Proximal Tubular Epithelial NRK52E Cells Research Articles

The overexpressed transcription factor RGPR-p117 suppresses the proliferation of normal rat kidney proximal tubular epithelial NRK-52E cells: Involvement of diverse signaling pathways

AimsRGPR-p117 was originally discovered as a novel transcription factor, which specifically binds to a nuclear factor I (NFI) consensus motif TTGGC(N)6CC in the promoter region of the regucalcin gene. RGPR-p117 is also called as Lztr2 and SEC16B. The role of RGPR-p117 in cell regulation is poorly understood. This study was undertaken to determine whether the overexpression of RGPR-p117 impacts the proliferation of normal rat kidney proximal tubular epithelial NRK-52E cells in vitro. Main methodsThe NRK-52E wild-type cells and RGPR-p117-overexpressing NRK-52E cells were cultured in DMEM containing fetal bovine serum. Key findingsThe overexpression of RGPR-p117 repressed colony formation and proliferation of NRK-52E cells. Interestingly, RGPR-p117 overexpression blocked cell proliferation promoted by culturing with Bay K 8644, a calcium-entry agonist, and phorbol 12-myristate 13-acetate, an activator of protein kinase C. The depressive effects of RGPR-p117 overexpression on cell proliferation were not occurred by culturing with various inhibitors of cell cycle and intracellular signaling processes. RGPR-p117 overexpression increased the translocation of RGPR-p117 into the nucleus of NRK-52E cells. Mechanistically, RGPR-p117 overexpression diminished the levels of Ras, PI3 kinase, Akt, mitogen-activated protein kinase, and mTOR, while it raised the levels of p53, Rb, p21, and regucalcin. Furthermore, RGPR-p117 overexpression protected cell death caused by apoptosis-inducing factors, suggesting that the suppressive effects of RGPR-p117 on cell growth are independent of cell death. SignificanceThe present study demonstrates that the overexpressed transcription factor RGPR-p117 suppresses cell proliferation via targeting diverse signaling processes, suggesting a role of RGPR-p117 in cell regulation.

Induction of Autophagy by Pterostilbene Contributes to the Prevention of Renal Fibrosis via Attenuating NLRP3 Inflammasome Activation and Epithelial-Mesenchymal Transition.

Chronic kidney disease (CKD) is recognized as a global public health problem. NLRP3 inflammasome activation has been characterized to mediate diverse aspect mechanisms of CKD through regulation of proinflammatory cytokines, tubulointerstitial injury, glomerular diseases, renal inflammation, and fibrosis pathways. Autophagy is a characterized negative regulation mechanism in the regulation of the NLRP3 inflammasome, which is now recognized as the key regulator in the pathogenesis of inflammation and fibrosis in CKD. Thus, autophagy is undoubtedly an attractive target for developing new renal protective treatments of kidney disease via its potential effects in regulation of inflammasome. However, there is no clinical useful agent targeting the autophagy pathway for patients with renal diseases. Pterostilbene (PT, trans-3,5-dimethoxy-4-hydroxystilbene) is a natural analog of resveratrol that has various health benefits including autophagy inducing effects. Accordingly, we aim to investigate underlying mechanisms of preventive and therapeutic effects of PT by reducing NLRP3 inflammasome activation and fibrosis through autophagy-inducing effects. The renal protective effects of PT were evaluated by potassium oxonate (PO)-induced hyperuricemia and high adenine diet-induced CKD models. The autophagy induction mechanisms and anti-fibrosis effects of PT by down-regulation of NLRP3 inflammasome are investigated by using immortalized rat kidney proximal tubular epithelial NRK-52E cells. To determine the role of autophagy induction in the alleviating of NLRP3 inflammasome activation and epithelial-mesenchymal transition (EMT), NRK-52E with Atg5 knockdown [NRK-Atg5-(2)] cells were applied in the study. The results indicated that PT significantly reduces serum uric acid levels, liver xanthine oxidase activity, collagen accumulation, macrophage recruitment, and renal fibrosis in CKD models. At the molecular levels, pretreatment with PT downregulating TGF-β-triggered NLRP3 inflammasome activation, and subsequent EMT in NRK-52E cells. After blockage of autophagy by treatment of Atg5 shRNA, PT loss of its ability to prevent NLRP3 inflammasome activation and EMT. Taken together, we suggested the renal protective effects of PT in urate nephropathy and proved that PT induces autophagy leading to restraining TGF-β-mediated NLRP3 inflammasome activation and EMT. This study is also the first one to provide a clinical potential application of PT for a better management of CKD through its autophagy inducing effects.

MiR-378 reduces mesangial hypertrophy and kidney tubular fibrosis via MAPK signalling.

The regulatory role of a novel miRNA, miR-378, was determined in the development of fibrosis through repression of the MAPK1 pathway, miR-378 and fibrotic gene expression was examined in streptozotocin (STZ)-induced diabetic mice at 18weeks or in unilateral ureteral obstruction (UUO) mice at 7days. miR-378 transfection of proximal tubular epithelial cells, NRK52E and mesangial cells was assessed with/without endogenous miR-378 knockdown using the locked nucleic acid (LNA) inhibitor. NRK52E cells were co-transfected with the mothers against decapentaplegic homolog 3 (SMAD3) CAGA reporter and miR-378 in the presence of transforming growth factor-β (TGF-β1) was assessed. Quantitative polymerase chain reaction (qPCR) showed a significant reduction in miR-378 (P<0.05) corresponding with up-regulated typeI collagen, typeIV collagen and α-smooth muscle actin (SMA) in kidneys of STZ or UUO mice, compared with controls. TGF-β1 significantly increased mRNA expression of typeI collagen (P<0.05), typeIV collagen (P<0.05) and α-SMA (P<0.05) in NRK52E cells, which was significantly reduced (P<0.05) following miR-378 transfection and reversed following addition of the LNA inhibitor of endogenous miR-378 Overexpression of miR-378 inhibited mesangial cell expansion and proliferation in response to TGF-β1, with LNA-miR-378 transfection reversing this protective effect, associated with cell morphological alterations. The protective function of MAPK1 on miR-378 was shown in kidney cells treated with the MAPK1 inhibitor, selumetinib, which inhibited mesangial cell hypertrophy in response to TGF-β1. Taken together, these results suggest that miR-378 acts via regulation of the MAPK1 pathway. These studies demonstrate the protective function of MAPK1, regulated by miR-378, in the induction of kidney cell fibrosis and mesangial hypertrophy.

Novel Protein RGPR-p117: New Aspects in Cell Regulation

RGPR-p117 was initially discovered as novel protein which binds to the nuclear factor I (NF1)-like motif TTGGC(N)6CC in the regucalcin gene promoter region (RGPR). RGPR-p117 is localized to the nucleus with stimulation of protein kinase C-related signaling process. Overexpression of RGPR-p117 has been shown to enhance regucalcin mRNA expression in the cloned normal rat kidney proximal tubularepithelial NRK52E cells in vitro. This process is mediated through phosphorylated RGPR-p117. Overexpression of RGPR-p117 was found to suppress apoptotic cell death induced after stimulation with various signaling factors in NRK52E cells, while it did not have an effect on cell proliferation. Moreover, RGPR-p117 was found to localize in the plasma membranes, mitochondria and microsomes, suggesting an involvement in the regulation of function of these organelles. After that, RGPR-p117 was renamed as Sec16B that is involved in the endoplasmic reticulum export. However, this is not suitable name with many findings of the role of RGPR-p117 in cell regulation. RGPR-p117 may play an essential role as transcription factor, and the elucidation of other roles in cell regulation will be expected.

Nuclear localization of regucalcin is enhanced in culture with protein kinase C activation in cloned normal rat kidney proximal tubular epithelial NRK52E cells

In this study we investigated whether the nuclear localization of regucalcin in cloned normal rat kidney tubular epithelial NRK52E cells is regulated after culture with hormonal signaling factors. Stable regucalcin/pCXN2 transfectants with subconfluent monolayers were further cultured for 24 or 48 h in a serum-free medium containing either vehicle, tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta1 (TGF-beta1), parathyroid hormone (PTH), phorbol 12-myristate 13-acetate (PMA), or other factors. Culture with TNF-alpha (1.0 ng/ml of medium) or TGF-beta1 (5.0 ng/ml) for 48 h caused a significant decrease in regucalcin mRNA levels in NRK52E cells (wild-type), while regucalcin mRNA levels were markedly increased in the presence of PMA (10(-6) M), an activator of protein kinase C, in wild-type cells. Immunocytochemical observation showed that HA-regucalcin was markedly localized in the nucleus of HA-regucalcin/ phCMV2-transfected cells. The nuclear localization was enhanced in culture with BS (5%), PTH (10(-7) M), Bay K 8644 (2.5x10(-6) M), or PMA (10(-6) M) for 24 or 48 h. Culture with staurosporine, an inhibitor of protein kinase C, caused a remarkable decrease in the localization of HA-regucalcin in the nucleus of HA-RGPR-p117/phCMV2-transfected cells with PMA. Culture with PMA (10(-6) M) for 24 or 48 h caused a remarkable increase in nuclear regucalcin protein levels. The effect of PMA in increasing nuclear regucalcin levels was completely absent in culture with staurosporine (10(-8) M). The nuclear localization of regucalcin in the stable regucalcin/pCXN2-transfected cells (transfectant) increased markedly as compared with that of wild-type cells, whereas the increase was less evident in the transfectants cultured with staurosporine. This study demonstrated that regucalcin localizes in the nucleus of cloned normal rat kidney proximal tubular epithelial NRK52E cells, and that its nuclear localization is enhanced through an intracellular signaling process which involves protein kinase C.

Overexpression of RGPR-p117 suppresses apoptotic cell death and its related gene expression in cloned normal rat kidney proximal tubular epithelial NRK52E cells

The novel protein RGPR-p117 was discovered as a regucalcin gene promoter region-related protein that binds to the TTGGC motif using a yeast one-hybrid system. The role of RGPR-p117 in cell function has not been fully clarified. This study was undertaken to determine whether overexpression of RGPR-p117 regulates various types of signaling factor-induced apoptotic cell death in the cloned normal rat kidney proximal tubular epithelial NRK52E cells. NRK52E cells (wild-type) or stable RGPR-p117/phCMV2-transfected cells (transfectant) were cultured in Dulbecco's modified Eagle's medium containing 5% bovine serum (BS). NRK52E cells with subconfluent monolayers were cultured for 24-72 h in a medium without BS. The presence of tumor necrosis factor-alpha (TNF-alpha; 1.0 or 10 ng/ml of medium), lipopolysaccharide (LPS; 0.1 or 1.0 microg/ml), Bay K 8644 (10(-6) or 10(-5) M), or thapsigargin (10(-8) or 10(-7) M) caused a significant decrease in the number of NRK52E wild-type cells or phCMV2-transfected (mock-type) cells. The effect of TNF-alpha, LPS, Bay K 8644, or thapsigargin in decreasing cell number was significantly suppressed in the presence of the caspase-3 inhibitor (10(-8) M) in wild-type cells cultured for 48 h. The effect of TNF-alpha, LPS, or Bay K 8644 in decreasing cell number was significantly inhibited in the transfectants, while the effect of thapsigargin on cell death was not inhibited in the transfectants. Culture with TNF-alpha or LPS caused DNA fragmentation in wild-type cells. These effects were significantly suppressed in the transfectants. The result of reverse transcription-polymerase chain reaction analysis using specific primers for the genes of apoptotic cell death-related proteins showed that IAP-1, FADD, caspase-8, caspase-9, and caspase-3 mRNA levels were significantly decreased in the transfectants, while Akt-1, Bid, Apaf-1, and glyceroaldehyde-3-phosphate dehydrogenase mRNA levels were not significantly altered in the transfectants. Culture with TNF-alpha, LPS, Bay K 8644, or thapsigargin caused a significant increase in Apaf-1 or caspase-3 mRNA levels. Such an effect was not seen in the transfectants. This study demonstrates that overexpression of RGPR-p117 has a suppressive effect on cell death and apoptosis induced by TNF-alpha, LPS, or Bay K 8644 whose actions are mediated through intracellular signaling pathways. This study also demonstrates that RGPR-p117 regulates the gene expression of apoptosis-related proteins.

Overexpression of RGPR-p117 induces the decrease in protein and DNA contents in cloned normal rat kidney proximal tubular epithelial NRK52E cells

A novel protein RGPR-p117 was discovered as a regucalcin gene promoter region-related protein that binds to the TTGGC motif. Regucalcin is known to regulate the intracellular signaling system in many cell types. RGPR-p117 has been shown to enhance the promoter activity of the regucalcin gene in cloned normal rat kidney proximal tubular epithelial NRK52E cells. The role of RGPR-p117 in cell function remains to be elucidated, however. This study was undertaken to determine whether overexpression of RGPR-p117 has an effect on cell proliferation, protein and DNA contents in NRK52E cells. NRK52E cells (wild-type) or stable RGPR-p117/phCMV2-transfected cells (transfectants) were cultured in Dulbecco's minimum essential medium containing 5% bovine serum (BS). RGPR-p117 was markedly expressed in the transfectants. NRK52E cells (wild-type) or transfectants were cultured for 24, 48, or 72 h in a medium containing 5% BS, and after subconfluency the cells were cultured for 24, 48, or 72 h in a medium without BS. Cell proliferation was not significantly changed in the transfectants as compared with that of wild-type cells. Protein and DNA contents in NRK52E cells were significantly decreased in the transfectants with cell proliferation in the presence of BS. When NRK52E cells with subconfluency were cultured for 24, 48, or 72 h in a medium without BS, the number of transfectant cells was not significantly changed compared with that of wild-type cells. Protein and DNA contents in NRK52E cells were significantly decreased in the transfectants cultured in a medium without BS after subconfluency. This study demonstrates that overexpression of RGPR-p117 induces the decrease in protein and DNA contents in NK52E cells, indicating its role in the regulation of cell function.

Overexpression of regucalcin suppresses cell response for tumor necrosis factor‐α or transforming growth factor‐β1 in cloned normal rat kidney proximal tubular epithelial NRK52E cells

The regulatory role of regucalcin on cell responses for tumor necrosis factor-alpha (TNF-alpha) or transforming growth factor-beta1 (TGF-beta1) was investigated using the cloned normal rat kidney proximal tubular epithelial NRK52E cells overexpressing regucalcin. NRK52E cells (wild type) and stable regucalcin (RC)/pCXN2-transfected cells (transfectant) were cultured for 72 h in a medium containing 5% bovine serum (BS) to obtain subconfluent monolayers. After culture, cells were further cultured for 24-72 h in medium without BS containing either vehicle, TNF-alpha (0.1 or 1.0 ng/ml of medium), or TGF-beta1 (1.0 or 5.0 ng/ml). Culture with TNF-alpha or TGF-beta1 caused a significant decrease in the number of wild-type cells. This decrease was significantly prevented in transfectants overexpressing regucalcin. Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent wild-type cells cultured with TNF-alpha (1.0 ng/ml) or TGF-beta1 (5.0 ng/ml). This DNA fragmentation was significantly suppressed in transfectants. TNF-alpha- or TGF-beta1-induced cell death was significantly prevented in culture with caspase-3 inhibitor (10(-8) M). Nitric oxide (NO) synthase activity in wild-type cells was significantly increased by addition of calcium chloride (10 microM) and calmodulin (5 microg/ml) into the enzyme reaction mixture. This increase was significantly suppressed in transfectants. Culture with TNF-alpha caused a significant increase in NO synthase activity in wild-type cells. The effect of TNF-alpha was not seen in transfectants. Culture with TGF-beta1 did not cause a significant increase in NO synthase activity in wild-type cells and transfectants. Culture with TNF-alpha or TGF-beta1 caused a remarkable increase in alpha-smooth muscle actin in wild-type cells. This increase was significantly prevented in transfectants. The expression of Smad 2 or NF-kappaB mRNAs was significantly increased in transfectants as compared with that of wild-type cells. Smad 3 or glyceroaldehyde-3-phosphate dehydrogenase (G3PDH) mRNA expression was not significantly changed in transfectants. NF-kappaB mRNA expression in wild-type cells was significantly increased with culture of TNF-alpha. Smad 2 mRNA expression was significantly enhanced in wild-type cells cultured with TGF-beta1. These effects of TNF-alpha or TGF-beta1 were not significantly enhanced in transfectants. This study demonstrates that overexpression of regucalcin has suppressive effects on cell responses which are mediated through intracellular signaling pathways of TNF-alpha or TGF-beta1 in kidney NRK52E cells.

Involvement of nuclear factor I-A1 in the regulation of regucalcin gene promoter activity in cloned normal rat kidney proximal tubular epithelial cells

Nuclear factor I-A1 (NF1-A1) can bind to the TTGGC motif in the rat regucalcin gene promoter region. This study was undertaken to determine whether NF1-A1 is involved in the enhancement of the rat regucalcin gene promoter activity using the -710/+18 LUC construct (wild-type) or -710/+18 LUC construct with the deletion of -523/-435 including the TTGGC motif (mutant) in cloned normal rat kidney proximal tubular epithelial NRK52E cells. Cells were transfected with the -710/+18 LUC construct vector or the -710/+18 LUC construct with the deletion of -523/-435. NRK52E cells (wild-type) or NRK52E cells transiently transfected with HA-NF1-A1/phCMV2 were cultured for 48 h in a medium containing either vehicle or BS (5%) in the presence or absence of various factors. HA-NF1-A1 was localized in the nucleus of wild-type cells. Luciferase activity was significantly increased as compared to that of wild-type cells. This increase was significantly enhanced in the presence of phorbol 12-myristate 13-acetate (PMA; 10(-6) M). Such an enhancement was not seen by culture with Bay K 8644 (10(-6) M) or dibutyryl cyclic AMP (10(-4) M). The increase in luciferase activity in NRK52E cells transfected with HA-NF1-A1 was not observed in the presence of dibucaine (10(-6) M), staurosporine (10(-9) M), or PD 98059 (10(-8) M), which is an inhibitor of various protein kinases. Such an inhibition was also seen in the presence of vanadate (10(-6) M) or okadaic acid (10(-6) M), an inhibitor of protein phosphatase. The increase in luciferase activity in NRK52E cells transfected with HA-NF1-A1/ phCMV2 was not seen in the mutant with deletion of -523/-435. The increase in luciferase activity in HA-NF1-A1/ phCMV2-transfected NRK52E cells was not significantly enhanced in the cells transiently co-transfected with HA-RGPR-p117/phCMV2, which could increase the regucalcin gene promoter activity using the -710/+18 LUC construct (wild-type). This study demonstrates that NF1-A1 enhances the regucalcin promoter activity which is related to the TTGGC motif, and that its enhancing effect is partly mediated through phosphorylation in NRK52E cells.

Overexpression of RGPR‐p117 enhances regucalcin gene promoter activity in cloned normal rat kidney proximal tubular epithelial cells: Involvement of TTGGC motif

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 N(6), 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.

Overexpression of regucalcin suppresses Ca2+‐sensing receptor and L‐type Ca2+ channel mRNA expression in cloned rat kidney proximal tubular NRK52E cells

The effect of regucalcin, a regulatory protein in intracellular signaling pathway [Yamaguchi M, Int J Mol Med 15:372–389, 2005 in Review], on various gene expressions was investigated using the cloned normal rat kidney proximal tubular epithelial NRK52E cells overexpressing regucalcin. NRK52E cells (wild type) and stable regucalcin /pCXN2 transfectants were cultured for 72 hours in medium containing 5% bovine serum to obtain subconfluent monolayers. After culture, cells were further cultured for 24–72 hours in serum free medium. Regucalcin was localized in the nucleus of wild type cells. This localization was markedly increased in transfectants. Overexpression of regucalcin caused a significant increase in rat outer medullary K+ channel mRNA expression, while it did not cause epithelial sodium channel, Na+-K+-ATPase, type II Na-Pi cotransporter, angiotensinogen and Na+/Ca+ exchanger mRNA expressions in transfectants. The expression of Ca2+-sensing receptor and L-type Ca2+ channel mRNA was markedly suppressed in transfectants. Thses decreases were changed in the presence of parathyroid hormone that can suppress the gene expression. This study demonstrates that regucalcin has a role in the regulation of gene expression for Ca2+-sensing receptor and L-type Ca2+ channel that is involved in intracellular Ca2+ transport in kidney cells.

Overexpression of regucalcin enhances its nuclear localization and suppressesL-type Ca2+ channel and calcium-sensing receptor mRNA expressions in cloned normal rat kidney proximal tubular epithelial NRK52E cells

The effect of regucalcin (RC), a regulatory protein in intracellular signaling pathway, on the gene expression of various mineral ion transport-related proteins was investigated using the cloned normal rat kidney proximal tubular epithelial NRK52E cells overexpressing RC. NRK52E cells (wild-type) and stable RC/pCXN2 transfectant were cultured for 72 h in medium containing 5% bovine serum (BS) to obtain subconfluent monolayers. After culture for 72 h, cells were further cultured 24-72 h in a medium containing either vehicle, aldosterone (10(-8) or 10(-7) M), or parathyroid hormone (PTH) (1-34) (10(-8) or 10(-7) M) without BS. RC was markedly localized in the nucleus of transfectants. Overexpression of RC caused a significant increase in rat outer medullary K(+) channel (ROMK) mRNA expression, while it caused a remarkable decrease in L-type Ca(2+) channel and calcium-sensing receptor (CaR) mRNA expressions. Overexpression of RC did not have an effect on epithelial sodium channel (ENaC), Na, K-ATPase (alpha-subunit), Type II Na-Pi cotransporter (NaPi-IIa), angiotensinogen, Na(+)-Ca(2+) exchanger, and glyceroaldehyde-3-phosphate dehydrogenase (G3PDH) mRNA expressions. Hormonal effect on gene expression, moreover, was examined. Culture with aldosterone (10(-8) or 10(-7) M) caused a significant increase in ENaC, Na, K-ATPase, and ROMK mRNA expressions in the wild-type cells. Those increases were weakened in the transfectants. Culture with PTH (10(-8) or 10(-7) M) significantly decreased NaPi-IIa mRNA expression in the wild-type cells. This effect was not altered in the transfectants. PTH significantly decreased angiotensinogen mRNA expression in the wild-type cells and the transfectants, while aldosterone had no effect. Culture with PTH (10(-8) or 10(-7) M) caused a significant decrease in L-type Ca(2+) channel and CaR mRNA expressions in the wild-type cells, while the hormone significantly increased Na(+)-Ca(2+) exchanger mRNA expression. The effects of PTH on L-type Ca(2+) channel, CaR, and Na(+)-Ca(2+) exchanger mRNA expressions were also seen in the transfectants. This study demonstrates that overexpression of RC caused a remarkable increase in its nuclear localization, and that it has suppressive effects on the gene expression of L-type Ca(2+) channel or CaR, which regulates intracellular Ca(2+) signaling, among various regulator proteins for mineral ions in NRK52E cells.

Overexpression of RGPR-p117 enhances regucalcin gene expression in cloned normal rat kidney proximal tubular epithelial cells

A novel protein RGPR-p117 was discovered as a regucalcin gene promoter region-related protein that binds to the TTGGC motif using a yeast one-hybrid system. Whether overexpression of RGPR-p117 can modulate gene expression in the cloned normal rat kidney proximal tubular epithelial NRK52E cells was investigated. NRK52E cells (wild-type) or HA-RGPR-p117/phCMV2-transfected NRK52E cells were cultured in Dulbecco's minimum essential medium (DMEM) containing 5% bovine serum (BS). Proliferation of NRK52E cells (wild-type) was not significantly altered by overexpression of HA-RGPR-p117. The expression of rat regucalcin, alpha-fetoprotein, albumin, glucokinase, 11beta-hydroxy-steroid dehydrogenase, phosphoenolpyruvate carboxykinase, which contains TTGGC motif in the promoter region of their genes, was seen in NRK52E cells (wild-type) by using reverse transcription-polymerase chain reaction (RT-PCR). Of these genes, regucalcin mRNA levels were significantly enhanced in transfectants. The expression of p21 or glycero-aldehyde-3-phosphate dehydrogenase mRNA was not significantly changed in transfectants. The results of Western blot analysis showed that regucalcin protein was significantly increased in transfectants. The enhancement of regucalcin mRNA expression in transfectants was significantly suppressed in the presence of staurosporine (10(-10) M), an inhibitor of protein kinase C. This enhancement was not significantly changed in the presence of dibucaine (10(-8) M), PD98059 (10(-8) M) or vanadate (10(-6) M). This study demonstrates that overexpression of RGPR-p117 enhances the expression of regucalcin mRNA and its protein level in NRK52E cells. RGPR-p117 may play a role as a transcriptional factor.

Nuclear localization of a novel protein, RGPR-p117, in cloned normal rat kidney proximal tubular epithelial cells

A novel protein RGPR-p117 was discovered as a regucalcin gene promoter region-related protein that binds to the TTGGC motif. The role of RGPR-p117 in cell function is unknown. The nuclear localization of RGPR-p117 was investigated using cloned normal rat kidney proximal tubular epithelial NRK52E cells in vitro. RGPR-p117 mRNA was expressed in NRK52E cells, and its expression was stimulated by culture with parathyroid hormone (10(-7) M) or phorbol 12-myristate (10(-6) M). RGPR-p117 was found to localize in the cytoplasm and nucleus with immunocytochemical and Western blot analysis using HA-RGPR-p117/phCMV2-transfected NRK52E cells. Overexpression of HA-RGPR-p117 was found to have a significant stimulatory effect on regucalcin mRNA expression in NRK52E cells. This study demonstrates that RGPR-p117 is localized in the nucleus of kidney cells, and may be involved in gene expression.

Overexpression of regucalcin suppresses cell proliferation of cloned normal rat kidney proximal tubular epithelial NRK52E cells.

The role of regucalcin, which is a regulatory protein in intracellular signaling pathway, in the regulation of cell proliferation was investigated by using the cloned normal rat kidney proximal tubular epithelial NRK52E cells over-expressing regucalcin. NRK52E cells were transfected with regucalcin (RC) pCXN2 vector and the multiple neomycin-resistant clones that stably overexpress regucalcin were selected. The regucalcin content of RC/pCXN2-transfected cells used in this study was about 21-fold as compared with that of the parental wild-type NRK52E cells. Wild-type NRK52E cells, pCXN2 vector-transfected cells (mock-type), and RC/pCXN2-transfected cells (transfectants) were cultured for 24, 48, and 72 h in the presence of bovine serum (5%). The cell numbers of wild- and mock-type were significantly increased with the time course of the culture. Cell numbers of transfectants were significantly suppressed as compared with that of wild- and mock-type. The decrease in cell number of wild-type cultured for 72 h in the presence of butyrate (8.3 x 10(-6) or 8.3 x 10(-5) M), rescovitine (10(-8) or 10(-7) M), or sulforaphane (10(-9) M), which is an inhibitor of the cell cycle, was not observed in transfectants. The effect of PD98059 (10(-8) M), staurosporine (10(-10) M) or dibucaine (10(-8)-10(-6) M), which is an inhibitor of protein kinases, in decreasing cell number of wild-type was not seen in transfectants. Moreover, the culture with wortmannin (10(-8) or 10(-7) M), an inhibitor of phosphatidylinositol 3 (PI3)-kinase, or Bay K 8644 (10(-8) or 10(-7) M), an agonist of calcium entry in cells, caused a significant decrease in cell number of the wild-type. This decrease was not observed in transfectants. The result of reverse transcription-polymerase chain reaction (RT-PCR) analysis using specific primers showed that c-jun and chk2 mRNA levels were significantly decreased in transfectant. p53 mRNA level was significantly increased in transfectants. The expression of c-myc, c-fos, cdc2, p21, and G3PDH mRNAs in transfectants was not significantly changed. This study demonstrates that overexpression of regucalcin has a suppressive effect on cell proliferation, which is mediated through various signaling pathways, in the cloned normal rat kidney proximal tubular epithelial NRK52E cells.

Overexpression of regucalcin suppresses apoptotic cell death in cloned normal rat kidney proximal tubular epithelial NRK52E cells: Change in apoptosis‐related gene expression

The effect of regucalcin, a regulatory protein in intracellular signaling pathway, on cell death and apoptosis was investigated using the cloned normal rat kidney proximal tubular epithelial NRK52E cells overexpressing regucalcin. NRK52E cells (wild type) and stable regucalcin (RC)/pCXN2 transfectants were cultured for 72 h in a medium containing 5% bovine serum (BS) to obtain subconfluent monolayers. After culture for 72 h, cells were further cultured for 24-72 h in a medium without BS containing either vehicle, tumor necrosis factor-alpha (TNF-alpha; 0.1 or 1.0 ng/ml of medium), lipopolysaccharide (LPS; 0.1 or 1.0 microg/ml), Bay K 8644 (10(-9)-10(-7) M), or thapsigargin (10(-9)-10(-7) M). The number of wild-type cells was significantly decreased by culture for 42-72 h in the presence of TNF-alpha (0.1 or 1.0 ng/ml), LPS (0.1 or 1.0 microg/ml), Bay K 8644 (10(-7)-10(-5) M), or thapsigargin (10(-8) or 10(-7) M). The effect of TNF-alpha (0.1 or 1.0 ng/ml), LPS (0.1 or 1.0 microg/ml), Bay K 8644 (10(-7)-10(-6) M), or thapsigargin (10(-7) M) in decreasing the number of wild-type cells cultured for 24-72 h was significantly prevented in transfectants overexpressing regucalcin. Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent wild-type cells cultured with LPS (1.0 microg/ml), Bay K 8644 (10(-7) M), or thapsigargin (10(-8) M) for 24 h, and this DNA fragmentation was significantly suppressed in transfectants. DNA fragmentation in adherent cells was not seen by culture with TNF-alpha (1.0 ng/ml). TNF-alpha-induced decrease in the number of wild-type cells was significantly prevented by culture with caspase-3 inhibitor (10(-8) M), while LPS- or Bay K 8644-induced decrease in cell number was significantly prevented by caspase-3 inhibitor or N omega-nitro-L-arginine methylester (NAME) (10(-5) M), an inhibitor of nitric oxide (NO) synthase. Thapsigargin-induced decrease in cell number was not prevented in the presence of two inhibitors. Bcl-2 and Akt-1 mRNA levels were significantly increased in transfectants cultured for 24 h as compared with those of wild-type cells, while Apaf-1, caspase-3, or glyceroaldehyde-3-phosphate dehydrogenase (G3PDH) mRNA expressions were not significantly changed in transfectants. Culture with TNF-alpha (1.0 ng/ml), LPS (1.0 microg/ml), Bay K 8644 (l0(-7) M), or thapsigargin (10(-8) M) caused a significant increase in caspase-3 mRNA levels in wild-type cells. LPS (1.0 microg/ml) significantly decreased Bcl-2 mRNA expression in the cells. Their effects on the gene expression of apoptosis-related proteins were not significantly changed in transfectants. This study demonstrates that overexpression of regucalcin has a suppressive effect on cell death and apoptosis induced by various factors which their action are mediated through many intracellular signaling pathways, and that it modulates the gene expression of apoptosis-related proteins.

Hormonal regulation on regucalcin mRNA expression in cloned normal rat kidney proximal tubular epithelial NRK52E cells

Regucalcin is a regulatory protein in cell signaling. This study was undertaken to determine whether regucalcin mRNA expresses in the cloned normal rat kidney proximal tubular epithelial NRK52E cells and its expression regulates due to hormones and cell signaling-related factors. Cells with subconfluency were cultured for 24, 48, or 72 h in a Dulbecco's modified Eagle medium supplemented with non-essential amino acid without bovine serum (BS). The result of Western blot analysis showed that regucalcin protein was present in the NRK52E cells. The expression of regucalcin mRNA in the cells was determined using reverse transcription-polymerase chain reaction (RT-PCR). Regucalcin mRNA expression in the NRK52E cells was significantly increased by culture with parathyroid hormone (PTH, 10(-8) or 10(-7) M), aldosterone (10(-8) or 10(-7) M), or dexamethasone (10(-8) M). The presence of 1,25-dihydroxyvitamin D(3) (1,25(OH)2D3, 10(-8) or 10(-7) M) or calcitonin (10(-9) or 10(-8) M) did not have a significant effect on regucalcin mRNA levels in the cells. Culture with dibutyryl cyclic AMP (DcAMP, 10(-5) or 10(-4) M) or phorbol 12-myristate 13-acetate (PMA, 10(-6) M), an activator of protein kinase C, caused a significant increase in regucalcin mRNA expression. The presence of staurosporine (10(-8) M) caused a significant decrease in regucalcin mRNA expression. Dibucaine (10(-7) M), PD98059 (10(-7) M), or vanadate (10(-6) or 10(-5) M) did not have an effect on regucalcin mRNA levels. The present study demonstrates that regucalcin mRNA and its protein are expressed in the cloned normal rat kidney proximal tubular epithelial NRK52E cells, and that the expression is enhanced by hormones which regulate ion transport in the proximal tubule.