EBF1 promotes triple-negative breast cancer progression by surveillance of the HIF1α pathway.

Early B cell factor 1 (EBF1) is a transcriptional factor with a variety of roles in cell differentiation and metabolism. However, the functional roles of EBF1 in tumorigenesis remain elusive. Here, we demonstrate that EBF1 is highly expressed in triple-negative breast cancer (TNBC). Furthermore, EBF1 has a pivotal role in the tumorigenicity and progression of TNBC. Moreover, we found that depletion of EBF1 induces extensive cell mitophagy and inhibits tumor growth. Genome-wide mapping of the EBF1 transcriptional regulatory network revealed that EBF1 drives TNBC tumorigenicity by assembling a transcriptional complex with HIF1α that fine-tunes the expression of HIF1α targets via suppression of p300 activity. EBF1 therefore holds HIF1α activity in check to avert extensive mitophagy-induced cell death. Our findings reveal a key function for EBF1 as a master regulator of mitochondria homeostasis in TNBC and indicate that targeting this pathway may offer alternative treatment strategies for this aggressive subtype of breast cancer.

STK39 promotes breast cancer invasion and metastasis by increasing SNAI1 activity upon phosphorylation.

SNAI1 is widely regarded as a master driver of epithelial-mesenchymal transition (EMT) and associated with breast cancer progression and metastasis. This pro-malignant role is strongly linked to posttranslational modification, especially phosphorylation, which controls its protein levels and subcellular localization. While multiple kinases are implicated in regulation of SNAI1 stability, the precise mechanism by which SNAI1 is stabilized in tumors remains to be fully elucidated. Methods: A series of in vitro and in vivo experiments were conducted to reveal the regulation of SNAI1 by Serine/Threonine Kinase 39 (STK39) and the role of STK39 in breast cancer metastasis. Results: We identified STK39, a member of Stem 20-like serine/threonine kinase family, as a novel posttranslational regulator that enhances the stability of SNAI1. Inhibition of STK39 via knockdown or use of a specific inhibitor resulted in SNAI1 destabilization. Mechanistically, STK39 interacted with and phosphorylated SNAI1 at T203, which is critical for its nuclear retention. Functionally, STK39 inhibition markedly impaired the EMT phenotype and decreased tumor cell migration, invasion, and metastasis both in vitro and in vivo. These effects were rescued by ectopic SNAI1 expression. In addition, depletion of STK39 dramatically enhanced sensitivity to chemotherapeutic agents. Conclusions: Our study demonstrated that STK39 is a key mediator of SNAI1 stability and is associated with the pro-metastatic cellular process, highlighting the STK39-SNAI1 signaling axis as promising therapeutic targets for treatments of metastatic breast cancer.

Tackle Epithelial-Mesenchymal Transition With Epigenetic Drugs in Cancer.

Epithelial-mesenchymal Transition (EMT) is a de-differentiation process in which epithelial cells lose their epithelial properties to acquire mesenchymal features. EMT is essential for embryogenesis and wound healing but is aberrantly activated in pathological conditions like fibrosis and cancer. Tumor-associated EMT contributes to cancer cell initiation, invasion, metastasis, drug resistance and recurrence. This dynamic and reversible event is governed by EMT-transcription factors (EMT-TFs) with epigenetic complexes. In this review, we discuss recent advances regarding the mechanisms that modulate EMT in the context of epigenetic regulation, with emphasis on epigenetic drugs, such as DNA demethylating reagents, inhibitors of histone modifiers and non-coding RNA medication. Therapeutic contributions that improve epigenetic regulation of EMT will translate the clinical manifestation as treating cancer progression more efficiently.

The combined effect of epigenetic inhibitors for LSD1 and BRD4 alters prostate cancer growth and invasion.

Epigenetic modifications play an important role in prostate tumor development and progression. Epigenetic drugs are emerging as effective modulators of gene expression that act on pathways potentially important in the control of cancer clinically. We investigated two different epigenetic modulating drugs, SP-2509 and JQ1, that target histone lysine demethylase 1 (LSD1), and bromodomain-containing protein (BRD), respectively and their combined effect in three different prostate cancer (PCa) types: 1) androgen receptor (AR)-positive and androgen-sensitive; 2) AR-positive but castration-resistant; and 3) androgen-nonresponsive. We found combined treatment provided a synergistic growth inhibition in castration-resistant PCa cells but knockdown of AR reduced sensitivity to both inhibitors in these cells. In the androgen-sensitive cell lines, AR knockdown attenuated sensitivity to the LSD1 inhibitor but not the JQ1 inhibitor. Strikingly, treatment with SP-2509 slightly, and JQ1 markedly increased invasion in PCa cells with high AR expression but decreased invasion in PCa cells with low/negative AR expression. Our results suggest that these two epigenetic drugs are novel and promising compounds for the development of PCa therapeutics, particularly for castration-resistant disease. However, due to the potential risks, including metastasis, caution must be exercised in the clinical setting.

Ubiquitin Regulation: The Histone Modifying Enzyme's Story.

Histone post-translational modifications influence many fundamental cellular events by regulating chromatin structure and gene transcriptional activity. These modifications are highly dynamic and tightly controlled, with many enzymes devoted to the addition and removal of these modifications. Interestingly, these modifying enzymes are themselves fine-tuned and precisely regulated at the level of protein turnover by ubiquitin-proteasomal processing. Here, we focus on recent progress centered on the mechanisms regulating ubiquitination of histone modifying enzymes, including ubiquitin proteasomal degradation and the reverse process of deubiquitination. We will also discuss the potential pathophysiological significance of these processes.

MicroRNA-124 reduces the pentose phosphate pathway and proliferation by targeting PRPS1 and RPIA mRNAs in human colorectal cancer cells.

BACKGROUND & AIMS: Cancer cells alter glucose metabolism to support their uncontrolled proliferation. Changes in microRNAs (miRNAs) have been associated with colorectal cancer (CRC) development and progression, but it is not clear whether they regulate metabolism in CRC cells. We aimed to identify miRNAs that alter glucose metabolism in CRC cells and to determine their effects on tumor development. METHODS: CRC tissues and matched nontumor tissues were collected from 78 patients for messenger RNA (mRNA) analysis and from 112 patients for immunohistochemical analysis at the Fudan University Shanghai Cancer Center from 2005 through 2007. We integrated data on 100 miRNAs previously identified as potential regulators of glucose metabolism in a high-throughput screen with data on 66 miRNAs that often are deregulated in CRC cells. miRNAs with the potential to regulate glucose metabolism in CRC cells were blocked with mimics, and effects on lactate production were measured in CRC cell lines. miRNAs and their targets were overexpressed from lentivirals in CRC cell lines (LoVo and HCT-116) or knocked down with small interfering RNAs. The cells were analyzed in proliferation and colony formation assays and for growth as xenograft tumors in mice. RESULTS: We identified 3 miRNAs that significantly inhibited lactate production in 3 CRC cell lines; miR124-3p (miR124) had the strongest effect. By using complementary DNA microarray analyses, we identified 67 mRNAs that were reduced in CRC cell lines that overexpressed miR124; the mRNAs encoding phosphoribosyl pyrophosphate synthetase 1 (PRPS1) and ribose-5-phosphate isomerase-A (RPIA) were found to be direct targets of miR124. Knockdown of PRPS1 and RPIA, as well as overexpression of miR124, each reduced glucose consumption and adenosine triphosphate in level CRC cells. Conversely, overexpression of PRPS1 or RPIA restored glycometabolism to these cells. RPIA and PRPS1 contribute to nucleotide metabolism and supply precursors for DNA and RNA biosynthesis. CRC cells that overexpressed miR124 or with knockdown of RPIA or PRPS1 had reduced DNA synthesis and proliferation, whereas cells incubated with an inhibitor of miR124 had significantly increased DNA synthesis and proliferation and formed more colonies. LoVo cells that overexpressed miR124 formed smaller xenograft tumors that controlled cells in mice, and had lower levels of PRPS1 and RPIA mRNA and protein. Compared with normal colorectal tissues, levels of miR124 were reduced significantly in CRC tissues from patients, whereas levels of PRPS1 and RPIA increased, which was associated with reduced patient survival times. CONCLUSIONS: miR124 inhibits DNA synthesis and proliferation by reducing levels of pentose phosphate pathway enzymes in CRC cells. Expression of miR124 and its targets correlate with survival times and might be used in prognosis.

MiR-199a-5p is negatively associated with malignancies and regulates glycolysis and lactate production by targeting hexokinase 2 in liver cancer.

UNLABELLED: Cancer cells possess a unique metabolic phenotype that allows them to preferentially utilize glucose through aerobic glycolysis. This phenomenon is referred to as the "Warburg effect." Accumulating evidence suggests that microRNAs (miRNAs), a class of small noncoding regulatory RNAs, interact with oncogenes/tumor suppressors and induce such metabolic reprograming in cancer cells. To systematically study the metabolic roles of miRNAs in cancer cells, we developed a gain-of-function miRNA screen in HeLa cells. Subsequent investigation of the characterized miRNAs indicated that miR-199a-5p acts as a suppressor for glucose metabolism. Furthermore, miR-199a-5p is often down-regulated in human liver cancer, and its low expression level was correlated with a low survival rate, large tumor size, poor tumor differentiation status, high tumor-node-metastasis stage and the presence of tumor thrombus of patients. MicroRNA-199a-5p directly targets the 3'-untranslated region of hexokinase 2 (HK2), an enzyme that catalyzes the irreversible first step of glycolysis, thereby suppressing glucose consumption, lactate production, cellular glucose-6-phosphate and adenosine triphosphate levels, cell proliferation, and tumorigenesis of liver cancer cells. Moreover, HK2 is frequently up-regulated in liver cancer tissues and associated with poor patient outcomes. The up-regulation of hypoxia-inducible factor-1α under hypoxic conditions suppresses the expression of miR-199a-5p and promotes glycolysis, whereas reintroduction of miR-199a-5p interferes with the expression of HK2, abrogating hypoxia-enhanced glycolysis. CONCLUSION: miR-199a-5p/HK2 reprograms the metabolic process in liver cancer cells and provides potential prognostic predictors for liver cancer patients.

MicroRNA-30d-5p inhibits tumour cell proliferation and motility by directly targeting CCNE2 in non-small cell lung cancer.

MicroRNAs (miRNAs) are small, single-stranded, non-coding RNA molecules that are dysregulated in many types of human cancers, although their precise functions in driving non-small cell lung cancer (NSCLC) are incompletely understood. In the present study, we found that miR-30d-5p, often downregulated in NSCLC tissues, significantly inhibited the growth, cell cycle distribution, and motility of NSCLC cells. Furthermore, we demonstrated that cyclin E2 (CCNE2), which was often upregulated in NSCLC tissues, was a direct target of miR-30d-5p. CCNE2 expression promoted the proliferation, invasion, and migration of NSCLC cells. In addition, the re-introduction of CCNE2 expression antagonised the inhibitory effects of miR-30d-5p on the capacity of NSCLC cells for proliferation and motility. Together, these results suggest that the miR-30d-5p/CCNE2 axis may contribute to NSCLC cell proliferation and motility, indicating miR-30d-5p as a potential therapeutic target for the treatment of NSCLC.

Integrated analysis of mutation data from various sources identifies key genes and signaling pathways in hepatocellular carcinoma.

BACKGROUND: Recently, a number of studies have performed genome or exome sequencing of hepatocellular carcinoma (HCC) and identified hundreds or even thousands of mutations in protein-coding genes. However, these studies have only focused on a limited number of candidate genes, and many important mutation resources remain to be explored. PRINCIPAL FINDINGS: In this study, we integrated mutation data obtained from various sources and performed pathway and network analysis. We identified 113 pathways that were significantly mutated in HCC samples and found that the mutated genes included in these pathways contained high percentages of known cancer genes, and damaging genes and also demonstrated high conservation scores, indicating their important roles in liver tumorigenesis. Five classes of pathways that were mutated most frequently included (a) proliferation and apoptosis related pathways, (b) tumor microenvironment related pathways, (c) neural signaling related pathways, (d) metabolic related pathways, and (e) circadian related pathways. Network analysis further revealed that the mutated genes with the highest betweenness coefficients, such as the well-known cancer genes TP53, CTNNB1 and recently identified novel mutated genes GNAL and the ADCY family, may play key roles in these significantly mutated pathways. Finally, we highlight several key genes (e.g., RPS6KA3 and PCLO) and pathways (e.g., axon guidance) in which the mutations were associated with clinical features. CONCLUSIONS: Our workflow illustrates the increased statistical power of integrating multiple studies of the same subject, which can provide biological insights that would otherwise be masked under individual sample sets. This type of bioinformatics approach is consistent with the necessity of making the best use of the ever increasing data provided in valuable databases, such as TCGA, to enhance the speed of deciphering human cancers.

Genome-wide screening identified that miR-134 acts as a metastasis suppressor by targeting integrin ß1 in hepatocellular carcinoma.

MicroRNAs (miRNAs) are small, single-stranded, non-coding RNAs that play pivotal roles in human cancer development and progression, such as tumor metastasis. Here, we identified the miRNAs that regulate hepatocellular carcinoma (HCC) cell migration by a high-throughput screening method using the classical wound-healing assay with time-lapse video microscopy and validation with a transwell migration assay. Eleven miRNAs (miR-134, -146b-3p, -188-3p, -525-3p, -661, -767-5p, -891a, -891b, -1244, -1247 and miR-1471) were found to promote or inhibit HCC cell migration. Further investigation revealed that miR-134 suppressed the invasion and metastasis of HCC cells in vitro and in vivo, and integrin beta 1 (ITGB1) was a direct and functional target gene of miR-134. Moreover, miR-134 inhibited the phosphorylation of focal adhesion kinase (FAK) and the activation of RhoA downstream of the ITGB1 pathway, thereby decreasing stress fiber formation and cell adhesion in HCC cells. In conclusion, we demonstrated that miR-134 is a novel metastasis suppressor in HCC and could be a potential therapeutic target for the treatment of HCC.

Activating mutations in PTPN3 promote cholangiocarcinoma cell proliferation and migration and are associated with tumor recurrence in patients.

BACKGROUND & AIMS: The pathogenesis of intrahepatic cholangiocarcinoma (ICC), the second most common hepatic cancer, is poorly understood, and the incidence of ICC is increasing worldwide. We searched for mutations in human ICC tumor samples and investigated how they affect ICC cell function. METHODS: We performed whole exome sequencing of 7 pairs of ICC tumors and their surrounding nontumor tissues to detect somatic alterations. We then screened 124 pairs of ICC and nontumor samples for these mutations, including 7 exomes. We compared mutations in PTPN3 with tumor recurrence in 124 patients and PTPN3 expression levels with recurrence in 322 patients (the combination of both in 86 patients). The functional effects of PTPN3 variations were determined by RNA interference and transgenic expression in cholangiocarcinoma cell lines (RBE, HCCC-9810, and Huh28). RESULTS: Based on exome sequencing, pathways that regulate protein phosphorylation were among the most frequently altered in ICC samples and genes encoding protein tyrosine phosphatases (PTPs) were among the most frequently mutated. We identified mutations in 9 genes encoding PTPs in 4 of 7 ICC exomes. In the prevalence screen of 124 paired samples, 51.6% of ICCs contained somatic mutations in at least 1 of 9 PTP genes; 41.1% had mutations in PTPN3. Transgenic expression of PTPN3 in cell lines increased cell proliferation, colony formation, and migration. PTPN3(L232R) and PTPN3(L384H), which were frequently detected in ICC samples, were found to be gain-of-function mutations; their expression in cell lines further increased cell proliferation, colony formation, and migration. ICC-associated variants of PTPN3 altered phosphatase activity. Patients whose tumors contained activating mutations or higher levels of PTPN3 protein than nontumor tissues had higher rates of disease recurrence than patients whose tumors did not have these characteristics. CONCLUSIONS: Using whole exome sequencing of ICC samples from patients, we found that more than 40% contain somatic mutations in PTPN3. Activating mutations in and high expression levels of PTPN3 were associated with tumor recurrence.

Heme oxygenase-1 induces 15-lipoxygenase expression during hypoxia-induced pulmonary hypertension.

We previously reported that 15-lipoxygenase (15-LO) induced by hypoxia catalyzed the conversion of arachidonic acid (AA) into 15-hydroxyeicosatetraenoic acid (15-HETE), which plays an essential role in the development of hypoxic pulmonary arterial hypertension (HPH). However, the mechanisms by which hypoxia up-regulated 15-LO are still unclear. Heme oxygenase-1 (HO-1), an oxygen-dependent enzyme regulating vascular tone and cell proliferation, was implicated in HPH and was promoted by hypoxia. Therefore, the present study was carried out to determine whether hypoxia induced the expression of 15-LO via the HO-1 pathway. To test this hypothesis, we studied the role of HO-1 in HPH and 15-LO/15-HETE expression We found increased right ventricular systolic pressure and pulmonary arteries (PAs) reactivity to vasoconstrictors as well as intima-to-media ratio of PAs in HO-1 overexpressing transgenic mice. Moreover, HO-1 up-regulated 15-LO transcription and translation as well as 15-HETE in both transgenic mice and cultured pulmonary arterial smooth muscle cells (PASMCs). Results from immunoprecipitation and immunocytochemistry showed the interaction and colocalization of HO-1 and 15-LO. Together, these data suggest that HO-1 is an important upstream mediator in the hypoxia-induced 15-LO up-regulation during HPH. Unveiling the relevance of HO-1 signaling in PHP provides attractive treatment targets for HPH.

Cellular mechanisms and intracellular signaling pathways for the modulation of eNOS in pulmonary arteries by 15-HETE.

The 15-hydroxyeicosatetraenoic acid (15-HETE), a lipid metabolite and vasoconstrictor, plays an important role in hypoxic contraction of pulmonary arteries (PAs) through working on smooth muscle cells (SMCs). Previous studies have shown that vascular endothelium is also involved in PAs tone regulation. However, little is known as to how the pulmonary artery endothelial cells (PAECs) are related to the 15-HETE-induced vasoconstriction and that which intracellular signaling systems are critical. To test this hypothesis, we examined PAs constriction in isolated rat PAs rings, the expression and activity of endothelial nitric oxide synthase (eNOS) with western blot, and nitric oxide (NO) production using the DAF-FM DA fluorescent indicator. The results showed that the 15-HETE-induced PAs constriction was diminished in endothelium-intact rings. In the presence of the eNOS inhibitor L-NAME, vasoconstrictor responses to KCl were greater than the control. The activation of eNOS was activated by Ca²âº released from intracellular stores and the PI3K/Akt pathway. Phosphorylations of the eNOS at Ser-1177 and Akt at Ser-473 were necessary for their activity. A prolonged 15-HETE treatment (30 min) led to a decrease in NO production by phosphorylation of eNOS at Thr-495, leading to augmentation of PAs constriction. Therefore, 15-HETE initially inhibited the PAs constriction through the endothelial NO system, and both Ca²âº and the PI3K/Akt signaling systems are required for the effects of 15-HETE on PAs tone regulation.

The microRNA-328 regulates hypoxic pulmonary hypertension by targeting at insulin growth factor 1 receptor and L-type calcium channel-α1C.

Chronic hypoxia is the most common cause of secondary pulmonary hypertension, for which the mechanisms are still unclear. Recent studies implicated an important role for microRNAs (miRNAs) in hypoxia-mediated responses in various cellular processes, including cell apoptosis and proliferation. Therefore, we hypothesized that these regulatory molecules might be implicated in the etiology of hypoxic pulmonary hypertension. Here we show that miRNA-328, a posttranscriptional regulator, was drastically downregulated in the pulmonary artery (PA) after a hypoxic assault. PA rings, Western blot, quantitative real-time PCR, in situ hybridization, and luciferase assay were used to investigate the role of miRNA-328 in hypoxic pulmonary hypertension. We found that hypoxia produced a significant inhibition of miRNA-328 expression, which was involved in PA vasoconstriction and remodeling. Overexpressing miRNA-328 in the transgenic mice remarkably decreased the right ventricular systolic pressure and PA wall thickness under both normoxia and hypoxia. MiRNA-328 inhibited L-type calcium channel-α1C expression through a miRNA-328 binding site within the 3' untranslational region of L-type calcium channel-α1C. The L-type calcium channel-α1C inhibition attenuated the PA response to KCl. Furthermore, miRNA-328 suppressed the insulin growth factor 1 receptor, ultimately leading to apoptosis of pulmonary arterial smooth muscle cells. The posttranscriptional repression of L-type calcium channel-α1C and insulin growth factor 1 receptor was further confirmed by luciferase reporter assay. These results showed that miRNA-328, an important protecting factor, plays a significant role in PA constriction and remodeling by regulating multiple gene targets in hypoxic pulmonary hypertension.

15-Hydroxyeicosatetraenoic acid (15-HETE) protects pulmonary artery smooth muscle cells from apoptosis via inducible nitric oxide synthase (iNOS) pathway.

15-Hydroxyeicosatetraenoic acid (15-HETE), one of many important metabolic products of arachidonic acid (AA) catalyzed by 15-lipoxygenase, plays an important role in pulmonary vascular smooth muscle remodeling. We have previously shown its unsubstituted effects on the apoptotic responses of pulmonary artery smooth muscle cells (PASMCs), but the underlying mechanisms are still poorly manifested. Previous studies have shown that inducible nitric oxide synthase (iNOS) plays an important protective role against sepsis-induced pulmonary apoptosis. Therefore, the purpose of this study is to determine whether 15-HETE anti-apoptotic process is mediated through the iNOS pathway in rat PASMCs. To test this hypothesis, we studied the contribution of iNOS to the 15-HETE induced anti-apoptotic responses using cell viability measurement, Western blot, mitochondrial potential analysis, nuclear morphology determination and TUNEL assay. Our results showed that both exogenous and endogenous 15-HETE up-regulated iNOS protein and mRNA expression and 15-HETE enhanced the cell survival, attenuated mitochondrial depolarization, up-regulated the expression of Bcl-2 and procaspase-3 in PASMCs under serum-deprived condition. These effects were reversed by iNOS inhibitor SMT or l-canavanine. Taken together, our data indicates that iNOS is a novel signaling transduction pathway, which is necessary for the effects of 15-HETE in protection PASMCs from apoptosis and may be an important mechanism underlying the treatment of pulmonary artery hypertension and also provides a novel therapeutic insight in future.

Hypoxia suppresses Kv 2.1 channel expression through endogenous 15-hydroxyeicosatetraenoic acid in rat pulmonary artery.

We have previously reported that hypoxia activates lung 15-lipoxygenase (15-LOX), which catalyzes arachidonic acid to produce 15-HETE, leading to constriction of neonatal rabbit pulmonary arteries. Hypoxia suppresses Kv2.1 channel expression. Although the Kv channel inhibition by hypoxia is likely to be mediated through 15-HETE, direct evidence is still lacking. To explore whether 15-LOX/15-HETE pathway contributes to the hypoxia-induced down-regulation of Kv2.1 channel, we performed studies using 15-LOX blockers, semi-quantitative PCR and western blot analysis. We found that Kv2.1 channel expression at the mRNA and protein levels was greatly up-regulated in pulmonary arterial smooth muscle cells (PASMCs) and pulmonary artery (PA) after blockade of endogenous 15-HETE under hypoxic condition. 15-HETE further decreased Kv2.1 channel expression in comparison with 12-HETE and 5-HETE in cultured PASMCs and PA under normoxic conditions. These data indicate that hypoxia suppresses Kv2.1 channel expression through endogenous 15-HETE in PA.

Role of the G-protein and tyrosine kinase--Rho/ROK pathways in 15-hydroxyeicosatetraenoic acid induced pulmonary vasoconstriction in hypoxic rats.

It has been previously reported by us that hypoxia activates lung 15-lipoxygenase (15-LO), which catalyzes arachidonic acid to 15-hydroxyeicosatetraenoic acid (15-HETE), leading to the constriction of pulmonary artery (PA). Rho-associated serine/threonine kinase (ROK), a downstream effector of small GTPase RhoA that may be modulated by G-protein and tyrosine kinase, plays an important role in smooth muscle contraction. However, whether the 15-HETE induced PA vasoconstriction involves the Rho/ROK pathway remains to be demonstrated. Therefore, we studied the contribution of ROK as well as G-protein and tyrosine kinase to the 15-HETE induced pulmonary vasoconstriction using PA ring technique, RNA interference technology, RP-HPLC, western blot and RT-PCR combined with the blockers. The hypoxia-induced expression of ROK is regulated by 15-HETE in rat PA smooth muscle cells (PASMCs), leading to vasoconstriction. The up-regulation of ROK expression caused by 15-HETE appears to be mediated by the G-protein and tyrosine kinase pathways. The translocation of ROK2 from the nucleus to the cytoplasm during hypoxia exposure relies on the mechanism for 15-HETE production. These results suggest that 15-HETE may mediate the up-regulation of ROK expression through G-protein and tyrosine kinase pathways under hypoxic condition, leading to PA vasoconstriction.

15-HETE protects rat pulmonary arterial smooth muscle cells from apoptosis via the PI3K/Akt pathway.

15-Hydroxyeicosatetraenoic acid (15-HETE), a metabolic product of arachidonic acid (AA), plays an important role in pulmonary vascular smooth muscle remodeling. Although its effects on the apoptotic responses are known, the underlying mechanisms are still poorly understood. Since Akt is a critical regulator of cell survival and vascular remodeling, there may be a crosstalk between 15-HETE anti-apoptotic process and PI3K/Akt survival effect in rat pulmonary arterial smooth muscle cells (PASMCs). To test this hypothesis, we studied the effect of 15-HETE on cell survival and apoptosis using Western blot, cell viability measurement, nuclear morphology determination, TUNEL assay and mitochondrial potential analysis. We found that activation of the PI3K/Akt signaling system was necessary for the 15-HETE to suppress PASMC apoptosis and improve cell survival. Our results indicated that 15-HETE inhibited the apoptotic responses of PASMCs, including morphological alterations, mitochondrial depolarization and the expression apoptosis-specific proteins. These effects were likely to be mediated through the activation of PI3K/Akt. Two downstream signal molecules of PI3K/Akt were identified. Both FasL and Bad were down-regulated by 15-HETE and 15-HETE phosphorylated Bad. These changes depended on the PI3K/Akt signaling pathway in PASMCs. Thus a signal transduction pathway was demonstrated which is necessary for the effects of 15-HETE in protection PASMCs from apoptosis.

Role of protein kinase C in 15-HETE-induced hypoxic pulmonary vasoconstriction.

The aim of the present study was to investigate the roles of protein kinase C (PKC) signal transduction pathway in the 15-hydroxyeicosatetraenoic acid (15-HETE)-induced down-regulation expression of K(V) 1.5, K(V) 2.1 and K(V) 3.4, and pulmonary vasoconstriction under hypoxia. Tension measurements on rat pulmonary artery (PA) rings, Western blots, semi-quantitative PCR and whole-cell patch-clamp technique were employed to investigate the effects of 15-HETE on PKC and K(V) channels. Hypericin (6.8 micromol/L), a PKC inhibitor, significantly attenuated the constriction of PA rings to 15-HETE under hypoxia. The down-regulation of K(V) 1.5, K(V) 2.1 and K(V) 3.4 channel expression and inhibition of whole-cell K currents (I(K)(V)) induced by 15-HETE were rescued and restored, respectively, by hypericin. These studies indicate that the PKC signal transduction pathway is involved in 15-HETE-induced rat pulmonary vasoconstriction under hypoxia. 15-HETE suppresses the expression of K(V) 1.5, K(V) 2.1 and K(V) 3.4 channels and inhibits I(K)(V) through the PKC signaling pathway in pulmonary arterial smooth muscle cells.