Protein kinase CK2 is frequently elevated in a variety of human cancers. The Notch1 signaling pathway has been implicated in stem cell maintenance and its aberrant activation has been shown in several types of cancer including lung cancer. Here we show, for the first time, that CK2α is a positive regulator of Notch1 signaling in lung cancer cell lines A549 and H1299. We found that Notch1 protein level was reduced after CK2α silencing. Down-regulation of Notch1 transcriptional activity was demonstrated after the silencing of CK2α in lung cancer cells. Furthermore, small-molecule CK2α inhibitor CX-4945 led to a dose-dependent inhibition of Notch1 transcriptional activity. Conversely, forced over-expression of CK2α resulted in an increase in Notch1 transcriptional activity. Finally, the inhibition of CK2α led to a reduced proportion of stem-like CD44+/CD24− cell population. Thus, we report that the inhibition of CK2α down-regulates Notch1 signaling and subsequently reduces a cancer stem-like cell population in human lung cancer cells. Our data suggest that CK2α inhibitors may be beneficial to the lung cancer patients with activated Notch1 signaling.
CK2α; Notch1; CD44+/CD24−; cancer stem cell; lung cancer
Recently, several studies have investigated the association between a newly reported rare functional single nucleotide polymorphism (SNP) in TP53 (rs78378222) and cancer risk but generated inconsistent findings. The present study further investigated this association with risk of melanoma, squamous cell carcinoma of head and neck (SCCHN) and lung cancer. Using subjects of non-Hispanic whites recruited for three large case-control studies, we genotyped the TP53 rs78378222 SNP in 1,329 patients with melanoma, 1,096 with SCCHN, 1,013 with lung cancer, and 3000 cancer-free controls. Overall, we did not observe any variant homozygotes in this study population, nor significant associations between the TP53 rs78378222AC genotype or C allele and risk for melanoma (P=0.680 and 0.682, respectively) and lung cancer (P=0.379 and 0.382, respectively) but a protection against SCCHN (P=0.008 and 0.008, respectively), compared with the AA genotype or A allele. An additional meta-analysis including 19,423 cancer patients and 54,050 control subjects did not support such a risk association either. Our studies did not provide statistical evidence of an association between this rare TP53 variant and increased risk of melanoma, nor of lung cancer, but a possible protection against SCCHN.
biomarker; genetic susceptibility; genotype; polymorphism
Inhalation anthrax is often described as a toxin-mediated disease. However, the toxemia model does not account for the high mortality of inhalation anthrax relative to other forms of the disease or for the pathology present in inhalation anthrax. Patients with inhalation anthrax consistently show extreme bacteremia and, in contrast to animals challenged with toxin, signs of sepsis. Rather than toxemia, we propose that death in inhalation anthrax results from an overwhelming bacteremia that leads to severe sepsis. According to our model, the central role of anthrax toxin is to permit the vegetative bacteria to escape immune detection. Other forms of B. anthracis infection have lower mortality because their overt symptoms early in the course of disease cause patients to seek medical care at a time when the infection and its sequelae can still be reversed by antibiotics. Thus, the sepsis model explains key features of inhalation anthrax and may offer a more complete understanding of disease pathology for researchers as well as those involved in the care of patients.
Sepsis; anthrax; lethal factor; edema factor; disseminated intravascular coagulation; Gram-positive
KCNQ4, a voltage-gated potassium channel, plays an important role in maintaining cochlear ion homeostasis and regulating hair cell membrane potential, both essential for normal auditory function. Mutations in the KCNQ4 gene lead to DFNA2, a subtype of autosomal dominant non-syndromic deafness that is characterized by progressive sensorineural hearing loss across all frequencies. Despite recent advances in the identification of pathogenic KCNQ4 mutations, the molecular etiology of DFNA2 is unknown. We report here that decreased cell surface expression and impaired conductance of the KCNQ4 channel are two mechanisms underlying hearing loss in DFNA2. In HEK293T cells, a dramatic decrease in cell surface expression was detected by immunofluorescent microscopy and confirmed by Western blot for the pathogenic KCNQ4 mutants L274H, W276S, L281S, G285C, G285S, G296S, and G321S, while their overall cellular levels remained normal. In addition, none of these mutations affected tetrameric assembly of KCNQ4 channels. Consistent with these results, all mutants showed strong dominant-negative effects on the wild type channel function. Most importantly, overexpression of HSP90β, a key component of the molecular chaperone network that controls the KCNQ4 biogenesis, significantly increased cell surface expression of the KCNQ4 mutants L281S, G296S, and G321S. KCNQ4 surface expression was restored or considerably improved in HEK293T cells mimicking the heterozygous condition of these mutations in DFNA2 patients. Finally, our electrophysiological studies demonstrated that these mutations directly compromise the conductance of the KCNQ4 channel, since no significant change in KCNQ4 current was observed after KCNQ4 surface expression was restored or improved.
KCNQ4; hearing loss; mutations; potassium channel; surface expression; molecular chaperone; trafficking
Stem cells have been shown to have the potential to provide a source of cells for applications to tissue engineering and organ repair. The mechanisms that regulate stem cell fate, however, mostly remain unclear. Mesenchymal stem cells (MSCs) are multipotent progenitor cells that are isolated from bone marrow and other adult tissues, and can be differentiated into multiple cell lineages, such as bone, cartilage, fat, muscles, and neurons. While previous studies have focused intensively on effects of chemical signals that regulate MSC commitment, the effects of physical/mechanical cues of the microenvironment on MSC fate determination have long been neglected. However, several studies provided evidence that mechanical signals, both direct and indirect, played important roles in regulating a stem cell fate. In this review, we summarize a number of recent studies on how cell adhesion and mechanical cues influence the differentiation of MSCs into specific lineages. Understanding how chemical and mechanical cues in the microenvironment orchestrate stem cell differentiation may provide new insights into ways to improve our techniques in cell therapy and organ repair.
microenvironment; cell adhesion; mechanical force; mesenchymal stem cell; differentiation
Cardiovascular diseases are the number one cause of death globally and are projected to remain the single leading cause of death. Treatment options abounds, though efficacy is limited. Recent studies attribute discrete and ephemeral benefits to adult stem cell therapies, indicating the urge to improve stem cell based-therapy. In the present study, we show that priming Mesenchymal Stem Cells (MSC) towards cardiomyogenic lineage enhances their beneficial effects in vivo as treatment option for acute phase myocardial infarction. MSC were primed using cardiomyogenic media for 4 days, after which peak expression of key cardiomyogenic genes are reached and protein expression of Cx-43 and sarcomeric α-actinin are observed. MSC and primed MSC (pMSC) were characterized in vitro and used to treat infarcted rats immediately after LAD occlusion. Echocardiography analysis indicated that MSC-treated myocardium presented discrete improvement in function, but it also showed that pMSC treatment lead to superior beneficial results, compared to undifferentiated MSC. Seven days after cell injection, MSC and pMSC could still be detected in myocardium. Connexin-43 expression was quantified through immunoblotting, and was superior in pMSC, indicating that this could be a possible explanation for the superior performance of pMSC therapy.
Adipose tissue stem cells; mesenchymal stem cells; myocardial infarction; cell therapy; connexin-43
Living cells are continuously exposed to mechanical cues, and can translate these signals into biochemical information (e.g. mechanotransduction). This process is crucial in many normal cellular functions, e.g. cell adhesion, migration, proliferation, and survival, as well as the progression of diseases such as cancer. Focal adhesions are the major sites of interactions between extracellular mechanical environments and intracellular biochemical signaling molecules/cytoskeleton, and hence focal adhesion proteins have been suggested to play important roles in mechanotransduction. Here, we overview the current molecular understanding in mechanotransduction occurring at focal adhesions. We also introduce recent studies on how extracellular matrix and mechanical microenvironments contribute to the development of cancer.
Mechanotransduction; Focal Adhesions; Extracellular Matrix; Integrin; Cancer; Invadopodia
Hydrogen sulfide (H2S) has recently been proposed as an endogenous mediator of inflammation and is present in human synovial fluid. This study determined whether primary human articular chondrocytes (HACs) and mesenchymal progenitor cells (MPCs) could synthesize H2S in response to pro-inflammatory cytokines relevant to human arthropathies, and to determine the cellular responses to endogenous and pharmacological H2S. HACs and MPCs were exposed to IL-1β, IL-6, TNF-α and lipopolysaccharide (LPS). The expression and enzymatic activity of the H2S synthesizing enzymes cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) were determined by Western blot and zinc-trap spectrophotometry, respectively. Cellular oxidative stress was induced by H2O2, the peroxynitrite donor SIN-1 and 4-hydroxynonenal (4-HNE). Cell death was assessed by 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Mitochondrial membrane potential (ΔΨm) was determined in situ by flow cytometry. Endogenous H2S synthesis was inhibited by siRNA-mediated knockdown of CSE and CBS and pharmacological inhibitors D,L-propargylglycine and aminoxyacetate, respectively. Exogenous H2S was generated using GYY4137. Under basal conditions HACs and MPCs expressed CBS and CSE and synthesized H2S in a CBS-dependent manner, whereas CSE expression and activity was induced by treatment of cells with IL-1β, TNF-α, IL-6 or LPS. Oxidative stress-induced cell death was significantly inhibited by GYY4137 treatment but increased by pharmacological inhibition of H2S synthesis or by CBS/CSE-siRNA treatment. These data suggest CSE is an inducible source of H2S in cultured HACs and MPCs. H2S may represent a novel endogenous mechanism of cytoprotection in the inflamed joint, suggesting a potential opportunity for therapeutic intervention.
arthritis; cystathionine-γ-lyase; cystathionine-β-synthase; GYY4137; apoptosis; oxidative stress
The role of hydrogen sulfide (H2S) in inflammation remains unclear with both pro- and anti-inflammatory actions of this gas described. We have now assessed the effect of GYY4137 (a slow-releasing H2S donor) on lipopolysaccharide (LPS)-evoked release of inflammatory mediators from human synoviocytes (HFLS) and articular chondrocytes (HAC) in vitro. We have also examined the effect of GYY4137 in a complete Freund’s adjuvant (CFA) model of acute joint inflammation in the mouse. GYY4137 (0.1–0.5 mM) decreased LPS-induced production of nitrite (NO2−), PGE2, TNF-α and IL-6 from HFLS and HAC, reduced the levels and catalytic activity of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and reduced LPS-induced NF-κB activation in vitro. Using recombinant human enzymes, GYY4137 inhibited the activity of COX-2, iNOS and TNF-α converting enzyme (TACE). In the CFA-treated mouse, GYY4137 (50 mg/kg, i.p.) injected 1 hr prior to CFA increased knee joint swelling while an anti-inflammatory effect, as demonstrated by reduced synovial fluid myeloperoxidase (MPO) and N-acetyl-β-D-glucosa-minidase (NAG) activity and decreased TNF-α, IL-1β, IL-6 and IL-8 concentration, was apparent when GYY4137 was injected 6 hrs after CFA. GYY4137 was also anti-inflammatory when given 18 hrs after CFA. Thus, although GYY4137 consistently reduced the generation of pro-inflammatory mediators from human joint cells in vitro, its effect on acute joint inflammation in vivo depended on the timing of administration.
hydrogen sulfide; GYY4137; inflammation; synoviocyte; cytokines; myeloperoxidase; lipopolysaccharide; Freund’s adjuvant; COX-2; TNF-alpha converting enzyme
Taste signaling molecules are found in the gastrointestinal (GI) tract suggesting they participate to chemosensing. We tested whether fasting and refeeding affect the expression of the taste signaling molecule, α-transducin (Gαtran), throughout the pig GI tract and the peptide content of Gαtran cells. The highest density of Gαtran-immunoreactive (IR) cells was in the pylorus, followed by the cardiac mucosa, duodenum, rectum, descending colon, jejunum, caecum, ascending colon and ileum. Most Gαtran-IR cells contained chromogranin-A. In the stomach, many Gαtran-IR cells contained ghrelin, whereas in the upper small intestine many were gastrin/cholecystokinin-IR and a few somatostatin-IR. Gαtran-IR and Gαgust-IR colocalized in some cells. Fasting (24h) resulted in a significant decrease in Gαtran-IR cells in the cardiac mucosa (29.3 ± 0.8 vs. 64.8 ± 1.3, P < 0.05), pylorus (98.8 ± 1.7 vs. 190.8 ± 1.9, P < 0.0l), caecum (8 ± 0.01 vs. 15.5 ± 0.5, P < 0.01), descending colon (17.8 ± 0.3 vs. 23 ± 0.6, P < 0.05) and rectum (15.3 ± 0.3 vs. 27.5 ± 0.7, P < 0.05). Refeeding restored the control level of Gαtran-IR cells in the cardiac mucosa. By contrast, in the duodenum and jejunum, Gαtran-IR cells were significantly reduced after refeeding, whereas Gαtran-IR cells density in the ileum was not changed by fasting/refeeding. These findings provide further support to the concept that taste receptors contribute to luminal chemosensing in the GI tract and suggest they are involved in modulation of food intake and GI function induced by feeding and fasting.
α-gustducin; taste receptors; enteroendocrine cells; chemosensing
Vascular endothelial growth factor (VEGF) is a critical angiogenic factor affecting endothelial cells, inflammatory cells and neuronal cells. In addition to its well-defined positive role in wound healing, pathological roles for VEGF have been described in cancer and inflammatory diseases (i.e. atherosclerosis, rheumatoid arthritis, inflammatory bowel disease, and osteoarthritis). Recently we showed that transcription factors LITAF and STAT6B affected the inflammatory response. The present study builds upon our previous results in testing the role of mouse LITAF and STAT6B in the regulation of VEGF-mediated processes. Cells co-transfected with a series of VEGF promoter deletions along with truncated forms of mLITAF and/or mSTAT6B identified a DNA binding site (between −338 and −305 upstream of the transcription site) important in LITAF and/or STAT6B-mediated transcriptional regulation of VEGF. LITAF and STAT6B corresponding protein sites were identified. In addition, siRNA-mediated knockdown of mLITAF and/or mSTAT6B leads to significant reduction of VEGF mRNA levels and inhibits LPS-induced VEGF secretion in mouse RAW 264.7 cells and. Furthermore, VEGF treatment of mouse macrophage or endothelial cells induces LITAF/STAT6B nuclear translocation and cell migration. To translate these observations in vivo, VEGF164-soaked matrigel were implanted in whole-body LITAF-deficient animals (TamLITAF−/−), wild-type mice silenced for STAT6B, and in respective control animals. Vessel formation was found significantly reduced in TamLITAF−/− as well as in STAT6B-silenced wild-type animals compared to control animals. The present data demonstrates that VEGF regulation by LITAF and/or STAT6B is important in angiogenesis signaling pathways and may be a useful target in the treatment of VEGF diseases.
regulation; VEGF; LITAF; STAT6B; interaction; angiogenesis
Inhibitors of PI3-K/Akt are currently being assessed clinically in patients with advanced RCC. Identification of therapeutic strategies that might enhance the efficacy of PI3-K/Akt inhibitors is therefore of great interest. As PI3-K inhibition would be expected to have many pro-apoptotic effects, we hypothesized that there may be unique synergy between PI3-K inhibitors and BH3-mimetics. Towards this end, we assessed the combination of the PI3K inhibitor LY 294002 and the Bcl-2 family inhibitor ABT-737 in RCC cell lines. We found that combinatorial treatment with these agents led to a significant increase in PARP cleavage and cell death in all RCC cell lines. The synergized cell death was correlated with decreased levels of Mcl-1 and XIAP, and increased levels in Bim, and appears critically dependent upon the activation of caspase 3 and 8. The enhanced lethality observed with the combination also appears dependent upon the regulation of XIAP, Mcl-1, and Bim levels. Our results suggest that the combination of PI3-K inhibitors with BH3-mimetics may be a viable therapeutic strategy in RCC.
PI3K; Bcl-2; apoptosis; renal carcinoma cell; XIAP
microRNAs (miRNAs) are small, regulatory noncoding RNAs that have potent effects on gene expression. Several miRNA are deregulated in cellular processes involved in human liver diseases and regulation of cellular processes. Recent studies have identified the involvement of miR-29 in hepatic fibrosis and carcinogenesis. Although several targets of miR-29 have been identified, there is limited information regarding the cell-type specific roles of miR-29 in the liver, and we sought to evaluate the role of this miRNA in hepatic pathobiology. We report the generation of a tissue–specific knock-out mouse to evaluate the role of miR-29 in hepatic fibrosis and carcinogenesis in response to injury. We hypothesized that miR-29 contributes to the hepatocyte driven response to chronic cellular injury that results in fibrosis. In support of this hypothesis, fibrosis and mortality were enhanced in miR29 knockout mice in response to carbon tetrachloride. Genome-wide gene expression analysis identified an over-representation of genes associated with fibrosis. The oncofetal RNA H19 was modulated in a miR-29 dependent manner following exposure to carbon tetrachloride in vivo. The impact of a hepatocyte specific miR-29 knock-out on survival following chronic hepatic injury in vivo implicates this miRNA as a potential target for intervention. These results provide evidence of the involvement of miR-29 in chronic hepatic injury, and suggest a role for deregulated hepatocyte expression of miR-29 in the response to hepatic injury, fibrosis and carcinogenesis.
fibrosis; liver cancer; transgenic
In this study we used a large nonhuman primate model, the baboon, to establish a stepwise protocol to generate CD34+ endothelial progenitor cells (EPCs) from embryonic stem cells (ESCs) and to demonstrate their reparative effects. Baboon ESCs were sequentially differentiated from embryoid body cultures for 9 days and then were specified into EPCs by culturing them in monolayer for 12 days. The resulting EPCs expressed CD34, CXCR4, and UEA-1, but neither CD31 nor CD117. The EPCs were able to form intact lumen structures when seeded on Matrigel, took up Dil-LDL, and responded to TNF-α. Angioblasts specified in EGM-2 medium and ECGS medium had 6.41±1.16% (n=3) and 9.32±3.73% CD34+ cells (n=3). The efficiency of generating CD34+ EPCs did not differ significantly from ECGS to EGM-2 culture media, however, angioblasts specified in ECGS medium expressed a higher percentage of CD34+/CXCR4+ cells (3.49 ±1.32%, n=3) than those specified in EGM-2 medium (0.49±0.52%, n=3). To observe their reparative capacity, we purified CD34+ progenitors after specification by EGM-2 medium; inoculated fluorescently labeled CD34+ EPCs into an arterial segment denuded of endothelium in an ex vivo system. After 14 days of ex vivo culture, the grafted cells had attached and integrated to the denuded surface; in addition, they had matured further and expressed terminally differentiated endothelial markers including CD31 and CD146. In conclusion, we have proved that specified CD34+ EPCs are promising therapeutic agents for repairing damaged vasculature.
Embryonic Stem Cells; Endothelial Differentiation; Specification and Maturation; Stem Cell Therapy; Nonhuman Primate Model
Mu opioid receptor (MOR) is the main site of interaction for major clinical analgesics, particularly morphine. MOR expression is regulated at the transcriptional and post-transcriptional levels. However, the protein expression of the MOR gene is relatively low and the translational control of MOR gene has not been well studied. The 5′-untranslated region (UTR) of the human MOR (OPRM1) mRNA contains four upstream AUG codons (uAUG) preceding the main translation initiation site. We mutated the four uAUGs individually and in combination. Mutations of the third uAUG, containing the same open reading frame, had the strongest inhibitory effect. The inhibitory effect caused by the third in-frame uAUG was confirmed by in vitro translation and receptor-binding assays. Toeprinting results showed that OPRM1 ribosomes initiated efficiently at the first uAUG, and subsequently re-initiated at the in-frame #3 uAUG and the physiological AUG site. This re-initiation resulted in negative expression of OPRM1 under normal conditions. These results indicate that re-initiation in MOR gene expression could play an important role in OPRM1 regulation.
human mu opioid receptor; post-transcriptional regulation; uORF
To determine the hypermethylation status of the promoter regions of tumor suppressor genes in normal breast tissues and identify the determinants of these epigenetic changes.
Questionnaires and breast tissues were collected from healthy women without a history of cancer and undergoing reduction mammoplasty (N=141). Methylation for p16INK4, BRCA1, ERα and RAR-β promoter regions from normal breast tissues were determined by methylation specific PCR. Associations were examined with chi-square and Fisher’s exact test as well as logistic regression. All statistical tests were two-sided.
p16INK4, BRCA1, ERα and RAR-β hypermethylation were identified in 31%, 17% 9% and 0% of the women, respectively. Women with BRCA1 hypermethylation had an eight-fold increase in the risk of ERα hypermethylation (p=0.007). p16INK4 hypermethylation was present in 28% of African-Americans, but 65% in European-Americans (p=0.02). There was an increased likelihood of p16INK4 or BRCA1 hypermethylation for women with family history of cancer (OR 2.3; 95%CI: 1.05–4.85 and OR 5.0; 95%CI: 1.55–15.81, respectively). ERα hypermethylation was associated with family history of breast cancer (OR 6.6; 95%CI: 1.58–27.71). After stratification by race, p16INK4 in European-Americans and BRCA1 hypermethylation in African-Americans were associated with family history of cancer (OR 3.8; 95%CI: 1.21–12.03 and OR 6.5; 95%CI: 1.33–31.32, respectively).
Gene promoter hypermethylation was commonly found in healthy breast tissues from women without cancer, indicating that these events are frequent and early lesions. Race and family history of cancer increase the likelihood of these early events.
p16INK4; BRCA1; ERα CpG islands hypermethylation; breast biology; family history of cancer
Injury to lens epithelial cells (LECs) leads to epithelial-mesenchymal transition (EMT) with resultant fibrosis. The tropomyosin (Tpm) family of cytoskeleton proteins is involved in regulating and stabilizing actin microfilaments. Aberrant expression of Tpms leads to abnormal morphological changes with disintegration of epithelial integrity. The EMT of LECs has been proposed as a major cause of posterior capsule opacification (PCO) after cataract surgery. Using in vivo rodent PCO and human cataractous LECs, we demonstrated that the aberrant expression of rat Tpm and human Tpm1α/2β suggested their association in remodeling of the actin cytoskeleton during EMT of LECs. Expression analysis from abnormally growing LECs after lens extraction revealed elevated expression of α-smooth muscle actin (α-SMA), a marker for EMT. Importantly, these cells displayed increased expression of Tpm1α/2β following EMT/PCO formation. Expression of Tpm1α/2β was upregulated in LECs isolated from cataractous lenses of Shumiya Cataract Rats (SCRs), compared to noncataractous lenses. Also, LECs from human patients with nuclear cataract and anterior subcapsular fibrosis (ASF) displayed significantly increased expression of Tpm2β mRNA, suggesting that similar signaling invokes the expression of these molecules in LECs of cataractous SCR and human lenses. EMT was observed in LECs over-expressed with Tpm1α/2β, as evidenced by increased expression of α-SMA. These conditions were correlated with remodeling of actin filaments, possibly leading to EMT/PCO and ASF. The present findings may help clarify the condition of the actin cytoskeleton during morphogenetic EMT, and may contribute to development of Tpm-based inhibitors for postponing PCO and cataractogenesis.
epithelial-mesenchymal transition; tropomyosin; lens; posterior capsule opacity anterior subcapsular fibrosis; cataract
Tumor-induced myeloid-derived suppressor cells (MDSC) promote immune suppression and mediate tumor progression. However, the molecular basis for the generation of MDSC, which in mice co-express the CD11b+ and Gr-1+ cell surface markers remains unclear. Because CD11b+Gr-1+ cells expand during progressive tumor growth, this suggests that tumor-induced events alter signaling pathways that affect normal myeloid cell development. Interferon regulatory factor-8 (IRF-8), a member of the IFN-γ regulatory factor family, is essential for normal myelopoiesis. We therefore examined whether IRF-8 modulated tumor-induced CD11b+Gr-1+ cell development or accumulation using both implantable (4T1) and transgenic (MMTV-PyMT) mouse models of mammary tumor growth. In the 4T1 model, both splenic and bone marrow-derived CD11b+Gr-1+ cells of tumor-bearing mice displayed a marked reduction in IRF-8 expression compared to control populations. A causal link between IRF-8 expression and the emergence of tumor-induced CD11b+Gr-1+ cells was explored in vivo using a double transgenic (dTg) mouse model designed to express transgenes for both IRF-8 and mammary carcinoma development. Despite the fact that tumor growth was unaffected, splenomegaly, as well as the frequencies and absolute numbers of CD11b+Gr-1+ cells were significantly lower in dTg mice when compared with single transgenic tumor-bearing mice. Overall, these data reveal that IRF-8 plays an important role in tumor-induced development and/or accumulation of CD11b+Gr-1+ cells, and establishes a molecular basis for the potential manipulation of these myeloid populations for cancer therapy.
IRF-8; myeloid-derived suppressor cells; tumor progression; hematopoiesis
The histone deacetylase (HDAC) inhibitor trichostatin A (TSA) has recently been shown to inhibit deleterious effects of cytokines on β-cells, but it is unable to protect β-cells from death due to its own cytotoxicity. Herein, we investigated novel HDAC inhibitors for their cytoprotective effects against IL-1β-induced damage to isolated β-cells. We report that three novel compounds (THS-73–44, THS-72–5 and THS-78–5) significantly inhibited HDAC activity and increased the acetylation of histone H4 in isolated β-cells. Further, these compounds exerted no toxic effects on metabolic cell viability in these cells. However, among the three compounds tested, only THS-78–5 protected against IL-1β-mediated loss in β-cell viability. THS-78–5 was also able to attenuate IL-1β-induced inducible nitric oxide synthase expression and subsequent NO release. Our data also indicate that the cytoprotective properties of THS-78–5 against IL-1β-mediated effects may, in part, be due to inhibition of IL-1β-induced transactivation of nuclear factor κB (NF-κB) in these cells. Together, we provide evidence for a novel HDAC inhibitor with a significant potential to prevent IL-1β-mediated effects on isolated β-cells. Potential implications of these findings in the development of novel therapeutics to prevent deleterious effects of cytokines and the onset of autoimmune diabetes are discussed.
HDAC inhibitors; pancreatic β-cell; inducible nitric oxide synthase; NO release; NF-κB
Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system (CNS) affects cross-talk between the individual cell types of the neurovascular unit, which then contributes to disruption of the blood–brain barrier (BBB) and the development of neurological dysfunctions. While the toxicity of HIV-1 on neurons, astrocytes, and brain endothelial cells has been widely studied, there are no reports addressing the influence of HIV-1 on pericytes. Therefore, the purpose of this study was to evaluate whether pericytes can be infected with HIV-1 and how such an infection affects the barrier function of brain endothelial cells. Our results indicate that human brain pericytes express the major HIV-1 receptor CD4 and coreceptors CXCR4 and CCR5. We also determined that HIV-1 can replicate, though at a low level, in human brain pericytes as detected by HIV-1 p24 ELISA. Pericytes were susceptible to infection with both the X4-tropic NL4-3 and R5-tropic JR-CSF HIV-1 strains. Moreover, HIV-1 infection of pericytes resulted in compromised integrity of an in vitro model of the BBB. These findings indicate that human brain pericytes can be infected with HIV-1 and suggest that infected pericytes are involved in the progression of HIV-1-induced CNS damage.
HIV-1; pericytes; blood–brain barrier; neurovascular unit
Lack of an adequate experimental model has hindered the ability to fully understand scleroderma (SSc) pathogenesis. Current SSc research is based on the study of cultured fibroblasts from skin biopsies. In depth characterization of the SSc fibroblast phenotype is hindered by the limited lifespan and heterogeneity of these cells. The goal of this study was to isolate high collagen-producing fibroblasts from SSc biopsies and extend their lifespan with hTERT immortalization to enable characterization of their phenotype. Fibroblasts from two pairs of closely matched normal and SSc biopsies were infected with an hTERT lentivirus. Infected colonies were isolated, cultured into clonal cell lines and analysed with respect to profibrotic gene expression. The mRNA levels of nine profibrotic genes were measured by quantitative real-time PCR. Protein levels were assessed by Western blot. The hTERT SSc clones were heterogeneous with regards to expression of the profibrotic genes measured. A subset of the SSc clones showed elevated expression levels of collagen I, connective tissue growth factor and thrombospondin 1 mRNA, while expression of other genes was not significantly changed. Elevated expression of collagen I protein and mRNA was correlative with elevated expression of connective tissue growth factor. Several hTERT clones expressed high levels of pSmad1, Smad1 and TGF-βRI indicative of altered TGF-β signalling. A portion of SSc clones expressed several profibrotic genes. This study demonstrates that select characteristics of the SSc phenotype are expressed in a subset of activated fibroblasts in culture. The clonal SSc cell lines may present a new and useful model to investigate the mechanisms involved in SSc fibrosis.
scleroderma; fibroblast; hTERT; fibrosis; collagen; TGF-β; Smad1
Previous studies have shown that the transforming growth factor (TGF)β/Alk1/Smad1 signaling pathway is constitutively activated in a subset of systemic sclerosis (SSc) fibroblasts and this pathway is a critical regulator of CCN2 gene expression. Caveolin-1 (cav-1), an integral membrane protein and the main component of caveolae, has also been implicated in SSc pathogenesis. This study was undertaken to evaluate the role of caveolin-1 in Smad1 signaling and CCN2 expression in healthy and SSc dermal fibroblasts. We show that a significant subset of SSc dermal fibroblasts has up-regulated cav-1 expression in vitro, and that cav-1 up-regulation correlates with constitutive Smad1 phosphorylation. In addition, basal levels of phospho-Smad1 were down-regulated after inhibition of cav-1 in SSc dermal fibroblasts. Caveolin-1 formed a protein complex with Alk1 in dermal fibroblasts, and this association was enhanced by TGFβ. By using siRNA against cav-1 and adenoviral cav-1 overexpression we demonstrate that activation of Smad1 in response to TGFβ requires cav-1 and that cav-1 is sufficient for Smad-1 phosphorylation. We also show that cav-1 is a positive regulator of CCN2 gene expression, and that it is required for the basal and TGFβ-induced CCN2 levels. In conclusion, this study has revealed an important role of cav-1 in mediating TGFβ/Smad1 signaling and CCN2 gene expression in healthy and SSc dermal fibroblasts.
scleroderma; caveolin-1; Smad1; CCN2
Combined morphological, immunocytochemical, biochemical and molecular genetic studies were performed on skeletal muscle, heart muscle and liver tissue of a 16-months boy with fatal liver failure. The pathological characterization of the tissues revealed a severe depletion of mtDNA (mitochondrial DNA) that was most pronounced in liver, followed by a less severe, but still significant depletion in skeletal muscle and the heart. The primary cause of the disease was linked to compound heterozygous mutations in the polymerase γ (POLG) gene (DNA polymerase γ; A467T, K1191N). We present evidence, that compound heterozygous POLG mutations lead to tissue selective impairment of mtDNA replication and thus to a mosaic defect pattern even in the severely affected liver. A variable defect pattern was found in liver, muscle and heart tissue as revealed by biochemical, cytochemical, immunocytochemical and in situ hybridization analysis. Functionally, a severe deficiency of cytochrome-c-oxidase (cox) activity was seen in the liver. Although mtDNA depletion was detected in heart and skeletal muscle, there was no cox deficiency in these tissues. Depletion of mtDNA and microdissection of cox-positive or negative areas correlated with the histological pattern in the liver. Interestingly, the mosaic pattern detected for cox-activity and mtDNA copy number fully aligned with the immunohistologically revealed defect pattern using Pol γ, mtSSB- and mtTFA-antibodies, thus substantiating the hypothesis that nuclear encoded proteins located within mitochondria become unstable and are degraded when they are not actively bound to mtDNA. Their disappearance could also aggravate the mtDNA depletion and contribute to the non-homogenous defect pattern.
depletion of mtDNA; polymerase γ; mitochondrial single stranded binding protein(mtSSB); mitochondrial transcription factor A (mtTFA); liver failure; in situ hybridization
PV1 is an endothelial specific protein with structural roles in the formation of diaphragms in endothelial cells of normal vessels. PV1 is also highly expressed on endothelial cells of many solid tumors. Based on in vitro data, PV1 is thought to actively participate in angiogenesis. In order to test whether PV1 has a function in tumor angiogenesis and in tumor growth in vivo, we have treated pancreatic tumor bearing mice by single dose intratumoral delivery of lentiviruses encoding for two different shRNAs targeting murine PV1. We find that PV1 downregulation by shRNAs inhibits the growth of established tumors derived from two different human pancreatic adenocarcinoma cell lines (AsPC-1 and BxPC-3). The effect observed is due to downregulation of PV1 in the tumor endothelial cells of host origin, PV1 being specifically expressed in tumor vascular endothelial cells and not in cancer or other stromal cells. There are no differences in vascular density of tumors treated or not with PV1 shRNA and gain and loss of function of PV1 in endothelial cells does not modify either their proliferation or migration, suggesting that tumor angiogenesis is not impaired. Together, our data argue that down regulation of PV1 in tumor endothelial cells results in the inhibition of tumor growth via a mechanism different from inhibiting angiogenesis.
angiogenesis; fenestrae; vesiculo-vacuolar organelles; caveolae; pancreatic cancer; transendothelial channels; tumor microenvironment
Study of physiologic angiogenesis and associated signaling mechanisms in adult heart has been limited by the lack of a robust animal model. We investigated thyroid hormone induced sprouting angiogenesis and the underlying mechanism. Hypothyroidism was induced in C57BL/6J mice by feeding with propylthiouracil (PTU). One year of PTU treatment induced heart failure. Both 12 weeks- (young) and 1 year-PTU (middle age) treatment caused a remarkable capillary rarefaction observed in capillary density. Three-days Triiodothyronine (T3) treatment significantly induced cardiac capillary growth in hypothyroid mice. In cultured left ventricle (LV) tissues from PTU treated mice, T3 also induced robust sprouting angiogenesis where pericyte-wrapped endothelial cells formed tubes. The in vitro T3 angiogenic response was similar in mice pretreated with PTU for periods ranging from 1.5–12 months. Besides bFGF and VEGF164, PDGF-BB was the most robust angiogenic growth factor, which stimulated notable sprouting angiogenesis in cultured hypothyroid LV tissues with increasing potency but had little effect on tissues from euthyroid mice. T3 treatment significantly increased PDGF receptor beta (PDGFR-β) protein levels in hypothyroid heart. PDGFR inhibitors blocked the action of T3 both on sprouting angiogenesis in cultured LV tissue and on capillary growth in vivo. Additionally, activation of Akt signaling mediated in T3-induced angiogenesis was blocked by PDGFR inhibitor and neutralizing antibody. Our results suggest that hypothyroidism leads to cardiac microvascular impairment and rarefaction with increased sensitivity to angiogenic growth factors. T3 induced cardiac sprouting angiogenesis in adult hypothyroid mice was associated with PDGF-BB, PDGFR-β, and downstream activation of Akt.
thyroid hormone; sprouting angiogenesis; pericyte; PDGF; Akt