OBJECTIVE: Uterine leiomyomas are the most common tumor of the uterus. But the molecular causes of uterine leiomyoma remain unclear. We conducted the current investigation in order to elucidate the molecular mechanisms in the development of uterine leiomyoma. METHODS: We employed a new and accurate reverse transcription-polymerase chain reaction (RT-PCR) method that involved annealing control primers (ACPs) to identify the genes that are differently expressed in uterine leiomyoma. RESULTS: Using 120 ACPs, we identified and sequenced 14 differently expressed genes (DEGs) in uterine leiomyoma compared with normal myometrium. Basic Local Alignment Search Tool (BLAST) searches were performed to examine the known functions of these genes associated with uterine leiomyoma. We confirmed differently expressed patterns in more cases using the RT-PCR method. We also detected two novel genes, Tenascin-X and Leukemia Inhibitory Factor Receptor (LIFR), which had not yet been reported to have any functions associated with uterine leiomyoma. RT-PCR confirmation shows that both of these two genes are down-regulated in uterine leiomyoma. CONCLUSION: Our results suggest that Tenascin-X and LIFR may play a role in the development of uterine leiomyoma. Although further studies are required to establish the precise mechanisms with which these genes are involved in the genesis of uterine leiomyoma, the present research is significant in that it is the first study which detects down-regulated novel genes in uterine leiomyoma using the ACP system.
Animals ; Female ; Leiomyoma ; Leukemia ; Leukemia Inhibitory Factor ; Mice ; Myometrium ; Receptors, OSM-LIF ; Tenascin ; Uterus

OBJECTIVETo study the effects of Box-3 region of the leukemia inhibitory factor receptor (LIFR) alpha-chain cytoplasmic domain on the proliferation and differentiation of HL-60 cells.

METHODSExpression vector of gp190CT3 was constructed and expressed in HL-60 cells. The expression level of gp190CT3 was assayed by immunocytochemistry. The growth of wild type and gp190CT3 transfected HL-60 cells were examined under microscope. The PCNA levels were assayed by Western blot, and the levels of CD15 by flow cytometry.

RESULTSThe gp190CT3 transfected HL-60 cells were enlarged in size and their proliferation was slower than that of wild type. The expression level of PCNA was down-regulated while the level of CD15 up-regulated in transfected HL-60 cells as compared with that of the wild type cells.

CONCLUSIONThe Box-3 region of the leukemia inhibitory factor receptor alpha-chain cytoplasmic domain (gp190CT3) participates the LIFR signal transduction in inhibiting the growth and inducing the differentiation of HL-60 cells.

Binding Sites ; genetics ; Blotting, Western ; Cell Differentiation ; genetics ; physiology ; Cell Proliferation ; Genetic Vectors ; genetics ; HL-60 Cells ; Humans ; Immunohistochemistry ; Lewis X Antigen ; metabolism ; Proliferating Cell Nuclear Antigen ; metabolism ; Receptors, OSM-LIF ; genetics ; metabolism ; Transfection

OBJECTIVETo screen the differentially expressed miRNAs and their target genes in adipogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs) to better understand the mechanism for regulating the balance between osteoblast and adipocyte differentiation.

METHODSCultured hMSCs were induced for adipogenic differentiation, and at 0, 7, 14, and 21 days of induction, the cells were examined for miRNA and mRNA expression profiles using miRNA chip and transcriptome sequencing (RNA-seq) techniques. Correlation analysis was carried out for the miRNAs and mRNAs of potential interest. The databases including TargetScan, PicTar and miRanda were used to predict the target genes of the differentially expressed miRNA.

RESULTSThe expression of miR-140-5p was down-regulated and leukemia inhibitory factor receptor (LIFR) expression increased progressively during adipogenic differentiation of hMSCs, showing a negative correlation between them. Target gene prediction using the 3 databases identified LIFR as the target gene of miR-140-5p.

CONCLUSIONmiRNA-140-5p may play an important role by regulating its target gene LIFR during adipogenic differentiation of hMSCs.

Adipocytes ; cytology ; Adipogenesis ; Cell Differentiation ; Cells, Cultured ; Down-Regulation ; Humans ; Leukemia Inhibitory Factor Receptor alpha Subunit ; metabolism ; Mesenchymal Stromal Cells ; cytology ; MicroRNAs ; genetics ; Oligonucleotide Array Sequence Analysis ; Osteoblasts ; cytology ; RNA, Messenger ; Transcriptome

BACKGROUND: The recognition of bronchial asthma as an inflammatory disease led to a search for soluble markers that would be useful in assessing airway inflammation. Interleukin-6 (IL-6) is a representative proinflammatory cytokine that has been shown to be connected with various inflammatory diseases. IL-6 acts via specific receptors that consist of the IL-6 binding glycoprotein gp80 and the signal transducer gp130. In the search for markers of airway inflammation, we investigated the role of soluble interleukin-6 receptor (sIL-6R) and IL-6 in acute asthma. METHODS: Serum levels of sIL-6R and IL-6 were measured in 78 acute asthmatics, in 15 patients with asymptomatic asthma and in 10 healthy control subjects by a specific ELISA using a murine antihuman IL-6R, IL-6 mAb (Quantikine sIL-6R, IL-6). RESULTS: Serum levels of IL-6 in acute asthmatics significantly exeeded those of control subjects. Those of sIL-6R in acute asthmatics were also significantly increased compared to those of control subjects. The serum concentration of IL-6 obtained in acute asthmatics was elevated as compared with the asymptomatic asthmatics. However, Association between eosinophilic count / IgE and IL-6 / sIL-6R in acute asthma could not found. CONCLUSION: Our results suggest that IL-6 may be involved in the pathogenesis of acute asthma and serum levels of IL-6 and sIL-6R may reflect the severity of airway inflammation.
Asthma* ; Cytokine Receptor gp130 ; Enzyme-Linked Immunosorbent Assay ; Eosinophils ; Glycoproteins ; Humans ; Immunoglobulin E ; Inflammation ; Interleukin-6*

gp130-mediated signaling is involved in both chondrogenesis and osteogenesis, but its direct role in the formation of embryonic Meckel's cartilage and associated mandibular development has not yet been elucidated. In this study, we examined the influence of gp130 ablation on the developing mandibular Meckel's cartilage by evaluating the morphological and histological changes as well as the gene expression patterns in developing embryonic gp130-/- mice. The ablation of the gp130 gene showed no change in region-specific collagen mRNA expression except for a slight delay in its expression but caused shortened embryonic Meckel's cartilage, delayed hypertrophic chondrocyte maturation and subsequent bony replacement with characteristic bending of the intramandibular Meckel's cartilage. The bending of Meckel's cartilage led to a narrow mandibular arch at the rostral area with poor cortical plate formation. These findings indicate that gp130-mediated signaling is important for the normal morphogenesis of Meckel's cartilage and subsequent mandibular development.
Animals ; Body Patterning ; Cartilage/embryology/metabolism/*physiology ; Collagen ; Cytokine Receptor gp130/genetics/*physiology ; Mandible/embryology/metabolism/*physiology ; Mice ; Mice, Knockout

The second nonligand binding chain of IL-6 receptor (IL-6R), the membrane glycoprotein with 130 kD mol wt (gp130), is responsible for the signal transduction of IL-6 biological activity. Our experiments indicated that the rat gp130 molecule, which was expressed in the rat acute myeloid leukemia cell line R2, could associate with the complex of rhIL-6 and membrane human IL-6R molecule and transduce the inhibition-inducing signal on R2 cells. In the present study, antisense oligonucleotides of rat gp130 were synthesized and uptaked into the R2 cells. Then the effects of the antisense or sense nucleic acids of rat gp130 on the inhibition induced by rhIL-6 in the R2 cell were investigated. Our results show that the antisense oligonucleotide of rat gp130 blocked the inhibitory effect of rhIL-6 on the R2 cells by (45 +/- 7)% at the optimal concentration
Acute Disease ; Animals ; Antigens, CD ; genetics ; Cell Division ; drug effects ; Cytokine Receptor gp130 ; Drug Interactions ; Interleukin-6 ; antagonists & inhibitors ; pharmacology ; Leukemia, Myeloid ; pathology ; Membrane Glycoproteins ; antagonists & inhibitors ; genetics ; Oligonucleotides, Antisense ; chemical synthesis ; chemistry ; pharmacology ; Rats ; Tumor Cells, Cultured

Mouse embryonic stem cells (ES cells) are pluripotent in that they can give rise to almost all the cell types in vitro and in vivo. Also, they can sustain self-renewal in vitro owing to symmetrical mitosis, i.e., only the cell number increases while the daughter cells remain pluripotent. Self-renewal and pluripotency of ES cells are under stringent regulation of several signaling pathways. Activation of either JAK-STAT3 or PI3K, the downstream cascade of gp130, can maintain the self-renewal of ES cells, while phosphorylation of another gp130-related branch, SHP2-Ras-ERK, drives the differentiation. BMP2/4-mediated signaling is capable of suppressing the differentiation of ES cells in collaboration with activated JAK-STAT3 under serum free culture conditions. Other signaling such as Wnt also contributes to the self-renewal of ES cells. Generally, the network, which is composed of various signaling pathways, modulates the self-renewal and differentiation of mouse ES cells precisely. This review focuses on the role of gp130 in proliferation of mouse ES cells including inhibitory effect of JAK-STAT3 pathway activation on differentiation of mouse ES cells, maintenance effect of PI3K pathway activation on self-renewal of ES cells, promotive effect of SHP-2-Ras-ERK pathway activation on differentiation of ES cells, and influence of other signaling pathways on self-renewal of mouse ES cells, including maintenance effect of BMP combination with LIF under serum free culture conditions on self-renewal of ES cells and promotive effect of Wnt pathway activation on self-renewal of ES cells.
Animals ; Cell Differentiation ; physiology ; Cell Proliferation ; Cell Survival ; Cells, Cultured ; Cytokine Receptor gp130 ; metabolism ; Embryonic Stem Cells ; cytology ; physiology ; Janus Kinase 1 ; metabolism ; Leukemia Inhibitory Factor ; metabolism ; Mice ; STAT3 Transcription Factor ; metabolism ; Signal Transduction ; physiology

The goal of this study was to investigate the regulatory effect of angiotensin converting enzyme 2 (ACE2) on cellular inflammation caused by avian infectious bronchitis virus (IBV) and the underlying mechanism of such effect. Vero and DF-1 cells were used as test target to be exposed to recombinant IBV virus (IBV-3ab-Luc). Four different groups were tested: the control group, the infection group[IBV-3ab-Luc, MOI (multiplicity of infection)=1], the ACE2 overexpression group[IBV-3ab Luc+pcDNA3.1(+)-ACE2], and the ACE2-depleted group (IBV-3ab-Luc+siRNA-ACE2). After the cells in the infection group started to show cytopathic indicators, the overall protein and RNA in cell of each group were extracted. real-time quantitative polymerase chain reaction (RT-qPCR) was used to determine the mRNA expression level of the IBV nucleoprotein (IBV-N), glycoprotein 130 (gp130) and cellular interleukin-6 (IL-6). Enzyme linked immunosorbent assay (ELISA) was used to determine the level of IL-6 in cell supernatant. Western blotting was performed to determine the level of ACE2 phosphorylation of janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3). We found that ACE2 was successfully overexpressed and depleted in both Vero and DF-1 cells. Secondly, cytopathic indicators were observed in infected Vero cells including rounding, detaching, clumping, and formation of syncytia. These indicators were alleviated in ACE2 overexpression group but exacerbated when ACE2 was depleted. Thirdly, in the infection group, capering with the control group, the expression level of IBV-N, gp130, IL-6 mRNA and increased significantly (P < 0.05), the IL-6 level was significant or extremely significant elevated in cell supernatant (P < 0.05 or P < 0.01); the expression of ACE2 decreased significantly (P < 0.05); protein phosphorylation level of JAK2 and STAT3 increased significantly (P < 0.05). Fourthly, comparing with the infected group, the level of IBV-N mRNA expression in the ACE2 overexpression group had no notable change (P > 0.05), but the expression of gp130 mRNA, IL-6 level and expression of mRNA were elevated (P < 0.05) and the protein phosphorylation level of JAK2 and STAT3 decreased significantly (P < 0.05). In the ACE2-depleted group, there was no notable change in IBV-N (P > 0.05), but the IL-6 level and expression of mRNA increased significantly (P < 0.05) and the phosphorylation level of JAK2 and STAT3 protein decreased slightly (P > 0.05). The results demonstrated for the first time that ACE2 did not affect the replication of IBV in DF-1 cell, but it did contribute to the prevention of the activation of the IL-6/JAK2/STAT3 signaling pathway, resulting in an alleviation of IBV-induced cellular inflammation in Vero and DF-1 cells.
Animals ; Chlorocebus aethiops ; Humans ; Interleukin-6/genetics* ; Janus Kinase 2/pharmacology* ; Infectious bronchitis virus/metabolism* ; STAT3 Transcription Factor/metabolism* ; Angiotensin-Converting Enzyme 2/pharmacology* ; Cytokine Receptor gp130/metabolism* ; Vero Cells ; Signal Transduction ; Inflammation ; RNA, Messenger

OBJECTIVETo observe the effect of Chinese herbs for supplementing Shen and strengthening bone (HB) on myelogenic osteoclasts formation, and gene expression of interleukin-6 (IL-6), IL-6 receptor (IL-6R) and gp130 in bone marrow.

METHODSSeventy-two healthy female SD rats of 3 months, were randomly divided into three groups, 24 in the sham-operated group (A), 24 in the ovariectomized group (B) and 24 in the after ovariectomy HB treated group (C). Bone marrow cells of 6 rats from each group were respectively collected and cultured at four time points (2nd, 4th, 6th and 12th weeks after operation). After 6 days of culture, the bone marrow cells were differentiated by Wright-Giemsa stain and TRAP stain, and total RNA in them was extracted by TRIZOL.

RESULTSBeginning from the 2nd week, the osteoclasts formation in Group B was higher than that in Group A (P < 0.05), and IL-6, IL-6R gene expression significantly increased in Group B (P < 0.05 or P < 0.01). These changes reached the peak in the 4th to 6th week, with the level maintained to the 12th week. As for comparison of Group B and C, the above-mentioned changes were significantly weakened in the latter (P < 0.05 or P < 0.01). No significant change of gp130 gene expression revealed in the whole course in either group.

CONCLUSIONHB could inhibit the myelogenic osteoclasts formation in ovariectomized rats, this effect may be correlated with, partially at least, its inhibitory effect on the over-expressed IL-6 and IL-6R gene expression in myelocytes after ovariectomy.

Animals ; Antigens, CD ; biosynthesis ; genetics ; Bone Marrow ; metabolism ; Cell Division ; drug effects ; Cells, Cultured ; Cytokine Receptor gp130 ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Female ; Granulocyte Precursor Cells ; metabolism ; Interleukin-6 ; biosynthesis ; genetics ; Isoflavones ; pharmacology ; Membrane Glycoproteins ; biosynthesis ; genetics ; Osteoblasts ; pathology ; Osteoporosis ; metabolism ; pathology ; Ovariectomy ; RNA ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, Interleukin-6 ; biosynthesis ; genetics

BACKGROUNDThe Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway and the extracellular signal-regulated kinases 1/2 (ERK1/2) pathway are the two major independent signal transduction pathways. However, it has recently been found that STAT3 may be negatively regulated by ERK1/2 in gp130-dependent signaling. Cardiotrophin-1 (CT-1), a potent novel hypertrophic cytokine, depends on gp130 to induce signaling and depends on STAT3 to exert hypertrophic effect. In this study, we examined whether STAT3 activity was negatively regulated by ERK1/2 during CT-1-induced signaling in rat cardiomyocytes and, if so, whether such crosstalk interfered with the hypertrophic effect of CT-1 and, furthermore, whether the mechanism underlying the crosstalk involved phosphorylation of serine 727 (S727) in STAT3.

METHODSThe activities of ERK1/2 and STAT3 were assessed by in-gel kinase assay and Western blot analysis, respectively. The role of S727 phosphorylation in the crosstalk between ERK1/2 and STAT3 was determined by a transient transfection study using a STAT3S727A mutant. Cardiomyocyte hypertrophy was evaluated by the cellular protein-to-DNA ratio and [(3)H]-leucine incorporation.

RESULTSCT-1 simultaneously activated both ERK1/2 and STAT3 in rat cardiomyocytes. Inhibition of ERK1/2 by U0126 resulted in an increase of CT-1-induced tyrosine phosphorylation of STAT3 and, consequently, the protein-to-DNA ratio and [(3)H]-leucine incorporation. Transient transfection of the cells with STAT3S727A had no significant effect on CT-1-induced tyrosine phosphorylation of STAT3.

CONCLUSIONSSTAT3 is activated by CT-1 in rat cardiomyocytes, but full activation is mitigated by the simultaneous activation of ERK1/2. The inhibition of ERK1/2 increases the activity of STAT3, which, in turn, enhances the hypertrophic effect of CT-1. The crosstalk between ERK1/2 and STAT3 is independent of the phosphorylation of the S727 in STAT3. Such crosstalk may contribute to the development of adequate cardiac hypertrophy.

Active Transport, Cell Nucleus ; Animals ; Antigens, CD ; metabolism ; Cardiomegaly ; chemically induced ; metabolism ; Cytokine Receptor gp130 ; Cytokines ; toxicity ; DNA-Binding Proteins ; physiology ; Membrane Glycoproteins ; metabolism ; Mitogen-Activated Protein Kinase 1 ; physiology ; Mitogen-Activated Protein Kinase 3 ; physiology ; Phosphorylation ; Rats ; Rats, Sprague-Dawley ; STAT3 Transcription Factor ; Trans-Activators ; physiology ; Tyrosine ; metabolism