The Kinase Insert Domain-containing Receptor (KDR) is Regulated by Shear Stress

Objective : Intimal hyperplasia develops in areas with low shear stress. The aim of the present study was to investigate if the mRNA expression of vascular endothelial growth factor (VEGF), Fms-like tyrosine kinase-1 receptor (Flt-1) and kinase insert domain-containing receptor (KDR) is regulated by shear stress. Design : Endothelial cells from human umbilical veins were in an in vitro system subjected to different levels of shear stress during 1 and 12 h. The mRNA expression of VEGF, Flt-1 and KDR was measured with RT-PCR. eNOS served as positive control and actin as housekeeping gene. Results : The KDR expression was isolated upregulated 3-4 times after 12 h exposure to high shear stress. Conclusion : The genetic expression of KDR is upregulated by shear stress and this response is time dependent.     

KDR 启动子/增强子靶向干扰磷脂酰肌醇3激酶信号通路对大鼠血管内皮细胞增殖和凋亡的影响

目的 针对大鼠血管内皮细胞(VEC)的磷脂酰肌醇3激酶B亚单位(Pik3cb),构建KDR特异性启动子/增强子真核表达载体,观察其对VEC增殖和凋亡的影响.方法 构建KDR特异性启动子/增强子真核表达载体并转染大鼠VEC.实验分5组,A组:正常VEC;B组:转染特异性KDR质粒,浓度2.0 mg/L;C组:转染非特异性巨细胞病毒(CMV)质粒,浓度2.0mg/L;D组:转染空质粒,浓度2.0 mg/L;E组:阳性对照渥曼青霉素,浓度50 nmol/L.分别于转染24、48、72 h后,实时荧光定量逆转录-聚合酶链反应(RT-qPCR)法检测各组细胞Pik3cb mRNA相对表达量,细胞计数试剂盒(CCK-8)和流式细胞仪检测各组细胞增殖抑制率和凋亡率.结果 转染24、48、72 h后,RT-qPCR法测得KDR组Pik3cb mRNA相对表达量分别为(54.82 ±2.77)％、(50.54±3.98)％和(35.47±4.83)％,均明显低于正常对照组(P＜0.05);CCK-8法测得KDR组细胞增殖抑制率分别为(21.98±2.25)％、(24.32±3.04)％和(26.38±5.06)％;流式细胞仪测得KDR组细胞凋亡率分别为(9.9±1.3)％、(31.0±7.4)％和(44.5±8.3)％,细胞增殖抑制率和凋亡率均明显高于正常对照组(P＜0.05).结论 KDR特异性启动子/增强子真核表达载体可通过下调磷脂酰肌醇3激酶(PI3K)信号通路特异性抑制VEC增殖并促使其凋亡.     

Activation of extracellular-signal regulated kinase (ERK1/2) by fluid shear is Ca(2+)- and ATP-dependent in MC3T3-E1 osteoblasts

To determine the role of Ca2+ signaling in activation of the Mitogen-Activated Protein Kinase (MAPK) pathway, we subjected MC3T3-E1 pre-osteoblastic cells to inhibitors of Ca2+ signaling during application of fluid shear stress (FSS). FSS only activated ERK1/2, rapidly inducing phosphorylation within 5 min of the onset of shear. Phosphorylation of ERK1/2 (pERK1/2) was significantly reduced when Ca2+i was chelated with BAPTA or when Ca2+ was removed from the flow media. Inhibition of both the L-type voltage-sensitive Ca2+ channel and the mechanosensitive cation-selective channel blocked FSS-induced pERK1/2. Inhibition of phospholipase C with U73122 significantly reduced pERK1/2. This inhibition did not result from blockage of intracellular Ca2+ release, but a loss of PKC activation. Recent data suggests a role of ATP release and purinergic receptor activation in mechanotransduction. Apyrase-mediated hydrolysis of extracellular ATP completely blocked FSS-induced phosphorylation of ERK1/2, while the addition of exogenous ATP to static cells mimicked the effects of FSS on pERK1/2. Two P2 receptors, P2Y2 and P2X7, have been associated with the anabolic responses of bone to mechanical loading. Using both iRNA techniques and primary osteoblasts isolated from P2X7 knockout mice, we found that the P2X7, but not the P2Y2, purinergic receptor was involved in ERK1/2 activation under FSS. These data suggest that FSS-induced ERK1/2 phosphorylation requires Ca2+-dependent ATP release, however both increased Ca2+i and PKC activation are needed for complete activation. Further, this ATP-dependent ERK1/2 phosphorylation is mediated through P2X7, but not P2Y2, purinergic receptors.     

Renin-stimulated TGF-beta1 expression is regulated by a mitogen-activated protein kinase in mesangial cells

Recent evidence indicates that renin itself might be profibrotic, independent of angiotensin II; however, the signaling system by which renin exerts a direct effect is not known. We tested the hypothesis that renin receptor activation, in turn, activates the extracellular-signal regulated kinase 1 and 2 (ERK1/2) of the mitogen-activated protein kinase system in mesangial cells. Recombinant rat renin induced a rapid phosphorylation of ERK1/2 and subsequent cell proliferation in a dose- and time-dependent manner. ERK1/2 activation by renin addition was not altered by angiotensin-converting enzyme inhibition or angiotensin receptor blockade. An ERK kinase inhibitor significantly reduced the renin-induced ERK1/2 phosphorylation and the subsequent increase in transforming growth factor-beta1 (TGF-beta1) and plasminogen activator inhibitor-1 mRNA expression. A small-inhibiting RNA, siRNA, against the renin receptor completely blocked ERK1/2 activation by rat renin. We conclude that renin induces ERK1/2 activation though a receptor-mediated, angiotensin II-independent mechanism in mesangial cells. This renin-activated pathway triggers cell proliferation along with TGF-beta1 and plasminogen activator inhibitor-1 gene expression. This system may play an important role in the overall profibrotic actions of renin.     

Protein kinase A (PKA) pathway is functionally linked to androgen receptor (AR) in the progression of prostate cancer

ObjectivesIn the present study, we investigated whether the cyclic adenosine monophosphate (cAMP)-activated protein kinase A (PKA) pathway may regulate the expression of AR and prostate-specific antigen (PSA) and whether there is a correlation between the expression of cAMP/PKA-associated genes and androgen receptor (AR) in patients with prostate cancer (CaP).Materials and methodsThe functional studies were performed in LNCaP and PC3 cell lines. Data on the mRNA expression of sets of genes in human clinical samples, including prostate tissues from organ donors, prostate primary cancer, and metastatic cancer, were extracted from the National Center for Biotechnology Informations Gene Expression Omnibus (GEO) database. Statistical tests were applied.ResultsWe showed that elevated levels of cAMP/PKA pathways induced an increased expression of AR and PSA proteins in LNCaP cells in the absence of androgen. A cAMP-associated phosphodiesterase-4 (PDE4) inhibitor, rolipram induced an up-regulation in AR expression, whereas a cAMP enhancer, forskolin increased PSA level without affecting AR expression. Forskolin treatment increased the level of PKA R1α in LNCaP cells, but remarkably inhibited R1α expression in aggressive PC3 cells. In patients with CaP, we found that the expression of genes encoding R1α and phosphodiesterase-4B was statistically significantly lower in the metastatic specimens than that in the primary CaP specimens or in the normal prostate tissues (P<0.01) and was reversely correlated with AR expression. Conversely, AR and PRKAR2B mRNA expressions were significantly higher in metastatic lesions than those in the primary CaP specimens or in the normal prostate tissues (P<0.01).ConclusionOur study revealed a novel mechanism to precisely define the functional and clinical interrelationship between the cAMP/PKA pathway and AR signaling in the development of androgen-independent growth of CaPs and metastasis progression.     

Plasminogen activator inhibitor-1 expression is regulated by the angiotensin type 1 receptor in vivo

BACKGROUND: The fibrinolytic system plays an important role in degrading fibrin-rich thrombi and in vascular and tissue remodeling. Elevated levels of plasminogen activator inhibitor-1 (PAI-1) can reduce the efficiency of the endogenous fibrinolytic system. Angiotensin (Ang) has been shown to regulate PAI-1 expression via the Ang type 1 (AT1) receptor in some tissues and via the AT4 receptor in cultured endothelium. The purpose of this study was to examine the tissue-specific pattern of PAI-1 expression in response to infusion of Ang II in vivo. METHODS: Adult male Sprague-Dawley rats (N = 5 in each group) were treated with four hours of intravenous infusions of Ang II or vehicle control while mean arterial pressure (MAP) was monitored: group 1, 600 ng/kg/min Ang II; group 2, Ang II + 10 mg/kg of the AT1 receptor antagonist (AT1RA) L158-809 q2 hour; group 3, Ang II + 0.01 to 0.1 mg/kg hydralazine as required to maintain normal blood pressure; and group 4, saline-infused controls. After infusion, tissue was harvested for Northern blotting, immunohistochemical analysis, and in situ hybridization. RESULTS: In group 1, Ang II infusion increased MAP from 105 +/- 8 to 160 +/- 9 mm Hg (mean +/- SE, P < 0. 01). Ang II induced increased expression of PAI-1 mRNA in all tissues examined from 5.1-fold in the heart, 9.7-fold in the kidney, 10.0-fold in the aorta, and up to 30.0-fold in the liver (all P < 0. 01 vs. control). While both AT1RA (group 3) and hydralazine (group 4) prevented Ang II-induced elevation in blood pressure, the Ang II-dependent expression of PAI-1 mRNA was reduced by only AT1 receptor blockade. CONCLUSIONS: We conclude that in the rat, PAI-1 is induced in a variety of tissues by Ang II directly through the AT1 receptor, independent of its effects on blood pressure.     

Protein kinase C activity is acutely regulated by plasma glucose concentration in human monocytes in vivo.

Activation of protein kinase C (PKC) by hyperglycemia is implicated in the pathogenesis of long-term diabetic complications. Monocyte activation and transformation into macrophages is a key step in the atherosclerotic process. Therefore, in this study, we sought to determine 1) the effect of hyperglycemia on monocyte PKC activity and on the distribution of Ca2+-dependent and diacylglycerol-sensitive PKC isoforms; and 2) whether the effects on these parameters are determined by hyperglycemia per se, independent of the diabetic state. The studies were performed in 19 type 2 diabetic patients and 14 control subjects. Plasma glucose concentration was higher and insulin sensitivity lower (both P < 0.01) in diabetic patients than in control subjects. Monocytes from diabetic patients showed similar cytosol PKC activity to those from control subjects but higher membrane PKC activity (78+/-6 vs. 50+/-5 pmol x min(-1) x mg(-1) protein; P < 0.01). A direct correlation was observed between fasting plasma glucose and membrane PKC activity (r2 = 0.4008, P = 0.0001). In contrast, a reciprocal correlation was observed between membrane PKC activity and insulin sensitivity index (r2 = 0.28, P < 0.05). Using immunoblotting analysis, we found that membrane beta2, but not alpha, isoform of PKC was more abundant in monocytes from diabetic patients. In diabetic patients, when euglycemia was acutely induced, membrane PKC activity decreased by approximately 42% and beta2 isoform by approximately 15%. In two normal subjects in whom hyperglycemia was induced, membrane PKC increased from 63 and 57 to 92 and 128.6 pmol x min(-1) x mg(-1) protein, respectively. This increase was associated with an increase in the membrane isoform beta2; alpha isoform was unchanged. We conclude that 1) monocytes express the glucose-sensitive beta2 isoform of PKC; 2) the prevailing plasma glucose acutely regulates the activity of the membrane PKC and the content of membrane PKC beta2 isoform; and 3) this effect appears to be a direct effect of glucose per se, since the phenomenon was observed in normal control subjects when hyperglycemia was induced. Monocyte PKC activation may account for the accelerated atherosclerosis of patients with type 2 diabetes.     

Binding of the renal epithelial cell line LLC-PK1 to laminin is regulated by protein kinase C.

The alpha6beta1 integrin heterodimer has been implicated in the mediation of renal epithelial cell binding to laminin, and it has been suggested that this binding is important for renal morphogenesis and development. Studies of nonrenal cells have suggested that the functional activity of alpha6beta1 integrin is regulated by protein kinase C (PKC) activity. In this study, the binding of a renal epithelial cell line, LLC-PK1, to laminin was characterized and the role of PKC activity in the modulation of binding was investigated. LLC-PK1 cells bound to laminin-coated surfaces in a time- and laminin concentration-dependent manner. Binding was strongly inhibited by anti-beta1 integrin antibodies and by anti-alpha6 integrin antibodies. Antibodies against alpha2 integrin and a3 integrin had little inhibitory effect. Cells bound to both whole laminin and laminin fragment E8, i.e., the fragment to which the alpha6beta1 integrin heterodimer binds. Exposure of cells to PKC activators for as little as 2 h enhanced cell binding to laminin approximately twofold, in a protein synthesis-dependent manner. PKC inhibitors antagonized this effect. PKC-stimulated binding was also inhibited by anti-beta1 integrin and anti-alpha6 integrin antibodies. PKC activation did not alter expression of beta1 integrin subunits at the cell surface after short time periods (2 to 4 h), but expression was increased after longer time periods (24 h). These results indicate that the renal epithelial cell line LLC-PK1 binds to laminin via the alpha6betal integrin heterodimer and binding is enhanced by PKC activation. The PKC-mediated enhancement of binding requires protein synthesis and is mediated in part by activation of surface alpha6beta1 integrin.     

Angiotensin II-mediated collagen production is inhibited by a transforming growth factor-beta receptor kinase inhibitor

Introduction. The purpose of this experiment is to determine if the pro-fibrotic effect of angiotensin II (AII) is mediated through a transforming growth factor-beta (TGF-beta) receptor kinase. Methods. Neonatal rat heart fibroblast were cultured and incubated with SB 431542 (an inhibitor of TGF-beta receptor kinase activity) or vehicle. Recombinant human TGF-beta or AII was then added to the cardiac fibroblasts, and cells were incubated for 48 h under the experimental conditions. The cells were then fixed and stained with Sirius red collagen stain. Sirius red was extracted and quantified by absorbance at 540 nm. Five replicates were performed in each group and compared using the unpaired Student’s t-test. Results. Addition of SB 431542 to cells treated with TGF-beta showed an absorbance of 0.14 ± 0.01 (mean ± SE) compared to treatment with TGF-beta alone which showed an absorbance of 0.19 ± 0.01 representing a 26% reduction in total collagen (P = 0.03). Cells treated with SB 431542 and AII showed an absorbance of 0.105 ± 0.004, while cells treated with AII alone showed an absorbance of 0.147 ± 0.008 representing a 28% reduction in total collagen (P < 0.01). Conclusion. Angiotensin II induced collagen production is mediated through a TGF-beta receptor kinase in neonatal rat heart fibroblasts. The TGF-beta receptor kinase-mediated effect of AII may be responsible for the increased fibrosis seen in the myocardium of patients with congestive heart failure and may provide a target for therapeutic intervention.     

The epithelial Mg2+ channel transient receptor potential melastatin 6 is regulated by dietary Mg2+ content and estrogens

The kidney is the principal organ responsible for the regulation of the body Mg(2+) balance. Identification of the gene defect in hypomagnesemia with secondary hypocalcemia recently elucidated transient receptor potential melastatin 6 (TRPM6) as the gatekeeper in transepithelial Mg(2+) transport, whereas its homolog, TRPM7, is implicated in cellular Mg(2+) homeostasis. The aim of this study was to determine the tissue distribution in mouse and regulation of TRPM6 and TRPM7 by dietary Mg(2+) and hormones. This study demonstrates that TRPM6 is expressed predominantly in kidney, lung, cecum, and colon, whereas TRPM7 is distributed ubiquitously. Dietary Mg(2+) restriction in mice resulted in hypomagnesemia and renal Mg(2+) and Ca(2+) conservation, whereas a Mg(2+)-enriched diet led to increased urinary Mg(2+) and Ca(2+) excretion. Conversely, Mg(2+) restriction significantly upregulated renal TRPM6 mRNA levels, whereas a Mg(2+) enriched diet increased TRPM6 mRNA expression in colon. Dietary Mg(2+) did not alter TRPM7 mRNA expression in mouse kidney and colon. In addition, it was demonstrated that 17beta-estradiol but not 1,25-dihydroxyvitamin D(3) or parathyroid hormone regulates TRPM6 renal mRNA levels. Renal TRPM7 mRNA abundance remained unaltered under these conditions. The renal TRPM6 mRNA level in ovariectomized rats was significantly reduced, whereas 17beta-estradiol treatment normalized TRPM6 mRNA levels. In conclusion, kidney, lung, cecum, and colon likely constitute the main sites of active Mg(2+) (re)absorption in the mouse. In addition, Mg(2+) restriction and 17beta-estradiol upregulated renal TRPM6 mRNA levels, whereas a Mg(2+)-enriched diet stimulated TRPM6 mRNA expression in colon, supporting the gatekeeper function of TRPM6 in transepithelial Mg(2+) transport.     

运动调控骨代谢的关键膜上受体—G蛋白偶联受体48

G蛋白偶联受体48(GPR48)作为一富含亮氨酸重复序列的膜上七次跨膜受体,其LRR结构域与R-spondin1或Norrin结合形成复合体后可作用于下游关键因子可调控骨质疏松、阿尔茨海默病、高血压等疾病发生。骨作为重要的生理学和生物力学组织,由成骨细胞(OB)和破骨细胞(OC)分别主导的骨形成和骨吸收之间的拮抗及协同调控骨组织代谢平衡。骨细胞是力学刺激敏感细胞,调节应力加载后的骨适应性反应,而运动训练对骨产生的力学刺激可翻译成结构级联性生化反应(Wnt、c AMP/PKA/Atf4、OPG/RANKL/RANK等途径稳态表达),调控骨形成和/或骨吸收。并且,GPR48通过R-spondin1可直接调控以上信号途径的激活状态。那么,GPR48能否通过下游级联信号途径从而在运动影响骨代谢中起分子介导作用呢?探究GPR48在运动影响骨代谢中的作用及其分子介导机制,希望能进一步完善运动影响骨代谢的分子机制信号网络并为骨疾病诊治提供新靶点和非药物干预方式。     

Bone regeneration is regulated by wnt signaling.

Tissue regeneration is increasingly viewed as reactivation of a developmental process that, when misappropriated, can lead to malignant growth. Therefore, understanding the molecular and cellular pathways that govern tissue regeneration provides a glimpse into normal development as well as insights into pathological conditions such as cancer. Herein, we studied the role of Wnt signaling in skeletal tissue regeneration. INTRODUCTION: Some adult tissues have the ability to regenerate, and among these, bone is one of the most remarkable. Bone exhibits a persistent, lifelong capacity to reform after injury, and continual bone regeneration is a prerequisite to maintaining bone mass and density. Even slight perturbations in bone regeneration can have profound consequences, as exemplified by conditions such as osteoporosis and delayed skeletal repair. Here, our goal was to determine the role of Wnts in adult bone regeneration. MATERIALS AND METHODS: Using TOPgal reporter mice, we found that damage to the skeleton instigated Wnt reporter activity, specifically at the site of injury. We used a skeletal injury model to show that Wnt inhibition, achieved through adenoviral expression of Dkk1 in the adult skeleton, prevented the differentiation of osteoprogenitor cells. RESULTS: As a result, injury-induced bone regeneration was reduced by 84% compared with controls. Constitutive activation of the Wnt pathway resulting from a mutation in the Lrp5 Wnt co-receptor results in high bone mass, but our experiments showed that this same point mutation caused a delay in bone regeneration. In these transgenic mice, osteoprogenitor cells in the injury site were maintained in a proliferative state and differentiation into osteoblasts was delayed. CONCLUSIONS: When considered together, these data provide a framework for understanding the roles of Wnt signaling in adult bone regeneration and suggest a feasible approach to treating clinical conditions where enhanced bone formation is desired.     

Cerebrovascular dysfunction in Zucker obese rats is mediated by oxidative stress and protein kinase C

Insulin resistance (IR) impairs vascular function in the peripheral and coronary circulations, but its effects on cerebral arteries are virtually unexplored. We examined the vascular responses of the basilar artery (BA) and its side branches through a cranial window in Zucker lean (ZL) and IR Zucker obese (ZO) rats. Nitric oxide (NO) and K+ channel-mediated dilator responses, elicited by acetylcholine, iloprost, cromakalim, and elevated [K+], were greatly diminished in the ZO rats compared with ZL rats. In contrast, sodium nitroprusside induced similar relaxations in the two experimental groups. Expressions of the K+ channel pore-forming subunits were not affected by IR, while endothelial NO synthase was upregulated in the ZO arteries compared with ZL arteries. Protein kinase C (PKC) activity and production of superoxide anion were increased in the cerebral arteries of ZO rats, and pretreatment with superoxide dismutase restored all examined dilator responses. In contrast, application of PKC inhibitors improved only receptor-linked NO-mediated relaxation, but not K+ channel-dependent responses. Thus, IR induces in ZO rats cerebrovascular dysfunction, which is mediated by oxidative stress and partly by PKC activation. The revealed impairment of NO and K+ channel-dependent dilator responses may be responsible for the increased risk of cerebrovascular events and neurodegenerative disorders in IR.     

The carboxyproxyl-derived spin trap (CP-H) is an appropriate detector-compound for oxidative stress

Reperfusion of ischemic tissue disturbs the balance between reactive oxygen species (ROS) and the cellular antioxidative defense. This imbalance is known as oxidative stress. In this study the spin trap 3-carboxy-2,2,5,5-tetramethylpyrrolin-1-hydroxide (CP-H) with its ESR-detectable paramagnetic analogue 3-carboxy-2,2,5,5-tetramethylpyrrolin-1-oxyl (?CP) was analyzed in vitro and in vivo. In preliminary in vitro experiments we studied the interaction of CP-H with reactive compounds like hydroxyl radicals (?OH) and alkylperoxyl radicals (ROO?) which are formed during organ reperfusion or tissue reoxygenation. The increase in the peak intensity of the ESR signal of the ?CP-radical was used as a measure for CP-H oxidation by the above-mentioned oxidizing radicals. It could be clearly shown that ?OH as well as ROO? induce CP-H oxidation. The intensity of the ESR signal (?CP) depends on the concentration of the applied oxidant. In a further set of in vitro experiments we analyzed some factors influencing the stability of the generated ?CP. Cellular reductants are able to interact with many radicals whereby their paramagnetic signal intensity decreases. We could show that glutathione (GSH) up to 5 mM does not influence ?CP concentration. On the other hand, ascorbate at a concentration of 0.6 mM significantly reduces 55% of ?CP within 60 min to the ESR-silent CP-H. At 1 mM ascorbate the ?CP derived ESR signal is reduced within 60 min by 90%. Lower concentrations of ascorbate (0.1–0.3 mM) do not significantly decrease signal intensity within 1 h. Homogenization of ischemic rat kidney in the presence of an air-equilibrated buffer obviously induces the formation of oxidizing radicals which in turn are able to convert diamagnetic CP-H into paramagnetic ?CP. The intensity of the formed ?CP was analyzed in a 600 g supernatant with ESR spectroscopy at 25°C. It could be demonstrated that at least 3.0 ± 0.5 μM ?CP is formed 15 min after starting tissue homogenization and reoxygenation. Subsequent measurements of the ?CP concentration indicated that its signal intensity continuously decreases. After 75 min a residual ?CP concentration of 0.7 ± 0.3 μM was monitored. Removal of mitochondria from the homogenate by centrifugation at 6,000g decelerates the disappearance of ?CP but does not block it completely. In summary it could be shown that the marker (CP-H) is able to indicate the formation of oxidizing radicals during reoxygenation of ischemic tissue. This method underestimates the amount of produced oxidizing radicals. One reason for this is the reduction of ?CP by some cellular reductants. Other reasons will be discussed. We assume that the used method allows a nearly real-time determination of radical production during organ reoxygenation.