Phosphatidylinositol 3-kinase activity is required for the expression of glial fibrillary acidic protein upon cAMP-dependent induction of differentiation in rat C6 glioma

Glial fibrillary acidic protein (GFAP) is an intermediate filament (IF) protein expressed upon maturation of astrocytes and upregulated during reactive astrogliosis. Its expression is modulated by several growth factors and hormones. Although an upregulation of intracellular cAMP is required for the induction of GFAP expression in astrocytes, little information is available on other downstream factors of the signal transduction pathways involved in the regulation of its expression. In this communication, we identified phosphatidylinositol 3-kinase (PI 3-K) as a necessary enzyme for GFAP expression in rat C6 glioma cells. Use of the specific PI 3-K inhibitors wortmannin and LY294002 and transfection of C6 cells with a dominant negative PI 3-K construct, resulting in a decrease of the enzymatic activity of PI 3-K, inhibited the cAMP-dependent expression of GFAP. Furthermore, confocal laser scanning microscopy demonstrated that inhibition of the PI 3-K activity by LY294002 or wortmannin concomitant with induction of differentiation changes the cellular distribution leading to a pericentrosomal localization of GFAP and an altered cell shape lacking process formation. We conclude that the expression and cellular distribution of GFAP is mediated through a PI 3-K-dependent mechanism.     

P2Y nucleotide receptor interaction with alpha integrin mediates astrocyte migration

Astrocytes become activated in response to brain injury, as characterized by increased expression of glial fibrillary acidic protein (GFAP) and increased rates of cell migration and proliferation. Damage to brain cells causes the release of cytoplasmic nucleotides, such as ATP and uridine 5'-triphosphate (UTP), ligands for P2 nucleotide receptors. Results in this study with primary rat astrocytes indicate that activation of a G protein-coupled P2Y(2) receptor for ATP and UTP increases GFAP expression and both chemotactic and chemokinetic cell migration. UTP-induced astrocyte migration was inhibited by silencing of P2Y(2) nucleotide receptor (P2Y(2)R) expression with siRNA of P2Y(2)R (P2Y(2)R siRNA). UTP also increased the expression in astrocytes of alpha(V)beta(3/5) integrins that are known to interact directly with the P2Y(2)R to modulate its function. Anti-alpha(V) integrin antibodies prevented UTP-stimulated astrocyte migration, suggesting that P2Y(2)R/alpha(V) interactions mediate the activation of astrocytes by UTP. P2Y(2)R-mediated astrocyte migration required the activation of the phosphatidylinositol-3-kinase (PI3-K)/protein kinase B (Akt) and the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathways, responses that also were inhibited by anti-alpha(V) integrin antibody. These results suggest that P2Y(2)Rs and their associated signaling pathways may be important factors regulating astrogliosis in brain disorders.     

Lactosylceramide stimulates aortic smooth muscle cell proliferation.

We have investigated the effects of various sphingolipids on aortic smooth muscle cell proliferation employing viable cell counting, [3H] thymidine incorporation into DNA and the release of lactate dehydrogenase. Assays for UDP Gal: GlcCer Bl-4 galactosyltransferase (GalT-2) in control and treated cells were pursued simultaneously. Lactosylceramide stimulated cell proliferation in the order of 5 fold. Antibody against LacCer but not GbOse3Cer blocked the proliferative effects of LacCer in these cells. This phenomena was specific for aortic smooth muscle cells as LacCer decreased cell viability of aortic endothelial cells and had no effect on pulmonary endothelial cells. D-PDMP inhibited the activity of GalT-2 in smooth muscle cells and markedly prevented cell proliferation. In contrast, L-PDMP stimulated the activity of GalT-2 in smooth muscle cells and stimulated cell proliferation. Antibody against GalT-2 inhibited cell proliferation. Our findings suggest that the activation of GalT-2 leads to increased LacCer levels, which in turn, may be involved in aortic smooth muscle cell proliferation.     

Tumor necrosis factor receptor-1 can function through a G alpha q/11-beta-arrestin-1 signaling complex

Tumor necrosis factor-alpha (TNFalpha) is a proinflammatory cytokine secreted from macrophages and adipocytes. It is well known that chronic TNFalpha exposure can lead to insulin resistance both in vitro and in vivo and that elevated blood levels of TNFalpha are observed in obese and/or diabetic individuals. TNFalpha has many acute biologic effects, mediated by a complex intracellular signaling pathway. In these studies we have identified new G-protein signaling components to this pathway in 3T3-L1 adipocytes. We found that beta-arrestin-1 is associated with TRAF2 (TNF receptor-associated factor 2), an adaptor protein of TNF receptors, and that TNFalpha acutely stimulates tyrosine phosphorylation of G alpha(q/11) with an increase in G alpha(q/11) activity. Small interfering RNA-mediated knockdown of beta-arrestin-1 inhibits TNFalpha-induced tyrosine phosphorylation of G alpha(q/11) by interruption of Src kinase activation. TNFalpha stimulates lipolysis in 3T3-L1 adipocytes, and beta-arrestin-1 knockdown blocks the effects of TNFalpha to stimulate ERK activation and glycerol release. TNFalpha also led to activation of JNK with increased expression of the proinflammatory gene, monocyte chemoattractant protein-1 and matrix metalloproteinase 3, and beta-arrestin-1 knockdown inhibited both of these effects. Taken together these results reveal novel elements of TNFalpha action; 1) the trimeric G-protein component G alpha(q/11) and the adapter protein beta-arrestin-1 can function as signaling molecules in the TNFalpha action cascade; 2) beta-arrestin-1 can couple TNFalpha stimulation to ERK activation and lipolysis; 3) beta-arrestin-1 and G alpha(q/11) can mediate TNFalpha-induced phosphatidylinositol 3-kinase activation and inflammatory gene expression.     

Species differences in reactivity of mouse and rat astrocytes in vitro

Reactive astrogliosis constitutes a major obstacle to neuronal regeneration and is characterized by rearrangement and upregulation of expression of cytoskeletal proteins, increased proliferation and hypertrophy. Many approaches have been attempted to mimic astrogliosis by inducing reactive astrocytes in vitro. Such research is usually performed using astrocytes derived from Mus musculus or Rattus norvegicus, and results compared between species on the assumption that these cells behave equivalently. Therefore, we compared reactivity between mouse and rat astrocytes in scratch wound assays to gain further insight into how comparable these cell culture models are. Proliferation and migration, as well as expression of the cytoskeletal proteins glial fibrillary acidic protein (GFAP) and vimentin, were compared by immunocytochemistry and immunoblot. Further, we investigated migration of proliferating cells by 5-ethynyl-2'-deoxyuridine staining. Substantial differences in GFAP expression and proliferation between astrocytes of the two species were found: rat astrocytes showed different cytoskeletal morphology, expressed significantly more GFAP and vimentin of different molecular size and were more proliferative than comparable mouse astrocytes. Our results suggest that rat and mouse astrocytes may respond differently to various reactivity-triggering stimuli, which needs to be considered when general conclusions are drawn regarding effects of factors regulating astrocyte reactivity.     

Src and Pyk2 mediate angiotensin II effects in cultured rat astrocytes

Angiotensin II (Ang II)-induced proliferation of rat astrocytes is mediated by multiple signaling pathways. In the present study, we investigated the role of non-receptor tyrosine kinases on Ang II-signaling and proliferation of astrocytes cultured from neonatal rat pups. Ang II stimulated astrocyte growth, ERK1/2 phosphorylation and the phosphorylation of Src and proline-rich tyrosine kinase-2 (Pyk2), in astrocytes obtained from brainstem and cerebellum. Pretreatment with 10 microM PP2, a selective Src inhibitor, inhibited Ang II stimulated ERK1/2 phosphorylation by 59% to 91% both in brainstem and cerebellum astrocytes. PP2 also inhibited Ang II induction of brainstem (76% inhibition) and cerebellar (64% inhibition) astrocyte growth. Similarly, pretreatment with 25 microM dantrolene, the Pyk2 inhibitor, attenuated ERK1/2 activity in brainstem (62% inhibition) and in cerebellum astrocytes (44% inhibition). Interestingly, inhibition of Pyk2 inhibited Ang II-induced Src activation suggesting that these two non-receptor tyrosine kinases may be acting in concert to mediate Ang II effects in astrocytes. In summary, we found that Ang II stimulates the non-receptor tyrosine kinases Src and Pyk2 which mediate Ang II-induced ERK1/2 activation leading to stimulation of astrocyte growth. In addition, these two tyrosine kinases may be interacting to regulate effects of the peptide in these cells.     

Class I phosphatidylinositol 3-kinase inhibitor LY294002 activates autophagy and induces apoptosis through p53 pathway in gastric cancer cell line SGC7901

We aimed to study the effects of LY294002, an inhibitor of class I phosphatidylinositol 3-kinase (PI3K), on proliferation, apoptosis, and autophagy in gastric cancer cell line SGC7901. In this study, we showed that LY294002 inhibited the viability of gastric cancer SGC7901 cells. We also showed that LY294002 increased the expression of microtubule-associated protein 1 light chain 3 (LC3), and increased monodansylcadaverine (MDC)-labeled vesicles. LY294002 activated autophagy by activating p53 and caspase-3, and induced apoptosis by up-regulatingp53 and p53-up-regulated modulator of apoptosis ( PUMA ). Therefore, LY294002 might induce cytotoxicity in SGC7901 cells through activation of p53 and the downstream point PUMA . These findings suggest that inhibition of the class I PI3K signaling pathway is a potential strategy for managing gastric cancers.     

Oscillatory Flow-induced Proliferation of Osteoblast-like Cells Is Mediated by ��v��3 and ��1 Integrins through Synergistic Interactions of Focal Adhesion Kinase and Shc with Phosphatidylinositol 3-Kinase and the Akt/mTOR/p70S6K Pathway

Interstitial flow in and around bone tissue is oscillatory in nature and affects the mechanical microenvironment for bone cell growth and formation. We investigated the role of oscillatory shear stress (OSS) in modulating the proliferation of human osteoblast-like MG63 cells and its underlying mechanisms. Application of OSS (0.5 ± 4 dynes/cm²) to MG63 cells induced sustained activation of phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR/p70S6K (p70S6 kinase) signaling cascades and hence cell proliferation, which was accompanied by increased expression of cyclins A and D1, cyclin-dependent protein kinases-2, -4, and -6, and bone formation-related genes (c-fos, Egr-1, and Cox-2) and decreased expression of p21^CIP1 and p27^KIP1. OSS-induced activation of PI3K/Akt/mTOR/p70S6K and cell proliferation were inhibited by specific antibodies or small interference RNAs of α_vβ₃ and β₁ integrins and by dominant-negative mutants of Shc (Shc-SH2) and focal adhesion kinase (FAK) (FAK(F397Y)). Co-immunoprecipitation assay showed that OSS induces sustained increases in association of Shc and FAK with α_vβ₃ and β₁ integrins and PI3K subunit p85, which were abolished by transfecting the cells with FAK(F397Y) or Shc-SH2. OSS also induced sustained activation of ERK, which was inhibited by the specific PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and was required for OSS-induced activation of mTOR/p70S6K and proliferation in MG63 cells. Our findings provide insights into the mechanisms by which OSS induces osteoblast-like cell proliferation through activation of α_vβ₃ and β₁ integrins and synergistic interactions of FAK and Shc with PI3K, leading to the modulation of downstream ERK and Akt/mTOR/p70S6K pathways.     

Growth hormone-stimulated insulin-like growth factor-1 expression in rainbow trout (Oncorhynchus mykiss) hepatocytes is mediated by ERK, PI3K-AKT, and JAK-STAT

Growth hormone (GH) initiates many of its growth-promoting actions by binding to GH receptors (GHR) and stimulating the synthesis and secretion of insulin-like growth factor-1 (IGF-1) from the liver and other sites. In this study, we used hepatocytes isolated from rainbow trout as a model system in which to determine the molecular signaling events of GH in fish. GH directly stimulated the phosphorylation of ERK, protein kinase B (Akt), a downstream target of phosphatidylinositol 3-kinase (PI3K), JAK2, and STAT5 in hepatocytes incubated in vitro. Activation of ERK, Akt, JAK2, and STAT5 was rapid, occurring within 5-10 min, and was concentration dependent. GH-induced ERK activation was completely blocked by the ERK pathway inhibitor, U0126, and the JAK2 inhibitor, 1,2,3,4,5,6-hexabromocyclohexane (Hex), and was partially blocked by the PI3K inhibitor LY294002. GH-stimulated Akt activation was completely blocked by LY294002 and Hex, but was not affected by U0126; whereas, STAT5 activation by GH was blocked only by Hex, and was not affected by either U0126 or LY294002. GH stimulated hepatic expression of IGF-1 mRNA as well as the secretion of IGF-1, effects that were partially or completely blocked by U0126, LY294002, and Hex. These results indicate that GHR linkage to the ERK, PI3K/Akt, or STAT pathways in trout liver cells requires activation of JAK2, and that GH-stimulated IGF-1 synthesis and secretion is mediated through the ERK, PI3K/Akt, and JAK-STAT pathways.     

Effect of 6-hydroxydopamine (6-OHDA) on proliferation of glial cells in the rat cortex and striatum: evidence for de-differentiation of resident astrocytes

Reactive astrogliosis is the universal response to any brain insult. It is characterized by cellular hypertrophy, up-regulation of the astrocyte marker glial fibrillary acidic protein (GFAP), and proliferation. The source of these proliferating cells is under intense debate. Progenitor cells derived from the subventricular zone (SVZ), cells positive for chondroitin sulfate proteoglycan (NG2(+)), and de-differentiated astrocytes have been proposed as the origin of proliferating cells following injury. We have analyzed the effect of intraventricular-applied 6-hydroxydopamine (6-OHDA) on the proliferation and morphology of astrocytes in rat cortex and striatum by means of immunohistochemistry and confocal laser microscopy. At 4 days post-lesion, GFAP expression increased markedly. A subpopulation of the GFAP(+) cells co-expressed Ki-67, indicating that these cells were proliferating. To investigate whether these cells (1) arose from migrating SVZ progenitor cells, (2) derived from NG2(+) progenitor cells, or (3) de-differentiated from resident astrocytes, we studied the expression of the migration marker doublecortin (Dcx), the oligodendrocyte progenitor marker NG2, and the progenitor markers Nestin and Pax6. The proliferating Ki-67(+) cells co-expressed Nestin and Pax6, whereas no co-expression of Ki-67 with NG2 or the migration marker Dcx was observed. Thus, resident astrocytes de-differentiate, in response to the intraventricular application of 6-OHDA, to a phenotype resembling radial glia cells, which represent transient astrocyte precursors during development. An understanding of the mechanisms of the de-differentiation of mature astrocytes might be useful for designing new approaches to cell therapy in neurodegenerative diseases such as Parkinson's disease.     

Centaurin-alpha1 is a phosphatidylinositol 3-kinase-dependent activator of ERK1/2 mitogen-activated protein kinases

Centaurin-alpha1 is known to be a phosphatidylinositol 3,4,5-triphosphate (PIP3)-binding protein that has two pleckstrin homology domains and a putative ADP ribosylation factor GTPase-activating protein domain. However, the physiological function of centaurin-alpha1 is still not understood. Here we have shown that transient expression of centaurin-alpha1 in COS-7 cells results in specific activation of ERK, and the activation is inhibited by co-expression of a dominant negative form of Ras. We have also found that a mutant form of centaurin-alpha1 that is unable to bind PIP3 fails to induce ERK activation and that a phosphatidylinositol 3-kinase inhibitor LY294002 inhibits centaurin-alpha1-dependent ERK activation. Furthermore, transient knockdown of centaurin-alpha1 by small interfering RNAs results in reduced ERK activation after epidermal growth factor stimulation in T-REx 293 cells. These results suggest that centaurin-alpha1 contributes to ERK activation in growth factor signaling, linking the PI3K pathway to the ERK mitogen-activated protein kinase pathway through its ability to interact with PIP3.     

p75NTR Promotes Astrocyte Proliferation in Response to Cortical Stab Wound

Astrogliosis after brain trauma can have a significant impact on functional recovery. However, little is known about the mechanisms underlying astrocyte proliferation and subsequent astrogliosis. In this study, we established a cortical stab wound injury mouse model and observed dramatic astrocyte activation and nerve growth factor receptor (p75NTR) upregulation near the lesion. We also found profound alterations in the cell cycle of astrocytes near the lesion, with a switch from a mitotically quiescent (G0) phase to the G2/M and S phases. However, no changes in the level of astrocyte apoptosis were observed. Cell cycle progression to the G2/M and S phases and CDK2 protein levels in response to cortical stab wound was inhibited after p75NTR knockdown in mouse astrocytes. Conversely, p75NTR overexpression in mouse astrocytes was sufficient in promoting cell cycle progression. In conclusion, our results suggested that p75NTR upregulation in astrocytes after brain injury induces cell cycle entry by promoting CDK2 expression and promoting astrocyte proliferation. Our findings provided a better understanding of astrocytic responses after cortical stab wound injury in mice.

     

MiR-144通过靶向抑制GRK5表达促进脊髓星形胶质细胞活化

已知miR-144与细胞活化和增殖有关,然而其具体分子机制尚不明确。本研究发现miR-144通过靶向GRK5促进脊髓星形胶质细胞的活化。运用real-time PCR检测脊髓损伤和正常大鼠的脊髓组织及其脊髓星形胶质细胞中miR-144的表达,发现与正常的组织和细胞相比,miR-144在脊髓损伤组织和星形胶质细胞中的表达水平显著降低;Western印迹检测到脊髓损伤大鼠的星形胶质细胞中GFAP蛋白的表达显著低于正常大鼠,而GRK5蛋白的表达高于正常大鼠;MTT分析结果显示转染miR-144可显著提高脊髓损伤大鼠的星形胶质细胞活性,但对细胞增殖无明显作用;酶活性试剂盒分析发现miR-144显著提高了SOD和GSH活性;生物学信息分析和萤光素酶报告基因检测结果显示miR-144能靶向结合GRK5,并下调GRK5的表达;MiR-144 mimic转染或miR-144 mimic与pcDNA-GRK5共转染脊髓损伤的星形胶质细胞,发现miR-144转染能通过激活NF-κB通路消除pcDNA-GRK5引起的细胞活化抑制。综上所述,miR-144通过靶定结合癌基因GRK5来促进脊髓星形胶质细胞细胞的活化。