Biomedical subjects

Limei Zhou

Publications and source records attributed to Limei Zhou.

9 recordsLinked to original sources

PubMed2006

Monocyte chemoattractant protein-1 induces a novel transcription factor that causes cardiac myocyte apoptosis and ventricular dysfunction.

Monocyte chemoattractant protein-1 (MCP-1; CCL2)-mediated inflammation plays a critical role in the development of ischemic heart disease (IHD). However, the gene expression changes caused by signal transduction, triggered by MCP-1 binding to its receptor CCR2, and their possible role in the development of IHD are not understood. We present evidence that MCP-1 binding to CCR2 induces a novel transcription factor (MCP-induced protein [MCPIP]) that causes cell death. Gene microarray analysis showed that when expressed in hiuman embryonic kidney 293 cells, MCPIP induced apoptotic gene families before causing cell death. Mutagenesis studies showed that the structural features required for transcription factor-like activity were also required for causing cell death. Activation of caspase-3 was detected after MCPIP transfection and Z-VAD-fmk partially inhibited cell death. Cardiomyocyte-targeted expression of MCP-1 in mice caused death by heart failure at 6 months of age. MCPIP expression increased in parallel with the development of ventricular dysfunction. In situ hybridization showed the presence of MCPIP transcripts in the cardiomyocytes and immunohistochemistry showed that MCPIP was associated with the cardiomyocyte nuclei of apoptotic cardiomyocytes. CCR2 expression in cardiomyocytes increased with the development of IHD. MCPIP production induced by MCP-1 binding to CCR2 in the cardiomyocytes is probably involved in the development of IHD in this murine model. MCPIP transcript levels were much higher in the explanted human hearts with IHD than with nonischemic heart disease. These results provide a molecular insight into how chronic inflammation and exposure to MCP-1 contributes to heart failure and suggest that MCPIP could be a potential target for therapeutic intervention.

Animals↗

PubMed2005

Transforming growth factor-beta induces airway smooth muscle hypertrophy.

Although smooth muscle hypertrophy is present in asthmatic airways, little is known about the biochemical pathways regulating airway smooth muscle protein synthesis, cell size, or accumulation of contractile apparatus proteins. We sought to develop a model of airway smooth muscle hypertrophy in primary cells using a physiologically relevant stimulus. We hypothesized that transforming growth factor (TGF)-beta induces hypertrophy in primary bronchial smooth muscle cells. Primary human bronchial smooth muscle cells isolated from unacceptable lung donor tissue were studied. Cells were seeded on uncoated plastic dishes at 50% confluence and TGF-beta was added. Experiments were performed in the absence of serum. TGF-beta increased cell size and total protein synthesis, expression of alpha-smooth muscle actin and smooth muscle myosin heavy chain, formation of actomyosin filaments, and cell shortening to acetylcholine. Further, TGF-beta increased airway smooth muscle alpha-actin synthesis in the presence of the transcriptional inhibitor actinomycin D, evidence that translational control is a physiologically important element of the observed hypertrophy. TGF-beta induced the phosphorylation of eukaryotic translation initiation factor-4E-binding protein, a signaling event specifically involved in translational control. Finally, two inhibitors of 4E-binding protein phosphorylation, the phosphoinositol 3-kinase inhibitor LY294002 and a phosphorylation site mutant of 4E-binding protein-1 that dominantly inhibits eukaryotic initiation factor-4E, each blocked TGF-beta-induced alpha-actin expression and cell enlargement. We conclude that TGF-beta induces hypertrophy of primary bronchial smooth muscle cells. Further, phosphorylation of 4E-binding protein is required for the observed hypertrophy.

Acetylcholine↗

PubMed2005

4E-binding protein phosphorylation and eukaryotic initiation factor-4E release are required for airway smooth muscle hypertrophy.

The molecular mechanisms of airway smooth muscle hypertrophy, a feature of severe asthma, are poorly understood. We previously established a conditionally immortalized human bronchial smooth muscle cell line with a temperature-sensitive SV40 large T antigen. Temperature shift and loss of large T cause G1-phase cell cycle arrest that is accompanied by increased airway smooth muscle cell size. In the present study, we hypothesized that phosphorylation of eukaryotic initiation factor-4E (eIF4E)-binding protein (4E-BP), which subsequently releases eIF4E and initiates cap-dependent mRNA translation, was required for airway smooth muscle hypertrophy. Treatment of cells with chemical inhibitors of PI 3-kinase and mammalian target of rapamycin blocked protein synthesis and cell growth while decreasing the phosphorylation of 4E-BP and increasing the binding of 4E-BP to eIF4E, consistent with the notion that 4E-BP1 phosphorylation and eIF4E function are required for hypertrophy. To test this directly, we infected cells with a retrovirus encoding a phosphorylation site mutant of 4E-BP1 (AA-4E-BP-1) that dominantly inhibits eIF4E. Upon temperature shift, cells infected with AA-4E-BP-1, but not empty vector, failed to undergo hypertrophic growth. We conclude that phosphorylation of 4E-BP, eIF4E release, and cap-dependent protein synthesis are required for hypertrophy of human airway smooth muscle cells.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Yersinia YopJ inhibits pro-inflammatory molecule expression in human bronchial epithelial cells.

Human bronchial epithelial cell pro-inflammatory molecule expression plays a role in the pathogenesis of airway diseases. We hypothesize that Yersinia outer protein-J (YopJ), a Yersinia virulence effector which inhibits mitogen activated protein (MAP) kinase kinases (MKKs), attenuates epithelial cell pro-inflammatory molecule expression. 16HBE14o-cells were co-transfected with cDNAs encoding Yersinia pseudotuberculosis YopJ or empty vector. Expression of YopJ reduced activation of extracellular signal regulated kinase (ERK)-2, Jun amino terminal kinase (JNK)-1 and IkappaB kinase (IKK)-beta. YopJ also blocked transactivation of NF-kappaB and AP-1 promoter sequences which has been shown to regulate chemokine expression. Finally, expression of YopJ reduced transcription from the IL-8, RANTES (regulated upon activation, normal T cell expressed and secreted) and intercellular adhesion molecule (ICAM)-1 promoters. We conclude that YopJ expression blocks the innate immune response in lung epithelial cells, the site of Yersinia pestis infection. Inhibition of bronchial epithelial cell responses by YopJ is consistent with the notion that MAP kinases regulates bronchial epithelial cell pro-inflammatory molecule expression.

Bacterial Proteins↗

PubMed2003

Human bronchial smooth muscle cell lines show a hypertrophic phenotype typical of severe asthma.

We developed clonal cell lines of human bronchial smooth muscle origin by retroviral transduction of temperature-sensitive simian virus 40 large tumor (T) antigen. These cells show increased growth potential at 33 degrees C, but on shift to the nonpermissive temperature (39 degrees C), they show diminished or arrested growth. In addition to the expected reduction in the level of large T antigen, cells shifted to 39 degrees C show increased expression of the cyclin-dependent kinase inhibitor p21(Waf1/Cip1), characteristic of cells arrested in G1 of the cell cycle. Shifted cells undergo a process of cell hypertrophy, as demonstrated by increased time of flight and forward scatter, as well as increased expression of the contractile proteins alpha-smooth muscle actin, myosin light chain kinase, and SM22. Changes in contractile protein expression were regulated primarily in a posttranscriptional manner. Phosphatidylinositol 3-kinase activity was increased in shifted cells, and chemical inhibition of phosphatidylinositol 3-kinase attenuated alpha-actin and myosin light-chain kinase expression. We have developed clonal cell lines of human bronchial smooth muscle origin that may be useful for the study of airway smooth muscle biology. Furthermore, we demonstrate that arrest of airway smooth muscle cell cycle traversal can induce cellular hypertrophy, which parallels changes observed in the airways of patients with severe asthma.

Antigens, Viral, Tumor↗

PubMed2003

Rhinovirus 16 3C protease induces interleukin-8 and granulocyte-macrophage colony-stimulating factor expression in human bronchial epithelial cells.

Rhinovirus (RV), a member of the Picornaviridae family, accounts for many virus-induced asthma exacerbations. RV induces airway cell chemokine expression both in vivo and in vitro. Because of the known interactions of proteases with cellular functions, we hypothesized that RV 3C protease is sufficient for cytokine up-regulation. A cDNA encoding RV16 3C protease was constructed by PCR amplification and transfected into 16HBE14o- human bronchial epithelial cells. 3C protease induced expression of both IL-8 and GM-CSF, as well as transcription from both the IL-8 and GM-CSF promoters. 3C expression also induced activator protein 1 and NF-kappaB transcriptional activation. Finally, mutation of IL-8 promoter AP-1 and NF-kappaB promoter sequences significantly reduced 3C-induced responses. Together, these data suggest expression of RV16 3C protease is sufficient to induce chemokine expression in human bronchial epithelial cells, and does so in an AP-1- and NF-kappaB-dependent manner.

3C Viral Proteases↗

PubMed2003

Mitogenic signaling pathways in airway smooth muscle.

Increased airway smooth muscle mass has been demonstrated in patients with asthma, bronchopulmonary dysplasia and most recently, cystic fibrosis. These observations emphasize the need for further knowledge of the events involved in airway smooth muscle mitogenesis and hypertrophy. Workers in the field have developed cell culture systems involving tracheal and bronchial myocytes from different species. An emergent body of literature indicates that mutual signal transduction pathways control airway smooth muscle cell cycle entry across species lines. This article reviews what is known about mitogen-activated signal transduction in airway myocytes. The extracellular signal regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI 3-kinase) pathways appear to be key positive regulators of airway smooth muscle mitogenesis; recent studies have also demonstrated specific roles for reactive oxygen and the JAK/STAT pathway. It is also possible that growth factor stimulation of airway smooth muscle concurrently elicits signaling through negative regulatory intermediates such as p38 mitogen-activated protein (MAP) kinase and protein kinase C (PKC) delta, conceivably as a defense against extreme growth.

Animals↗

PubMed2003

Regulation of airway epithelial cell NF-kappa B-dependent gene expression by protein kinase C delta.

Airway epithelial cells synthesize proinflammatory molecules such as IL-8, GM-CSF, RANTES, and ICAM-1, the expression of which is increased in the airways of patients with asthma. We investigated the regulation of these NF-kappa B-dependent genes by the novel protein kinase C (PKC) isoform PKC delta in 16HBE14o- human airway epithelial cells, focusing on IL-8 expression. Transient transfection with the constitutively active catalytic subunit of PKC delta (PKC delta-CAT), and treatment with bryostatin 1, an activator of PKC delta, each increased transcription from the IL-8 promoter, whereas overexpression of PKC epsilon had minor effects. Expression of a dominant negative PKC delta mutant (PKC delta-KR) or pretreatment of cells with rottlerin, a chemical PKC delta inhibitor, attenuated TNF-alpha- and phorbol ester-induced transcription from the IL-8 promoter. Bryostatin 1 treatment increased IL-8 protein abundance in primary airway epithelial cells. Selective activation of PKC delta by bryostatin also activated NF-kappa B, as evidenced by p65 RelA and p50 NF-kappa B1 binding to DNA, NF-kappa B trans-activation, and I kappa B degradation. The sufficiency of PKC delta to induce NF-kappa B nuclear translocation and binding to DNA was confirmed in a 16HBE14o- cell line inducibly expressing PKC delta-CAT under the tet-off system. Deletion of the NF-kappa B response element severely attenuated PKC delta-induced IL-8 promoter activity. Finally, PKC delta-CAT induced transcription from the GM-CSF, RANTES, and ICAM-1 promoters. Together these data suggest that PKC delta plays a key role in the regulation of airway epithelial cell NF-kappa B-dependent gene expression.

Cell Line↗

PubMed2003

Ras and mitogen-activated protein kinase kinase kinase-1 coregulate activator protein-1- and nuclear factor-kappaB-mediated gene expression in airway epithelial cells.

In 16HBE14o- human bronchial epithelial cells, maximal tumor necrosis factor (TNF)-alpha-induced interleukin (IL)-8 expression depends on the activation of two distinct signaling pathways, one constituted in part by activator protein (AP)-1 and the other by nuclear factor (NF)-kappaB. We examined the upstream signaling intermediates responsible for IL-8 and granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in this system, hypothesizing that p21 Ras and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase (MEKK)-1 function as common upstream activators of both the AP-1 and NF-kappaB pathways. TNF-alpha treatment induced both Ras and MEKK1 activation. Dominant-negative forms of Ras (N17Ras) and MEKK1 (MEKK1-KM) each inhibited TNF-alpha-induced transcription from IL-8 and GM-CSF promoters. Ras was required for maximal activation of extracellular signal-regulated kinase (ERK) and Jun amino terminal kinase (JNK) as well as AP-1 and NF-kappaB transcriptional activities, but not for activation of IkappaB kinase (IKK)-beta, an upstream activator of NF-kappaB. MEKK1 was required for maximal activation of ERK, JNK, and IKK, as well as for maximal AP-1 and NF-kappaB transcriptional activities. We conclude that Ras regulates TNF-alpha-induced chemokine expression by activating the AP-1 pathway and enhancing transcriptional function of NF-kappaB, whereas MEKK1 activates both the AP-1 and NF-kappaB pathways.

Bronchi↗