Characteristics and Outcomes of Obstetric Sepsis by Three Sets of Clinical Diagnostic Criteria: A Retrospective Study.

OBJECTIVE: Pregnancy-related mortality in the United States is the highest of all developed nations with a reported rate of 17 deaths per 100,000 live births in 2014 to 2017. Sepsis-related mortality is a major component of pregnancy-related mortality. Similar to nonpregnancy-related sepsis, the criteria for pregnancy-related sepsis are evolving. The purposes of this study were to compare three criteria for sepsis (Sepsis-2, Sepsis-3, California Maternal Quality Care Collaborative [CMQCC]) with one another and to determine patient outcomes using those three sets of criteria. STUDY DESIGN: Using the electronic medical record, we obtained granular data on all patients at University of Michigan Medical Center from July 10, 2009 to September 4, 2019 with suspected sepsis (blood cultures and administration of antibiotics) during pregnancy until the 42nd postpartum day. Agreement between the three criteria were assessed with kappa and shown by a Venn diagram. Groups were compared using standardized differences and chi square, rank sum, or independent t-tests. RESULTS: Of the 228 patients having sepsis by any criteria, 191 (83%) patients met the criteria for Sepsis-2, 131 (57%) for Sepsis-3, and 62 (27%) met criteria according to CMQCC. Agreement between the three criteria ranged from kappa = 0.13 (95% confidence interval [CI]: 0.09, 0.18) to kappa = 0.31 (95% CI: 0.23, 0.39). Patients who met CMQCC criteria tended to have more comorbidities and higher APACHE II (Acute Physiology And Chronic Health Evaluation) scores. Mortality (by 90 days) among the groups was low with 10 (4%) patients dying. Patients meeting criteria for CMQCC sepsis had higher mortality than the non-CMQCC patients with sepsis (10 vs. 2%, standardized difference = 0.31, p = 0.027). CONCLUSION: The agreement among Sepsis-2, Sepsis-3, and CMQCC diagnostic criteria is weak. CMQCC criteria identifies patients with sepsis at higher risk of death. KEY POINTS: · Agreements (kappa) between the three criteria are poor.. · CMQCC sepsis patients have more comorbidities.. · Endometritis was the most common cause of sepsis.. · CMQCC mortality was 10%; non-CMQCC mortality was 2%..

MicroRNA-mediated downregulation of K+ channels in 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 (KV1.5/KCNA5) in normal PASMC. Inhibition of miR-29b in IPAH-PASMC completely recovered K+ channel function and KV1.5 expression, while miR-138 and miR-222 had a partial or no effect. Luciferase assays further revealed that KV1.5 is a direct target of miR-29b. Additionally, overexpression of miR-29b in normal PASMC decreased large-conductance Ca2+-activated K+ (BKCa) channel currents and downregulated BKCa channel ß1 subunit (BKCaß1 or KCNMB1) expression, while inhibition of miR-29b in IPAH-PASMC increased BKCa channel activity and BKCaß1 levels. These data indicate upregulated miR-29b contributes at least partially to the attenuated function and expression of KV and BKCa channels in PASMC from patients with IPAH.

c-Jun NH2-teminal kinase 1 interacts with vitamin D receptor and affects vitamin D-mediated inhibition of cancer cell proliferation.

BACKGROUND: Vitamin D is a chemopreventive agent that acts against colorectal carcinogenesis in vivo and in vitro through vitamin D receptor (VDR). Previous studies showed that stress-activated protein kinase JNKs (c-Jun NH2-terminal kinases) and p38 cooperated to activate VDR and increase vitamin D3-dependent growth inhibition in breast cancer cells. This study is to determine whether vitamin D-mediated inhibition of cell proliferation is associated with JNK1 in colorectal cancer cells. METHODS AND RESULTS: Human colon cancer cells were treated with calcitriol, an active vitamin D3. The results showed that calcitriol significantly inhibited cell proliferation and caused cell cycle arrest in HT29 cells, which was associated with induction of phosphorylated JNK1 (p-JNK). The induction of VDR and p-JNK by calcitriol was also observed in Caco-2 cells. Furthermore, VDR expression was significantly downregulated in JNK1-/- mouse intestinal epithelial cells, and VDR reporter activity was reduced in JNK1-/- mouse embryonic fibroblasts (MEFs). However, increasing activated JNK1 upregulated VDR expression and transcriptional activity in vitro. Moreover, JNK1 co-localized with VDR in nuclei and cytoplasm and physically bound together. Reduced expression of JNK1 and VDR in HT29 and Caco-2 cells and JNK1 absence in JNK1-/- MEFs attenuated calcitriol-mediated inhibition of cell proliferation. CONCLUSION: JNK1 physically and functionally interacted with VDR and positively regulated VDR expression at transcriptional and translational levels, which influenced calcitriol-mediated inhibition of cancer cell proliferation.

Pathogenic role of calcium-sensing receptors in the development and progression of pulmonary hypertension.

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.

Notch Activation of Ca(2+) Signaling in the Development of Hypoxic Pulmonary Vasoconstriction and Pulmonary 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.

Activation of Notch signaling by short-term treatment with Jagged-1 enhances store-operated Ca(2+) entry in human pulmonary arterial smooth muscle cells.

Notch signaling plays a critical role in controlling proliferation and differentiation of pulmonary arterial smooth muscle cells (PASMC). Upregulated Notch ligands and Notch3 receptors in PASMC have been reported to promote the development of pulmonary vascular remodeling in patients with pulmonary arterial hypertension (PAH) and in animals with experimental pulmonary hypertension. Activation of Notch receptors by their ligands leads to the cleavage of the Notch intracellular domain (NICD) to the cytosol by γ-secretase; NICD then translocates into the nucleus to regulate gene transcription. In this study, we examined whether short-term activation of Notch functionally regulates store-operated Ca(2+) entry (SOCE) in human PASMC. Treatment of PASMC with the active fragment of human Jagged-1 protein (Jag-1) for 15-60 min significantly increased the amplitude of SOCE induced by passive deletion of Ca(2+) from the intracellular stores, the sarcoplasmic reticulum (SR). The Jag-1-induced enhancement of SOCE was time dependent: the amplitude was maximized at 30 min of treatment with Jag-1, which was closely correlated with the time course of Jag-1-mediated increase in NICD protein level. The scrambled peptide of Jag-1 active fragment had no effect on SOCE. Inhibition of γ-secretase by N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT) significantly attenuated the Jag-1-induced augmentation of SOCE. In addition to the short-term effect, prolonged treatment of PASMC with Jag-1 for 48 h also markedly enhanced the amplitude of SOCE. These data demonstrate that short-term activation of Notch signaling enhances SOCE in PASMC; the NICD-mediated functional interaction with store-operated Ca(2+) channels (SOC) may be involved in the Jag-1-mediated enhancement of SOCE in human PASMC.

A sequence variant in the phospholipase C epsilon C2 domain is associated with esophageal carcinoma and esophagitis.

A single-nucleotide polymorphism (rs2274223: A5780G:His1927Arg) in the phospholipase C epsilon gene (PLCϵ) was recently identified as a susceptibility locus for esophageal cancer in Chinese subjects. To determine the underlying mechanisms of PLCϵ and this SNP in esophageal carcinogenesis, we analyzed PLCϵ genotypes, expression, and their correlation in esophageal cancer cell lines, non-transformed esophageal cells, 58 esophageal squamous cell carcinomas and 10,614 non-cancer subjects from China. We found that the G allele (AG or GG) was associated with increased PLCϵ mRNA and protein expression in esophageal cancer tissues and in esophageal cancer cell lines. G allele was also associated with higher enzyme activity, which might be associated with increased protein expression. Quantitative analysis of the C2 domain sequences revealed that A:G allelic imbalance was strongly linked to esophageal malignancy. Moreover, the analysis of 10,614 non-cancer subjects demonstrated that the G allele was strongly associated with moderate to severe esophagitis in the subjects from the high-incidence areas of China (OR 6.03, 95% CI 1.59-22.9 in high-incidence area vs. OR 0.74, 95% CI 0.33-1.64 in low-incidence area; P = 0.008). In conclusion, the PLCϵ gene, particularly the 5780G allele, might play a pivotal role in esophageal carcinogenesis via upregulating PLCϵ mRNA, protein, and enzyme activity, and augmenting inflammatory process in esophageal epithelium. Thus, 5780G allele may constitute a promising biomarker for esophageal squamous cell carcinoma risk stratification, early detection, and progression prediction.

Dihydropyridine Ca(2+) channel blockers increase cytosolic [Ca(2+)] by activating Ca(2+)-sensing receptors in pulmonary arterial smooth muscle cells.

RATIONALE: 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 an important stimulus for PASMC proliferation and pulmonary vascular remodeling. The dihydropyridine Ca(2+) channel blockers, such as nifedipine, have been used for treatment of idiopathic pulmonary arterial hypertension (IPAH). OBJECTIVE: Our previous study demonstrated that the Ca(2+)-sensing receptor (CaSR) was upregulated and the extracellular Ca(2+)-induced increase in [Ca(2+)](cyt) was enhanced in PASMC from patients with IPAH and animals with experimental pulmonary hypertension. Here, we report that the dihydropyridines (eg, nifedipine) increase [Ca(2+)](cyt) 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 [Ca(2+)](cyt) 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 [Ca(2+)](cyt), whereas overexpression of CaSR in normal PASMC conferred the nifedipine-induced rise in [Ca(2+)](cyt). Other dihydropyridines, nicardipine and Bay K8644, had similar augmenting effects on the CaSR-mediated increase in [Ca(2+)](cyt) in IPAH-PASMC; however, the nondihydropyridine blockers, such as diltiazem and verapamil, had no effect on the CaSR-mediated rise in [Ca(2+)](cyt). CONCLUSIONS: The dihydropyridine derivatives increase [Ca(2+)](cyt) by potentiating the activity of CaSR in PASMC independently of their blocking (or activating) effect on Ca(2+) channels; therefore, it is possible that the use of dihydropyridine Ca(2+) channel blockers (eg, nifedipine) to treat IPAH patients with upregulated CaSR in PASMC may exacerbate pulmonary hypertension.

Selenium and sulindac are synergistic to inhibit intestinal tumorigenesis in Apc/p21 mice.

BACKGROUND: Both selenium and non-steroidal anti-inflammatory drug (NSAID) sulindac are effective in cancer prevention, but their effects are affected by several factors including epigenetic alterations and gene expression. The current study was designed to determine the effects of the combination of selenium and sulindac on tumor inhibition and the underlying mechanisms. RESULTS: We fed the intestinal tumor model Apc/p21 mice with selenium- and sulindac-supplemented diet for 24 weeks, and found that the combination of selenium and sulindac significantly inhibited intestinal tumorigenesis, in terms of reducing tumor incidence by 52% and tumor multiplicities by 80% (p<0.01). Mechanistic studies revealed that the combination of selenium and sulindac led to the significant induction of the expression of p27 and p53 and JNK1 phosphorylation, and led to the suppression of ß-catenin and its downstream targets. Impressively, the data also showed that demythelation on p21 promoter was associated with tumor inhibition by the combination of selenium and sulindac. CONCLUSIONS: The selenium is synergistic with sulindac to exert maximal effects on tumor inhibition. This finding provides an important chemopreventive strategy using combination of anti-cancer agents, which has a great impact on cancer prevention and has a promising translational potential.

Enhanced Ca(2+)-sensing receptor function in idiopathic pulmonary arterial hypertension.

RATIONALE: 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). OBJECTIVE: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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.

Decorin-mediated inhibition of colorectal cancer growth and migration is associated with E-cadherin in vitro and in mice.

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, p27(kip1) and E-cadherin and an upregulation of ß-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.

Loss of JNK2 increases intestinal tumor susceptibility in Apc1638+/- mice with dietary modulation.

A recent study has shown that c-Jun NH2-terminal kinases (JNKs) 2 interacts with and inhibits ß-catenin signaling in vitro. To determine the role of genetic interaction between JNK2 and ß-catenin in vivo and to elucidate JNK2-mediated intestinal carcinogenesis, we crossed the JNK2-/- mice with Apc1638+/- mice that carry inactivated Apc allele and develop intestinal tumor due to ß-catenin activation. We found that the introduction of mutant JNK2 into Apc1638+/- mice did not increase intestinal tumorigenesis when the mice were fed a defined AIN-76A control diet. However, loss of JNK2 significantly increased animal body weight in the Apc/JNK2+/- and Apc/JNK2-/- mice. Surprisingly, JNK2 loss was synergistic with a Western-style high-risk diet (high fat and phosphate and low calcium and vitamin D) to accelerate intestinal tumorigenesis. Tumor number increased to 3.56 from 1.89 (on AIN-76A diet) in the Apc/JNK2+/- mice (P<0.01) and increased to 4.14 from 1.92 (on AIN-76A diet) in the Apc/JNK2-/- mice (P<0.01) although there was a slight increase of tumor formation in Apc/JNK2+/+ mice. Intestinal tumorigenesis in Apc/JNK2 double-mutant mice with high-risk diet modulation was associated with ß-catenin signaling, peroxisome proliferator-activated receptor-γ and inflammation pathway. Collectively, we concluded that JNK2 may function in controlling fat metabolism and loss of JNK2 increases the risk of obesity, the latter synergizes with high-fat diet to increase intestinal tumor susceptibility. This data strongly suggests the importance of JNK2 in intestinal carcinogenesis and the importance of dietary manipulation for cancer prevention in the population whose JNK2 is inactivated.

Spectral and thermodynamic properties of methanobactin from γ-proteobacterial methane oxidizing bacteria: a case for copper competition on a molecular level.

Methanobactin (mb) is a low molecular mass copper-binding molecule analogous to iron-binding siderophores. The molecule is produced by many methanotrophic or methane oxidizing bacteria (MOB), but has only been characterized to date in one MOB, Methylosinus trichosporium OB3b. To explore the potential molecular diversity in this novel class of metal binding compound, the spectral (UV-visible, fluorescent, and electron paramagnetic resonance) and thermodynamic properties of mb from two γ-proteobacterial MOB, Methylococcus capsulatus Bath and Methylomicrobium album BG8, were determined and compared to the mb from the α-proteobacterial MOB, M. trichosporium OB3b. The mb from both γ-proteobacterial MOB differed from the mb from M. trichosporium OB3b in molecular mass and spectral properties. Compared to mb from M. trichosporium OB3b, the extracellular concentrations were low, as were copper-binding constants of mb from both γ-proteobacterial MOB. In addition, the mb from M. trichosporium OB3b removed Cu(I) from the mb of both γ-proteobacterial MOB. Taken together the results suggest mb may be a factor in regulating methanotrophic community structure in copper-limited environments.

Functional and physical interaction between the selenium-binding protein 1 (SBP1) and the glutathione peroxidase 1 selenoprotein.

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.

PTPH1 dephosphorylates and cooperates with p38gamma MAPK to increase ras oncogenesis through PDZ-mediated interaction.

Protein phosphatases are believed to coordinate with kinases to execute biological functions, but examples of such integrated activities, however, are still missing. In this report, we have identified protein tyrosine phosphatase H1 (PTPH1) as a specific phosphatase for p38gamma mitogen-activated protein kinase (MAPK) and shown their cooperative oncogenic activity through direct binding. p38gamma, a Ras effector known to act independent of its phosphorylation, was first shown to require its unique PDZ-binding motif to increase Ras transformation. Yeast two-hybrid screening and in vitro and in vivo analyses further identified PTPH1 as a specific p38gamma phosphatase through PDZ-mediated binding. Additional experiments showed that PTPH1 itself plays a role in Ras-dependent malignant growth in vitro and/or in mice by a mechanism depending on its p38gamma-binding activity. Moreover, Ras increases both p38gamma and PTPH1 protein expression and there is a coupling of increased p38gamma and PTPH1 protein expression in primary colon cancer tissues. These results reveal a coordinative oncogenic activity of a MAPK with its specific phosphatase and suggest that PDZ-mediated p38gamma/PTPH1 complex may be a novel target for Ras-dependent malignancies.

Transcriptional regulation and biological functions of selenium-binding protein 1 in colorectal cancer in vitro and in nude mouse xenografts.

BACKGROUND: 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. METHODOLOGY AND PRINCIPAL FINDINGS: 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. CONCLUSION AND SIGNIFICANCE: These data demonstrate that SBP1 has tumor suppressor functions that are inhibited in colorectal cancer through epigenetic silencing.

p38alpha antagonizes p38gamma activity through c-Jun-dependent ubiquitin-proteasome pathways in regulating Ras transformation and stress response.

p38 MAPK family consists of four isoform proteins (alpha, beta, gamma, and delta) that are activated by the same stimuli, but the information about how these proteins act together to yield a biological response is missing. Here we show a feed-forward mechanism by which p38alpha may regulate Ras transformation and stress response through depleting its family member p38gamma protein via c-Jun-dependent ubiquitin-proteasome pathways. Analyses of MAPK kinase 6 (MKK6)-p38 fusion proteins showed that constitutively active p38alpha (MKK6-p38alpha) and p38gamma (MKK6-p38gamma) stimulates and inhibits c-Jun phosphorylation respectively, leading to a distinct AP-1 regulation. Depending on cell type and/or stimuli, p38alpha phosphorylation results in either Ras-transformation inhibition or a cell-death escalation that invariably couples with a decrease in p38gamma protein expression. p38gamma, on the other hand, increases Ras-dependent growth or inhibits stress induced cell-death independent of phosphorylation. In cells expressing both proteins, p38alpha phosphorylation decreases p38gamma protein expression, whereas its inhibition increases cellular p38gamma concentrations, indicating an active role of p38alpha phosphorylation in negatively regulating p38gamma protein expression. Mechanistic analyses show that p38alpha requires c-Jun activation to deplete p38gamma proteins by ubiquitin-proteasome pathways. These results suggest that p38alpha may, upon phosphorylation, act as a gatekeeper of the p38 MAPK family to yield a coordinative biological response through disrupting its antagonistic p38gamma family protein.

Stress-induced c-Jun-dependent Vitamin D receptor (VDR) activation dissects the non-classical VDR pathway from the classical VDR activity.

Vitamin D receptor (VDR) is a ligand-dependent transcription factor that mediates vitamin D(3)-induced gene expression. Our previous work has established that stress MAPK signaling stimulates VDR expression (Qi, X., Pramank, R., Wang, J., Schultz, R. M., Maitra, R. K., Han, J., DeLuca, H. F., and Chen, G. (2002) J. Biol. Chem. 277, 25884-25892) and VDR inhibits cell death in response to p38 MAPK activation (Qi, X., Tang, J., Pramanik, R., Schultz, R. M., Shirasawa, S., Sasazuki, T., Han, J., and Chen, G. (2004) J. Biol. Chem. 279, 22138-22144). Here we show that c-Jun is essential for VDR expression and VDR in turn inhibits c-Jun-dependent cell death by non-classical mechanisms. In response to stress c-Jun is recruited to the Vdr promoter before VDR protein expression is induced. The necessary and sufficient role of c-Jun in VDR expression was established by the fact that c-Jun knock-out decreases VDR expression, whereas c-Jun restoration recovers its activity. Existence of the non-classical VDR pathway was suggested by a requirement of both c-Jun and VDR in stress-induced VDR activity and further demonstrated by VDR inhibiting c-Jun-dependent cell death independent of its classical transcriptional activity and independent of vitamin D(3). c-Jun is also required for vitamin D(3)-induced classical VDR transcriptional activity by a mechanism likely involving physical interactions between c-Jun and VDR proteins. These results together reveal a non-classical mechanism by which VDR acts as a c-Jun/AP-1 target gene to modify c-Jun activity in stress response through increased protein expression independent of classical transcriptional regulations.

p38gamma mitogen-activated protein kinase integrates signaling crosstalk between Ras and estrogen receptor to increase breast cancer invasion.

Ras is believed to stimulate invasion and growth by different effector pathways, and yet, the existence of such effectors under physiologic conditions has not been shown. Estrogen receptor (ER), on the other hand, is both anti-invasive and proliferative in human breast cancer, with mechanisms for these paradoxical actions remaining largely unknown. Our previous work showed an essential role of p38gamma mitogen-activated protein kinase in Ras transformation in rat intestinal epithelial cells, and here, we show that p38gamma integrates invasive antagonism between Ras and ER to increase human breast cancer invasion without affecting their proliferative activity. Ras positively regulates p38gamma expression, and p38gamma in turn mediates Ras nonmitogenic signaling to increase invasion. Expression of the Ras/p38gamma axis, however, is trans-suppressed by ER that inhibits invasion and stimulates growth also by distinct mechanisms. Analysis of ER and its cytoplasmic localized mutant reveals that ER additionally binds to p38gamma protein, leading to its specific down-regulation in the nuclear compartment. A p38gamma-antagonistic activity of ER was further shown in a panel of breast cancer cell lines and was shown independent of estrogens by both ER depletion and ER expression. These results revealed that both Ras and ER use distinct pathways to regulate breast cancer growth and invasion, and that p38gamma specifically integrates their antagonistic activity to stimulate cell invasion. Selective targeting of p38gamma-dependent invasion pathways may be a novel strategy to control breast cancer progression.