Proangiogenic action of thyroid hormone is fibroblast growth factor-dependent and is initiated at the cell surface. (33/5946)

The effects of thyroid hormone analogues on modulation of angiogenesis have been studied in the chick chorioallantoic membrane model. Generation of new blood vessels from existing vessels was increased 3-fold by either l-thyroxine (T4; 10(-7) mol/L) or 3,5,3'-triiodo-l-thyronine (10(-9) mol/L). T4-agarose reproduced the effects of T4, and tetraiodothyroacetic acid (tetrac) inhibited the effects of both T4 and T4-agarose. Tetrac itself was inactive and is known to block actions of T4 on signal transduction that are initiated at the plasma membrane. T4 and basic fibroblast growth factor (FGF2) were comparably effective as inducers of angiogenesis. Low concentrations of FGF2 combined with submaximal concentrations of T4 produced an additive angiogenic response. Anti-FGF2 inhibited the angiogenic effect of T4. The proangiogenic effects of T4 and FGF2 were blocked by PD 98059, a mitogen-activated protein kinase (MAPK) pathway inhibitor. Endothelial cells (ECV304) treated with T4 or FGF2 for 15 minutes demonstrated activation of MAPK, an effect inhibited by PD 98059 and the protein kinase C inhibitor CGP41251. Reverse transcription-polymerase chain reaction of RNA extracted from endothelial cells treated with T4 revealed increased abundance of FGF2 transcript at 6 to 48 hours, and after 72 hours, the medium of treated cells showed increased FGF2 content, an effect inhibited by PD 98059. Thus, thyroid hormone is shown to be a proangiogenic factor. This action, initiated at the plasma membrane, is MAPK dependent and mediated by FGF2.  (+info)

Estradiol abrogates apoptosis in breast cancer cells through inactivation of BAD: Ras-dependent nongenomic pathways requiring signaling through ERK and Akt. (34/5946)

Estrogens such as 17-beta estradiol (E(2)) play a critical role in sporadic breast cancer progression and decrease apoptosis in breast cancer cells. Our studies using estrogen receptor-positive MCF7 cells show that E(2) abrogates apoptosis possibly through phosphorylation/inactivation of the proapoptotic protein BAD, which was rapidly phosphorylated at S112 and S136. Inhibition of BAD protein expression with specific antisense oligonucleotides reduced the effectiveness of tumor necrosis factor-alpha, H(2)O(2), and serum starvation in causing apoptosis. Furthermore, the ability of E(2) to prevent tumor necrosis factor-alpha-induced apoptosis was blocked by overexpression of the BAD S112A/S136A mutant but not the wild-type BAD. BAD S112A/S136A, which lacks phosphorylation sites for p90(RSK1) and Akt, was not phosphorylated in response to E(2) in vitro(.) E(2) treatment rapidly activated phosphatidylinositol 3-kinase (PI-3K)/Akt and p90(RSK1) to an extent similar to insulin-like growth factor-1 treatment. In agreement with p90(RSK1) activation, E(2) also rapidly activated extracellular signal-regulated kinase, and this activity was down-regulated by chemical and biological inhibition of PI-3K suggestive of cross talk between signaling pathways responding to E(2). Dominant negative Ras blocked E(2)-induced BAD phosphorylation and the Raf-activator RasV12T35S induced BAD phosphorylation as well as enhanced E(2)-induced phosphorylation at S112. Chemical inhibition of PI-3K and mitogen-activated protein kinase kinase 1 inhibited E(2)-induced BAD phosphorylation at S112 and S136 and expression of dominant negative Ras-induced apoptosis in proliferating cells. Together, these data demonstrate a new nongenomic mechanism by which E(2) prevents apoptosis.  (+info)

Phospholipase C delta-4 overexpression upregulates ErbB1/2 expression, Erk signaling pathway, and proliferation in MCF-7 cells. (35/5946)

BACKGROUND: The expression of the rodent phosphoinositide-specific phospholipase C delta-4 (PLCdelta4) has been found to be elevated upon mitogenic stimulation and expression analysis have linked the upregulation of PLCdelta4 expression with rapid proliferation in certain rat transformed cell lines. The human homologue of PLCdelta4 has not been extensively characterized. Accordingly, we investigate the effects of overexpression of human PLCdelta4 on cell signaling and proliferation in this study. RESULTS: The cDNA for human PLCdelta4 has been isolated and expressed ectopically in breast cancer MCF-7 cells. Overexpression of PLCdelta4 selectively activates protein kinase C-phi and upregulates the expression of epidermal growth factor receptors EGFR/erbB1 and HER2/erbB2, leading to constitutive activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway in MCF-7 cells. MCF-7 cells stably expressing PLCdelta4 demonstrates several phenotypes of transformation, such as rapid proliferation in low serum, formation of colonies in soft agar, and capacity to form densely packed spheroids in low-attachment plates. The growth signaling responses induced by PLCdelta4 are not reversible by siRNA. CONCLUSION: Overexpression or dysregulated expression of PLCdelta4 may initiate oncogenesis in certain tissues through upregulation of ErbB expression and activation of ERK pathway. Since the growth responses induced by PLCdelta4 are not reversible, PLCdelta4 itself is not a suitable drug target, but enzymes in pathways activated by PLCdelta4 are potential therapeutic targets for oncogenic intervention.  (+info)

Multiple aspects of the phenotype of mammary epithelial cells transformed by expression of activated M-Ras depend on an autocrine mechanism mediated by hepatocyte growth factor/scatter factor. (36/5946)

Multiple aspects of the transformed phenotype induced in a murine mammary epithelial cell line scp-2 by expression of activated G22V M-Ras, including maintainance of cell number at low density, anchorage-independent growth, invasion of Matrigel, and secretion of matrix metalloproteinases (MMP) 2 and 9, were dependent on an autocrine mechanism. Conditioned medium from dense cultures of scp-2 cells expressing G22V M-Ras, but not from parental cells, induced activation of Erk and Akt in cells expressing G22V M-Ras, maintained the cell number and promoted anchorage-independent growth of cells expressing G22V M-Ras (although not the parental cells), and induced scattering of MDCK cells. The latter activities were blocked by neutralizing antibodies to hepatocyte growth factor/scatter factor (HGF/SF) and could be mimicked by HGF/SF. Anti-HGF/SF antibodies also inhibited invasion of Matrigel, and the production of MMP-2 and MMP-9, together with urokinase-type plasminogen activator, was secreted by G22V M-Ras scp-2 cells but not by parental cells. Invasion of Matrigel was blocked by an inhibitor of MMPs, BB94, and by the mitogen-activated protein kinase kinase 1/2 kinase inhibitor PD98059 but was only marginally affected by the phosphatidylinositol 3-kinase inhibitor LY294002. Autocrine HGF/SF was thus critical for expression of key features of the phenotype of mammary epithelial cells transformed by expression of activated M-Ras.  (+info)

The insulin-like growth factor-I (IGF-I) receptor kinase inhibitor NVP-ADW742, in combination with STI571, delineates a spectrum of dependence of small cell lung cancer on IGF-I and stem cell factor signaling. (37/5946)

Stem cell factor (SCF)/Kit and insulin-like growth factor-I (IGF-I)/IGF-I receptor (IGF-IR) autocrine loops play a prominent role in the growth of small cell lung cancer (SCLC). Previous data suggested that IGF-I protects cells from apoptosis induced by STI571, an efficient inhibitor of Kit signal transduction, by activating the critical phosphatidylinositol 3-kinase-Akt pathway. To determine if inhibition of IGF-IR signaling would be therapeutically relevant in SCLC, the activity of a novel kinase inhibitor of IGF-IR, NVP-ADW742 (Novartis Pharma AG, Basel, Switzerland), was characterized. Pretreatment of the H526 cell line with NVP-ADW742 inhibited IGF-IR signaling and growth with IC(50) values between 0.1 and 0.4 micro M. SCF-mediated Kit phosphorylation and Akt activation were inhibited with IC(50) values in the 1-5 micro M range. However, NVP-ADW742 affected neither hepatocyte growth factor-mediated Akt activation nor activity of constitutively active Akt. The therapeutic potential of NVP-ADW742 was assessed by determining its effect on growth of several SCLC cell lines in serum. These studies clearly delineated two populations of cell lines as determined by differential sensitivity to NVP-ADW742. One population, which lacks active SCF/Kit autocrine loops, was inhibited with IC(50) values between 0.1 and 0.5 micro M. A second population, which has active SCF/Kit autocrine loops, was inhibited with IC(50) values in the 4-7 micro M range. When these cell lines were treated with a combination of STI571 and NVP-ADW742, no advantage was seen in the former group, whereas, in the latter group, a clearly synergistic response to the combination was seen when growth, apoptosis, or Akt activation was assessed. These data demonstrate that NVP-ADW742 is a potent and selective IGF-IR kinase inhibitor that can efficiently inhibit the growth of cells that are highly dependent on IGF-I signaling. However, for optimal growth inhibition of SCLC cells with an active SCF/Kit autocrine loop, a combination of a Kit inhibitor (STI571) and an IGF-IR inhibitor (NVP-ADW742) appears to be necessary. These observations suggest that, in tumors in which critical signal transduction pathways can be activated by alternative receptors, optimal therapy may require inhibition of multiple receptors.  (+info)

PI3 kinase is important for Ras, MEK and Erk activation of Epo-stimulated human erythroid progenitors. (38/5946)

BACKGROUND: Erythropoietin is a multifunctional cytokine which regulates the number of erythrocytes circulating in mammalian blood. This is crucial in order to maintain an appropriate oxygen supply throughout the body. Stimulation of primary human erythroid progenitors (PEPs) with erythropoietin (Epo) leads to the activation of the mitogenic kinases (MEKs and Erks). How this is accomplished mechanistically remained unclear. RESULTS: Biochemical studies with human cord blood-derived PEPs now show that Ras and the class Ib enzyme of the phosphatidylinositol-3 kinase (PI3K) family, PI3K gamma, are activated in response to minimal Epo concentrations. Surprisingly, three structurally different PI3K inhibitors block Ras, MEK and Erk activation in PEPs by Epo. Furthermore, Erk activation in PEPs is insensitive to the inhibition of Raf kinases but suppressed upon PKC inhibition. In contrast, Erk activation induced by stem cell factor, which activates c-Kit in the same cells, is sensitive to Raf inhibition and insensitive to PI3K and PKC inhibitors. CONCLUSIONS: These unexpected findings contrast with previous results in human primary cells using Epo at supraphysiological concentrations and open new doors to eventually understanding how low Epo concentrations mediate the moderate proliferation of erythroid progenitors under homeostatic blood oxygen levels. They indicate that the basal activation of MEKs and Erks in PEPs by minimal concentrations of Epo does not occur through the classical cascade Shc/Grb2/Sos/Ras/Raf/MEK/Erk. Instead, MEKs and Erks are signal mediators of PI3K, probably the recently described PI3K gamma, through a Raf-independent signaling pathway which requires PKC activity. It is likely that higher concentrations of Epo that are induced by hypoxia, for example, following blood loss, lead to additional mitogenic signals which greatly accelerate erythroid progenitor proliferation.  (+info)

Aldosterone potentiates angiotensin II-induced signaling in vascular smooth muscle cells. (39/5946)

BACKGROUND: In a double-transgenic human renin and human angiotensinogen rat model, we found that mineralocorticoid receptor (MR) blockade ameliorated angiotensin II (Ang II)-induced renal and cardiac damage. How Ang II and aldosterone (Ald) might interact is ill defined. METHODS AND RESULTS: We investigated the effects of Ang II (10(-7) mol/L) and Ald (10(-7) mol/L) on extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling in vascular smooth muscle cells (VSMCs) with Western blotting and confocal microscopy. Ang II induced ERK 1/2 and JNK phosphorylation by 2 minutes. Ald achieved the same at 10 minutes. Ang II+Ald had a potentiating effect by 2 minutes. Two oxygen radical scavengers and the epidermal growth factor receptor (EGFR) antagonist AG1478 reduced Ang II-, Ald-, and combination-induced ERK1/2 phosphorylation. Preincubating the cells with the MR blocker spironolactone (10(-6) mol/L) abolished Ang II-induced ROS generation, EGFR transactivation, and ERK1/2 phosphorylation. CONCLUSIONS: Ald potentiates Ang II-induced ERK-1/2 and JNK phosphorylation. Oxygen radicals, the MR, and the EGFR play a role in early signaling induced by Ang II and Ald in VSMCs. These in vitro data may help explain the effects of MR blockade on Ang II-induced end-organ damage in vivo.  (+info)

Activated protein C induces endothelial cell proliferation by mitogen-activated protein kinase activation in vitro and angiogenesis in vivo. (40/5946)

Activated protein C (APC), a natural anticoagulant, has recently been demonstrated to activate the mitogen-activated protein kinase (MAPK) pathway in endothelial cells in vitro. Because the MAPK pathway is implicated in endothelial cell proliferation, it is possible that APC induces endothelial cell proliferation, thereby causing angiogenesis. We examined this possibility in the present study. APC activated the MAPK pathway, increased DNA synthesis, and induced proliferation in cultured human umbilical vein endothelial cells dependent on its serine protease activity. Antibody against the endothelial protein C receptor (EPCR) inhibited these events. Early activation of the MAPK pathway was inhibited by an antibody against protease-activated receptor-1, whereas neither late and complete activation of the MAPK pathway nor endothelial cell proliferation were inhibited by this antibody. APC activated endothelial nitric oxide synthase (eNOS) via phosphatidylinositol 3-kinase-dependent phosphorylation, followed by activation of protein kinase G, suggesting that APC bound to EPCR might activate the endothelial MAPK pathway by a mechanism similar to that of VEGF. APC induced morphogenetic changes resembling tube-like structures of endothelial cells, whereas DIP-APC did not. When applied topically to the mouse cornea, APC clearly induced angiogenesis in wild-type mice, but not in eNOS knockout mice. These in vitro events induced by APC might at least partly explain the angiogenic activity in vivo. This angiogenic activity of APC might contribute to maintain proper microcirculation in addition to its antithrombotic activity.  (+info)