. Fig. 6 B shows that m1, m2, and m3 fragments were able to compete for protein binding with radiolabeled wild type probe ( lanes 5-10), whereas m4 and m5 fragments were not ( lanes 11-14), suggesting that the region of the promoter necessary for protein binding is present within a 12-base pair region at the 3`-end of the NRR-C` fragment (see the bold underlined region between positions -241 and -229 of Fig. 2A). Qualitatively similar results were seen in assays using nuclei from non-NGF-treated PC12 cells (data not shown). Interestingly, gel mobility shift assays using radiolabeled probes corresponding to each of these mutated fragments showed that all five were able to bind nuclear proteins to some extent in the absence of any competitor DNA (data not shown). This suggests that the mutations in m4 and m5 reduced, rather than abolished, protein binding affinity.

Heterologous Promoter Experiments Using the NRR-C Fragment

The mobility shift assays and 5` deletion analysis show that an element within the NRR-C region is necessary for NGF responsiveness. To show that this element is also sufficient for NGF responsiveness, the NRR-C was cloned into a vector containing the basal thymidine kinase promoter with an added AP1 site driving expression of the CAT reporter gene. The NRR-C was inserted in either the forward ``AS{NRR-C(F)}'' or reverse ``AS{NRR-C(R)}'' alignment in order to determine whether it was equally active in either orientation. PC12 cells were then transiently transfected with these plasmids or the parental AS plasmid and treated for 24 h with culture medium containing either NGF or EGF, and then the cultures were harvested for CAT enzymatic activity. Fig. 7shows that a single copy of the NRR-C region in the forward orientation was sufficient to induce a 2-fold increase of CAT activity by NGF, but not EGF. In the reverse orientation, however, this region failed to induce a statistically significant increase in CAT activity by either NGF or EGF in the three times this experiment was performed. Similar results were obtained using the NRR-ABC fragment in either the forward or reverse orientation (data not shown). Together, these results indicate that the NRR-C region is both necessary and sufficient to function as an orientation-specific NGF-responsive element.


Figure 7: The NRR-C region of the ST-1 promoter confers NGF responsiveness to a heterologous promoter. PC12 cells were transiently transfected with a plasmid containing the NRR-C in either the forward AS{NRR-C(F)} or reverse AS{NRR-C(R)} orientation upstream of the basal thymidine kinase (TK) promoter driving expression of the reporter gene CAT. As a negative control, cells were transfected with the pAS vector alone. The p715 vector was used as a positive control. CAT enzymatic activity was assayed for PC12 cells that were either untreated ( C; open bars), treated with 50 ng/ml NGF ( N; black bars), or 5 ng/ml EGF ( E; stippled bars). Results from Student's t test analysis between bracketed samples is indicated. NS, not statistically significant ( i.e. p < 0.05).




DISCUSSION

NGF has been shown to be critical for the normal development and maintenance of the nervous system in the embryo and for the survival of neurons in the adult, presumably via its effects on gene expression (2, 53) . While many of the initial events in the NGF signaling pathway have been identified (9, 10, 11, 14, 54, 55, 56, 57, 58, 59) , little is known about nuclear events that regulate growth factor-specific changes in gene expression.

In this paper, we identify a 60-bp NGF-responsive region in the ST-1 promoter that is both necessary and sufficient for NGF responsiveness. When this region was deleted from the promoter, the remaining portions failed to display NGF inducibility; when this region was placed next to a heterologous promoter, it was sufficient to confer NGF inducibility. The orientation of this 60-bp region was also found to be important for heterologous gene inducibility, since experiments reversing the orientation of this region failed to display significant levels of NGF induction. This is consistent with the fact that this 60-bp region lacks palindromic sequences of appreciable length, which are often associated with regulatory elements which have biological activities in either the forward or reverse orientations. Using various mutants of this region, we showed that the protein binding site most likely included sequences within a 12-bp region of the ST-1 promoter. Mobility shift assays confirmed, moreover, that this region bound a nuclear protein or proteins expressed by PC12 cells. Interestingly, this protein(s) seemed to be expressed in both untreated and NGF-treated cells, since the mobility shift patterns of nuclei isolated from both treatments were indistinguishable. These similar mobility shift patterns may also indicate that the binding of accessory proteins to the preformed complex is not necessary for ST-1 induction. If this were the case, one might have expected supershifted bands or other differences in the patterns of bands between untreated and NGF-treated cells. Alternatively, post-transcriptional modifications of this nuclear protein(s), such as phosphorylation, may be associated with the mechanism of ST-1-induction by NGF. This is consistent with observations that protein kinase A or C activators, such as forskolin and phorbol esters, augments the NGF induction of ST-1 in PC12 cells (60) . The notion that phosphorylation is involved in the NGF induction of ST-1 is also consistent with observations that activation of the cytoplasmic kinases src, ras, and raf seem to be necessary for the NGF induction of neuronal differentiation in PC12 cells, including the expression of ST-1 (61, 62, 63, 64, 65) . These proto-oncogenes are believed to act as upstream regulators of the microtubule-associated protein kinase cascade leading to the phosphorylation of critical regulatory proteins in the nucleus (66, 67, 68) .

Several other NGF-responsive elements have also been described, but all differ significantly from sequences present in the ST-1 promoter region described here. These NGF-responsive elements include a fat-specific element (TH-FSE) in the tyrosine hydroxylase promoter that binds c- fos as part of the nucleoprotein complex (30) and a unique novel negative regulatory element in the peripherin gene (31) . In this latter case, derepression of peripherin gene expression also involved a less well-defined ``distal positive element'' within a fairly large 370-bp region of the promoter. These studies did not, however, test whether these elements conferred NGF responsiveness to a heterologous promoter. This analysis was included, however, in a recent report of a 50-bp NGF-responsive region of the neuropeptide Y promoter (32) . This NGF-responsive region in the neuropeptide Y promoter also bound a transcription factor present in both untreated and NGF-treated PC12 cells, and the patterns of shifted bands were similar in NGF-treated and untreated cultures. Comparison of the neuropeptide Y and the ST-1 NGF-responsive regions showed no significant sequence similarities, which may indicate that the transcription factors recognizing the two NGF-responsive elements also differ. Although the 12-bp sequence in the ST1 promoter contains a relatively high A + T content, it doesn't seem to correspond to known homeodomain binding protein consensus sequences, which are typically A + T-rich (69) .

We also report here that the AP1 site at position -71 regulates basal levels of ST-1 expression, at least in these transient transfection assays. Promoter constructs with point mutations in the AP1 site show, for example, large reductions in basal levels of CAT expression in transiently transfected PC12 cells, yet these cells still displayed statistically significant NGF inducibility. Although the absolute levels of this induction were attenuated with the mutated AP1 site, the relative levels of NGF inducibility remained unaffected in cells transfected with the mutated AP1 site. Further evidence that the AP1 site is not involved in NGF responsiveness is that c- fos and c- jun, two transcription factors which bind to this site, are also induced by EGF in PC12 cells, yet EGF fails to elicit ST-1 expression (15, 16, 18, 19, 20) . In any case, it should be noted that basal levels of endogenous ST-1 gene expression in PC12 cells are normally nondetectable (28, 60) , and, therefore, the notion that the AP1 site regulates basal level of expression may only apply to the truncated ( i.e. 753 bp) ST-1 promoter region used in these studies.

In contrast to PC12 cells, the AP1 site in rat-1 fibroblasts seems to play a critical role in the induction of ST-1 gene expression by both EGF and PDGF (41) . The AP1 site was also implicated in ST-1 gene expression in a polyomavirus-transformed rat embryonic cell line, in which the negative regulation of ST-1 mRNA levels by retinoic acid was shown to be mediated by the same AP1 site (70) . The fact that NGF induction of ST-1 gene expression in rat PC12 cells does not seem to be dependent on the AP1 site would suggest, therefore, that the tissue-specific mechanisms of regulation differ significantly. On the other hand, it is interesting to note that phorbol esters, although they cannot induce ST-1 themselves, can augment the NGF induction of ST-1 in PC12 cells (60) . If this phorbol ester augmentation is mediated via the AP1 site, it would suggest that this element acts to modulate, rather than activate, gene expression.

The progressive deletion mutants of the ST-1 promoter also allowed us to determine the contribution of other potential transcriptional elements to NGF inducibility. There is, for example, an NGFI-A site at position -400 in the ST-1 promoter. This site is believed to bind the NGF-IA protein (also known as zif 268, Egr-1, Krox 24, and Tis 8), which is a zinc finger DNA binding protein rapidly induced by either NGF or EGF in PC12 cells (15, 71, 72) . Removal of this site, however, was found not to have an appreciable effect on the NGF inducibility of CAT expression in transient transfection assays, suggesting that this NGFI-A site is not involved. This conclusion is consistent with earlier observations in which addition of the kinase inhibitor staurosporine to PC12 cells was found to have little effect on NGF induction of the NGFI-A, yet completely blocked the NGF induction of ST-1 mRNA expression and neurite extension (60) .

In sum, we have shown that the ST-1 gene regulation by NGF is mediated by an element present in a 12-bp region of the ST-1 proximal promoter. Future studies characterizing the core element within this region, as well as studies identifying transcription factors responsible for mediating activation of the transcription apparatus, should provide important information about how the biological effects of NGF are manifested at the level of gene expression in the nervous system.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant NS27886 (to G. C.) and a fellowship from the N. L. Tartar Research Fund (to S. deS.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) M13012.

§
To whom correspondence and reprint requests should be addressed: Dept. of Cell Biology & Anatomy (L-215), School of Medicine, Oregon Health Sciences University, Portland, OR 97201-3098. Tel.: 503-494-7362; Fax: 503-494-4253; E-mail: cimentg@ohsu.edu.

The abbreviations used are: NGF, nerve growth factor; EGF, epidermal growth factor; PDGF, platelet-derived growth factor; ST-1, stromelysin-1; bp, base pair(s); CAT, chloramphenicol acetyltransferase; PCR, polymerase chain reaction; TK, thymidine kinase; AP1, activator protein 1; NRR, NGF-responsive region.


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