U2AF65 enhances milk synthesis and growth of bovine mammary epithelial cells by positively regulating the mTOR‐SREBP‐1c signalling pathway

U2 snRNP auxiliary factor 65 kDa (U2AF65) is a splicing factor that promotes prespliceosome assembly. The function of U2AF65 in alternative splicing has been identified; however, the essential physiological role of U2AF65 remains poorly understood. In this study, we investigated the regulatory role of U2AF65 in milk synthesis and growth of bovine mammary epithelial cells (BMECs). Our results showed that U2AF65 localizes in the nucleus. Treatment with amino acids (Met and Leu) and hormones (prolactin and β‐estradiol) upregulated the expression of U2AF65 in these cells. U2AF65 overexpression increased the synthesis of β‐casein, triglycerides, and lactose; increased cell viability; and promoted proliferation of BMECs. Furthermore, our results showed that U2AF65 positively regulated mTOR phosphorylation and expression of mature mRNA of mTOR and SREBP‐1c. Collectively, our findings demonstrate that U2AF65 regulates the mRNA expression of signalling molecules (mTOR and SREBP‐1c) involved in milk synthesis and growth of BMECs, possibly via controlling the splicing and maturation of these mRNAs. U2 snRNP auxiliary factor 65 kDa (U2AF65) is a splicing factor that promotes prespliceosome assembly. The essential physiological role of U2AF65 remains poorly understood. In the present study, we confirmed that U2AF65 functions as a positive regulator of milk synthesis in and proliferation of bovine mammary epithelial cells via the mTOR‐SREBP‐1c signalling pathway. Therefore, our study uncovers the regulatory role of U2AF65 in milk synthesis and cell proliferation.     

Kp-10 promotes bovine mammary epithelial cell proliferation by activating GPR54 and its downstream signaling pathways

It has been reported that the proliferation and apoptosis of mammary epithelial cells affect milk production. Therefore, ensuring adequate mammary epithelial cells is expected to enhance milk production. This study is devoted to studying the effects of kisspeptin-10 (Kp-10), a peptide hormone composed of 10 amino acids, on bovine mammary epithelial cell (bMEC) proliferation and exploring the underlying mechanism of its action. bMECs were treated with various concentrations of Kp-10 (1, 10, 100, and 1,000 nM), and 100 nM Kp-10 promoted the proliferation of the bMECs. Kp-10 promoted the cell cycle transition from G1 to the S and G2 phases, increased the protein levels of Cyclin D1 and Cyclin D3, and reduced the expression levels of the p21 gene. This study also showed that inhibition of G protein-coupled receptor 54 (GPR54), AKT, mTOR, and ERK1/2 reduced the proliferation of the bMECs that had been induced by Kp-10. In addition, Kp-10 decreased the complexes formed by Rb and E2F1 and increased the expression levels of the E2F1 target genes. These results indicate that Kp-10 promotes bMEC proliferation by activating GPR54 and its downstream signaling pathways.     

GlyRS is a new mediator of amino acid-induced milk synthesis in bovine mammary epithelial cells

Amino acids are required for the mammalian target of rapamycin (mTOR) signaling pathway and milk synthesis in bovine mammary epithelial cells (BMECs). However, the mechanism through which amino acids activate this pathway is largely unknown. Here we show that glycyl-tRNA synthetase (GlyRS) mediates amino acid-induced activation of the mTOR-S6K1/4EBP1 pathway, and milk protein and fat synthesis in BMECs. Among 19 aminoacyl-tRNA synthetases, only the mRNA expression of GlyRS and Leucyl-tRNA synthetase (LeuRS) were significantly increased by several amino acids including Met and Leu. We then observed that GlyRS knockdown abolished the stimulation of Met on milk protein and fat synthesis in BMECs, whereas GlyRS overexpression led to more significantly increased milk synthesis in cells treated with Met. By western blotting and qualitative real time-polymerase chain reaction analysis (qRT-PCR) analysis, we next revealed that GlyRS is required for amino acid-induced activation of the mTOR-S6K1/4EBP1 pathway. Thus, this study establishes that GlyRS mediates amino acid-induced activation of the mTOR pathway, thereby regulating milk protein and fat synthesis.     

Cyclase‐associated protein 1 is a key negative regulator of milk synthesis and proliferation of bovine mammary epithelial cells

Adenylyl cyclase‐associated protein (CAP) is a highly conserved protein. Previous reports have suggested that CAP1 may be a negative regulator of cellular proliferation, migration, and adhesion and the development of cell carcinomas. The molecular mechanism of CAP1 regulation of downstream pathways, as well as how CAP1 is regulated by environmental stimuli and upstream signalling, is not well understood. In this present study, we assessed the role of CAP1 in milk synthesis and proliferation of bovine mammary epithelial cells. Using gene overexpression and silencing methods, CAP1 was found to negatively regulate milk synthesis and proliferation of cells via the PI3K‐mTOR/SREBP‐1c/Cyclin D1 signalling pathway. Hormones, such as prolactin and oestrogen, and amino acids, such as methionine and leucine, stimulate MMP9 expression and trigger CAP1 degradation, and thus, abrogate its inhibition of synthesis of milk protein, fat, and lactose by and proliferation of bovine mammary epithelial cells. The results of our study help deepen our understanding of the regulatory mechanisms underlying milk synthesis and aid in characterizing the molecular mechanisms of CAP1. Previous reports have suggested that CAP1 is a negative regulator of cellular proliferation and anabolism, but the molecular mechanisms are largely unknown. In this present study, we identified CAP1 as a negative regulator of milk synthesis and proliferation of bovine mammary epithelial cells. Our results will deepen our understanding of the regulatory mechanisms underlying milk synthesis and aid in exploring the molecular mechanisms of CAP1.     

Knockdown of mediator subunit Med19 suppresses bladder cancer cell proliferation and migration by downregulating Wnt/β‐catenin signalling pathway

Mediator complex subunit 19 (Med19), a RNA polymerase II‐embedded coactivator, is reported to be involved in bladder cancer (BCa) progression, but its functional contribution to this process is poorly understood. Here, we investigate the effects of Med19 on malignant behaviours of BCa, as well as to elucidate the possible mechanisms. Med19 expression in 15 BCa tissues was significantly higher than adjacent paired normal tissues using real‐time PCR and Western blot analysis. Immunohistochemical staining of 167 paraffin‐embedded BCa tissues was performed, and the results showed that high Med19 protein level was positively correlated with clinical stages and histopathological grade. Med19 was knocked down in BCa cells using short‐hairpin RNA. Functional assays showed that knocking‐down of Med19 can suppress cell proliferation and migration in T24, UM‐UC3 cells and 5637 in vitro, and inhibited BCa tumour growth in vivo. TOP/FOPflash reporter assay revealed that Med19 knockdown decreased the activity of Wnt/β‐catenin pathway, and the target genes of Wnt/β‐catenin pathway were down‐regulated, including Wnt2, β‐catenin, Cyclin‐D1 and MMP‐9. However, protein levels of Gsk3β and E‐cadherin were elevated. Our data suggest that Med19 expression correlates with aggressive characteristics of BCa and Med19 knockdown suppresses the proliferation and migration of BCa cells through down‐regulating the Wnt/β‐catenin pathway, thereby highlighting Med19 as a potential therapeutic target for BCa treatment.     

Cyclin T1 overexpression induces malignant transformation and tumor growth

Human PTEFb is a protein kinase composed by CDK9 and Cyclin T that controls the elongation phase of RNA Pol II. This complex also affects the activation and differentiation program of lymphoid cells. In this study we found that several head and neck tumor cell lines overexpress PTEFb. We also established that Cyclin T1 is able to induce transformation in vitro, as we determined by foci and colony formation assays. Nu/nu mice s.c. injected with stable transfected Cyclin T1 cells (NIH 3T3 Cyclin T1) developed tumors faster than animals injected with control cells (NIH 3T3 b-gal). In vitro, NIH 3T3 Cyclin T1 cells show increased proliferation and CDK4-Rb phosphorylation. Even more, silencing E2F1 expression (shRNA E2F1) in NIH 3T3 cells resulted in a dramatic inhibition of Cyclin T1-induced foci. All these data demonstrate for the first time the Cyclin T1 oncogenic function and suggest a role for this protein in controlling cell cycle probably via Rb/E2F1 pathway.     

Interaction of profilin‐1 and F‐actin via a β‐arrestin‐1/JNK signaling pathway involved in prostaglandin E2‐induced human mesenchymal stem cells migration and proliferation

Although many previous reports have examined the function of prostaglandin E₂ (PGE₂) in the migration and proliferation of various cell types, the role of the actin cytoskeleton in human mesenchymal stem cells (hMSCs) migration and proliferation has not been reported. The present study examined the involvement of profilin‐1 (Pfn‐1) and filamentous‐actin (F‐actin) in PGE₂‐induced hMSC migration and proliferation and its related signal pathways. PGE₂ (10^?6 M) increased both cell migration and proliferation, and also increased E‐type prostaglandin receptor 2 (EP2) mRNA expression, β‐arrestin‐1 phosphorylation, and c‐Jun N‐terminal kinase (JNK) phosphorylation. Small interfering RNA (siRNA)‐mediated knockdown of β‐arrestin‐1 and JNK (‐1, ‐2, ‐3) inhibited PGE₂‐induced growth of hMSCs. PGE₂ also activated Pfn‐1, which was blocked by JNK siRNA, and induced F‐actin level and organization. Downregulation of Pfn‐1 by siRNA decreased the level and organization of F‐actin. In addition, specific siRNA for TRIO and F‐actin‐binding protein (TRIOBP) reduced the PGE₂‐induced increase in hMSC migration and proliferation. Together, these experimental data demonstrate that PGE₂ partially stimulates hMSCs migration and proliferation by interaction of Pfn‐1 and F‐actin via EP2 receptor‐dependent β‐arrestin‐1/JNK signaling pathways. J. Cell. Physiol. 226: 559–571, 2011. © 2010 Wiley‐Liss, Inc.     

DNAJA1 positively regulates amino acid-stimulated milk protein and fat synthesis in bovine mammary epithelial cells

Several cellular processes, including the recovery of misfolded proteins, the folding of polypeptide chains, transit of polypeptides across the membrane, construction and disassembly of protein complexes, and modulation of protein control, are carried out by DnaJ homolog subfamily A member 1 (DNAJA1), which belongs to the DnaJ heat-shock protein family. It is unknown if DNAJA1 regulates the production of milk in bovine mammary epithelium cells (BMECs). Methionine and leucine increased DNAJA1 expression and nuclear location, as seen by us. In contrast to DNAJA1 knockdown, overexpression of DNAJA1 boosted the production of milk proteins and fats as well as mammalian target of rapamycin (mTOR) and sterol regulatory element binding protein-1c (SREBP-1c). As a result of amino acids, mTOR and SREBP-1c gene expression are stimulated, and DNAJA1 is a positive regulator of BMECs' amino acid-induced controlled milk protein and fat production.     

FER1L4/miR‐372/E2F1 works as a ceRNA system to regulate the proliferation and cell cycle of glioma cells

Long non‐coding RNAs have recently become a key regulatory factor for cancers, whereas FER1L4, a newly discovered long non‐coding RNA, has been mostly studied in gastric carcinoma and colon cancer cases. The functions and molecular mechanism of FER1L4 have been rarely reported in glioma malignant phenotypes. In this study, it was found that the expression of LncRNA FER1L4 is upregulated in high‐grade gliomas than in low‐grade cases and that a high expression of LncRNA FER1L4 predicts poor prognosis of gliomas. Meanwhile, in vitro study suggests that expression of FER1L4 with SiRNA knockdown obviously suppresses cell cycle and proliferation. It is further demonstrated by experiments that the FER1L4 knockdown suppresses growth of in vivo glioma. Besides, it is found in our study that LncRNA FER1L4 expression is positively correlated with E2F1 mRNA expression. After knockdown of FER1L4 expression, E2F1 expression is significantly down‐regulated, whereas the expression of miR‐372 is significantly up‐regulated; the up‐regulation of miR‐372 leads to significant down‐regulation of FER1L4 and E2F1 expression. In addition, it is also found that FER1L4 can be used as competitive endogenous RNA to interact or bind with miR‐371 and thereby up‐regulate E2F1, thus promoting the cycle and proliferation of glioma cells. It may be one of the molecular mechanisms in which FER1L4 plays its oncogene‐like role in gliomas.     

The apolipoprotein A‐I mimetic peptide,D‐4F,restrains neointimal formation through heme oxygenase‐1 up‐regulation

D‐4F, an apolipoprotein A‐I (apoA‐I) mimetic peptide, possesses distinctly anti‐atherogenic effects. However, the biological functions and mechanisms of D‐4F on the hyperplasia of vascular smooth muscle cells (VSMCs) remain unclear. This study aimed to determine its roles in the proliferation and migration of VSMCs. In vitro, D‐4F inhibited VSMC proliferation and migration induced by ox‐LDL in a dose‐dependent manner. D‐4F up‐regulated heme oxygenase‐1 (HO‐1) expression in VSMCs, and the PI3K/Akt/AMP‐activated protein kinase (AMPK) pathway was involved in these processes. HO‐1 down‐regulation with siRNA or inhibition with zinc protoporphyrin (Znpp) impaired the protective effects of D‐4F on the oxidative stress and the proliferation and migration of VSMCs. Moreover, down‐regulation of ATP‐binding cassette transporter A1 (ABCA1) abolished the activation of Akt and AMPK, the up‐regulation of HO‐1 and the anti‐oxidative effects of D‐4F. In vivo, D‐4F restrained neointimal formation and oxidative stress of carotid arteries in balloon‐injured Sprague Dawley rats. And inhibition of HO‐1 with Znpp decreased the inhibitory effects of D‐4F on neointimal formation and ROS production in arteries. In conclusion, D‐4F inhibited VSMC proliferation and migration in vitro and neointimal formation in vivo through HO‐1 up‐regulation, which provided a novel prophylactic and therapeutic strategy for anti‐restenosis of arteries.     

Both farnesyl diphosphate synthase genes are involved in the production of alarm pheromone in the green peach aphid Myzus persicae

Farnesyl diphosphate synthase (FPPS) catalyzes the formation of FPP, providing the precursor for the biosynthesis of (E)‐β‐farnesene (EβF) in plants, but it is unknown if FPPS supplies the precursor for the biosynthesis of EβF, the major component of aphid alarm pheromone, though our previous studies support the hypothesis that EβF is synthesized by the aphid itself. Here, we used two cohorts of the green peach aphid Myzus persicae separately, reared on pepper plant and artificial diet to test the correlations among droplet emission, EβF quantity, and FPPS gene expression. It was found that the proportion of aphids emitting cornicle droplets and the quantity of EβF per milligram of aphid were both significantly different between the two cohorts, which were positively correlated with the expression of the two FPPS genes ( MpFPPS1/ 2) in M. persicae. These results were further confirmed by RNAi‐mediated knockdown of MpFPPS1/ 2. Specifically, knockdown of MpFPPS1/ 2 imposed no significant cost on the survival of aphid but remarkably increased the number of offspring per aphid; most importantly, knockdown of MpFPPS1/ 2 significantly reduced the proportion of aphids emitting droplets and the quantity of EβF calculated as per the weight of aphid. Our results suggest that both FPPS genes are involved in the production of EβF in M. persicae and cornicle droplet emission is closely associated with the EβF release in the aphid.     

葡萄糖6-磷酸脱氢酶调控细胞周期蛋白E1和细胞周期蛋白依赖性激酶2促进细胞增殖及其在肾透明细胞癌中的预后价值

肾透明细胞癌(clear cell renal cell carcinoma,ccRCC)是一种转移率高、预后差的细胞代谢性疾病,对其有效诊疗及预后分子标志物的研究十分重要。葡萄糖6-磷酸脱氢酶(glucose 6-phosphatedehydrogenase, G6PD)在ccRCC中高表达,并提示患者不良预后,其促进ccRCC细胞增殖的分子机制有待进一步揭示。本研究发现,降低G6PD可抑制细胞周期G₁/S期转化并显著抑制ccRCC细胞增殖。G6PD可在细胞水平调控G₁/S期转化及增殖相关因子Cyclin D1,CDK4,CDK6,Cyclin E1和CDK2基因表达。TCGA数据库分析结果表明,ccRCC 中Cyclin D1,Cyclin E1 和 CDK2的mRNA 水平显著升高,而CDK4表达无明显差异,CDK6表达却显著降低。相关性分析结果显示,G6PD与Cyclin D1呈显著负相关(P<0.0001),G6PD与CDK4,CDK6之间无显著相关性(P>0.05),G6PD与Cyclin E1(P<0.0001)以及CDK2(P<0.05)显著正相关。进一步免疫组化检测结果表明,Cyclin E1和 CDK2在ccRCC肿瘤组织中表达显著升高。生存预后分析结果显示,Cyclin D1高表达提示ccRCC患者整体预后更为良好,CDK4和CDK6表达水平在ccRCC患者总生存率预测中无意义;而Cyclin E1和CDK2高表达均可提示ccRCC患者预后不良。进一步细胞水平检测发现,Cyclin E1、CDK2表达降低可显著逆转G6PD促进ccRCC细胞增殖的能力。综上,与增殖相关因子Cyclin D1,CDK4和CDK6相比,G6PD有可能通过促进Cyclin E1和CDK2表达升高而发挥促进 ccRCC肿瘤细胞增殖的作用,并且这3者的异常高表达有望成为ccRCC患者不良预后的独立生存预测因素。     

Specificity and cooperativity at β-lactamase position 104 in TEM-1/BLIP and SHV-1/BLIP interactions

Establishing a quantitative understanding of the determinants of affinity in protein–protein interactions remains challenging. For example, TEM‐1/β‐lactamase inhibitor protein (BLIP) and SHV‐1/BLIP are homologous β‐lactamase/β‐lactamase inhibitor protein complexes with disparate K_d values (3 nM and 2 μM, respectively), and a single substitution, D104E in SHV‐1, results in a 1000‐fold enhancement in binding affinity. In TEM‐1, E104 participates in a salt bridge with BLIP K74, whereas the corresponding SHV‐1 D104 does not in the wild type SHV‐1/BLIP co‐structure. Here, we present a 1.6 Å crystal structure of the SHV‐1 D104E/BLIP complex that demonstrates that this point mutation restores this salt bridge. Additionally, mutation of a neighboring residue, BLIP E73M, results in salt bridge formation between SHV‐1 D104 and BLIP K74 and a 400‐fold increase in binding affinity. To understand how this salt bridge contributes to complex affinity, the cooperativity between the E/K or D/K salt bridge pair and a neighboring hot spot residue (BLIP F142) was investigated using double mutant cycle analyses in the background of the E73M mutation. We find that BLIP F142 cooperatively stabilizes both interactions, illustrating how a single mutation at a hot spot position can drive large perturbations in interface stability and specificity through a cooperative interaction network. Proteins 2011. © 2011 Wiley‐Liss, Inc.