Growth of LLC-PK1 renal cells is mediated by EGR-1 up-regulation of G protein alpha i-2 protooncogene transcription.

The early growth response zinc finger transcription factor (EGR-1) and the heterotrimeric guanine nucleotide binding protein encoded by the protooncogene G alpha i-2 each play pivotal roles in signaling pathways that control cell growth and differentiation. The G alpha i-2 gene 5'-flanking region contains a putative binding site (5'-CGCCCCCGC-3') for EGR-1 that may allow it to be a target gene for EGR-1 mitogenic signaling. We now demonstrate in LLC-PK1 renal cells the temporal expression of EGR-1 protein by immunoblotting and immunocytochemistry coincident with the maximal activation of the G alpha i-2 gene during cell growth. To determine whether G alpha i-2 or EGR-1 influence epithelial cell growth, LLC-PK1 cells were transiently transfected with plasmids encoding cDNAs for G alpha i-2 (pRSV G alpha i-2) or EGR-1 (pRSV EGR-1) driven by a viral Rous sarcoma promoter enhancer to overexpress each protein. Following transfection, cell growth was examined in media containing either 10 or 0.1% fetal bovine serum. Only cells transfected with plasmids encoding G alpha i-2 and EGR-1 had growth rates greater than that of serum replete cohorts. To assess whether EGR-1 was contributing to the transcriptional activation of the G alpha i-2 gene, cells were cotransfected with pRSV EGR-1 and plasmids encoding firefly luciferase reporter genes fused to 5'-flanking areas of the G alpha i-2 gene containing either the EGR-1 binding site or a mutated EGR-1 binding site (5'-AAAAACCGC-3'). A 320% enhancement of G alpha i-2 transcription was found only in LLC-PK1 cells following their transfection with plasmids that contained both the EGR-1 binding site and overexpressed EGR-1 protein. Utilizing mobility shift assays, which compared nuclear extracts from cells before and after cell polarization, a probe containing the EGR-1 motif detected induced nuclear protein complexes during transcriptional activation of the G alpha i-2 gene. An anti-EGR-1 antibody specifically retarded the mobility of the induced nuclear complexes, indicating that the EGR-1 protein was a component of these complexes. These data provide direct evidence for a novel mitogenic signaling pathway coupling proximal signaling events that activate EGR-1 gene expression to a target protooncogene G alpha i-2 that is participatory for growth and differentiation in renal cells.

The early growth response zinc finger transcription factor (EGR-1) and the heterotrimeric guanine nucleotide binding protein encoded by the protooncogene Gai-2 each play pivotal roles in signaling pathways that control cell growth and differentiation. The Gai-2 gene 5"flanking region contains a putative binding site (5'-CGCCCCCGC-3') for EGR-1 that may allow it to be a target gene for EGR-1 mitogenic signaling. We now demonstrate in LLC-PK1 renal cells the temporal expression of EGR-1 protein by immunoblotting and immunocytochemistry coincident with the maximal activation of the Gai-2 gene during cell growth. To determine whether Gai-2 or EGR-1 influence epithelial cell growth, LLC-PK1 cells were transiently transfected with plasmids encoding cDNAs for Gai-2 (pRSV Gai-2) or EGR-1 (pRSV EGR-1) driven by a viral Rous sarcoma promoter enhancer to overexpress each protein. Following transfection, cell growth was examined in media containing either 10 or 0.1% fetal bovine serum. Only cells transfected with plasmids encoding Gai-2 and EGR-1 had growth rates greater than that of serum replete cohorts. To assess whether EGR-1 was contributing to the transcriptional activation of the Gai-2 gene, cells were cotransfected with pRSV EGR-1 and plasmids encoding firefly luciferase reporter genes fused to 5"flanking areas of the Gai-2 gene containing either the EGR-1 binding site or a mutated EGR-1 binding site (B'-AAAAACCGC-3'). A 320% enhancement of Gai-2 transcription was found only in LLC-PK1 cells following their transfection with plasmids that contained both the EGR-1 binding site and overexpressed EGR-1 protein. Utilizing mobility shift assays, which compared nuclear extracts from cells before and after cell polarization, a probe containing the EGR-1 motif detected induced nuclear protein complexes during transcriptional activation of the Gai-2 gene. An anti-EGR-1 antibody specifically retarded the mobility of the induced nuclear complexes, indicating that the EGR-1 protein was a component of these complexes. These data provide direct evidence for a novel mitogenic signaling pathway coupling proximal signaling events that activate EGR-1 gene expression to a tar-DK-42543, American Heart Association Grant 13-553-890, and Estab-* This work was supported by National Institutes of Health Grant lished Investigatorship award 94003090 ( t o L. E.) and National Institutes of Health Grants F32 DK-08838 (to J. D. F.), DK-02271 (to T. B. K.), and DK-38452 (to L. E. and D. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18  Heterotrimeric guanine nucleotide binding proteins (G proteins) play an essential role in transmembrane signaling by coupling receptors with enzyme and ion transport processes in mammalian cells (1). These regulatory proteins are composed of individual a, D, and y subunits that are encoded by super gene families that have been conserved by eukaryotes throughout evolution (2). Most of the transducing activities of G proteins in mammalian cells are associated with the state o f activation of the a subunit, which is involved in GDP/GTP exchange and GTP hydrolysis (3).
Recently, it has been established that G proteins alter cell growth or differentiation by participation in growth factor receptor signaling pathways that converge in the nucleus to alter gene expression. Pertussis toxin, which decouples a subset of G proteins (G,), attenuates both Gi inhibition of adenylyl cyclase in platelets (4) and mitogenic responses to serum, thrombin, bombesidgastrin-activating peptide, and vasopressin (5). Both pertussis toxin effects can be mimicked by antibodies to Gai.2 subunits (6). Mutations in G q p comparable with Ha-ras GP21 that decrease GTPase activity are found in tumors of the adrenal cortex and ovary (7). Such mutations, which convert the GCY,.~ gene into the oncogene gip2, induce increased growth and oncogenic transformation in Rat-1 cells (8). Increased growth may be a consequence of persistent activation of pathways coupled to mitogen-activated protein kinase (9). In both Dictyostelium (10,11,12) and Drosophila (13), differentiation events are dependent on G protein-a subunit expression. Similarly, it has been recently demonstrated that the GCX,.~ subunit interacts with pathways required for differentiation of F9 teratocarcinoma cells (14). Importantly, even modest repression of Gai.2 expression is associated with renal morphologic abnormalities in transgenic mice (15).
We have utilized the polarized renal epithelial cell LLC-PK, as a model to study G, protein-regulated functions. These studies indicated that only two G, isoforms, Gi-2 and Gi-3, are detected in these cells, which, respectively, are involved in the regulation of hormone-stimulated adenylyl cyclase and constitutive proteoglycan secretion through the Golgi complex (16,17). In polarized LLC-PK, cells, the Gi-3 isoform is found in Golgi and apical membranes, whereas the Gi-2 isoform is found at the basolateral membrane consistent with the location of their physiological effects (16,17). We demonstrated that the genes encoding both Gi subunits are transcriptionally activated in these cells in a coordinated manner during growth and differentiation but differ in response to glucocorticoids and CAMP (18,19,20).
In the present study, we examine in mitotically active predifferentiated LLC-PK, cells the molecular mechanism for the activation of the Gai-2 gene. We now demonstrate that the expression of the early growth response gene 1 (EGR-l),' also known as TIS-8, Krox24,Zif!268, and NGFI-A (21), is increased in dividing LLC-PK1 cells, which temporally coincides with the maximal transcriptional activation of the Gai-2 gene. An increase in LLC-PK1 growth rates during culture is the physiological consequence of overexpression of each gene product in these cells. We also demonstrate that the enhancer area in the 5'-flanking region of the Gai-2 gene contains an EGR-1 motif (5'-CGCCCCCGC-3') (211, which is both the site for both EGR-1 binding and activation of the Gai-2 gene during renal cell growth. These studies provide evidence for the first example of a novel mitogenic signaling pathway coupling proximal signaling events that activate EGR-1 gene expression to a target protooncogene Gai-2 that is participatory for growth and differentiation in renal cells.

EXPERIMENTAL PROCEDURES
Cell Culture Wild type LLC-PK, cells are a polarized epithelial cell line derived from pig kidney. Cells were grown as confluent monolayers and maintained in Dulbecco's modified Eagle's medium containing 10 or 0.1% fetal calf serum in a 5% CO, atmosphere as previously described (16). Cells were plated a t a density of 1 x 106/10 cmz achieving confluence a t approximately culture day 7.
Transient Transfections-Plasmids were transfected in equimolar amounts into LLC-PK, cells by the calcium phosphate precipitation as previously described (13). Optimum transfection efficiency was obtained by the addition of 20 pg of total plasmid DNA per 55-em2 p10 plate (Falcon), followed by incubation for 20 h without glycerol shock. When required, this amount of DNA was achieved by the addition of "carrier plasmid" Bluescript I1 KS+. Transfection efficiency was normalized by cotransfection with 2.5 pg of pSV2Apap, a plasmid carrying a human placental alkaline phosphatase reporter gene driven by a Rous sarcoma virus promoter (generously provided by T. Kadesch, University of Pennsylvania). Zkansfection Assays 4 S 9 8 h after transfection, LLC-PK, cells were washed twice in phosphate-buffered saline (without calcium or magnesium) and then lysed by the addition of 1.0 ml of lysis buffer A (1% Triton, 25 m M glycylglycine, pH 7.8, 15 m~ MgSO,, 4 m M EGTA, and 1 m M fresh dithiothreitol). Scraped lysates were transferred to Eppendorfmicrofuge tubes and centrifuged at 10,000 x g for 5 min a t 4 "C. The supernatants were transferred to fresh Eppendorf tubes and briefly vortexed prior to each assay.
In some experiments, cell number ofeach plate was determined by direct cell count of trypsinized cells utilizing inverted phase microscopy.
Firefly Luciferase and Human Placental Alkaline Phosphatase Assays-These were performed as previously described (20). Results are expressed as percent increase f S.E. in luciferase activity normalized for heat-insensitive alkaline phosphatase activity. Data were analyzed by paired Student's t test.
Protein Assay-This was performed by the dye binding assay of Bradford as described by the manufacturer (Bio-Rad).

Mobility ShiB Assays
Nuclear Extract Preparation-Nuclear proteins were extracted from LLC-PK, cells as described by Saatcioglu et al. (22).
BindingAssays"6 pg of nuclear extract was preincubated for 30 min in the presence of 32P end-labeled double-stranded DNA, 4 Zn Vitro Danslation Assays-These were performed as previously described (23) utilizing T3 polymerase to drive transcription of a 2.1kilobase EGR-1 cDNA subcloned into Bluescript for use in a rabbit reticulocyte translation system as described by the manufacturer (Promega).
Zmmunoblotting a n d Immunofluorescence of EGR-1 LLC-PK, cells were washed twice in phosphate-buffered saline (without calcium or magnesium) and then lysed by the addition of 1.0 ml of lysis buffer A. Scraped lysates were solubilized by boiling in sample buffer (1% SDS, 30 m M Tris, pH 6.8,12% glycerol) and loaded onto a 10% acrylamide gel with 150 pg of protein loaded per lane. Following SDSpolyacrylamide gel electrophoresis, proteins were transferred onto Immobilon membrane (Millipore), and the membrane was then stained with Coomassie Blue to ensure that all lanes contained equivalent amounts of transferred protein. The destained membrane was then blocked in blotting buffer (5% nonfat dry milk in 20 m M Tris, pH 7.4, with 0.15 M NaCl and 1% Triton X-loo), incubated with either preimmune or immune rabbit IgG anti-EGR-1 Wi 21 (alpha 1) (which detects the carboxyl-terminal non-zinc finger region of the EGR-1 protein) or AS rabbit antiserum (which only detects Gai-2 as Gai-1 and is not expressed in LLC-PK1 cells) (DuPont NEN), diluted IilOOO in blotting buffer, and washed. EGR-I-and Gai-2-bound proteins were reacted with an enhanced chemiluminescent detection system as described by the manufacturer (Amersham Corp.) followed by autoradiography.
LLC-PK, cells plated on glass coverslips were fmed for immunofluorescent staining on days 1-7. Cells were fixed in 4% paraformaldehyde for 1 h, permeabilized in Triton X-100 for 4 min, and then incubated in phosphate-buffered saline containing 0.1% bovine serum albumin for 5 min to reduce nonspecific background staining. The cells were incubated for 2 h in anti-EGR-1 Wi 21 (el) or preimmune rabbit IgG at 1:lOO dilutions, washed three times in 0.1% bovine serum albumin in phosphate-buffered saline, and then incubated for 1 h with goat anti-rabbit IgG conjugated to fluorescein isothiocyanate (Kirkegaard and Perry). Cells were washed three times in phosphate-buffered saline, mounted in 100 m M Tris-HC1, glycerol (50:50), 2% n-propyl gallate, pH 8, and viewed on an Olympus BHS photomicroscope equipped for epifluorescence.

Autoradiography
For mobility shift and immunoblotting studies, the dried gels or membranes were autoradiographed with Kodak X-AR film at -80 "C for 0.5-96 h with Cronex lightning plus intensifylng screens (Dupont NEN). Quantification of signals was performed by densitometry of the autoradiograms with an LKB ultroscan XL enhanced laser densitometer (Pharmacia Biotech Inc.).

EGR-1 Is
Maximally Expressed during the Growth of LLC-PK, Cells-During culture, LLC-PK, cells differentiate from a rounded cell type to a fully polarized epithelium. Prior to their polarization and tight junction formation, these cells undergo several rounds of cell division. In other cell types, the Gai-2 protooncogene is participatory in pathways essential for growth and differentiation. We have previously demonstrated maximal activation of the Gai-2 gene during growth of LLC-PK1 cells (19). However, the transactivating factors required for transcriptional activation of the Gai-2 gene during growth have yet to be identified in any cell type. The EGR-1 gene encodes a zinc finger transcription factor of the Cys2-His2 subclass that is rapidly induced by a variety of agents during induction of cellular proliferation (21). The EGR-1 gene is rapidly activated in renal tissues following ischemia and hypertrophy (24,25). Following activation, the expressed EGR-1 protein is primarily detected in the nuclei of thick ascending limbs and To determine whether EGR-1 also participates in mitogenic events that precede the acquisition of the polarized phenotype in renal cells, we utilized an antihody raised to the carboxylterminal non-zinc finger region of the EGR-I protein to detect its presence in LLC-PK1 crlls. As seen in Fig. 1, immunofluorescence of dividing nonconfluent nonpolarized cells with this antihody revealed a bright nuclear staining pattern in virtually every cell. By contrast, in fully polarized confluent cells this staining pattern was rarely detected.

I.I,C-PK,
Crlls-We previously documrntrd in rapidlv dividing nonpolarized cells a 135-hp enhancrr arra in t h r -200 to -33.5 rrk6on of the Gtri-2 gene. This rrpjon contains a putativr hinding sitr (5'-CGCCCCCGC-3') for thc EGJI-I transcription factor that may provide a genomic signaling pathway for mitogrnrsis. To assess whether EGR-I was contrihuting to the transcriptional activation of t h r Gtri-2 gene, crlls wrrc cotransfrctrd with the pRSV EGR-1 plasmid and plasmids rncoding firefly lucifwasr reporter genes fused to 5"flanking arras of t h r ( h i -2 grnr with (M14) or without thr EGR-I sitr (mutatrd 5714 and 514). As seen in Fig. 4, a 32W+ rnhancrmrnt of Chi-2 transcription was only found in renal cells following thrir transfrction with the M I 4 plasmid. which containrd hoth an intact EGR-1 hinding site and also overexprrssrd a functional JX;K-I protrin. Thrsr data suggested that thr EGR-I protrin was contrihut.ing to thr temporal transcriptional activation of t h r Gwi-2 grnr in L I X - Identification of thr EGR-1 Prolcin os n cornponvnt nf n n Induced Nuclcnr 7'rnnscription Cornp1r.r drrring 7hn.sc.riptional Activation of the Gtri-2 Grnv-To drtermine whrthrr thr EGR-1 protein was directly contrihuting to the activation of t h r Gai-2 gene, a douhle-stranded 23-hp D N A srgmrnt drrivcd from the 5"flanking sequence of thr grne, which also containrd the EGR-1 consensus sequence rFi'-ATCCGCC(7C;CCCCC(;C-CGTCGGG-3'1, was synthesizrd and "P end-lahrlrd for dirrct binding studies in mobility shift assavs. In rtitro translatrd EGR-1 protein was utilized to nssrss its capability for hinding the EGR-1 consensus sequence. A s s r r n in Fig. SA, this prohr bound a component of the non-programmrd rrticulocytr lysate as we have previously describrd (2.3,. Howrvrr, the probr sptcifically bound the slower mobility EGR-1 protrin. Furthrrmore, immune rabhit IgG anti-EGR-1 Wi 21 (alpha 1 ) (which detects the non-zinc finger carboxyl-terminal rrgion of the EGR-1 protein) but not preimmune rahhit I K r could specifically retard the mobility of this complex.
To confirm that the EGR-1 protein was acting dirrctly by binding its cognate site and not the region 5' to thr EGR-I binding site in the M14 construct (sre Fig. 4, lorc-rr pnncl ), we assessed the ability of this area with thr mutatrcl E(;R-I binding site (!5'-AAAAACCGC-3') to hind t h r EGR-I protrin. As seen in Fig. 5B, probes containing rithrr thr mutatrd EGR-I binding site or the 5'-flanking arra of M14 and the mutatrd EGR-1 binding site displayed indistinguishahlr hinding of hoth the non-programmed and programmed reticulocyte lysate. These data demonstrated that hoth the mutatrd EGR-I sitr and the region 5' to the EGR-1 binding site in MI4 wrrc incapable of binding the EGR-1 protein. Thrsr findings sugg't.sted that the EGR-1 protein might be acting by R dirrct intrraction with its cognate site to activate thr Gmi-2 gcne..
To examine this possibility. we comparrd nuclrar rxtracts from LLC-PK1 cells that were eithrr activrly dividing and nonpolarized (culture days 1-2) or rrlatively quiescent and fully polarized (culture day 8). As seen in Fig. 6, thr hinding patterns of nuclear extracts from culture days 1-2 w w c strikingly different from those on culturr day 8. The 2.7-h~ ECR-I prohr detected two prominent induced complrxes in nuclrar rxtracts from culture days 1-2. By contrast. the samr probr drtected three distinct complexes in nuclear extracts from culture day 8. The upper complex had slightly lower mohility as comparrd with the upper complex in days 1-2 nuclear extracts, a srcond slower mobility complex was also drtrcted that was not srrn in nuclear extracts of culture days 1-2. and a third nuclc.ar complex, whose mobility was comparahlr with that sern in nuclrar extracts in days 1-2, was much less intense. Thrse data confirmed that nuclear extracts from culturr day 8 as comparrd with culture days 1-2 contained diffrring D N A hinding proteins that interact with the EGR-1 consrnsus srquencr

( 5 ' -
Based on our immunoqytochemical studics. it would br anticipated that the EGR-1 protein should he present in nuclrar extracts of actively dividing LLC-PKI crlls on culturr days 1-2. To determine whether EGR-1 was one of thr protrins intrrarting with the EGR-1 probe, nuclear extracts from culturr days 1-2 and day 8 were prrincuhatrd with immune rahhit tional slower mobility complex could be easily detected in extracts from culture days 1-2. By contrast, a n additional slower mobility complex was barely detected in extracts from culture day 8. These data demonstrated that one component of these nuclear complexes was the EGR-1 protein. Detectability of this protein was consistent with its pattern of maximal expression in dividing LLC-PK1 cells that also have a corresponding activation of the Gai-2 gene. DISCUSSION We have previously demonstrated in renal cells t h a t t h e G q Z gene is transcriptionally activated during growth and differentiation and is subsequently repressed to a basal state following the achievement of cell polarity. The activity of this gene is directly reflected in the expression of its gene products during this period (19). We had suggested that the necessity for a genetic control mechanism that prevents the strong constitutive activation of the Gai-2 gene in fully differentiated renal epithelia may be required to maintain normal intracellular signaling and also to repress normal growth pathways, thereby preventing oncogenic transformation. To directly test this possibility, LLC-PK1 cells were transiently transfected with a plasmid encoding the cDNA for Gai-2 fused to the viral Rous sarcoma promoter enhancer to constitutively overexpress Gai-2 proteins. Although only 10-20% of these cells were transiently transfected, this was sufficient to confer to the overall cell population growth rates that were, respectively, 2.8 times faster than cells transfected with Bluescript in 10% FBS. Furthermore, although LLC-PK1 cells normally require 10% FBS t o grow efficiently in culture, high growth rates were maintained for cells grown in 0.1% FBS that were transfected with plasmids encoding Gai-2. These findings are consistent with other studies that indicate that Gai-2 can couple receptors and effectors in signaling pathways for growth and differentiation in fibroblasts (6) and F9 teratocarcinoma cells (14). Mutations from to Cys or His, which decrease GTPase activity in Gai.z, have been found in tumors of the adrenal cortex and ovary, which convert this gene into the oncogene gip2 (7). Transfection of gip2 into Rat-1 cells induces their oncogenic transformation (5, 8). Increased growth and oncogenic transformation in this setting is, in part, attributable to persistent activation of downstream effectors such as mitogen-activated protein kinase (9). Importantly, stoichiometric control of Gai-2 protein is also critical for normal intracellular signaling, as overexpression of wild type Gai.2 subunits can also induce on-cogenic transformation in Rat-la cells (5). Conversely, the serum-stimulated mitogenic response of mouse Balbk3T3 fibroblasts can be inhibited by either pertussis toxin or their direct injection with antibodies against Gai.z subunits (6). Recent studies suggest that even modest repression of Gai.2 expression in vivo is associated with renal morphologic abnormalities in transgenic mice (15).
In our previous studies, an enhancer region (position -200 to -335) in the Gai-2 gene was identified in a deletion series used to define the gene promoter (18). Interestingly, enhancer activity was found in this area only in proliferating cells prior to cell polarization. These studies suggested that the transcription factor or factors necessary for transactivation of the gene might be induced by mitogenic signaling pathways. Inspection of the enhancer area revealed a motif (5'-CGCCCCCGC-3') that could serve as a perfect binding site for the EGR-1 zinc finger transcription factor. The gene encoding this transcription factor is rapidly induced by a variety of agents that induce cellular proliferation (21) and has recently been implicated in signaling pathways essential for the differentiation of macrophages (26) and cardiac myocytes (27). In renal tissues, the EGR-1 gene is rapidly activated following ischemia and hypertrophy (24). Following EGR-1 gene activation, the expressed protein is primarily detected in the nuclei of thick ascending limbs and principal cells of the collecting ducts in the cortex and medulla (25).
To determine whether this transcription factor could be playing a role in Gai-2 gene transactivation in LLC-PK1 renal cells, we first demonstrated that it was expressed in this cell type. In fully confluent quiescent LLC-PK1 cells, the EGR-1 protein was not detected either by immunoblotting or by immunocytochemistry. However, in actively dividing cells prior to polarization, the EGR-1 protein was easily detected in the nuclei of these cells in a pattern identical t o that seen in renal tissues following ischemic and hypertrophy. Immunoblotting indicated that the relative content of this protein was highest in dividing cells and declined rapidly to undetectable levels in differentiated cells that had achieved polarity. Importantly, EGR-1 protein expression coincided with the same pattern of expression that we have previously documented for Gai-2 proteins in this cell type during culture. If the pattern of EGR-1 protein expression contributed to LLC-PK1 cell mitogenesis during culture, we reasoned that overexpression of this protein might affect growth in this cell type by stimulating endogenous Gai-2 gene expression. Although only 10-20% of these cells were transiently transfected, this was sufficient to confer to the overall programmed lysate comprted with 20 n g of Rlurscript I1 ks; Innr 5-7, prohe with EGR-1 programmrd lysatr compctrd with 5, IO, and 20 n g of cold prohe, respectively; lnnr X , prohe with EGR-I programmed lysate prrincuhatrd with prrimmune rahhit I s , ; lnna 9, prohe with ECR-I Wi 21 (alpha 11. R . the 28-hp DNA segment containing the EGR-1 programmed lysate prcincuhnted with immune rahhit IgG anti-IXR-1 consensus sequence was compared with a 28-hp DNA segment with thr ECK-1 sequrnce mutated hy adenosines in mobility shift assays (5'-A T T C C C C C~C C G C C G T C C : ( ; G -9 ' ) and a 6 2 -h   population growth rates that were initially 1.7 timrs faster than cells transfected with Rluescript in IO' + FRS. As with cells transfected with plasmids to overexpress Gtri-2, accelerated growth rates could he maintained despite reduction ofserum in the culture media to O.lp+.
Although EGR-1 might have many potential target genes. we reasoned that EGR-1 might be acting to up-replate Chi-2 grne expression to account for increased p-owth in dividing renal cells. To examine this pnssihility, we cotransfected plasmids to overexpress EGR-1 (pRSV EGR-11, and plasmids that contained the Gtri-2 5"flanking srquence with EGR-1 hinding site (5'-CGCCCCCGC-.'J') (1v141, a mutated EGR-1 site (.5'-AAAAACCGC-3') (mutated M14). or a n EGR-I site fully deleted ( M 4 ) fused to a firefly lucifrrasr reporter gene. In our previous studies, maximal activation of the &ti-2 grne was found 24-48 h after recovery from transfection when cells were at approximately 10-203 confluence. At latrr time periods. as the cells became more confluent, Gui-2 transcriptional activity fell. Interestingly, in these later culture timrs enhancer activity decreased, and as cells hecamr polarizrd this rrgion actually repressed Grri-2 transcription (data not shown). Our initial experiments at 48 h following transfection drmonstrated a 20-50% increase in Gtri-2 transcription only in cells transfected with pRSV EGR-1 plasmids fthat overexpress EGR-I 1 and >TI4 reporter plasmids that contained the EGR-1 binding site. However, by delaying these experimrnts for an additional24 h when cells hecame 30-40'; confluent, a consistent .72OC; activation was seen in these cells. By contrast, no significant increase in Gtri-2 transcription was found in cells that were transfected with pRSV EGR-1 plasmids with reporter plasmids that contained the mutated EGR-1 binding site or had the site deleted. One interpretation of these findings is that EGR-1 expression was maximal in cells that were 10-20% confluent, hence additional EGR-1 protein would not appreciably contribute to further activation of the Gai-2 gene. However, as the cells became more confluent and their endogenous EGR-1 protein levels fell, there would be a corresponding decrease in Gai-2 gene transcription in all cells except those that were transfected with plasmids to overexpress EGR-1. Cells transfected with pRSV EGR-1 would maintain high EGR-1 protein levels allowing for the persistent maximal activation of the Gai-2 gene.
To verify that EGR-1 was directly contributing to the activation of the Gai-2 gene, a 23-bp DNA fragment of the 60-bp gene enhancer region containing the central EGR-1 binding motif was synthesized for use in mobility shift assays with in vitro translated EGR-1 protein. This probe was able to specifically bind EGR-1 protein. Furthermore, this complex could be recognized by rabbit antibody generated against the non-zinc finger carboxyl-terminal region of the EGR-1 protein. Two additional DNA segments were synthesized to confirm that the EGR-1 protein was only binding its cognate site in the 60-bp enhancer region. EGR-1 binds its cognate DNA site by participation of three distinct zinc fingers (28). Finger 1 binds near the 5'-end of the primary strand (5'-CGCCCCCGC-3'), finger 2 binds near the center (5'-CGCCCCCGC-3'), and finger 3 binds near the 3'-end of the primary strand (5'-CGCCCCCGC-3'). A mutated DNA segment corresponding to the EGR-1 probe was therefore synthesized, which differed by five 5"adenosine substitutions (5'-AAAAACCGC-3') to prevent binding of fingers 1 and 2. We demonstrated that this probe was unable t o bind in vitro translated EGR-1 protein in mobility shift assays. Furthermore, utilizing a primer derived from the upper strand 5 ' 4 -2 sequence of M14 with the bottom strand primer of the mutated EGR-1 probe, a DNA segment was derived from the M14 plasmid that was also unable to bind in vitro translated EGR-1 protein in mobility shift assays, which demonstrated that the area 5' to the EGR-1 binding site in M14 was unable to bind EGR-1. These studies complemented our functional studies, which suggested that increased transcription of the Gai-2 gene by EGR-1 overexpression was occurring specifically through its cognate binding site and not in the region 5' to this site.
To confirm that this discrete area of the gene participated in a n EGR-1-mediated transcriptional response, nuclear extracts were prepared from cells 18-24 h or 8 days following culture for use in the mobility shift assay. These studies indicated that nuclear extracts from culture day 8 as compared with culture days 1-2 contained differing DNA binding proteins that interact with the EGR-1 consensus sequence (5'-CGCCCCCGC-3'). Furthermore, the preincubation of these extracts with an antibody that specifically recognizes EGR-1 confirmed that the induced complexes from cells 18-24 h contained the EGR-1 protein as evidenced by the production of a slower mobility "super shifted" nuclear complex. Detectability of this protein was consistent with its pattern of maximal expression in dividing LLC-PK1 cells that also have a corresponding activation of the Gai-2 gene.
It was also apparent from these studies that the temporal deactivation of the Gai-2 gene in fully polarized LLC-PK1 cells taken from day 8 of culture was not simply mediated by the loss Protein ai-2 Subunit Gene 27509 of EGR-1 protein. Our studies suggested that EGR-1 was replaced with a lower molecular weight protein complex or complexes that efficiently bind the EGR-1 motif. A candidate transacting factor that may act as a repressor in this context is the Wilms' tumor protein (WTl), which has greater than 65% amino acid sequence similarity with EGR-1 in its zinc finger region (21). Importantly, WT1 has recently been shown to efficiently bind the EGR-1 motif (29) and act as a repressor of gene transcription either independently or in concert with the tumor suppressor factor p53 in several cell types (30). We are currently investigating whether WT1 andor p53 can act as a repressor of Gai-2 gene expression in renal cells.
We believe these studies provide evidence for a novel mitogenic pathway in renal cells that potentially link proximal signaling events that induce EGR-1 gene expression to activation of a genomic target essential for growth and differentiation in this cell type, the Gai-2 protooncogene. Upon further examination, this pathway may provide significant insights into the molecular events involved in renal hypertrophy, nephrogenesis, and oncogenesis.