Quansheng Lu

Dopamine 5 receptor mediates Ang II type 1 receptor degradation via a ubiquitin-proteasome pathway in mice and human cells

Hypertension is a multigenic disorder in which abnormal counterregulation between dopamine and Ang II plays a role. Recent studies suggest that this counterregulation results, at least in part, from regulation of the expression of both the antihypertensive dopamine 5 receptor (D5R) and the prohypertensive Ang II type 1 receptor (AT1R). In this report, we investigated the in vivo and in vitro interaction between these GPCRs. Disruption of the gene encoding D5R in mice increased both blood pressure and AT1R protein expression, and the increase in blood pressure was reversed by AT1R blockade. Activation of D5R increased the degradation of glycosylated AT1R in proteasomes in HEK cells and human renal proximal tubule cells heterologously and endogenously expressing human AT1R and D5R. Confocal microscopy, Förster/fluorescence resonance energy transfer microscopy, and fluorescence lifetime imaging microscopy revealed that activation of D5R initiated ubiquitination of the glycosylated AT1R at the plasma membrane. The regulated degradation of AT1R via a ubiquitin/proteasome pathway by activation of D5R provides what we believe to be a novel mechanism whereby blood pressure can be regulated by the interaction of 2 counterregulatory GPCRs. Our results therefore suggest that treatments for hypertension might be optimized by designing compounds that can target the AT1R and the D5R.

Response Gene to Complement 32, a Novel Regulator for Transforming Growth Factor-β-induced Smooth Muscle Differentiation of Neural Crest Cells

We previously developed a robust in vitro model system for vascular smooth muscle cell (VSMC) differentiation from neural crest cell line Monc-1 upon transforming growth factor-β (TGF-β) induction. Further studies demonstrated that both Smad and RhoA signaling are critical for TGF-β-induced VSMC development. To identify downstream targets, we performed Affymetrix cDNA array analysis of Monc-1 cells and identified a gene named response gene to complement 32 (RGC-32) to be important for the VSMC differentiation. RGC-32 expression was increased 5-fold after 2 h and 50-fold after 24 h of TGF-β induction. Knockdown of RGC-32 expression in Monc-1 cells by small interfering RNA significantly inhibited the expression of multiple smooth muscle marker genes, including SM α-actin (α-SMA), SM22α, and calponin. Of importance, the inhibition of RGC-32 expression correlated with the reduction of α-SMA while not inhibiting smooth muscle-unrelated c-fos gene expression, suggesting that RGC-32 is an important protein factor for VSMC differentiation from neural crest cells. Moreover, RGC-32 overexpression significantly enhanced TGF-β-induced α-SMA, SM22α, and SM myosin heavy chain promoter activities in both Monc-1 and C3H10T1/2 cells. The induction of VSMC gene promoters by RGC-32 appears to be CArG-dependent. These data suggest that RGC-32 controls VSMC differentiation by regulating marker gene transcription in a CArG-dependent manner. Further studies revealed that both Smad and RhoA signaling are important for RGC-32 activation.

D1-Like Receptors Regulate NADPH Oxidase Activity and Subunit Expression in Lipid Raft Microdomains of Renal Proximal Tubule Cells

NADPH oxidase (Nox)–dependent reactive oxygen species production is implicated in the pathogenesis of cardiovascular diseases, including hypertension. We tested the hypothesis that oxidase subunits are differentially regulated in renal proximal tubules from normotensive and spontaneously hypertensive rats. Basal Nox2 and Nox4, but not Rac1, in immortalized renal proximal tubule cells and brush border membranes were greater in hypertensive than in normotensive rats. However, more Rac1 was expressed in lipid rafts in cells from hypertensive rats than in cells from normotensive rats; the converse was observed with Nox4, whereas Nox2 expression was similar. The D1-like receptor agonist fenoldopam decreased Nox2 and Rac1 protein in lipid rafts to a greater extent in hypertensive than in normotensive rats. Basal oxidase activity was 3-fold higher in hypertensive than in normotensive rats but was inhibited to a greater extent by fenoldopam in normotensive (58±3.3%) than in hypertensive rats (31±5.2%; P<0.05; n=6 per group). Fenoldopam decreased the amount of Nox2 that coimmunoprecipitated with p67phox in cells from normotensive rats. D1-like receptors may decrease oxidase activity by disrupting the distribution and assembly of oxidase subunits in cell membrane microdomains. The cholesterol-depleting reagent methyl–β-cyclodextrin decreased oxidase activity and cholesterol content to a greater extent in hypertensive than in normotensive rats. The greater basal levels of Nox2 and Nox4 in cell membranes and Nox2 and Rac1 in lipid rafts in hypertensive rats than in normotensive rats may explain the increased basal oxidase activity in hypertensive rats.

Upregulation of Renal Sodium Transporters in D5 Dopamine Receptor–Deficient Mice

D5 dopamine receptor (D5R)-deficient (D5−/−) mice have hypertension that is aggravated by an increase in sodium intake. The present experiments were designed to test the hypothesis that a dysregulation of renal sodium transporters is related to the salt sensitivity in D5−/− mice. D5R was expressed in the renal proximal tubule, thick ascending limb, distal convoluted tubule, and cortical and outer medullary collecting ducts in D5+/+ mice. On a control Na+ diet, renal protein expressions of NKCC2 (sodium-potassium-2 chloride cotransporter), sodium chloride cotransporter, and &agr; and &ggr; subunits of the epithelial sodium channel were greater in D5−/− than in D5+/+ mice. Renal renin abundance and urine aldosterone levels were similar but renal angiotensin II type 1 receptor (AT1R) protein expression was increased in D5−/− mice. An elevated Na+ diet increased further the elevated blood pressure of D5−/− mice but did not affect the normal blood pressure of D5+/+ mice. The increased levels of NKCC2, sodium chloride cotransporter, and &agr; and &ggr; subunits of the epithelial sodium channel persisted with the elevated Na+ diet and unaffected by chronic AT1R blockade (losartan) in D5−/− mice. The expressions of proximal sodium transporters NHE3 (sodium hydrogen exchanger type 3) and NaPi2 (sodium phosphate cotransporter type 2) were increased by the elevated Na+ diet in D5−/− mice; the increased expression of NHE3 but not NaPi2 was abolished by AT1R blockade. Our findings suggest that the increased protein expression of sodium transporters/channels in distal nephron segments may be the direct consequence of the disruption of D5R, independent of the renin–angiotensin aldosterone system.

Identification of RanBP 9/10 as Interacting Partners for Protein Kinase C (PKC) γ/δ and the D1 Dopamine Receptor: Regulation of PKC-Mediated Receptor Phosphorylation

We reported previously that ethanol treatment regulates D1 receptor phosphorylation and signaling in a protein kinase C (PKC) δ- and PKCγ-dependent fashion by a mechanism that may involve PKC isozyme-specific interacting proteins. Using a PKC isozyme-specific coimmunoprecipitation approach coupled to mass spectrometry, we report the identification of RanBP9 and RanBP10 as novel interacting proteins for both PKCγ and PKCδ. Both RanBP9 and RanBP10 were found to specifically coimmunoprecipitate with both PKCγ and PKCδ; however, this association did not seem to mediate the ethanol regulation of the PKCs. It is noteworthy that the D1 receptor was also found to specifically coimmunoprecipitate with RanBP9/10 from human embryonic kidney (HEK) 293T cells and with endogenous RanBP9 from rat kidney. RanBP9 and RanBP10 were also found to colocalize at the cellular level with the D1 receptor in both kidney and brain tissue. Although overexpression of RanBP9 or RanBP10 in HEK293T cells did not seem to alter the kinase activities of either PKCδ or PKCγ, both RanBP proteins regulated D1 receptor phosphorylation, signaling, and, in the case of RanBP9, expression. Specifically, overexpression of either RanBP9 or RanBP10 enhanced basal D1 receptor phosphorylation, which was associated with attenuation of D1 receptor-stimulated cAMP accumulation. Moreover, treatment of cells with select PKC inhibitors blocked the RanBP9/10-dependent increase in basal receptor phosphorylation, suggesting that phosphorylation of the receptor by PKC is regulated by RanBP9/10. These data support the idea that RanBP9 and RanBP10 may function as signaling integrators and dictate the efficient regulation of D1 receptor signaling by PKCδ and PKCγ.

Unique role of NADPH oxidase 5 in oxidative stress in human renal proximal tubule cells

NADPH oxidases are the major sources of reactive oxygen species in cardiovascular, neural, and kidney cells. The NADPH oxidase 5 (NOX5) gene is present in humans but not rodents. Because Nox isoforms in renal proximal tubules (RPTs) are involved in the pathogenesis of hypertension, we tested the hypothesis that NOX5 is differentially expressed in RPT cells from normotensive (NT) and hypertensive subjects (HT). We found that NOX5 mRNA, total NOX5 protein, and apical membrane NOX5 protein were 4.2±0.7-fold, 5.2±0.7-fold, and 2.8±0.5-fold greater in HT than NT. Basal total NADPH oxidase activity was 4.5±0.2-fold and basal NOX5 activity in NOX5 immunoprecipitates was 6.2±0.2-fold greater in HT than NT (P=<0.001, n=6–14/group). Ionomycin increased total NOX and NOX5 activities in RPT cells from HT (P<0.01, n=4, ANOVA), effects that were abrogated by pre-treatment of the RPT cells with diphenylene-iodonium or superoxide dismutase. Silencing NOX5 using NOX5-siRNA decreased NADPH oxidase activity (−45.1±3.2% vs. mock-siRNA, n=6–8) in HT. D1-like receptor stimulation decreased NADPH oxidase activity to a greater extent in NT (−32.5±1.8%) than HT (−14.8±1.8). In contrast to the marked increase in expression and activity of NOX5 in HT, NOX1 mRNA and protein were minimally increased in HT, relative to NT; total NOX2 and NOX4 proteins were not different between HT and NT, while the increase in apical RPT cell membrane NOX1, NOX2, and NOX4 proteins in HT, relative to NT, was much less than those observed with NOX5. Thus, we demonstrate, for the first time, that NOX5 is expressed in human RPT cells and to greater extent than the other Nox isoforms in HT than NT. We suggest that the increased expression of NOX5, which may be responsible for the increased oxidative stress in RPT cells in human essential hypertension, is caused, in part, by a defective renal dopaminergic system.

D5 dopamine receptor decreases NADPH oxidase, reactive oxygen species and blood pressure via heme oxygenase-1.

D5 dopamine receptor (D5R) knock-out mice (D5−/−) have a higher blood pressure (BP) and higher reactive oxygen species (ROS) production than their D5R wild-type littermates (D5+/+). We tested the hypothesis that the high BP and increased ROS production in D5−/− mice may be caused by decreased heme oxygenase-1 (HO-1) expression and activity. We found that renal HO-1 protein expression and HO enzyme activity were decreased (65 and 50%, respectively) in D5−/− relative to D5+/+ mice. A 24 h of administration of hemin, an HO-1 inducer, increased HO-1 expression and HO activity (6.8- and 1.9-fold, respectively) and normalized the increased ROS production and BP in D5−/− mice. Expression of HO-1 protein and HO activity were increased (2.3- and 1.5-fold, respectively) in HEK cells that heterologously expressed human wild-type D5R (HEK-hD5R), but not the empty vector-transfected HEK-293 cells. Fenoldopam (Fen), a D5R agonist, increased HO activity (3 h), HO-1 protein expression, HO-1 and D5R colocalization and co-immunoprecipitation in HEK-hD5R cells. Cellular NADPH oxidase activity was decreased by 35% in HEK-hD5R that was abrogated with silencing of the heme oxygenase 1 gene (HMOX1). HMOX1 siRNA also impaired the ability of Fen to decrease NADPH oxidase activity in HEK-hD5R cells. In summary, the D5R positively regulates HO-1 through direct protein/protein interaction in the short-term and by increasing HO-1 protein expression in the long-term. The impaired D5R regulation of HO-1 and ROS production contributes to the pathogenesis of hypertension in D5−/− mice.