IFITM3/STAT3 axis promotes glioma cells invasion and is modulated by TGF-β

Molecular Biology Reports - Glioma is the most aggressive primary brain tumor. We have previously provided evidence that IFITM3 promoted glioma cells migration. However, the mechanism of how IFITM3...     

Na,K-ATPase α2 activity in mammalian skeletal muscle T-tubules is acutely stimulated by extracellular K+

The Na,K-ATPase α2 isoform is the predominant Na,K-ATPase in adult skeletal muscle and the sole Na,K-ATPase in the transverse tubules (T-tubules). In quiescent muscles, the α2 isozyme operates substantially below its maximal transport capacity. Unlike the α1 isoform, the α2 isoform is not required for maintaining resting ion gradients or the resting membrane potential, canonical roles of the Na,K-ATPase in most other cells. However, α2 activity is stimulated immediately upon the start of contraction and, in working muscles, its contribution is crucial to maintaining excitation and resisting fatigue. Here, we show that α2 activity is determined in part by the K⁺ concentration in the T-tubules, through its K⁺ substrate affinity. Apparent K⁺ affinity was determined from measurements of the K_1/2 for K⁺ activation of pump current in intact, voltage-clamped mouse flexor digitorum brevis muscle fibers. Pump current generated by the α2 Na,K-ATPase, Ip, was identified as the outward current activated by K⁺ and inhibited by micromolar ouabain. Ip was outward at all potentials studied (−90 to −30 mV) and increased with depolarization in the subthreshold range, −90 to −50 mV. The Q₁₀ was 2.1 over the range of 22–37°C. The K_1/2,K of Ip was 4.3 ± 0.3 mM at −90 mV and was relatively voltage independent. This K⁺ affinity is lower than that reported for other cell types but closely matches the dynamic range of extracellular K⁺ concentrations in the T-tubules. During muscle contraction, T-tubule luminal K⁺ increases in proportion to the frequency and duration of action potential firing. This K_1/2,K predicts a low fractional occupancy of K⁺ substrate sites at the resting extracellular K⁺ concentration, with occupancy increasing in proportion to the frequency of membrane excitation. The stimulation of preexisting pumps by greater K⁺ site occupancy thus provides a rapid mechanism for increasing α2 activity in working muscles.     

K562 cell proliferation is modulated by PLCβ1 through a PKCα-mediated pathway

Phospholipase C β1 (PLCβ1) is known to play an important role in cell proliferation. Previous studies reported an involvement of PLCβ1 in G₀-G₁/S transition and G₂/M progression in Friend murine erythroleukemia cells (FELC). However, little has been found about its role in human models. Here, we used K562 cell line as human homologous of FELC in order to investigate the possible key regulatory role of PLCβ1 during cell proliferation of this human cell line. Our studies on the effects of the overexpression of both these isoforms showed a specific and positive connection between cyclin D3 and PLCβ1 in K562 cells, which led to a prolonged S phase of the cell cycle and a delay in cell proliferation. In order to shed light on this mechanism, we decided to study the possible involvement of protein kinases C (PKC), known to be direct targets of PLC signaling and important regulators of cell proliferation. Our data showed a peculiar decrease of PKCα levels in cells overexpressing PLCβ1. Moreover, when we silenced PKCα, by RNAi technique, in order to mimic the effects of PLCβ1, we caused the same upregulation of cyclin D3 levels and the same decrease of cell proliferation found in PLCβ1-overexpressing cells. The key features emerging from our studies in K562 cells is that PLCβ1 targets cyclin D3, likely through a PKCα-mediated-pathway, and that, as a downstream effect of its activity, K562 cells undergo an accumulation in the S phase of the cell cycle.     

Susceptibility to collagen-induced arthritis is modulated by TGFβ responsiveness of T cells

The objective of our study was to determine the regulatory effects that endogenous transforming growth factor β (TGFβ) exerts on T cells in the pathogenesis of collagen-induced arthritis (CIA). CIA was induced in transgenic mice expressing a dominant negative TGFβ type II receptor in T cells under the control of the human CD2 promoter. Clinical and histological arthritis scores were determined and experiments on disease induction and the healing phase of disease were performed. The proliferation and cytokine production of draining lymph node cells in vitro were analyzed. Transgenic mice were more susceptible to induction of CIA. The overall incidence was higher in transgenic mice than in wild-type mice (57% vs 35%, P < 0.05). Affected transgenic animals displayed a significantly higher clinical (4.5 ± 0.6 vs 1.67 ± 0.19, P = 0.001) and histological arthritis score (8.01 ± 0.9 vs 4.06 ± 1.1, P < 0.05). Draining lymph node cells of transgenic mice secreted more tumor necrosis factor α and IFNγ and proliferated more vigorously in response to collagen type II and upon CD3/CD28 costimulation in vitro. Therefore, the regulation of T cells by endogenous TGFβ is important for the maintenance of joint integrity after arthritis induction. Defects in TGFβ-signalling as a susceptibility factor for rheumatoid arthritis may warrant further investigation.     

Transforming growth factor-β1 is constitutively secreted by Chinese hamster ovary cells and is functional in human cells

Chinese hamster ovary (CHO) cells are widely used for the production of recombinant proteins for clinical use as well as academic research. They are particularly important for the production of glycoproteins where bacteria cannot be used. TGFβ1 is a potent cytokine highly conserved across species with multiple immunological and non-immunological effects. We have discovered that CHOK1, the CHO clone most commonly used by the pharmaceutical industry, constitutively secretes latent TGFβ1 and that this hamster TGFβ1 is active on human cells inducing profound immunological effects. As far as we are aware, the production of TGFβ1 by CHOK1 cells has not been reported before in the literature. As TGFβ1 exerts powerful and pleiotropic effects on diverse cell types, and as CHO cells are used to produce a large number of clinical and non-clinical products, our findings are highly relevant to studies that rely on recombinant proteins.     

IFN-α production by plasmacytoid dendritic cells stimulated with RNA-containing immune complexes is promoted by NK cells via MIP-1β and LFA-1

Several systemic autoimmune diseases display a prominent IFN signature. This is caused by a continuous IFN-α production by plasmacytoid dendritic cells (pDCs), which are activated by immune complexes (ICs) containing nucleic acid. The IFN-α production by pDCs stimulated with RNA-containing IC (RNA-IC) consisting of anti-RNP autoantibodies and U1 small nuclear ribonucleoprotein particles was recently shown to be inhibited by monocytes, but enhanced by NK cells. The inhibitory effect of monocytes was mediated by TNF-α, PGE(2), and reactive oxygen species, but the mechanisms for the NK cell-mediated increase in IFN-α production remained unclear. In this study, we investigated the mechanisms whereby NK cells increase the RNA-IC-induced IFN-α production by pDCs. Furthermore, NK cells from patients with systemic lupus erythematosus (SLE) were evaluated for their capacity to promote IFN-α production. We found that CD56(dim) NK cells could increase IFN-α production >1000-fold after RNA-IC activation, whereas CD56(bright) NK cells required costimulation by IL-12 and IL-18 to promote IFN-α production. NK cells produced MIP-1α, MIP-1β, RANTES, IFN-γ, and TNF-α via RNA-IC-mediated FcγRIIIA activation. The IFN-α production in pDCs was promoted by NK cells via MIP-1β secretion and LFA-mediated cell-cell contact. Moreover, NK cells from SLE patients displayed a reduced capacity to promote the RNA-IC-induced IFN-α production, which could be restored by exogenous IL-12 and IL-18. Thus, different molecular mechanisms can mediate the NK cell-dependent increase in IFN-α production by RNA-IC-stimulated pDCs, and our study suggests that the possibility to therapeutically target the NK-pDC axis in IFN-α-driven autoimmune diseases such as SLE should be investigated.     

Estrogen receptor alpha is essential for the proliferation of prostatic smooth muscle cells stimulated by 17β-estradiol and insulin-like growth factor 1

Estradiol (E₂) and its receptor estrogen receptor alpha (ERα) are implicated in the pathology of stromal‐predominant benign prostatic hyperplasia (BPH). Insulin‐like growth factor 1(IGF1) is produced primarily by stromal cells in the prostate. Recent study showed that E₂‐mediated regulation of IGF1 in mouse uterus requires the DNA binding ability of ERα. However, the crosstalk between ERα and IGF1 in the prostate has not been addressed yet. Therefore, in this study we employed mouse prostatic smooth muscle cells (PSMCs) as a model to demonstrate that E₂ stimulated the proliferation of PSMCs and up‐regulated the expression of IGF1 and its receptor IGF1R as well as cyclin D1 in PSMCs, all of which could be inhibited by shRNA‐mediated knockdown of ERα. Furthermore, we found that exogenous IGF1 could not promote cell proliferation and cyclin D1 expression in PSMCs subjected to shRNA‐mediated knockdown of ERα. Interestingly, blockage of IGF1R by antibody could inhibit E₂‐stimulated PSMCs proliferation. Altogether our present study provides the first line of evidence that there is crosstalk between ERα and IGF1 signaling in PSMCs proliferation in which ERα up‐regulates the expression of IGF1 and IGF1R, and IGF1 signaling is indispensable to mediate downstream effects of E₂ and ERα. Copyright © 2011 John Wiley & Sons, Ltd.     

GTP-binding protein-independent potentiation by mastoparan of IL-1β-induced nitric oxide release from insulin-secreting HIT-T15 cells

Our recent data implicated small molecular weight G-proteins (e.g., H-Ras) in interleukin 1beta (IL 1beta)-induced metabolic dysfunction and apoptotic demise of the islet beta cell (Tannous et al., Biochem Pharmacol 2001; 62:1459-1468, Kowluru and Morgan, Biochem Pharmacol, 2002; 63:1027-1035, Chen et al. Biochem Pharmacol, 2003; 66:1681-1694). Recently, we have shown that mastoparan, a tetradecapeptide from wasp venom, has been shown to directly activate islet endogenous G-proteins and regulate islet function (Amin et al., Endocrinology 2003; 144: 4508-4518). Herein, we investigated potential contributory roles, if any, of mastoparan (Mas)-sensitive G-proteins in IL-induced nitric oxide (NO) release from insulin-secreting HIT-T15 cells. While, ineffective by itself, Mas significantly potentiated IL-induced NO release from HIT-T15 cells. Interestingly, Mas-17, an inactive analog of Mas, also potentiated IL-induced NO release, suggesting that the potentiating effect of Mas may not involve activation of specific G-proteins. Such potentiating effects on IL-induced NO release were also demonstrable in the presence of another polycationic compound, melittin. Together, these findings suggest that Mas-induced potentiation of IL-induced NO release may in part be due to its amphiphilic and polycationic nature. These data also warrant caution in the use of Mas to study its regulation of cellular function without the use of an appropriate negative control, such as Mas-17.     

Cleavage of chitinous elicitors from the ectomycorrhizal fungus Hebeloma crustuliniforme by host chitinases prevents induction of K+ and Cl− release, extracellular alkalinization and H2O2 synthesis of Picea abies cells

Rapid reactions comprising efflux of K⁺ and Cl⁻, phosphorylation of a 63-kDa protein (pp63), extracellular alkalinization and synthesis of H₂O₂ are equally induced in cells of Picea abies (L.) Karst. by chitotetraose, colloidal chitin and cell wall elicitors from the ectomycorrhizal fungus Hebeloma crustuliniforme (Bull. ex Fries.) Quél. an ectomycorrhizal partner of spruce. Cleavage of fungal cell wall elicitors and of artificial chitin elicitors to monomeric and dimeric fragments by apoplasmic spruce chitinases (36-kDa class I chitinase, pI 8.0, and 28-kDa chitinase, pI 8.7; EC 3.2.1.14) equally prevented induction of these rapid reactions. Also, N-acetylglucosamine oligomers and elicitors from the fungal cell walls showed a similar dependence of their activity on the degree of polymerisation. From these results it is suggested that, during ectomycorrhiza formation, only some of the chitin-derived elicitors reach their receptors at the plant plasma membrane, initiating reactions of the hypersensitive response in the host cells. The remaining fungal elicitors will be degraded to varying extents by wall-localized chitinases of the host root, reducing the defence reactions of the plant and allowing symbiotic interactions of both organisms. Received: 6 January 1997 / Accepted: 14 March 1997     

Modulation of islet G-proteins, α-glucosidehydrolase inhibition and insulin release stimulated by various secretagogues

Guanine nucleotide-binding proteins (G-proteins) are known to act as important modulators of insulin release from the islets of Langerhans. We have recently found that the deoxynojirimycin-derivative emiglitate, a recognized inhibitor of intestinal -glucosidehydrolase activity, is a powerful inhibitor of glucose-induced insulin release. With the use of isolated mouse islets the present investigation was performed in a primary attempt to elucidate whether this inhibitory mechanism in some way was linked to the -cell G-protein system. Treatment of freshly isolated islets with pertussis toxin (PTX), which is known to inactivate the G_i-proteins, abolished the inhibitory effect of the ₂-adrenoceptor agonist clonidine on insulin release stimulated by the phosphodiesterase inhibitor IBMX in the presence of the protein kinase C activator TPA and even changed it into an increase. Emiglitate did not display any inhibitory action on insulin release induced by these secretagogues. Similarly, clonidine-induced inhibition of glucose stimulated insulin release was reversed by PTX. However, PTX did not influence the suppressive action of emiglitate on glucose-induced insulin secretion. In contrast, the adenylate cyclase activator forskolin totally abolished the inhibitory effect of emiglitate, but not that of the glucose analogue mannoheptulose, on glucose-induced insulin release. Moreover, the stimulatory effect of forskolin and cholera toxin (CTX) (activator of G_s-proteins) on the secretion of insulin was markedly enhanced in the presence of emiglitate. In conclusion, our results suggest that the inhibitory effect of emiglitate on glucose-induced insulin release is not directly related to the G_j-proteins, but most likely exerted solely through the selective suppression of lysosomal -glucosidehydrolase activity, a step in between the proximal and the distal G_i-proteins, in glucose-induced stimulus-secretion mechanisms. Our data also suggests that the inhibitory action of emiglitate on glucose stimulated insulin release can be compensated for by an increased sensitivity of the cyclic AMP-protein kinase A pathway. Hence, emiglitate might indirectly elicit an increased activity of the G_s-proteins to facilitate the secretory process.     

Conversion of pancreatic α cells into insulin-producing cells modulated by β-cell insufficiency and supplemental insulin administration

The emergence of bihormonal (BH) cells expressing insulin and glucagon has been reported under diabetic conditions in humans and mice. Whereas lineage tracing studies demonstrated that glucagon-producing α cells can be reprogrammed into BH cells, the underlying dynamics of the conversion process remain poorly understood. In the present study, we investigated the identities of pancreatic endocrine cells by genetic lineage tracing under diabetic conditions. When β-cell ablation was induced by alloxan (ALX), a time-dependent increase in BH cells was subsequently observed. Lineage tracing experiments demonstrated that BH cells originate from α cells, but not from β cells, in ALX-induced diabetic mice. Notably, supplemental insulin administration into diabetic mice resulted in a significant increase in α-cell-derived insulin-producing cells that did not express glucagon. Furthermore, lineage tracing in Ins2^Akita diabetic mice demonstrated a significant induction of α-to-β conversion. Thus, adult α cells have plasticity, which enables them to be reprogrammed into insulin-producing cells under diabetic conditions, and this can be modulated by supplemental insulin administration.     

U-73122 inhibits carbachol-induced increases in [Ca2+]i,IP3, and insulin release in β-TC3 cells

We studied the effects of the aminosteroid U-73122, a putative phospholipase C (PLC) inhibitor, on carbachol-induced increases in insulin release, [Ca²⁺]_i, and IP₃ in β-TC3 cells. Carbachol (0.1–100 μM) increased [Ca²⁺]_i and carbachol (0.1–1000 μM) increased insulin release dose-dependently. Carbachol (100 μM) also increased inositol 1,4,5-trisphosphate (IP₃) production. U-73122 (2–12 νM) inhibited the effects of carbachol on [Ca²⁺]_i and insulin release in a dose-dependent manner, and at the highest dose studied (12 μM) it abolished or greatly attenuated all three effects of carbachol. In contrast, U-73343 (12 μM), the analog of U-73122 that does not inhibit PLC, only inhibited the effect of carbachol on [Ca²⁺]_i by 20% and did not inhibit the effect of carbachol on insulin release. Since carbachol increased IP₃, [Ca²⁺]_i, and insulin release by activating PLC, these results suggested that U-73122 inhibits phospholipase C-depenent processes in β-TC3 cells.     

The release of soluble VAP-1/SSAO by 3T3-L1 adipocytes is stimulated by isoproterenol and low concentrations of TNFα

Plasma level of the protein VAP-1/SSAO (Vascular Adhesion Protein-1/Semicarbazide-Sensitive Amine Oxidase) is increased in diabetes and/or obesity and may be related to vascular complications associated to these pathologies. The aim of this work was to complete a preceding study where we described the role played by some hormones or metabolites, implicated in diabetes and/or obesity, in the regulation of the release of VAP-1/SSAO by 3T3-L1 adipocytes. Here we focused on the previously observed effect produced by TNFalpha in the release of VAP-1/SSAO and studied the effect of a beta-adrenergic compound, isoproterenol. Both compounds stimulated the release of VAP-1/SSAO to the culture medium but had a different effect on the VAP-1/SSAO membrane form. While TNFalpha produced a decrease on VAP-1/SSAO membrane form content, isoproterenol did not modify it. We thus observed two different ways of regulation of the release of VAP-1/SSAO by 3T3-L1 adipocytes by metabolites implicated in diabetes and adipose tissue physiopathology. Our work permits a better understanding of this increased plasma VAP-1/SSAO levels observed in diabetes.     

In vitro angiogenesis is modulated by the mechanical properties of fibrin gels and is related to αvβ3 integrin localization

Summary This study deals with the role of the mechanical properties of matrices in in vitro angiogenesis. The ability of rigid fibrinogen matrices with fibrin gels to promote capillarylike structures was compared. The role of the mechanical properties of the fibrin gels was assessed by varying concentration of the fibrin gels. When the concentration of fibrin gels was decreased from 2 mg/ml to 0.5 mg/ml, the capillarylike network increased. On rigid fibrinogen matrices, capillarylike structures were not formed. The extent of the capillarylike network formed on fibrin gels having the lowest concentration depended on the number of cells seeded. The dynamic analysis of capillarylike network formation permitted a direct visualization of a progressive stretching of the 0.5 mg/ml fibrin gels. This stretching was not observed when fibrin concentration increases. This analysis shows that 10 h after seeding, a prearrangement of cells into ringlike structures was observed. These ringlike structures grew in size. Between 16 and 24 h after seeding, the capillarylike structures were formed at the junction of two ringlike structures. Analysis of the α_vβ₃ integrin localization demonstrates that cell adhesion to fibrinogen is mediated through the α_vβ₃ integrin localized into adhesion plaques. Conversely, cell adhesion to fibrin shows a diffuse and dot-contact distribution. We suggest that the balance of the stresses between the tractions exerted by the cells and the resistance of the fibrin gels triggers an angiogenic signal into the intracellular compartment. This signal could be associated with modification in the α_vβ₃ integrin distribution.