PDGF-BB induces vascular smooth muscle cell expression of high molecular weight FGF-2, which accumulates in the nucleus

Basic fibroblast growth factor (FGF-2) and platelet-derived growth factor (PDGF) are implicated in vascular remodeling secondary to injury. Both growth factors control vascular endothelial and smooth muscle cell proliferation, migration, and survival through overlapping intracellular signaling pathways. In vascular smooth muscle cells PDGF-BB induces FGF-2 expression. However, the effect of PDGF on the different forms of FGF-2 has not been elucidated. Here, we report that treatment of vascular aortic smooth muscle cells with PDGF-BB rapidly induces expression of 20.5 and 21 kDa, high molecular weight (HMW) FGF-2 that accumulates in the nucleus and nucleolus. Conversely, PDGF treatment has little or no effect on 18 kDa, low-molecular weight FGF-2 expression. PDGF-BB-induced upregulation of HMW FGF-2 expression is controlled by sustained activation of extracellular signal-regulated kinase (ERK)-1/2 and is abolished by actinomycin D. These data describe a novel interaction between PDGF-BB and FGF-2, and indicate that the nuclear forms of FGF-2 may mediate the effect of PDGF activity on vascular smooth muscle cells.     

Neuroprotective effects of Argon are mediated via an ERK‐1/2 dependent regulation of heme‐oxygenase‐1 in retinal ganglion cells

Retinal ischemia and reperfusion injuries (R‐IRI) damage neuronal tissue permanently. Recently, we demonstrated that Argon exerts anti‐apoptotic and protective properties. The molecular mechanism remains unclear. We hypothesized that Argon inhalation exert neuroprotective effects in rats retinal ganglion cells (RGC) via an ERK‐1/2 dependent regulation of heat‐shock proteins. Inhalation of Argon (75 Vol%) was performed after R‐IRI on the rats′ left eyes for 1 h immediately or with delay. Retinal tissue was harvested after 24 h to analyze mRNA and protein expression of heat‐shock proteins ?70, ?90 and heme‐oxygenase‐1, mitogen‐activated protein kinases (p38, JNK, ERK‐1/2) and histological changes. To analyze ERK dependent effects, the ERK inhibitor PD98059 was applicated prior to Argon inhalation. RGC count was analyzed 7 days after injury. Statistics were performed using anova . Argon significantly reduced the R‐IRI‐affected heat‐shock protein expression (p < 0.05). While Argon significantly induced ERK‐1/2 expression (p < 0.001), inhibition of ERK‐1/2 before Argon inhalation resulted in significantly lower vital RGCs (p < 0.01) and increase in heme‐oxygenase‐1 (p < 0.05). R‐IRI‐induced RGC loss was reduced by Argon inhalation (p < 0.001). Immunohistochemistry suggested ERK‐1/2 activation in Müller cells. We conclude, that Argon treatment protects R‐IRI‐induced apoptotic loss of RGC via an ERK‐1/2 dependent regulation of heme‐oxygenase‐1.

     

Translocation of Exogenous FGF1 and FGF2 Protects the Cell against Apoptosis Independently of Receptor Activation

FGF1 and FGF2 bind to specific cell-surface tyrosine kinase receptors (FGFRs) and activate intracellular signaling that leads to proliferation, migration or differentiation of many cell types. Besides this classical mode of action, under stress conditions, FGF1 and FGF2 are translocated in a receptor-dependent manner via the endosomal membrane into the cytosol and nucleus of the cell. However, despite many years of research, the role of translocated FGF1 and FGF2 inside the cell remains unclear. Here, we reveal an anti-apoptotic activity of intracellular FGF1 and FGF2, which is independent of FGFR activation and downstream signaling. We observed an inhibition of cell apoptosis induced by serum starvation or staurosporine upon treatment with exogenous FGF1 or FGF2, despite the presence of highly potent FGFR inhibitors. Similar results were found when the tyrosine kinase of FGFR1 was completely blocked by a specific mutation. Moreover, the anti-apoptotic effect of the growth factors was abolished by known inhibitors of the translocation of FGF1 and FGF2 from the endosomes to the interior of the cell. Interestingly, FGF2 showed higher anti-apoptotic activity than FGF1. Since FGF2 is not phosphorylated by PKCδ and is present inside the nucleus longer than is FGF1, we speculated that the different activities could reflect their diverse nuclear export kinetics. Indeed, we observed that FGF1 mutations preventing binding to nucleolin and therefore phosphorylation in the nucleus affect the anti-apoptotic activity of FGF1. Taken together, our data indicate that the translocation of FGF1 and FGF2 protects cells against apoptosis and promotes cell survival.     

H2S transiently blocks IL‐6 expression in rheumatoid arthritic fibroblast‐like synoviocytes and deactivates p44/42 mitogen‐activated protein kinase

Sulfur bath therapy represents the oldest form of treatment for patients with different types of rheumatic disorders. However, scientific reports about the beneficial effects of this form of therapy are controversial, rare and of poor scientific quality. Also, little is known about the role and underlying molecular mechanisms of H₂S. Therefore, this topic encouraged us to investigate the influence of H₂S on fibroblasts isolated from the synovial membrane of RA (rheumatoid arthritis) patients. FLSs (fibroblast‐like synoviocytes) were treated with different concentrations of an exogenous H₂S donor (NaHS). At defined time points, secretion of IL‐6 was quantified by ELISA. Activation/deactivation of MAPKs (mitogen‐activated protein kinases), p38 and p44/42 MAPK (ERK1/2) were confirmed by Western blot experiments. FLSs constitutively express and secrete large quantities of IL‐6 and IL‐8. Data provided prove that, in FLSs, constitutive as well as IL‐1β‐induced expression of IL‐6 is transiently and partially down‐regulated by the short treatment of cells with low concentrations of NaHS. Another key finding is that H₂S deactivates p44/42 MAPK (ERK1/2). Long‐term exposure of FLSs to H₂S provides stimulatory effects, leading to reinforced activation of p38 MAPK and ERK1/2 accompanied by upregulation of IL‐6 expression. Presented data seem of importance for studying (patho‐) physiological functions of H₂S and also for re‐evaluating sulfur spa therapy as one of the oldest forms of therapy for rheumatic disorders.     

The CYP2E1 inhibitor DDC up-regulates MMP-1 expression in hepatic stellate cells via an ERK1/2- and Akt-dependent mechanism

DDC (diethyldithiocarbamate) could block collagen synthesis in HSC (hepatic stellate cells) through the inhibition of ROS (reactive oxygen species) derived from hepatocyte CYP2E1 (cytochrome P450 2E1). However, the effect of DDC on MMP-1 (matrix metalloproteinase-1), which is the main collagen degrading matrix metalloproteinase, has not been reported. In co-culture experiments, we found that DDC significantly enhanced MMP-1 expression in human HSC (LX-2) that were cultured with hepatocyte C3A cells either expressing or not expressing CYP2E1. The levels of both proenzyme and active MMP-1 enzyme were up-regulated in LX-2 cells, accompanied by elevated enzyme activity of MMP-1 and decreased collagen I, in both LX-2 cells and the culture medium. H₂O₂ treatment abrogated DDC-induced MMP-1 up-regulation and collagen I decrease, while catalase treatment slightly up-regulated MMP-1 expression. These data suggested that the decrease in ROS by DDC was partially responsible for the MMP-1 up-regulation. ERK1/2 (extracellular signal-regulated kinase 1/2), Akt (protein kinase B) and p38 were significantly activated by DDC. The ERK1/2 inhibitor (U0126) and Akt inhibitor (T3830) abrogated the DDC-induced MMP-1 up-regulation. In addition, a p38 inhibitor (SB203580) improved MMP-1 up-regulation through the stimulation of ERK1/2. Our data indicate that DDC significantly up-regulates the expression of MMP-1 in LX-2 cells which results in greater MMP-1 enzyme activity and decreased collagen I. The enhancement of MMP-1 expression by DDC was associated with H₂O₂ inhibition and coordinated regulation by the ERK1/2 and Akt pathways. These data provide some new insights into treatment strategies for hepatic fibrosis.     

Insulin-like growth factor (IGF)-II regulates CCAAT/enhancer binding protein alpha expression via phosphatidyl-inositol 3 kinase in human hepatoblastoma cell lines

To reveal growth factor and its signal pathway to CCAAT/enhancer binding protein alpha (C/EBPalpha) in hepatocyte differentiation, we used Huh-6 and HepG2, human hepatoblastoma (HBL) cell lines that maintain the expression of genes in hepatoblasts and remain at that stage of differentiation. Insulin-like growth factor (IGF)-II, hepatocyte growth factor (HGF), and dexamethasone (Dex) stimulated HBL cells for Northern blot analysis. Bromodeoxyuridine (BrdU) up-take assay and Western blot analysis on albumin was performed to unveil proliferation and differentiation activity of IGF-II. C/EBPalpha and phosphorylation of Akt were analyzed by Western blot analysis. LY294002 and wortmannin, specific inhibitors of PI3 kinase, and PD98059, a specific inhibitor of mitogen-activated protein (MAP) kinase, were used to examine the signaling pathway of C/EBPalpha upregulated by IGF-II. Luciferase assay was performed to study the promoter activity of C/EBPalpha. Actinomycin D was used to analyze half-life of C/EBPalpha mRNA. IGF-II up-regualted C/EBPalpha by Northern blot and Western blot while HGF and Dex did not by Northern blot. IGF-II promoted proliferation and differentiation by BrdU up-take assay and Western blot analysis on albumin. Akt phosphorylated by IGF-II, suggested that phosphatidyl-inositol (PI) 3 kinase mediated the signaling pathway of IGF-II. LY294002 and wortmannin suppressed expression of C/EBPalpha. IGF-II activated the promoter activity and prolonged half-life of mRNA, suggesting that IGF-II activated promoter and stabilized mRNA. LY294002 and wortmannin suppressed the promoter activity of C/EBPalpha while PD98059 did not, suggesting that activation of the promoter was mediated by PI3 kinase.     

Gastrin increases its own synthesis in gastrointestinal cancer cells via the CCK2 receptor

The involvement of the gastrointestinal hormone gastrin in the development of gastrointestinal cancer is highly controversial. Here we demonstrate a positive-feedback loop whereby gastrin, acting via the CCK2 receptor, increases its own expression. Such an autocrine loop has not previously been reported for any other gastrointestinal hormone. Gastrin promoter activation was dependent on the MAP kinase pathway and did not involve Sp1 binding sites or epidermal growth factor receptor transactivation. As the treatment of gastrointestinal cancer cells with amidated gastrin led to increased expression of non-amidated gastrins, the positive-feedback loop may contribute to the sustained increase in circulating gastrins observed in colorectal cancer patients.     

Nerve growth factor-induced expression of the GTP cyclohydrolase I gene via Ras/MEK pathway in PC12D cells

Neurotrophins are essential for the development and survival of the catecholaminergic neurons. GTP cyclohydrolase I (GCH) is the first and rate-limiting enzyme in the biosynthesis of 5,6,7,8-tertahydrobiopterin (BH4), the required cofactor for tyrosine hydroxylase. Previously, we reported that TH requires the Ras/mitogen-activated protein kinase kinase (MEK) pathway for its induction by nerve growth factor (NGF). Here, we examined intracellular signals required for NGF-induced expression of the GCH gene in PC12D cells. The activity of GCH was increased up to 5-fold after the NGF treatment, and the increase was repressed by pretreatment with U0126, an MEK1/2 inhibitor, but not with protein kinase A (PKA), phosphoinositide 3-kinase (PI3K), p38 mitogen-activated protein kinase (MAPK), and c-Jun NH2-terminal kinase (JNK) inhibitors. Induction of GCH mRNA by NGF was also abolished by pretreatment with U0126. The human GCH promoter activity was significantly enhanced by NGF treatment. Deletion analysis showed that the 465-bp 5'-flanking region is responsible for NGF-enhanced promoter activity. These data suggest that the Ras-MEK pathway is required for coordinate expression of the GCH and TH genes induced by neurotrophins.     

Activation of the MAPKs ERK1/2 by cell swelling in turbot hepatocytes

Background information. Activation of MAPKs (mitogen‐activated protein kinases), in particular ERK1/2 (extracellular‐signal‐regulated kinase 1/2), has been reported to take place in a large variety of cell types after hypo‐osmotic cell swelling. Depending on cell type, ERK1/2 phosphorylation can then serve or not the RVD (regulatory volume decrease) process. The present study investigates ERK1/2 activation after aniso‐osmotic stimulations in turbot hepatocytes and the potential link between phosphorylation of these proteins and RVD. Results. In turbot hepatocytes, Western‐blot analysis shows that a hypo‐osmotic shock from 320 to 240 mOsm·kg^?1 induced a rapid increase in ERK1/2 phosphorylation, whereas a hyper‐osmotic shock from 320 to 400 mOsm·kg^?1 induced no significant change in the phosphorylation of these proteins. The hypo‐osmotic‐induced ERK1/2 phosphorylation was significantly prevented when hypo‐osmotic shock was performed in the presence of the specific MEK (MAPK/ERK kinase) inhibitor PD98059 (100 μM). In these conditions, the RVD process was not altered, suggesting that ERK1/2 did not participate in this process in turbot hepatocytes. Moreover, the hypo‐osmotic‐induced activation of ERK1/2 was significantly prevented by breakdown of extracellular ATP by apyrase (10 units·ml^?1), by inhibition of purinergic P₂ receptors by suramin (100 μM) or by calcium depletion using EGTA (1 mM) and thapsigargin (1 μM). Conclusions. In turbot hepatocytes, hypo‐osmotic swelling but not hyper‐osmotic shrinkage induced the activation of ERK1/2. However, these proteins do not seem to be involved in the RVD process. Their hypo‐osmotic‐induced activation is partially due to cascades of signalling events triggered by the binding of released ATP on purinergic P₂ receptors and requires the presence of calcium.     

Microarray and phosphokinase screenings leading to studies on ERK and JNK regulation of connective tissue growth factor expression by angiotensin II 1a and bradykinin B2 receptors in Rat1 fibroblasts

Rat1 fibroblasts stably transfected with the rat angiotensin II (AngII) AT1a and bradykinin (BK) B2 receptor cDNAs gained the ability to bind Ang II and BK. Wild-type Rat1 cells bound neither ligand. Exposure to either effector led to characteristic Galphai and Galphaq signal cascades, the release of arachidonic acid (ARA), and the intracellular accumulation of inositol phosphates (IP). Microarray analyses in response to BK or AngII showed that both receptors markedly induce the CCN family genes, CTGF (CCN2) and Cyr61 (CCN1), as well as the vasculature-related genes, Cnn1 and Egr1. Real time PCR confirmed the increased expression of connective tissue growth factor (CTGF) mRNA. Combined sequence-based analysis of gene promoter regions with statistical prevalence analyses identified CREB, SRF, and ATF-1, downstream targets of ERK, and JNK, as prominent products of genes that are regulated by ligand binding to the BK or AngII receptors. The binding of AngII or BK markedly stimulated the phosphorylation and thus the activation of ERK2, JNK, and p38MAPK. A BKB2R and an AT1aR chimera which displayed only negligible G-protein-related signaling were constructed. Both mutant receptors continued to activate these kinases and stimulate CTGF expression. Inhibitors of ERK1/2 and JNK but not p38MAPK inhibited the BK- and AngII-stimulated expression of CTGF in cells expressing either the WT or mutant receptors, illustrating that ERK and JNK participate in the control of CTGF expression in a manner that appears to be independent of G-protein. Conversely, addition of BK or AngII to the cell line expressing WT AT1aR and BKB2R downregulated the expression of collagen alpha1(I) (COL1A1) mRNA. However, these effectors did not have this effect in cells expressing the mutant receptors. Thus, a robust G-protein related response is necessary for BK or AngII to affect COL1A1 expression.     

LncRNA TNK2‐AS1 regulated ox‐LDL‐stimulated HASMC proliferation and migration via modulating VEGFA and FGF1 expression by sponging miR‐150‐5p

Long non‐coding RNAs (lncRNAs) have been indicated for the regulatory roles in cardiovascular diseases. This study determined the expression of lncRNA TNK2 antisense RNA 1 (TNK2‐AS1) in oxidized low‐density lipoprotein (ox‐LDL)‐stimulated human aortic smooth muscle cells (HASMCs) and examined the mechanistic role of TNK2‐AS1 in the proliferation and migration of HASMCs. Our results demonstrated that ox‐LDL promoted HASMC proliferation and migration, and the enhanced proliferation and migration in ox‐LDL‐treated HASMCs were accompanied by the up‐regulation of TNK2‐AS1. In vitro functional studies showed that TNK2‐AS1 knockdown suppressed cell proliferation and migration of ox‐LDL‐stimulated HASMCs, while TNK2‐AS1 overexpression enhanced HASMC proliferation and migration. Additionally, TNK2‐AS1 inversely regulated miR‐150‐5p expression via acting as a competing endogenous RNA (ceRNA), and the enhanced effects of TNK2‐AS1 overexpression on HASMC proliferation and migration were attenuated by miR‐150‐5p overexpression. Moreover, miR‐150‐5p could target the 3’ untranslated regions of vascular endothelial growth factor A (VEGFA) and fibroblast growth factor 1 (FGF1) to regulate FGF1 and VEGFA expression in HASMCs, and the inhibitory effects of miR‐150‐5p overexpression in ox‐LDL‐stimulated HASMCs were attenuated by enforced expression of VEGFA and FGF1. Enforced expression of VEGFA and FGF1 also partially restored the suppressed cell proliferation and migration induced by TNK2‐AS1 knockdown in ox‐LDL‐stimulated HASMCs, while the enhanced effects of TNK2‐AS1 overexpression on HASMC proliferation and migration were attenuated by the knockdown of VEGFA and FGF1. Collectively, our findings showed that TNK2‐AS1 exerted its action in ox‐LDL‐stimulated HASMCs via regulating VEGFA and FGF1 expression by acting as a ceRNA for miR‐150‐5p.