Search

Background Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) produces the coronavirus disease of 2019 (COVID-19), primarily presenting with respiratory symptoms, including cough, shortness of breath, etc. Respiratory failure can present similarly to a COVID-19 infection, and COVID-19 infection can cause respiratory failure. Thus, it is important to study respiratory failure, COVID-19, and the interaction between the two in hopes of improving patient outcomes. In this study, we compared mortality rates in patients admitted with COVID-19, respiratory failure, or both. Mortality rates in our study populations were further scrutinized based on patient age. Materials and methods Respiratory failure and COVID-19 data were collected via the electronic medical records system at Freeman Health System, a 410-bed, rural hospital, in Neosho and Joplin, Missouri, from April 2020 through December 2021. The patient population included all patients admitted to the hospital with a diagnosis of COVID-19 or respiratory failure, as defined by the International Classification of Disease, Tenth Revision (ICD-10). Patients with or without COVID-19, with or without respiratory failure, and patients with respiratory failure with COVID-19 were included. Results There was a significant increase in mortality (17.28%) in patients with COVID-19 and respiratory failure (P1) compared to patients with COVID-19 who did not have respiratory failure (P2). No significance was found when comparing patients with COVID-19 and respiratory failure (P1) and patients with respiratory failure without COVID-19 (P3) (p value=0.4921). In contrast, when divided based on age, we found a significant increase in mortality in patients 65 and older with COVID-19 and respiratory failure compared to patients 65 and older with respiratory failure who did not have COVID-19 (P5). There were no significant mortality increases in other comparisons. Conclusion When comparing patient populations within the Freeman Health System, patients with COVID-19 and respiratory failure had similar mortality rates as those with respiratory failure without COVID-19, while patients with only COVID-19 had a markedly reduced mortality rate, relatively. The higher mortality rates in patients with only respiratory failure when compared to patients with both respiratory failure and COVID-19 indicate that the presence of respiratory failure likely plays a bigger role in the inflammatory response that reduces one's chance of survival in this setting. Furthermore, age was shown to be a significant risk factor as patients aged 65 and older showed a greater mortality rate when patients had both COVID-19 and respiratory failure compared to patients with both conditions below the age of 65. The decrease in immune response that results in older patients is likely the largest contributing factor along with the increased likelihood of patients in this population also having more comorbidities, further decreasing the chance of survival. Future studies can investigate alternate treatment plans for patients aged 65 and older who are at higher risk of mortality with COVID-19 and respiratory failure., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2023, Kneller et al.)

Neurodegeneration is characterized by loss of neurons causing changes that lead individuals to debilitating conditions; the most common of this condition is the Alzheimer's disease. It has been related that enriched environment (EE) induces experience‑dependent plasticity mechanisms, improving the performance of the animals in learning and memory tests. This study evaluated the effects of EE on histological parameters of the cerebellum in rats that received intracerebroventricular streptozotocin. In the standard environment, streptozotocin (STZ) promoted a significant increase between the gaps in the Purkinje layer of approximately 20%. On the other hand, in an enriched environment, the control result (EE) was similar to the result under streptozotocin effect (STZEE). In the standard environment (SE) group a 26% significant reduction in Purkinje cell density was observed under STZ presence. By analyzing the results of the density of Purkinje cells under the effect of streptozotocin in a standard environment (STZSE) against the density of the layer of Purkinje cells also under the effect of streptozotocin in an enriched environment (STZEE), a significant reduction of approximately 76% in density was observed of Purkinje cells in standard environment (STZSE), the mean number of Purkinje cells in enriched environments was not reduced, despite of STZ. According to the results, treatment with STZ and exposure to EE did not change the cerebellum general morphology/cytoarchitecture, hence was no significant difference in the layers thickness. These facts demonstrate that the enriched environment appears to protect the Purkinje cells layer of cerebellum from possible degeneration.

Signal transduction pathways post-translationally regulating nucleotide metabolism remain largely unknown. Guanosine monophosphate reductase (GMPR) is a nucleotide metabolism enzyme that decreases GTP pools by converting GMP to IMP. We observed that phosphorylation of GMPR at Tyr267 is critical for its activity and found that this phosphorylation by ephrin receptor tyrosine kinase EPHA4 decreases GTP pools in cell protrusions and levels of GTP-bound RAC1. EPHs possess oncogenic and tumor-suppressor activities, although the mechanisms underlying switches between these two modes are poorly understood. We demonstrated that GMPR plays a key role in EPHA4-mediated RAC1 suppression. This supersedes GMPR-independent activation of RAC1 by EPHA4, resulting in a negative overall effect on melanoma cell invasion and tumorigenicity. Accordingly, EPHA4 levels increase during melanoma progression and inversely correlate with GMPR levels in individual melanoma tumors. Therefore, phosphorylation of GMPR at Tyr267 is a metabolic signal transduction switch controlling GTP biosynthesis and transformed phenotypes., Competing Interests: Declaration of interests The authors declare no competing financial interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Physiological changes in GTP levels in live cells have never been considered a regulatory step of RAC1 activation because intracellular GTP concentration (determined by chromatography or mass spectrometry) was shown to be substantially higher than the in vitro RAC1 GTP dissociation constant (RAC1-GTP Kd). Here, by combining genetically encoded GTP biosensors and a RAC1 activity biosensor, we demonstrated that GTP levels fluctuating around RAC1-GTP Kd correlated with changes in RAC1 activity in live cells. Furthermore, RAC1 co-localized in protrusions of invading cells with several guanylate metabolism enzymes, including rate-limiting inosine monophosphate dehydrogenase 2 (IMPDH2), which was partially due to direct RAC1-IMPDH2 interaction. Substitution of endogenous IMPDH2 with IMPDH2 mutants incapable of binding RAC1 did not affect total intracellular GTP levels but suppressed RAC1 activity. Targeting IMPDH2 away from the plasma membrane did not alter total intracellular GTP pools but decreased GTP levels in cell protrusions, RAC1 activity, and cell invasion. These data provide a mechanism of regulation of RAC1 activity by local GTP pools in live cells., (© 2021. The Author(s).)

Resistance to the proteasome inhibitor bortezomib (BTZ) represents a major obstacle in the treatment of multiple myeloma (MM). The contribution of lipid metabolism in the resistance of MM cells to BTZ is mostly unknown. Here we report that levels of fatty acid elongase 6 (ELOVL6) were lower in MM cells from BTZ-nonresponsive vs BTZ-responsive patients and in cultured MM cells selected for BTZ resistance compared with parental counterparts. Accordingly, depletion of ELOVL6 in parental MM cells suppressed BTZ-induced endoplasmic reticulum (ER) stress and cytotoxicity, whereas restoration of ELOVL6 levels in BTZ-resistant MM cells sensitized them to BTZ in tissue culture settings and, as xenografts, in a plasmacytoma mouse model. Furthermore, for the first time, we identified changes in the BTZ-induced lipidome between parental and BTZ-resistant MM cell lines underlying a functional difference in their response to BTZ. We demonstrated that restoration of ELOVL6 levels in BTZ-resistant MM cells resensitized them to BTZ largely via upregulation of ELOVL6-dependent ceramide species, which was a prerequisite for BTZ-induced ER stress and cell death in these cells. Our data characterize ELOVL6 as a major clinically relevant regulator of MM cell resistance to BTZ, which can emerge from the impaired ability of these cells to alter ceramide composition in response to BTZ., (© 2021 by The American Society of Hematology.)

Transforming growth factor (TGF)-β1, a main profibrogenic cytokine in the progression of idiopathic pulmonary fibrosis (IPF), induces differentiation of pulmonary fibroblasts to myofibroblasts that produce high levels of collagen, leading to concomitantly loss of lung elasticity and function. Recent studies implicate the importance of microRNAs (miRNAs) in IPF but their regulation and individual pathological roles remain largely unknown. We used both RNA sequencing and quantitative RT-PCR strategies to systematically study TGF-β1-induced alternations of miRNAs in human lung fibroblasts (HFL). Our data show that miR-133a was significantly upregulated by TGF-β1 in a time- and concentration-dependent manner. Surprisingly, miR-133a inhibits TGF-β1-induced myofibroblast differentiation whereas miR-133a inhibitor enhances TGF-β1-induced myofibroblast differentiation. Interestingly, quantitative proteomics analysis indicates that miR-133a attenuates myofibroblast differentiation via targeting multiple components of TGF-β1 profibrogenic pathways. Western blot analysis confirmed that miR-133a down-regulates TGF-β1-induced expression of classic myofibroblast differentiation markers such as ɑ-smooth muscle actin (ɑ-SMA), connective tissue growth factor (CTGF) and collagens. miRNA Target Searcher analysis and luciferase reporter assays indicate that TGF-β receptor 1, CTGF and collagen type 1-alpha1 (Col1a1) are direct targets of miR-133a. More importantly, miR-133a gene transferred into lung tissues ameliorated bleomycin-induced pulmonary fibrosis in mice. Together, our study identified TGF-β1-induced miR-133a as an anti-fibrotic factor. It functions as a feed-back negative regulator of TGF-β1 profibrogenic pathways. Thus, manipulations of miR-133a expression may provide a new therapeutic strategy to halt and perhaps even partially reverse the progression of IPF.

Although antioxidants promote melanoma metastasis, the role of reactive oxygen species (ROS) in other stages of melanoma progression is controversial. Moreover, genes regulating ROS have not been functionally characterized throughout the entire tumor progression in mouse models of cancer. To address this question, we crossed mice-bearing knock-out of Klf9, an ubiquitous transcriptional regulator of oxidative stress, with two conditional melanocytic mouse models: Braf CA mice, where Braf V600E causes premalignant melanocytic hyperplasia, and Braf CA /Pten -/- mice, where Braf V600E and loss of Pten induce primary melanomas and metastases. Klf9 deficiency inhibited premalignant melanocytic hyperplasia in Braf CA mice but did not affect formation and growth of Braf CA /Pten -/- primary melanomas. It also, as expected, promoted Braf CA /Pten -/- metastasis. Treatment with antioxidant N-acetyl cysteine phenocopied loss of Klf9 including suppression of melanocytic hyperplasia. We were interested in a different role of Klf9 in regulation of cell proliferation in Braf CA and Braf CA /Pten -/- melanocytic cells. Mechanistically, we demonstrated that BRAF V600E signaling transcriptionally upregulated KLF9 and that KLF9-dependent ROS were required for full-scale activation of ERK1/2 and induction of cell proliferation by BRAF V600E . PTEN depletion in BRAF V600E -melanocytes did not further activate ERK1/2 and cell proliferation, but rendered these phenotypes insensitive to KLF9 and ROS. Our data identified an essential role of KLF9-dependent ROS in BRAF V600E signaling in premalignant melanocytes, offered an explanation to variable role of ROS in premalignant and transformed melanocytic cells and suggested a novel mechanism for suppression of premalignant growth by topical antioxidants.

Dysregulation of microRNAs (miRNAs) contributes to epithelial-mesenchymal transition (EMT) of cancer, but the pathological roles of miRNAs in airway EMT of lung diseases remains largely unknown. We performed sequencing and real-time PCR analysis of the miRNA expression profile of human airway epithelial cells undergoing EMT, and revealed miR-133a to be one of the most common up-regulated miRNAs. MiR-133a was previously reported to be persistently up-regulated in airway epithelial cells of smokers. We found that mice exposed to cigarette smoke (CS) showed airway hyper-responsiveness, a typical symptom occurring in CS-related lung diseases, up-regulation of miR-133a and EMT marker protein N-cadherin in airway epithelium. Importantly, miR-133a overexpression induces airway epithelial cells to undergo spontaneous EMT via down-regulation of grainyhead-like 2 (GRHL2), an epithelial specific transcriptional factor. Loss of GRHL2 causes down-regulation of epithelial splicing regulatory protein 1 (ESRP1), a central coordinator of alternative splicing processes that are critical in the regulation of EMT. Down-regulation of ESRP1 induces isoform switching of adherens junction-associated protein p120-catenin, and leads to the loss of E-cadherin. Our study is the first to demonstrate that up-regulated miR-133a orchestrates airway EMT via alternative splicing processes, which points to novel therapeutic possibilities for the treatment of CS-related lung disease.

Transcription factor XBP1s, activated by endoplasmic reticulum (ER) stress in a dose-dependent manner, plays a central role in adaptive unfolded protein response (UPR) via direct activation of multiple genes controlling protein refolding. Here, we report that elevation of ER stress above a critical threshold causes accumulation of XBP1s protein sufficient for binding to the promoter and activation of a gene encoding a transcription factor KLF9. In comparison to other XBP1s targets, KLF9 promoter contains an evolutionary conserved lower-affinity binding site that requires higher amounts of XBP1s for activation. In turn, KLF9 induces expression of two regulators of ER calcium storage, TMEM38B and ITPR1, facilitating additional calcium release from ER, exacerbation of ER stress, and cell death. Accordingly, Klf9 deficiency attenuates tunicamycin-induced ER stress in mouse liver. These data reveal a role for XBP1s in cytotoxic UPR and provide insights into mechanisms of life-or-death decisions in cells under ER stress., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Polyamine inhibition for cancer therapy is, conceptually, an attractive approach but has yet to meet success in the clinical setting. The aryl hydrocarbon receptor (AHR) is the central transcriptional regulator of the xenobiotic response. Our study revealed that AHR also positively regulates intracellular polyamine production via direct transcriptional activation of 2 genes, ODC1 and AZIN1, which are involved in polyamine biosynthesis and control, respectively. In patients with multiple myeloma (MM), AHR levels were inversely correlated with survival, suggesting that AHR inhibition may be beneficial for the treatment of this disease. We identified clofazimine (CLF), an FDA-approved anti-leprosy drug, as a potent AHR antagonist and a suppressor of polyamine biosynthesis. Experiments in a transgenic model of MM (Vk*Myc mice) and in immunocompromised mice bearing MM cell xenografts revealed high efficacy of CLF comparable to that of bortezomib, a first-in-class proteasome inhibitor used for the treatment of MM. This study identifies a previously unrecognized regulatory axis between AHR and polyamine metabolism and reveals CLF as an inhibitor of AHR and a potentially clinically relevant anti-MM agent.

Oncogenic transcription factor FOXQ1 has been implicated in promotion of multiple transformed phenotypes in carcinoma cells. Recently, we have characterized FOXQ1 as a melanoma tumor suppressor that acts via repression of N-cadherin gene, and invasion and metastasis. Here we report that FOXQ1 induces differentiation in normal and transformed melanocytic cells at least partially via direct transcriptional activation of MITF gene, melanocytic lineage-specific regulator of differentiation. Importantly, we demonstrate that pigmentation induced in cultured melanocytic cells and in mice by activation of cAMP/CREB1 pathway depends in large part on FOXQ1. Moreover, our data reveal that FOXQ1 acts as a critical mediator of BRAF V600E -dependent regulation of MITF levels, thus providing a novel link between two major signal transduction pathways controlling MITF and differentiation in melanocytic cells.

Alveolar rhabdomyosarcoma (aRMS) is an aggressive subtype of the most common soft tissue cancer in children. A hallmark of aRMS tumors is incomplete myogenic differentiation despite expression of master myogenic regulators such as MyoD. We previously reported that histone methyltransferase KMT1A suppresses MyoD function to maintain an undifferentiated state in aRMS cells, and that loss of KMT1A is sufficient to induce differentiation and suppress malignant phenotypes in these cells. Here, we develop a chemical compound screening approach using MyoD-responsive luciferase reporter myoblast cells to identify compounds that alleviate suppression of MyoD-mediated differentiation by KMT1A. A screen of pharmacological compounds yielded the topoisomerase I (TOP1) poison camptothecin (CPT) as the strongest hit in our assay system. Furthermore, treatment of aRMS cells with clinically relevant CPT derivative irinotecan restores MyoD function, and myogenic differentiation in vitro and in a xenograft model. This differentiated phenotype was associated with downregulation of the KMT1A protein. Remarkably, loss of KMT1A in CPT-treated cells occurs independently of its well-known anti-TOP1 mechanism. We further demonstrate that CPT can directly inhibit KMT1A activity in vitro . Collectively, these findings uncover a novel function of CPT that downregulates KMT1A independently of CPT-mediated TOP1 inhibition and permits differentiation of aRMS cells., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Aposematic signals as well as body behaviours may be important anti-predator defences. Species of the genus Melanophryniscus are characterised by having toxic lipophilic alkaloids in the skin and for presenting a red ventral colouration, which can be observed when they perform the behaviour called the unken reflex. Both the reflex behaviour and the colouration pattern are described as defence mechanisms. However, there are currently no studies testing their effectiveness against predators. This study aimed to test experimentally if both ventral conspicuous colouration and the unken reflex in Melanophryniscus cambaraensis function as aposematic signals against visually oriented predators (birds). We simulated the species studied using three different clay toad models as follows: (a) in a normal position with green coloured bodies, (b) in the unken reflex position with green coloured body and extremities and (c) in the unken reflex position with a green body and red extremities. Models were distributed on a known M. cambaraensis breeding site and in the adjacent forest. More than half of the attacks on the models were from birds; however, there was no preference for any model type. Thus, just the presence of the red colour associated with the motionless unken reflex position does not seem to prevent attacks from potential predators. It is possible that the effective aposematic signal in Melanophryniscus is achieved through the unken reflex movement together with the subsequent exhibition of the warning colouration and the secretion of toxins.

Background: Virtually all prostate cancer deaths occur due to obtaining the castration-resistant phenotype after prostate cancer cells escaped from apoptosis and/or growth suppression initially induced by androgen receptor blockade. TNF-related apoptosis-inducing ligand (TRAIL) was an attractive cancer therapeutic agent due to its minimal toxicity to normal cells and remarkable apoptotic activity in tumor cells. However, most localized cancers including prostate cancer are resistant to TRAIL-induced apoptosis, thereby creating a therapeutic challenge of inducing TRAIL sensitivity in cancer cells. Herein the effects of cyproterone acetate, an antiandrogen steroid, on the TRAIL-induced apoptosis of androgen receptor-negative prostate cancer cells are reported., Methods: Cell apoptosis was assessed by both annexin V/propidium iodide labeling and poly (ADP-ribose) polymerase cleavage assays. Gene and protein expression changes were determined by quantitative real-time PCR and western blot assays. The effect of cyproterone acetate on gene promoter activity was determined by luciferase reporter assay., Results: Cyproterone acetate but not AR antagonist bicalutamide dramatically increased the susceptibility of androgen receptor-negative human prostate cancer PC-3 and DU145 cells to TRAIL-induced apoptosis but no effects on immortalized human prostate stromal PS30 cells and human embryonic kidney HEK293 cells. Further investigation of the TRAIL-induced apoptosis pathway revealed that cyproterone acetate exerted its effect by selectively increasing death receptor 5 (DR5) mRNA and protein expression. Cyproterone acetate treatment also increased DR5 gene promoter activity, which could be abolished by mutation of a consensus binding domain of transcription factor CCAAT-enhancer-binding protein homologous protein (CHOP) in the DR5 gene promoter. Cyproterone acetate increases CHOP expression in a concentration and time-dependent manner and endoplasmic reticulum stress reducer 4-phenylbutyrate could block cyproterone acetate-induced CHOP and DR5 up-regulation. More importantly, siRNA silencing of CHOP significantly reduced cyproterone acetate-induced DR5 up-regulation and TRAIL sensitivity in prostate cancer cells., Conclusions: Our study shows a novel effect of cyproterone acetate on apoptosis pathways in prostate cancer cells and raises the possibility that a combination of TRAIL with cyproterone acetate could be a promising strategy for treating castration-resistant prostate cancer.

Background: Master transcription factor MyoD can initiate the entire myogenic gene expression program which differentiates proliferating myoblasts into multinucleated myotubes. We previously demonstrated that histone methyltransferase KMT1A associates with and inhibits MyoD in proliferating myoblasts, and must be removed to allow differentiation to proceed. It is known that pro-myogenic signaling pathways such as PI3K/AKT and p38α MAPK play critical roles in enforcing associations between MyoD and transcriptional activators, while removing repressors. However, the mechanism which displaces KMT1A from MyoD, and the signals responsible, remain unknown., Methods: To investigate the role of p38α on MyoD-mediated differentiation, we utilized C2C12 myoblast cells as an in vitro model. p38α activity was either augmented via overexpression of a constitutively active upstream kinase or blocked via lentiviral delivery of a specific p38α shRNA or treatment with p38α/β inhibitor SB203580. Overexpression of KMT1A in these cells via lentiviral delivery was also used as a system wherein terminal differentiation is impeded by high levels of KMT1A., Results: The association of KMT1A and MyoD persisted, and differentiation was blocked in C2C12 myoblasts specifically after pharmacologic or genetic blockade of p38α. Conversely, forced activation of p38α was sufficient to activate MyoD and overcome the differentiation blockade in KMT1A-overexpressing C2C12 cells. Consistent with this finding, KMT1A phosphorylation during C2C12 differentiation correlated strongly with the activation of p38α. This phosphorylation was prevented by the inhibition of p38α. Biochemical studies further revealed that KMT1A can be a direct substrate for p38α. Importantly, chromatin immunoprecipitation (ChIP) studies show that the removal of KMT1A-mediated transcription repressive histone tri-methylation (H3K9me3) from the promoter of the Myogenin gene, a critical regulator of muscle differentiation, is dependent on p38α activity in C2C12 cells. Elevated p38α activity was also sufficient to remove this repressive H3K9me3 mark. Moreover, ChIP studies from C2C12 cells show that p38α activity is necessary and sufficient to establish active H3K9 acetylation on the Myogenin promoter., Conclusions: Activation of p38α displaces KMT1A from MyoD to initiate myogenic gene expression upon induction of myoblasts differentiation.

Background: Pirfenidone was recently approved for treatment of idiopathic pulmonary fibrosis. However, the therapeutic dose of pirfenidone is very high, causing side effects that limit its doses and therapeutic effectiveness. Understanding the molecular mechanisms of action of pirfenidone could improve its safety and efficacy. Because activated fibroblasts are critical effector cells associated with the progression of fibrosis, this study investigated the genes that change expression rapidly in response to pirfenidone treatment of pulmonary fibroblasts and explored their contributions to the anti-fibrotic effects of pirfenidone., Methods: We used the GeneChip microarray to screen for genes that were rapidly up-regulated upon exposure of human lung fibroblast cells to pirfenidone, with confirmation for specific genes by real-time PCR and western blots. Biochemical and functional analyses were used to establish their anti-fibrotic effects in cellular and animal models of pulmonary fibrosis., Results: We identified Regulator of G-protein Signaling 2 (RGS2) as an early pirfenidone-induced gene. Treatment with pirfenidone significantly increased RGS2 mRNA and protein expression in both a human fetal lung fibroblast cell line and primary pulmonary fibroblasts isolated from patients without or with idiopathic pulmonary fibrosis. Pirfenidone treatment or direct overexpression of recombinant RGS2 in human lung fibroblasts inhibited the profibrotic effects of thrombin, whereas loss of RGS2 exacerbated bleomycin-induced pulmonary fibrosis and mortality in mice. Pirfenidone treatment reduced bleomycin-induced pulmonary fibrosis in wild-type but not RGS2 knockout mice., Conclusions: Endogenous RGS2 exhibits anti-fibrotic functions. Upregulated RGS2 contributes significantly to the anti-fibrotic effects of pirfenidone.

G protein-coupled receptors (GPCRs) are important regulators of cell functions in asthma. We recently reported that regulator of G-protein signaling (RGS) 2, a selective modulator of Gq-coupled GPCRs, is a key regulator of airway hyper-responsiveness (AHR), the pathophysiologic hallmark of asthma. Because RGS2 protein levels in airway cells were significantly lower in patients with asthma compared with patients without asthma, we further investigated the potential pathological importance of RGS2 repression in asthma. The human RGS2 gene maps to chromosome 1q31. We first screened patients with asthma for RGS2 gene promoter single-nucleotide polymorphisms (SNPs) and found significant differences in the distribution of two RGS2 SNPs (A638G, rs2746071 and C395G, rs2746072) between patients with asthma and nonasthmatic subjects. These two SNPs are always associated with each other and have the same higher prevalence in patients with asthma (65%) as compared with nonasthmatic subjects (35%). Point mutations corresponding to these SNPs decrease RGS2 promoter activity by 44%. The importance of RGS2 down-regulation was then determined in an acute IL-13 mouse model of asthma. Intranasal administration of IL-13 in mice also decreased RGS2 expression in lungs by ∼50% and caused AHR. Although naive RGS2 knockout (KO) mice exhibit spontaneous AHR, acute IL-13 exposure further increased AHR in RGS2 KO mice. Loss of RGS2 also significantly enhanced IL-13-induced mouse airway remodeling, including peribronchial smooth muscle thickening and fibrosis, without effects on goblet cell hyperplasia or airway inflammation in mice. Thus, genetic variations and increased inflammatory cytokines can lead to RGS2 repression, which exacerbates AHR and airway remodeling in asthma.

Mitochondria are highly dynamic and undergo constant fusion and fission that are essential for maintaining physiological functions of cells. Although dysfunction of mitochondria has been implicated in tumorigenesis, little is known about the roles of mitochondrial dynamics in metastasis, the major cause of cancer death. In the present study, we found a marked upregulation of mitochondrial fission protein dynamin-related protein 1 (Drp1) expression in human invasive breast carcinoma and metastases to lymph nodes. Compared with non-metastatic breast cancer cells, mitochondria also were more fragmented in metastatic breast cancer cells that express higher levels of total and active Drp1 and less mitochondrial fusion protein 1 (Mfn1). Silencing Drp1 or overexpression of Mfn1 resulted in mitochondria elongation or clusters, respectively, and significantly suppressed metastatic abilities of breast cancer cells. In contrast, silencing Mfn proteins led to mitochondrial fragmentation and enhanced metastatic abilities of breast cancer cells. Interestingly, these manipulations of mitochondrial dynamics altered the subcellular distribution of mitochondria in breast cancer cells. For example, silencing Drp1 or overexpression of Mfn1 inhibited lamellipodia formation, a key step for cancer metastasis, and suppressed chemoattractant-induced recruitment of mitochondria to lamellipodial regions. Conversely, silencing Mfn proteins resulted in more cell spreading and lamellipodia formation, causing accumulation of more mitochondria in lamellipodia regions. More importantly, treatment with a mitochondrial uncoupling agent or adenosine triphosphate synthesis inhibitor reduced lamellipodia formation and decreased breast cancer cell migration and invasion, suggesting a functional importance of mitochondria in breast cancer metastasis. Together, our findings show a new role and mechanism for regulation of cancer cell migration and invasion by mitochondrial dynamics. Thus targeting dysregulated Drp1-dependent mitochondrial fission may provide a novel strategy for suppressing breast cancer metastasis.

RNA processing and transport are mediated by cotranscriptionally assembled ribonucleoprotein (RNP) complexes. RNPs have been postulated to help specify coordinated gene expression, but the requirements for specific RNP complexes in mammalian development and tissue homeostasis have not been extensively evaluated. THO is an evolutionarily conserved RNP complex that links transcription with nuclear export. THO is not essential for Saccharomyces cerevisiae viability, but it is essential for early mouse embryonic development. Embryonic lethality has limited the characterization of THO requirements in adult tissues. To overcome this limitation, a mouse model has been generated that allows widespread inducible deletion of Thoc1, which encodes an essential protein subunit of THO. Widespread Thoc1 deletion disrupts homeostasis within the small intestine but does not have detectable effects in other epithelial tissues such as the related mucosa of the large intestine. Thoc1 loss compromises the proliferation and lineage-generating capacity of small intestinal stem cells, disrupting the supply of differentiated cells in this rapidly renewing tissue. These findings demonstrate that the effects of THO deficiency in the adult mouse are tissue and cell type dependent.

Upon apoptotic stimuli, lysosomal proteases, including cathepsins and chymotrypsin, are released into cytosol due to lysosomal membrane permeabilization (LMP), where they trigger apoptosis via the lysosomal-mitochondrial pathway of apoptosis. Herein, the mechanism of LMP was investigated. We found that caspase 8-cleaved Bid (tBid) could result in LMP directly. Although Bax or Bak might modestly enhance tBid-triggered LMP, they are not necessary for LMP. To study this further, large unilamellar vesicles (LUVs), model membranes mimicking the lipid constitution of lysosomes, were used to reconstitute the membrane permeabilization process in vitro. We found that phosphatidic acid (PA), one of the major acidic phospholipids found in lysosome membrane, is essential for tBid-induced LMP. PA facilitates the insertion of tBid deeply into lipid bilayers, where it undergoes homo-oligomerization and triggers the formation of highly curved nonbilayer lipid phases. These events induce LMP via pore formation mechanisms because encapsulated fluorescein-conjugated dextran (FD)-20 was released more significantly than FD-70 or FD-250 from LUVs due to its smaller molecular size. On the basis of these data, we proposed tBid-PA interactions in the lysosomal membranes form lipidic pores and result in LMP. We further noted that chymotrypsin-cleaved Bid is more potent than tBid at binding to PA, inserting into the lipid bilayer, and promoting LMP. This amplification mechanism likely contributes to the culmination of apoptotic signaling.

The dnaN159 allele encodes a temperature-sensitive mutant form of the β sliding clamp (β159). SOS-induced levels of DNA polymerase IV (Pol IV) confer UV sensitivity upon the dnaN159 strain, while levels of Pol IV ∼4-fold higher than those induced by the SOS response severely impede its growth. Here, we used mutations in Pol IV that disrupted specific interactions with the β clamp to test our hypothesis that these phenotypes were the result of Pol IV gaining inappropriate access to the replication fork via a Pol III*-Pol IV switch relying on both the rim and cleft of the clamp. Our results clearly demonstrate that Pol IV relied on both the clamp rim and cleft interactions for these phenotypes. In contrast to the case for Pol IV, elevated levels of the other Pols, including Pol II, which was expressed at levels ∼8-fold higher than the normal SOS-induced levels, failed to impede growth of the dnaN159 strain. These findings suggest that the mechanism used by Pol IV to switch with Pol III* is distinct from those used by the other Pols. Results of experiments utilizing purified components to reconstitute the Pol III*-Pol II switch in vitro indicated that Pol II switched equally well with both a stalled and an actively replicating Pol III* in a manner that was independent of the rim contact required by Pol IV. These results provide compelling support for the Pol III*-Pol IV two-step switch model and demonstrate important mechanistic differences in how Pol IV and Pol II switch with Pol III*.

Regulator of G-protein signaling 4 (RGS4), an intracellular modulator of G-protein coupled receptor (GPCR)-mediated signaling, is regulated by multiple processes including palmitoylation and proteasome degradation. We found that co-expression of DHHC acyltransferases (DHHC3 or DHHC7), but not their acyltransferase-inactive mutants, increased expression levels of RGS4 but not its Cys2 to Ser mutant (RGS4C2S). DHHC3 interacts with and palmitoylates RGS4 but not RGS4C2S in vivo. Palmitoylation prolongs the half-life of RGS4 by over 8-fold and palmitoylated RGS4 blocked α(1A)-adrenergic receptor-stimulated intracellular Ca(2+) mobilization. Together, our findings revealed that DHHC proteins could regulate GPCR-mediated signaling by increasing RGS4 stability., (Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Aberrant signaling through G-protein coupled receptors promotes metastasis, the major cause of breast cancer death. We identified regulator of G-protein signaling 4 (RGS4) as a novel suppressor of breast cancer migration and invasion, important steps of metastatic cascades. By blocking signals initiated through G(i)-coupled receptors, such as protease-activated receptor 1 and CXC chemokine receptor 4, RGS4 disrupted Rac1-dependent lamellipodia formation, a key step involved in cancer migration and invasion. RGS4 has GTPase-activating protein (GAP) activity, which inhibits G-protein coupled receptor signaling by deactivating G-proteins. An RGS4 GAP-deficient mutant failed to inhibit migration and invasion of breast cancer cells in both in vitro assays and a mouse xenograft model. Interestingly, both established breast cancer cell lines and human breast cancer specimens showed that the highest levels of RGS4 protein were expressed in normal breast epithelia and that RGS4 down-regulation by proteasome degradation is an index of breast cancer invasiveness. Proteasome blockade increased endogenous RGS4 protein to levels that markedly inhibit breast cancer cell migration and invasion, which was reversed by an RGS4-targeted short hairpin RNA. Our findings point to the existence of a mechanism for posttranslational regulation of RGS4 function, which may have important implications for the acquisition of a metastatic phenotype by breast cancer cells. Preventing degradation of RGS4 protein should attenuate aberrant signal inputs from multiple G(i)-coupled receptors, thereby retarding the spread of breast cancer cells and making them targets for surgery, radiation, and immune treatment.

Excessive activation of G-protein-coupled receptor (GPCR) and receptor tyrosine kinase (RTK) pathways has been linked to prostate cancer metastasis. Rac activation by guanine nucleotide exchange factors (GEFs) plays an important role in directional cell migration, a critical step of tumor metastasis cascades. We found that the upregulation of P-Rex1, a Rac-selective GEF synergistically activated by Gbetagamma freed during GPCR signaling, and PIP3, generated during either RTK or GPCR signaling, strongly correlates with metastatic phenotypes in both prostate cancer cell lines and human prostate cancer specimens. Silencing endogenous P-Rex1 in metastatic prostate cancer PC-3 cells selectively inhibited Rac activity and reduced cell migration and invasion in response to ligands of both epidermal growth factor receptor and G-protein-coupled CXC chemokine receptor 4. Conversely, expression of recombinant P-Rex1, but not its 'GEF-dead' mutant, in non-metastatic prostate cancer cells, such as CWR22Rv1, increased cell migration and invasion through Rac-dependent lamellipodia formation. More importantly, using a mouse xenograft model, we showed that the expression of P-Rex1, but not its mutant, induced lymph node metastasis of CWR22Rv1 cells without an effect on primary tumor growth. Thus, by functioning as a coincidence detector of chemotactic signals from both GPCRs and RTKs, P-Rex1-dependent activation of Rac promotes prostate cancer metastasis.

Radioligand binding studies have detected alpha1A- and alpha1B-adrenergic receptors (AR) in rat heart, but the ligands available for these studies lack the sensitivity and specificity needed to map possible differences in alpha1-AR subtype expression. We therefore used competitive reverse transcriptase-polymerase chain reaction (RT-PCR) techniques to measure steady-state amounts of alpha1-AR messenger RNA (mRNA) subtypes in tissue dissected from several regions of rat heart. We detected mRNA for alpha1A-, alpha1B-, and alpha1D-AR in each region. Irrespective of the alphaAR subtype, the total number of alpha1-AR transcripts has the following regional rank order: left ventricular papillary muscle > left ventricle > left atrium > apex > right ventricle > ventricular septum > right atria. Among the regions, the fractional contribution of alpha1A-, alpha1B-, and alpha1D-AR mRNA to the total amount of alpha1-AR displays considerable variability. The alpha1B-AR mRNA accounts for >50% of the total alpha1-AR mRNA in all regions except the ventricular septum. There are also significant percentages of alpha1A-AR in each region, especially in the papillary muscle (48%) and ventricular septum (48%). The alpha1D-AR mRNA transcripts are found in comparatively low numbers; their highest levels (18% of total) were found in the right ventricle. These differences in alpha1-AR mRNA expression may contribute to the observed regional differences in myocardial responses to alpha1-AR agonists and antagonists.

The use of molecular markers to identify quantitative trait loci (QTLs) affecting agriculturally important traits has become a key approach in plant genetics-both for understanding the genetic basis of these traits and to help design novel plant improvement programs. In the study reported here, we mapped QTLs (and evaluated their phenotypic effects) associated with seven major traits (including grain yield) in a cross between two widely used elite maize inbred lines, B73 and Mo17, in order to explore two important phenomena in maize genetics-heterosis (hybrid vigor) and genotype-by-environment (G x E) interaction. We also compared two analytical approaches for identifying QTLs, the traditional single-marker method and the more recently described interval-mapping method. Phenotypic evaluations were made on 3168 plots (nearly 100,000 plants) grown in three states. Using 76 markers that represented 90-95% of the maize genome, both analytical methods showed virtually the same results in detecting QTLs affecting grain yield throughout the genome, except on chromosome 6. Fewer QTLs were detected for other quantitative traits measured. Whenever a QTL for grain yield was detected, the heterozygote had a higher phenotype than the respective homozygote (with only one exception) suggesting not only overdominance (or pseudooverdominance) but also that these detected QTLs play a significant role in heterosis. This conclusion was reinforced by a high correlation between grain yield and proportion of heterozygous markers. Although plant materials were grown and measured in six diverse environments (North Carolina, Iowa and Illinois) there was little evidence for G x E interaction for most QTLs.

The alpha 2-agonist, guanabenz, increases Na and water excretion with little change in renal blood flow, while clonidine has been reported to cause antinatriuresis proportional to renal vasoconstriction. We hypothesized that clonidine-mediated renal vasoconstriction involves alpha 1-receptors. Dose-response curves were constructed correlating decreases in renal blood flow with doses of phenylephrine, clonidine, and guanabenz injected as boluses into renal arteries of anesthetized dogs. Changes in renal blood flow before and after prazosin, yohimbine or verapamil were recorded. Guanabenz was a 10-fold weaker vasoconstrictor than clonidine, but was still effective when alpha 1-receptors were blocked. Tenfold differences were found between phenylephrine, clonidine, and guanabenz for dependence on alpha 1-receptors. Denervation did not significantly shift the curves. Verapamil markedly attenuated only clonidine and guanabenz, which supported their dependence on calcium channels for vascular smooth muscle contraction. Thus, postsynaptic alpha 2-adrenoceptors can contribute to renal vasoconstriction in the dog but the receptors are either less numerous on the vasculature or are less efficiently coupled to contractile elements than are alpha 1-receptors. Clonidine constricts the renal vasculature through both adrenoceptor subtypes.

Peptides of the pro-opiocortin class produce pronounced cardiovascular and natriuretic actions. We have investigated the acute cardiovascular effects of one of the most potent members of this class, gamma 2-melanocyte stimulating hormone (gamma 2-MSH), in rats. Pressor actions of gamma 2-MSH administered systemically were eliminated by ganglionic blockade with chlorisondamine. Peripheral cholinergic blockade failed to affect either the pressor or cardioaccelerator responses to gamma 2-MSH. Administration of gamma 2-MSH (2.0-10.0 micrograms) produced vasoconstriction primarily in the mesenteric and hindlimb vascular beds, while the renal bed showed little response. Infusions of phenylephrine produced pressor responses similar to those found with gamma 2-MSH, which were accompanied by a decrease in heart rate and vasoconstriction in the mesenteric and renal vascular beds. Hemodynamic changes produced by gamma 2-MSH and phenylephrine were blocked or attenuated by alpha 1-adrenergic receptor blockade with prazosin. Direct injection of gamma 2-MSH into the renal artery produced an acute renal vasoconstriction that was not attenuated by alpha 1-adrenergic or ganglionic blockade. These findings and the results of previous publications are consistent with the hypothesis that gamma 2-MSH may produce a centrally mediated activation of the sympathetic nervous system, have direct vasoconstriction actions on the renal vasculature, and inhibit baroreceptor function to produce an increase in blood pressure without an accompanying bradycardia.