help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Matsushita, M.
Right arrow Articles by Hirata, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Matsushita, M.
Right arrow Articles by Hirata, Y.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*CALCIUM COMPOUNDS
*CALCIUM, ELEMENTAL
Endocrinology Vol. 144, No. 5 1825-1831
Copyright © 2003 by The Endocrine Society

Urotensin II is an Autocrine/Paracrine Growth Factor for the Porcine Renal Epithelial Cell Line, LLCPK1

Mika Matsushita, Masayoshi Shichiri, Nozomi Fukai, Naoko Ozawa, Takanobu Yoshimoto, Nobuyuki Takasu and Yukio Hirata

Department of Clinical and Molecular Endocrinology (M.M., M.S., N.F., N.O., T.Y., Y.H.), Tokyo Medical and Dental University Graduate School, Tokyo, 113-8519, Japan; and Second Department of Internal Medicine (N.T.), University of Ryukyus, Okinawa 903-0215, Japan

Address all correspondence and requests for reprints to: Yukio Hirata, M.D., Department of Clinical and Molecular Endocrinology, Tokyo Medical and Dental University Graduate School, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8519, Japan. E-mail: yhirata.cme{at}tmd.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Urotensin-II (UII), a cyclic dodecapeptide with potent cardiovascular effects, has recently been shown to be abundantly expressed in the human kidney and excreted in human urine. To investigate whether UII acts as an autocrine/paracrine growth factor for renal epithelial cells, we have studied the effects of human UII (hUII) on DNA synthesis, cytosolic free Ca2+ concentration ([Ca2+]i), ERK activation, and protooncogene (c-myc) expression in a porcine renal epithelial cell line (LLCPK1). hUII stimulated [3H]thymidine uptake into quiescent cells in a dose-dependent manner (10-9 to 10-7 M); this effect was inhibited by a protein kinase C inhibitor (GF109203X), a MAPK kinase inhibitor (PD98059), and a calcium channel blocker (nicardipine). Neither phosphatidyl inositol-3 kinase inhibitors (LY294002, wortmannin) nor p38 kinase inhibitor (SB203580) affected the hUII-induced DNA syntheses. hUII rapidly (within 5 min) and dose-dependently (10-9 to 10-7 M) increased [Ca2+]i in fura-2-loaded cells. hUII also caused a rapid and transient activation of ERK1/2 and induction of c-myc. LLCPK1 cells expressed UII mRNA and its receptor GPR14 mRNA, as determined by RT-PCR, and released UII-like immunoreactivity into media. Neutralization of endogenous UII by anti-hUII antibody, but not nonimmune serum, significantly suppressed DNA synthesis. These data suggest that hUII is an autocrine/paracrine growth factor for renal epithelial cells via activation of both protein kinase C and ERK1/2 pathways as well as Ca2+ influx via voltage-dependent Ca2+ channels.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
UROTENSIN-II (UII) is a potent vasoconstrictor peptide originally isolated from fish spinal cords (1). Its human homolog has subsequently been cloned and characterized (2). The predicted human urotensin II (hUII) consists of 11 amino acid residues with a cyclic hexapeptide conserved among many species. Although the vasoconstrictive potency of hUII on rat thoracic aorta is greater than that of any other known peptides, it shows differential vasoconstrictor responses in various blood vessels of many species, suggesting its divergent species and regional differences (3, 4, 5). hUII has been shown to be an endogenous ligand for an orphan G protein-coupled receptor (GPCR), GPR14 (3, 4, 6, 7). GPR14 mRNA is exclusively expressed in the neuronal and the cardiovascular tissues (3, 4, 8, 9).

The expression of UII mRNA in human, rat, and mouse central nervous system was strongest in the medulla oblongata of the brain and the spinal cord (2, 3, 4), but hUII mRNA was also detectable in several human peripheral tissues, such as kidney, spleen, and adrenal glands, by dot blot analysis (2). However, a recent study (4) has revealed, by Northern blot analysis, that hUII mRNA was expressed most abundantly in human kidney, and little, if any, hUII mRNA was expressed in other human peripheral tissues. We have recently confirmed that both hUII and GPR14 mRNAs are more abundantly expressed in human kidney tissues than those in cardiovascular tissues, as quantified by real-time quantitative RT-PCR (10). Furthermore, we have also demonstrated that significant amounts of hUII are excreted in human urine, possibly secreted from renal tubular cells (10). However, there is no information yet available as to whether UII is actually produced by and secreted from renal tubular cells.

It has recently been reported that hUII stimulated the proliferation of vascular smooth muscle cells (11) and that the effect was potentiated by oxidized low-density lipoprotein (12). These data suggest that hUII, in addition to its vasoconstrictive effect, acts as a potent mitogen, which may be involved in the progression of atherosclerosis. However, it remains unknown whether UII has any biological activities on renal tubular cells.

These observations led us to examine whether renal epithelial cells express UII mRNA and secrete UII-like immunoreactivity (LI) and whether UII has any mitogenic action on these cells in an autocrine/paracrine fashion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Synthetic hUII and rabbit polyclonal anti-hUII antibody were obtained from Peptide Institute (Osaka, Japan); PD98059 from New England Biolabs, Inc. (Beverly, MA); GF109203X from Calbiochem-Novabiochem (La Jolla, CA); SB203580, LY294002, and wortmannin from Sigma (St. Louis, MO); nicardipine from Yamonouchi Phermaceutical (Tokyo, Japan); phosphorylated forms of ERK1/2 (Thr202/Tyr204) E10 monoclonal antibody from Cell Signaling Technology (Beverly, MA); nonphosphorylated anti-ERK2 SC-154 polyclonal antibody from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); peroxidase-linked antirabbit and antimouse IgG antibodies from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA); and [3H]thymidine (SA, 6.7 Ci/mM), [{alpha}-32P]deoxy-CTP (SA, 111 TBq/mM), and [{gamma}-32P]ATP (SA, 37 MBq/mM) from Amersham International (Tokyo, Japan).

Cell culture
LLCPK1 cells were cultured in DMEM containing 10% fetal calf serum at 37 C in a humidified atmosphere of 95% air-5% CO2 as previously described (13).

ERK1/2 activity
Cellular ERK1/2 activity was determined as previously described (14, 15). In brief, quiescent LLCPK1 cells grown on a 12-well plate were stimulated with hUII at 37 C in serum-free DMEM for the indicated times, and the reaction was terminated by the replacement of medium with ice-cold lysis buffer. The sample was centrifuged at 25,000 x g for 20 min at 4 C, and the supernatant was assayed for p42/p44 MAPK activity using an assay kit (Amersham Life Science, Piscataway, NJ).

Western blotting analysis was performed as previously described (14, 16) using cells grown on a six-well plate. SDS-PAGE gels were transferred onto nitrocellulose, which was blocked with Blotto (5% dried nonfat milk in PBS), and incubated with polyclonal phospho-specific MAPK antibody (1:2000) that recognizes ERK1/2 only when catalytically activated by phosphorylation at Tyr202/Tyr204, or with polyclonal rabbit anti-ERK2 antibody (1:1000) that recognizes both the phosphorylated and nonphosphorylated form, followed by horseradish peroxidase-conjugated antirabbit IgG antiserum. Signals were visualized using the ECL chemiluminescence system (Amersham).

Mitogenic assay
DNA synthesis was assessed by incorporation of [3H]thymidine into cells as previously described (14). In brief, after preincubation with serum-free DMEM for 48 h, the quiescent cells were incubated with hUII for 20 h, after which 1 µCi [3H]thymidine was added, and the cells were further incubated for 4 h. Trichloroacetic acid-insoluble radioactivity was then measured with a liquid scintillation counter, 1900TR (Packard BioScience, Meriden, CT).

Determination of intracellular calcium concentration ([Ca2+]i)
Measurement of [Ca2+]i was determined by the Ca2+-fura-2 fluorescence method as previously described (17). After incubation in serum-free DMEM for 48 h, cells were trypsinized and incubated with 4 mM fura-2 acetoxymethyl ester (Dojindo Chemical Laboratory, Kumamoto, Japan) at 37 C for 20 min in buffered physiological salt solution. The Ca2+-fura-2 fluorescence of the suspended cells was measured by a spectrofluorometer (CAF-100, Japan Spectroscopic Co., Tokyo, Japan) using excitation at 340 and 380 nm and emission at 500 nm. [Ca2+]i values were determined according to the method of Grynkiewicz et al. (18).

RT-PCR
Total RNA was extracted from LLCPK1 cells by using RNA zolB (Tel-Test, Friendswood, TX), and cDNA was synthesized with a first-strand cDNA synthesis kit (Amersham Pharmacia Biotech). Amplification of porcine c-myc and UII transcripts was performed using specific primers for c-myc (forward primer, 5'-AGAGAAGCTGGCCTCCTACC-3'; and reverse primer, 5'-GGCGGAGGAGAGCAGAGAGT-3') and UII (forward primer, 5'-GGATCTCAGGGAAGCAGATG-3'; and reverse primer, 5'-GCCCACGTTTCTTGTATGGT-3'), respectively. Real-time quantification of porcine c-myc levels in quiescent LLCPK1 cells was performed using LightCycler (Roche Molecular Biochemicals, Mannheim, Germany)-based real-time quantitative RT-PCR essentially as described (10, 17). TaqStart antibody (CLONTECH Laboratories, Inc., Palo Alto, CA) was used to prevent generation of nonspecific amplification products. The fluorescence data were quantitatively analyzed by using serial dilution of control samples included in each reaction to produce a standard curve. For verification of the melting curve results, the PCRs were examined by 1.5% agarose gel electrophoresis. Conventional RT-PCR of GPR14 mRNA was performed using two sets of primers (forward primer, 5'-GCAACCCTCAACAGCTCCTG-3'; and reverse primer, 5'-AAGTGCCACTCCTTGGTGAC-3') and (forward primer, 5'-TGCTGTACCTGCTCAGCATC-3'; and reverse primer, 5'-AAGTCCAGGCCGAAGAGCAC-3'), each amplifying a 266-bp and an 81-bp product, respectively.

RIA and extraction
RIA for hUII was performed as previously described (10). Briefly, samples or standard synthetic hUII (Peptide Institute Inc.) and rabbit anti-hUII serum (final dilution, 1:10,000; Peptide Institute Inc.) were incubated at 4 C for 24 h, followed by the late addition of [125I]-hUII (~10,000 cpm) and further incubation at 4 C for 24 h. Separation of bound from free ligands was performed by the double-antibody method. Serum-free conditioned media from cultured LLCPK1 cells was extracted as previously described (10). In brief, supernatant acidified with trifluoroacetic acid was loaded onto a Sep-Pak C18 cartridge (Millipore-Waters Inc., Milford, MA), and the adsorbed materials were eluted with 70% acetonitrile/0.1% trifluoroacetic acid. The coefficients of variation of interassay and intraassay were less than 10%.

Statistical analysis
All results are expressed as the mean ± SEM. Statistical analysis was performed by Wilcoxon’s t test or ANOVA; P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Growth-promoting effect
hUII stimulated DNA synthesis of quiescent LLCPK1 cells in a dose-dependent manner (10-9 to 10-7 M; Fig. 1AGo); its approximate ED50 was 10-8 M. DNA synthesis stimulated by hUII (10-7 M) was inhibited by a MAPK kinase (MEK)-1 inhibitor (PD98059, 10-5 M), a protein kinase C (PKC) inhibitor (GF109203X, 10-6 M), and a calcium-channel antagonist (nicardipine, 10-6 M) but not by either a p38 inhibitor (SB203580, 10-5 M) or phosphatidylinositol-3 (PI-3) kinase inhibitors (wortmannin, 10-6 M; or LY294002, 2 x 10-6 M; Fig. 2Go).



View larger version (12K):
[in this window]
[in a new window]
  		Figure 1. Effects of hUII on DNA synthesis and intracellular Ca2+ concentrations of LLCPK1 cells. Quiescent cells were incubated with hUII at the indicated concentrations. A, [3H]Thymidine radioactivity incorporated into cells was determined. B, The fura-2-loaded cells were stimulated with various concentrations of hUII for measurement of [Ca2+]i, and peak [Ca2+]i values after 1 min are shown; basal levels were 167 + 12 nM. Each point with bar shows the mean ± SEM (n = 4). *, P < 0.05 vs. control.

 


View larger version (19K):
[in this window]
[in a new window]
  		Figure 2. Effects of several compounds on hUII-induced DNA synthesis in LLCPK1 cells. Quiescent cells pretreated with or without PD98059 (10-5 M), GF109203X (10-8 M), nicardipine (10-6 M), LY294002 (2 x 10-6 M), wortmannin (10-6 M), and SB203580 (10-6 M) were incubated with hUII (10-7 M) for measurement of [3H]thymidine incorporation (n = 4). *, P < 0.05 vs. control.

 
Cytosolic free Ca2+ concentrations ([Ca2+]i)
hUII induced sustained increases in [Ca2+]i of quiescent LLCPK1 cells in a dose-dependent manner (10-9 to 10-7 M; Fig. 1BGo). These effects were inhibited by pretreatment with nicardipine (10-6 M) and 5 mM EGTA (data not shown). The hUII-induced [Ca2+]i increase was only partially suppressed by pretreament with PD98059 (2.5 x 10-5 M) but not by GF109203X or SB203580 (data not shown).

ERK1/2 activation
To determine whether the growth-promoting signal by hUII involves ERK activation, ERK1/2 was determined by immunoblotting with monoclonal antibody that selectively recognizes Tyr202/Tyr204-ERKs as well as by enzymatic assay. hUII induced a rapid (within 5 min) and transient tyrosine phosphorylation of ERK1/2 (Fig. 3Go). hUII also caused a transient ERK1/2 activation, peaking at 5 min, followed by a gradual decline to the basal levels by 30 min (Fig. 4AGo). Stimulation of ERK1/2 activity by hUII was dose-dependent (10-11 to 10-7 M; Fig. 4BGo). The effects were completely blocked by PD98059 (10-5 M) and GF109203X (10-6 M), but not by SB203580 or nicardipine. These data suggest that hUII induced phosphorylation and activation of ERK1/2 via a PKC-dependent manner, independently from PI-3 kinase pathway and Ca2+ influx via dihydropyridine (DHP)-sensitive Ca2+ channels.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 3. Stimulation of p42/p44 MAPK by hUII in LLCPK1 cells. Quiescent cells were stimulated with hUII (10-7 M) for the times indicated. Immunoblotting was performed using anti-phosphorylated p42/p44 MAPK (upper panel) and unphosphorylated anti-ERK2 (lower panel). Arrows denote phosphorylated p42/p44 MAPK at 42 and 44 kDa, respectively.

 


View larger version (15K):
[in this window]
[in a new window]
  		Figure 4. Effect of hUII on p42/p44 MAPK activity in LLCPK1. A, Quiescent cells were incubated with hUII (10-7 M) for the times indicated. B, Quiescent cells were pretreated without ({bullet}) or with PD98059 (10-5 M; {blacksquare}), GF109203X (10-6 M; {circ}), SB203580 (10-6 M; {square}), and incubated for 5 min with hUII at the indicated concentrations; p42/p44 MAPK activity was determined by enzymatic assay. Each point represents mean ± SEM (n = 4). *, P < 0.05 vs. control.

 
Induction of c-myc protooncogene
To examine whether hUII induces protooncogene, c-myc, we quantified c-myc mRNA levels by real-time quantitative RT-PCR. hUII rapidly (within 1 min) and transiently induced c-myc mRNA levels, which peaked after 2 min (Fig. 5Go).



View larger version (12K):
[in this window]
[in a new window]
  		Figure 5. Effect of hUII on c-myc induction in LLCPK1 cells. Quiescent cells were incubated with hUII (10-7 M) for the indicated time, and porcine c-myc transcripts were quantified by real-time quantitative RT-PCR. *, P < 0.05 vs. control.

 
Expression of UII mRNA and protein
The extracted conditioned media from LLCPK1 cells by SepPack C18 was subjected to hUII RIA. The dilution curve generated by the extracted media was parallel to that of synthetic hUII (Fig. 6Go), suggesting the presence of UII-LI in the media. UII-LI secreted by LLCPK1 cells (106 cells) was calculated to be 1.6 ng/24 h. RT-PCR using total RNA from LLCPK1 cells revealed a 142-bp band corresponding to porcine UII transcript (Fig. 7Go).



View larger version (16K):
[in this window]
[in a new window]
  		Figure 6. RIA of hUII. A serial dilution curve of extracted supernatant from cultured LLCPK1 is compared with a standard curve by synthetic hUII. A typical example of RIA is shown.

 


View larger version (20K):
[in this window]
[in a new window]
  		Figure 7. Expression of UII and GPR14 by LLCPK1 cells. A, RT-PCR of total RNA from LLCPK1 cells with or without reverse transcription. Arrows indicate the 142-bp band corresponding to the size of porcine UII transcripts. B, RT-PCR of total RNA from LLCPK1 cells using two different sets of human primers. Arrows indicate 266-bp and 81-bp bands corresponding to the sizes of GPR14 transcripts.

 
Expression of GPR14 mRNA
RT-PCR using two independent human GPR14 primers revealed a single band corresponding to the size of human GPR14 mRNA (Fig. 7Go). Partial sequencing of the amplified products revealed homology to human GPR14, indicating the expression of GPR14 in porcine LLCPK1 cells.

Effect of anti-hUII antibody on mitogenesis
Because the LLCPK1 cell line synthesizes and secretes hUII-LI into culture media, whereas exogenous hUII stimulates DNA synthesis of LLCPK1 cells, we reasoned that UII may function as an autocrine/paracrine growth factor for LLCPK1 cells. Addition of rabbit anti-hUII antiserum (dilution at 1:100) significantly suppressed DNA synthesis of LLCPK1 cells under serum-free condition, whereas the addition of normal rabbit serum (1:100) was without effect (Fig. 8Go), suggesting its role as an autocrine/paracrine growth-promoting factor.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 8. Inhibition of DNA synthesis in LLCPK1 cells by anti-hUII antibody. Quiescent cells were incubated with or without anti-hUII antibody or control normal rabbit serum (NRS) for 20 h, and [3H]thymidine incorporation was measured (n = 4). *, P < 0.05 vs. control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we have shown for the first time that hUII has a potent mitogenic effect on a porcine renal epithelial cell line (LLCPK1). The hUII-stimulated DNA synthesis appears to be mediated via activation of PKC and ERK1/2 as well as Ca2+ influx, because PKC inhibitor (GF109203X), MEK-1 inhibitor (PD98059), and DHP-sensitive calcium-channel antagonist (nicardipine) completely blocked the mitogenic effect by hUII. However, DNA synthesis stimulated by hUII was not inhibited by PI-3 kinase inhibitors (wortmannin and LY294002) or a p38 kinase inhibitor (SB203580), thus excluding the involvement of both p38 kinase and PI-3 kinase in hUII-induced mitogenesis. The present study has further shown that hUII dose-dependently stimulated ERK1/2 activity and increased [Ca2+]i in LLCPK1 cells. The mitogenic property of hUII is further strengthened by the result that hUII potently induced c-myc protooncogene, an immediate early response gene related to cell growth and differentiation (19). Taken together, it is suggested that UII exerts its mitogenic effect on LLCPK1 cells via activation of both PKC and ERK1/2 pathways as well as Ca2+ influx mediated via DHP-sensitive Ca2+ channels.

GPR14, a member of an orphan GPCR family, has recently been identified as a specific receptor for UII (3, 4). Although the porcine GPR14 sequence is not yet available in the databases, we PCR-amplified porcine GPR14 cDNA using human GPR14 primers and confirmed its homology to human GPR14 by sequencing amplified products. Thus, our results indicate that porcine renal epithelial cells (LLCPK1) express GPR14 mRNA with functional UII receptors linked to signal transduction and mitogenic response.

GPR14 has been shown to be coupled to Ca2+ mobilization (3, 4), but the detailed signaling pathways triggered by UII stimulation have been poorly understood. It has been shown that the hUII-induced arterial smooth muscle contraction is linked to phospholipase C-mediated generation of inositol phosphates (20) as well as activation of the small GTP protein Rho and its downstream effector Rho kinase (11). Stimulation of PLC and Gq signaling in general is coupled to rapid and transient increase in [Ca2+]i, which is usually related to contraction of vascular smooth muscle cells (21, 22), whereas influx of extracellular Ca2+ leading to sustained increase in [Ca2+]i is closely linked to mitogenesis activity (23). The present study has clearly shown that the sustained influx of extracellular Ca2+ via DHP-sensitive Ca2+ channels by hUII plays pivotal roles in its mitogenic effect in LLCPK1 cells.

ERK1/2 is activated by a variety of receptor tyrosine kinases and GPCRs (24). GPCR agonists, such as angiotensin II (25) and endothelin-1 (26), can recruit adaptor proteins to induce activation of ras-dependent ERK1/2 in a similar fashion as receptor tyrosine kinases. Alternatively, PKC can activate Raf-1, a MEK-1 kinase, thereby leading to ERK1/2 activation (27). The present study has clearly demonstrated that both MEK-1 inhibitor (PD98059) and PKC inhibitor (GF109203X) similarly and completely blocked the hUII-stimulated ERK1/2 activity as well as DNA synthesis in LLCPK1 cells. Our data are comparable to those of a recent study (12) showing that hUII-induced proliferation of vascular smooth muscle cells is equally blocked by MEK-1 and PKC inhibitors. Taken together, the mitogenic action by UII is most likely mediated via a PKC-dependent ERK1/2 pathway, although its exact signal transduction remains to be determined.

Our present study has provided direct evidence that porcine renal tubular cells (LLCPK1) express UII mRNA, as demonstrated by RT-PCR. Furthermore, the apparent parallelism of the conditioned media to standard hUII in the present RIA suggests that porcine UII secreted from renal epithelial cells is immunologically similar to that of human UII. In fact, LLCPK1 cells (106 cells) secrete approximately 1.6 ng UII-LI during 24 h under serum-free conditions; the concentrations (~10-9 M) appear to be effective enough to stimulate ERK1/2 activity, increase [Ca2+]i, and result in cell proliferation. Finally, the present neutralization experiments have clearly shown that the anti-hUII antibody significantly suppressed DNA synthesis in LLCPK1 cells. These results, in conjunction with the concomitant expression of both UII and GPR14 by the cells, lend strong support to the notion that endogenous UII secreted by renal tubular cells acts on itself and/or the nearby cells as an autocrine/paracrine growth factor.

In our previous study (10), hUII mRNA was expressed most abundantly in the kidney among certain human tissues examined, a finding that is in agreement with another study showing that the kidney is the major site of its synthesis in humans (4). It has been reported that several other vasoactive peptides, such as atrial natriuretic peptide (28), endothelin-1 (29), and adrenomedullin (30), are excreted in human urine. We have also observed that the urinary hUII clearance exceeds the glomerular filtration rate in normal subjects, and even higher urinary hUII excretion was observed in patients with renal tubular disorders (10), suggesting the impaired reabsorption and/or the enhanced secretion of hUII by the injured tubules. Thus, a significant amount of hUII-LI excreted in human urine is likely to be derived from the renal tubular cells. These observations, together with the present results, lead us to speculate that UII secreted from renal tubules may act on tubular cells to repair at the site of tubular injury in an autocrine/paracrine fashion.

In conclusion, we have demonstrated that UII is an autocrine/paracrine growth factor for renal epithelial cell line (LLCPK1) via activation of both PKC and ERK1/2 pathways as well as Ca2+ influx via DHP-sensitive Ca2+ channels. However, the physiological and pathophysiological roles of UII in renal tissues remain to be determined.


   Footnotes
 
This study was supported in part by a grant-in-aid from the Ministry of Education, Science, Sports, Culture and Technology of Japan, and by the Mitsukoshi Foundation.

Abbreviations: [Ca2+]i, Intracellular calcium concentration; DHP, dihydropyridine; hUII, human UII; GPCR, G protein-coupled receptor; LI, -like immunoreactivity; MEK, MAPK kinase; PI-3, phosphatidylinositol-3; PKC, protein kinase C; UII, urotensin-II.

Received January 7, 2003.

Accepted for publication January 29, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Pearson D, Shively JE, Clark BR, Geschwind I, Barkley M, Nishioka RS, Bern HA 1980 Urotensin II: a somatostatin-like peptide in the caudal neurosecretory system of fishes. Proc Natl Acad Sci USA 77:5021–5024[Abstract/Free Full Text]
  2. Coulouarn Y, Lihrmann I, Jegou S, Anouar Y, Tostivint H, Beauvillain JC, Conlon JM, Bern HA, Vaudry H 1998 Cloning of the cDNA encoding the urotensin II precursor in frog and human reveals intense expression of the urotensin II gene in motoneurons of the spinal cord. Proc Natl Acad Sci USA 95:15803–15808[Abstract/Free Full Text]
  3. Ames RS, Sarau HM, Chambers JK, Willette RN, Aiyar NV, Romanic AM, Louden CS, Foley JJ, Sauermelch CF, Coatney RW, Ao Z, Disa J, Holmes SD, Stadel JM, Martin JD, Liu WS, Glover GI, Wilson S, McNulty DE, Ellis CE, Elshourbagy NA, Shabon U, Trill JJ, Hay DW, Ohlstein EH, Bergsma DJ, Douglas SA 1999 Human urotensin-II is a potent vasoconstrictor and agonist for the orphan receptor GPR14. Nature 401:282–286[CrossRef][Medline]
  4. Nothacker H-P, Wang Z, McNeill AM, Saito Y, Merten S, O’Dowd B, Duckleds SP, Civelli O 1999 Identification of the natural ligand of an orphan G-protein-coupled receptor involved in the regulation of vasoconstriction. Nat Cell Biol 1:383–385[CrossRef][Medline]
  5. MacLean MR, Alexander D, Stirrat A, Gallagher M, Douglas SA, Ohlstein EH, Morecroft I, Polland K 2000 Contractile responses to human urotensin-II in rat and human pulmonary arteries: effect of endothelial factors and chronic hypoxia in the rat. Br J Pharmacol 130:201–204[CrossRef][Medline]
  6. Liu Q, Pong SS, Zeng Z, Zhang Q, Howard AD, Williams Jr DL, Davidoff M, Wang R, Austin CP, McDonald TP, Bai C, George SR, Evans JF, Caskey CT 1999 Identification of urotensin II as the endogenous ligand for the orphan G-protein-coupled receptor GPR14. Biochem Biophys Res Commun 266:174–178[CrossRef][Medline]
  7. Mori M, Sugo T, Abe M, Shimomura Y, Kurihara M, Kitada C, Kikuchi K, Shintani Y, Kurokawa T, Onda H, Nishimura O, Fujino M 1999 Urotensin II is the endogenous ligand of a G-protein-coupled orphan receptor, SENR (GPR14). Biochem Biophys Res Commun 265:123–129[CrossRef][Medline]
  8. Marchese A, Heiber M, Nguyen T, Heng HH, Saldivia VR, Cheng R, Murphy PM, Tsui LC, Shi X, Gregor P 1995 Cloning and chromosomal mapping of three novel genes, GPR9, GPR10, and GPR14, encoding receptors related to interleukin 8, neuropeptide Y, and somatostatin receptors. Genomics 29:335–244[CrossRef][Medline]
  9. Tal M, Ammar DA, Karpuj M, Krizhanovsky V, Naim M, Thompson DA 1995 A novel putative neuropeptide receptor expressed in neural tissue, including sensory epithelia. Biochem Biophys Res Commun 209:752–759[CrossRef][Medline]
  10. Matsushita M, Shichiri M, Imai T, Iwashina M, Tanaka H, Takasu N, Hirata Y 2001 Co-expression of urotensin II and its receptor (GPR14) in human cardiovascular and renal tissues. J Hypertens 19:2185–2190[CrossRef][Medline]
  11. Sauzeau V, Le ME, Bertoglio J, Scalbert E, Pacaud P, Loirand G 2001 Human urotensin II-induced contraction and arterial smooth muscle cell proliferation are mediated by RhoA and Rho-kinase. Circ Res 88:1102–1104[Abstract/Free Full Text]
  12. Watanabe T, Pakala R, Katagiri T, Benedict CR 2001 Synergistic effect of urotensin II with mildly oxidized LDL on DNA synthesis in vascular smooth muscle cells. Circulation 104:16–18[Abstract/Free Full Text]
  13. Shichiri M, Hirata Y, Emori T, Ohta K, Nakajima T, Sato K, Sato A, Marumo F 1989 Secretion of endothelin and related peptides from renal epithelial cell lines. FEBS Lett 253:203–206[CrossRef][Medline]
  14. Iwasaki H, Eguchi S, Shichiri M, Marumo F, Hirata Y 1998 Adrenomedullin as a novel growth-promoting factor for cultured vascular smooth muscle cells: role of tyrosine kinase-mediated mitogen-activated protein kinase activation. Endocrinology 139:3432–3441[Abstract/Free Full Text]
  15. Shichiri M, Sedivy JM, Marumo F, Hirata Y 1998 Endothelin-1 is a potent survival factor for c-Myc-dependent apoptosis. Mol Endocrinol 12:172–180
  16. Shichiri M, Kato H, Doi M, Marumo F, Hirata Y 1999 Induction of Max by adrenomedullin and calcitonin gene-related peptide antagonizes endothelial apoptosis. Mol Endocrinol 13:1353–1363[Abstract/Free Full Text]
  17. Shichiri M, Hirata Y 2001 Anti-angiogenesis signals by endostatin. FASEB J 15:1044–1053[Abstract/Free Full Text]
  18. Grynkiewicz G, Poenie M, Tsien RY 1985 A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450[Abstract/Free Full Text]
  19. Cole MD 1986 The myc oncogene: its role in transformation and differentiation. Annu Rev Genet 20:361–384[CrossRef][Medline]
  20. Saetrum OO, Nothacker H, Ehlert FJ, Krause DN 2000 Human urotensin II mediates vasoconstriction via an increase in inositol phosphates. Eur J Pharmacol 406:265–271[CrossRef][Medline]
  21. Haller H, Elliott HL 1996 Review: the central role of calcium in the pathogenesis of cardiovascular disease. J Hum Hypertens 10:143–155[Medline]
  22. Di SJ, Nelson SR, Kaplan N 1997 Protein tyrosine phosphorylation in smooth muscle: a potential coupling mechanism between receptor activation and intracellular calcium. Proc Soc Exp Biol Med 214:285–301[Abstract]
  23. Schachter M 1997 Calcium antagonists and atherosclerosis. Int J Cardiol 62:S9–S15
  24. Schlessinger J 2000 Cell signaling by receptor tyrosine kinases. Cell 103:211–225[CrossRef][Medline]
  25. Eguchi S, Numaguchi K, Iwasaki H, Matsumoto T, Yamakawa T, Utsunomiya H, Motley ED, Kawakatsu H, Owada KM, Hirata Y, Marumo F, Inagami T 1998 Calcium-dependent epidermal growth factor receptor transactivation mediates the angiotensin II-induced mitogen-activated protein kinase activation in vascular smooth muscle cells. J Biol Chem 273:8890-8896[Abstract/Free Full Text]
  26. Iwasaki H, Eguchi S, Ueno H, Marumo F, Hirata Y 1999 Endothelin-mediated vascular growth requires p42/p44 mitogen-activated protein kinase and p70 S6 kinase cascades via transactivation of epidermal growth factor receptor. Endocrinology 140:4659–4668[Abstract/Free Full Text]
  27. Liebmann C 2001 Regulation of MAP kinase activity by peptide receptor signalling pathway: paradigms of multiplicity. Cell Signal 13:777–785[CrossRef][Medline]
  28. Marumo F, Sakamoto H, Ando K, Ishigami T, Kawakami M 1986 A highly sensitive radioimmunoassay of atrial natriuretic peptide (ANP) in human plasma and urine. Biochem Biophys Res Commun 137:231–236[CrossRef][Medline]
  29. Ohta K, Hirata Y, Shichiri M, Kanno K, Emori T, Tomita K, Marumo F 1991 Urinary excretion of endothelin-1 in normal subjects and patients with renal disease. Kidney Int 39:307–311[Medline]
  30. Sato K, Imai T, Iwashina M, Marumo F, Hirata Y 1998 Secretion of adrenomedullin by renal tubular cell lines. Nephron 78:9–14[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Nephrol Dial TransplantHome page
A. Mosenkis, T. M. Danoff, N. Aiyar, J. Bazeley, and R. R. Townsend
Human Urotensin II in the plasma of anephric subjects
Nephrol. Dial. Transplant., April 1, 2007; 22(4): 1269 - 1270.
[Full Text] [PDF]

Home page
 HypertensionHome page
T. Watanabe, T. Suguro, T. Kanome, Y.-i. Sakamoto, S. Kodate, T. Hagiwara, S. Hongo, T. Hirano, M. Adachi, and A. Miyazaki
Human Urotensin II Accelerates Foam Cell Formation in Human Monocyte-Derived Macrophages
Hypertension, October 1, 2005; 46(4): 738 - 744.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
T. Djordjevic, R. S. BelAiba, S. Bonello, J. Pfeilschifter, J. Hess, and A. Gorlach
Human Urotensin II Is a Novel Activator of NADPH Oxidase in Human Pulmonary Artery Smooth Muscle Cells
Arterioscler. Thromb. Vasc. Biol., March 1, 2005; 25(3): 519 - 525.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Matsushita, M.
Right arrow Articles by Hirata, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Matsushita, M.
Right arrow Articles by Hirata, Y.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*CALCIUM COMPOUNDS
*CALCIUM, ELEMENTAL

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals