Altered expression of Bcl-2 and Bax in follicles within dehydroepiandrosterone-induced polycystic ovaries in rats

PCOS (polycystic ovary syndrome) is a heterogeneous disease characterized by hyperandrogenaemia, hirsutism, oligo- or amenorrhea, insulin resistance and anovulation. The aim of the present study was to evaluate if the balance between the ovarian expression of Bax (proapoptotic protein) and Bcl-2 (antiapoptotic protein) is altered in a PCOS model developed in rats by DHEA (dehydroepiandrosterone) administration. In addition, the ovarian morphology and the circulating progesterone levels were evaluated. Histological studies confirmed the presence of follicular cysts, atretic follicles and the absence of corpora lutea in the ovaries from the PCOS group and a significant decrease in circulating progesterone levels. Immunohistochemical studies showed that the expression of Bcl-2 and Bax were mainly localized in granulosa cells of AFs (antral follicles) in both groups. Bax expression was greater in preantral and AFs from PCOS ovarian sections than in the controls. In contrast, intense Bcl-2 immunostaining was observed in the control AFs, while Bcl-2 protein was either absent in PFs (preantral follicles) or weakly expressed in AFs from PCOS rats. These results were partially confirmed by Western studies. Data revealed that the ovarian level of Bcl-2 protein was lower in PCOS than in the control and that there were no differences in Bax ovarian levels between groups. However, Bax/Bcl-2 ratio was significantly higher in PCOS group than in the control group. In conclusion, an increase in ovarian apoptosis through an imbalance among the Bcl-2 family members may be involved in the transformation of growing follicles in cystic follicles in the ovaries from DHEA-induced PCOS rats.     

Design,synthesis and biological evaluation of bifunctional inhibitors of membrane type 1 matrix metalloproteinase (MT1-MMP)

Collagen degradation and proMMP-2 activation are major functions of MT1-MMP to promote cancer cell invasion. Since both processes require MT1-MMP homodimerization on the cell surface, herein we propose that the use of bifunctional inhibitors of this enzyme could represent an innovative approach to efficiently reduce tumor growth. A small series of symmetrical dimers derived from previously described monomeric arylsulfonamide hydroxamates was synthesized and tested in vitro on isolated MMPs. A nanomolar MT1-MMP inhibitor, compound 6, was identified and then submitted to cell-based assays on HT1080 fibrosarcoma cells. Dimer 6 reduced MT1-MMP-dependent proMMP-2 activation, collagen degradation and collagen invasion in a dose-dependent manner with better results even compared to its monomeric analogue 4. This preliminary study suggests that dimeric MT1-MMP inhibitors might be further developed and exploited as an alternative tool to reduce cancer cell invasion.     

Contrasting expression of membrane metalloproteinases, MT1-MMP and MT3-MMP, suggests distinct functions in skeletal development

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is the most ubiquitous and widely studied of the membrane-type metalloproteinases (MT-MMPs). It was thus surprising to find no published data on chicken MT1-MMP. We report here the characterization of the chicken gene. Its low sequence identity with the MT1-MMP genes of other species, high GC content, and divergent catalytic domain explains the absence of data and our difficulties in characterizing the gene. The absence of structural features in the chicken gene that have been suggested to be critical for the activation of MMP-2 by MT1-MMP; for the effect of MT1-MMP on cell migration and for the recycling of MT1-MMP suggest these features are either not essential or that MT1-MMP does not perform these functions in chickens. Comparison of the expression of chicken MT1-MMP with MT3-MMP and with MMP-2 and MMP-13 has confirmed the previously recognized co-expression of MT1-MMP with MMP-2 and MMP-13 in fibrous and vascular tissues, particularly those surrounding the developing long bones in other species. By contrast, MT3-MMP expression differs markedly from that of MT1-MMP and of both MMP-2 and MMP-13. MT3-MMP is expressed by chondrocytes of the developing articular surface. Similar expression patterns of this group of MT-MMPs and MMPs have been observed in mouse embryos and suggest distinct and specific functions for MT1-MMP and MT3-MMP in skeletal development.     

Phyllanthus urinaria induces the Fas receptor/ligand expression and ceramide-mediated apoptosis in HL-60 cells

Phyllanthus urinaria (P. urinaria), a widely used herb medicine, was tested for the anticancer effect on human myeloid leukemia cells in this study. The water extract of P. urinaria induced the apoptosis of HL-60 cells as demonstrated by morphological change, DNA fragmentation and increased caspase-3 activity. However, normal human peripheral mononuclear cells remained viable under the same treatment. The P. urinaria-induced apoptosis of HL-60 cells was associated with the increased Bax gene expression and decreased Bcl-2 gene expression. In addition, the gene expressions of Fas receptor and Fas ligand, but not p53, were also induced in HL-60 cells dose- and time-dependently. The inhibitor of ceramide synthase, fumonisin B1, completely suppressed the apoptosis induced by P. urinaria and this inhibitory effect of fumonisin B1 could be eliminated by the addition of ceramide. It indicated that the activity of ceramide synthase is critical for the P. urinaria-induced apoptosis in HL-60 cells. The P. urinaria-induced apoptosis in HL-60 cells is mediated through a ceramide-related pathway.     

Membrane type-1 matrix metalloproteinase (MT1-MMP) processing of pro-alphav integrin regulates cross-talk between alphavbeta3 and alpha2beta1 integrins in breast carcinoma cells

We have recently demonstrated that in breast carcinoma MCF7 cells MT1-MMP processes the alphav, alpha3, and alpha5 integrin precursors generating the respective mature S-S-linked heavy and light alpha-chains. The precursor of alpha2 integrin subunit was found resistant to MT1-MMP proteolysis. The processing of the alphav subunit by MT1-MMP facilitated alphavbeta3-dependent adhesion, activation of FAK signaling pathway, and migration of MCF7 cells on vitronectin. To elucidate further the effects of MT1-MMP on cellular integrins, we examined the functional activity of alpha5beta1 and alpha2beta1 integrins in MCF7 cells expressing MT1-MMP. Either expression of MT1-MMP alone or its coexpression with alphavbeta3 failed to affect the functionality of alpha5beta1 integrin, and adhesion of cells to fibronectin. MT1-MMP, however, profoundly affected the cross-talk involving alphavbeta3 and alpha2beta1 integrins. In MT1-MMP-deficient cells, integrin alphavbeta3 suppressed the functional activity of the collagen-binding alpha2beta1 integrin receptor and diminished cell adhesion to type I collagen. Coexpression of MT1-MMP with integrin alphavbeta3 restored the functionality of alpha2beta1 integrin and, consequently, the ability of MCF7 cells to adhere efficiently to collagen. We conclude that the MT1-MMP-controlled cross-talk between alphavbeta3 and alpha2beta1 integrins supports binding of aggressive, MT1-MMP-, and alphavbeta3 integrin-expressing malignant cells on type I collagen, the most common substratum of the extracellular matrix.     

Soluble Fas/Apo-1 (CD95) levels during T cell activation in the presence of acute myelogenous leukemia accessory cells; contributions from local release and variations in systemic levels

Fas (CD95/Apo-1) exists both in membrane-bound and in biologically active soluble (s) forms. Ligation of membrane-expressed Fas can induce apoptosis, and Fas-mediated signaling seems to be involved in T-cell-induced apoptosis of human acute myelogenous leukemia (AML) blasts. The local release of sFas by AML blasts may then function as a protective mechanism by competing with membrane-bound Fas for binding sites on the common Fas ligand (FasL). sFas was released by AML blasts during in vitro culture, and this release was modulated by several cytokines that can be secreted by activated T cells. Increased levels of sFas could be detected during in vitro activation of T cells in the presence of native AML accessory cells, and this was observed both for (i) mitogenic activation of CD4⁺ and CD8⁺ T cell clones derived from acute leukemia patients with therapy-induced leukopenia and (ii) allostimulated activation of T cells derived from normal donors. However, local in vivo levels of sFas will also be influenced by variations in systemic levels. High serum levels of sFas were detected in acute leukemia patients during chemotherapy-induced cytopenia, but these levels decreased during complicating bacterial infections. In contrast, serum levels of sFasL were normal in leukopenic patients. The present results support the hypothesis that local release of sFas can function as a protective mechanism against AML-reactive T cells, but the effects of this local release are, in addition, modulated by variations in systemic levels of sFas (but not sFasL). Received: 9 March 2000 / Accepted: 25 May 2000     

Spatial and temporal expression profiles suggest the involvement of gelatinase A and membrane type 1 matrix metalloproteinase in amphibian metamorphosis

The matrix metalloproteinases (MMPs) are a family of proteases capable of degrading various components of the extracellular matrix (ECM). Among them, the membrane type MMP–1 (MT1–MMP) has been shown to participate in the activation of MMP gelatinase A (GelA), suggesting that they may function together in development and pathogenesis. Here, we have investigated the spatiotemporal expression profiles of Xenopus laevis MT1–MMP and GelA genes during thyroid-hormone-dependent metamorphosis. We have focused our studies on two organs: (1) the intestine, which undergoes first the degeneration of the tadpole epithelium through apoptosis and then the development of adult epithelium and other tissues, and (2) the tail, which completely resorbs through programmed cell death. We show that both MT1–MMP and GelA are upregulated in the intestine and tail when both organs undergo metamorphosis. Within the organs, MT1–MMP and GelA are coexpressed in the connective tissues during both natural and thyroid-hormone-induced metamorphosis. In addition, MT1–MMP (but not GelA) is also expressed in the longitudinal muscle cells of the metamorphosing intestine. These results suggest that MT1–MMP and GelA function together in the ECM degradation or remodeling associated with metamorphosis and that MT1–MMP has additional GelA–independent roles in the development of adult longitudinal muscle in the intestine. This research was supported by the Intramural Research Program of the National Institute of Child Health and Human Development, NIH. T. Hasebe and H. Matsuda were supported in part by JSPS (NIH) fellowships.     

Distinctive functions of membrane type 1 matrix-metalloprotease (MT1-MMP or MMP-14) in lung and submandibular gland development are independent of its role in pro-MMP-2 activation

Membrane type 1-matrix metalloprotease (MT1-MMP or MMP-14) is a major activator of pro-MMP-2 and is essential for skeletal development. We show here that it is required for branching morphogenesis of the submandibular gland but not the lung. Instead, in the lung, it is essential for postnatal development of alveolar septae. Lung development in Mmp14-/- mice is arrested at the prealveolar stage with compensatory hyperinflation of immature saccules. Mmp2-/- mice lacked comparable defects in the lung and submandibular gland, suggesting that MT1-MMP acts via mechanisms independent of pro-MMP-2 activation. Since the developmental defects in the lung are first manifest around the time of initial vascularization (E16.5), we investigated the behavior of pulmonary endothelial cells from Mmp14+/+ and Mmp14-/- mice. Endothelial cells from lungs of 1-week-old Mmp14-/- mice show reduced migration and formation of three-dimensional structures on Matrigel. Since pulmonary septal development requires capillary growth, the underlying mechanism of pulmonary hypoplasia in Mmp14-/- mice may be defective angiogenesis, supporting a model in which angiogenesis is a critical rate-limiting step for acquisition of pulmonary parenchymal mass.     

Down‐regulated lncRNA HOTAIR alleviates polycystic ovaries syndrome in rats by reducing expression of insulin‐like growth factor 1 via microRNA‐130a

It has been found that long noncoding RNA HOTAIR, microRNA‐130a (miR‐130a) and insulin‐like growth factor 1 (IGF1) expression are associated with ovarian cancer, thus, we hypothesised that the HOTAIR/miR‐130a/IGF1 axis might associate with endocrine disorders and biological behaviours of ovarian granulosa cells in rat models of polycystic ovary syndrome (PCOS). PCOS rat models were established by injection of dehydro‐isoandrosterone, followed by treatment of si‐HOTAIR, oe‐HOTAIR, miR‐130a mimics or miR‐130a inhibitors. Serum hormonal levels were determined to evaluate endocrine conditions. The effect of HOTAIR and miR‐130a on activities of isolated ovarian granulosa cells was assessed, as well as the involvement of IGF1.In the ovarian tissues and granulosa cells of PCOS rat models, highly expressed HOTAIR and IGF1 and poorly expressed miR‐130a were identified. In response to oe‐HOTAIR, serum levels of E₂, T and LH were increased and serum levels of FSH were reduced; the proliferation of granulosa cells was reduced and apoptosis was promoted; notably, expression of miR‐130a was reduced while expression of IGF1 was increased. The treatment of si‐HOTAIR reversed the situation. Furthermore, the binding of HOTAIR to miR‐130a and targeting relationship of miR‐130a and IGF1 were confirmed. LncRNA HOTAIR up‐regulates the expression of IGF1 and aggravates the endocrine disorders and granulosa cell apoptosis through competitive binding to miR‐130a in rat models of PCOS. Based on our finding, we predict that competitive binding of HOTAIR to miR‐130a may act as a novel target for the molecular treatment of PCOS.     

Follicular expression of c-Kit/SCF and inhibin-alpha in mouse ovary during development.

The mechanism of development of the ovarian follicles has been largely unknown. We performed an immunohistochemical (IHC) study to determine the follicular expressions of c-kit, SCF, and inhibin-alpha at different developmental stages in mouse ovary. Ovaries were obtained from 14 and 16 days post coitum and 2, 7, and 21 days post partum (dpp) mice. IHC for c-kit, SCF, and inhibin-alpha was carried out. c-Kit and SCF were expressed on oogonia regardless of the developmental stage. Immunoreactive c-kit and SCF antigens were expressed on oocytes of primordial and primary follicles of neonate mouse ovaries. In 21 dpp mouse ovary, the expression of c-kit/SCF in oocytes gradually decreased as the follicles developed. c-Kit/SCF was expressed strongly in oocytes of preantral follicles and weakly in granulosa and thecal cells. Inhibin-alpha was mainly expressed on granulosa cells of preantral and early antral follicles of the 21 dpp mouse ovaries. These findings suggest that the IHC expression of c-kit/SCF proteins is specific in all developmental stages of ovarian follicles and is decreased after the follicle starts to grow. The expression of inhibin-alpha is negatively correlated with the expression of c-kit/SCF in the ovarian follicles in mice.     

Differential regulation of membrane type 1-matrix metalloproteinase activity by ERK 1/2- and p38 MAPK-modulated tissue inhibitor of metalloproteinases 2 expression controls transforming growth factor-beta1-induced pericellular collagenolysis

Acquisition of matrix metalloproteinase-2 (MMP-2) activity is temporally associated with increased migration and invasiveness of cancer cells. ProMMP-2 activation requires multimolecular complex assembly involving proMMP-2, membrane type 1-MMP (MT1-MMP, MMP-14), and tissue inhibitor of metalloproteinases-2 (TIMP-2). Because transforming growth factor-beta1 (TGF-beta1) promotes tumor invasion in advanced squamous cell carcinomas, the role of TGF-beta1 in the regulation of MMP activity in a cellular model of invasive oral squamous cell carcinoma was examined. Treatment of oral squamous cell carcinoma cells with TGF-beta1 promoted MMP-dependent cell scattering and collagen invasion, increased expression of MMP-2 and MT1-MMP, and enhanced MMP-2 activation. TGF-beta1 induced concomitant activation of ERK1/2 and p38 MAPK, and kinase inhibition studies revealed a negative regulatory role for ERK1/2 in modulating acquisition of MMP-2 activity. Thus, a reciprocal effect on proMMP-2 activation was observed whereupon blocking ERK1/2 phosphorylation promoted proMMP-2 activation and MT1-MMP activity, whereas inhibiting p38 MAPK activity decreased proteolytic potential. The cellular mechanism for the control of MT1-MMP catalytic activity involved concurrent reciprocal modulation of TIMP-2 expression by ERK1/2 and p38 MAPKs, such that inhibition of ERK1/2 phosphorylation decreased TIMP-2 production, and down-regulation of p38 MAPK activity enhanced TIMP-2 synthesis. Further, p38 MAPK inhibition promoted ERK1/2 phosphorylation, providing additional evidence for cross-talk between MAPK pathways. These observations demonstrate the complex reciprocal effects of ERK1/2 and p38 MAPK in the regulation of MMP activity, which could complicate the use of MAPK-specific inhibitors as therapeutic agents to down-regulate the biologic effects of TGF-beta1 on pericellular collagen degradation and tumor invasion.     

Membrane expression of DR4, DR5 and caspase-8 levels, but not Mcl-1, determine sensitivity of human myeloma cells to Apo2L/TRAIL

The improved recombinant form of the death ligand Apo2L/TRAIL (Apo2L/TRAIL.0) is not cytotoxic for normal human cells and is a good candidate for the therapy of multiple myeloma (MM), a B-cell neoplasia that remains incurable. We have analyzed the molecular determinants of myeloma sensitivity to Apo2L/TRAIL.0 in a number of MM cell lines, the mechanisms of resistance and a possible way of overcoming it. Expression of one death receptor for Apo2L/TRAIL (DR4 or DR5) is sufficient to transduce death signals, though DR5 was more efficient when both receptors were present. Membrane expression of decoy receptors (DcR1, DcR2) and intracellular levels of c-FLIP(L), XIAP and Mcl-1 were not predictive of resistance to Apo2L/TRAIL. Inhibition of Mcl-1 degradation did not prevent Apo2L/TRAIL-induced apoptosis. In IM-9 cells, resistance was associated to a reduced caspase-8 expression. U266 cells, though expressing significant levels of DR4 and caspase-8, were nevertheless resistant to Apo2L/TRAIL. This resistance could be overcome by co-treatment with valproic acid (VPA), a histone deacetylase inhibitor. VPA caused the redistribution of DR4 to plasma membrane lipid rafts and restored DR4 signaling. Overexpression of Mcl-1 in U266 cells did not prevent Apo2L/TRAIL cytotoxicity in VPA-sensitized cells. These results, taken together, support the possible use of Apo2L/TRAIL.0 in the treatment of MM.     

Oxidative phosphorylation in Azotobacter vinelandii. Energy-linked pH changes and fluorescence changes of atebrin and 1-anilinonaphthalene-8-sulphonate

1. Phosphorylating particles from Azotobacter vinelandii show a rapid, respiration-induced reversible increase in pH of the suspending medium; this is not found with non-phosphorylating particles.2. The observed pH response requires the presence of low concentrations of Mg²⁺ or of higher concentrations of Na⁺ or K⁺.3. Between 40 and 10 °C the rates of proton influx and efflux have similar temperature coefficients; below 10 °C the effect of temperature is greater on proton efflux.4. The kinetics of the energy-linked enhancement of fluorescence 1-anilinonaphthalene-8-sulphonate are slower than that of the quenching of the fluorescence of atebrin.     

Modulation of early growth response gene 1 and interleukin-8 expression by ribotoxin deoxynivalenol (vomitoxin) via ERK1/2 in human epithelial intestine 407 cells

Epithelium senses external toxic insults transmit the sentinel signals into the cells to reprogram a broad range of mucosal responses like epithelial inflammatory diseases. The purpose of this study was to test the hypothesis that ribotoxin DON evokes the epithelial sentinel signals which contributes to the pro-inflammatory cytokine interleukin-8 in human epithelial cells. Treatment with DON elevated interleukin-8 production in the human epithelial intestine 407 cells. Particularly, ribotoxin DON markedly elevated the phosphorylated extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) mitogen-activated protein kinase (MAPK) which mediated DON-induced interleukin-8 (IL-8) production. DON-activated ERK1/2 also mediated the production of early growth response gene 1 (EGR-1) in the epithelial cell line and EGR-1 had the positive regulatory effect on the interleukin-8 production in the human epithelial cells. Taken all, DON-activated MAPK sentinel signals of the epithelial cells which promoted interleukin-8 production and its positive modulator EGR-1. These findings will provide insight into the possible mechanism associated with the early epithelial inflammation by ribotoxic insults.     

Disruption of the cellulose synthase gene, AtCesA8/IRX1, enhances drought and osmotic stress tolerance in Arabidopsis

Two allelic Arabidopsis mutants, leaf wilting 2-1 and leaf wilting 2-2 (lew2-1 and lew2-2 ), were isolated in a screen for plants with altered drought stress responses. The mutants were more tolerant to drought stress as well as to NaCl, mannitol and other osmotic stresses. lew2 mutant plants accumulated more abscisic acid (ABA), proline and soluble sugars than the wild type. The expression of a stress-inducible marker gene RD29A, a proline synthesis-related gene P5CS (pyrroline-5-carboxylate synthase) and an ABA synthesis-related gene SDR1 (alcohol dehydrogenase/reductase) was higher in lew2 than in the wild type. Map-based cloning revealed that the lew2 mutants are new alleles of the AtCesA8/IRX1 gene which encodes a subunit of a cellulose synthesis complex. Our results suggest that cellulose synthesis is important for drought and osmotic stress responses including drought induction of gene expression.     

大鼠肢体缺血/再灌注后肝脏 HO-1表达的变化及其意义

目的:探讨肢体缺血/再灌注(I/R)致肝脏损伤时肝组织诱导型血红素氧合酶(HO-1)表达的变化及其意义.方法:夹闭大鼠双侧股动脉根部4 h、开放2～24 h,制备肢体I/R模型.RT-PCR检测肝组织HO-1 mRNA表达的变化,免疫组化染色法观察HO-1蛋白在肝内的生成与分布.对肢体I/R大鼠应用锌原卟啉抑制其体内HO-1活性后,光镜观察其肝组织的病理变化.结果:肢体I/R后肝组织HO-1 mRNA的表达水平显著高于各对照组,再灌12 h表达至峰值,至再灌24 h仍显著高于各对照组(P＜0.01).肢体I/R组肝组织内出现大量弥散分布的HO-1阳性肝细胞,抑制HO-1活性,使肢体I/R组肝组织损伤明显加重.结论:肢体I/R损伤可诱导肝细胞HO-1基因表达上调,所诱生的HO-1对肝细胞具有保护效应.     

Effects of food deprivation and high fat diet on immunoreactive dynorphin A(1–8) levels in brain regions of Zucker rats

The levels of immunoreactive dynorphin A(1–8) (ir-DYN8) were measured in discrete brain regions of lean Zucker rats subjected to food deprivation for 72 hr and to a high fat diet, and in fatty Zucker rats after food deprivation for 72 hr. Fatty rats showed higher concentrations of ir-DYN8 in the cortex and midbrain, when compared to lean rats fed a stock diet ad lib. Food deprivation increased ir-DYN8 levels in the cortex of lean rats and fatty rats and in the hippocampus of fatty rats, but decreased its content in the striatum of lean rats and in the midbrain of fatty rats. The high fat diet increased ir-DYN8 levels in the cortex and midbrain of lean rats. These results suggest that ir-DYN8 levels in extrahypothalamic structures of Zucker rats could be differentially modified under conditions of hereditary obesity and dietary manipulations.