Dopamine D₂ receptors' effects on renal inflammation are mediated by regulation of PP2A function

Abstract

Lack or downregulation of the dopamine D₂ receptor (D₂R) results in increased renal expression of injury markers and proinflammatory factors that is independent of a blood pressure increase. This study aimed to determine the mechanisms involved in the regulation of renal inflammation by D₂Rs. Silencing D₂Rs in mouse renal proximal tubule cells increased the expression of the proinflammatory TNF-α, monocyte chemoattractant protein-1 (MCP-1), and IL-6. D₂R downregulation also increased Akt phosphorylation and activity, and glycogen synthase kinase-3β (GSK3β) phosphorylation and cyclin D1 expression, downstream targets of Akt; however. phosphatidylinositol 3-kinase (PI3K) activity was not affected. Conversely, D₂R stimulation decreased Akt and GSK3β phosphorylation and cyclin D1 expression. Increased phospho-Akt, in the absence of increased PI3K activity, may result from decreased Akt dephosphorylation. Inhibition of protein phosphatase 2A (PP2A) with okadaic acid reproduced the effects of D₂R downregulation on Akt, GSK3β, and cyclin D1. The PP2A catalytic subunit and regulatory subunit PPP2R2C coimmunoprecipitated with the D₂R. Basal phosphatase activity and the expression of PPP2R2C were decreased by D₂R silencing that also blunted the increase in phosphatase activity induced by D₂R stimulation. Similarly, silencing PPP2R2C also increased the phosphorylation of Akt and GSK3β. Moreover, downregulation of PPP2R2C resulted in increased expression of TNF-α, MCP-1, and IL-6, indicating that decreased phosphatase activity may be responsible for the D₂R effect on inflammatory factors. Indeed, the increase in NF-κB reporter activity induced by D₂R silencing was blunted by increasing PP2A activity with protamine. Our results show that D₂R controls renal inflammation, at least in part, by modulation of the Akt pathway through effects on PP2A activity/expression.

independent of innervation, the kidney synthesizes dopamine that is important in the regulation of renal function and blood pressure (1). The renal dopaminergic system also regulates the production of reactive oxygen species and the inflammatory reaction (2, 3, 11, 52, 53, 56), both involved in the development of renal injury and ultimately in the induction and maintenance of hypertension (17). Mice with intrarenal dopamine deficiency have increased oxidative stress, as well as increased inflammatory and tubular injury markers in the kidney (55), and decreased renal dopamine production aggravates angiotensin II-mediated renal injury (52).

We have shown that the dopamine D₂ receptor (D₂R) in the kidney has a direct and significant role in regulating the mechanisms involved in the development of renal inflammation and injury, as well as in blood pressure control (2, 28). Mice with a lack or reduced level of D₂R expression and function in the kidney have increased renal expression of proinflammatory and decreased expression of anti-inflammatory cytokines/chemokines, as well as histological and functional evidence of renal inflammation and injury. These findings suggest that the D₂R has protective effects in the kidney by limiting the inflammatory reaction and that impaired function of the D₂R results in renal inflammation and organ damage (56).

Renal proximal tubule cells (RPTCs) produce both pro- and anti-inflammatory cytokines and chemokines (40), which are secreted across their apical and basolateral membranes (49) and contribute to the development and progression of glomerular and tubular injury. However, the factors that regulate cytokine production in these cells are incompletely understood. We have shown that the D₂R is one of the factors that regulate cytokine production in mouse RPTCs. Silencing the D₂R in RPTCs in culture increased the expression of proinflammatory factors while D₂R stimulation reduced the inflammatory effects of angiotensin II (56). Moreover human RPTCs bearing single nucleotide polymorphisms of the D₂R that result in decreased expression of the receptor have increased expression of inflammatory factors and a profibrotic phenotype (23).

The present study was aimed at determining the mechanisms involved in the D₂R regulation of cytokine production in mouse RPTCs. Our results show that the protective effects of the D₂R on renal inflammation are, at least in part, mediated by modulation of the Akt pathway, through effects on protein phosphatase 2A (PP2A) activity and expression.

MATERIALS AND METHODS

Cell culture.

Undifferentiated mouse renal cells were cultured from progenitor kidney cells, kindly supplied by Dr. Ulrich Hopfer (School of Medicine, Case Western Reserve University), isolated from mouse embryo kidneys and maintained following the procedure described by Woost et al. (50). The cells that differentiated to mouse RPTCs were maintained in DMEM/F-12 (Invitrogen) with 10% fetal bovine serum, 100 IU/ml penicillin, 100 IU/ml streptomycin, and 250 μg/ml amphotericin B. Cells with low passage numbers (<18) were used to avoid the confounding effects of cellular senescence. Mouse RPTCs were cultured to 60–70% confluence and transfected (Hyperfect, Qiagen, Valencia, CA) with non-silencing small interfering (si) RNA (30 nmol/l; All stars, Qiagen), Drd2 siRNA (30 nmol/l, Qiagen), or PP2A catalytic subunit and regulatory subunit (PPP2R2C) siRNA (30 nmol/l, Qiagen). The cells were harvested after 72 h of treatment. The cell lines tested negative for Mycoplasma infection.

D₂R-deficient mice.

The original F2 hybrid strain (129/SvXC57BL/6J, Oregon Health Sciences University) that contained the mutated Drd2 allele (D₂^−/−) was bred onto the C57BL/6J background for >20 generations (28). All animal-related studies were approved by the Institutional Animal Care and Use Committee. D₂^−/− mice and their wild-type littermates (D₂^+/+) were studied at 6–8 mo of age.

Coimmunoprecipitation.

Coimmunoprecipitation was performed using an Immunoprecipitation kit (protein G, Roche Applied Science, Indianapolis, IN). The mouse RPTC lysates were prepared using lysis buffer (50 mm Tris·HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate) with proteinase inhibitors. Equal amounts of cell lysates (500 μg protein) were mixed with normal rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA) as a negative control, monoclonal anti-D₂R (Abnova, Walnut, CA), monoclonal anti-PPP2R2C (Novus Biologicals, Littleton, CO), or monoclonal PP2A catalytic subunit (Millipore, Billerica, MA). Protein G-agarose beads (Roche) were added and incubated overnight. The immune complexes were pelleted out, and the bound proteins were eluted using 30 μl of Laemmli buffer. The samples were subjected to immunoblotting using a polyclonal anti-PPP2R2C antibody (Novus Biologicals), polyclonal anti-PP2A catalytic subunit antibody (Upstate Biotechnology, Lake Placid, NY), or polyclonal anti-D2R (Millipore).

Immunoblotting.

Mouse RPTC lysates were subjected to immunoblotting as described previously (2, 23, 56). The primary antibodies used were rabbit polyclonal p-Akt (Thr 308, Abcam, Cambridge, MA); rabbit polyclonal anti-Akt (Abcam); rabbit polyclonal anti-D₂R (Millipore); monoclonal anti-glycogen synthase kinase-3β (GSK3β; Cell Signaling Technology, Danvers, MA); rabbit polyclonal anti-p-GSK3β (Cell Signaling Technology); monoclonal anti-cyclin D1 (Cell Signaling Technology); rabbit polyclonal anti-TNF-α (Abcam); rabbit polyclonal anti-monocyte chemoattractant protein-1 (MCP-1; Millipore); rabbit polyclonal anti-IL-6 (Abcam); polyclonal anti-GAPDH (Sigma-Aldrich, St. Louis, MO); monoclonal anti-PPP2R2C (Novus Biologicals); and polyclonal anti-PP2A catalytic subunit (Millipore). The densitometry values were corrected by the expression of GAPDH and are shown as the percentage of the mean density of the control group.

Quantitative real-time PCR.

Total RNA was purified using the RNeasy RNA Extraction Mini kit (Qiagen, Valencia, CA). RNA samples were converted into first-strand cDNA using an RT2 First Strand kit, following the manufacturer's protocol (Qiagen). Quantitative gene expression was analyzed by real-time PCR, performed on an ABI Prism 7900 HT (Applied Biosystems, Foster City, CA). The assay used gene-specific primers (Qiagen) and the SYBR Green real-time PCR detection method (Qiagen) and was performed as described in the manufacturer's manual. Primers used were as follows: TNF-α: PPM03113F; MCP-1: PPM03151F; and IL-6 and GAPDH: PPM02946E. Data were analyzed using the ΔΔCt method (29).

Whole cell phosphorylation assay.

Phosphatase activity was measured using the SensoLyte FDP Protein Phosphatase Assay Kit (AnaSpec, Freemont, CA), which is optimized to detect protein phosphatase activity using 3,6-fluorescein diphosphate (FDP) as a fluorogenic phosphatase substrate, following the manufacturer's procedures. Mouse RPTCs were treated with vehicle or okadaic acid (2 nM) for 60 min. Another set of mouse RPTCs was transfected with mouse D₂R siRNA or non-silencing siRNA and grown to 90% confluence and then treated with the D₂R agonist quinpirole (1 μM) at three time points (0, 30, and 60 min). Thereafter, the cells were lysed and protein phosphatase-containing lysates were placed in 50 μl/well and mixed the same volume of FDP reaction solution by shaking gently for 30 s. The signal of fluorescein as the final hydrolytic product of FDP was read by a Victor³ multilabel reader at Ex/Em = 485 ± 20/528 ± 20 nm. All assays were performed in duplicate.

Akt kinase assay.

The Akt kinase activity was measured using an AKT Kinase Assay Kit (Cell Signaling Technology). The D₂R- transfected cells were washed twice in cold phosphate-buffered saline and then lysed on ice. The extracts were centrifuged to remove the cellular debris. Equal amounts of lysates (300 μg protein) were incubated with gentle rocking at 4°C overnight with immobilized anti-Akt antibody cross-linked to agarose hydrazide beads. After Akt was selectively immunoprecipitated from the cell lysates, the immunoprecipitated products were washed and the samples were resuspended in 50 μl of kinase assay buffer containing 200 μm ATP and 1 μg of GSK3α fusion protein, according to the manufacturer's instructions. The kinase reaction was allowed to proceed at 30°C for 30 min and stopped by the addition of Laemmli SDS sample buffer. The reaction products were resolved by 10% SDS-PAGE followed by Western blotting with an anti-phospho-GSK3α/β antibody and anti-horseradish peroxidase-conjugated anti-rabbit antibody. All assays were performed in duplicate.

Phosphatidylinositol 3-kinase assay.

Mouse RPTCs grown at 60–80% confluence were washed in ice-cold buffer and lysed in 200 μl ice-cold lysis buffer. Phosphatidylinositol 3-kinase (PI3K) was immunoprecipitated with 5 μl of rabbit antibody against full-length PI3K (Millipore), which precipitates the p110 catalytic subunit of PI3K, and 60 μl of protein A-Sepharose beads (Roche Applied Science). PI3K activity in the immunoprecipitates was measured with a PI3K ELISA (Echelon Biosciences, Salt Lake City, UT), according to the manufacturer's instructions. Briefly, immunoprecipitated enzyme and PI(4,5)P₂ substrate were incubated for 1 h at room temperature in the reaction buffer. The kinase reaction was stopped by pelleting the beads by centrifugation and incubation overnight at 4°C with a PI(3,4,5)P₃ detector protein, then added to the PI(3,4,5)P₃-coated microplate for 1 h for competitive binding. A peroxidase-linked secondary detection reagent and colorimetric detection (absorbance was measured at 450 nm) was used to detect PI(3,4,5)P₃ detector protein binding to the plate. The colorimetric signal is inversely proportional to the amount PI(3,4,5)P₃ produced by PI3K.

Reporter assay.

NF-κB activation was analyzed via the transient expression of an NF-κB luciferase reporter system (Cignal Reporter Assay, Qiagen). Cells were treated with D₂R-specific siRNA or non-silencing siRNA, as described above. After 48 h, the cells were treated with the PP2A activator protamine (5 μM) or vehicle and seeded for transfection with the NF-κB luciferase reporter construct. The assay was performed following the manufacturer's procedures.

Statistical analysis.

The data are expressed as means ± SE. Comparisons between two groups used the Student's t-test. One-way ANOVA followed by the Newman-Keuls test was used to assess significant differences in more than two groups. P < 0.05 was considered significant.

RESULTS

D₂R regulates the expression of proinflammatory factors and Akt activity in mouse proximal tubule cells.

In mouse RPTCs, treatment with D₂R siRNA significantly decreased D₂R expression compared with cells treated with non-silencing siRNA (Fig. 1). Downregulation of the D₂R increased the mRNA expression of TNF-α, MCP-1, and IL-6 in these cells (Fig. 1), as we have reported previously in mouse renal cortex (56).

Fig. 1.

The dopamine D₂ receptor (D₂R) inhibits expression of proinflammatory factors. Expression of D₂R determined by Western blotting and expression of TNF-α, monocyte chemoattractant protein-1 (MCP-1), and IL-6 mRNA in mouse renal proximal tubule cells (RPTCs) transfected with non-silencing small interference RNA (NS siRNA) or D₂R siRNA is shown. After 48 h, the cells were washed and lysed. mRNA expression was determined by qRT-PCR. Results were corrected for expression of GAPDH mRNA and expressed as fold-change compared with the expression in NS-transfected cells. *P < 0.05 vs. NS; n = 4/group.

The PI3K-Akt pathway is one of the main pathways involved in the inflammatory response (10). We found that Akt phosphorylation and activity were higher in mouse RPTCs transfected with D₂R siRNA than in cells transfected with non-silencing siRNA. The ratio of phosphorylated Akt to total Akt was also increased in the renal cortex of D₂^−/− mice compared with D₂^+/+ littermates (245 ± 30 vs .100 ± 10%; P < 0.03). Phosphorylation of GSK3β and expression of cyclin D1, downstream targets of Akt, were also increased in mouse RPTCs, indicating activation of the Akt pathway. However, this finding did not correlate with PI3K activity in mouse RPTCs, in which the D₂R was silenced: PI3K activity was not different between RPTCs with silenced D₂R and those with no D₂R downregulation (Fig. 2A). To confirm that the effects on Akt are not dependent on PI3K activity, mouse RPTCs with silenced D₂Rs were treated with the PI3K inhibitor LY294002. LY 294002 had no significant effect on the increased phosphorylation of Akt and GSK3β (Fig. 2B), indicating that the D₂R regulates Akt function independently of PI3K activity.

Fig. 2.

The D₂R regulates Akt activity. A: mouse RPTCs were transfected with NS siRNA or D₂R siRNA. After 72 h, the cells were washed and lysed. Protein expression of Akt, phosphorylated Akt (p-Akt), glycogen synthase kinase-3β (GSK3β), phosphorylated GSK3β (p- GSK3β), and cyclin D1 were quantified by immunoblotting. Akt and PI3K activities were determined by enzymatic assays. Inset: 1 set of immunoblots. Results of p-Akt/Akt, Akt activity, p-GSK3 β/GSK3β, phosphatidylinositol 3-kinase (PI3K) activity, and cyclin D1/actin are expressed as percentage of NS siRNA. *P < 0.05 vs. NS siRNA; n = 4/group. B: mouse RPTCs were transfected with NS siRNA or D₂R siRNA for 48 h after which they were treated for 24 h with vehicle or LY294002 (1 μM). Protein expression of Akt, p-Akt, GSK3β, and p-GSK3β were quantified by immunoblotting. Results of p-Akt/Akt and p-GSK3 β/GSK3β are expressed as percentage of NS siRNA. Black vertical lines in the blots denote rearrangement to better reflect the results shown in the bar graphs. *P < 0.05 vs. NS siRNA; n = 3–4/group. C: mouse RPTCs were treated with the D₂R agonist quinpirole for the indicated time periods. Protein expression of Akt, p-Akt, GSK3β, p-GSK3β, and cyclin D1 were determined as above. Results of p-Akt/Akt, Akt activity, p-GSK3 β/GSK3β, PI3K activity, and cyclin D1/actin are expressed as percentage of time 0. *P < 0.05 vs. time 0; n = 4/group.

In contrast, treatment of mouse RPTCs with quinpirole, a D₂R agonist, decreased Akt phosphorylation as well as activity as shown by decreased GSk3β phosphorylation and expression of cyclin D1 (Fig. 2C).

D₂R regulates protein phosphatase activity.

Akt activity is the result of the balance between its phosphorylation-activation by PI3K-mediated signaling and its dephosphorylation by protein phosphatases (19). Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase that has been shown to negatively regulate Akt activity in several tissues (4, 30, 47) and is expressed in renal proximal tubules (54). Inhibition of PP2A activity with okadaic acid, at a concentration that does not affect other phosphatases (2 nM) (12), decreased total protein phosphatase activity by 28% and increased phosphorylated Akt and GSK3β, indicating that PP2A is involved in Akt dephosphorylation in mouse RPTCs (Fig. 3A). These results demonstrate that PP2A is involved in the regulation of the Akt-GSK3β pathway. To determine whether D₂R function affects PP2A activity, we treated mouse RPTCs with a D₂R agonist. The stimulation of D₂Rs resulted in a significant (22%) increase in total phosphatase activity at 15 min of agonist treatment (Fig. 3B). In contrast, silencing the D₂R in these cells decreased basal phosphatase activity by 20% and abolished the increase in activity induced by the D₂R agonist (Fig. 3B). These results indicate that D₂R function is associated with an increase in PP2A phosphatase activity.

Fig. 3.

The D₂R regulates phosphatase activity. A: mouse RPTCs were treated with vehicle or with the PP2A inhibitor okadaic acid (2 nM). Phosphatase activity was assayed using a fluorogenic substrate as described in materials and methods. Protein expression of Akt, p-Akt, GSK3β, and p-GSK3β was quantified by immunoblotting. Inset: 1 set of immunnoblots. Results are expressed as percentage of vehicle. *P < 0.05 vs. vehicle; n = 5/group. B: mouse RPTCs transfected with NS siRNA or D₂R siRNA were treated with quinpirole (1 μM) or vehicle for the indicated time periods. Phosphatase activity was determined as above. Results are expressed as percentage of NS siRNA at time 0. *P < 0.05 vs. basal 0; n = 4/group.

D₂R regulates PPP2R2C, a regulatory subunit of PP2A.

PP2A consists of a family of heterotrimers composed of one scaffolding (A), one catalytic (C), and one regulatory (B) subunit. There are several regulatory B subunits of which PPP2R2C, a γ-isoform of the subunit B55 subfamily, has been reported to associate with Akt selectively, regulating phosphorylation of Akt at Thr-308 (25). We evaluated the interaction between the D₂R and PP2A catalytic subunit and PPP2R2C. Total mouse RPTC lysates were immunoprecipitated using a monoclonal anti-D₂R antibody, and the eluates were subsequently immunoblotted with a PPP2R2C or a PP2A catalytic subunit antibody. As shown in Fig. 4A, one band was visualized, which corresponded to PPP2R2C, as a similar band was obtained when the whole cell lysate was immunoprecipitated with anti-PPP2R2C antibody. No coimmunoprecipitation was observed when normal rabbit IgG was used as the immunoprecipitant. Similarly, the PP2A catalytic subunit also coimmunoprecipitated with the D₂R although to a lesser extent (Fig. 4B). Total cell lysates were also immunoprecipitated using anti D₂R, PPP2R2C, and PP2A catalytic subunit antibodies and immunoblotted for the D₂R. Both PPP2R2C and PP2A immunoprecipitated the D₂R, but PP2A did it to a lesser extent. As shown by the bands obtained, ∼25% of the total D2R was immunoprecipitated by PPP2R2C but only <15% was immunoprecipitated by PP2A (Fig. 4C).

Fig. 4.

The D₂R interacts with protein phosphatase 2A (PP2A). A: whole RPTC lysates were immunoprecipitated with anti-D₂R antibody and immunostained with anti-PP2A regulatory subunit PPP2R2C. negative control (NC), mouse IgG; positive control (PC), anti-PPP2R2C and whole cell lysate. B: whole RPTC lysates were immunoprecipitated with anti-D₂R antibody anti-PP2A catalytic subunit. NC: mouse IgG; PC, anti-PP2A catalytic subunit. C: whole cell lysates were immunoprecipitated with anti-D₂R antibody, anti-PP2A catalytic subunit and anti-PPP2R2C, and immunoblotted with anti-D₂R. NC, mouse IgG. These experiments were repeated 3 times with similar results. D and E: mouse RPTCs were transfected with NS siRNA or D₂R siRNA for 72 h. Protein expression of PPP2R2C and PP2A catalytic subunit was quantified by immunoblotting. Inset: 1 set of immunoblots. Results are expressed as percentage of NS siRNA. *P < 0.05 vs. NS siRNA; n = 4/group.

We next assessed the effect of the D₂R on the expression of PPP2R2C and the PP2A catalytic subunit by silencing the D₂R in mouse RPTCs. We found that PPP2R2C expression was decreased in mouse RPTCs transfected with Drd2 siRNA for 72 h (Fig. 4D). However, silencing the D₂R did not modify the expression of the PP2A catalytic subunit (Fig. 4E). These results may be taken to suggest that D₂R and PPP2R2C physically and functionally interact in mouse RPTCs. Further experimental proof is necessary to confirm D2R and PPP2R2C physical interaction.

PPP2R2C silencing results in increased Akt phosphorylation and expression of proinflammatory factors.

Treatment of mouse RPTCs with PPP2R2C siRNA significantly decreased PPP2R2C expression (Fig. 5A). The expression of phosphorylated Akt (2.2-fold), GSK3β (2-fold), and cyclin D1(1.7-fold) was increased in mouse RPTCs transfected with PPP2R2C siRNA for 48 h compared with mouse RPTCs transfected with no-silencing siRNA (Fig. 5A), indicating that PPP2R2C is important in the dephosphorylation of Akt.

Fig. 5.

Silencing PPP2R2C increases Akt activity and proinflammatory factors. A and B: mouse RPTCs were transfected with NS siRNA and PPP2R2C siRNA. Protein expressions of D₂R, Akt, p-Akt, GSK3β, p-GSK3β, and cyclin D1 (A) and TNF-α, MCP-1, and IL-6 (B) were quantified by immunoblotting. Inset: 1 set of immunnoblots Results are expressed as percentage of NS siRNA. *P < 0.05 vs. NS siRNA; n = 5/group.

We next determined whether PPP2R2C silencing and the associated increase in the Akt-GSK3β pathway reproduce the effects of D₂R silencing on the expression of proinflammatory factors. The expression of TNF-α (1.4-fold), MCP-1 (2.1-fold), and IL-6 (1.3-fold) was increased in mouse RPTCs transfected with PPP2R2C siRNA for 48 h (Fig. 5B).

Increasing PP2A activity blunts the increase in NF-κb reporter activity induced by D₂R silencing.

We have shown that in mouse RPTCs D₂R silencing increases NF-κB reporter activity (56). To determine the role of PP2A in the increase in NF-κB reporter activity induced by D₂R silencing, we used a PP2A activator (protamine, 5 μM) to increase PP2A activity. The stimulation of PP2A activity did not affect NF-κB reporter activity in cells treated with non-silencing siRNA but almost completely abolished the increase in the activity in cells with D₂R downregulation (Fig. 6), indicating that for the most part the increase in NF-κB reporter activity was related to the decreased PP2A activity.

Fig. 6.

Increasing PP2A activity blunts the increase in NF-κB activity induced by D₂R silencing. Mouse RPTCs were transfected with NS siRNA or D₂R siRNA for 48 h and treated with vehicle or protamine (5 μM, PP2A activator). NF-κB activation was analyzed via the transient expression of an NF-κB-luciferase reporter system by reverse transfection. Results are expressed as fold-activation compared with vehicle treatment. *P < 0.05 vs. NS siRNA; n = 4/group.

DISCUSSION

The results of this study show that the protective effects of the D₂R on renal production of inflammatory factors are exerted at least in part through enabling Akt dephosphorylation by PP2A. Decrease or lack of D₂R function leads to enhanced Akt phosphorylation. Downregulation of the D₂R is also associated with decreased expression of the PP2A regulatory subunit PPP2R2C that may physically interact with the D₂R, indicating that the D₂R is required for normal renal PP2A activity, termination of Akt signaling (Akt dephosphorylation), and inhibition of NF-κB (Fig. 7).

Fig. 7.

Schematic pathway of the protective effects of the D₂R on renal inflammation. The protective effects of the D₂R on renal production of inflammatory factors are exerted at least in part through enabling Akt dephosphorylation by PP2A. Decrease or lack of D₂R function leads to enhanced Akt phosphorylation. Downregulation of the D₂R is also associated with decreased expression of the PP2A regulatory subunit PPP2R2C that physically interacts with the D₂R, indicating that the D₂R is required for normal renal PP2A activity, termination of Akt signaling (Akt dephosphorylation), and inhibition of NF-κB.

The PI3K-Akt pathway is one of the pathways involved in the activation of NF-κB. Akt is activated by phosphorylation at its threonine (Thr 308) and serine (Ser 473) residues (35). Activated Akt activates IkB kinase, which phosphorylates IkB-α, the endogenous inhibitor of NF-κB, leading to the release and nuclear translocation of NF-κB (33). Activation and nuclear translocation of the transcription factor NF-κB are two of the mechanisms that regulate the expression of numerous genes related to inflammation such as cytokines/chemokines (27). NF-κB activation has been documented in vivo and in vitro in intrinsic glomerular cells, such as podocytes and mesangial, tubular, and endothelial cells, in renal injury or after exposure to inflammatory stimuli. Many stimuli activate canonical NF-κB in cultured renal cells to regulate the transcription of multiple proinflammatory molecules (16). In renal cells, NF-κB is activated by TNF-α and mediates the inflammatory response to TNF-α and IL-1 (37), generating a positive-feedback loop of activation (13).

Downregulation of the D₂R in mouse RPTCs is associated with increased NF-κB activation and expression of proinflammatory factors that are dependent on NF-κB activity, consistent with increased Akt phosphorylation and activity, demonstrated by effects on downstream targets. Because both RPTCs and microdissected renal proximal tubules, in culture, produce significant amounts of dopamine from l-DOPA present in the serum added to the medium (20), the D₂R in RPTCs is activated even in the absence of ligand added to the medium. The D₂R may also be constitutively active (24). In the pituitary gland, D₂R constitutively inhibits prolactin secretion (39).

Akt is dephosphorylated and inactivated by PP2A. PP2A is a multimeric phosphoprotein phosphatase composed of one scaffolding, one catalytic, and one regulatory (B) subunit (22, 30, 31, 48). The regulatory subunit determines subcellular localization, substrate specificity, signaling complexes, fine-tuning of enzyme activity, and controls the access of the substrate to the catalytic pocket of the catalytic subunit (22, 43, 48, 51). The diversity of PP2A regulatory subunits allows multiple patterns of assembly conferring spatial and temporal precision to PP2A-mediated dephosphorylation, and distinct cellular functions to different forms of PP2A (21, 43). Inactivation of Akt by dopamine can be prevented by inhibiting PP2A (4). In mouse RPTCs, inhibition of PP2A increased the phosphorylation of Akt and GSK3β, indicating that in these cells Akt activity is regulated by this phosphatase. Furthermore, D₂R stimulation increased PP2A activity, suggesting that in renal cells D₂R modulates the Akt-GSK3β pathway through regulation of PP2A activity. In the mouse striatum (4, 8, 18) and pituitary lactotrophs (35), rat frontal cortex (45), culture undifferentiated human neuroblastoma cells (14), gastric cancer cells (15), and zebrafish brain (44), D₂R stimulation results in the inactivation of Akt. However, there are several reports of activation of the Akt-GSK3β pathway by D₂R stimulation (9, 32, 36). Beaulieu et al. (6) have proposed that this apparent discrepancy may arise from the intrinsic temporal dynamics of the signaling mechanisms involved, with Akt inactivation mediated by β-arrestin occurring at periods later than responses mediated by G proteins and associated with Akt activation.

The D₂R facilitates Akt inactivation by promoting the formation of a signaling complex with Akt, β-arrestin 2, and PP2A (4, 5, 7). Consistent with this proposed pathway, we found that D₂R coimmunoprecipitated with both the catalytic and PPP2R2C regulatory subunits of PP2A. D₂R downregulation also resulted in decreased PPP2R2C expression, presumably by increased degradation, but did not affect the expression of the catalytic subunit. Moreover, downregulation of PPP2R2C increased phosphorylated Akt and Akt-GSK3β pathway activity reproducing the effects of D₂R silencing, suggesting that the effects of D₂R downregulation on the expression of proinflammatory factors in mouse RPTCs are related to PP2A/PPP2R2C activity. It is possible that in the absence of the D₂R the assembly of the signaling complex and consequently its function are impaired. Further evidence of the role of PP2A was provided by studying the effects of increasing PP2A activity on NF-κB promoter activity. As we have reported (56), D₂R downregulation increases NF-κB promoter activity in mouse RPTCs. This increase is almost completely reversed when PP2A activity is increased, supporting the hypothesis that the protective effects of D₂R on renal inflammation are mediated by PP2A/PPP2R2C regulation of Akt phosphorylation.

There is some evidence that the D₂Rs have protective effects in organs other than the kidney. In the respiratory tract, D₂R activation inhibits neurogenic inflammation (34). GLC756, a D₁R antagonist/D₂R agonist, inhibited TNF-α release from rat mast cells, suggesting that it reduced inflammation triggered by activated cells (26). In a mouse model of amyotrophic lateral sclerosis, a D₂R agonist suppresses glial inflammation and moderates the progression of the disease (46). Lack of the D₂R is associated with an inflammatory response in multiple central nervous system regions resulting from the hyperresponse of astrocytes to immune stimuli, indicating that astrocytic D₂R activation normally suppresses neuroinflammation. The D₂R modulation of the innate immune response in astrocytes is mediated by αB-crystalin and is also associated with increased GSK3β but not Akt phosphorylation (41).

Individuals carrying D₂R polymorphisms that result in decreased expression of the D2R may be more vulnerable to renal injury (23) when challenged with an insult such as elevated blood pressure. The results of this study may provide the basis for targeted pharmacological treatment to ameliorate the insult, which should result in decreased prevalence of renal injury and chronic kidney disease.

GRANTS

The work was funded by National Institutes of Health Grants P01HL074940, P01HL068686, R01HL092196, R37HL023081, R01DK039308, and R01DK090918.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Author contributions: Y.Z. and S.C. performed experiments; Y.Z. and S.C. analyzed data; Y.Z. and X.J. interpreted results of experiments; Y.Z., X.J., and S.C. prepared figures; Y.Z., X.J., C.Q., and S.C. drafted manuscript; C.Q., P.A.J., and I.A. edited and revised manuscript; C.Q., P.A.J., and I.A. approved final version of manuscript; I.A. provided conception and design of research.