Lo-Chun Au

Insulin Up-Regulates Heme Oxygenase-1 Expression in 3T3-L1 Adipocytes via PI3-Kinase- and PKC-Dependent Pathways and Heme Oxygenase-1–Associated MicroRNA Downregulation

Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme catabolism, has antioxidant, antiinflammatory, and antiapoptotic effects in many physiological systems. HO-1 activity in obese mice is lower than in controls, and a sustained increase in HO-1 protein levels ameliorates insulin resistance and compensatory hyperinsulinemia. In the present study, we explored the regulatory effect of insulin on HO-1 expression in 3T3-L1 adipocytes and the underlying mechanism. We investigated the time- and dose-effect of insulin on HO-1 expression in 3T3-L1 adipocytes. Using specific inhibitors acting on insulin signaling pathways, we clarified the involvement of insulin downstream signaling molecules in insulin-regulated HO-1 expression. We also investigated the involvement of microRNAs (miRNAs) in insulin-regulated HO-1 expression using microarray and real-time RT-PCR assays. In an in vivo study, we performed insulin/glucose coinfusion in rats to increase circulating insulin levels for 8 h, then measured adipocyte HO-1 expression. Insulin caused a significant increase in HO-1 expression that was time- and dose-dependent, and this effect was blocked by inhibition of phosphatidylinositol 3 (PI3)-kinase activation using LY294002 (50 μM) or of protein kinase C activation using Ro-318220 (2 μM), but not by an Akt inhibitor, triciribine (10 μM). Furthermore, incubation of 3T3-L1 adipocytes with 100 nm insulin resulted in a significant decrease in levels of the miRNAs mir-155, mir-183, and mir-872, and this effect was also blocked by pretreatment with LY294002 or Ro-318220, but not triciribine. An in vivo study in rats showed that 8 h of a hyperinsulinemic euglycemic state resulted in a significant increase in adipocyte HO-1 expression. In conclusion, insulin increases HO-1 protein expression in 3T3-L1 adipocytes via PI3-kinase and protein kinase C-dependent pathways and miRNAs down-regulation.

Production and characterization of bioactive recombinant resistin in Escherichia coli

Type 2 diabetes, characterized by peripheral target tissue resistance to insulin, is epidemic in industrialized countries and is strongly associated with obesity. The protein hormone, resistin, secreted specifically by the adipose tissues, is found to antagonize insulin action upon glucose uptake and may serve as an important role between human obesity and insulin resistance. Here, we report the production of bioactive recombinant resistin in Escherichia coli. cDNA of resistin was obtained by RT-PCR from mRNA of mouse differentiated NIH/3T3-L1 cells. The cDNA of mature resistin was inserted in the pQE-31 vector and the recombinant plasmid was transferred into E. coli JM109. After IPTG induction, the rec. resistin found in the inclusion body was dissolved in 6 M guanidine-HCl in the presence of 10 mM beta-mercaptoethanol. The His-tag containing protein was purified by Ni-NTA column to 95% homogeneity. After a quasi-static-like refolding process, the secondary structure of the rec. resistin was elucidated by circular dichroism which indicated that the protein was composed of 34.3% alpha-helix, 8.9% beta-sheet, 23.4% beta-turn, and 31.2% unordered structure. No disulfide-linked homodimers were formed in SDS-PAGE analysis under non-reducing conditions. The rec. resistin showed a dose-dependent antagonizing action against insulin in [3H]-2-deoxy-glucose transport in a broad range from 1 ng ml(-1) to 10 microg ml(-1) of resistin. A suppression of 85% of transport was achieved at the dosage of 10 microg ml(-1). This result may indicate that the rec. resistin does not need to form homodimers to establish its bioactivity. The rec. resistin will be useful for exploring the biological functions of this newly discovered hormone.

Short-Term Regulation of Tumor Necrosis Factor-α-Induced Lipolysis in 3T3-L1 Adipocytes Is Mediated through the Inducible Nitric Oxide Synthase/Nitric Oxide-Dependent Pathway

TNF-alpha has several effects on adipocytes that may be related to the development of type 2 diabetes in obese subjects. Many studies demonstrated that long-term treatment with TNF-alpha increases lipolysis in adipocytes. However, the short-term (<4 h) effects of TNF-alpha on lipolysis have not been well investigated. The aim of this study was to investigate the short-term regulatory mechanism of TNF-alpha-induced lipolysis in 3T3-L1 adipocytes. Well-differentiated 3T3-L1 adipocytes were used. Lipolysis was determined by measuring glycerol release. Expression of inducible nitric oxide (iNOS) and nitric oxide (NO) production were measured, respectively, by Western blots and the Griess reagent. A selective iNOS inhibitor (s-ethylisothiourea . HBr), an adenylyl cyclase inhibitor (SQ22536), and a guanylyl cyclase inhibitor (LY83583) were used to investigate the involvement of iNOS, cAMP, and cGMP in TNF-alpha-induced lipolysis. Transient transfection with iNOS short hairpin RNA was performed to confirm the involvement of iNOS in TNF-alpha-induced lipolysis. Phosphorylation of hormone-sensitive lipase (HSL) was measured by immunoprecipitation and Western blotting. Results showed that short-term TNF-alpha treatment significantly increased lipolysis, iNOS expression, and NO production in a time- and dose-dependent manner. Furthermore, treatment with the NO donor S-nitroso-N-acetylpenicillamine also stimulated lipolysis and HSL phosphorylation in 3T3-L1 adipocytes. Moreover, pretreatment with inhibitors of iNOS and guanylate cyclase, but not an adenylate cyclase inhibitor, abolished TNF-alpha-induced lipolysis and HSL phosphorylation. Suppression of TNF-alpha-induced iNOS expression using short hairpin RNA significantly reduced TNF-alpha-induced lipolysis. In conclusion, short-term TNF-alpha treatment induces lipolysis in 3T3-L1 adipocytes by increasing iNOS expression and NO production, which activates the guanylyl cyclase/cGMP-dependent pathway and induces phosphorylation of HSL.

Specific repression of mutant K-RAS by 10-23 DNAzyme: sensitizing cancer cell to anti-cancer therapies.

Point mutations of the Ras family are frequently found in human cancers at a prevalence rate of 30%. The most common mutation K-Ras(G12V), required for tumor proliferation, survival, and metastasis due to its constitutively active GTPase activity, has provided an ideal target for cancer therapy. 10-23 DNAzyme, an oligodeoxyribonucleotide-based ribonuclease consisting of a 15-nucleotide catalytical domain flanked by two target-specific complementary arms, has been shown to effectively cleave the target mRNA at purine-pyrimidine dinucleotide. Taking advantage of this specific property, 10-23 DNAzyme was designed to cleave mRNA of K-Ras(G12V)(GGU-->GUU) at the GU dinucleotide while left the wild-type (WT) K-Ras mRNA intact. The K-Ras(G12V)-specific 10-23 DNAzyme was able to reduce K-Ras(G12V) at both mRNA and protein levels in SW480 cell carrying homozygous K-Ras(G12V). No effect was observed on the WT K-Ras in HEK cells. Although K-Ras(G12V)-specific DNAzymes alone did not inhibit proliferation of SW480 or HEK cells, pre-treatment of this DNAzyme sensitized the K-Ras(G12V) mutant cells to anti-cancer agents such as doxorubicin and radiation. These results offer a potential of using allele-specific 10-23 DNAzyme in combination with other cancer therapies to achieve better effectiveness on cancer treatment.

Potential effect of resistin on the ET-1-increased reactions of blood pressure in rats and Ca2+ signaling in vascular smooth muscle cells.

Resistin and endothelin‐1 (ET‐1) are upregulated in people with type II diabetes mellitus, central obesity, and hypertension. ET‐1 signaling is involved in Ca2+‐contraction coupling and related to blood pressure regulation. The aim of this study is to investigate the role of resistin on ET‐1‐increased blood pressure and Ca2+ signaling. The blood pressure and cytosolic Ca2+ of vascular smooth muscle cells (VSMCs) of Sprague–Dawley rats were detected. The data demonstrated that resistin accelerated and prolonged ET‐1‐induced increases in blood pressure and had significant effects on ET‐1‐increased Ca2+ reactions. Resistin‐enhanced ET‐1‐increased Ca2+ reactions were reversed by blockers of store‐operated Ca2+ entry (SOCE) and extracellular‐signal‐regulated kinase (ERK). The endogenous expression of Orai and stromal interaction molecular (STIM) were characterized in the VSMCs. Furthermore, resistin‐enhanced ET‐1 Ca2+ reactions and the resistin‐dependent activation of SOCE were abolished under STIM1‐siRNA treatment, indicating that STIM1 plays an important role in resistin‐enhanced ET‐1 Ca2+ reactions in VSMCs. Resistin appears to exert effects on ET‐1‐induced Ca2+ increases by enhancing the activity of ERK‐dependent SOCE (STIM1‐partcipated), and may accelerate and prolong ET‐1‐increased blood pressure via the same pathway. J. Cell. Physiol. 227: 1610–1618, 2012.

A Multisampling Reporter System for Monitoring MicroRNA Activity in the Same Population of Cells

MicroRNAs (miRNAs) downregulate gene expression by binding to the partially complementary sites in the 3′ untranslated region (UTR) of target mRNAs. Several methods, such as Northern blot analysis, quantitative real-time RT-PCR, microarray, and the luciferase reporter system, are commonly used to quantify the relative level or activity of miRNAs. The disadvantage of these methods is the requirement for cell lysis, which means that several sets of wells/dishes of cells must be prepared to monitor changes in miRNA activity in time-course studies. In this study, we developed a multisampling reporter system in which two secretable bioluminescence-generating enzymes are employed, one as a reporter and the other as an internal control. The reporters consist of a pair of vectors containing the Metridia luciferase gene, one with and one without a duplicated miRNA targeting sequence at their 3′UTR, while the other vector coding for the secreted alkaline phosphatase gene is used as an internal control. This method allows miRNA activity to be monitored within the same population of cells over time by withdrawing aliquots of the culture medium. The practicability and benefits of this system are addressed in this report.

An endothelin type A receptor-expressing cell to characterize endothelin-1 binding and screen antagonist.

Endothelin-1 (ET-1) induces contraction of vascular smooth muscle through binding to endothelin type A receptor (ET(A)R). COS-7 cells stably expressing high levels of the ET(A)R were established (designated COS-7(ET(A)R)). The COS-7(ET(A)R) cell bound [(125)I]ET-1 with a K(d) of 932+/-161 pM and a B(max) of 74+/-13 fmol/2x10(5) cells. [(125)I]ET-1 binding was inhibited by ET-1 and the ET(A)R antagonist BQ-610, but not by the endothelin type B receptor (ET(B)R) antagonist BQ-788. In clones expressing two ET(A)R mutants containing D46N or R53Q substitutions in the first extracellular domain of the receptor, [(125)I]ET-1 binding activity was dramatically reduced. This suggests that these single amino acid substitutions alter the three-dimensional structure of the ligand-binding domain of the ET(A)R. Using COS-7(ET(A)R) cell, we showed that Ca(2+) or Mg(2+) was essential for ET-1 binding to the ET(A)R and that ET-1 treatment induced postreceptor signaling, that is, intracellular accumulation of cyclic AMP (cAMP) and Ca(2+) mobilization. The COS-7(ET(A)R) established in this study will be a useful tool for screening ET-1 antagonists for treating hypertension.

Prunellae Spica Extract Contains Antagonists for Human Endothelin Receptors

Endothelin-1 (ET-1) is a powerful vasoconstrictor that contributes to blood pressure elevation. The biological effects of ETs are mediated by two receptors, namely, endothelin type A receptor (ET(A)R) and endothelin type B receptor (ET(B)R). Chinese herbal medicines (CHM) with antagonist activity for these two receptors were screened by establishing stable clones of CHO-K1 cells expressing high levels of human ET(A)R and ET(B)R, namely CHO-ET(A)R and CHO-ET(B)R.The aqueous extract of Prunellae Spica (P1) inhibited the binding of (125)I-ET-1 to ET(A)R and ET(B)R in CHO-ET(A)R and CHO-ET(B)R cells, respectively. P1 suppressed the ET-1-induced mobilization of intracellular Ca(2+) . Through the alcohol fractionation of P1, the antagonists of human ET(A)R and ET(B)R were found to belong to different, separable ingredients and the antagonist of ET(A)R is more soluble in alcohol. The two antagonists were also effective in the test on human primary cells, HASMC and HUVEC. P1 successfully prevented the development of ET-1-associated hypertension in rats without further purification. These results indicate the presence of anti-hypertensive ingredients in P. Spica extract, at least through the inactivation of ET(A)R and/or ET(B)R.

Selective Inhibition of p53 Dominant Negative Mutation by shRNA Resulting in Partial Restoration of p53 Activity

The tumor suppressor gene p53 is the most frequently mutated gene found in human cancer. The majority of p53 mutations are missense mutations occurring within the DNA binding domain. Some of these mutations exhibit dominant negative effects (e.g., p53 R273H) that disrupt the normal function of wild-type p53. It is of therapeutic interest to determine if the normal function of p53 can be restored when the dominant negative effect is selectively inhibited. In this study, we tested this possibility using small hairpin RNA (shRNA) to specifically reduce the level of p53 R273H. The antisense strand of shRNA is fully complementary to mRNA of p53 R273H, but leaves a mismatch base in the middle of the duplex to wild-type p53 mRNA. Both wild-type p53 and mutant p53 R273H were transiently expressed in p53-null H1299 lung cancer cells. The shRNA we designed selectively reduced the mRNA level of p53 R273H, but had no RNA interference or antisense effects on wild-type mRNA. As a result, the transactivation activity of p53 was partially restored. In this report, we provide a new strategy for studying functional alterations for point mutation or single nucleotide polymorphism, and treating some dominant mutant-derived diseases.