Glypican-5 stimulates rhabdomyosarcoma cell proliferation by activating Hedgehog signaling

Glypican-5 (GPC5) is one of the six members of the glypican family. It has been previously reported that GPC5 stimulates the proliferation of rhabdomyosarcoma cells. In this study, we show that this stimulatory activity of GPC5 is a result of its ability to promote Hedgehog (Hh) signaling. We have previously shown that GPC3, another member of the glypican family, inhibits Hh signaling by competing with Patched 1 (Ptc1) for Hh binding. Furthermore, we showed that GPC3 binds to Hh through its core protein but not to Ptc1. In this paper, we demonstrate that GPC5 increases the binding of Sonic Hh to Ptc1. We also show that GPC5 binds to both Hh and Ptc1 through its glycosaminoglycan chains and that, unlike GPC3, GPC5 localizes to the primary cilia. Interestingly, we found that the heparan sulfate chains of GPC5 display a significantly higher degree of sulfation than those of GPC3. Based on these results, we propose that GPC5 stimulates Hh signaling by facilitating/stabilizing the interaction between Hh and Ptc1.     

HMGB1 enhances embryonic neural stem cell proliferation by activating the MAPK signaling pathway

Neural stem cells (NSCs) are involved in neural tube formation. As the high-mobility group box 1 (HMGB1) protein is involved in neurulation and is present at elevated levels in neural tube defects (NTDs) induced by hyperthermia, we have now investigated the effects of HMGB1 on proliferation, differentiation, and MAPK signaling pathways of NSCs in vitro. We constructed a lentivirus vector with HMGB1 siRNA and used it to infect NSCs. Down-regulation of HMGB1 expression was confirmed. Proliferation of NSCs was determined by MTS and nestin/BrdU double-labeling. Differentiation of NSCs was assessed using β-tubulinIII and GFAP. Knockdown of HMGB1 significantly suppressed NSC proliferation but hardly affected differentiation, which was regulated by decreased expression of MAPK signaling pathways. Thus, HMGB1 has beneficial effects on neurulation and may serve as a new target for the prevention of NTDs.     

Acupuncture induces adenosine in fibroblasts through energy metabolism and promotes proliferation by activating MAPK signaling pathway via adenosine3 receptor

Acupuncture has many advantages in the treatment of certain diseases as opposed to drug therapy. Besides, adenosine has been revealed to affect cellular progression including proliferation. Therefore, this study aimed at exploring the mechanism involving acupuncture stress and adenosine in fibroblast proliferation. The fibroblasts from fascia tissues of the acupoint area (Zusanli) were stimulated by different levels of stress, different concentrations of adenosine, and agonist or antagonist of A₃ receptor (A₃R) to investigate the effect of stress stimulation, adenosine, and adenosine-A₃R inhibition on fibroblasts. Then, the fibroblasts were treated with stress stimulation of 200 kPa or/and mitogen-activated protein kinase (MAPK) blocker. We revealed that stress stimulation and the binding of adenosine and A₃R promoted fibroblast proliferation in the fascial tissue, increased the expression of immune-related factors, adenosine and A₃R, and activated the MAPK signaling pathway. MAPK signaling pathway also directly affected the expression of adenosine, A₃R, and immune-related factors. Stress stimulation and adenosine treatment upregulated A₃R expression, and then activated the MAPK signaling pathway, which could in turn upregulate expression of adenosine, A₃R and immune-related factors, and promote cell proliferation. Adenosine is shown to form a positive feedback loop with the MAPK signaling pathway. Collectively, stress stimulation in vitro induces the increase of adenosine in fibroblasts through the energy metabolism and activation of the MAPK signaling pathway through A₃R, ultimately promoting fibroblast proliferation.     

蛋白激酶C-细胞外信号调节激酶1/2通路介导精氨酸升压素对成年大鼠心肌成纤维细胞的促增殖作用

精氨酸升压素(arginine vasopressin, AVP)是高血压和心力衰竭时激活的神经体液和血流动力学因子,同时,它还具有直接的生长刺激作用.我们以往的研究显示AVP可诱导新生大鼠心肌成纤维细胞(cardiac fibroblasts, CFs)增殖.本研究旨在进一步观察AVP是否对成年大鼠CFs具有促增殖作用,并探计其机制.采用组织块法培养成年大鼠CFs,用[3H]-TdR掺入法和流式细胞仪方法观察AVP作用下CFs的DNA合成和细胞周期分布.根据特异性底物髓磷脂基质蛋白(myelin basic protein, MBP)的磷酸化水平测定细胞外信号调节激酶1/2 (extracellular signal-regulated kinase 1/2, ERK1/2)的活性.用Western blot检测ERK1/2的磷酸化和p27Kip1、细胞周期蛋白D1、 A、 E的表达.结果显示,AVP(0.1μmol/L)可促进成年大鼠CFs的DNA合成,该作用可被V1受体拮抗剂d(CH2)5[Tyr2(Me),Arg8]-vasopressin (0.1μmol/L)阻断,而不受V2受体拮抗剂desglycinamide [d(CH2)5, D-Ile2, Ile4, Arg8]-vasopressin (0.1μmol/L)的影响.AVP可激活ERK1/2,用蛋白激酶C(protein kinase C, PKC)激动剂佛波酯(phorbol 12-myristate 13-acetate, PMA, 30nmol/L, 5min)急性刺激可模拟该作用,而PMA持续慢性作用(2.5μmol/L,24h)耗竭PKC后则抑制AVP对ERK1/2的激活.AVP可抑制p27Kip1的蛋白表达,升高细胞周期蛋白D1、 A和E的表达,同时促进细胞周期由G0/G1期进入S期.ERK1/2抑制剂PD98059 (30μmol/L)阻断AVP对DNA合成、p27Kip1、细胞周期蛋白D1、A和E蛋白表达的作用,并抑制细胞周期进程.以上结果表明,AVP可促进成年大鼠CFs增殖,该作用由V1受体和PKC-ERK1/2通路介导.AVP可通过ERK1/2调控p27Kip1、细胞周期蛋白D1、A和E的表达,从而促进成年大鼠CFs的细胞周期进程.     

Notch1 signaling stimulates proliferation of immature cardiomyocytes

The identification of the molecular mechanisms controlling cardiomyocyte proliferation during the embryonic, fetal, and early neonatal life appears of paramount interest in regard to exploiting this information to promote cardiac regeneration. Here, we show that the proliferative potential of neonatal rat cardiomyocytes is powerfully stimulated by the sustained activation of the Notch pathway. We found that Notch1 is expressed in proliferating ventricular immature cardiac myocytes (ICMs) both in vitro and in vivo, and that the number of Notch1-positive cells in the heart declines with age. Notch1 expression in ICMs paralleled the expression of its Jagged1 ligand on non-myocyte supporting cells. The inhibition of Notch signaling in ICMs blocked their proliferation and induced apoptosis; in contrast, its activation by Jagged1 or by the constitutive expression of its activated form using an adeno-associated virus markedly stimulated proliferative signaling and promoted ICM expansion. Maintenance or reactivation of Notch signaling in cardiac myocytes might represent an interesting target for innovative regenerative therapy.     

MicroRNA-21 promotes the proliferation and inhibits apoptosis in Eca109 via activating ERK1/2/MAPK pathway

The aim of this study was to investigate how miR-21 promotes proliferation and inhibits apoptosis in esophageal squamous cell carcinoma (ESCC). MTT, wound healing assay and cell cycle showed that proliferation and migration of ESCC cell line Eca109 cells were increased in miR-21 mimics group, and decreased in anti-miR-21 Oligonucleotide (AMO) group after transfection into Eca109 cells with miR-21 mimics, AMO and scramble sequence, respectively. Cell apoptosis assay indicated that cell apoptosis can be obviously inhibited by overexpression of miR-21 and promoted by downregulation of miR-21. Meanwhile, western-blot results showed that p-ERK1/2 expression was elevated in miR-21 mimics group, whereas decreased in AMO group. Furthermore, the ERK1/2, a key component of MAPK signaling pathway, was knocked down, and overexpressed successfully using shRNA-ERK1/2 and overexpressing plasmids containing full length cDNA of ERK1/2, respectively. It was observed that shRNA-ERK1/2 can significantly decreased the level of miR-21 expression, while overexpression of ERK1/2 can up-regulate expression of miR-21. As further confirmation, Eca109 cells were treated with gradient concentration of U0126, a kind of MEK inhibitor, and expression of miR-21 was subsequently examined. It was found that U0126 can significantly decreased endogenous expression of miR-21. In parallel, U0126 decreased cell proliferation, migration and increased the apoptosis in Eca109 cells, with the expression of miR-21 being reduced significantly in U0126 group as compared with control groups. Our findings indicated that miR-21 promoted the proliferation, migration and inhibited apoptosis of Eca109 cells through activating ERK1/2/MAPK pathway, and that targeting miR-21 could be a promising therapeutic strategy in ESCC.     

The MT2 receptor stimulates axonogenesis and enhances synaptic transmission by activating Akt signaling

The MT2 receptor is a principal type of G protein-coupled receptor that mainly mediates the effects of melatonin. Deficits of melatonin/MT2 signaling have been found in many neurological disorders, including Alzheimer''s disease, the most common cause of dementia in the elderly, suggesting that preservation of the MT2 receptor may be beneficial to these neurological disorders. However, direct evidence linking the MT2 receptor to cognition-related synaptic plasticity remains to be established. Here, we report that the MT2 receptor, but not the MT1 receptor, is essential for axonogenesis both in vitro and in vivo. We find that axon formation is retarded in MT2 receptor knockout mice, MT2-shRNA electroporated brain slices or primary neurons treated with an MT2 receptor selective antagonist. Activation of the MT2 receptor promotes axonogenesis that is associated with an enhancement in excitatory synaptic transmission in central neurons. The signaling components downstream of the MT2 receptor consist of the Akt/GSK-3β/CRMP-2 cascade. The MT2 receptor C-terminal motif binds to Akt directly. Either inhibition of the MT2 receptor or disruption of MT2 receptor-Akt binding reduces axonogenesis and synaptic transmission. Our data suggest that the MT2 receptor activates Akt/GSK-3β/CRMP-2 signaling and is necessary and sufficient to mediate functional axonogenesis and synaptic formation in central neurons.Synaptic circuits are established at the sites of axon–dendritic, axon–somatic or axon–axonal contact, in which functional axonogenesis is a critical step.¹ Axonogenesis can be regulated by many intracellular signals that involve cytoskeletal rearrangements,² local protein degradation,³ as well as diffusional barriers.⁴ Additionally, several extracellular neurotrophic factors and hormones have also been shown to have a role in axon guidance and synaptic formation in central neurons.^{5, 6} To date, the role of melatonin and its receptors in axonogenesis remains unclear. Most of the biological functions of melatonin are mediated by its two receptors, MT1 and MT2 receptors, both of them belong to the G protein-coupled receptor (GPCR) subfamily and are widely expressed throughout the central nervous system (CNS).⁷ Activation of the MT2 receptor in response to melatonin is critical for controlling circadian rhythms⁷ and regulation of slow wave sleep.^{8, 9} Early studies have shown that activation of the MT2 receptor in the retina reduces the release of dopamine, while dopamine inhibits growth cone motility and neurite outgrowth during embryonic development,^{10, 11} suggesting the involvement of the MT2 receptor in functional axonogenesis. In mutant mice with deficient expression of the MT2 gene, the induction of long-term potentiation (LTP) of excitatory synaptic transmission is impaired, and this impairment is closely related to deficits in learning.¹² In the hippocampus, the MT2 receptor inhibits GABA_A receptor-mediated current,¹³ which is implicated in the synaptic transmission. In Alzheimer''s disease, expression of the MT2 receptor is significantly reduced, especially in the hippocampus.^{12, 14, 15} A partial agonist of the MT2 receptor, UCM765, exhibits anxiolytic-like properties by increasing the time spent in the open arm of an elevated plus maze test, and by reducing the latency to eat in a novel environment in the novelty suppressed feeding test, suggesting its role in anxiety.¹⁶ Together, these findings suggest that the MT2 receptor links the signaling cascades that mediate learning and memory formation, one of the important biological functions of melatonin;¹⁷ however, the cellular and molecular events underlying this linkage are yet to be established.Dissociated hippocampal neurons have been commonly used as an excellent in vitro model in the study of axon development and synaptic transmission because they maintain morphological, functional and molecular characteristics of the hippocampal neurons in vivo.¹⁸ In dissociated hippocampal neurons, the transition for axon formation and maturation involves the following five stages:¹⁹ stage 1 neurons (~2 to 4 h after plating) display abundant lamellipodia and filopodia that develop into several immature short neurites at stage 2 (~12 to 24 h); polarization occurs at stage 3 (~24 to 48 h), in which a single neurite initiates a rapid elongation to become the axon while others acquire dendritic identity; stage 4 (~3–4 days) is characterized by rapid outgrowth of axon and dendrites; and at stage 5 (7 days onwards), the maturation of axon and dendrites is essential for functional synapse formation.^{20, 21} In the present study, we have identified a novel role for the MT2 receptor in functional axonogenesis and show that activation of the MT2 receptor is crucial for functional axonogenesis and synaptic transmission in central neurons. Using fluorescence resonance energy transfer (FRET) imaging combined with peptide blocking assays, we have identified Akt as an interacting partner and a substrate of the MT2 receptor. Activation of the MT2 receptor-Akt signaling cascade promotes the formation of functional synapses in the hippocampus, whereas inhibition of the MT2 receptor arrests axonogenesis and synaptic transmission. Given the implications of the MT2 receptor in learning and memory, we propose that targeting MT2 receptor-Akt signaling may be a feasible strategy for stimulating functional synaptic circuit assembly.     

LPS regulate ERK1/2-dependent signaling in cardiac fibroblasts via PKC-mediated MKP-1 induction

Activation of MAPK pathways by angiotensin II (Ang II) is important for cardiac fibroblast (CFB) proliferation and migration. Activity of MAP-kinases is closely controlled by a group of dual-specific MAP kinase phosphatases (MKPs). Lipopolysaccharides (LPS) and cytokines are elevated in patients with heart failure and may contribute to disease progression. In this study, we investigate the effect of LPS on Ang II-induced CFB function. Pretreatment of CFBs with LPS (1 microg/mL; 30 min) almost completely inhibited Ang II-induced DNA-synthesis and inhibited Ang II directed chemotaxis by more than 80%. Compared to controls, LPS pretreatment significantly reduced phosphorylation levels of ERK1/2- and p38 MAPK and induced MKP-1 levels. Silencing MKP-1 with antisense oligodesoxynucleotides reversed the antimitogenic effect of LPS on Ang II-induced CFB DNA-synthesis and migration. Induction of MKP-1 by LPS was inhibited by the protein kinase C (PKC)-inhibitor calphostin C, but not by the ERK1/2-pathway inhibitor PD98059, suggesting that PKC but not ERK1/2 is required for LPS-mediated MKP-1 induction in CFBs. Our data demonstrate that LPS have direct cellular effects in CFBs through an inhibition of Ang II-induced MAPK activity via PKC-mediated induction of MKP-1. This might be relevant with regard to the decreased MAPK activity and increased levels in MKPs reported during chronic heart failure in humans.     

Shedding of syndecan-1 by stromal fibroblasts stimulates human breast cancer cell proliferation via FGF2 activation

The cell surface heparan sulfate proteoglycan syndecan-1 is induced in stromal fibroblasts of breast carcinomas and participates in a reciprocal feedback loop, which stimulates carcinoma cell growth in vitro and in vivo. To define the molecular mechanism of carcinoma growth stimulation, a three-dimensional co-culture model was developed that combines T47D breast carcinoma cells with immortalized human mammary fibroblasts in collagen gels. By silencing endogenous syndecan-1 induction with short interfering RNA and expressing mutant murine syndecan-1 constructs, it was determined that carcinoma cell mitogenesis required proteolytic shedding of syndecan-1 from the fibroblast surface. The paracrine growth signal was mediated by the syndecan-1 heparan lfate chains rather than the ectodomain of the core protein and required fibroblast growth factor 2 and stroma-derived factor 1. This paracrine pathway may provide an opportunity for the therapeutic disruption of stromaepithelial signaling.     

Overexpression of fucosyltransferase IV promotes A431 cell proliferation through activating MAPK and PI3K/Akt signaling pathways

Lewis Y (LeY) is a carbohydrate tumor‐asssociated antigen. The majority of cancer cells derived from epithelial tissue express LeY type difucosylated oligosaccharide. Fucosyltransferase IV (FUT4) is an essential enzyme that catalyzes the synthesis of LeY oligosaccharide. Our previous studies have shown that FUT4 overexpression promotes A431 cell proliferation, but the mechanism is still largely unknown. Herein, we investigated the role of the mitogen‐activated protein kinases (MAPKs) and phosphoinositide‐3 kinase (PI3K)/Akt signaling pathways on FUT4‐induced cell proliferation. Results show that overexpression of FUT4 increases the phosphorylation of ERK1/2, p38 MAPK, and PI3K/Akt. Inhibitors of PI3K (LY294002 and Wortmannin) prevented the phosphorylation of ERK1/2, p38 MAPK, and Akt PI3K). Moreover, phosphorylation of Akt is abolished by inhibitors of ERK1/2 (PD98059) and p38 MAPK (SB203580). These data suggested that FUT4 not only activates MAPK and PI3K/Akt signals, but also promotes the crosstalk among these signaling pathways. In addition, FUT4‐induced stimulation of cell proliferation correlates with increased cell cycle progression by promoting cells into S‐phase. The mechanism involves in increased expression of cyclin D1, cyclin E, CDK 2, CDK 4, and pRb, and decreased level of cyclin‐dependent kinases inhibitors p21 and p27, which are blocked by the inhibitors of upstream signal molecules, MAPK and PI3K/Akt. In conclusion, these studies suggest that FUT4 regulates A431 cell growth through controlling cell cycle progression via MAPK and PI3K/Akt signaling pathways. J. Cell. Physiol. 225: 612–619, 2010. © 2010 Wiley‐Liss, Inc.     

Interleukin-1 stimulates cytokines, prostaglandin E2 and matrix metalloproteinase-1 production via activation of MAPK/AP-1 and NF-kappaB in human gingival fibroblasts

Interleukin-1 (IL-1) plays a crucial role in the immunopathological responses involved with tissue destruction in chronic inflammatory diseases, such as periodontal disease, as it stimulates host cells including fibroblasts to produce various inflammatory mediators and catabolic factors. We comprehensively investigated the involvement of mitogen-activated protein kinases (MAPKs)/activator protein-1 (AP-1) and IkappaB kinases (IKKs)/IkappaBs/nuclear factor-kappaB (NF-kappaB) in IL-1beta-stimulated IL-6, IL-8, prostaglandin E(2) (PGE(2)) and matrix metalloproteinase-1 (MMP-1) production by human gingival fibroblasts (HGF). Three MAPKs, extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun N-terminal kinase (JNK), which were simultaneously activated by IL-1beta, mediated subsequent c-fos and c-jun mRNA expression and DNA binding of AP-1 at different magnitudes. IKKalpha/beta/IkappaB-alpha/NF-kappaB was also involved in the IL-1 signaling cascade. Further, IL-1beta stimulated HGF to produce IL-6, IL-8, PGE(2) and MMP-1 via activation of the 3 MAPKs and NF-kappaB, as inhibitors of each MAPK and NF-kappaB significantly suppressed the production of IL-1beta-stimulated factors, though these pathways might also play distinct roles in IL-1beta activities. Our results strongly suggest that the MAPKs/AP-1 and IKK/IkappaB/NF-kappaB cascades cooperatively mediate the IL-1beta-stimulated synthesis of IL-6, IL-8, PGE(2) and MMP-1 in HGF.     

CSNK2B contributes to colorectal cancer cell proliferation by activating the mTOR signaling

The function of Casein kinase 2 beta (CSNK2B) in human malignancies has drawn increasing attention in recent years. However, its role in colorectal cancer (CRC) remains unclear. In the present study, we aimed to explore the expression and biological functions of CSNK2B in CRC. Public gene expression microarray data from online database and immunohistochemistry analysis demonstrated that CSNK2B was highly expressed in CRC tissues than in normal tissues. In vitro and in vivo cellular functional experiments showed that increased CSNK2B expression promoted CRC cell viability and tumorigenesis of CRC. Further western blots and rescue experiments confirmed that CSNK2B promoted CRC cell proliferation mainly by activating the mTOR signaling pathway. These findings identified CSNK2B as a novel oncogene contributing to the development of CRC.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12079-021-00619-1.