Novel Variants in the Putative Peroxisome Proliferator‐activated Receptor γ Promoter and Relationships with Obesity in Men

Yet unidentified variants within the peroxisome proliferator‐activated receptor γ (PPARγ) 2 promoter may explain the inconsistent reports on associations between variants in the coding region and obesity or diabetes. Thus, we examined the putative PPARγ2 promoter (?3371 to +43 bp) for variants in 83 subjects with obesity or type 2 diabetes. We identified eight variants, seven of which were novel, including ?792A>G, ?816C>T, ?882T>C, ?1505G>A, ?1881C>T, ?1884T>A, ?2604T>C, and ?2953A>G. The variants ?816C>T, ?1505G>A, ?1881C>T, and ?2604T>C were in total linkage disequilibrium, and there was a high degree of linkage disequilibrium between several of the novel variants and Pro12Ala. The novel variants were, together with Pro12Ala and 1431C>T, examined for relationships with obesity among 234 men with early‐onset obesity with a BMI at age ～20 years of 33.2 ± 2.5 kg/m² and 323 nonobese men with a BMI of 21.7 ± 2.5 kg/m², who were also reexamined after ～29 years. The prevalence of the identified variants was not significantly different between the two groups, and the variants did not affect changes in BMI over time. In conclusion, the identified novel variants in the PPARγ2 promoter region do not explain the reported discrepancies in the association of previously identified variants with obesity and type 2 diabetes.     

The Expression of Peroxisome Proliferator‐Activated Receptor γ in Pig Fetal Tissue and Primary Stromal‐Vascular Cultures

Objective: This study was designed to determine when peroxisome proliferator‐activated receptor γ (PPARγ) is expressed in developing fetal adipose tissue and stromal‐vascular adipose precursor cells derived from adipose tissue. In addition we examined developing tissue for CCAAT/enhancer‐binding protein β (C/EBPβ) expression to see if it was correlated with PPARγ expression. Pituitary function and hormones involved with differentiation (dexamethasone and retinoic acid) were also tested for their effects on PPARγ expression to determine if hormones known to affect differentiation also effect PPARγ expression in vivo and in cell culture. Research Methods and Procedures: Developing subcutaneous adipose tissues from the dorsal region of the fetal pig were collected at different gestation times and assayed using Western blot analysis to determine levels of PPARγ and C/EBPβ. Hypophysectomy was performed on 75‐day pig fetuses and tissue samples were then taken at 105 days for Western blot analysis. Adipose tissue was also taken from postnatal pigs to isolate stromal‐vascular (S‐V) cells. These adipose precursor cells were grown in culture and samples were taken for Western blot analysis to determine expression levels of PPARγ. Results: Our results indicate that PPARγ is expressed as early as 50 days of fetal development in adipose tissue and continues through 105 days. Expression of PPARγ was found to be significantly enhanced in adipose tissue from hypophysectomized fetuses at 105 days of fetal development (p < 0.05). C/EBPβ was not found in 50‐ or 75‐day fetal tissues and was found only at low levels in 105‐day tissues. C/EBPβ was not found in hypophysectomized (hypoxed) 105‐day tissue where PPARγ was elevated. S‐V cells freshly isolated from adipose tissue of 5‐ to 7‐day postnatal pigs showed the expression of PPARγ1. When S‐V cells were cultured, both PPARγ1 and 2 were expressed after the first day and continued as cells differentiated. High concentrations of retinoic acid decreased PPARγ expression in early S‐V cultures (p < 0.05). Discussion: Our data indicate that PPARγ is expressed in fetal adipose tissue very early before distinct fat cells are observed and can be expressed without the expression of C/EBPβ. The increase in PPARγ expression after hypophysectomy may explain the increase in fat cell size under these conditions. Adipose precursor cells (S‐V cells) from 5‐ to 7‐day postnatal pigs also express PPARγ in the tissue before being induced to differentiate in culture. Thus S‐V cells from newborn pig adipose tissue are probably more advanced in development than the 3T3‐L1 cell model. S‐V cells may be in a state where PPARγ and C/EBPα are expressed but new signals or vascularization are needed before cells are fully committed and lipid filling begins.     

Antagonizing peroxisome proliferator‐activated receptor γ facilitates M1‐to‐M2 shift of microglia by enhancing autophagy via the LKB1–AMPK signaling pathway

Microglia‐mediated neuroinflammation plays a dual role in various brain diseases due to distinct microglial phenotypes, including deleterious M1 and neuroprotective M2. There is growing evidence that the peroxisome proliferator‐activated receptor γ (PPARγ) agonist rosiglitazone prevents lipopolysaccharide (LPS)‐induced microglial activation. Here, we observed that antagonizing PPARγ promoted LPS‐stimulated changes in polarization from the M1 to the M2 phenotype in primary microglia. PPARγ antagonist T0070907 increased the expression of M2 markers, including CD206, IL‐4, IGF‐1, TGF‐β1, TGF‐β2, TGF‐β3, G‐CSF, and GM‐CSF, and reduced the expression of M1 markers, such as CD86, Cox‐2, iNOS, IL‐1β, IL‐6, TNF‐α, IFN‐γ, and CCL2, thereby inhibiting NFκB–IKKβ activation. Moreover, antagonizing PPARγ promoted microglial autophagy, as indicated by the downregulation of P62 and the upregulation of Beclin1, Atg5, and LC3‐II/LC3‐I, thereby enhancing the formation of autophagosomes and their degradation by lysosomes in microglia. Furthermore, we found that an increase in LKB1–STRAD–MO25 complex formation enhances autophagy. The LKB1 inhibitor radicicol or knocking down LKB1 prevented autophagy improvement and the M1‐to‐M2 phenotype shift by T0070907. Simultaneously, we found that knocking down PPARγ in BV2 microglial cells also activated LKB1–AMPK signaling and inhibited NFκB–IKKβ activation, which are similar to the effects of antagonizing PPARγ. Taken together, our findings demonstrate that antagonizing PPARγ promotes the M1‐to‐M2 phenotypic shift in LPS‐induced microglia, which might be due to improved autophagy via the activation of the LKB1–AMPK signaling pathway.     

Dihydromyricetin enhances glucose uptake by inhibition of MEK/ERK pathway and consequent down‐regulation of phosphorylation of PPARγ in 3T3‐L1 cells

Accumulating evidence suggests that inhibition of mitogen‐activated protein kinase signalling can reduce phosphorylation of peroxisome proliferator‐activated receptor γ (PPARγ) at serine 273, which mitigates obesity‐associated insulin resistance and might be a promising treatment for type 2 diabetes. Dihydromyricetin (DHM) is a flavonoid that has many beneficial pharmacological properties. In this study, mouse fibroblast 3T3‐L1 cells were used to investigate whether DHM alleviates insulin resistance by inhibiting PPARγ phosphorylation at serine 273 via the MEK/ERK pathway. 3T3‐L1 pre‐adipocytes were differentiated, and the effects of DHM on adipogenesis and glucose uptake in the resulting adipocytes were examined. DHM was found to dose dependently increase glucose uptake and decrease adipogenesis. Insulin resistance was then induced in adipocytes using dexamethasone, and DHM was shown to dose and time dependently promote glucose uptake in the dexamethasone‐treated adipocytes. DHM also inhibited phosphorylation of PPARγ and ERK. Inhibition of PPARγ activity with GW9662 potently blocked DHM‐induced glucose uptake and adiponectin secretion. Interestingly, DHM showed similar effects to PD98059, an inhibitor of the MEK/ERK pathway. DHM acted synergistically with PD98059 to improve glucose uptake and adiponectin secretion in dexamethasone‐treated adipocytes. In conclusion, our findings indicate that DHM improves glucose uptake in adipocytes by inhibiting ERK‐induced phosphorylation of PPARγ at serine 273.     

Tumour necrosis factor‐α promotes liver ischaemia‐reperfusion injury through the PGC‐1α/Mfn2 pathway

Tumour necrosis factor (TNF)‐α has been considered to induce ischaemia‐reperfusion injury (IRI) of liver which is characterized by energy dysmetabolism. Peroxisome proliferator–activated receptor‐γ co‐activator (PGC)‐1α and mitofusion2 (Mfn2) are reported to be involved in the regulation of mitochondrial function. However, whether PGC‐1α and Mfn2 form a pathway that mediates liver IRI, and if so, what the underlying involvement is in that pathway remain unclear. In this study, L02 cells administered recombinant human TNF‐α had increased TNF‐α levels and resulted in down‐regulation of PGC‐1α and Mfn2 in a rat liver IRI model. This was associated with hepatic mitochondrial swelling, decreased adenosine triphosphate (ATP) production, and increased levels of reactive oxygen species (ROS) and alanine aminotransferase (ALT) activity as well as cell apoptosis. Inhibition of TNF‐α by neutralizing antibody reversed PGC‐1α and Mfn2 expression, and decreased hepatic injury and cell apoptosis both in cell culture and in animals. Treatment by rosiglitazone sustained PGC‐1α and Mfn2 expression both in IR livers, and L02 cells treated with TNF‐α as indicated by increased hepatic mitochondrial integrity and ATP production, reduced ROS and ALT activity as well as decreased cell apoptosis. Overexpression of Mfn2 by lentiviral‐Mfn2 transfection decreased hepatic injury in IR livers and L02 cells treated with TNF‐α. However, there was no up‐regulation of PGC‐1α. These findings suggest that PGC‐1α and Mfn2 constitute a regulatory pathway, and play a critical role in TNF‐α‐induced hepatic IRI. Inhibition of the TNF‐α or PGC‐1α/Mfn2 pathways may represent novel therapeutic interventions for hepatic IRI.     

Antidiabetic‐Like Effects of Naringenin‐7‐O‐glucoside from Edible Chrysanthemum ‘Kotobuki’ and Naringenin by Activation of the PI3K/Akt Pathway and PPARγ

Obesity is directly associated with cancer, cardiovascular injury, hypertension, and type 2 diabetes. To date, Yamamoto identified that hot water extracts of edible Chrysanthemum (EC) induced cell size reduction, up‐regulation of adiponectin expression, and glucose absorption inhibition in 3T3‐L1 cells during adipocyte differentiation. Furthermore, EC showed antidiabetic effects such as improvement in insulin resistance and the down‐regulation of the blood glucose level and liver lipid content in type 2 diabetes model mice. In this study, we attempted to identify the antidiabetic components in EC. The methanol fraction from EC that showed relatively strong biological activity was purified by chromatography to obtain acacetin‐7‐O‐glucoside, apigenin‐7‐O‐glucoside, kaempferol‐7‐O‐glucoside, and naringenin‐7‐O‐glucoside. Among the isolated compounds and their aglycones, naringenin (NA) and naringenin‐7‐O‐glucoside (NAG) up‐regulated the intracellular accumulation of lipid and adiponectin‐secretion and down‐regulated the diameter of 3T3‐L1 cells during adipocyte differentiation. Because the PPARγ antagonist BADGE and PI3K/Akt inhibitors wortmannin and LY29004 inhibited the intracellular lipid accumulation by NA and NAG associated with adipogenesis, it was considered that NA and NAG showed the above‐mentioned activities via the activation of PPARγ as well as phosphorylation of the PI3K/Akt pathway.     

IFN‐γ inhibits basal and α‐MSH‐induced melanogenesis

Inflammatory cytokines are closely related to pigmentary changes. In this study, the effects of IFN‐γ on melanogenesis were investigated. IFN‐γ inhibits basal and α‐MSH‐induced melanogenesis in B16 melanoma cells and normal human melanocytes. MITF mRNA and protein expressions were significantly inhibited in response to IFN‐γ. IFN‐γ inhibited CREB binding to the MITF promoter but did not affect CREB phosphorylation. Instead, IFN‐γ inhibited the association of CBP and CREB through the increased association between CREB binding protein (CBP) and STAT1. These findings suggest that IFN‐γ inhibits both basal and α‐MSH‐induced melanogenesis by inhibiting MITF expression. The inhibitory action of IFN‐γ in α‐MSH‐induced melanogenesis is likely to be associated with the sequestration of CBP via the association between CBP and STAT1. These data suggest that IFN‐γ plays a role in controlling inflammation‐ or UV‐induced pigmentary changes.     

PPAR‐β facilitating maturation of hepatic‐like tissue derived from mouse embryonic stem cells accompanied by mitochondriogenesis and membrane potential retention

Relatively little is known about mitochondria metabolism in differentiating embryonic stem (ES) cells. Present research focused on several elements of cellular energy metabolism in hepatic‐like tissue derived from mouse ES cells. We demonstrated that mitochondrial location patterns and mitochondrial membrane potential (ΔΨ_m) existed in subsequent differentiation of the tissue. Mitochondriogenesis appeared at the early stage and kept a normal ΔΨ_m in differentiated mature hepatocytes. Peroxisome proliferator‐activated receptor‐α (PPAR‐α) expression was transitorily increased at the beginning, and kept a relatively low level later, which accompanied by expression of PPAR‐γ coactivator (PGC)‐1α, a master regulator of mitochondrial biogenesis. PPAR‐β expression showed robust up‐regulation in the late differentiation course. Enhanced co‐expressions of PPAR‐β and albumin with catalysis of UDP‐glucuronosyltransferases (UGTs) were observed at mature stage. While PPAR‐γ expression changed little before and after differentiation. Mitochondriogenesis could be accelerated by PPAR‐α specific agonist WY14643 and abolished by its antagonist GW6471 at the early stage. Neither of them affected mitochondrial ΔΨ_m and albumin generation in the differentiated hepatocytes. Furthermore, maturation of hepatic‐like tissue and mitochondriogenesis in hepatocyte could be efficiently stimulated by PPAR‐β specific agonist L165041 and abolished by PPAR‐β specific antagonist GSK0660, but not affected by PPAR‐γ specific agonist GW1929. In conclusion, the derived hepatic tissue morphologically possessed cellular energy metabolism features. PPAR‐α seemed only necessary for early mitochondriogenesis, while less important for ΔΨ_m retention in the mature tissue derived. The stimulation of PPAR‐β but not ‐γ enhanced hepatogenesis, hepatocytes maturation, and mitochondriogenesis. PPAR‐β took an important role in cellular energy metabolism of hepatogenesis. J. Cell. Biochem. 109: 498–508, 2010. © 2009 Wiley‐Liss, Inc.     

CD38 deficiency suppresses adipogenesis and lipogenesis in adipose tissues through activating Sirt1/PPARγ signaling pathway

It has been recently reported that CD38 was highly expressed in adipose tissues from obese people and CD38‐deficient mice were resistant to high‐fat diet (HFD)‐induced obesity. However, the role of CD38 in the regulation of adipogenesis and lipogenesis is unknown. In this study, to explore the roles of CD38 in adipogenesis and lipogenesis in vivo and in vitro, obesity models were generated with male CD38^?/? and WT mice fed with HFD. The adipocyte differentiations were induced with MEFs from WT and CD38^?/? mice, 3T3‐L1 and C3H10T1/2 cells in vitro. The lipid accumulations and the alternations of CD38 and the genes involved in adipogenesis and lipogenesis were determined with the adipose tissues from the HFD‐fed mice or the MEFs, 3T3‐L1 and C3H10T1/2 cells during induction of adipocyte differentiation. The results showed that CD38^?/? male mice were significantly resistant to HFD‐induced obesity. CD38 expressions in adipocytes were significantly increased in WT mice fed with HFD, and the similar results were obtained from WT MEFs, 3T3‐L1 and C3H10T1/2 during induction of adipocyte differentiation. The expressions of PPARγ, AP2 and C/EBPα were markedly attenuated in adipocytes from HFD‐fed CD38^?/? mice and CD38^?/? MEFs at late stage of adipocyte differentiation. Moreover, the expressions of SREBP1 and FASN were also significantly decreased in CD38^?/? MEFs. Finally, the CD38 deficiency‐mediated activations of Sirt1 signalling were up‐regulated or down‐regulated by resveratrol and nicotinamide, respectively. These results suggest that CD38 deficiency impairs adipogenesis and lipogenesis through activating Sirt1/PPARγ‐FASN signalling pathway during the development of obesity.     

Angiotensin II receptor blocker telmisartan enhances running endurance of skeletal muscle through activation of the PPAR‐δ/AMPK pathway

Clinical trials have shown that angiotensin II receptor blockers reduce the new onset of diabetes in hypertensives; however, the underlying mechanisms remain unknown. We investigated the effects of telmisartan on peroxisome proliferator activated receptor γ (PPAR‐δ) and the adenosine monophosphate (AMP)‐activated protein kinase (AMPK) pathway in cultured myotubes, as well as on the running endurance of wild‐type and PPAR‐δ‐deficient mice. Administration of telmisartan up‐regulated levels of PPAR‐δ and phospho‐AMPKα in cultured myotubes. However, PPAR‐δ gene deficiency completely abolished the telmisartan effect on phospho‐AMPKαin vitro. Chronic administration of telmisartan remarkably prevented weight gain, enhanced running endurance and post‐exercise oxygen consumption, and increased slow‐twitch skeletal muscle fibres in wild‐type mice, but these effects were absent in PPAR‐δ‐deficient mice. The mechanism is involved in PPAR‐δ‐mediated stimulation of the AMPK pathway. Compared to the control mice, phospho‐AMPKα level in skeletal muscle was up‐regulated in mice treated with telmisartan. In contrast, phospho‐AMPKα expression in skeletal muscle was unchanged in PPAR‐δ‐deficient mice treated with telmisartan. These findings highlight the ability of telmisartan to improve skeletal muscle function, and they implicate PPAR‐δ as a potential therapeutic target for the prevention of type 2 diabetes.     

Characterization of an Importin α/β‐recognized nuclear localization signal in β‐dystroglycan

β‐dystroglycan (β‐DG) is a widely expressed transmembrane protein that plays important roles in connecting the extracellular matrix to the cytoskeleton, and thereby contributing to plasma membrane integrity and signal transduction. We previously observed nuclear localization of β‐DG in cultured cell lines, implying the existence of a nuclear targeting mechanism that directs it to the nucleus instead of the plasma membrane. In this study, we delineate the nuclear import pathway of β‐DG, characterizing a functional nuclear localization signal (NLS) in the β‐DG cytoplasmic domain, within amino acids 776–782. The NLS either alone or in the context of the whole β‐DG protein was able to target the heterologous GFP protein to the nucleus, with site‐directed mutagenesis indicating that amino acids R⁷⁷⁹ and K⁷⁸⁰ are critical for NLS functionality. The nuclear transport molecules Importin (Imp)α and Impβ bound with high affinity to the NLS of β‐DG and were found to be essential for NLS‐dependent nuclear import in an in vitro reconstituted nuclear transport assay; cotransfection experiments confirmed the dependence on Ran for nuclear accumulation. Intriguingly, experiments suggested that tyrosine phosphorylation of β‐DG may result in cytoplasmic retention, with Y⁸⁹² playing a key role. β‐DG thus follows a conventional Impα/β‐dependent nuclear import pathway, with important implications for its potential function in the nucleus. J. Cell. Biochem. 110: 706–717, 2010. © 2010 Wiley‐Liss, Inc.     

The Peroxisome Proliferator‐Activated Receptor α L162V Mutation Is Associated with Reduced Adiposity

Objective: To determine the contribution of the peroxisome proliferator‐activated receptor α (PPARα) L162V mutation to the variation of several indexes of body fatness obtained from healthy adults who participated in the Quebec Family Study. Research Methods and Procedures: The PPARα L162V mutation was determined by a mismatch polymerase chain reaction method. Adiposity phenotypes were obtained by standardized anthropometric measurements, underwater weighing technique, and computed tomography. Results: For all adiposity phenotypes, subjects carrying the V162 allele had lower values compared with L162 homozygotes (HMZs) [BMI (kg/m²): 27.8 ± 7.6 vs. 26.0 ± 5.6, p < 0.05; percentage body fat: 28.5 ± 10.7 vs. 25.7 ± 10.1, p < 0.05; waist circumference (cm): 89.0 ± 18.1 vs. 85.7 ± 15.8, p = 0.07; total computed tomography abdominal fat areas (cm²): 406 ± 221 vs. 359 ± 192, p = 0.15; means ± SD for L162 HMZs vs. V162 carriers, respectively]. Differences in cross‐sectional abdominal adipose tissue areas and waist circumference were abolished after adjustment for total body fat mass. Similar trends were observed when results were analyzed by gender, although associations seemed stronger in women. The odds ratio of having a BMI above 30 kg/m² reached 1.77 (1.02; 3.07, 95% confidence intervals) for L162 HMZs. This risk could be considered marginal on an individual basis, but because 85% of the subjects are affected by this small risk, the impact on the population is important. Discussion: The PPARα V162 allele is associated with reduced adiposity and has a substantial population‐attributable risk.     

Regulation of interleukin‐1β by interferon‐γ is species specific,limited by suppressor of cytokine signalling 1 and influences interleukin‐17 production

Reports describing the effect of interferon‐γ (IFNγ) on interleukin‐1β (IL‐1β) production are conflicting. We resolve this controversy by showing that IFNγ potentiates IL‐1β release from human cells, but transiently inhibits the production of IL‐1β from mouse cells. Release from this inhibition is dependent on suppressor of cytokine signalling 1. IL‐1β and Th17 cells are pathogenic in mouse models for autoimmune disease, which use Mycobacterium tuberculosis (MTB), in which IFNγ and IFNβ are anti‐inflammatory. We observed that these cytokines suppress IL‐1β production in response to MTB, resulting in a reduced number of IL‐17‐producing cells. In human cells, IFNγ increased IL‐1β production, and this might explain why IFNγ is detrimental for multiple sclerosis. In mice, IFNγ decreased IL‐1β and subsequently IL‐17, indicating that the adaptive immune response can provide a systemic, but transient, signal to limit inflammation.     

寡核苷酸芯片技术用于检测过氧化物酶体活化物激活受体基因多态性

 通过寡核苷酸芯片技术检测PPARα基因Leu162Val、Val227Ala多态性和PPARγ Pro12Ala的基因多态性,建立一种快速、简便、准确的方法,为研究非酒精性脂肪性肝病的发病机制、临床诊断和治疗提供依据.收集人体外周血标本,提取DNA进行PCR扩增,设计相应的探针和引物,制备检测芯片,PCR产物与芯片杂交后,扫描芯片并分析结果.PCR产物进行测序验证.寡核苷酸芯片技术检测PPARα基因Leu162Val、Val227Ala多态性和PPARγ Pro12Ala基因多态性结果与测序结果一致.寡核苷酸芯片技术检测非酒精性脂肪性肝病(NAFLD)密切相关的PPAR基因多态性快速、准确,值得临床推广和应用.     

α1 adrenoceptor activation by norepinephrine inhibits LPS‐induced cardiomyocyte TNF‐α production via modulating ERK1/2 and NF‐κB pathway

Cardiomyocyte tumour necrosis factor α (TNF‐α) production contributes to myocardial depression during sepsis. This study was designed to observe the effect of norepinephrine (NE) on lipopolysaccharide (LPS)‐induced cardiomyocyte TNF‐α expression and to further investigate the underlying mechanisms in neonatal rat cardiomyocytes and endotoxaemic mice. In cultured neonatal rat cardiomyocytes, NE inhibited LPS‐induced TNF‐α production in a dose‐dependent manner. α₁‐ adrenoceptor (AR) antagonist (prazosin), but neither β₁‐ nor β₂‐AR antagonist, abrogated the inhibitory effect of NE on LPS‐stimulated TNF‐α production. Furthermore, phenylephrine (PE), an α₁‐AR agonist, also suppressed LPS‐induced TNF‐α production. NE inhibited p38 phosphorylation and NF‐κB activation, but enhanced extracellular signal‐regulated kinase 1/2 (ERK1/2) phosphorylation and c‐Fos expression in LPS‐treated cardiomyocytes, all of which were reversed by prazosin pre‐treatment. To determine whether ERK1/2 regulates c‐Fos expression, p38 phosphorylation, NF‐κB activation and TNF‐α production, cardiomyocytes were also treated with U0126, a selective ERK1/2 inhibitor. Treatment with U0126 reversed the effects of NE on c‐Fos expression, p38 mitogen‐activated protein kinase (MAPK) phosphorylation and TNF‐α production, but not NF‐κB activation in LPS‐challenged cardiomyocytes. In addition, pre‐treatment with SB202190, a p38 MAPK inhibitor, partly inhibited LPS‐induced TNF‐α production in cardiomyocytes. In endotoxaemic mice, PE promoted myocardial ERK1/2 phosphorylation and c‐Fos expression, inhibited p38 phosphorylation and IκBα degradation, reduced myocardial TNF‐α production and prevented LPS‐provoked cardiac dysfunction. Altogether, these findings indicate that activation of α₁‐AR by NE suppresses LPS‐induced cardiomyocyte TNF‐α expression and improves cardiac dysfunction during endotoxaemia via promoting myocardial ERK phosphorylation and suppressing NF‐κB activation.     

β‐arrestin2/miR‐155/GSK3β regulates transition of 5′‐azacytizine‐induced Sca‐1‐positive cells to cardiomyocytes

Stem‐cell antigen 1–positive (Sca‐1+) cardiac stem cells (CSCs), a vital kind of CSCs in humans, promote cardiac repair in vivo and can differentiate to cardiomyocytes with 5′‐azacytizine treatment in vitro. However, the underlying molecular mechanisms are unknown. β‐arrestin2 is an important scaffold protein and highly expressed in the heart. To explore the function of β‐arrestin2 in Sca‐1+ CSC differentiation, we used β‐arrestin2–knockout mice and overexpression strategies. Real‐time PCR revealed that β‐arrestin2 promoted 5′‐azacytizine‐induced Sca‐1+ CSC differentiation in vitro. Because the microRNA 155 (miR‐155) may regulate β‐arrestin2 expression, we detected its role and relationship with β‐arrestin2 and glycogen synthase kinase 3 (GSK3β), another probable target of miR‐155. Real‐time PCR revealed that miR‐155, inhibited by β‐arrestin2, impaired 5′‐azacytizine‐induced Sca‐1+ CSC differentiation. On luciferase report assay, miR‐155 could inhibit the activity of β‐arrestin2 and GSK3β, which suggests a loop pathway between miR‐155 and β‐arrestin2. Furthermore, β‐arrestin2‐knockout inhibited the activity of GSK3β. Akt, the upstream inhibitor of GSK3β, was inhibited in β‐arrestin2‐Knockout mice, so the activity of GSK3β was regulated by β‐arrestin2 not Akt. We transplanted Sca‐1+ CSCs from β‐arrestin2‐knockout mice to mice with myocardial infarction and found similar protective functions as in wild‐type mice but impaired arterial elastance. Furthermore, low level of β‐arrestin2 agreed with decreased phosphorylation of AKT and increased phophorylation of GSK3β, similar to in vitro findings. The β‐arrestin2/miR‐155/GSK3β pathway may be a new mechanism with implications for treatment of heart disease.