Overexpression of RGPR-p117 Enhances Regucalcin Gene Promoter Activity in Cloned Normal Rat Kidney Proximal Tubular Epithelial Cells: Involvement of TTGGC Motif

Abstract A novel protein RGPR-p117 was discovered as regucalcin gene promoter region-related protein that binds to the TTGGC motif using a yeast one-hybrid system. RGPR-p117 is localized in the nucleus of kidney cells, and overexpression of RGPR-p117 can modulate regucalcin protein and its mRNA expression in the cloned normal rat kidney proximal tubular epithelial NRK52E cells. This study was undertaken to determine whether overexpression of RGPR-p117 enhances the regucalcin promoter activity using the —710/+18 LUC construct (wild-type) or —710/+18 LUC construct (mutant) with deletion of —523/—435 including TTGGC motif. NRK52E cells (wild-type) or stable HA-RGPR-p117/ phCMV2-transfected cells (transfectant) were cultured in Dulbecco’s minimum essential medium (DMEM) containing 5% bovine serum (BS). Wild-type cells or transfectants were transfected with the —710/+18 LUC construct vector or the —710/ +18 LUC construct with deletion of —523/—435. Wild-type cells or transfectants with subconfluency were cultured for 48 h in a DMEM medium containing either vehicle, BS (5%), or parathyroid hormone (1–34) (PTH; 10—7 M). Luciferase activity in wild-type cells was significantly increased with culture of BS or PTH. This increase was significantly blocked in the presence of various protein kinase inhibitors (staurosporine and PD 98059). Luciferase activity in transfectants was significantly increased as compared with that of wild-type cells in the absence of BS or PTH. The increase in luciferase activity in transfectants was completely decreased in mutant with deletion of —523/—435 sequence of regucalcin promoter. This was also seen using the —710/+18 LUC construct with deletion of —523/—503 sequence containing TTGGC motif. The increase in luciferase activity in transfectants was not significantly enhanced with culture of BS (5%), PTH (10—7 M), Bay K 8644 (10—6 M), phorbol 12-myristate 13-acetate (PMA; 10—6 M), or N6, 2′-dibutyryl cyclic adenosine 3′, 5′-monophosphate (DcAMP; 10—4 M). The increase in luciferase activity in transfectants was completely inhibited with culture of dibucaine (10—6 M), staurosporine (10—9 M), PD 98059 (10—8 M), wortmannin (10—8 M), genistein (10—6 M), vanadate (10—6 M), or okadaic acid (10—6 M) which are inhibitors of various kinases and protein phosphatases. This study demonstrates that RGPR-p117 can enhance the regucalcin promoter activity which is related to the NF-1 consensus sequences including TTGGC motif, and that its enhancing effect is partly mediated through phosphorylation and dephosphorylation in NRK52E cells. J. Cell. Biochem. 99: 589–597, 2006. © 2006 Wiley-Liss, Inc.

Key words: RGPR-p117; regucalcin gene promoter region-related protein; regucalcin; transcription factor; normal rat kidney proximal tubular epithelial cells (NRK52E)

RGPR-p117 was discovered as a regucalcin gene promoter region-related protein [Misawa and Yamaguchi, 2001]. Regucalcin has been demonstrated to play a multifunctional role as regulatory protein in the intracellular signaling system of many cell types [Yamaguchi, 2000a,b, 2005; in review]. Regucalcin gene expression has been regulated by various signaling factors [Murata and Yamaguchi, 1998, 1999; Misawa and Yamaguchi, 2000]. The nuclear factor binding site, which contains a nuclear factor I(NF-1)-like motif, TTGGC(N)6CC, is present in the promoter region of the rat regucalcin gene [Misawa and Yamaguchi, 2000, 2002a]. An unknown protein that binds to the TTGGC sequence in the promoter region of the rat regucalcin gene was identified using a yeast one-hybrid system [Misawa and Yamaguchi, 2001]. The protein was termed as a regucalcin promoter region-related protein (RGPR-p117) [Misawa and Yamaguchi, 2001].

RGPR-p117 is conserved in various verte- brate species, including human, rat, mouse, bovine, rabbit, and chicken [Misawa and Yama- guchi, 2001; Sawada and Yamaguchi, 2005a], indicating a great conservation of RGPR-p117 genes throughout evolution. The entire human RGPR-p117 cDNA consists of 3,989 bp, which contains an open reading frame (ORF) of 3,180 bp, encoding a protein of 1,060 amino-acid residues [Misawa and Yamaguchi, 2001]. A comparison of human RGPR-p117 sequence with the genomic sequence database indicates that the gene consists of at least 26 exons, spanning ~41 kb, and localized on human chromosome 1q25.2 [Misawa and Yamaguchi, 2001]. RGPR-p117 mRNA is ubiquitously expressed in many tissues including liver, kidney, heart, and brain of rats [Misawa and Yamaguchi, 2002b], and stably expressed for the physiologic change in rat liver [Yamaguchi et al., 2003].

RGPR-p117 is suggested to play a role in nuclear function. RGPR-p117 has been found to localize in the cytoplasm and nucleus with immunocytochemical and Western blot analysis using HA-RGPR-p117/phCMV2-trans- fected normal rat kidney proximal tubular epithelial NRK52E cells [Sawada et al., 2005]. Overexpression of RGPR-p117 enhances the expression of regucalcin mRNA and its protein level in NRK52E cells [Sawada and Yamaguchi, 2005b]. RGPR-p117 may play a role as a transcriptional factor that is related to TTGGC motif.

This study was undertaken to determine whether overexpression of RGPR-p117 en- hances the regucalcin promoter activity using the —710/+18 LUC construct (including TTG- GC sequences) in NRK52E cells and HA-RG- PR-p117/phCMV2-transfected NRK52E cells. We found that RGPR-p117 can enhance the regucalcin promoter activity that is involved in the NF-1 consensus sequences including TTGGC motif in NRK52E cells.

MATERIALS AND METHODS
Chemicals

Non-essential amino acid solution, bovine serum (BS), and penicillin-streptomycin solu- tion (5,000 U/ml penicillin; 5,000 mg/ml strepto- mycin) were obtained from Gibco Laboratories (Grand Island, NY). Dulbeco’s modified Eagle’s medium (DMEM), staurosporine, PD98059, di- bucaine, Bay K 8644, PTH [synthetic human PTH (1–34)], aldosterone, DcAMP (N6, 2′-dibutyryl cyclic adenosine 3′, 5′ monophosphate), phorbol 12-myristate 13-acetate (PMA), wortmannin, genistein, and okadaic acid were obtained from Sigma Chemical (St. Louis, MO). Vanadate and other chemicals were purchased from Wako Pure Chemical Co. (Osaka, Japan). pGL3-basic vector and pRL-TK vector were purchased from Promega (Madison, WI). Reagents used were dissolved in distilled water or ethanol, and some reagents were passed through ion exchange resin to remove metal ions.

Cell Culture

The cloned normal rat kidney proximal tubular epithelial cells (NRK52E; 1 × 105 cells) [Rice et al., 2003] were maintained in DMEM supplemented with 5% BS, non-essential amino acids solution, 50 U/ml penicillin, and 50 mg/ml streptomycin in humidified 5% CO2/95% air at 378C to obtain subconfluent monolayers.

Selection of Stable Transfectant

Expression plasmid for HA-tag fused rat RGPR-p117, HA-RGPR-p117/phCMV2 was
constructed as described previously [Sawada et al., 2005]. NRK52E cells were grown on 35- mm dishes to approximately 70% confluence and transfected with HA-RGPR-p117/phCMV2 vector using the synthetic cationic lipid compo- nents, a Lipofectamine reagent (Invitrogen), according to the manufacturer’s instructions. At 48 h after transfection, neomycin (0.8 mg/ml Geneticin G418, Sigma) was added to culture medium for selection and transfected cells were plated at limiting dilution. Multiple surviving clones were isolated, transferred to 35-mm dishes, and grown in the medium without neomycin. HA-RGPR-p117 was stably expre- ssed in the transfectants. In experiments, transfectants were cultured for 72 h in DMEM containing 5% BS.

RT-PCR Analysis

Total RNAs were prepared using Trizol regent (Invitrogen), according to the manufac- turer’s instruction [Chomczynski and Sacci, 1987]. RT-PCR was performed with a TitanTM One-Tube RT-PCR kit (Roche Molecular Bio- chemicals) as recommended by the supplier. Primers for amplification of rat regucalcin cDNA [Shimokawa and Yamaguchi, 1993] were: 5′-GGAGGCTATGTTGCCACCATTGGA
-3′ (sense strand, position 292–314 of cDNA sequence) and 5′-CCCTCCAAAGCAGCATGA AGTTG-3′ (antisense strand, position 849–827). For semiquantative PCR, glyceraldehyde-3- phosphate dehydrogenase (G3PDH) was used as an internal control to evaluate total RNA input. Primers for amplification of G3PDH were: 5′-GATTTGGCCGTATCGGACGC-3′ (sense strand) and 5′-CTCCTTGGAGGCCAT- GTAGG-3′ (antisense strand). The pair oligo- nucleotide primers were designed to amplify a 977 bp sequence from the mRNA of G3PDH. RT- PCR was performed using reaction mixture (20 ml) containing 2 mg of total RNAs, supplied RT-PCR buffer, TitanTM enzyme mixture (AMV and Expand High Fidelity), 0.2 mM dNTP, 5 mM DTT, 5U RNase inhibitor, and 0.3 mM primers. Samples were incubated at 508C for 30 min, and then amplified for 35 cycles under the following conditions; denaturation for 30 sec at 948C, annealing for 30 sec at 60, and extension for 60 sec for 688C. The amplified products were separated by electrophoresis on 1.5% agarose gel and visualized by ethidium bromide staining.

Construction of the Reporter Gene Plasmids

The reporter gene plasmids were generated with subcloning of restriction fragment from
—710/+18 TA vector [Misawa and Yamaguchi, 2000]. The DNA fragment —710/+18 of the rat regucalcin gene was prepared from a vector by KpnI/XhoI restriction digestion, and then cloned into the pGL3-basic promoterless plasmid containing the firefly luciferase gene [Murata and Yamaguchi, 1998]. This wild-type promoter construct was named the —710/+18 LUC (WT). The DNA fragment (—710/—523) was amplified by two primers 5′-AGGTACC- GAATTCCTGACTGATCTTT-3′ and 5′AATGA GCTCCAGGCAAAGAACATTCTAT-3′, anddigested by EcoRI and Sac I, and the fragment was ligated into EcoRI and Sac I site of —710/ +18 LUC (WT), and its construct was named Mut 1. The DNA fragment (—710/—523) amplified by 5′-AGGTACCGAATTCCTGACT- GATCTTT-3′ and 5′-ATGGATCCCACAGG-
CAAAGAACA-3′ was digested by EcoRI and BamHI. The DNA fragment (—505/+18) ampli- fied by 5′-AGGATCCTCATGCAAGGAAGCA- AG-3′ and 5′-ACACTCGAGGGTTGTAATGACTCCTGGC-3′ was digested by BamHI and XhoI. These two DNA fragments were ligated into EcoRI and XhoI site of —710/+18 LUC (WT), and the plasmid was named Mut 2. The DNA fragment (—710/—505) amplified by 5′- AGGTACCGAATTCCTGACTGATCTTT-3′ and 5′-ATGAGCTCGTTTGGCAGGGAGCCTTG-3′ was digested by EcoRI and SacI. The fragment was ligated into EcoRI and SacI site of —710/+18 LUC (WT), and the plasmid was named Mut 3.

Cell Culture and Transfection

The cloned normal rat kidney proximal tubular epithelial NRK52E cells were cultured as previously described [Nakagawa and Yama- guchi, 2005]. For the transfection experiments, the cells were grown on 24-well plate to approximately 70% confluence and washed once with serum-free DMEM. Either 300 ng of pGL3- basic plasmid or an equivalent molar amount of test plasmid was co-transfected into NRK52E cells along with 300 ng of pRL-TK plasmid using the synthetic cationic lipid component, a lipo- fectamin reagent, according to the manufac- turer’s instructions (Invitorogen). The pRL-TK vector containing the Renilla luciferase gene under control of the herpes simplex virus thymidine kinase promoter was used as an internal control for differences in transfection efficiency and cell number. For analysis of regulation of the regucalcin promoter by signal- ing factors, the transfected cells were main- tained for 48 h in serum-free DMEM containing either vehicle, 5% BS, hormones, or various inhibitors. After culture, the transfectants were lysed, and the luciferase activity in the cell lysates was measured using dual-luciferase reporter assay system (Promega).

Statistical Analysis

The significance of difference between values was estimated by Student’s t test. A P-value of less than 0.05 was considered significant. Also, we used a multiway ANOVA multiple compar- ison to compare the treatment groups.

RESULTS

Effects of Hormones on Regucalcin mRNA Expression in NRK52E Cells
NRK52E cells (wild-type) or stably HA- RGPR-p117/phCMV2-transfected cells (trans- fectants) were cultured for 72 h in medium with BS (5%), and the cells with subconfluency were cultured for 24 h in the presence of BS (5%). The expression of regucalcin mRNA was sig- nificantly increased in transfectants as com- pared with that of wild-type cells (Fig. 1A), indicating that overexpression of RGPR-p117 enhances regucalcin mRNA, and its protein expressions in NRK52E cells as shown pre- viously [Sawada and Yamaguchi, 2005b]. When wild-type cells or transfectants with subcon- fluency were cultured for 24 or 48 h in a medium containing either vehicle, PTH (10—7 M), aldosterone (10—5 M), or PMA (10—6 M) in the absence of BS, regucalcin mRNA expression in wild-type cells (Fig. 1B), or transfectants (Fig. 1C) was significantly increased in the presence of PTH or PMA. G3PDH mRNA levels were not chang- ed in transfectants (data not shown).

Involvement of the NF-1 Consensus Sequences Including TTGGC Motif in Regucalcin Promoter in NRK52E Cells Overexpressing RGPR-p117

To clarify whether the NF-1 consensus sequences including TTGGC motif in the reg- ucalcin promoter region is involved in the enhancement of regucalcin mRNA expression in NRK52E cells overexpressing RGPR-p117, NRK52E cells wild-type, or stable transfectants with subconfluency were transfected with either —710/+18 LUC construct (WT) or chi- meric constructs containing serial deletions [—523/—435 (mutant 1), —523/—503 (mutant 2), or —504/—435 (mutant 3)] of the regucalcin promoter region (Fig. 2A). The cells were cultured for 48 h in medium without BS. Luciferase activity in NRK52E cells in stable NRK52E cells overexpressing RGPR-p117 (transfectants) was significantly increased as compared with that of wild-type cells with the
—710/+18 LUC construct (Fig. 2B). Luciferase activity in the mutant 1, 2, or 3 was not significantly changed as compared with that of wild-type cells with the —710/+18 construct. In transfectants overexpressing RGPR-p117, how- ever, mutant 1 or 2 was unresponsive. The enhancement of luciferase activity in the trans- fectants overexpressing RGPR-p117 was seen in mutant 3. These results indicate that the nucleotide —523/—503 containing TTGGC motif in the regucalcin promoter region are essential in the enhancement of regucalcn promoter activity in NRK52E cells overexpressing RGPR-p117.

Effects of Signaling Factors on Regucalcin Promoter Activity in NRK52E Cells Overexpressing RGPR-p117

The effects of signaling factors on the reg- ucalcin promoter activity in NRK52E cells (wild-type) or stable RGPR-p117-overexpres- sing transfectants transfected with the —710/ +18 LUC construct (WT) or the —523/—435 deletion (Mut 1) was examined. Wild-type cells or transfectants with subconfluency were cul- tured for 48 h in a medium containing either vehicle, BS (5%), PTH (10—7 M), Bay K 8644 (10—6 M), PMA (10—6 M), or DcAMP (10—4 M). In wild-type cells, culture with BS or PTH caused a significant increase in luciferase activity in the cells transfected with the —710/+18 LUC con- struct (WT) (Fig. 3). This increase was not seen in Mut 1. The presence of PMA caused a significant increase in luciferase activity in wild-type cells transfected with the —710/+18 LUC construct (Fig. 4). Such an increase was not seen in the presence of Bay K 8644 or DcAMP. Luciferase activity in transfectants overexpressing RGPR-p117 was not signifi- cantly enhanced with culture of BS, PTH, Bay K 8644, PMA, or DcAMP (Figs. 3,4). The enzyme activity in transfectants in the presence or absence of various signaling factors was not enhanced in the Mut 1 (Figs. 3,4).

It is speculated that the effect of RGPR-p117 in enhancing the regucalcin promoter activity, which is related to NF-1 consensus sequences, may be mediated through the recruitment of phosphorylation and dephosphorylation, which is regulated by intracellular signaling factor in NRK52E cells.

In conclusion, it has been demonstrated that overexpression of RGPR-p117 can enhance the regucalcin promoter activity which is related to the NF-1 consensus sequences including TTGGC motif in the cloned normal rat kidney proximal tubular epithelial NRK52E cells.