Cell Type- and Isotype-Specific Expression and Regulation of β-Tubulins in Primary Olfactory Ensheathing Cells and Schwann Cells In Vitro

Olfactory ensheathing cells (OECs) and Schwann cells (SCs) are closely-related cell types with regeneration-promoting properties. Comparative gene expression analysis is particularly relevant since it may explain cell type-specific effects and guide the use of each cell type into special clinical applications. In the present study, we focused on β-tubulin isotype expression in primary adult canine glia as a translational large animal model. β-tubulins so far have been studied mainly in non-neuronal tumors and implied in tumorigenic growth. We show here that primary OECs and SCs expressed βII–V isotype mRNA. Interestingly, βIII-tubulin mRNA and protein expression was high in OECs and low in SCs, while fibroblast growth factor-2 (FGF-2) induced its down-regulation in both cell types to the same extent. This was in contrast to βV-tubulin mRNA which was similarly expressed in both cell types and unaltered by FGF-2. Immunocytochemical analysis revealed that OEC cultures contained a higher percentage of βIII-tubulin-positive cells compared to SC cultures. Addition of FGF-2 reduced the number of βIII-tubulin-positive cells in both cultures and significantly increased the percentage of cells with a multipolar morphology. Taken together, we demonstrate cell type-specific expression (βIII) and isotype-specific regulation (βIII, βV) of β-tubulin isotypes in OECs and SCs. While differential expression of βIII-tubulin in primary glial cell types with identical proliferative behaviour argues for novel functions unrelated to tumorigenic growth, strong βIII-tubulin expression in OECs may help to explain the specific properties of this glial cell type.     

Exposure to Inflammatory Cytokines IL-1β and TNFα Induces Compromise and Death of Astrocytes; Implications for Chronic Neuroinflammation

Background

Astrocytes have critical roles in the human CNS in health and disease. They provide trophic support to neurons and are innate-immune cells with keys roles during states-of-inflammation. In addition, they have integral functions associated with maintaining the integrity of the blood-brain barrier.

Methods

We have used cytometric bead arrays and xCELLigence technology to monitor the to monitor the inflammatory response profiles and astrocyte compromise in real-time under various inflammatory conditions. Responses were compared to a variety of inflammatory cytokines known to be released in the CNS during neuroinflammation. Astrocyte compromise measured by xCELLigence was confirmed using ATP measurements, cleaved caspase 3 expression, assessment of nuclear morphology and cell death.

Results

Inflammatory activation (IL-1β or TNFα) of astrocytes results in the transient production of key inflammatory mediators including IL-6, cell surface adhesion molecules, and various leukocyte chemoattractants. Following this phase, the NT2-astrocytes progressively become compromised, which is indicated by a loss of adhesion, appearance of apoptotic nuclei and reduction in ATP levels, followed by DEATH.The earliest signs of astrocyte compromise were observed between 24-48h post cytokine treatment. However, significant cell loss was not observed until at least 72h, where there was also an increase in the expression of cleaved-caspase 3. By 96 hours approximately 50% of the astrocytes were dead, with many of the remaining showing signs of compromise too. Numerous other inflammatory factors were tested, however these effects were only observed with IL-1β or TNFα treatment.

Conclusions

Here we reveal direct sensitivity to mediators of the inflammatory milieu. We highlight the power of xCELLigence technology for revealing the early progressive compromise of the astrocytes, which occurs 24-48 hours prior to substantive cell loss. Death induced by IL-1β or TNFα is relevant clinically as these two cytokines are produced by various peripheral tissues and by resident brain cells.     

TNF-α Induces Apoptosis Through JNK/Bax-Dependent Pathway in Differentiated,but not Naïve PC12 Cells

Differentiated PC12 cells have been used widely as a model for the analysis of neuronal degeneration. Some evidences showed that differentiated PC12 cells were more sensitive than naïve PC12 against apoptosis stimuli. However, the apoptosis mechanism of both types of PC12 cells was not fully known. In this study, the signaling pathways involved in tumor necrosis factor-α (TNF-α)-induced apoptosis in living differentiated and naïve PC12 cells were investigated using confocal microscope for the first time. Our results showed that during TNF-α-induced apoptosis, Bax translocation to mitochondria and cytochrome C (Cyt c) release from mitochondria were observed in differentiated PC12 cells, but not in naïve PC12 cells. Furthermore, the mRNA levels of bim, c-Jun N-terminal protein kinase 1 and 2 (JNK1 and JNK2) increased noticeably in differentiated PC12 cells. The apoptosis induced by TNF-α was inhibited by Z-IETD-fmk (specific inhibitor of caspase-8) but not SP600125 (specific inhibitor of JNK) in naïve PC12 cells. While in differentiated PC12 cells, the process of apoptosis could only be inhibited effectively by Z-IETD-fmk and SP600125 co-treatment, and SP600125 inhibited the Bax translocation to mitochondria implying that JNK mediated activation of Bax. The experimental data strongly demonstrated that TNF-α induced apoptosis through JNK/Bax-dependent pathway in differentiated, but not naïve PC12 cells.     

Reactive Astrocytes in Glial Scar Attract Olfactory Ensheathing Cells Migration by Secreted TNF-α in Spinal Cord Lesion of Rat

Background

After spinal cord injury (SCI), the formation of glial scar contributes to the failure of injured adult axons to regenerate past the lesion. Increasing evidence indicates that olfactory ensheathing cells (OECs) implanted into spinal cord are found to migrate into the lesion site and induce axons regeneration beyond glial scar and resumption of functions. However, little is known about the mechanisms of OECs migrating from injection site to glial scar/lesion site.

Methods and Findings

In the present study, we identified a link between OECs migration and reactive astrocytes in glial scar that was mediated by the tumor necrosis factor-α (TNF-α). Initially, the Boyden chamber migration assay showed that both glial scar tissue and reactive astrocyte-conditioned medium promoted OECs migration in vitro. Reactive astrocyte-derived TNF-α and its type 1 receptor TNFR1 expressed on OECs were identified to be responsible for the promoting effect on OECs migration. TNF-α-induced OECs migration was demonstrated depending on activation of the extracellular signal-regulated kinase (ERK) signaling cascades. Furthermore, TNF-α secreted by reactive astrocytes in glial scar was also showed to attract OECs migration in a spinal cord hemisection injury model of rat.

Conclusions

These findings showed that TNF-α was released by reactive astrocytes in glial scar and attracted OECs migration by interacting with TNFR1 expressed on OECs via regulation of ERK signaling. This migration-attracting effect of reactive astrocytes on OECs may suggest a mechanism for guiding OECs migration into glial scar, which is crucial for OECs-mediated axons regrowth beyond the spinal cord lesion site.     

Effect of Some Growth Factors on Tissue Transglutaminase Overexpression Induced by β-Amyloid in Olfactory Ensheathing Cells

Herein, we assessed in a particular glial cell type, called olfactory ensheathing cells (OECs), the effect of some growth factors (GFs) on tissue transglutaminase (TG2) overexpression induced by amyloid-beta (Aβ) with native full-length peptide 1–42 or by fragments, 25–35 or 35–25, as control. Previously, we demonstrated that TG2 overexpression induced by some stressors was down-regulated by GFs exposure in OECs. To monitor cell viability, an MTT test was used, while TG2 expression was examined using immunocytochemical and Western blot analysis. We also considered the involvement of the TG2-mediated apoptotic pathway. Vimentin expression was evaluated as well. Reactive oxygen species and reduced glutathione levels were utilized to test the oxidative intracellular status. Lactate dehydrogenase released into the medium, as a marker of necrotic cell death, was evaluated. We found that in OECs exposed to Aβ(1–42) or Aβ(25–35) for 24 h, TG2 expression increased, and we observed that the protein appeared prevalently localized in the cytosol. The pre-treatment with GFs, basic fibroblast growth factor (bFGF) or glial-derived neurotrophic factor (GDNF), down-regulated the TG2 level, which was prevalently limited to the nuclear compartment. Vimentin expression and caspase cleavage showed a significant enhancement in Aβ(1–42) and Aβ(25–35) exposed cells. The pre-treatment with bFGF or GDNF was able to restore the levels of the proteins to control values, and the intracellular oxidative status modified by the exposure to Aβ(1–42) or Aβ(25–35). Our data suggest that both bFGF or GDNF could be an innovative mechanism to contrast TG2 expression, which plays a key role in Alzheimer’s disease.     

In Utero and Lactational Lanthanum Exposure Induces Olfactory Dysfunction Associated with Downregulation of βIII-tubulin and Olfactory Marker Protein in Young Rats

We evaluated the role of βIII-tubulin in the morphology of olfactory receptor neuron (ORN) and olfactory dysfunction in offspring caused by prenatal and postnatal lanthanum exposure. Pregnant rats were exposed to 0.25% lanthanum chloride in drinking water from gestational day (GD) 7 until postnatal day 21. From postnatal day 23 until postnatal day 28, pups were examined with buried food pellet and olfactory maze test. The ultrastructural features of ORNs in the olfactory epithelium (OE) were observed by transmission electron microscope. The expression of βIII-tubulin and olfactory marker protein (OMP) in the tissue sections and homogenates of OE were, respectively, measured by immunodetection and western blot. Behavioral analysis of olfaction showed that lanthanum chloride exposure induced olfactory dysfunction. Offsprings exposed to lanthanum chloride showed enlarged ORN knobs and a decreased number of cilia. In addition, the levels of OMP and βIII-tubulin expression in lanthanum chloride exposure offsprings significantly decreased. Developmental lanthanum exposure could impair olfaction, and this deficit may be attributed to the downregulation of βIII-tubulin and OMP in the OE.     

Conditioned Medium of Wnt/β-Catenin Signaling-Activated Olfactory Ensheathing Cells Promotes Synaptogenesis and Neurite Growth In Vitro

Olfactory ensheathing cells (OECs), the major glia cells in the olfactory system, have been extensively studied because of their ability to promote axonal growth and regeneration. Whether it could facilitate synaptogenesis is an important, but remains as yet an unanswered question. We have identified a subgroup of Wnt signaling-activated OECs, spatiotemporal distribution of which in the olfactory bulb suggests a role for these cells in both axonal growth and synaptogenesis. In the present study, we explored this possibility in vitro. OECs were primarily cultured, in which Wnt signaling was activated by overexpressing β-catenin, and inhibited by dominant negative TCF4. Neurite growth and synaptogenesis were assessed by co-culturing neurons with conditioned medium from control OECs (cOECs CM), Wnt/β-catenin signaling-activated OECs (wOECs CM), or Wnt signaling-inhibited OECs (wiOECs). The results showed that although cOECs CM enhances axonal growth, wOECs CM exhibited a stronger axonal growth-promoting effect, than cOECs CM. More importantly, wOECs CM stimulates synatpogenesis, demonstrated by the expression of Synaptophysin and whole-cell patch clamp recording. In contrast, both cOECs CM and wiOECs CM do not affect synaptogenesis. Our data, for the first time, demonstrated that, in comparison with regularly cultured OECs, wOECs CM are more effective in enhancing axonal growth, and can promote synaptogenesis, probably by secreting factors. These results suggest a potential application of wOECs for treating spinal cord injury.     

TNF-α as an Autocrine Mediator and its Role in the Activation of Schwann Cells

In the peripheral nervous system (PNS), tumor necrosis factor-alpha (TNF-α) derived from activated Schwann cells (SCs) play a critical role as a pleiotropic mediator. In this study, we examined the function of TNF-α as an inflammatory mediator in SCs activation. TNF-α exhibits its biological effect through two distinct surface receptors, TNF receptor 1 (TNFR1) and TNFR2. We show here that cultured SCs express both TNFR1 and TNFR2, and that activation of these receptors by TNF-α promotes expression of TNF-α. Meanwhile, TNF-α also increased the production of other inflammatory mediators. Furthermore, TNF-α is involved in the induction of apoptosis through binding to TNFR in SCs. The activation of SCs by lipopolysaccharide (LPS) is partially mediated by SCs-derived TNF-α. These findings suggest the existence of a positive feedback loop in the activation of SC via TNF-α. This loop may be involved in the prolonged activation of SCs. Acute or chronic stimulation of TNF-α by SC at sites of PNS inflammation may be critical in determining whether TNF-α has activational, inflammatory, or cytotoxic effects on these cells. Yongwei Qin and Chun Cheng contributed equally to this work.     

HIF-1α Contributes to Proliferation and Invasiveness of Neuroblastoma Cells via SHH Signaling

The aim of this study was to investigate the effects of hypoxia-inducible factor-1α (HIF-1α) on the proliferation, migration and invasion of neuroblastoma (NB) cells and the mechanisms involved. We here initially used the real-time polymerase chain reaction (real-time PCR), Western blotting and immunohistochemistry (IHC) to detect the expression of HIF-1α and components of the sonic hedgehog (SHH) signaling pathway in NB cells and human specimens. Subsequently, cell proliferation, migration and invasion were analyzed using the cell counting assay, wound healing assay and Transwell system in two types of human NB cell lines, SH-SY5Y and IMR32. In addition, the role of HIF-1α in NB cells growth was determined in a xenograft nude mouse model. We found that the level of HIF-1α was significantly upregulated during NB progression and was associated with the expression of two components of SHH signaling, SHH and GLI1. We next indicated that the proliferation, migration and invasiveness of SH-SY5Y and IMR32 cells were significantly inhibited by HIF-1α knockdown, which was mediated by small interfering RNAs (siRNAs) targeting against its mRNA. Furthermore, the growth of NB cells in vivo was also suppressed by HIF-1α inhibition. Finally, the pro-migration and proliferative effects of HIF-1α could be reversed by disrupting SHH signaling. In conclusion, our results demonstrated that upregulation of HIF-1α in NB promotes proliferation, migration and invasiveness via SHH signaling.     

Chronic Cadmium Exposure Accelerates the Development of Atherosclerosis and Induces Vascular Dysfunction in the Aorta of ApoE−/− Mice

Biological Trace Element Research - Cadmium exposure is related to cardiovascular diseases, including hypertension, atherosclerosis, increased oxidative stress, endothelial dysfunction, and...     

γδ T Cells Are Reduced and Rendered Unresponsive by Hyperglycemia and Chronic TNFα in Mouse Models of Obesity and Metabolic Disease

Epithelial cells provide an initial line of defense against damage and pathogens in barrier tissues such as the skin; however this balance is disrupted in obesity and metabolic disease. Skin γδ T cells recognize epithelial damage, and release cytokines and growth factors that facilitate wound repair. We report here that hyperglycemia results in impaired skin γδ T cell proliferation due to altered STAT5 signaling, ultimately resulting in half the number of γδ T cells populating the epidermis. Skin γδ T cells that overcome this hyperglycemic state are unresponsive to epithelial cell damage due to chronic inflammatory mediators, including TNFα. Cytokine and growth factor production at the site of tissue damage was partially restored by administering neutralizing TNFα antibodies in vivo. Thus, metabolic disease negatively impacts homeostasis and functionality of skin γδ T cells, rendering host defense mechanisms vulnerable to injury and infection.     

Activation of PyMT in β Cells Induces Irreversible Hyperplasia,but Oncogene-Dependent Acinar Cell Carcinomas When Activated in Pancreatic Progenitors

It is unclear whether the cellular origin of various forms of pancreatic cancer involves transformation or transdifferentiation of different target cells or whether tumors arise from common precursors, with tumor types determined by the specific genetic alterations. Previous studies suggested that pancreatic ductal carcinomas might be induced by polyoma middle T antigen (PyMT) expressed in non-ductal cells. To ask whether PyMT transforms and transdifferentiates endocrine cells toward exocrine tumor phenotypes, we generated transgenic mice that carry tetracycline-inducible PyMT and a linked luciferase reporter. Induction of PyMT in β cells causes β-cell hyperplastic lesions that do not progress to malignant neoplasms. When PyMT is de-induced, β cell proliferation and growth cease; however, regression does not occur, suggesting that continued production of PyMT is not required to maintain the viable expanded β cell population. In contrast, induction of PyMT in early pancreatic progenitor cells under the control of Pdx1 produces acinar cell carcinomas and β-cell hyperplasia. The survival of acinar tumor cells is dependent on continued expression of PyMT. Our findings indicate that PyMT can induce exocrine tumors from pancreatic progenitor cells, but cells in the β cell lineage are not transdifferentiated toward exocrine cell types by PyMT; instead, they undergo oncogene-dependent hyperplastic growth, but do not require PyMT for survival.     

Engagement of SIRPα Inhibits Growth and Induces Programmed Cell Death in Acute Myeloid Leukemia Cells

Background

Recent studies show the importance of interactions between CD47 expressed on acute myeloid leukemia (AML) cells and the inhibitory immunoreceptor, signal regulatory protein-alpha (SIRPα) on macrophages. Although AML cells express SIRPα, its function has not been investigated in these cells. In this study we aimed to determine the role of the SIRPα in acute myeloid leukemia.

Design and Methods

We analyzed the expression of SIRPα, both on mRNA and protein level in AML patients and we further investigated whether the expression of SIRPα on two low SIRPα expressing AML cell lines could be upregulated upon differentiation of the cells. We determined the effect of chimeric SIRPα expression on tumor cell growth and programmed cell death by its triggering with an agonistic antibody in these cells. Moreover, we examined the efficacy of agonistic antibody in combination with established antileukemic drugs.

Results

By microarray analysis of an extensive cohort of primary AML samples, we demonstrated that SIRPα is differentially expressed in AML subgroups and its expression level is dependent on differentiation stage, with high levels in FAB M4/M5 AML and low levels in FAB M0–M3. Interestingly, AML patients with high SIRPα expression had a poor prognosis. Our results also showed that SIRPα is upregulated upon differentiation of NB4 and Kasumi cells. In addition, triggering of SIRPα with an agonistic antibody in the cells stably expressing chimeric SIRPα, led to inhibition of growth and induction of programmed cell death. Finally, the SIRPα-derived signaling synergized with the activity of established antileukemic drugs.

Conclusions

Our data indicate that triggering of SIRPα has antileukemic effect and may function as a potential therapeutic target in AML.     

TNF-α Induces Epithelial-Mesenchymal Transition of Renal Cell Carcinoma Cells via a GSK3β-Dependent Mechanism

TNF-α is a cytokine with antitumorigenic property. In contrast, low dose, chronic TNF-α production by tumor cells or stromal cells may promote tumor growth and metastasis. Serum levels of TNF-α are significantly elevated in renal cell carcinoma (RCC) patients. Here, we showed that TNF-α induced epithelial-mesenchymal transition (EMT) and promoted tumorigenicity of RCC by repressing E-cadherin, upregulating vimentin, activating MMP9, and invasion activities. In addition, TNF-α treatment inhibited glycogen synthase kinase 3β (GSK-3β) activity through serine-9 phosphorylation mediated by the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway in RCC cells. Inhibition of PI3K/AKT by LY294002 reactivated GSK-3β and suppressed the TNF-α-induced EMT of RCC cells. Inactivation of GSK-3β by LiCl significantly increased MMP9 activity and EMT of RCC cells. Activation of GSK-3β by transduction of constitutively active GSK-3β into RCC cells suppressed TNF-α-mediated anchorage-independent growth in soft agar and tumorigenicity in nude mice. Overexpression of a kinase-deficient GSK-3β, in contrast, potentiated EMT, anchorage-independent growth and drastically enhanced tumorigenicity in vivo. Most importantly, a 15-fold inactivation of GSK-3β activity, 3-fold decrease of E-cadherin, and 2-fold increase of vimentin were observed in human RCC tumor tissues. These results indicated that inactivation of GSK-3β plays a pivotal role in the TNF-α-mediated tumorigenesis of RCC. Mol Cancer Res; 10(8); 1109-19. ?2012 AACR.     

PGC-1α Is a Key Regulator of Glucose-Induced Proliferation and Migration in Vascular Smooth Muscle Cells

Background

Atherosclerosis is a complex pathological condition caused by a number of mechanisms including the accelerated proliferation of vascular smooth muscle cells (VSMCs). Diabetes is likely to be an important risk factor for atherosclerosis, as hyperglycemia induces vascular smooth muscle cell (VSMC) proliferation and migration and may thus contribute to the formation of atherosclerotic lesions. This study was performed to investigate whether PGC-1α, a PPARγ coactivator and metabolic master regulator, plays a role in regulating VSMC proliferation and migration induced by high glucose.

Methodology/Principal Findings

PGC-1α mRNA levels are decreased in blood vessel media of STZ-treated diabetic rats. In cultured rat VSMCs, high glucose dose-dependently inhibits PGC-1α mRNA expression. Overexpression of PGC-1α either by infection with adenovirus, or by stimulation with palmitic acid, significantly reduces high glucose-induced VSMC proliferation and migration. In contrast, suppression of PGC-1α by siRNA mimics the effects of glucose on VSMCs. Finally, mechanistic studies suggest that PGC-1α-mediated inhibition of VSMC proliferation and migration is regulated through preventing ERK1/2 phosphorylation.

Conclusions/Significance

These results indicate that PGC-1α is a key regulator of high glucose-induced proliferation and migration in VSMCs, and suggest that elevation of PGC-1α in VSMC could be a useful strategy in preventing the development of diabetic atherosclerosis.     

Methadone but not Morphine Inhibits Lubiprostone-Stimulated Cl− Currents in T84 Intestinal Cells and Recombinant Human ClC-2, but not CFTR Cl− Currents

In clinical trials, methadone, but not morphine, appeared to prevent beneficial effects of lubiprostone, a ClC-2 Cl^? channel activator, on opioid-induced constipation. Effects of methadone and morphine on lubiprostone-stimulated Cl^? currents were measured by short circuit current (Isc) across T84 cells. Whole cell patch clamp of human ClC-2 (hClC-2) stably expressed in HEK293 cells and in a high expression cell line (HEK293EBNA) as well as human CFTR (hCFTR) stably expressed in HEK293 cells was used to study methadone and morphine effects on recombinant hClC-2 and hCFTR Cl^? currents. Methadone but not morphine inhibited lubiprostone-stimulated Isc in T84 cells with half-maximal inhibition at 100 nM. Naloxone did not affect lubiprostone stimulation or methadone inhibition of Isc. Lubiprostone-stimulated Cl^? currents in hClC-2/HEK293 cells, but not forskolin/IBMX-stimulated Cl^? currents in hCFTR/HEK293 cells, were inhibited by methadone, but not morphine. HEK293EBNA cells expressing hClC-2 showed time-dependent, voltage-activated, CdCl₂-inhibited Cl^? currents in the absence (control) and the presence of lubiprostone. Methadone, but not morphine, inhibited control and lubiprostone-stimulated hClC-2 Cl^? currents with half-maximal inhibition at 100 and 200–230 nM, respectively. Forskolin/IBMX-stimulated hClC-2 Cl^? currents were also inhibited by methadone. Myristoylated protein kinase inhibitor (a specific PKA inhibitor) inhibited forskolin/IBMX- but not lubiprostone-stimulated hClC-2 Cl^? currents. Methadone caused greater inhibition of lubiprostone-stimulated currents added before patching (66.1 %) compared with after patching (28.7 %). Methadone caused inhibition of lubiprostone-stimulated Cl^? currents in T84 cells and control; lubiprostone- and forskolin/IBMX-stimulated recombinant hClC-2 Cl^? currents may be the basis for reduced efficacy of lubiprostone in methadone-treated patients.