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Am J Physiol Renal Physiol 294: F982-F989, 2008. First published February 13, 2008; doi:10.1152/ajprenal.00483.2007
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Molecular mechanism of ADP-ribosyl cyclase activation in angiotensin II signaling in murine mesangial cells

Seon-Young Kim,1 Rukhsana Gul,1 So-Young Rah,1 Suhn Hee Kim,2 Sung Kwang Park,3 Mie-Jae Im,1 Ho Jeong Kwon,4 and Uh-Hyun Kim1,5

Departments of 1Biochemistry, 2Physiology, and 3Internal Medicine and 5Institute of Cardiovascular Research, Chonbuk National University Medical School, Jeonju; and 4Department of Biotechnology, College of Engineering, Yonsei University, Seoul, Republic of Korea

Submitted 16 October 2007 ; accepted in final form 5 February 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
ADP-ribosyl cyclase (ADPR-cyclase) produces a Ca2+-mobilizing second messenger cyclic ADP-ribose (cADPR) from NAD+. In this study, we investigated the molecular basis of ADPR-cyclase activation and the following cellular events in angiotensin II (ANG II) signaling in mouse mesangial cells (MMCs). Treatment of MMCs with ANG II induced an increase in intracellular Ca2+ concentrations through a transient Ca2+ release via an inositol 1,4,5-trisphosphate receptor and a sustained Ca2+ influx via L-type Ca2+ channels. The sustained Ca2+ signal, but not the transient Ca2+ signal, was blocked by a cADPR antagonistic analog, 8-bromo-cADPR (8-Br-cADPR), and an ADPR-cyclase inhibitor, 4,4'-dihydroxyazobenzene (DHAB). In support of the results, ANG II stimulated cADPR production in a time-dependent manner, and DHAB inhibited ANG II-induced cADPR production. Application of pharmacological inhibitors revealed that activation of ADPR-cyclase by ANG II involved ANG II type 1 receptor, phosphoinositide 3-kinase, protein tyrosine kinase, and phospolipase C-{gamma}1. Moreover, DHAB as well as 8-Br-cADPR abrogated ANG II-mediated Akt phosphorylation, nuclear translocation of nuclear factor of activated T cell, and uptake of [3H]thymidine and [3H]leucine in MMCs. These results demonstrate that ADPR-cyclase in MMCs plays a pivotal role in ANG II signaling for cell proliferation and protein synthesis.

cyclic ADP-ribose; intracellular Ca2+ concentration

A TYPE II TRANSMEMBRANE GLYCOPROTEIN, CD38, represents mammalian ADP-ribosyl cyclase (ADPR-cyclase) and is involved in T cell activation (18) and oxytocin secretion, which is closely associated with social behavior (20), and acts mainly as an NAD glycohydrolase therewith regulating intracellular NAD+ levels (2, 40). Studies have also suggested that ADPR-cyclase(s) other than CD38 may exist in kidney, brain, and heart (3, 28), including various cells (3, 4, 27, 28, 40, 41). In addition, a bisphenyl compound has been shown to inhibit kidney ADPR-cyclase, but not CD38, with a high potency (27). ADPR-cyclases generate a Ca2+-mobilizing second messenger, cyclic ADP-ribose (cADPR), from NAD+ (8, 13, 22). The second messenger, cADPR, increases intracellular Ca2+ concentration ([Ca2+]i) through release of Ca2+ from intracellular stores via ryanodine receptor and/or Ca2+ influx through plasma membrane Ca2+ channels (16, 22, 28, 29). Production of cADPR by ADPR-cyclases including CD38 is stimulated by various G protein-coupled receptors (GPCRs) such as receptors for adrenaline (15), angiotensin II (ANG II) (6), acetylcholine (26), and interleukin-8 (IL-8) (29). However, except for IL-8-mediated CD38 activation in lymphokine-activated killer (LAK) cells (29), the molecular mechanism of ADPR-cyclase activation by other GPCRs has not been clarified.

The major renin-angiotensin system effector, ANG II, plays critical roles in cell growth, vascular contraction, migration, and salt water retention (1, 7, 21). Effects of ANG II are mediated by at least two structurally and pharmacologically distinct ANG II type 1 and 2 receptors (AT1R and AT2R, respectively) (7, 21). The physiological and pathophysiological effects of ANG II are mainly exerted by AT1R activation (12, 21, 33) via complex interacting signaling pathways involving primary stimulation of phospholipase C (PLC) and Ca2+ mobilization and secondary activation of protein tyrosine kinase (PTK), extracellular signal-regulated kinases-1 and -2 (Erk1/2), and phosphatidylinositol 3-kinase (PI3K)-dependent kinase Akt (12, 21, 30, 33). ADPR-cyclase activation or cADPR-mediated [Ca2+]i increase by ANG II has also been observed in rat neonatal cardiomyocytes (17) and afferent arterioles (6). However, the molecular mechanism(s) of ADPR-cyclase activation in ANG II signaling remains elusive.

In this study, we elucidated the molecular basis of ADPR-cyclase activation in ANG II signaling in murine mesangial cells (MMCs) utilizing various pharmacological inhibitors, including an inhibitor of kidney ADPR-cyclase. We also examined the cellular responses involving ADPR-cyclase. The results showed that activation of ADPR-cyclase by ANG II involves a sequential activation of PI3K, PTK, and PLC{gamma}1 and induces cell proliferation and protein synthesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents and antibodies. 4,4'-Dihydroxyazobenzene (DHAB), ANG II, LY294002, genistein, 8-bromo-cADPR (8-Br-cADPR), nicotinamide guanine dinucleotide (NGD+), and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Wortmannin, Akt inhibitor, 1L6-hydroxymethyl-chiro-inositol-2-(R)-2-O-methyl-3-O-octadecyl-sn-glycerocarbonate, BAPTA-AM, xestospongin C, BAY K 8644, and PD123319 were purchased from Calbiochem (Darmstadt, Germany). Losartan was purchased from Merck (Whitehouse Station, NJ). Antibodies were obtained from following sources: anti-phospho-Akt (Ser473) monoclonal antibody (mAb), anti-Akt polycolonal antibody (pAb), anti-phospho-Erk1/2 (Thr202/Tyr204) pAb, anti-Erk1/2 pAb, and anti-tyrosine mAb from Cell Signaling Technology (Beverly, MA) and anti-nuclear factor of activated T cell-3 (anti-NFAT3) mAb from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-PLC{gamma}1 mAb was kindly provided by S. G. Rhee (Ewha Women's University, Seoul, South Korea). Horseradish peroxidase-conjugated anti-mouse IgG, anti-goat IgG, and anti-rabbit IgG were purchased from Santa Cruz Biotechnology.

Culture of MMCs. SV40-transformed MMCs (MES-13) were obtained from America Type Culture Collection (ATCC; Rockville, MD) and maintained in DMEM containing 5% fetal bovine serum (FBS), 0.25 µg/ml amphotericin B, 100 units/ml penicillin, and 100 units/ml streptomycin in a humidified incubator at 37°C in presence of 5% CO2 and 95% air. Cells were passaged three times per week.

Measurement of intracellular cADPR concentration and ADPR-cyclase activity. Level of cADPR was measured using a cyclic enzymatic assay as described previously (11, 29). ADPR-cyclase activity was measured using NGD+ as a substrate. Samples were incubated with NGD+ (200 µM) in 0.1 M sodium phosphate buffer (pH 7.2) at 37°C for 10 min. Fluorescence of cGDPR in the solution was determined at excitation/emission wavelengths of 297/410 nm (Hitachi F-2000 fluorescence spectrophotometer).

Measurement of [Ca2+]i. Changes in [Ca2+]i in MMCs were determined as described previously (29). MMCs grown to near confluence were made quiescent by serum deprivation overnight at 37°C. Starved cells were incubated with 5 µM Fluo-3 AM (Molecular Probe, Eugene, OR) in HBSS (2 mM CaCl2, 145 mM NaCl, 5 mM KCl, 1 mM MgCl2, 5 mM D-glucose, and 20 mM HEPES, pH 7.3) at 37°C for 40 min. The cells were washed three times with HBSS. Changes in [Ca2+]i in MMCs were determined at 488 nm excitation/530 nm emission by an air-cooled argon laser system. The emitted fluorescence at 530 nm was collected using a photomultiplier. One image every 4 s for 400 s was scanned using a confocal microscope (Nikon, Japan). For the calculation of [Ca2+]i, the method of Tsien et al. (37) was used with the following equation: [Ca2+]i = Kd(F – Fmin)/(Fmax – F), where dissociation constant (Kd) is 450 nM for Fluo-3, and F is the observed fluorescence levels. Each tracing was calibrated for the maximal intensity (Fmax) by addition of ionomycin (8 µM) and for the minimal intensity (Fmin) by addition of EGTA (50 mM) at the end of each measurement.

Cell proliferation and protein synthesis assay. [3H]thymidine and [3H]leucine incorporation experiments were conducted as described previously (1). Cells were incubated in the serum-free medium with or without 150 nM ANG II in the presence of various concentrations of DHAB for 48 h, and were incubated with 1 µCi of [3H]thymidine or 2 µCi of [3H]leucine for 6 h at 37°C. The cells were then washed once with phosphate-buffered saline (PBS), then treated with ice-cold 5% trichloroacetic acid (TCA) at 4°C for 15 min, and then washed twice in 5% TCA. The acid-insoluble material was dissolved in 2 N NaOH at room temperature and counted for radioactivity by liquid scintillation counting. All experiments were performed in triplicate.

Immunoblotting. Protein extraction and immunoblotting of MMCs were performed as previously described (29). Cytosolic and nuclear fractions were prepared with Pierce NE-PER nuclear and cytoplasmic extraction kit according to the manufacturer's directions (Pierce, Rockford, IL). Proteins (20 µg/lane) were resolved on 10 or 12% SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF; GE Healthcare, Little Chalfont, Buckinghamshire, UK) membranes. Blots were incubated with primary antibodies (phospho-Akt, Akt, phospho-Erk1/2, and Erk1/2, 1:2,500 dilution; NFAT3, 1:1,000 dilution) overnight at 4°C. The blots were rinsed four times with TTBS (Tris-buffered saline with Tween-20) and incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000 dilution of each antibody) for 1 h at room temperature. The binding of the antibodies was visualized using an enhanced chemiluminescence (ECL) system (Bio-Rad, Munich, Germany). Protein concentration was determined using a Bio-Rad protein assay kit, and known concentrations of bovine serum albumin (BSA) were used as the standard.

Immunoprecipitation. Immunoprecipitation of MMC lysates was performed as previously described (29). In brief, cell lysates (600 µg/lane) precleared with protein G-agarose were incubated with anti PLC{gamma}1 mAb at 4°C for 12 h, and then protein G-agarose was added for an additional 1 h. Immunoprecipitates were washed four times with cell lysis buffer (1% Triton X-100, 145 mM NaCl, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, protease inhibitor cocktail, and 20 mM HEPES, pH 7.3) and boiled for 10 min. The proteins immunoprecipitated were resolved by 8% SDS-PAGE and transferred to PVDF membranes. After blocking with 5% skim milk in TTBS, the membrane was treated with an anti-tyrosine mAb (1:2,500 dilution), followed by horseradish peroxidease-conjugaed anti-mouse IgG (1:5,000 dilution).

Statistical analysis. All immunoreactive and phosphorylated signals were analyzed by densitometric scanning (Fuji Photo Film, Tokyo, Japan). Data represent means ± SE of at least three separate experiments. Statistical comparisons were performed using one-way ANOVA followed by Scheffé's test. Statistical significance of difference between groups was determined using the Student's t-test. Statistical significance was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
ANG II induces a sustained rise of [Ca2+]i and production of intracellular cADPR through AT1R. Treatment of MMCs with ANG II (150 nM) induced a sustained rise of [Ca2+]i in the cells (Fig. 1A). The ANG II-mediated rise of [Ca2+]i was mediated by the AT1R, because pretreatment of the cells with losartan (1 µM), an AT1R antagonist, completely abolished the rise of [Ca2+]i, whereas pretreatment with PD123319 (1 µM), an AT2R antagonist, had no effect (Fig. 1, A and B). To examine whether ANG II signaling involves ADPR-cyclase, ANG II-mediated production of cADPR was measured. As shown in Fig. 1C, the production of cADPR was increased by ANG II, reaching maximum level at 60 s and subsequently decreasing with time. ANG II-mediated production of cADPR was significantly abrogated with losartan, but not by PD123319 (Fig. 1D). These data suggest that ANG II stimulates ADPR-cyclase and induces a sustained rise of [Ca2+]i through AT1R.


Figure 1
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  		Fig. 1. Effect of ANG II on the rise of intracellular Ca2+ concentration ([Ca2+]i) and production of intracellular cyclic ADP-ribose (cADPRi) is mediated by ANG II type 1 receptor (AT1R) in mouse mesangial cells (MMCs). A: increases of [Ca2+]i by ANG II were inhibited by pretreatment with losartan (1 µM) but not with PD123319 (1 µM). B: changes in [Ca2+]i at 110 s of A (n = 18). C: kinetics of cADPRi production by ANG II. D: ANG II-stimulated cADPRi production was inhibited with losartan but not with PD123319. Values are means ± SE of 3 independent experiments. *P < 0.01 vs. basal [Ca2+]i or [cADPR]i; #P < 0.01 vs. ANG II.

 
ANG II-induced increase of [Ca2+]i in MMCs requires the activation of ADPR-cyclase. To investigate whether the increase of intracellular Ca2+ induced by ANG II/AT1R entails activation of ADPR-cyclase/cADPR production, an antagonistic cADPR analog, 8-Br-cADPR, and a kidney ADPR-cyclase inhibitor, DHAB, were utilized. As expected, treatment of MMCs with ANG II generated a sustained Ca2+ increase (Fig. 2A ). Pretreatment with 8-Br-cADPR or DHAB abrogated the late phase of the ANG II-induced Ca2+ signals but not the initial spiky Ca2+ rise (Fig. 2, A and B). In support of the data, DHAB inhibited ANG II-mediated cADPR production (Fig. 2C). These results indicate that activation of ADPR-cyclase and, in turn, cADPR production evokes the late-phase Ca2+ mobilization of ANG II-induced Ca2+ signals in MMCs.


Figure 2
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  		Fig. 2. ADPR-cyclase activation by ANG II induces a sustained Ca2+ entry. A: inhibition of ANG II-induced Ca2+ signals by an antagonistic cADPR [8-bromo-cADPR; (8-)Br-cADPR; 100 µM], and dihydroxyazobenzene (DHAB; 10 nM). B: changes in [Ca2+]i of A at 110 s (n = 17). C: ANG II-stimulated cADPR production was inhibited by DHAB. Values are means ± SE of 3 independent experiments. *P < 0.01 vs. basal [Ca2+]i or [cADPR]i; #P < 0.01 vs. ANG II.

 
ANG II induces a sustained rise of [Ca2+]i through opening of L-type Ca2+ channels in MMCs. ANG II induces Ca2+ release from endoplasmic reticulum/sarcoplasmic reticulum and Ca2+ entry through L-type Ca2+ channels via PI3K (31, 39). Pretreatment of MMCs with nifedipine, an L-type Ca2+ channel blocker, or absence of extracellular Ca2+ resulted in complete suppression of the late phase of Ca2+ signal, maintaining the initial spiky Ca2+ rise (Fig. 3, A and B). ANG II-induced Ca2+ signals were completely inhibited by pretreatment with PLC inhibitor (U73122 [GenBank] ), PI3K inhibitor (LY294002), or inositol 1,4,5-trisphosphate receptor (IP3R) blocker (xestospongin C) (Fig. 3, C and D). These results suggest that PLC and PI3K regulate IP3-mediated Ca2+ release, and that the initial spiky Ca2+ rise is a requisite for the late phase of Ca2+ influx via L-type Ca2+ channels. A previous study indicated that ANG II-induced elevation of [Ca2+]i is mediated by PLC{gamma}1 activated by genistein-sensitive PTK (31). Therefore, we evaluated ANG II-stimulated phosphorylation of PLC{gamma}1 with or without genistein, wortmannin, or DHAB. As presented in Fig. 3, E and F, ANG II stimulated tyrosine phosphorylation of PLC{gamma}1, and the tyrosine phosphorylation was completely inhibited by genistein or wortmannin but not by DHAB.


Figure 3
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  		Fig. 3. ANG II-induced elevation of [Ca2+]i and stimulation of ADPR-cyclase involves activation of phosphatidylinositol 3-kinase (PI3K), phospholipase C (PLC), and L-type Ca2+ channels. A: changes in [Ca2+]i mediated by ANG II in the presence or absence of extracellular Ca2+ or by nifedipine (Nife; 10 µM). B: changes in [Ca2+]i at 110 s of A (n = 18). C: ANG II-induced rise of [Ca2+]i was abolished by LY294002 (Ly; 10 µM), U73122 (10 µM), or xestospongin C (Xe C; 2 µM). D: levels of [Ca2+]i at 110 sec of B (n = 20). E: ANG II-mediated tyrosine phosphorylation of PLC{gamma}1 was inhibited by wortmannin (Wort) and genistein (Gen) but not DHAB. F: densitometric analysis of tyrosine phosphorylation levels of PLC{gamma}1 induced by ANG II. G: ANG II-stimulated cADPR production was inhibited by various inhibitors but not Akt inhibitor: wortmannin (Wort; 100 nM), LY294002 (10 µM), U73122 (10 µM), xestospongin C (2 µM), genistein (Gen; 120 µM), and Akt inhibitor (10 µM). H: MMCs were treated with ANG II in the presence or absence of extracellular Ca2+ or after pretreatment with BAPTA-AM (BAPTA; 10 µM). Values are means ± SE. *P < 0.01 vs. basal; #P < 0.01 vs. ANG II.

 
The above data indicate that PLC{gamma}1, PI3K, and IP3 regulate ANG II-mediated Ca2+ signals. An involvement of these molecules in the activation of ADPR-cyclase by ANG II was examined by determining cADPR formation. The inhibitors of PI3K (wortmannin and LY294002), PLC (U73122 [GenBank] ), IP3R (xestospongin C), ADPR-cyclase (DHAB), and PTK (genistein) blocked completely the ANG II-mediated cADPR formation (Fig. 3G). However, an Akt inhibitor did not inhibit the cADPR formation (Fig. 3G). In addition, examination of the Ca2+ effect on the ANG II-mediated cADPR formation showed that depletion of intracellular Ca2+ with BAPTA inhibited the cADPR formation, whereas in the absence of extracellular Ca2+, the cADPR formation was increased to the control level measured in the presence of extracellular Ca2+ (Fig. 3H). These results together suggest that PI3K, PTK, and PLC{gamma}1 are the upstream effectors of ADPR-cyclase in the ANG II signaling pathway, and that activation of ADPR-cyclase requires an intracellular Ca2+ rise, probably the IP3R-mediated Ca2+ rise.

ANG II-induced Akt phosphorylation requires activation of ADPR-cyclase and intracellular Ca2+. To define the effectors, which were stimulated by ADPR-cyclase/cADPR signaling, ANG II-mediated phosphorylation of either Akt or Erk1/2 was evaluated by determining phospho-Akt and phospho-Erk1/2. Treatment of MMCs with ANG II increased the levels of phospho-Akt and phospho-Erk1/2 in a time-dependent manner (Fig. 4, A and B). Pretreatment of the cells with DHAB significantly reduced the level of phospho-Akt but not the level of phospho-Erk1/2 (Fig. 4, C and D). Involvement of ADPR-cyclase signaling in ANG II-induced Akt phosphorylation was further ascertained by using inhibitors of the upstream effectors of ADPR-cyclase. As shown in Fig. 4, E and F, ANG II-mediated Akt phosphorylation was inhibited by LY294002, U73122 [GenBank] , and depletion of intracellular Ca2+ with BAPTA. In the presence of extracellular Ca2+, 8-Br-cADPR inhibited ANG II-stimulated Akt phosphorylation, and cADPR additively enhanced ANG II-induced Akt phosphorylation (Fig. 4, G and H). Moreover, cADPR alone was also able to increase the Akt phosphorylation. On the other hand, neither ANG II nor cADPR stimulated Akt phosphorylation in the absence of extracellular Ca2+ (Fig. 4, I and J). These results suggest that the ADPR-cyclase/cADPR-mediated Ca2+ signal is required for Akt phosphorylation by ANG II.


Figure 4
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  		Fig. 4. ANG II-mediated phosphorylation of Akt, but not Erk, is downstream of ADPR-cyclase signaling, and phosphorylation of Akt requires a rise in [Ca2+]i via cADPR. A: time course of Akt and Erk1/2 phosphorylation induced by ANG II in MMCs. B: densitometric analysis of phospho-Akt and phospho-Erk1/2. C: effect of DHAB on ANG II-induced phosphorylation of Akt and Erk1/2. D: relative ratio of phospho-Akt (or phospho-Erk1/2) to Akt (or Erk1/2) is arbitrarily presented as 1-fold. E: effects of various blockers on ANG II-induced Akt phosphorylation. MMCs were preincubated with LY294002 (10 µM), BAPTA-AM (10 µM), or U73122 (10 µM) for 30 min before stimulation with ANG II for 1 min. F: densitometry analysis of phospho-Akt. G: cADPR additively increases phospho-Akt levels stimulated by ANG II in the presence of extracellular Ca2+ (1.2 mM). H: densitometry analysis of phospho-Akt. I: absence of extracellular Ca2+ abolishes phosphorylation of Akt by ANG II or cADPR. J: densitometry analysis of phospho-Akt. Data are means ± SE of 3 independent experiments. *P < 0.05 vs. basal level of phospho-Akt; {dagger}P < 0.05 vs. basal level of phospho-Erk1/2; #P < 0.05 vs. phospho-Akt by ANG II.

 
DHAB inhibits ANG II-induced NFAT translocation into nuclear. In various cell types including mesangial cells (MCs), an increase in [Ca2+]i induces nuclear translocation of NFATs via activation of calcineurin, a calcium-dependent serine/threonine phosphatase (9, 25, 35, 38, 42). We therefore examined NFAT3 activation, since this transcription factor is known to be activated by ANG II (38). ANG II increased the level of NFAT3 in the nuclear fraction, whereas the level in the cytosol fraction was reciprocally decreased (Fig. 5, A and B). The increase of nuclear NFAT3 level and the decrease of cytosolic NFAT3 level were inhibited by DHAB, calcineurin inhibitor FK506, and wortmannin (Fig. 5, A and B), indicating that the sustained Ca2+ increase by ADPR-cyclase signaling induces NFAT3 activation in MMCs. We also examined whether NFAT activation by ANG II induced cell proliferation or protein synthesis. ANG II increased incorporation of [3H]thymidine and [3H]leucine into MMCs, and DHAB inhibited the incorporation in a dose-dependent manner (Fig. 5, C and D). The ANG II-induced cell proliferation (Fig. 5E) and protein synthesis (Fig. 5F) were inhibited by FK506, wortmannin, and 8-Br-ADPR but not by the Akt inhibitor. These results indicate that ADPR-cyclase/cADPR play roles in ANG II-mediated NFAT3 activation, resulting in induction of cell proliferation and protein synthesis.


Figure 5
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  		Fig. 5. DHAB inhibits ANG II-induced nuclear factor of activated T cell (NFAT) nuclear translocation. A: NFAT3 levels in nuclear and cytosolic fractions were changed in response to ANG II in the presence of FK506, DHAB, or wortmannin. B: densitometric analysis of NFAT levels. *P < 0.01 vs. basal (no drug addition) in cytosolic fraction (no drug addition); #P < 0.05 vs. ANG II (control); {dagger}P < 0.01 vs. control (no drug addition); §P < 0.01 vs. ANG II in nucleus fraction. C: DHAB inhibited ANG II-mediated DNA synthesis. D: DHAB inhibited protein synthesis in a dose-dependent manner. E and F: ANG II-mediated DNA synthesis or protein synthesis was blocked by FK506, wortmannin, or 8-Br-cADPR but not Akt inhibitor. Values are means ± SE of 3 independent experiments. *P < 0.05 vs. control (no drug addition); #P < 0.05 vs. ANG II.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our present studies have elucidated the molecular basis of ADPR-cyclase activation in ANG II signaling and the cellular responses induced by ADPR-cyclase signaling in MMCs. The results for the first time revealed that activation of ADPR-cyclase by AT1R in MMCs is triggered by a sequential activation of PI3K, PTK, PLC{gamma}1, and IP3-mediated Ca2+ increase (Fig. 6). Moreover, the results showed that the activation of ADPR-cyclase by ANG II induces a sustained Ca2+ signal via L-type Ca2+ channels and upregulates NFAT3 nuclear translocation and Akt phosphorylation, including cell proliferation and protein synthesis (Fig. 6). These cellular events mediated by ANG II were attenuated by ADPR-cyclase inhibitor DHAB as well as cADPR antagonist 8-Br-cADPR, suggesting that ADPR-cyclase in MMCs plays a crucial role in ANG II signaling.


Figure 6
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  		Fig. 6. Schematic model of ADPR-cyclase activation and cellular responses involving ADPR-cyclase in ANG II signaling pathway. Stimulation of AT1R by ANG II leads to sequential activation of PI3K, protein tyrosine kinase (PTK), and PLC{gamma}1, in turn causing a Ca2+ release by inositol 1,4,5-trisphosphate receptor (IP3R) from endoplasmic reticulum (ER), resulting in activation of ADPR-cyclase. Activation of ADPR-cyclase induces Ca2+ influx via L-type calcium channels, Akt phosphorylation, NFAT nuclear translocation, cell proliferation, and protein synthesis.

 
Assessment of ANG II-mediated Ca2+ signals showed that ANG II induces two phases of [Ca2+]i rise: the first phase is a transient rise of [Ca2+]i via IP3R through sequential activation of PI3K, PTK, and PLC{gamma}1, and the second phase is a sustained rise of [Ca2+]i via L-type Ca2+ channels through cADPR production by the activation of ADPR-cyclase. Moreover, the initial IP3R-mediated Ca2+ signal appears to be a prerequisite for the second-phase Ca2+ rise, since pretreatment with IP3R blocker xestospongin C and the inhibitors of PI3K, PTK, and PLC completely abolished ANG II-mediated Ca2+ signals. Along the same lines, ANG II-dependent activation of L-type Ca2+ channels via PI3K{gamma} (39) and PI3K-mediated activation of PLC{gamma}1 (31) have been observed in smooth muscle cells and endothelial cells. The molecular mechanism of the activation of L-type Ca2+ channels by cADPR remains completely unknown. However, studies have shown that cADPR induces Ca2+ influx in various cells (13, 16, 28, 29). ANG II-mediated Ca2+ influx through transient receptor potential channels (TRPC) in human glomerular MCs (5, 34) and direct binding of cADPR to transient receptor potential melastatin 2 (TRPM2) (1, 36) have been reported. In addition, a study has indicated that L-type Ca2+ channels are not expressed in MMCs (32). However, we clearly observed the increase in [Ca2+]i in MMCs by KCl as well as by an L-type channel agonist BAY K 8644 (data not shown), and several reports have also indicated the expression of L-type Ca2+ channels in MCs (14, 19, 24).

Examination of the downstream effectors of ADPR-cyclase in ANG II signaling revealed that ADPR-cyclase activation resulted in increases in nuclear NFAT3 level and Akt phosphorylation. The NFAT3 activation, but not Akt activation, by ANG II stimulated cell proliferation and protein synthesis. However, it was of an interest to observe that ANG II-induced Akt phosphorylation required not only PI3K activity but also elevated [Ca2+]i, which was mediated by activation of ADPR-cyclase/cADPR production. In support of the results, Akt phosphorylation has been shown to require both PI3K activity and increased [Ca2+]i in smooth muscle cells (23). Moreover, PI3K-independent activation of Akt/protein kinase B in rat glomerular MCs by ANG II has also been reported (10). Exact cellular responses of Akt in glomerular MCs should be examined in the future.

Finally, biochemical properties of kidney ADPR-cyclase are different from those of prototype ADPR-cyclase, CD38 (3, 4, 27, 28, 41). Discovery of a potent inhibitor of kidney ADPR-cyclase has made possible the elucidation of the involvement of ADPR-cyclase/cADPR in ANG II signaling in MMCs. Our findings have indicated that ADPR-cyclase in MMCs may play a central role in switching the external signaling events to cell proliferation and protein synthesis. Our study may also provide a new therapeutic target, namely ADPR-cyclase, in ANG II-induced pathogenesis in kidney.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Korea Science and Engineering Foundation Grant R01-2005-000-10933-0 (U.-H. Kim and H. J. Kwon) and by the National Research Laboratory Program of the Ministry of Science and Technology (No. M10400000292-06J0000-29210) (S. K. Park). S.-Y. Kim and M.-J. Im were the recipients of Brain Korea 21 Program of the Ministry of Education of Korea. S.-Y. Rah was supported by the Chonbuk National University Postdoctoral Fellow Program (2007) and R. Gul by the foreigner support program of the Korea Research Foundation (KRF-2006-211-E0003).


   ACKNOWLEDGMENTS
 
We thank Dr. S. G. Rhee (Ewha Women's University, Seoul, South Korea) for providing anti-PLC{gamma}1 monoclonal antibody.


   FOOTNOTES
 

Address for reprint requests and other correspondence: U.-H. Kim, Dept. of Biochemistry, Chonbuk National Univ. Medical School, Keum-am dong, Jeonju, 561-182, Republic of Korea (e-mail: uhkim{at}chonbuk.ac.kr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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