Down‐regulation of mir‐27b promotes angiogenesis and fibroblast activation through activating PI3K/AKT signaling pathway

To study the effects of mir‐27b on angiogenesis and fibroblast activation and to explore its further mechanism. Humanmicrovascular endothelial cell (HMEC)‐1 and humannormal skin fibroblast (BJ) cells were treated with mir‐27b inhibitor negative control reagent, mir‐27b inhibitor, LY294002, and mir‐27b inhibitor + LY294002, respectively. 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) was used to detect the T‐cell proliferation. The migration ability was detected by Scratch assays. The angiogenesis of HMEC‐1 cells was observed by in vitro tube formation assay. The mRNA and protein expression of vascular endothelial growth factor (VEGF) in HMEC‐1 cells and the mRNA and protein expression of collagen I, collagen III, α‐SMA, and MMP1 in BJ cells were detected by quantitativereal‐time polymerase chain reaction (qRT‐PCR) and Western blot, respectively. Meanwhile, the PI3K/protein kinase B (AKT) pathway‐related proteins were also detected by Western blot. The proliferation, migration, angiogenesis, the mRNA and protein expression of VEGF and the protein expression of p‐PI3K and p‐AKT in HMEC‐1 cells were increased after treated with mir‐27b inhibitor. Meanwhile, the proliferation, migration, and the protein expression of collagen I, collagen III, α‐SMA, MMP1, p‐PI3K, and p‐AKT in BJ cells were increased after treated with mir‐27b inhibitor. However, the angiogenesis and fibroblast activation of mir‐27b inhibitor was reversed by LY294002, and the activate effect to PI3K/AKT pathway was also inhibited. Down‐regulation of mir‐27b could promote angiogenesis and fibroblast activation, and its mechanism is related to activate PI3K/AKT signaling pathway.     

LY294002, a PI3K pathway inhibitor,prevents leptin‐induced adverse effects on spermatozoa in Sprague‐Dawley rats

This study examined the effects of PI3K and AMPK signalling pathway inhibitors on leptin‐induced adverse effects on rat spermatozoa. Sprague‐Dawley rats, aged 14–16 weeks, were randomised into control, leptin‐, leptin + dorsomorphin (AMPK inhibitor)‐, and leptin+LY294002 (PI3K inhibitor)‐treated groups with six rats per group. Leptin was given once daily for 14 days via the intraperitoneal (i.p.) route at a dose of 60 ug kg^?1 body weight. Rats in the leptin and inhibitor‐treated groups received concurrently either dorsomorphin (5 mg kg^?1 day^?1) or LY294002 (1.2 mg kg^?1 day^?1) i.p. for 14 days. Controls received 0.1 ml of normal saline. Upon completion, sperm count, sperm morphology, seminiferous tubular epithelial height (STEH), seminiferous tubular diameter (STD), 8‐hydroxy‐2‐deoxyguanosine (8‐OHdG) and phospho‐Akt/total Akt ratio were estimated. Data were analysed using ANOVA. Sperm count, STEH and STD were significantly lower, while the percentage of spermatozoa with abnormal morphology and the level of 8‐OHdG were significantly higher in rats treated with leptin and leptin + dorsomorphin when compared to those in controls and LY294002‐treated rats. Testicular phospho‐Akt/total Akt ratio was significantly higher in leptin and leptin + LY294002‐treated rats. In conclusion, LY294002 prevents leptin‐induced changes in rat sperm parameters, suggesting the potential role of the PI3K signalling pathway in the adverse effects of leptin on sperm parameters.     

Leucine‐rich glioma inactivated 3 promotes HaCaT keratinocyte migration

Our finding that human skin expresses leucine‐rich glioma inactivated 3 (LGI3) raises the question of the function of this cytokine in keratinocytes. We have shown that LGI3 stimulates human HaCaT keratinocyte migration without affecting viability or proliferation. Western blot analysis showed that LGI3 induced focal adhesion kinase activation, Akt phosphorylation, and glycogen synthase kinase 3β (GSK3β) phosphorylation in these cells. Using the scratch wound assay and a modified Boyden chamber, we found that LY294002, a selective phosphatidylinositol 3‐kinase inhibitor, and LiCl, a selective GSK3β inhibitor, abolished LGI3‐induced cell migration. We tested β‐catenin levels after LGI3 treatment because the Akt‐GSK3β pathway regulates β‐catenin accumulation, and β‐catenin promotes cell migration. LGI3 treatment increased β‐catenin protein and nuclear localization, whereas LY294002 prevented LGI3‐induced focal adhesion kinase and Akt activation as well as β‐catenin accumulation. Overall, these data suggest that LGI3 stimulates HaCaT cell migration following β‐catenin accumulation through the Akt pathway.     

肝癌细胞VEGF转录调控的信号传导通路

目的研究磷脂酰肌醇3-激酶(PI3K)和p42／p44丝裂源激活的蛋白激酶(MAPK)在肝癌细胞血管内皮生长因子(VEGF)转录调控中的作用。方法应用缺氧诱导剂氯化钴或重组人表皮生长因子(EGF)刺激HepG2细胞中VEGF的转录，在此过程中应用PI3K特异性阻断剂LY294002或p42／p44 MAPK特异性阻断剂PD98059干预，采用半定量逆转录PCR检测VEGF mRNA表达的变化。结果氯化钴或EGF可以诱导HepG2细胞中VEGF的表达。PI3K阻断剂LY294002可以在一定范围内浓度依赖性地抑制VEGF mRNA的表达，而p42／p44 MAPK阻断剂PD98059对VEGF mRNA的表达无抑制作用。结论肝癌细胞VEGF的转录调控受PI3K通路调控，而不受p42／p44 MAPK调控。     

Insulin protects against damage to pulmonary endothelial tight junctions after thermal injury: Relationship with zonula occludens‐1, F‐actin,and AKT activity

Intensive insulin therapy during critical illness protects the endothelium and thereby prevents organ failure. This study tested the hypothesis that insulin directly affects the attenuation of burn injury‐induced damage to pulmonary endothelial tight junction and investigated the underlying mechanisms. Sprague Dawley rats with severe burn injury were randomized to treatment with insulin dissolved in normal saline (maintenance of blood glucose at a level between 5.0 and 7.0 mmol/L) or normal saline alone (in vivo treatment). Pulmonary damage was evaluated. Rat pulmonary microvascular endothelial cells were treated with 20% burn serum or 20% burn serum + insulin (in vitro treatment). Selected cultures were pretreated with phosphatidylinositol 3‐kinase/protein kinase B (AKT) inhibitor (LY294002). Permeability was assessed by migration of bovine serum albumin across cell monolayers. Cells were stained with rhodamine phalloidin and were examined. Cell extracts were obtained to assess zonula occludens‐1, occludin, and phosphorylated AKT levels by immunoblotting. Treatment with insulin attenuated the pulmonary edema, hemorrhage, and inflammatory cell infiltration of rats with severe burn injury. Burn serum significantly enhanced monolayer permeability to albumin, whereas treatment with insulin (10^?7 mol/L) limited this effect. Meanwhile, insulin (10^?7 mol/L) reduced burn serum‐induced F‐actin stress fiber formation and decreased zonula occludens‐1 expression. LY294002 decreased cytoplasmic AKT phosphorylation and inhibited the protection effects of insulin. Through the phosphatidylinositol 3‐kinase/AKT pathway, insulin independent of glucose toxicity can attenuate increased pulmonary endothelial permeability induced by burn injury. The effect is attributed to the attenuation of the architectural disruption of protein components of the endothelial tight junction. This result is useful in inhibiting multiple organ failure after burn injury.     

PI-3K和p38MAPK通路在EGF诱导PC-3细胞环氧化酶-2表达上调中的作用

目的:研究p38丝裂原激活蛋白激酶(p38MAPK)和磷脂酰肌醇-3激酶(PI/3K)通路在表皮生长因子(EGF)诱导的激素非依赖性前列腺癌(hormone-refractory prostate cancer,HRPC)PC-3细胞环氧化酶2(cyclooxygen-ase-2,COX-2)表达上调中的作用。方法:MTT法检测EGF(0μg/L)、EGF(10μg/L)、EGF(10μg/L)+PI-3K阻断剂(LY294002,20μmol/L)、EGF(10μg/L)+p38MAPK阻断剂(SC203580,20μmol/L)处理后的细胞增殖情况。RT-PCR和Western印迹测定上述处理24h后PC-3细胞COX-2的表达变化,ELISA测定细胞培养液中前列腺素E2(PGE2)的变化。结果:LY294002和SC203580明显抑制EGF刺激后的PC-3细胞增殖(P<0.05)及EGF诱导的COX-2上调和PGE2生成(P<0.05)。结论:PI-3K通路和p38MAPK通路可能参与了EGF诱导的PC-3细胞COX-2的表达上调。     

Sphingosine-1-phosphate stimulates smooth muscle cell migration through galpha(i)- and pi3-kinase-dependent p38(MAPK) activation

BACKGROUND: Sphingosine-1-phosphate (S-1-P) is an extracellular mediator released in response to vessel injury. S-1-P binds to G-protein-coupled receptors, which can be Galpha(i)-, Galpha(q)-, or G(12/13)-linked. This study examines the role of p38 mitogen-activated protein kinase (p38(MAPK)) in vascular smooth muscle cell migration after stimulation with S-1-P, and pathways leading to p38(MAPK) activation. S-1-P has previously been shown to stimulate migration of vascular smooth muscle cells (VSMCs) in vitro through ERK1/2 and G(i). We hypothesized that S-1-P-induced VSMC migration is also dependent on p38(MAPK) activation through a G(i)-coupled extracellular receptor and phosphoinositide 3-kinase (PI3-K). METHODS: VSMCs were cultured in vitro. A linear wound assay was performed in the presence of S-1-P and inhibitors of p38(MAPK) (SB203580) or epidermal growth factor (EGF) receptor kinase (AG1478). Chemotaxis stimulated by S-1-P was also assayed in a modified Boyden chamber with and without SB203580 pretreatment. Western blotting was performed to examine p38(MAPK) activation in response to S-1-P with and without SB203580, AG1478, or inhibitors of G(i) (pertussis toxin), PI3-K (Wortmannin and LY294002), or MEK1 (PD98059). Western blotting and immunoprecipitation for targets of p38(MAPK) (MAPKAP kinase-2) and PI3-K (Akt) were also performed.S-1-P stimulated migration of VSMCs in both wound and Boyden transwell assays. This migration was inhibited by SB203580 to the level of control, whereas AG478 had no effect. RESULTS: S-1-P stimulated activation of p38(MAPK) that peaked at 10 min, as well as activation of MAPKAP kinase-2. Activation of p38(MAPK) was significantly inhibited by SB203580, pertussis toxin, Wortmannin, and LY294002, but not by PD98059 or AG1478; MAPKAP kinase-2 activation was inhibited by SB203580. Akt was activated by S-1-P at 3 to 5 min; this response was inhibited by Wortmannin and LY294002, but not by SB203580 or pertussis toxin. CONCLUSIONS: S-1-P induced VSMC migration through a G(i)-linked and a PI3-K coupled, p38(MAPK)- dependent process. PI3-K appears to function upstream of p38(MAPK), but was not G(i)-dependent. S-1-P-stimulated activation of p38(MAPK) does not signal via transactivation of the EGF receptor. Understanding signal transduction will allow targeted molecular interventions to treat the response of a vessel to injury.     

Pancreatic cancer cell proliferation is phosphatidylinositol 3-kinase dependent

BACKGROUND: Genetic mutations found in pancreatic cancer (K-ras, p16, p53) lead to inappropriate cellular proliferation. Mitogens stimulate proliferation via the phosphatidylinositol 3-kinase (PI3K)- and/or the p44/42-mitogen-activated protein kinase [p44/42-MAPK or extracellular signal-regulated kinase (ERK)] signaling pathways. We examined whether inhibition of either PI3K or ERK could limit proliferation in human pancreatic cancer. METHODS: Proliferation was stimulated in quiescent human pancreatic cancer cell lines (BxPC3 and Panc-1) by 10% fetal calf serum (FCS). In certain samples, PD98059 (an ERK inhibitor) or LY294002 (a PI3K inhibitor) was also added. AKT phosphorylation (indicating PI3K activity) and ERK phosphorylation (ERK activation) were determined by Western blot. Cell viability was determined by MTT assay. Cell cycle progression and apoptosis were determined by flow cytometry. A two-tailed t test was used for statistical analysis of the data (significance P < 0.05). RESULTS: LY294002 inhibited the PI3K pathway without affecting ERK activation in response to serum. PD98059 inhibited the ERK pathway specifically. In both BxPC-3 and Panc-1 cell lines, LY294002 inhibited serum-induced proliferation. This was associated with G(1) cell cycle arrest and with an increase in the rate of apoptosis. PD98059 inhibited proliferation only in BxPC3 cells, and to a lesser degree than did LY294002. CONCLUSIONS: PI3K signaling appears to be necessary for G(1)-to-S phase progression and proliferation in pancreatic cancer cells. ERK plays a lesser role in mitogen-induced proliferation. Pharmacological inhibition of PI3K may decrease proliferation, increase apoptosis, and potentially confer therapeutic benefit in pancreatic cancer.     

Regulation of BMP2‐induced intracellular calcium increases in osteoblasts

Although bone morphogenetic protein‐2 (BMP2) is a well‐characterized regulator that stimulates osteoblast differentiation, little is known about how it regulates intracellular Ca²⁺ signaling. In this study, intracellular Ca²⁺ concentration ([Ca²⁺]_i) upon BMP2 application, focal adhesion kinase (FAK) and Src activities were measured in the MC3T3‐E1 osteoblast cell line using fluorescence resonance energy transfer‐based biosensors. Increase in [Ca²⁺]_i, FAK, and Src activities were observed during BMP2 stimulation. The removal of extracellular calcium, the application of membrane channel inhibitors streptomycin or nifedipine, the FAK inhibitor PF‐573228 (PF228), and the alkaline phosphatase (ALP) siRNA all blocked the BMP2‐stimulated [Ca²⁺]_i increase, while the Src inhibitor PP1 did not. In contrast, a gentle decrease of endoplasmic reticulum calcium concentration was found after BMP2 stimulation, which could be blocked by both streptomycin and PP1. Further experiments revealed that BMP2‐induced FAK activation could not be inhibited by PP1, ALP siRNA or the calcium channel inhibitor nifedipine. PF228, but not PP1 or calcium channel inhibitors, suppressed ALP elevation resulting from BMP2 stimulation. Therefore, our results suggest that BMP2 can increase [Ca²⁺]_i through extracellular calcium influx regulated by FAK and ALP and can deplete ER calcium through Src signaling simultaneously. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1725–1733, 2016.     

Epidermal growth factor activation of intestinal glutamine transport is mediated by mitogen-activated protein kinases

Glutamine is an essential nutrient for gut functions, but the regulation of its uptake by intestinal mucosal cells is poorly understood. Given the pivotal role of epidermal growth factor (EGF) in regulating gut metabolism, growth, and differentiation, this in vitro study was designed to investigate the intracellular signaling pathways involved in the regulation of EGF-mediated intestinal glutamine transport in intestinal epithelia. Continuous incubation with EGF (>30 hours, 100 ng/ml) stimulated glutamine transport activity across intestinal epithelial Caco-2 cell apical membrane. Exposure to EGF for 48 hours resulted in an increase in transport activity (50%) and glutamine transport system B gene ATB⁰ mRNA levels (ninefold). EGF stimulated glutamine transport activity by increasing the glutamine transporter maximal velocity (V_max) without altering the transporter apparent affinity (Km). Furthermore, EGF stimulated both intracellular protein kinase C and mitogen-activated protein kinase MEK1/2 activities. The EGF-stimulated glutamine transport activity was attenuated individually by the specific protein kinase C inhibitor chelerythrine chloride and the mitogen-activated protein kinase MEK1 inhibitor PD 98059. These data suggest that EGF activates glutamine transport activity across intestinal epithelial membrane via a signaling mechanism that involves activation of protein kinase C and the mitogen-activated protein kinase MEK1/2 cascade. EGF activates glutamine transport via alterations in transporter mRNA levels and the number of functional copies of transporter units. Presented at the Forty-Third Annual Meeting of The Society for Surgery of the Alimentary Tract, San Francisco, California, May 19–22, 2002 (poster presentation).     

Adipose‐derived stem cells and keratinocytes in a chronic wound cell culture model: the role of hydroxyectoine

Chronic wounds represent a major socio‐economic problem in developed countries today. Wound healing is a complex biological process. It requires a well‐orchestrated interaction of mediators, resident cells and infiltrating cells. In this context, mesenchymal stem cells and keratinocytes play a crucial role in tissue regeneration. In chronic wounds these processes are disturbed and cell viability is reduced. Hydroxyectoine (HyEc) is a membrane protecting osmolyte with protein and macromolecule stabilising properties. Adipose‐derived stem cells (ASC) and keratinocytes were cultured with chronic wound fluid (CWF) and treated with HyEc. Proliferation was investigated using MTT test and migration was examined with transwell‐migration assay and scratch assay. Gene expression changes of basic fibroblast growth factor (b‐FGF), vascular endothelial growth factor (VEGF), matrix metalloproteinases‐2 (MMP‐2) and MMP‐9 were analysed by quantitative real‐time polymerase chain reaction (qRT‐PCR). CWF significantly inhibited proliferation and migration of keratinocytes. Addition of HyEc did not affect these results. Proliferation capacity of ASC was not influenced by CWF whereas migration was significantly enhanced. HyEc significantly reduced ASC migration. Expression of b‐FGF, VEGF, MMP‐2 and MMP‐9 in ASC, and b‐FGF, VEGF and MMP‐9 in keratinocytes was strongly induced by chronic wound fluid. HyEc enhanced CWF induced gene expression of VEGF in ASC and MMP‐9 in keratinocytes. CWF negatively impaired keratinocyte function, which was not influenced by HyEc. ASC migration was stimulated by CWF, whereas HyEc significantly inhibited migration of ASC. CWF induced gene expression of VEGF in ASC and MMP‐9 in keratinocytes was enhanced by HyEc, which might partly be explained by an RNA stabilising effect of HyEc.     

Bone marrow mesenchymal stem cells undergo nemosis and induce keratinocyte wound healing utilizing the HGF/c-Met/PI3K pathway

We previously showed cell–cell contacts of human dermal fibroblasts to induce expression of the hepatocyte growth factor/scatter factor (HGF) in a process designated as nemosis. Now we report on nemosis initiation in bone marrow mesenchymal stem cells (BMSCs). Because BMSCs are being used increasingly in cell transplantation therapy we aimed to demonstrate a functional effect and benefit of BMSC nemosis for wound healing. Nemotic and monolayer cells were used to stimulate HaCaT keratinocyte migration in a scratch-wound healing assay. Both indicators of nemosis, HGF production and cyclooxygenase-2 expression, were induced in BMSC spheroids. When compared with a similar amount of cells as monolayer, nemotic cells induced keratinocyte in vitro scratch-wound healing in a concentration-dependent manner. The HGF receptor, c-Met, was rapidly phosphorylated in the nemosis-stimulated keratinocytes. Nemosis-induced in vitro scratch-wound healing was inhibited by an HGF-neutralizing antibody as well as the small molecule c-Met inhibitor, SU11274. HGF-induced in vitro scratch-wound healing was inhibited by PI3K inhibitors, wortmannin and LY294002, while LY303511, an inactive structural analogue of LY294002, had no effect. Inhibitors of the mitogen-activated protein kinases MEK/ERK1/2 (PD98059 and U0126), and p38 (SB203580) attenuated HGF-induced keratinocyte in vitro scratch-wound healing. We conclude that nemosis of BMSCs can induce keratinocyte in vitro scratch-wound healing, and that in this effect signaling via HGF/c-Met is involved.     

结缔组织生长因子诱导人近端肾小管上皮细胞整合素连接激酶的表达及与激酶信号途径的关系

目的探讨结缔组织生长因子（CTGF）对人近端肾小管上皮细胞系HK-2整合素连接激酶（ILK）表达的影响，以及丝裂原激活蛋白激酶（MAPK）和磷脂酰肌醇3激酶（P13-K）途径对该因子表达的影响。方法用不同浓度的CTGF作用HK-2细胞24h及50μg／L的CTGF作用HK-2细胞不同时间，以实时PCR和Western印迹方法检测ILKmRNA及蛋白的表达。用信号通路特异性抑制剂预处理，观察其对CTGF的干预作用。结果CTGF呈时间及浓度依赖性诱导人近端肾小管上皮细胞ILK蛋白表达。5、20、50μg／L的CTGF可使ILK表达量分别增加为对照组的3．284、5．103、5．638倍。50μg／LCTGF使ILK的表达在6h开始升高，高峰在48h（为对照组5．740倍），MEK抑制剂PD98059和P13．K抑制剂LY294002显著降低CTGF诱导的HK-2细胞ILK基因和蛋白的表达（P均〈0．05）。p38MAPK抑制剂SB203580对CTGF诱导的ILK表达无显著影响。结论CTGF能诱导HK-2细胞ILK蛋白的表达，该作用可能与ERK1／2和P13-K信号途径激活有关。     

磷脂酰肌醇3激酶/蛋白激酶B/基质金属蛋白酶-9信号通路在芬太尼抑制人胃癌细胞侵袭和迁移中的作用

目的观察磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)/基质金属蛋白酶(MMP)-9信号通路在芬太尼抑制人胃癌MGC-803细胞侵袭和迁移中的作用。方法将人胃癌MGC-803细胞随机分为空白对照组(C组)、芬太尼组(F组),LY294002组(LY组),SB-3CT组(SB组),LY294002+芬太尼组(FLY组)和SB-3CT+芬太尼组(FSB组)。Transwell实验和划痕实验检测细胞侵袭迁移能力,实时荧光聚合酶链反应(RT-PCR)和蛋白质印迹法(Western blot)分别检测MMP-9和Akt的mRNA和蛋白质的表达,使用方差分析和独立样本t检验分析组间差异。结果F组胃癌细胞侵袭数低于C组(35.91±3.36比48.46±4.86,t=3.679,P<0.05)、迁移数低于C组(74.75±3.76比96.00±5.20,t=5.739,P<0.05)细胞迁移率(%)低于C组(14.29±4.29比22.43±3.37,t=3.591,P<0.05),Akt mRNA表达低于C组(0.77±0.02比1.00,t=8.110,P<0.05)、MMP-9 mRNA表达低于C组(0.87±0.08比1.00,t=2.967,P<0.05)、Akt-1蛋白表达低于C组(0.76±0.04比0.90±0.05,t=3.940,P<0.05)和MMP-9蛋白表达低于C组(0.76±0.05比0.94±0.04,t=4.774,P<0.05),差异均有统计学意义。FLY组胃癌细胞侵袭数低于LY组(18.45±4.86比33.62±4.13,t=4.999,P<0.05)、细胞迁移数低于LY组(42.79±3.56比71.79±7.60,t=5.986,P<0.05)和细胞迁移率(%)低于LY组(7.52±1.30比11.02±1.73,t=2.811,P<0.05),FLY组Akt mRNA表达低于LY组(0.60±0.05比0.76±0.17,t=3.468,P<0.05)、MMP-9 mRNA表达低于LY组(0.75±0.06比0.88±0.03,t=3.288,P<0.05)、Akt-1蛋白表达低于LY组(0.60±0.02比0.75±0.05,t=4.783,P<0.05)和MMP-9蛋白表达低于LY组(0.48±0.03比0.74±0.06,t=7.266,P<0.05),差异均有统计学意义。FSB组胃癌细胞侵袭数低于SB组(18.67±2.15比32.33±6.20,t=3.607,P<0.05)、细胞迁移数低于SB组(45.67±4.63比75.04±4.47,t=7.898,P<0.05)和细胞迁移率(%)低于SB组(7.09±1.69比14.39±2.06,t=4.745,P<0.05),FSB组Akt mRNA表达低于SB组(0.53±0.11比0.76±0.07,t=2.950,P<0.05)、MMP-9 mRNA低于SB组表达(0.63±0.07比0.84±0.01,t=3.687,P<0.05)、Akt-1蛋白表达低于SB组(0.61±0.03比0.75±0.04,t=4.573,P<0.05)和MMP-9蛋白表达低于SB组(0.53±0.07比0.72±0.05,t=3.730,P<0.05),差异均有统计学意义。芬太尼可降低细胞侵袭和迁移能力,MMP-9和p-Akt表达减少,且芬太尼联合LY294002或SB-3CT较单独用药时进一步加强了抑制效果。结论芬太尼抑制人胃癌MGC-803细胞侵袭和迁移的机制与抑制PI3K/Akt/MMP-9信号通路有关。     

ATP and adenosine act as a mitogen for osteoblast-like cells (MC3T3-E1)

Extracellular ATP, and to a lesser extent adenosine, an ATP metabolite, stimulated cell proliferation in osteoblast-like cells (MC3T3-E1). ATP increased cytosolic Ca²⁺ due to Ca²⁺ mobilization from intracellular storage in the same concentration range of the nucleotide as that effective for DNA synthesis, suggesting the mediation of the phospholipase C/Ca²⁺ system in the mitogenic action. Since adenosine induced no Ca²⁺ mobilization, P₂-purinergic receptor appears to be associated with ATP actions. The growth-promoting effect of ATP was not inhibited by H7, a protein kinase C inhibitor, and indomethacin, a cyclooxygenase inhibitor, indicating no involvement of activation of protein kinase C and production of prostaglandins in ATP-induced mitogenic signals. Either ATP or adenosine remarkably and synergistically potentiated platelet derived growth factor-induced DNA synthesis. These findings suggest that extracellular ATP and adenosine may play a physiological role in the regulation of bone formation. Received: 17 January 1995 / Accepted: 21 March 1995     

Extracellular matrix protects pancreatic beta-cells against apoptosis: role of short- and long-term signaling pathways

We have shown previously that culture of beta-cells on matrix derived from 804G cells and rich in laminin-5 improves their function. The purpose of this study was to investigate whether this matrix protects beta-cells against apoptosis and to elucidate signaling pathways involved. Matrix protected sorted rat beta-cells against apoptosis under standard conditions (11.2 mmol/l glucose, 10% serum), after serum deprivation (1% serum), and in response to interleukin-1beta (IL-1beta; 2 ng/ml), compared with control (poly-L-lysine [pLL]). Caspase-8 activity was reduced in cells cultured on matrix, whereas focal adhesion kinase (FAK), protein kinase B (PKB, or Akt), and extracellular signal-regulated kinase (ERK) phosphorylation was augmented. Treatment (4 h) with an anti-beta1 integrin antibody, with the ERK pathway inhibitor PD98059, and/or with the phosphatidylinositol 3-kinase inhibitor LY294002 augmented cell death on 804G matrix but not on pLL. In long-term assays (48 h), PD98059 but not LY294002 drastically augmented cell death on 804G matrix but did so to a lesser extent on pLL. The protein inhibitor of nuclear factor-kappaB (IkappaBalpha) was overexpressed in cells cultured 18 h on matrix with partial blockade by PD98059. In summary, this study provides evidence for activation of signaling pathways and gene expression by extracellular matrix leading to improved beta-cell survival.     

Vascular smooth muscle cell migration induced by domains of thrombospondin-1 is differentially regulated

Background

Thrombospondin-1 (TSP-1) stimulates vascular smooth muscle cell (VSMC) migration via defined intracellular signaling pathways. The aim of this study was to examine the signaling pathways whereby TSP-1 folded domains (amino-terminal [NH₂], procollagen homology [PCH], all 3 type 1 repeats [3TSR], and a single recombinant protein containing the 3rd type 2 repeat, the type 3 repeats, and the carboxyl-terminal [E3T3C1]) induce VSMC migration.

Methods

Quiescent VSMCs were pretreated with serum-free media or inhibitors: PP2 (c-Src), LY294002 (phosphatidylinositol 3-kinase), FPT (Ras), Y27632 (Rho kinase), SB202190 (p38 kinase), and PD98059 (extracellular signal-regulated kinase). Migration induced by serum-free media, TSP-1, NH₂, PCH, 3TSR, and E3T3C1 was assessed using a modified Boyden chamber.

Results

TSP-1, NH₂, 3TSR, and E3T3C1 induced VSMC chemotaxis (P < .05), but PCH did not (P > .05). PP2, FPT, SB202190, and PD98059 attenuated chemotaxis stimulated by TSP-1, NH₂, 3TSR, and E3T3C1 (P < .05). LY294002 inhibited TSP-1-induced and E3T3C1-induced (P < .05) but not NH₂-induced or 3TSR-induced (P > .05) chemotaxis. Y27632 inhibited NH₂-induced, 3TSR-induced, and E3T3C1-induced (P < .05) but not TSP-1-induced (P > .05) induced chemotaxis.

Conclusions

TSP-1 folded domains are differentially dependent on intracellular signaling pathways to induce migration.     

Thiazide‐Sensitive Na+‐Cl− Cotransporter (NCC) Gene Inactivation Results in Increased Duodenal Ca2+ Absorption,Enhanced Osteoblast Differentiation and Elevated Bone Mineral Density

Inactivation of the thiazide‐sensitive sodium chloride cotransporter (NCC) due to genetic mutations in Gitelman's syndrome (GS) or pharmacological inhibition with thiazide diuretics causes hypocalciuria and increased bone mineral density (BMD) with unclear extrarenal calcium (Ca²⁺) regulation. We investigated intestinal Ca²⁺ absorption and bone Ca²⁺ metabolism in nonsense Ncc Ser707X (S707X) homozygous knockin mice (Ncc^S707X/S707X mice). Compared to wild‐type and heterozygous knockin littermates, Ncc^S707X/S707X mice had increased intestinal absorption of ⁴⁵Ca²⁺ and expression of the active Ca²⁺ transport machinery (transient receptor potential vanilloid 6, calbindin‐D_9K, and plasma membrane Ca²⁺ ATPase isoform 1b). Ncc^S707X/S707X mice had also significantly increased Ca²⁺ content accompanied by greater mineral apposition rate (MAR) in their femurs and higher trabecular bone volume, cortical bone thickness, and BMD determined by μCT. Their osteoblast differentiation markers, such as bone alkaline phosphatase, procollagen I, osteocalcin, and osterix, were also significantly increased while osteoclast activity was unaffected. Analysis of marrow‐derived bone cells, either treated with thiazide or directly cultured from Ncc S707X knockin mice, showed that the differentiation of osteoblasts was associated with increased phosphorylation of mechanical stress‐induced focal adhesion kinase (FAK) and extracellular signal‐regulated kinase (ERK). In conclusion, NCC inhibition stimulates duodenal Ca²⁺ absorption as well as osteoblast differentiation and bone Ca²⁺ storage, possibly through a FAK/ERK dependent mechanism. © 2014 American Society for Bone and Mineral Research.