It has been shown that selenium-binding protein 1 (SBP1) is significantly downregulated in different human cancers. Its regulation and function have not yet been established.
We show that the SBP1 ...promoter is hypermethylated in colon cancer tissues and human colon cancer cells. Treatment with 5'-Aza-2'-deoxycytidine leads to demethylation of the SBP1 promoter and to an increase of SBP1 promoter activity, rescues SBP1 mRNA and protein expression in human colon cancer cells. Additionally, overexpression of SBP1 sensitizes colon cancer cells to H2O2-induced apoptosis, inhibits cancer cell migration in vitro and inhibits tumor growth in nude mice.
These data demonstrate that SBP1 has tumor suppressor functions that are inhibited in colorectal cancer through epigenetic silencing.
RATIONALE:A rise in cytosolic Ca concentration (Cacyt) in pulmonary arterial smooth muscle cells (PASMC) is an important stimulus for pulmonary vasoconstriction and vascular remodeling. Increased ...resting Cacyt and enhanced Ca influx have been implicated in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH).
OBJECTIVE:We examined whether the extracellular Ca-sensing receptor (CaSR) is involved in the enhanced Ca influx and proliferation in IPAH-PASMC and whether blockade of CaSR inhibits experimental pulmonary hypertension.
METHODS AND RESULTS:In normal PASMC superfused with Ca-free solution, addition of 2.2 mmol/L Ca to the perfusate had little effect on Cacyt. In IPAH-PASMC, however, restoration of extracellular Ca induced a significant increase in Cacyt. Extracellular application of spermine also markedly raised Cacyt in IPAH-PASMC but not in normal PASMC. The calcimimetic R568 enhanced, whereas the calcilytic NPS 2143 attenuated, the extracellular Ca-induced Cacyt rise in IPAH-PASMC. Furthermore, the protein expression level of CaSR in IPAH-PASMC was greater than in normal PASMC; knockdown of CaSR in IPAH-PASMC with siRNA attenuated the extracellular Ca-mediated Cacyt increase and inhibited IPAH-PASMC proliferation. Using animal models of pulmonary hypertension, our data showed that CaSR expression and function were both enhanced in PASMC, whereas intraperitoneal injection of the calcilytic NPS 2143 prevented the development of pulmonary hypertension and right ventricular hypertrophy in rats injected with monocrotaline and mice exposed to hypoxia.
CONCLUSIONS:The extracellular Ca-induced increase in Cacyt due to upregulated CaSR is a novel pathogenic mechanism contributing to the augmented Ca influx and excessive PASMC proliferation in patients and animals with pulmonary arterial hypertension.
Downregulated expression of K
channels and decreased K
currents in pulmonary artery smooth muscle cells (PASMC) have been implicated in the development of sustained pulmonary vasoconstriction and ...vascular remodeling in patients with idiopathic pulmonary arterial hypertension (IPAH). However, it is unclear exactly how K
channels are downregulated in IPAH-PASMC. MicroRNAs (miRNAs) are small non-coding RNAs that are capable of posttranscriptionally regulating gene expression by binding to the 3'-untranslated regions of their targeted mRNAs. Here, we report that specific miRNAs are responsible for the decreased K
channel expression and function in IPAH-PASMC. We identified 3 miRNAs (miR-29b, miR-138, and miR-222) that were highly expressed in IPAH-PASMC in comparison to normal PASMC (>2.5-fold difference). Selectively upregulated miRNAs are correlated with the decreased expression and attenuated activity of K
channels. Overexpression of miR-29b, miR-138, or miR-222 in normal PASMC significantly decreased whole cell K
currents and downregulated voltage-gated K
channel 1.5 (K
1.5/KCNA5) in normal PASMC. Inhibition of miR-29b in IPAH-PASMC completely recovered K
channel function and K
1.5 expression, while miR-138 and miR-222 had a partial or no effect. Luciferase assays further revealed that K
1.5 is a direct target of miR-29b. Additionally, overexpression of miR-29b in normal PASMC decreased large-conductance Ca
-activated K
(BK
) channel currents and downregulated BK
channel β1 subunit (BK
β1 or KCNMB1) expression, while inhibition of miR-29b in IPAH-PASMC increased BK
channel activity and BK
β1 levels. These data indicate upregulated miR-29b contributes at least partially to the attenuated function and expression of K
and BK
channels in PASMC from patients with IPAH.
An increase in cytosolic free Ca(2+) concentration (Ca(2+)cyt) in pulmonary arterial smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and a critical stimulation for PASMC ...proliferation and migration. Previously, we demonstrated that expression and function of calcium sensing receptors (CaSR) in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH) and animals with experimental pulmonary hypertension (PH) were greater than in PASMC from normal subjects and control animals. However, the mechanisms by which CaSR triggers Ca(2+) influx in PASMC and the implication of CaSR in the development of PH remain elusive. Here, we report that CaSR functionally interacts with TRPC6 to regulate Ca(2+)cyt in PASMC. Downregulation of CaSR or TRPC6 with siRNA inhibited Ca(2+)-induced Ca(2+)cyt increase in IPAH-PASMC (in which CaSR is upregulated), whereas overexpression of CaSR or TRPC6 enhanced Ca(2+)-induced Ca(2+)cyt increase in normal PASMC (in which CaSR expression level is low). The upregulated CaSR in IPAH-PASMC was also associated with enhanced Akt phosphorylation, whereas blockade of CaSR in IPAH-PASMC attenuated cell proliferation. In in vivo experiments, deletion of the CaSR gene in mice (casr(-/-)) significantly inhibited the development and progression of experimental PH and markedly attenuated acute hypoxia-induced pulmonary vasoconstriction. These data indicate that functional interaction of upregulated CaSR and upregulated TRPC6 in PASMC from IPAH patients and animals with experimental PH may play an important role in the development and progression of sustained pulmonary vasoconstriction and pulmonary vascular remodeling. Blockade or downregulation of CaSR and/or TRPC6 with siRNA or miRNA may be a novel therapeutic strategy to develop new drugs for patients with pulmonary arterial hypertension.
Hypoxic pulmonary vasoconstriction (HPV) is an important physiological response that optimizes the ventilation/perfusion ratio. Chronic hypoxia causes vascular remodeling, which is central to the ...pathogenesis of hypoxia-induced pulmonary hypertension (HPH). We have previously shown that Notch3 is up-regulated in HPH and that activation of Notch signaling enhances store-operated Ca(2+) entry (SOCE), an important mechanism that contributes to pulmonary arterial smooth muscle cell (PASMC) proliferation and contraction. Here, we investigate the role of Notch signaling in HPV and hypoxia-induced enhancement of SOCE. We examined SOCE in human PASMCs exposed to hypoxia and pulmonary arterial pressure in mice using the isolated perfused/ventilated lung method. Wild-type and canonical transient receptor potential (TRPC) 6(-/-) mice were exposed to chronic hypoxia to induce HPH. Inhibition of Notch signaling with a γ-secretase inhibitor attenuates hypoxia-enhanced SOCE in PASMCs and hypoxia-induced increase in pulmonary arterial pressure. Our results demonstrate that hypoxia activates Notch signaling and up-regulates TRPC6 channels. Additionally, treatment with a Notch ligand can mimic hypoxic responses. Finally, inhibition of TRPC6, either pharmacologically or genetically, attenuates HPV, hypoxia-enhanced SOCE, and the development of HPH. These results demonstrate that hypoxia-induced activation of Notch signaling mediates HPV and the development of HPH via functional activation and up-regulation of TRPC6 channels. Understanding the molecular mechanisms that regulate cytosolic free Ca(2+) concentration and PASMC proliferation is critical to elucidation of the pathogenesis of HPH. Targeting Notch regulation of TRPC6 will be beneficial in the development of novel therapies for pulmonary hypertension associated with hypoxia.
Previous studies have shown that decorin expression is significantly reduced in colorectal cancer tissues and cancer cells, and genetic deletion of the decorin gene is sufficient to cause intestinal ...tumor formation in mice, resulting from a downregulation of p21, p27kip1 and E-cadherin and an upregulation of β-catenin signaling Bi,X. et al. (2008) Genetic deficiency of decorin causes intestinal tumor formation through disruption of intestinal cell maturation. Carcinogenesis, 29, 1435-1440. However, the regulation of E-cadherin by decorin and its implication in cancer formation and metastasis is largely unknown. Using a decorin knockout mouse model (Dcn
−/− mice) and manipulated expression of decorin in human colorectal cancer cells, we found that E-cadherin, a protein that regulates cell-cell adhesion, epithelial-mesenchymal transition and metastasis, was almost completely lost in Dcn−/−
mouse intestine, and loss of decorin and E-cadherin accelerated colon cancer cell growth and invasion in Dcn−/−
mice. However, increasing decorin expression in colorectal cancer cells attenuated cancer cell malignancy, including inhibition of cancer cell proliferation, promotion of apoptosis and importantly, attenuation of cancer cell migration. All these changes were linked to the regulation of E-cadherin by decorin. Moreover, overexpression of decorin upregulated E-cadherin through increasing of E-cadherin protein stability as E-cadherin messenger RNA and promoter activity were not affected. Co-immunoprecipitation assay showed a physical binding between decorin and E-cadherin proteins. Taken together, our results provide direct evidence that decorin-mediated inhibition of colorectal cancer growth and migration are through the interaction with and stabilization of E-cadherin.
A rise in cytosolic Ca(2+) concentration (Ca(2+)(cyt)) in pulmonary arterial smooth muscle cells (PASMC) is an important stimulus for pulmonary vasoconstriction and vascular remodeling. Increased ...resting Ca(2+)(cyt) and enhanced Ca(2+) influx have been implicated in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH).
We examined whether the extracellular Ca(2+)-sensing receptor (CaSR) is involved in the enhanced Ca(2+) influx and proliferation in IPAH-PASMC and whether blockade of CaSR inhibits experimental pulmonary hypertension.
In normal PASMC superfused with Ca(2+)-free solution, addition of 2.2 mmol/L Ca(2+) to the perfusate had little effect on Ca(2+)(cyt). In IPAH-PASMC, however, restoration of extracellular Ca(2+) induced a significant increase in Ca(2+)(cyt). Extracellular application of spermine also markedly raised Ca(2+)(cyt) in IPAH-PASMC but not in normal PASMC. The calcimimetic R568 enhanced, whereas the calcilytic NPS 2143 attenuated, the extracellular Ca(2+)-induced Ca(2+)(cyt) rise in IPAH-PASMC. Furthermore, the protein expression level of CaSR in IPAH-PASMC was greater than in normal PASMC; knockdown of CaSR in IPAH-PASMC with siRNA attenuated the extracellular Ca(2+)-mediated Ca(2+)(cyt) increase and inhibited IPAH-PASMC proliferation. Using animal models of pulmonary hypertension, our data showed that CaSR expression and function were both enhanced in PASMC, whereas intraperitoneal injection of the calcilytic NPS 2143 prevented the development of pulmonary hypertension and right ventricular hypertrophy in rats injected with monocrotaline and mice exposed to hypoxia.
The extracellular Ca(2+)-induced increase in Ca(2+)(cyt) due to upregulated CaSR is a novel pathogenic mechanism contributing to the augmented Ca(2+) influx and excessive PASMC proliferation in patients and animals with pulmonary arterial hypertension.
Selenium-binding protein (SBP) 1 is present in reduced levels in several cancer types as compared with normal tissues, and lower levels are associated with poor clinical prognosis. Another ...selenium-containing protein, glutathione peroxidase 1 (GPX1), has been associated with cancer risk and development. The interaction between these representatives of different classes of selenoproteins was investigated. Increasing SBP1 levels in either human colorectal or breast cancer cells by transfection of an expression construct resulted in the reduction of GPX1 enzyme activity. Increased expression of GPX1 in the same cell types resulted in the transcriptional and translational repression of SBP1, as evidenced by the reduction of SBP1 messenger RNA and protein and the inhibition of transcription measured using an SBP1 reporter construct. The opposing effects of SBP1 and GPX1 on each other were also observed when GPX1 was increased by supplementing the media of these tissue culture cells with selenium, and the effect of selenium on SBP1 was shown to be GPX1 dependent. Decreasing or increasing GPX1 levels in colonic epithelial cells of mice fed a selenium-deficient, -adequate or -supplemented diet resulted in the opposing effect on SBP1 levels. These data are explained in part by the demonstration that SBP1 and GPX1 form a physical association, as determined by coimmunoprecipitation and fluorescence resonance energy transfer assay. The results presented establish an interaction between two distinct selenium-containing proteins that may enhance the understanding of the mechanisms by which selenium and selenoproteins affect carcinogenesis in humans.
RATIONALE:An increase in cytosolic free Ca concentration (Cacyt) in pulmonary arterial smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and an important stimulus for ...PASMC proliferation and pulmonary vascular remodeling. The dihydropyridine Ca channel blockers, such as nifedipine, have been used for treatment of idiopathic pulmonary arterial hypertension (IPAH).
OBJECTIVE:Our previous study demonstrated that the Ca-sensing receptor (CaSR) was upregulated and the extracellular Ca-induced increase in Cacyt was enhanced in PASMC from patients with IPAH and animals with experimental pulmonary hypertension. Here, we report that the dihydropyridines (eg, nifedipine) increase Cacyt by activating CaSR in PASMC from IPAH patients (in which CaSR is upregulated), but not in normal PASMC.
METHODS AND RESULTS:The nifedipine-mediated increase in Cacyt in IPAH-PASMC was concentration dependent with a half maximal effective concentration of 0.20 µmol/L. Knockdown of CaSR with siRNA in IPAH-PASMC significantly inhibited the nifedipine-induced increase in Cacyt, whereas overexpression of CaSR in normal PASMC conferred the nifedipine-induced rise in Cacyt. Other dihydropyridines, nicardipine and Bay K8644, had similar augmenting effects on the CaSR-mediated increase in Cacyt in IPAH-PASMC; however, the nondihydropyridine blockers, such as diltiazem and verapamil, had no effect on the CaSR-mediated rise in Cacyt.
CONCLUSIONS:The dihydropyridine derivatives increase Cacyt by potentiating the activity of CaSR in PASMC independently of their blocking (or activating) effect on Ca channels; therefore, it is possible that the use of dihydropyridine Ca channel blockers (eg, nifedipine) to treat IPAH patients with upregulated CaSR in PASMC may exacerbate pulmonary hypertension.
This article explores the complexities of creating an archive - in our case, a digital archive of eighteenth-century manuscript letters, The Elizabeth Montagu Correspondence Online (EMCO). Elizabeth ...Montagu was one of the most prominent and well-connected women in eighteenth-century polite society. EMCO faces a variety of challenges. Firstly, the project aims to trace all extant letters in different libraries and public/private collections; secondly, it seeks to amalgamate the extant correspondence into one digital repository and a comprehensive inventory; thirdly, it mobilises a team of scholars to transcribe, annotate and develop a critical apparatus; fourthly, EMCO seeks to develop digital tools that foster novel methods of scholarly research and debate. Taking recent scholarship on board, this article concludes by reflecting on the complexities of marrying a data-rich digital edition with literary methodologies that allow both close reading and analysis of the scope and materiality of the archive and its objects.
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