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Endocrinology Vol. 140, No. 12 5659-5668
Copyright © 1999 by The Endocrine Society

ARTICLES

Prolactin Regulation of pim-1 Expression: Positive and Negative Promoter Elements1

Kurt E. Borg, Mingyu Zhang, David Hegge, Ruth L. Stephen, Donna J. Buckley, Nancy S. Magnuson and Arthur R. Buckley

Department of Pharmacology & Toxicology (K.E.B., D.H.), University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58202; College of Pharmacy (M.Z., R.L.S., D.J.B., A.R.B.) and Department of Molecular and Cellular Physiology, College of Medicine (A.R.B.), University of Cincinnati Medical Center, Cincinnati, Ohio 45267; and Department of Microbiology and Immunology (N.S.M.), Washington State University, Pullman, Washington 99164

Address all correspondence and requests for reprints to: Arthur R. Buckley, College of Pharmacy, University of Cincinnati Medical Center, 3223 Eden Avenue, P.O. Box 670004, Cincinnati, Ohio 45267-0004. E-mail: Arthur.Buckley{at}uc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The lactogen-dependent rat Nb2 lymphoma is a useful model to investigate PRL signaling pathways that lead to regulation of gene transcription. A primary mechanism coupled to PRL receptor (PRLR) activation in Nb2 cells involves phosphorylation by Jak-family tyrosine kinases of one or more signal transducers and activators of transcription (Stat) factors which subsequently bind to {gamma}-interferon activation sequences (GAS) within promoter regions of target genes. However, it is presently unclear whether this mechanism is operative as a means for regulating PRL-induced gene expression to the exclusion of other signaling pathways. Previously, we reported that PRL directly stimulated rapid expression of the protooncogene, pim-1, at the mRNA and protein levels in lactogen-dependent Nb2–11 cells. In the present study, experiments were conducted to evaluate signaling mechanisms by which PRL regulates transcription of pim-1. Toward this end, a 1,268-bp segment upstream of the transcription initiation site of the 5'-pim-1 promoter and a series of deletion mutants were ligated upstream of the chloramphenicol acetylase transferase (CAT) gene in an expression vector that was introduced into FDC/Nb2 cells, a premyeloid line that stably expresses the intermediate form of the PRLR. Analysis of PRL-treated cultures indicated that two elements [distal (DE), -427 to -336 bp and proximal (PE), -104 to -1] but not several GAS or GAS-like sequences were required for hormone activation of the pim-1 promoter. Moreover, treatment of Nb2–11 cells with PRL activated protein binding to these elements assessed by gel mobility shift assay. Deoxyribonuclease I (DNase I) protection experiments revealed a motif containing a nuclear factor-1 (NF-1, -224 to -217 bp) half-site that was hydrolyzed when exposed to extracts from PRL-treated cells but protected by proteins from unstimulated cells. Gel mobility shift analysis of this sequence showed decreased protein binding after PRL stimulation. It is concluded that the PRLR initiates pim-1 transcription by a mechanism that involves transcriptional activation by factors that stimulate the DE- and PE-sites and derepress a NF-1-containing element. Moreover, this mechanism appears to be independent of an interaction between Stat transcription factors and GAS-like elements present within the promoter.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE RAT Nb2 T-cell lymphoma, originally described by Gout et al. (1), is a widely employed paradigm for investigation of PRL receptor (PRLR)-coupled signaling mechanisms linked to transcriptional regulation of lactogen-responsive genes. The parental Nb2 cell line was found to require PRL (or other lactogens) for stimulation of proliferation and cell survival (1). Quiescent cultures, arrested in the early G1 phase of the cell cycle, can be obtained by incubating the cells in the absence of lactogen for a period of 18–24 h. Subsequent reintroduction of PRL stimulates partially synchronous cell cycle progression. Prolonged culturing of the cells in the absence of hormone leads to activation of the cell death mechanism similar to the demonstrated effect of growth factor deprivation on apoptosis in other factor-dependent cell systems (2).

Lactogen-stimulated Nb2 cells have been used to investigate PRLR signaling to several signal transduction pathways, some of which have been linked to gene transcription. Thus, PRLR ligation has been reported to activate the tyrosine kinases, Janus kinase 2 (Jak2) (3, 4, 5), p59fyn (6), and ZAP-70 (7, 8), the serine-threonine kinases, mitogen-activated protein kinase [MAPK (9)], protein kinase C (10), S6 kinase (11), and phosphatidylinositol 3-kinase (12), as well as guanine nucleotide-associated proteins, Ras (13, 14) and p95vav (15). While it is likely that signals from several of these early signaling mediators coalesce into one or more common pathways, the precise step-wise mechanisms leading to transcription of PRL-responsive genes are incompletely understood.

Based upon the sequence of its extracellular domain, the PRLR is recognized as a member of the hematopoietin/cytokine receptor superfamily, which includes receptors for various interleukins, colony-stimulating factors, interferons, as well as GH (16, 17). Ligand-mediated activation of these receptors has been shown to rapidly activate one or more members of Jak family receptor-associated tyrosine kinases (3, 4, 5). Once activated, these kinases phosphorylate members of the signal transducers and activators of transcription (Stat)-family of transcription factors (reviewed in Ref. 18). Activated Stats dimerize, translocate to the nucleus, and bind specifically to promoter regions of responsive genes. The PRLR has been shown to signal through Jak2 with subsequent activation of Stats 1, 3, and 5a (19, 20, 21, 22). Once activated, these Stats, as homo- or heterodimers, bind to {gamma}-interferon activation sequences (GAS) in PRL-responsive genes (23, 24). Thus, the Jak/Stat signaling pathway has been implicated in the regulation of several PRL-inducible genes in Nb2 cells and in mammary gland.

The protooncogene, pim-1, encodes a highly conserved serine-threonine kinase that is primarily expressed in germ and hematopoietic cells (25, 26). Originally identified in malignant lymphomas (27), pim-1 is induced by cytokines in lymphocytes in which its elevated expression is associated with proliferation (28, 29) and suppression of apoptosis (30). We previously reported that PRL stimulation of Nb2–11 cells caused rapid induction of pim-1 mRNA expression, which was coupled to enhanced synthesis of its protein product. In the PRL-independent Nb2-SFJCD1 subline, the protooncogene was found to be constitutively expressed; its expression was further enhanced by PRL treatment (31). In this line, pretreatment with the diet-derived differentiating agent, butyrate, reduced pim-1 mRNA expression (32). Since butyrate transiently reverted Nb2-SFJCD1 cells to a PRL-dependent phenotype and enhanced their sensitivity to glucocorticoid-induced apoptosis (32, 33), this observation suggested that the survival advantage conferred by PRL in lactogen-dependent Nb2 cells may reflect its stimulation of pim-1 transcription and translation. In studies using Nb2–11 and butyrate-pretreated Nb2-SFJCD1 cells, we observed that PRL-mediated suppression of apoptosis correlated precisely with pim-1 expression (2, 33). Based on these observations, we concluded that Pim-1-catalyzed phosphorylation of key intermediates may mediate apoptosis-suppression observed after PRL stimulation in Nb2 cells.

The signaling mechanism leading from PRLR activation to induction of pim-1 transcription is currently unknown. The 5'-promoter region of the pim-1 gene is G+C rich and lacks TATA or CAAT elements, reminiscent of housekeeping genes constitutively expressed at low levels (34, 35). Recent evidence has indicated that signaling through the Jak/Stat pathway may contribute to transcriptional regulation of pim-1 in hematopoietic cells (36, 37, 38), although activation of protein kinase C and associated downstream transcription factors may also participate (39). In the present study, we investigated the mechanism by which PRL provokes pim-1 expression. We show that the PRLR signals to two approxiately 100-bp promoter sequences that positively regulate its transcription while an element that binds nuclear factor-1 (NF-1) appears to repress gene expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
The PRL-dependent rat T-lymphoma, Nb2–11, provided by Peter Gout (Vancouver, British Columbia, Canada), has been extensively characterized and was cultured as described (1). Cells were maintained in Fischer’s medium supplemented with 10% FBS and 10% horse serum (Summit Biotechnology, Fort Collins, CO), 100 µM 2-mercaptoethanol, penicillin (50 U/ml), and streptomycin (50 µg/ml) (maintenance medium). For analysis of PRL-stimulated transcription factor binding to specific pim-1 promoter oligonucleotides, cells were rendered quiescent by culturing for 18–24 h in maintenance medium containing 10% nonlactogenic gelding serum (ICN Biochemicals, Inc., Irvine, CA), and then incubated with 20 ng/ml of ovine PRL (oPRL-20, National Hormone and Pituitary Program, Bethesda, MD) for varying time periods.

Transient transfection experiments to evaluate pim-1 promoter activity were conducted in FDC/Nb2 cells, a premyeloid cell line that stably expresses the intermediate form of the PRLR (40). Stock cultures of FDC/Nb2 cells (a gift from L-y Yu-Lee, Houston, TX) were maintained in RPMI-1640, 25 mM HEPES supplemented with 10% FBS, and PRL (20 ng/ml).

Proliferation of FDC/Nb2 cells
Before cell proliferation experiments, FDC/Nb2 cells were rendered quiescent by preincubation for 24 h in nonmitogenic, RPMI-1640 medium supplemented with 10% nonlactogenic gelding serum. Quiescent cells were incubated in sextuplicate with increasing concentrations of PRL or recombinant mouse interleukin-3 (IL-3) (R&D Systems, Minneapolis, MN) for 48 h. The effect of cytokine stimulation on cell population density was determined using automated cell counting (Coulter Corp., Hialeah, FL).

Northern blotting procedures
Total RNA was isolated from 2.5 x 107 FDC/Nb2 cells cultured in 25-cm2 flasks using RNAzol-B (Tel-Test, Friendswood, TX) and quantitated spectrophotometrically. The RNA was denatured in formaldehyde and fractionated on 1% agarose gels, and then transferred to GeneScreen Plus (DuPont Merck Pharmaceutical Co., Wilmington, DE). Equal loading per lane was verified by ethidium bromide staining of 18S and 28S ribosomal RNA, which was visualized and photographed under UV illumination. Isolated pim-1 cDNA inserts were labeled with [32P]deoxy-CTP (New England Nuclear, Boston, MA) using the random primer method of Feinberg and Vogelstein (41). Hybridization and wash procedures were conducted using the methods of Church and Gilbert (42).

Promoter/reporter studies
To evaluate potential PRL-regulated regions within the pim-1 promoter, experiments were conducted using a chloramphenicol acetyltransferase (CAT) promoter/reporter system. The CAT vector used, composed of a 1,644-bp XbaI/BamHI fragment from pCAT-Basic (Promega Corp., Madison, WI) which included the entire CAT gene and SV40 T antigen sequence, was ligated into the XbaI/BamHI site of pUC19 (New England Biolabs, Inc., Beverly, MA; designated pUC19-CAT).

Deletion mutants of the pim-1 promoter were generated by endonuclease digestion of a 1.4-kb Swa I/PstI segment with AflIII, BamHI, XhoI, ClaI, Nar I, or BglI concurrently with PstI, which produced 5'-mutants of 1,268 bp, 749 bp, 521 bp, 428 bp, 337 bp, and 104 bp, respectively, upstream of the transcription initiation site (Fig. 2Go). The PstI site is located at the 3'-end, +37 bp. Mutants were ligated into the PstI site of pUC19-CAT using T4 DNA ligase followed by blunting of the remaining noncompatible ends with DNA polymerase I (Klenow) followed by a second ligation.



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  		Figure 2. Schematic diagram of the 5'-pim-1 promoter. Deletion mutants of the promoter were generated by digestion with restriction endonucleases or PCR amplification using promoter-specific primers and ligated into the pUC19-CAT expression vector as described in Materials and Methods. Nucleotide numbering is relative to the transcription initiation sequence. GL1–4, GAS-like sequence.

 
Several mutants were also generated by PCR amplification of specific regions of the pim-1 promoter. Primers for PCR were designed to include 5'-restriction sites for HindIII (+ primer) and PstI (- primer) for subsequent digestion of PCR products to facilitate ligation into pUC19-CAT. All mutants (PCR and endonuclease generated) were sequenced using the dideoxynucleotide method (Thermosequenase, Amersham Pharmacia Biotech, Inc., Arlington Heights, IL) for sequence verification and orientation.

The CAT constructs (50 µg), together with a cytomegalovirus (CMV)-ß-galactosidase plasmid (30 µg; pCMVß, CLONTECH Laboratories, Inc. Palo Alto, CA) were cotransfected into FDC/Nb2 cells. Cells (1 x 107) suspended in maintenance medium were transferred to an electroporation cuvette. Electroporation was conducted using an ECM 600 instrument (300 V/950 µF, 20 sec, Genetronics, Inc., San Diego, CA). After overnight recovery in RPMI-1640 supplemented with 25 mM HEPES and 1% lactogen-free gelding serum, cells were cultured in the presence of PRL (100 ng/ml) for 48 h, and then harvested for subsequent analysis of CAT and ß-galactosidase (ß-gal) activities.

CAT and ß-gal determinations
Cell pellets were lysed by three freeze/thaw cycles in ice-cold 0.25 M Tris-Cl (pH 7.5). Lysates were centrifuged at 13,000 x g for 5 min. Supernatants were assessed for ß-gal activity, and then heat inactivated at 65 C for 10 min before conducting CAT analysis.

To determine ß-gal activity, cell lysates were mixed with 100 x Mg solution (0.1 M MgCl2, 4.5 M ß-mercaptoethanol), o-nitrophenyl-ß-D-galactopyranoside (4 mg/ml), and 0.1 M sodium phosphate (pH 7.5). Reactions were incubated at 37 C for 30 min and terminated by addition of 1 M Na2CO3, and the absorbance at 420 nm was determined. Volumes of cell lysates used for CAT analysis were normalized to the ß-gal activities obtained.

Activity of CAT was assessed by incubating cell lysates for 60 min with [1,2-14C]chloramphenicol (100 µCi/ml, 103 mCi/mmol, ICN Pharmaceuticals, Inc., Costa Mesa, CA), acetyl-coenzyme A (CoA), and 1 M Tris-Cl (pH 7.5) in a final volume of 150 µl. Reactions were terminated by adding 1 ml of ethyl acetate. Samples were mixed and centrifuged, and the ethyl acetate layers were transferred to fresh tubes. After evaporating to dryness, the samples were resuspended in ethyl acetate, spotted onto flexible silica gel plates (Baker Flex 1B; 20 x 20 cm; J.T. Baker, Inc., Phillipsburg, NJ), and developed by TLC in 19:1 chloroform/methanol (vol/vol). Plates were dried and exposed to x-ray film (Hyperfilm, Amersham Pharmacia Biotech). Acetylated and nonacetylated chloramphenicol was excised from the plates and radioactivity was determined by scintillation counting. Percent conversion was calculated using the formula:

in which %C = percent conversion from nonacetylated to acetylated chloramphenicol; acetylCPM = counts per min in acetylated chloramphenicol; acetylCPMPV = background counts per min of CAT activity from FDC/Nb2 cells transfected with a promoterless control (pUC19-CAT vector); and nonacetylCPM = counts per min in nonacetylated chloramphenicol.

Data are expressed as fold increase in CAT activity in PRL-treated cells compared with transfectants incubated in the absence of hormone. Controls included: the promoterless pUC19-CAT vector ± PRL (negative control); recombinant CAT enzyme (positive control to verify conditions for CAT analysis); and transfection with pCDNA3.1-CAT (positive control for transfection with a constitutive promoter).

DNA mobility shift assays
Nb2–11 cells (2–3 x 107) were lysed using a glass Dounce homogenizer in a buffer containing 20 mM HEPES (pH 7.5), 1.5 mM MgCl2, 0.2 mM EDTA, 0.2 mM dithiothreitol, 0.4 M NaCl, 20% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5 mM leupeptin. Lysates were centrifuged and the protein content of supernatants was determined using the Bradford reagent (Bio-Rad Laboratories, Inc. Hercules, CA). Assays were performed using a GELSHIFT kit (Stratagene, La Jolla, CA). Cell lysates (10 µg of total protein) were incubated with 32P-labeled oligonucleotides corresponding to the pim-1 DE (-427 to -336 bp), PE (-104 to +37), a pim-1 GAS-like element (Table 1Go, GL2), the IRF-1 GAS element (kindly provided by L-y Yu-Lee), or an oligonucleotide containing the NF-1 half-site (5'-CTCTGGTTGGCTGGAGTAGCGCTGG-3') on ice for 30 min in a poly(dI-dC)·(dI-dC) (Sigma, St. Louis, MO)-containing buffer. Specificity of binding was determined by incubation of lysates in the presence of excess unlabeled DE, PE, NF-1, heat shock element (HSE), SP-1, NFkB, or AP-2 (Life Technologies, Inc., Gaithersburg, MD) oligonucleotides. After incubation, the reactions were fractionated on SDS-polyacrylamide gels, dried, and exposed to x-ray film.


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  		Table 1. The presence and locations relative to the transcription initiation sequence of GAS and GAS-like (GL) elements present in the -1700 bp sequence of the pim-1 promoter

 
DNase I protection assay
Nuclei were prepared as previously described (43) by resuspending the cells in hypotonic buffer [0.4 mM KH2PO4 (pH 7.4), 2.5 mM MgCl2, 0.5 mM EGTA], and incubating on ice for 1 min. After the addition of hypertonic buffer (hypotonic buffer containing 1.6 M sucrose) the cells were centrifuged at 750 x g for 10 min. Swollen cells were disrupted in a buffer containing 94 mM KH2PO4 (pH 7.4), 2.5 mM MgCl2, 0.32 M sucrose, 0.5 mM EGTA, and 50 µg/ml leupeptin and again centrifuged at 750 x g. Buffer was removed and extracts of the nuclear pellets were used for determination of DNase I protection.

Nuclear extracts were preincubated on ice for 10 min in a buffer containing 12.5 mM HEPES (pH 7.5), 50 mM KCl, 10% glycerol, 0.005% Nonidet P-40, 0.5 mM ZnSO4, and 0.5 mM dithiothreitol and poly(dI:dC). The 32P-labeled (~125,000 cpm, ICN Biomedicals, Inc.) ClaI-PstI promoter segment (-427 to +37 bp) was added and the samples returned to ice for 25 min. An equal volume of 10 mM MgCl2 and 5 mM CaCl2 was added 1 min before DNase I treatment. Samples were incubated for 1 min at room temperature in the presence of DNase I before 37 C stop solution (12.5 mM EDTA, 12.5 µg/ml Proteinase K, 125 µg/ml yeast transfer RNA, 0.1% SDS) was added. After incubation at 37 C for an additional 15 min, the samples were extracted with phenol-chloroform-isoamyl alcohol (25:24:1), chloroform-isoamyl alcohol (24:1), extensively washed by ethanol precipitation, and resolved on 6% acrylamide gels with 50% urea. DNA sequencing analysis was performed in parallel in each experiment.

Statistical analyses
All experiments were replicated at least three times. Cell density and CAT activities presented represent mean ± SEM of results obtained from three independent experiments. Data were analyzed by ANOVA followed by the Fisher’s least significant difference analysis posttest. Representative autoradiographs are presented (mobility gel shift and DNase I protection analyses) from experiments replicated at least three times with similar results.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PRL signaling through the DE- and PE-pim-1 promoter elements
To investigate PRL signaling to activation of pim-1 transcription, promoter/reporter studies were conducted using the premyeloid cell line, FDC-P1. This cell line has been extensively employed to investigate signaling mechanisms coupled to type 1 cytokine receptors including erythropoietin, IL-2, IL-3, granulocyte-macrophage colony-stimulating factor, GH, and PRL (40, 44, 45, 46, 47, 48, 49). Each of these cytokines/hormones has been linked to activation of the Jak2/Stat and MAPK pathways that are expressed in FDC-P1 cells (44, 45, 46, 47, 48, 49). The cell line used in the present study was previously transfected with the intermediate form of the PRLR and stably expresses this protein [FDC/Nb2 (40)]. Originally dependent on IL-3 for growth, the FDC/Nb2 line can be maintained in long term culture in medium supplemented with PRL in the absence of IL-3. The results presented in Fig. 1AGo demonstrate PRL responsiveness of this cell line. Addition of either PRL or IL-3 to quiescent FDC/Nb2 cells stimulated proliferation in a concentration-dependent manner. Maximal population growth was observed with 1 and 0.1 ng/ml of PRL and IL-3, respectively. Thus, in addition to the PRLR, this line also expresses signaling intermediates required for PRL-stimulated cell proliferation. In addition to proliferation, PRL also stimulates rapid accumulation of pim-1 mRNA in these cells (Fig. 1BGo). The 2.8-kb transcript was observed in exponentially proliferating FDC/Nb2 cells (Log) and undetectable in quiescent cultures (GA). Stimulation with PRL (10 ng/ml) markedly increased the level of pim-1 mRNA, which reached maximal levels within 2 h. Notably, this pattern of pim-1 expression in FDC/Nb2 cells is nearly identical to that observed in the PRL-stimulated Nb2–11 line (31). Therefore, not only does PRL stimulate proliferation of FDC/Nb2 cells just as it does in the Nb2 line, it also provokes expression of PRL-responsive genes with strikingly similar kinetics. These observations indicated that the FDC/Nb2 subline is a suitable system with which to investigate PRL-regulated pim-1 expression.



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  		Figure 1. Effect of PRL on FDC/Nb2 cells. A, Quiescent cells (initial concentration: 1.9–2.5 x 105 cells/ml) were incubated in the presence of PRL or IL-3 for 48 h. Population density of the cultures was determined by electronic cell counting. Results are presented as means ± SEM of sextuplicate wells per cytokine concentration from an experiment replicated three times. When not visible, error bars are obscured by the symbols. B, Quiescent cells (2 x 107) were incubated with 10 ng/ml of PRL and harvested at the times indicated. Total RNA was prepared and pim-1 Northern blot analysis conducted as described in Materials and Methods. A representative autoradiograph from an experiment conducted three times is presented. Log, Exponentially proliferating cells; GA, quiescent cells.

 
A series of deletion mutants were constructed from a 1.4-kb segment of the 5'-pim-1 promoter (Fig. 2Go). Ligated upstream of CAT in pUC19-CAT, these were cotransfected with a ß-gal plasmid into FDC/Nb2 cells. The effect of PRL (100 ng/ml) on promoter activity was determined after 48 h. Transfectants incubated in the absence of PRL served as controls. In cells transfected with the -1,268-bp portion of the promoter, PRL-induced CAT activity to a level >40-fold (P < 0.001) above that observed in control cells (Fig. 3Go). Truncation of the promoter to -749 or -521 bp did not reduce the effect of PRL to induce CAT activity. Notably, sequence analysis of the pim-1 promoter revealed several GAS-like sequences (Table 1Go) that represent potential sites for interaction with Stat transcription factors activated by upstream Jaks. However, the observation that elimination of these elements did not decrease PRL-stimulated CAT activity suggests that these sequences are most likely not required for transcriptional activation of the pim-1 promoter.



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  		Figure 3. Effect of PRL on pim-1 promoter activity. Various deletion mutants of the pim-1 promoter in pUC19-CAT and a plasmid containing the ß-gal gene were transfected into FDC/Nb2 cells. After recovery, cells were cultured in the presence or absence of PRL (100 ng/ml) for 48 h. CAT activity of cell lysates was determined as described in Materials and Methods. Results are expressed as fold increase in PRL-treated cells compared with identically transfected cultures in the absence of hormone. Results are presented as the mean ± SEM of three independent experiments. *, P < 0.01.

 
The -427 bp construct contained two approximately 100-bp elements (DE, -427 to -336 and PE, -104 to -1) that have been linked to constitutive pim-1 expression in another T-cell line (50). PRL stimulation of FDC/Nb2 cells transfected with this construct exhibited similar CAT activity as that observed when the -1,268 bp sequence was introduced. However, deletion of the DE (-336 bp construct) reduced PRL-induced CAT activity. Moreover, PRL treatment of cells transfected with a promoter mutant containing the DE but not the PE (-699 to -279 bp) resulted in significantly elevated CAT activity. Together, these observations suggest that the DE is likely activated by a PRLR-coupled signaling mechanism leading to activation of one or more transcription factors that regulate pim-1 expression.

PRL stimulation of cells transfected with the -336 bp mutant which contained the PE or with the PE-only construct (-104 bp) produced equivalent CAT activity at levels nearly 20-fold above those observed in control cultures. Since PRL-responsiveness was retained in cells transfected with only the PE, activation of this element is a likely component of hormone-induced transcription of the protooncogene.

Since the DE and PE appeared important for PRL signaling to pim-1 gene expression, electrophoretic mobility gel-shift analysis was conducted to determine whether proteins present in PRL-stimulated Nb2–11 cells bound to these promoter elements. In these experiments, quiescent Nb2–11 cells were stimulated with PRL (20 ng/ml) and harvested after various time intervals through 2 h. As shown in Fig. 4Go, incubation of lysates obtained from exponentially proliferating cells (Log, lane 1) with a 32P-labeled DE resulted in formation of a DNA-protein complex that migrated as a readily detected single band while addition of lysates from quiescent cells (GA, lane 2) shifted migration to a lesser extent. Stimulation of quiescent Nb2–11 cultures with PRL increased protein binding to the DE, which reached maximal levels within 1.5 h. This effect of PRL was specific since addition of excess unlabeled DE (lanes 8 and 9) competitively inhibited binding while incubation with unlabeled, irrelevant oligonucleotides (lanes 10–13) failed to block DNA-protein complex formation. Importantly, addition of the unlabeled PE (lanes 14 and 15) inhibited DE binding, suggesting that factors present in the lysates may bind to sequences common to both regions of the pim-1 promoter.



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  		Figure 4. Gel mobility shift assay of the DE in PRL-stimulated Nb2–11 cells. Quiescent (GA) Nb2–11 were stimulated with PRL (20 ng/ml) and harvested at the times indicated. Electrophoretic mobility of nuclear protein preparations was determined by SDS-PAGE subsequent to incubation with 32P-labeled DE as described in Materials and Methods. Specificity of protein binding to the DE was determined by including: 1) 5 ng unlabeled DE or 2) 100 ng of unlabeled DE; 60 ng of HSE; 100 ng each of SP-1, AP-2, or NFkB; or 3) 5 ng of unlabeled PE or 4) 200 ng of unlabeled PE in the incubation mixture. A representative autoradiograph of an experiment replicated four times is presented. Log, Exponentially proliferating cells.

 
Similar experiments were conducted to assess transcription factor binding to the PE. As shown in Fig. 5Go, proliferating Nb2–11 cells (Log, lane 1) contained proteins that shifted the mobility of 32P-labeled PE, resulting in several protein-DNA complexes that were reduced in quiescent cells (GA, lane 2). Stimulation with PRL increased PE binding in a time-dependent manner that reached maximal levels within 2 h. Binding to the PE was specific since it was competitively inhibited by the addition of excess unlabeled PE (lanes 8 and 9) but not by non-PE sequences (lanes 10–13). Importantly, addition of DE competitively inhibited PE binding consistent with the hypothesis that common transcription factors, activated by PRL, may bind to each sequence.



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  		Figure 5. Gel mobility shift assay of the PE in PRL-stimulated Nb2–11 cells. Quiescent (GA) Nb2–11 were stimulated with PRL (20 ng/ml) and harvested at the times indicated. Electrophoretic mobility of nuclear protein preparations was determined by SDS-PAGE subsequent to incubation with 32P-labeled PE as described in Materials and Methods. Specificity of protein binding to the DE was determined by including: 1) 10 or 2) 200 ng of unlabeled DE; 210 ng of HSE; 175 ng each of SP-1, AP-2, or NFkB; or 3) 5 or 4) 175 ng of unlabeled DE in the incubation mixture. A representative autoradiograph of an experiment replicated four times is presented. Log, Exponentially proliferating cells.

 
Stimulation of the PRLR has been shown to activate Stat transcription factors that bind to GAS elements in hormone-responsive genes. Since one complete GAS and several GAS-like sequences are contained within the pim-1 promoter (Table 1Go), it was important to evaluate whether PRL provoked protein binding to these regions in a gel mobility shift assay. Therefore, the effect of PRL on binding to a GAS-like element present at -923 to -914 bp was evaluated. As shown in Fig. 6BGo, exposure of Nb2–11 cells to PRL (20 ng/ml) did not significantly alter protein binding to the pim-1-GAS-like oligonucleotide compared with untreated, control cultures. In contrast, hormone treatment increased binding to the GAS element present in the IRF-1 gene promoter (Fig. 6AGo) as well as to the DE (Fig. 6CGo). Therefore, just as indicated by results from promoter/reporter experiments, GAS-related elements present within the -1268 promoter are most likely not necessary for PRL-stimulated pim-1 gene transcription.



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  		Figure 6. Gel mobility shift of GAS and GL sequences in PRL-stimulated Nb2–11 cells. Nuclear proteins obtained from quiescent and PRL-treated (20 ng/ml) Nb2–11 cells were harvested at the times indicated and incubated with 32P-labeled IRF-1 (A), GL2 (B), or the DE (C). Electrophoretic mobility of the resulting complexes was resolved by SDS-PAGE as described in Materials and Methods. Specificity of protein binding was determined by the addition of excess unlabeled IRF-1 (105 ng), GL2 (105 ng), and DE (100 ng) to the incubation mixture. A representative autoradiograph of an experiment replicated four times is presented.

 
Derepression of pim-1 transcription by PRL
Signaling to the pim-1 promoter was further evaluated by DNase I protection analysis. For these experiments, the -427 bp segment (Fig. 2Go) was incubated with nuclear protein obtained from PRL-dependent Nb2–11 and -independent Nb2-SFJCD1 cells and DNase I; the resulting DNA fragments were resolved electrophoretically. Regions of protection were directly compared with the DNA sequence of this construct. In the absence of added nuclear protein, DNase I digestion of the -427 promoter mutant produced the fragmentation pattern shown in Fig. 7AGo, lane 1. Addition of nuclear protein derived from exponentially proliferating Nb2-SFJCD1 (lane 2), Nb2–11 (lane 3), or Nb2–11 cells exposed to PRL for 2 h (lanes 4 and 7) did not alter this pattern. However, nuclear protein from quiescent Nb2–11 cells (lanes 5 and 6) protected a site within the DNA segment from hydrolysis indicating that it likely contained a bound protein that blocked accessibility to the enzyme (Fig. 7Go, arrow). Sequencing of this fragment revealed a NF-1 half-site within the protected DNA segment (Fig. 7BGo), suggesting that its binding may repress pim-1 promoter activity. Moreover, treatment with PRL appeared to derepress this site; the element was no longer bound by a factor that recognized the NF-1 element that allowed gene transcription.



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  		Figure 7. PRL stimulation derepresses binding to a NF-1 recognition half-site. A, The ClaI–PstI segment of the pim-1 promoter was incubated with DNase I only (lane 1), or with added nuclear protein obtained from exponentially proliferating Nb2-SFJCD1 (lane 2) or Nb2–11 (lane 3) cells or quiescent Nb2–11 (lanes 5 and 6) or Nb2–11 cells cultured for 2 h in the presence of PRL (20 ng/ml, lanes 4 and 7). Segment of DNase I protection is indicated (arrow). B, DNA sequencing of the protected region revealed a NF-1 half-site which is schematically represented.

 
To further assess the relationship between the NF-1 element and PRL stimulation in Nb2–11 cells, the promoter element identified in the DNase I protection experiments was subjected to gel mobility shift analysis. Incubation of a [32P]NF-1 oligonucleotide with lysates obtained from quiescent cells markedly shifted its migration (Fig. 8Go). However, addition of proteins from PRL-treated Nb2–11 cells caused a time-dependent attenuation of NF-1 oligonucleotide migration that reached maximally reduced levels within 2–4 h. Protein binding was specific since it was competitively inhibited by the addition of excess unlabeled NF-1, while an irrelevant oligonucleotide (SP-1) did not reduce protein binding to the NF-1-containing element. Notably, addition of an oligonucleotide containing an AP-2 recognition element also reduced NF-1 binding, suggesting that this or a related transcription factor may also regulate the pim-1 promoter.



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  		Figure 8. Gel mobility shift of NF-1 site in PRL-stimulated Nb2–11 cells. Quiescent Nb2–11 cells were stimulated with PRL (20 ng/ml) and harvested at the times indicated (h). Nuclear proteins were incubated with a 32P-labeled NF-1 oligonucleotide and resolved by SDS-PAGE as described in Materials and Methods. Specificity of protein binding to the NF-1 oligonucleotide was determined by including: 1) 112 or 2) 224 ng of unlabeled NF-1 (L) or (H), respectively; or 3) 150 ng each of SP-1 or AP-2 oligonucleotides in the incubation mixture. A representative autoradiograph of an experiment replicated three times is presented.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previously, we demonstrated that PRL treatment of lactogen-dependent Nb2–11 cells resulted in a rapid and substantial increase in pim-1 mRNA expression and protein translation (31). The results from other studies demonstrated that this effect of PRL on pim-1 correlated with the capability of the hormone to suppress apoptosis in cells deprived of growth factor or treated with glucocorticoids (2, 33). In this report, we demonstrate that PRL stimulates pim-1 transcription by a mechanism that involves at least two distinct promoter elements, the DE and PE. Moreover, evidence is presented that suggests that PRL-provoked transcription of pim-1 may also reflect derepression of an NF-1-like motif located between these elements. Notably, while the 1,268-bp promoter segment investigated contains several GAS and GAS-like elements, each with the potential to bind Stat family transcription factors, the results presented herein suggest that alternative PRL-stimulated pathways can regulate pim-1 transcription.

The regulation of pim-1 expression in several hematopoietic cell lines was previously investigated (50). Using B-, T-, and myeloid-lineage cell lines, two functional promoter regions, the PE and DE that appeared to regulate constitutive pim-1 expression were identified (50). However, while transfection of FDC/Nb2 cells with promoter constructs containing these elements did not produce appreciable constitutive expression, their elimination markedly reduced PRL-stimulated promoter activity. Notably, the transcription factors, SP-1 and AP-2, which were suggested by Meeker et al. (50) to regulate pim-1 expression by binding to the PE and AP-2 together with a novel factor, Pim-1 promoter factor-348, appeared to bind to the DE in their study. The results presented here suggest that the PE and DE may share DNA binding factors; specific binding to each was inhibited when the other sequence was included as a competitor in gel mobility shift experiments. Moreover, PRL has been previously reported to activate SP-1 binding to DNA in Nb2 cells (51). However, addition of excess SP-1 or AP-2 oligonucleotides did not reduce protein binding to either the DE or PE, suggesting that additional transcription factors may be shared between the two promoter elements.

In addition to the DE and PE, the results suggest that pim-1 gene expression may also be regulated by silencing of its promoter. DNase I analysis of the -427 to +37 bp segment revealed an area of protection from -227 to -216 bp when it was incubated with extracts from unstimulated cells that was susceptible to enzymatic digestion upon exposure to extracts obtained from PRL-treated cultures. Moreover, the promoter construct that lacked this sequence (Fig. 2Go, -699 to -279 bp) yielded the highest level of PRL-stimulated CAT activity observed when it was introduced into FDC/Nb2 cells. Sequence analysis of the protected site revealed a consensus NF-1 half-site motif. Further evaluation by gel mobility shift analysis demonstrated a time-dependent decrease in protein binding to this motif after exposure to PRL. These observations suggest that in quiescent Nb2 cells, a NF-1-like factor, bound to the pim-1 promoter, may silence its transcription similar to its repressive effects on myelin basic protein (52), rat GH (53), von Willebrand factor (54), and glutathione transferase P (55) genes. Treatment of the cultures with PRL may relieve NF-1-mediated repression contributing to enhanced pim-1 transcription.

Recent evidence has indicated that a primary signaling mechanism coupled to PRLR-regulated gene expression is the Jak/Stat pathway. Thus, PRL induced expression of several responsive genes in Nb2 cells, including IRF-1, reflects upstream tyrosyl phosphorylation of Jak2 and activation of Stat transcription factors that mediate gene expression by binding to GAS promoter elements (18). In addition, others have provided evidence suggesting that this mechanism may regulate pim-1 expression in several hematopoietic systems. For example, interferon-{gamma} caused tyrosyl phosphorylation of Stat 1{alpha} and its binding to a GAS sequence contained within the pim-1 promoter in the growth factor-dependent myeloid cell line, MO7e (36). In an immature mast cell line, B6M, IL-3, which activates the Jak2/Stat 5 (56, 57) pathway as well as Ras/MAPK (58), provoked pim-1 mRNA expression (59). Finally, transfection of CD16/Jak2 fusion proteins that produce constitutive activation of the kinase increased expression of bcl-2 as well as pim-1 in IL-3-dependent Ba/F3 cells (37). Therefore, a plausible hypothesis to explain the action of PRL on pim-1 gene expression included activation of Jak/Stat, resulting in subsequent transcriptional activation of this protooncogene.

Examination of the proximal 1,700-bp segment of the pim-1 promoter (50) revealed five GAS or GAS-like elements each with potential to interact with Stat transcription factors. However, systematic elimination of each of these sequences did not reduce promoter activation by PRL in CAT transfection experiments, suggesting that these elements are not required for hormone-mediated transcriptional activation. Further support for this supposition was provided by gel mobility shift results, which indicated that one GAS-related element failed to bind additional protein upon PRL stimulation. While at least one GAS element (-1,435 to -1,424 bp) was absent from the constructs transfected into FDC/Nb2 cells, PRL-stimulated cells exhibited levels of CAT activity significantly elevated above controls, indicating its sensitivity to hormonal activation. It is possible that additional Stat factor recognition elements may be present upstream of the promoter sequence investigated in the present study; however, the results presented here suggest that other transcription factors are most likely required for PRL-stimulated pim-1 gene expression.

A second signaling pathway activated as a consequence of PRLR stimulation is the Ras/MAPK cascade. We and others have shown that PRL rapidly increased Ras-GTP and MAPK activity in cultured Nb2 cells and in other hormone-responsive systems (9, 13, 14, 60, 61). In addition to these critical components, PRL has also been shown to increase membrane association of phosphorylated SHC and caused its binding to growth factor receptor bound-2 and son of sevenless (13, 60, 61), key signaling proteins leading to Ras activation, as well as to activate Raf-1 (62). Recently, we demonstrated that tyrosyl phosphorylation of the TCR-associated kinase, ZAP-70, was an early event following ligation of the PRLR in Nb2 cells and in human peripheral blood lymphocytes and thymocytes (7, 8). This observation linked signaling mechanisms coupled to the PRLR to those stimulated by TCR activation. Importantly, the adapter protein, SHC, which has recently been reported to associate with activated ZAP-70, can serve as its substrate, and thereby couple signaling events provoked by TCR stimulation to the Ras pathway (63). Therefore, since activation of the pim-1 promoter may not require Stat family transcription factors, we suggest that in Nb2 cells, PRL transmits signals leading to its transcription through ZAP-70 which, in turn, may be coupled to Ras/MAPK activation via SHC. Once activated, MAPK translocates to the nucleus (14) and phosphorylates transcription factors that activate specific elements within the DE and PE as well as derepress the NF-1 binding motif (Fig. 9Go). Future work will focus on determining the identity of the PRL-responsive trans-acting factors and their specific DNA binding motifs.



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  		Figure 9. Proposed mechanism of PRL-induced pim-1 transcription in Nb2–11 cells. Activation of the PRLR by ligation and homodimerization leads to activation of the Jak2/Stat and Ras/MAPK pathways. Activated MAPK translocates to the nucleus and phosphorylates transcription factors (TF*) that bind to the DE and PE. NF-1 is released from the 5'-pim-1 promoter to initiate transcription.

 


   Acknowledgments
 
The authors are grateful to Dr. Li-yuan Yu-Lee (Baylor University, Houston, TX) for generously providing the FDC/Nb2 cell line and IRF-1 GAS oligonucleotides.


   Footnotes
 
1 Supported by NIH Grant DK-53452, Grant 95B089 from the American Institute for Cancer Research, and Grant 91–37206-6867 from the US Department of Agriculture. Back

Received May 20, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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