Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The integrity of the blood-brain barrier (BBB) is critical for normal brain function. Neuropathological abnormalities in AIDS patients have been associated with perivascular HIV-infected macrophages, gliosis, and abnormalities in the permeability of the BBB. The processes by which HIV causes these pathological conditions are not well understood. To characterize the mechanism by which HIV-1 Tat protein modulates human brain microvascular endothelial cell (HBMEC) functions, we studied the effects of HIV-1 Tat in modulating HBMEC apoptosis and permeability. Treatment of HBMEC with HIV-1 Tat led to Flk-1/KDR and Flt-4 receptor activation and the release of NO. The protein levels of endothelial NO synthase (NOS) and inducible NOS were increased by HIV-1 Tat stimulation. Importantly, HIV-1 Tat caused apoptosis of HBMEC, as evidenced by changes in the cleavage of poly(A)DP-ribose polymerase, DNA laddering, and incorporation of fluorescein into the nicked chromosomal DNA (TUNEL assay). HIV-1 Tat-mediated apoptosis in HBMEC was significantly inhibited in the presence of N-nitro-L-arginine methyl ester (an inhibitor of NOS) and wortmannin (a phosphoinositol 3-kinase inhibitor). Furthermore, HIV-1 Tat treatment significantly increased HBMEC permeability, and pretreatment with both N-nitro-L-arginine methyl ester and wortmannin inhibited the Tat-induced permeability. Taken together, these results indicate that dysregulated production of NO by HIV-1 Tat plays a pivotal role in brain endothelial injury, resulting in the irreversible loss of BBB integrity, which may lead to enhanced infiltration of virus-carrying cells across the BBB.
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PMID:HIV-1 Tat-mediated apoptosis in human brain microvascular endothelial cells. 1259 91

Vascular endothelial growth factors (VEGFs) and their receptors have emerged as central regulators of the angiogenic process. However, involvement of VEGF-B, one of these factors, in angiogenesis remains obscure. Mice received subcutaneous injection of Matrigel alone or Matrigel with human recombinant protein rhVEGF-B167 or with rhVEGF-A165. After 14 days, cell ingrowth in the Matrigel plug was increased by 2.0- and 2.5-fold in rhVEGF-B167-treated and rhVEGF-A165-treated mice, respectively (P<0.01), in association with a raise in phospho-Akt/Akt (1.8-fold, P<0.01) and endothelial NO synthase (eNOS) (1.80- and 1.60-fold, respectively; P<0.05) protein levels measured by Western blot. VEGF-B-induced cell ingrowth was impaired by treatment with NOS inhibitor (NG-nitro-l-arginine methyl ester; L-NAME, 10 mg/kg per day). Treatment with neutralizing antibody directed against the VEGF-B receptor VEGF-R1 (anti-VEGFR1, 10 microg) completely abrogated VEGF-B-related effects. Proangiogenic effect of VEGF-B was confirmed in a mouse model of surgically induced hindlimb ischemia. Plasmids containing human form of VEGF-A (phVEGF-A165) or VEGF-B (phVEGF-B167 or phVEGF-B186) were administered by in vivo electrotransfer. Angiographic score at day 28 showed significant improvement in ischemic/nonischemic leg ratio by 1.4- and 1.5-fold in mice treated with phVEGF-B167 and phVEGF-B186, respectively (P<0.05). Laser Doppler perfusion data also evidenced a 1.5-fold increase in phVEGF-B167-treated and phVEGF-B186-treated mice (P<0.05). Such an effect was associated with an upregulation of phospho-Akt/Akt and eNOS protein levels in the ischemic legs and was hampered by treatment with anti-VEGFR1. This study demonstrates for the first time that VEGF-B, in part through its receptor VEGF-R1, promotes angiogenesis in association with an activation of Akt and eNOS-related pathways.
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PMID:Vascular endothelial growth factor-B promotes in vivo angiogenesis. 1288 72

Medullary thyroid carcinoma (MTC), a neoplasm of thyroid C-cells, is characterized by dominant activating mutations in the RET proto-oncogene. Currently therapy is restricted to surgical removal of all neoplastic tissue lacking alternative forms of treatment such as chemotherapy or radiotherapy. Therefore MTC is a particularly attractive target for gene therapeutic approaches. Many promising gene therapy strategies have been used in various animal models of MTC, showing enhanced antitumoral efficacy, and these will hopefully extend our current standard of care in the future. These approaches can tentatively be subdivided into four groups: (a) Inhibition of oncogenic RET signaling, (b) suicide gene therapy, (c) immunotherapy, and (d) combination of immunotherapy and suicide approaches. To block oncogenic signal transduction dominant-negative RET mutants were delivered into tumor cells and found to possess strong antineoplastic activity, including tumor growth suppression and increased animal survival. Suicide gene therapeutic approaches applied to MTC treatment featured either gene transfer of herpes simplex virus thymidine kinase with concomitant application of ganciclovir or delivery of nitric oxide synthase II. Here antitumor effects were attributed to the occurrence of substantial bystander activities. Immunotherapy approaches comprised stimulation of immune response by delivery of interleukin 2 or 12. Finally, treatment with herpes simplex virus thymidine kinase/ganciclovir in combination with interleukin 2 was found to be superior over either treatment alone. This review discusses the various gene therapeutic approaches applied to MTC treatment in detail, gives an overview on the diverse vector systems used to achieve efficient transduction of thyroid cancer cells, and points out the strategies employed to accomplish target cell selective gene expression thereby contributing to enhanced safety of gene therapy for MTC
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PMID:Gene therapeutic approaches for medullary thyroid carcinoma treatment. 1281 13

Using clonal derivatives of spontaneous mammary tumours in C3H/HeJ mice, we had earlier shown that tumour-derived nitric oxide (NO), resulting from endothelial type (e) NO synthase (NOS) expression by tumour cells, promoted tumour growth and metastasis by multiple mechanisms: stimulation of tumour cell invasiveness, migration and angiogenesis. Our present study examined the signaling mechanisms underlying NO-mediated promotion of tumour cell migration in a highly metastatic and high eNOS-expressing C3H/HeJ mammary tumour cell line, C3L5. C3L5 cell migration was reduced in the presence of N(G)-nitro-L-arginine methyl ester (L-NAME, NOS inhibitor) in a concentration-dependent manner and restored in the additional presence of excess L-arginine (NOS substrate), confirming a migration-promoting role of endogenous NO. Migratory capacity of C3L5 cells was reduced after treatment with the guanylate cyclase (GC) inhibitor 1-H-[1,2,4]oxadiaxolo[4,3-a]quinolalin-1-one (ODQ) and restored in the additional presence of 8-bromoguanosine 3'5'-cyclic monophosphate (8-Br cGMP, cGMP analogue), demonstrating a pivotal role for GC in C3L5 cell migration. Mitogen-activated protein kinase kinase (MAPKK; MEK) inhibitor, UO126, blocked migration, demonstrating MEK involvement in C3L5 cell migration. Furthermore, both ODQ and UO126 blocked migration-restoring effects of L-arginine in L-NAME-treated cells, indicating that GC and MAPK pathways are required for endogenous NO-mediated migratory responses. Similarly, L-NAME reduced and additional treatment with excess L-arginine or sodium nitroprusside (SNP, NO donor) stimulated phosphorylation of extracellular signal-regulated kinases (ERK(1/2)), demonstrating a role for endogenous and exogenous NO in ERK(1/2) activation. ODQ inhibited ERK(1/2) activation, whereas 8-Br cGMP stimulated ERK(1/2) phosphorylation in L-NAME-treated cells, indicating that cGMP is a downstream effector of NOS for ERK(1/2) activation. Finally, both ODQ and UO126 blocked the capacity of L-arginine to restore ERK(1/2) phosphorylation in L-NAME-treated cells, demonstrating that GC and MEK are both required for endogenous NO-mediated MAPK activation. Together, these results indicate sequential activation of NOS, GC and MAPK pathways in mediating signals for C3L5 cell migration, an essential step in invasion and metastasis. Since NOS activity is positively associated with human breast cancer progression, the present results are relevant for development of therapeutic modalities for this disease.
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PMID:Nitric oxide-mediated promotion of mammary tumour cell migration requires sequential activation of nitric oxide synthase, guanylate cyclase and mitogen-activated protein kinase. 1284 43

Rapid uterine vasodilatation after estrogen administration is believed to be mediated by endothelial production of nitric oxide (NO) via endothelial NO synthase (eNOS). However, the mechanism(s) by which estrogen activates eNOS in uterine artery endothelial cells (UAEC) is unknown. In this study, we observed that estradiol-17beta (E2) and E2-BSA rapidly (<2 min) increased total NOx production in UAEC in vitro. This was associated with rapid eNOS phosphorylation and activation but was unaltered by pretreatment with actinomycin-D. Estrogen receptor-alpha protein was detectable in isolated plasma membrane proteins by immunoblotting, and E2-BSA-fluorescein isothiocyanate binding was evident on the plasma membrane of UAEC. E2 did not mobilize intracellular Ca2+, but E2 and ionomycin in combination induced greater eNOS phosphorylation than either E2 or ionomycin alone. E2 did not stimulate rapid Akt phosphorylation. E2 stimulated rapid ERK2/1 activation in a time- and dose-dependent manner, with maximal responses observed at 5-10 min with E2 (10 nm to 1 microm) treatment. Acute activation of eNOS and NOx production by E2 could be inhibited by PD98059 but not by LY294002. When E2-BSA was applied, similar responses in NOx production, eNOS, and ERK2/1 activation to those of E2 were achieved. In addition, E2 and E2-BSA-induced ERK2/1 activation and ICI 182,780 could inhibit NOx production by E2. Thus, acute activation of eNOS to produce NO in UAEC by estrogen is at least partially through an ERK pathway, possibly via estrogen receptor localized on the plasma membrane. This pathway may provide a novel mechanism for NO-mediated rapid uterine vasodilatation by estrogen.
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PMID:Membrane estrogen receptor-dependent extracellular signal-regulated kinase pathway mediates acute activation of endothelial nitric oxide synthase by estrogen in uterine artery endothelial cells. 1451 34

The retinoid N-(4-hydroxyphenyl)retinamide (4-HPR also known as fenretinide) is a potent inducer of apoptosis in breast cancer cells. We observed a 4.5-fold reduction in 4-HPR-mediated apoptosis in MCF-7 breast cancer cells transfected with HER2/neu (MCF-7/HER2) as compared with the parental MCF-7 (MCF-7/WT) cells. Blocking HER2/neu with trastuzumab (Herceptin) led to a six-fold increase in 4-HPR-induced apoptosis in HER2/neu-overexpressing cells. These data indicate that HER2/neu reduces the sensitivity of breast cancer cells to 4-HPR. We showed previously that nitric oxide (NO) is essential for 4-HPR to induce apoptosis in breast cancer cells. The inhibitory effects of the 4-HPR and trastuzumab combination correlated with the amount of NO produced in HER2/neu-overexpressing cells. When a NO synthase (NOS) inhibitor was used to block NO production, decreased apoptosis by the 4-HPR and trastuzumab combination was observed. Furthermore, 4-HPR-mediated NOSII expression was lower in MCF-7/HER2 than MCF-7/WT cells, but was increased by trastuzumab in HER2/neu-overexpressing cells. Here we report the novel findings that HER2/neu reduces the ability of 4-HPR to induce apoptosis in breast cancer cells, and that one mechanism by which HER2/neu increases the resistance of breast cancer cells to 4-HPR is by decreasing NOSII-mediated NO production.
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PMID:HER2/neu reduces the apoptotic effects of N-(4-hydroxyphenyl)retinamide (4-HPR) in breast cancer cells by decreasing nitric oxide production. 1455 87

The effects of EDTA on the expression and topologic localization of mitogen-activated protein (MAP) kinases (ERK, JNK, and p38), along with nitric oxide synthase (NOS), I-KappaB, and p53 were examined to elucidate the host response provoked by the intravaginal application of a female controlled drug delivery system (FcDDS) containing a spermicidal/microbicidal agent and EDTA. Immunohistochemical and immunoblotting studies were conducted to identify and quantitate the EDTA-inducible proteins in vaginal mucosa. The content of nitrite, which is one of the primary stable breakdown products of nitric oxide (NO), was determined to correlate the expression of NOS with NO formation in HeLa cervical carcinoma cell line. The immunohistochemical study demonstrated that the modulation of the calcium gradient by EDTA activated MAP kinases (ERK and JNK) in the rabbit vaginal mucosa. The results of Western immunoblot study demonstrated differential expression of MAP kinases (ERK and JNK) with EDTA treatment, whereas the expression of NOS and NF-KappaB was not affected by EDTA. There was no significant difference in nitrite production in the HeLa cell line upon exposure to EDTA compared with the control, which was consistent with the results of the Western blot study. The results of this work support that the regulation of MAP kinase was affected by calcium, which is controlled by chelation activity of EDTA. The specific tissue responses exerted by the loading components of a biomaterial-based system should be fully taken into consideration for its intravaginal application.
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PMID:EDTA-induced activation of Ca-regulated proteins in the vaginal mucosa. 1466 Dec 61

Patency of the fetal ductus arteriosus (DA) is maintained in an environment of low relative oxygen tension and a preponderance of vasodilating forces. In addition to prostaglandins, nitric oxide (NO), a potent vasodilator in the pulmonary and systemic vasculatures, has been implicated in regulation of the fetal DA. To further define the contribution of NO to DA patency, the expression and function of NO synthase (NOS) isoforms were examined in the mouse DA on days 17-19 of pregnancy and after birth. Our results show that endothelial NOS (eNOS) is the predominant isoform expressed in the mouse DA and is localized in the DA endothelium by in situ hybridization. Despite rapid constriction of the DA after birth, eNOS expression levels were unchanged throughout the fetal and postnatal period. Pharmacological inhibition of prostaglandin vs. NO synthesis in vivo showed that the preterm fetal DA on day 16 is more sensitive to NOS inhibition than the mature fetal DA on day 19, whereas prostaglandin inhibition results in marked DA constriction on day 19 but minimal effects on the day 16 DA. Combined prostaglandin and NO inhibition caused additional DA constriction on day 16. The contribution of vasa vasorum to DA regulation was also examined. Immunoreactive platelet endothelial cell adhesion molecule and lacZ tagged FLK1 localized to DA endothelial cells but revealed the absence of vasa vasorum within the DA wall. Similarly, there was no evidence of vasa vasorum by vascular casting. These studies indicate that eNOS is the primary source of NO in the mouse DA and that vasomotor tone of the preterm fetal mouse DA is regulated by eNOS-derived NO and is potentiated by prostaglandins. In contrast to other species, mechanisms for DA patency and closure appear to be independent of any contribution of the vasa vasorum.
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PMID:Patency of the preterm fetal ductus arteriosus is regulated by endothelial nitric oxide synthase and is independent of vasa vasorum in the mouse. 1514 32

Nitric oxide (NO) is a powerful angiogenic mediator acting downstream of vascular endothelial growth factor (VEGF). Both the endothelial NO synthase (eNOS) and the VEGFR-2 receptor colocalize in caveolae. Because the structural protein of these signaling platforms, caveolin, also represses eNOS activity, changes in its abundance are likely to influence the angiogenic process in various ways. In this study, we used mice deficient for the caveolin-1 gene (Cav-/-) to examine the impact of caveolae suppression in a model of adaptive angiogenesis obtained after femoral artery resection. Evaluation of the ischemic tissue perfusion and histochemical analyses revealed that contrary to Cav+/+ mice, Cav-/- mice failed to recover a functional vasculature and actually lost part of the ligated limbs, thereby recapitulating the effects of the NOS inhibitor L-NAME administered to operated Cav+/+ mice. We also isolated endothelial cells (ECs) from Cav-/- aorta and showed that on VEGF stimulation, NO production and endothelial tube formation were dramatically abrogated when compared with Cav+/+ ECs. The Ser1177 eNOS phosphorylation and Thr495 dephosphorylation but also the ERK phosphorylation were similarly altered in VEGF-treated Cav-/- ECs. Interestingly, caveolin transfection in Cav-/- ECs redirected the VEGFR-2 in caveolar membranes and restored the VEGF-induced ERK and eNOS activation. However, when high levels of recombinant caveolin were reached, VEGF exposure failed to activate ERK and eNOS. These results emphasize the critical role of caveolae in ensuring the coupling between VEGFR-2 stimulation and downstream mediators of angiogenesis. This study also provides new insights to understand the paradoxical roles of caveolin (eg, repressing basal enzyme activity but facilitating activation on agonist stimulation) in cardiovascular pathophysiology.
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PMID:Caveolin-1 expression is critical for vascular endothelial growth factor-induced ischemic hindlimb collateralization and nitric oxide-mediated angiogenesis. 1520 64

While many endothelial cell lines exist, few are of human origin with characteristics close to the parent endothelial cell. We derived a subline (HUVEC-CS) of immortalized human umbilical vein endothelial cells (HUVEC-C) that proliferate in standard growth media and exhibit positive acetylated low-density lipoprotein (AcLDL) uptake, express eNOS, CD31 and ve-cadherin, and spontaneously form capillary-like structures when grown on Matrigel. HUVEC-CS also maintain endothelial cell characteristics at the level of mitogenesis, kinase activation and vasodilator production. Like primary HUVEC cells, HUVEC-CS express many of the key proteins necessary for vasodilator production, including epithelial nitric oxide synthase (eNOS), HSP 90, cav-1 and -2, cPLA2, and COX-1 and -2. Prostaglandin I synthase (PGIS) was not detectable by Western blot analysis, consistent with primary HUVEC in which PGI2 production is minimal. Receptors were detected for angiotensin II (AII), bradykinin, ATP and growth factors. ATP induced a dose- and time-dependent rise in the intracellular free Ca2+ concentration ([Ca2+]i). Initially, ATP stimulates P2Y receptors rather than P2X receptors, as demonstrated by the inability of ATP to initiate a Ca2+ response subsequent to emptying of the internal Ca2+ stores by thapsigargin. AII, bradykinin, epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) also caused a rise in [Ca2+]i in a subset of the cells. ATP, basic fibroblastic growth factor (bFGF), EGF and VEGF induced mitogenesis and caused a rise in ERK 2 activation within 10 min. L-Arginine to L-citrulline conversion assays showed that ATP, EGF and VEGF induced a significant rise in eNOS activity, and this correlates with an ability to induce Ca2+ mobilization and ERK 2 activation. In conclusion, HUVEC-CS are indeed endothelial cells and appear to be functionally very similar to primary HUVEC. These cells will prove a valuable tool for future studies in both basic and therapeutic sciences.
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PMID:Functional characterization of HUVEC-CS: Ca2+ signaling, ERK 1/2 activation, mitogenesis and vasodilator production. 1535 Jan 90


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