Gyun Jee Song

Mitochondrial DNA integrity and copy number in sperm from infertile men

OBJECTIVE To determine the integrity and copy number of mitochondrial DNA (mtDNA) in spermatozoa from infertile men. We also compared mtDNA integrity to nuclear DNA integrity. DESIGN Observational. SETTING Tertiary care center. PATIENT(S) Semen samples were collected from 57 men, whose wives were undergoing in vitro fertilization, including 24 men with normal semen parameters. MAIN OUTCOME MEASURE(S) Quantitative long polymerase chain reaction (PCR) and real-time PCR were used to measure mtDNA integrity and copy number in spermatozoa. We also measured nuclear DNA integrity of spermatozoa with flow cytometry after acridine orange staining. RESULT(S) A significant increase in mtDNA copy number and a decrease in mtDNA integrity were observed in the spermatozoa samples from patients with abnormal semen parameters compared to the patients with normal semen parameters. The integrity and copy number of mtDNA were significantly correlated with sperm count. CONCLUSION(S) Mitochondrial DNA quantity and quality may serve as useful diagnostic markers of sperm quality in infertile men.

Aquaporin 1, Nox1, and Ask1 mediate oxidant-induced smooth muscle cell hypertrophy.

AIMS Reactive oxygen species (ROS)-mediated intracellular signalling is well described in the vasculature, yet the precise roles of ROS in paracrine signalling are not known. Studies implicate interstitial ROS hydrogen peroxide (H(2)O(2)) in vascular disease, and plasma H(2)O(2) levels in the micromolar range are detectable in animal models and humans with hypertension. Recently, H(2)O(2) was shown to cross biological membranes of non-vascular cells via aquaporin (Aqp) water channels. Previous findings suggest that H(2)O(2) activates NADPH oxidase (Nox) enzymes in vascular cells and apoptosis signal-regulating kinase 1 (Ask1) in non-vascular cells. We hypothesized that extracellular H(2)O(2) induces smooth muscle cell (SMC) hypertrophy by a mechanism involving Aqp1, Nox1, and Ask1. METHODS AND RESULTS Treatment of rat aortic SMCs (rASMC) with exogenous H(2)O(2) resulted in a concentration-dependent increase in Nox-derived superoxide (O(2)(•-)), determined by L-012 chemiluminescence, cytochrome c and electron paramagnetic resonance. Nox1 was verified as the source of O(2)(·-) by siRNA. Aqp1 siRNA attenuated H(2)O(2) cellular entry and H(2)O(2)-induced O(2)(•-) production. H(2)O(2) treatment increased Ask1 activation and induced rASMC hypertrophy in a Nox1-dependent mechanism. Adenoviral-dominant-negative Ask1 attenuated H(2)O(2)-induced rASMC hypertrophy and adenoviral overexpression of Ask1 augmented it. CONCLUSION Our results demonstrate for the first time that extracellular H(2)O(2), at pathophysiological concentrations, stimulates rASMC Nox1-derived O(2)(•-), subsequent Ask1 activation and SMC hypertrophy. The data demonstrate a novel pathway by which H(2)O(2) enters vascular cells via aquaporins and activates Nox, leading to hypertrophy, and provide multiple novel targets for combinatorial therapeutics development targeting hypertrophy and vascular disease.

Phosphorylation of the Endogenous Thyrotropin-releasing Hormone Receptor in Pituitary GH3 Cells and Pituitary Tissue Revealed by Phosphosite-specific Antibodies

To study phosphorylation of the endogenous type I thyrotropin-releasing hormone receptor in the anterior pituitary, we generated phosphosite-specific polyclonal antibodies. The major phosphorylation site of receptor endogenously expressed in pituitary GH3 cells was Thr365 in the receptor tail; distal sites were more phosphorylated in some heterologous models. β-Arrestin 2 reduced thyrotropin-releasing hormone (TRH)-stimulated inositol phosphate production and accelerated internalization of the wild type receptor but not receptor mutants where the critical phosphosites were mutated to Ala. Phosphorylation peaked within seconds and was maximal at 100 nm TRH. Based on dominant negative kinase and small interfering RNA approaches, phosphorylation was mediated primarily by G protein-coupled receptor kinase 2. Phosphorylated receptor, visualized by immunofluorescence microscopy, was initially at the plasma membrane, and over 5-30 min it moved to intracellular vesicles in GH3 cells. Dephosphorylation was rapid (t½ ∼ 1 min) if agonist was removed while receptor was at the surface. Dephosphorylation was slower (t½ ∼ 4 min) if agonist was withdrawn after receptor had internalized. After agonist removal and dephosphorylation, a second pulse of agonist caused extensive rephosphorylation, particularly if most receptor was still on the plasma membrane. Phosphorylated receptor staining was visible in prolactin- and thyrotropin-producing cells in rat pituitary tissue from untreated rats and much stronger in tissue from animals injected with TRH. Our results show that the TRH receptor can rapidly cycle between a phosphorylated and nonphosphorylated state in response to changing agonist concentrations and that phosphorylation can be used as an indicator of receptor activity in vivo.

Endogenous Parathyroid Hormone-Related Protein Regulates the Expression of PTH Type 1 Receptor and Proliferation of Vascular Smooth Muscle Cells

The PTH type 1 receptor (PTH1R) and PTHrP are expressed in vessels, where they contribute to regulating vascular smooth muscle cell (VSMC) function. Elevated PTHrP levels in VSMC are often associated with hyperplasia. In contrast, exogenous PTHrP, acting through the PTH1R, inhibits VSMC proliferation. In this study, we investigated the regulation of PTH1R expression by endogenous PTHrP and the associated effects on VSMC proliferation. Blocking binding of secreted PTHrP fragments to the PTH1R by treatment with either an antagonist or an antibody against PTHrP, and inhibition of PTHrP expression by small interfering RNA significantly increased PTH1R expression. Interestingly, treatment of the cells with a PTHrP analog (Bpa(1)-PTHrP) that activates the PTH1R without inducing its internalization had the same effect on receptor expression. To examine the association between receptor expression and the antiproliferative effect of N-terminal fragments of PTHrP, VSMC were treated with exogenous PTHrP (1-36) acutely and chronically to induce receptor down-regulation. Stimulation of VSMC with exogenous PTHrP (1-36) significantly reduced cell proliferation during the first 18 h of treatment but was no longer effective after 3 d, a time when PTH1R was down-regulated. In contrast, treatment with the noninternalizing agonist Bpa(1)-PTHrP strongly inhibited cell proliferation at all time points. In conclusion, our study show that PTHrP, after its intracellular processing and secretion, promotes down-regulation of the PTH1R in VSMC, thereby regulating cell proliferation in an auto/paracrine fashion. This regulatory mechanism may have important implication during vascular remodeling, in particular in the development of neointima after arterial injury, where PTHrP overexpression occurs.

Dimerization of the thyrotropin-releasing hormone receptor potentiates hormone-dependent receptor phosphorylation

The G protein-coupled thyrotropin (TSH)-releasing hormone (TRH) receptor forms homodimers. Regulated receptor dimerization increases TRH-induced receptor endocytosis. These studies test whether dimerization increases receptor phosphorylation, which could potentiate internalization. Phosphorylation at residues 355–365, which is critical for internalization, was measured with a highly selective phospho-site-specific antibody. Two strategies were used to drive receptor dimerization. Dimerization of a TRH receptor-FK506-binding protein (FKBP) fusion protein was stimulated by a dimeric FKBP ligand. The chemical dimerizer caused a large increase in TRH-dependent phosphorylation within 1 min, whereas a monomeric FKBP ligand had no effect. The dimerizer did not alter phoshorylation of receptors lacking the FKBP domain. Dimerization of receptors containing an N-terminal HA epitope also was induced with anti-HA antibody. Anti-HA IgG strongly increased TRH-induced phosphorylation, whereas monomeric Fab fragments had no effect. Anti-HA antibody did not alter phosphorylation in receptors lacking an HA tag. Furthermore, two phosphorylation-defective TRH receptors functionally complemented one another and permitted phosphorylation. Receptors with a D71A mutation in the second transmembrane domain do not signal, whereas receptors with four Ala mutations in the 355–365 region signal normally but lack phosphorylation sites. When D71A- and 4Ala-TRH receptors were expressed alone, neither underwent TRH-dependent phosphorylation. When they were expressed together, D71A receptor was phosphorylated by G protein-coupled receptor kinases in response to TRH. These results suggest that the TRH receptor is phosphorylated preferentially when it is in dimers or when preexisting receptor dimers are driven into microaggregates. Increased receptor phosphorylation may amplify desensitization.

EBP50 inhibits the anti-mitogenic action of the parathyroid hormone type 1 receptor in vascular smooth muscle cells

Parathyroid hormone-related protein (PTHrP) and the parathyroid hormone type 1 receptor (PTH1R) are important regulators of vascular remodeling. PTHrP expression is associated to increased proliferation of vascular smooth muscle cells (VSMC). In contrast, signaling via the PTH1R inhibits cell growth. The mechanisms regulating the dual effect of PTHrP and PTH1R on VSMC proliferation are only partially understood. In this study we examined the role of the adaptor protein ezrin-radixin-moesin-binding phosphoprotein (EBP50) on PTH1R expression, trafficking, signaling and control of A10 cell proliferation. In normal rat vascular tissues, EBP50 was restricted to the endothelium with little expression in VSMC. EBP50 expression significantly increased in VSMC following angioplasty in parallel with PTHrP. Interestingly, PTHrP was able to induce EBP50 expression. In the clonal rat aortic smooth muscle cell line A10, EBP50 increased the recruitment of PTH1R to the cell membrane and delayed its internalization in response to PTHrP(1-36). This effect required an intact C-terminal motif in the PTH1R. In naïve A10 cells, PTHrP(1-36) stimulated cAMP production but not intracellular calcium release. In contrast, PTHrP(1-36) induced both cAMP and calcium signaling in A10 cells over-expressing EBP50. Finally, EBP50 attenuated the induction of p27(kip1) and the anti-proliferative effect of PTHrP(1-36). In summary, this study demonstrates the dynamic expression of EBP50 in vessels following injury and the effects of EBP50 on PTH1R function in VSMC. These findings highlight one of the mechanisms leading to increased VSMC proliferation and have important implication in the understanding of the molecular events leading to restenosis.

The scaffolding protein EBP50 promotes vascular smooth muscle cell proliferation and neointima formation by regulating Skp2 and p21(cip1).

Objective— The Ezrin-radixin–moesin–binding phosphoprotein 50 (EBP50) is a scaffolding protein known to regulate ion homeostasis in the kidney and intestine. Previous work showed that EBP50 expression increases after balloon injury in rat carotids. This study was designed to determine the role of EBP50 on vascular smooth muscle cells (VSMC) proliferation and the development of neointimal hyperplasia. Methods and Results— Wire injury was performed in wild type (WT) and EBP50 knockout (KO) mice. Two weeks after injury, neointima formation was 80% lower in KO than in WT mice. Proliferation of KO VSMC was significantly lower than WT cells and overexpression of EBP50 increased VSMC proliferation. Akt activity and expression of S-phase kinase protein2 decreased in KO cells resulting in the stabilization of the cyclin-dependent kinase inhibitor, p21cip1. Consequently, KO cells were arrested in G0/G1 phase. Consistent with these observations, p21cip1 was detected in injured femoral arteries of KO but not WT mice. No differences in apoptosis between WT and KO were observed. Conclusion— EBP50 is critical for neointima formation and induces VSMC proliferation by decreasing S-phase kinase protein2 stability, thereby accelerating the degradation of the cell cycle inhibitor p21cip1.

EBP50 Promotes Focal Adhesion Turnover and Vascular Smooth Muscle Cells Migration

The ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a PDZ-containing scaffolding protein that regulates a variety of physiological functions. In the vasculature, EBP50 promotes neointima formation following arterial injury. In this study the role of EBP50 on vascular smooth muscle cell (VSMC) migration was characterized. The spreading and motility of primary VSMC isolated from EBP50 knockout (KO) mice were significantly reduced compared to wild-type (WT) cells. EBP50-null VSMC had fewer and larger focal adhesions than wild-type cells. Assembly and disassembly of focal adhesion-assessed by live-cell total internal reflection fluorescence imaging-in response to epidermal growth factor (EGF) were significantly reduced in KO cells. Immunoprecipitation experiments showed that EBP50 interacts with EGF receptor via the PDZ2 domain and with focal adhesion kinase (FAK) via the C-terminal ERM domain. EBP50 promoted the formation of a complex containing both EGF receptor and FAK. Phosphorylation of Tyr-925 of FAK in response to EGF was significantly reduced in KO cell compared to WT cells. The residence time of FAK in focal adhesions-determined by fluorescence recovery after photobleaching-was increased in WT cells. Collectively, these studies indicate that EBP50, by scaffolding EGF receptor and FAK, facilitates activation of FAK, focal adhesion turnover, and migration of VSMC.

Ezrin-Radixin-Moesin-binding Phosphoprotein 50 (EBP50) and Nuclear Factor-κB (NF-κB) A FEED-FORWARD LOOP FOR SYSTEMIC AND VASCULAR INFLAMMATION

Background: EBP50 is a scaffolding protein that is critical during vascular remodeling. Results: EBP50 expression is induced by inflammatory stimuli and potentiates NF-κB activation and inflammation. Conclusion: EBP50 and NF-κB participate in a feed-forward loop leading to increased macrophage activation and enhanced response of vascular cells to inflammation. Significance: This work reveals a novel mechanism regulating systemic and vascular inflammation. The interaction between vascular cells and macrophages is critical during vascular remodeling. Here we report that the scaffolding protein, ezrin-binding phosphoprotein 50 (EBP50), is a central regulator of macrophage and vascular smooth muscle cells (VSMC) function. EBP50 is up-regulated in intimal VSMC following endoluminal injury and promotes neointima formation. However, the mechanisms underlying these effects are not fully understood. Because of the fundamental role that inflammation plays in vascular diseases, we hypothesized that EBP50 mediates macrophage activation and the response of vessels to inflammation. Indeed, EBP50 expression increased in primary macrophages and VSMC, and in the aorta of mice, upon treatment with LPS or TNFα. This increase was nuclear factor-κB (NF-κB)-dependent. Conversely, activation of NF-κB was impaired in EBP50-null VSMC and macrophages. We found that inflammatory stimuli promote the formation of an EBP50-PKCζ complex at the cell membrane that induces NF-κB signaling. Macrophage activation and vascular inflammation after acute LPS treatment were reduced in EBP50-null cells and mice as compared with WT. Furthermore, macrophage recruitment to vascular lesions was significantly reduced in EBP50 knock-out mice. Thus, EBP50 and NF-κB participate in a feed-forward loop leading to increased macrophage activation and enhanced response of vascular cells to inflammation.

Phosphorylation of Ezrin-Radixin-Moesin-binding Phosphoprotein 50 (EBP50) by Akt Promotes Stability and Mitogenic Function of S-phase Kinase-associated Protein-2 (Skp2)

Background: The PDZ scaffolding protein EBP50 promotes Akt-dependent cell proliferation through the S-phase kinase associated protein-2 Skp2. Results: Akt phosphorylates threonine 156 of EBP50, allosterically promoting the binding of Skp2 to the first PDZ domain of EBP50. Conclusion: The interaction with EBP50 regulates the localization and stability of Skp2 and promotes cell proliferation. Significance: These studies define a regulatory mechanism contributing to Akt-dependent cell proliferation. The regulation of the cell cycle by the ubiquitin-proteasome system is dependent on the activity of E3 ligases. Skp2 (S-phase kinase associated protein-2) is the substrate recognition subunit of the E3 ligase that ubiquitylates the cell cycle inhibitors p21cip1 and p27kip1 thus promoting cell cycle progression. Increased expression of Skp2 is frequently observed in diseases characterized by excessive cell proliferation, such as cancer and neointima hyperplasia. The stability and cellular localization of Skp2 are regulated by Akt, but the molecular mechanisms underlying these effects remain only partly understood. The scaffolding protein Ezrin-Binding Phosphoprotein of 50 kDa (EBP50) contains two PDZ domains and plays a critical role in the development of neointimal hyperplasia. Here we report that EBP50 directly binds Skp2 via its first PDZ domain. Moreover, EBP50 is phosphorylated by Akt on Thr-156 within the second PDZ domain, an event that allosterically promotes binding to Skp2. The interaction with EBP50 causes cytoplasmic localization of Skp2, increases Skp2 stability and promotes proliferation of primary vascular smooth muscle cells. Collectively, these studies define a novel regulatory mechanism contributing to aberrant cell growth and highlight the importance of scaffolding function of EBP50 in Akt-dependent cell proliferation.