Differential effect of FGF and PDGF on cell proliferation and migration in osteoblastic cells

It has been known that growth factors such as fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF) can promote proliferation and migration in a variety of cell types including osteoblastic cells. However, the mechanism underlying their action has not been clearly defined. The present study was undertaken to examine the effect of FGF and PDGF on cell proliferation and migration and to determine the role of extracellular signal-regulated kinase (ERK) and Akt in action of FGF and PDGF in osteoblastic cells. FGF enhanced proliferation in a dose- and time-dependent manner, whereas it did not affect cell migration. FGF induced a transient activation of ERK, but not Akt, which was inhibited by an inhibitor of MEK, the upstream kinase of ERK, but not by inhibitors of PI3K/Akt (LY294002), epidermal growth factor receptor (EGFR, AG1478), and Src (PP2). FGF-induced proliferation was inhibited by inhibitors of MEK/ERK and Src pathways. Exposure of cells to FGF stimulated transition of cell cycle from the G1 phase to S phase and increased phosphorylation of Rb. FGF-induced phosphorylation of Rb was attenuated by inhibitors of MEK/ERK and Src pathways. Cell migration studies indicated that PDGF stimulated migration, but it had no effect on cell proliferation. PDGF induced activation of ERK and Akt. The ERK activatin was inhibited by the Src inhibitor and the Akt activation was inhibited by inhibitors of EGFR and Src. PDGF-induced migration was inhibited by inhibitors of MEK/ERK, PI3K/Akt, EGFR and Src pathways. Taken together, these findings suggest that the MEK/ERK and Src pathways play an important role in the FGF-induced proliferation and signaling pathways involving MEK/ERK, EGFR, Src and PI3K/Akt mediate the PDGF-induced migration. These data are of importance in understanding the roles of these growth factors in osteoblastic cell proliferation and migration.     

Calpain-mediated regulation of the distinct signaling pathways and cell migration in human neutrophils

We studied the mechanisms underlying calpain inhibition-mediated human neutrophil migration. MAPKs, including ERK, p38, and JNK, MEK1/2, MAPK kinase 3/6 (MKK3/6), PI-3K/Akt, c-Raf, and p21-activated kinase (PAK; an effector molecule of Rac) were rapidly (within 30 s) activated in neutrophils upon exposure to calpain inhibitors (PD150606 and N-acetyl-Leu-Leu-Nle-CHO) but not PD145305 (inactive analog of PD150606). Following activation of these pathways, neutrophils displayed active migration (chemotaxis), which was sustained for more than 45 min. The studies with pharmacological inhibitors suggest that calpain inhibition-mediated neutrophil migration is mediated by activation of MEK/ERK, p38, JNK, PI-3K/Akt, and Rac. NSC23766 (Rac inhibitor) and pertussis toxin (PTX) suppressed calpain inhibitor-induced phosphorylation of distinct signaling molecules (PAK, c-Raf, MEK1/2, ERK, MKK3/6, p38, JNK, and Akt) as well as cell migration, suggesting that the PTX-sensitive G protein and Rac axis may be a possible key target of calpain inhibitors. Differentiated neutrophil-like HL-60 cells but not undifferentiated cells displayed cell migration and activation of MAPKs and PI-3K/Akt on calpain inhibition. These findings suggest that constitutively active calpain negatively regulates activation of the distinct signaling pathways and cell migration in resting neutrophils, and this regulatory system develops during differentiation into mature neutrophils.     

Sequential activation of Rap1 and Rac1 small G proteins by PDGF locally at leading edges of NIH3T3 cells

Moving cells form protrusions, such as filopodia and lamellipodia, and focal complexes at leading edges, which eventually enhance cell movement. The Rho family small G proteins, Rac1, Cdc42 and RhoA, are involved in the formation of these leading edge structures. We investigated the role of another small G protein Rap1 in the platelet-derived growth factor (PDGF)-induced formation of leading edge structures and cell movement. Upon stimulation of NIH3T3 cells by PDGF, leading edge structures were formed and Necl-5, integrin α_Vβ₃, and PDGF receptor were accumulated at leading edges. Rap1, upstream regulators of Rap1 such as Crk and C3G, and a downstream effector RalGDS, were accumulated at peripheral ruffles over lamellipodia. Over-expression of Rap1GAP, which inactivates Rap1, and knockdown of Rap1 inhibited the PDGF-induced formation of leading edge structures, accumulation of these molecules, and cell movement. In addition, Rap1 activation subsequently induced accumulation of Rac1, Vav2 and PAK at peripheral ruffles, which was inhibited by Rap1GAP and knockdown of Rap1. These results indicate that Rap1, activated by PDGF, is recruited to leading edges and that Rac1 is thereby activated locally at peripheral ruffles. This process is pivotal for the PDGF-induced formation of leading edge structures and cell movement.     

Prediction of Sphingosine 1-Phosphate-Stimulated Endothelial Cell Migration Rates Using Biochemical Measurements

The ability to predict endothelial cell migration rates may aid in the design of biomaterials that endothelialize following implantation. However, the complexity of the signaling response to migration-promoting stimuli such as sphingosine 1-phosphate (S1P) makes such predictions quite challenging. A number of signaling pathways impact S1P-mediated cell migration, including the Akt and Src pathways, which both affect activation of the small GTPase Rac. Rac activation promotes the formation of lamellipodia, and thus should be intimately linked to cell migration rates. In immortalized endothelial cells, expression of proteins that inhibit Akt, Src, and Rac (PTEN, CSK, and β2-chimaerin, respectively) was decreased using RNA interference, resulting in increases in the basal level of activation of Akt, Src, and Rac. Cells were scrape-wounded and stimulated with 1 μM S1P. The timecourse of Akt, Src, and Rac activation was followed over 2 h in the perturbed cells, while migration into the scrape wound was measured over 6 h. Rac activation at 120 min post-stimulation was highly correlated with the mean migration rate of cells, but only in cells stimulated with S1P. Using partial least squares regression, the migration rate of cells into the scrape wound was found to be highly correlated with the magnitude of the early Akt peak (e.g., 5–15 min post-stimulation). These results demonstrated that biochemical measurements might be useful in predicting rates of endothelial cell migration.     

Partial characterization of a novel mitogen-activated protein kinase/extracellular signal-regulated kinase activator in airway smooth-muscle cells

We demonstrated previously that in bovine tracheal myocytes, pretreatment with either forskolin or histamine significantly reduces both platelet-derived growth factor (PDGF)- and epidermal growth factor- induced Raf-1 activation but fails to inhibit extracellular signal-regulated kinase (ERK) activation substantially, evidence of a Raf-1-independent ERK activation pathway. To identify Raf-1-independent upstream signaling intermediates of mitogen-activated protein kinase/ERK kinase-1 (MEK1), the dual-function kinase required and sufficient for ERK activation in these cells, lysates from forskolin and PDGF-treated bovine tracheal myocytes were resolved using ion exchange chromatography. Kinase activity for MEK1 was assessed by in vitro phosphorylation assay. In all experiments, the major peak of MEK1 phosphorylation activity was detected in fractions 18 through 26 (80 to 160 mM NaCl), with the peak fraction eluting at a NaCl concentration of 140 mM. The ability of these fractions to activate MEK1 was confirmed by examining the phosphorylation of myelin basic protein, a known substrate for ERKs, in the presence of functional MEK1 and ERK1. Fractions containing kinase activity were also probed with antibodies against MEK kinase-1, Raf-1, A-Raf, B-Raf, Mos, and Tpl-2. None of these proteins was detected in fractions containing peak kinase activity, suggesting the presence of a novel PDGF-stimulated, forskolin-insensitive MEK1 kinase. Further separation of fractions holding peak MEK phosphorylation activity by gel filtration suggested an apparent molecular mass of 40 to 45 kD. We conclude that PDGF-induced activation of MEK1 in bovine tracheal myocytes is mediated at least in part by a novel kinase.     

Src regulates phorbol 12-myristate 13-acetate-activated PKC-induced migration via Cas/Crk/Rac1 signaling pathway in glioblastoma cells

In this study, we demonstrate that phorbol 12-myristate 13-acetate (PMA)-activated protein kinase C (PKC) induced migration in A172 glioblastoma cells via Src. PMA treatment induced tyrosine phosphorylation of Crk-associated substrate (Cas) and formation of a complex with Crk, followed by Rac1 activation, a downstream effector of Cas/Crk complex. These effects were blocked by a tyrosine kinase inhibitor (PP2) or Src small interfering RNA (siRNA), indicating that Src was involved in the PMA-induced activation of Cas/Crk/Rac1 signaling pathway. An immunohistochemical study showed that after PMA treatment, Cas, Crk and Rac1 translocated into lamellipodia. Tyrosine phosphorylated Cas was also detected at the periphery of the cells, where focal complexes were prominent. These results indicated that signaling of Cas, Crk and Rac1 might be involved in PMA-induced cytoskeletal reorganization. Translocation of Rac1 to the cell membrane is known to be dependent on phosphorylation of tyrosine-221 residue of Crk. We demonstrated that PMA induced phosphorylation of Crk, and this phosphorylation was blocked by PP2 or Src siRNA. These results indicated that Src might regulate the subcellular localization of Rac1 through phosphorylation of Crk. We propose that PMA-induced migration was dependent on activation of PKC/Src/Cas/Crk/Rac1 signaling pathway via modulating cytoskeletal reorganization during glioblastoma cell migration.     

PI3K/Akt and ERK1/2 signalling pathways are involved in endometrial cell migration induced by 17beta-estradiol and growth factors

Cell motility and invasion are crucial events for endometrial cells, not only for the establishment of pathological states but also during the physiological tissue remodelling that occurs during the menstrual cycle and embryo implantation. We have characterized these phenomena in endometrial stromal cells evaluating cell migration-specific stimuli and the biochemical pathways involved. Ability of endometrial cells to migrate on collagen type IV substrate was evaluated by means of chemotaxis experiments. Modulation of this phenomenon by different growth factors and steroid hormones and their ability to activate extracellular signal-regulated protein kinase (ERK) and phosphatidylinositol 3 kinase (PI3K)/Akt signalling in this context were examined. Platelet-derived growth factor (PDGF)-BB, epidermal growth factor and fibroblast growth factor-2 as chemoattractant agents stimulated basal migration of endometrial stromal cells through the rapid activation of both ERK1/2 and PI3K/Akt signalling pathways. Experiments using wortmannin and PD98059, specific inhibitors of the PI3K/Akt and ERK1/2 activity, respectively, showed that the activation of both pathways is required for growth-factor-induced cell motility responses. Similarly, 17beta-estradiol (10(-6)-10(-8) M) could enhance both constitutive and PDGF-induced migration of the cells and their rapid treatment with the hormone significantly increased phosphorylation of ERK1/2 and Akt. Conversely, progesterone did not interfere with the basal migration but inhibits the PDGF-induced motility of this cell type. Rapid activation of intracellular signalling cascades ERK1/2 and PI3K/Akt by growth factors and estrogens is involved in the migration of normal endometrial stromal cells.     

Transforming growth factor-β stimulates IL-1β-induced monocyte chemoattractant protein-1 expression in human synovial cells via the ERK/AP-1 pathway

Objective and Design: Transforming growth factor- β (TGF-β) has not only a fibrogenic role, but also monocyte/ macrophage chemotactic properties in a synovial joint. However, little is known about the effects of TGF-β on monocyte chemoattractant protein-1 (MCP-1) expression in human synovial cells under inflammatory status. The aim of this study was to determine whether TGF-modulates MCP-1 production under the chronic inflammation, and to elucidate the cell signaling mechanism involved. Materials and methods: Human synovial cells were exposed to IL-1β, which mimics the environment of chronic inflammation. Production of MCP-1 protein and expression of MCP-1 mRNA were determined by ELISA and real-time PCR. Results: TGF-β upregulated the expression of MCP-1 mRNA and protein with or without IL-1β. TGF-β and IL-1β synergistically enhanced MCP-1 gene expression, and an AP-1 binding site was involved in the signal transduction. In addition, MEK inhibitor completely suppressed TGF-β-induced MCP-1 production. Conclusions: TGF-β and IL-1β synergistically enhance MCP-1 gene expression through the activation of the MEK/ERK1/2 pathways, which leads to AP-1 activation. The impairment of MCP-1 regulation by TGF-β in resident synovial cells might represent an important mechanism of chronic inflammation and tissue fibrosis in a synovial joint. MCP-1 should be considered a valid target for therapeutic intervention. Received 22 September 2005; returned for revision 3 February 2006; accepted by J. Skotnicki 29 May 2006     

Retinoic acid inhibits airway smooth muscle cell migration

Airway remodeling in chronic asthma is characterized by increased smooth muscle mass that is associated with the reduction of the bronchial lumen as well as airway hyperresponsiveness. The development of agents that inhibit smooth muscle growth is therefore of interest for therapy to prevent asthma-associated airway remodeling. All-trans retinoic acid (ATRA) suppresses growth of vascular smooth muscle cells (SMCs) from the systemic and pulmonary circulation. The present study investigated the effects of ATRA on human bronchial (airway) SMCs. Human bronchial SMCs were found to express mRNAs for retinoic acid receptor (RAR)-alpha, -beta, -gamma, and retinoid X receptor (RXR)-alpha, -beta, but not RXR-gamma. Although ATRA was not effective in inhibiting proliferation or in inducing apoptosis in airway SMCs, we found that ATRA (0.2-2 microM) inhibited the SMC migration in response to platelet-derived growth factor (PDGF), as determined in a modified Boyden chamber assay. Both RAR and RXR agonists also blocked PDGF-induced airway SMC migration. ATRA also inhibited PDGF-induced actin reorganization associated with migration. PDGF-induced actin reorganization and migration were blocked by inhibitors of phosphatidylinositol 3 kinase (PI3K) and Akt. However, migration was blocked by inhibitors of the MEK/ERK pathway, with no effect on cytoskeletal reorganization. ATRA suppressed PDGF-induced Akt activation without influencing ERK activation. RAR was found to form protein-protein interactions with the p85 PI3K subunit. These results suggest that retinoic acid inhibits airway SMC migration through the modulation of the PI3K/Akt pathway.     

Interaction and localization of Necl-5 and PDGF receptor beta at the leading edges of moving NIH3T3 cells: Implications for directional cell movement

It was previously shown that platelet-derived growth factor (PDGF) receptor physically and functionally interacts with integrin α_vβ₃, effectively inducing cell movement. We previously showed that Necl-5, originally identified as a poliovirus receptor, interacts with integrin α_vβ₃ and enhances its clustering and the formation of focal complexes at the leading edges of moving cells, resulting in an enhancement of cell movement. We showed here that Necl-5 additionally interacts with PDGF receptor in NIH3T3 cells and regulates the interaction between PDGF receptor and integrin α_vβ₃, effectively inducing directional cell movement. PDGF receptor co-localized with Necl-5 and integrin α_vβ₃ at peripheral ruffles over lamellipodia, which were formed at the leading edges of moving cells in response to PDGF, but not at the focal complexes under these ruffles, whereas Necl-5 and integrin α_vβ₃ co-localized at these focal complexes. The clustering of these three molecules at peripheral ruffles required the activation of integrin α_vβ₃ by vitronectin and the PDGF-induced activation of the small G protein Rac and subsequent re-organization of the actin cytoskeleton. These results indicate a key role of Necl-5 in directional cell movement by physically and functionally interacting with both integrin α_vβ₃ and PDGF receptor.     

Central role of protein kinase Cepsilon in constitutive activation of ERK1/2 and Rac1 in the malignant cells of hairy cell leukemia

We have previously identified the presence of Ras/Raf-independent constitutive activation of extracellular signal-regulated kinase (ERK) in the hairy cells (HCs) of hairy cell leukemia. The aim of the present study was to characterize the signaling components involved in this activation and their relationship to the reported activation of Rac1. We found that both Rac1 and ERK activation in HCs are downstream of active Src and protein kinase C (PKC). Inhibition with toxin B showed that Rac1 plays no role in ERK activation in HCs. However, toxin B inhibited p60src and the Rac1-GEF Vav, demonstrating a positive feedback/activation of p60src by Rac1. Treatment with specific small interfering RNA for various PKC isoforms, or with PKC isoform-specific inhibitors, demonstrated a central role for PKCepsilon in the constitutive activation of Rac1 and ERK in HCs. PKCepsilon and active ERK were mutually associated and co-localized with mitochondria in HCs. Furthermore, active PKCepsilon was nitrated on tyrosine, pointing to a reactive oxygen species-dependent mechanism of activation. By being involved in activation of ERK and Rac1, PKCepsilon plays roles in both the survival of HCs and in the cytoskeletal dynamics responsible for the distinctive morphology and tissue homing of these cells. Our study therefore describes novel aspects of signaling important for the pathogenesis of hairy cell leukemia.     

Inhibition of airway smooth muscle adhesion and migration by the disintegrin domain of ADAM-15

Disintegrin and metalloprotease proteins (ADAMs) are membrane-anchored glycoproteins involved in cell adhesion, cell fusion, protein ecto-domain shedding, and intracellular signaling. We examined whether the disintegrin domain of ADAM-15 (named ddADAM-15) containing an Asp-Gly-Asp (RGD) integrin-binding motif could interfere with airway smooth muscle cell (ASMC) adhesion and migration. Recombinant ddADAM-15 adhered to human ASMCs with saturation kinetics, and was beta(1)-integrin dependent. ddADAM-15 inhibited the binding of fibrinogen but not of fibronectin to ASMCs. ddADAM-15 also inhibited platelet-derived growth factor (PDGF)-induced ASMC migration, and this was reversed by an anti-beta(1)-integrin antibody. PDGF induced the activation of phosphoinositol-3-kinase (PI3K) and p38 mitogen-activated protein kinase (MAPK), and selective inhibitors of these kinases inhibited PDGF-induced ASMC migration. ddADAM-15 did not inhibit PDGF-induced activation of PI3K or p38, thereby excluding these kinase pathways as a mechanism by which ddADAM-15 inhibits ASMC migration. ADAM-15 mRNA and protein were expressed under basal conditions, and both gene and protein expression were inhibited by PDGF. In summary, ddADAM-15 inhibits ASMC adhesion and migration through the beta(1)-integrin, without modulating signaling pathways involved in ASMC migratory responses.     

Pivotal role of phosphoinositide-3 kinase in regulation of cytotoxicity in natural killer cells

The mitogen-activated protein kinase-extracellular signal-regulated kinase signaling element (MAPK-ERK) plays a critical role in natural killer (NK) cell lysis of tumor cells, but its upstream effectors were previously unknown. We show that inhibition of phosphoinositide-3 kinase (PI3K) in NK cells blocks p21-activated kinase 1 (PAK1), MAPK kinase (MEK) and ERK activation by target cell ligation, interferes with perforin and granzyme B movement toward target cells and suppresses NK cytotoxicity. Dominant-negative N17Rac1 and PAK1 mimic the suppressive effects of PI3K inhibitors, whereas constitutively active V12Rac1 has the opposite effect. V12Rac1 restores the activity of downstream effectors and lytic function in LY294002- or wortmannin-treated, but not PD98059-treated, NK cells. These results document a specific PI3K-->Rac1-->PAK1-->MEK-->ERK pathway in NK cells that effects lysis.     

Activation of Rac1-PI3K/Akt is required for epidermal growth factor-induced PAK1 activation and cell migration in MDA-MB-231 breast cancer cells

Epidermal growth factor (EGF) may increase cell motility,an event implicated in cancer cell invasion and metastasis.However,the underlying mechanisms for EGF-induced cell motility remain elusive.In this study,we found that EGF treatment could activate Ras-related C3 botulinum toxin substrate 1 (Rac1),PI3K/Akt and p21actived kinase (PAK1) along with cell migration.Ectopic expression of PAK1 K299R,a dominant negative PAK1 mutant,could largely abolish EGF-induced cell migration.Blocking PI3K/Akt signalling with LY294002 or Akt siRNA remarkably inhibited both EGF-induced PAK1 activation and cell migration.Furthermore,expression of dominant-negative Rac1 (T17N) could largely block EGF-induced PI3K/Akt-PAK1 activation and cell migration.Interestingly,EGF could induce a significant production of ROS,and N-acetyl-L-cysteine,a scavenger of ROS which abolished the EGF-induced ROS generation,cell migration,as well as activation of PI3K/Akt and PAK,but not Rac1.Our study demonstrated that EGF-induced cell migration involves a cascade of signalling events,including activation of Rac1,generation of ROS and subsequent activation of PI3K/Akt and PAK1.     

Dominant-negative soluble PDGF-beta receptor inhibits hepatic stellate cell activation and attenuates liver fibrosis

Hepatic fibrogenesis is a consequence of hepatic stellate cells that become activated and transdifferentiate into a myofibroblastic phenotype with the ability to proliferate and synthesize large quantities of extracellular matrix components. In this process, platelet-derived growth factor (PDGF) is the most potent stimulus for hepatic stellate cell proliferation and migration, and is overexpressed during active hepatic fibrogenesis. This cytokine binds to the PDGF receptor type beta, activates Ras and sequentially propagates the stimulatory signal sequentially via phosphorylation of Raf-1, MEK and the extracellular-signal regulated kinases ERK1/ERK2. Hepatic injury is associated with both increased autocrine PDGF signaling and upregulation of PDGF receptor. In this study, we report that a dominant-negative soluble PDGF-beta receptor consisting of a chimeric IgG containing the extracellular portion of the PDGF receptor type beta blocks HSC activation and attenuates fibrogenesis induced by ligation of the common bile duct in rats. In culture-activated hepatic stellate cells, the soluble receptor blocks phosphorylation of endogenous PDGF receptor, phosphorylation of the ERK1/EKR2 signal and reduces proliferative activities of HSC. In vivo, both the delivery of the purified soluble PDGF antagonist and the administration of adenoviruses expressing the artificial transgene were able to reduce significantly the expression of collagen and alpha-smooth muscle actin. Our results demonstrate that PDGF plays a critical role in the progression and initiation of experimental liver fibrogenesis, and suggest that early anti-PDGF intervention should have a therapeutical impact on the treatment of liver fibrogenesis.     

Trichostatin A,a histone deacetylase inhibitor,induces synergistic cytotoxicity with chemotherapy via suppression of Raf/MEK/ERK pathway in urothelial carcinoma

In this study, we aimed to investigate the antitumor effects of trichostatin A (TSA), an antifungal antibiotic that inhibits histone deacetylase (HDAC) family of enzymes, alone or in combination with anyone of the three chemotherapeutic agents (cisplatin, gemcitabine, and doxorubicin) for the treatment of human urothelial carcinoma (UC). Two high-grade human UC cell lines (T24 and NTUB1) were used. Cytotoxicity and apoptosis were assessed by MTT assay and flow cytometry, respectively. The expression of phospho-c-Raf, phospho-MEK1/2, and phospho-ERK1/2 was measured by western blotting. ERK siRNA knockdown and the specific MEK inhibitor U0126 were used to examine the role of Raf/MEK/ERK signaling pathway in combined cytotoxicity of TSA and chemotherapy. TSA co-treatment with any one of the three chemotherapeutic agents induced synergistic cytotoxicity (combination index <?1) and concomitantly suppressed chemotherapeutic drug-induced activation of Raf-MEK-ERK pathway. Combination of ERK siRNA knockdown and treatment with the specific MEK inhibitor (U0126) enhanced the cytotoxic effects of the chemotherapy on UC cells. These observations were confirmed in a xenograft nude mouse model. Moreover, activated Raf/MEK/ERK pathway was observed in human bladder UC specimens from patients with chemoresistant status. In conclusion, TSA elicits a synergistic cytotoxic response in combination with chemotherapy via targeting the Raf/MEK/ERK pathway. TSA elicits synergistic cytotoxic response in combination with three DNA-damaging drugs (cisplatin, gemcitabine, and doxorubicin). Activated Raf/MEK/ERK pathway is involved in chemoresistant mechanism of UC. Combining chemotherapeutic agents with HDAC inhibitor (TSA) or with targeting Raf/MEK/ERK pathway is promising to circumvent chemoresistance in UCs.     

叶酸通过下调ERK1/2信号通路抑制血管平滑肌细胞增殖和迁移

目的:探讨叶酸(folic acid,FA)对血管平滑肌细胞(VSMCs)增殖和迁移的影响及其机制。方法:取SD大鼠的主动脉,采用组织贴块法培养VSMCs,随机分组进行实验。采用CCK-8和Ed U法检测叶酸对VSMCs活力和增殖能力的影响。采用划痕实验和Transwell法检测叶酸对VSMCs迁移和侵袭的影响。采用Western blot法检测细胞增殖核抗原(PCNA)蛋白表达以及血小板源性生长因子受体(PDGFR)和细胞外信号调节激酶1/2(ERK1/2)蛋白的磷酸化水平。结果:叶酸抑制血小板源性生长因子(PDGF)诱导的VSMCs的活力,并呈浓度依赖性(P0.05)。叶酸抑制PDGF诱导的VSMCs的迁移,并呈浓度依赖性(P0.05)。叶酸降低PCNA表达和PDGFR磷酸化水平,并抑制PDGF激活的ERK1/2信号通路。结论:叶酸降低PDGF诱导的VSMCs PCNA和p-PDGFR蛋白水平,下调ERK1/2信号通路,从而抑制VSMCs的增殖和迁移。     

Signalling mechanisms in sphingosine 1-phosphate-promoted mesangial cell proliferation

BACKGROUND: The bioactive sphingolipid sphingosine 1-phosphate (S1P) is formed by the activation of sphingosine kinase (SPHK) in diverse stimuli, such as platelet-derived growth factor (PDGF). S1P acts not only as an extracellular mediator but also as an intracellular second messenger, resulting in the proliferation of various different types of cells. However, the signal transduction mechanism in S1P-induced proliferation of mesangial cells is poorly known. RESULTS: We examined the signalling mechanisms by which S1P and dihydro-S1P (DHS1P), another S1P receptor agonist, induce mesangial cell proliferation. We first observed that exogenous S1P/DHS1P had additive effects on the PDGF-promoted proliferation of mesangial cells. Treatment of mesangial cells with pertussis toxin almost completely inhibited S1P- and DHS1P-induced, and slightly inhibited PDGF-induced cell proliferation. Additionally, the ERK kinase inhibitor PD98059 partially blocked the proliferation of mesangial cells induced by all these ligands. N,N-dimethylsphingosine, a competitive inhibitor of SPHK, reduced PDGF-induced mesangial cell proliferation, whereas over-expression of SPHK promoted it. We also revealed that PDGF induces SPHK mRNA expression and SPHK activity, suggesting that SPHK, which links the PDGF to the S1P signalling cascade, is, at least in part, involved in PDGF-induced mesangial cell proliferation. Moreover, we found that extracellular S1P stimulates two S1P receptors, EDG3 and EDG5, which leads to cell proliferation and survival. CONCLUSIONS: The data show that S1P-induced mesangial cell proliferation is mediated by EDG-dependent and -independent signalling pathways. S1P may cooperate with PDGF to increase the proliferation of mesangial cells during pathophysiological processes.