Participation of growth factor signal transduction pathways in estradiol facilitation of female reproductive behavior

Estradiol (E(2)) regulates female reproductive behavior (lordosis) by acting on estrogen-sensitive neurons. We recently showed that E(2) facilitation of lordosis behavior requires concurrent activation of brain IGF-I receptors. The present study confirmed this finding and sought to identify the downstream signaling pathways involved in estrogen/IGF-I priming of lordosis. Intracerebroventricular infusions of a selective IGF-I receptor antagonist were administered to ovariectomized rats every 12 h beginning 1 h before the first of two daily E(2) injections. IGF-I receptor blockade partially inhibits lordosis if the antagonist is infused throughout the 2-d estrogen treatment period but not if it is administered only during the first or last 12 h of estrogen treatment. Because E(2) and IGF-I can activate phosphatidylinositol-3-kinase (PI3K) and MAPK, we infused agents that block PI3K and/or MAPK activity as described above. Both PI3K inhibitors (wortmannin and LY294002) and MAPK inhibitors (PD98059 and U0126) partially attenuate lordosis when administered during estrogen priming. None of these drugs modifies lordosis if they are infused only once, during the last 12 h of estrogen treatment. When both wortmannin and PD98059 are infused during E(2) priming, lordosis behavior is completely abolished. These data suggest that activation of both PI3K and MAPK by E(2) and IGF-I mediates hormonal facilitation of lordosis behavior.     

Activation of adenosine deaminase in MCF-7 cells through IGF-estrogen receptor alpha crosstalk

Adenosine deaminase (ADA) regulates cellular levels of adenosine and deoxyadenosine, and 17beta-estradiol (E(2)) induces ADA mRNA in MCF-7 human breast cancer cells. IGF-I also induces ADA gene expression in these cells, and induction of this response through IGF activation of estrogen receptor alpha (ERalpha) was further investigated. IGF and other polypeptide growth factors induce reporter gene expression in MCF-7 cells cotransfected with ERalpha expression plasmid and pADA211, a construct containing the -211 to +11 region of the ADA gene promoter which is required for high basal and E(2)-inducible activity. Deletion analysis of this promoter demonstrates that IGF activates ERalpha/Sp1 interactions with multiple GC-rich sites in the promoter and this response is abrogated in cells transfected with ERalpha containing mutations at Ser(118) or Ser(163). IGF induces both MAPK (mitogen-activated protein kinase) and PI3-K (phosphatidylinositol-3-kinase) phosphorylation cascades in MCF-7 cells; however, using a series of inhibitors and dominant negative constructs, our results show that induction of ADA by IGF activation of ERalpha/Sp1 is dependent on the MAPK signaling pathway.     

Early expression of myocardial HIF-1alpha in response to mechanical stresses: regulation by stretch-activated channels and the phosphatidylinositol 3-kinase signaling pathway

Vascular endothelial growth factor (VEGF) expression is upregulated by hypoxia-inducible factor-1 (HIF-1) in ischemic tissues and growing tumors. Normally, HIF-1 activity depends on the amount of HIF-1alpha subunit, which is tightly regulated by the oxygen tension. In the myocardium, VEGF expression has been shown to be induced under nonhypoxic conditions by mechanical stresses. However, the cellular mechanism of stress-mediated VEGF induction remains unclear. Therefore, we examined the possible involvement of HIF-1 in stress-mediated VEGF induction in rat hearts. In this study, we increased the left ventricular wall tension using 3 different methods, namely by inducing regional ischemia, by expanding an intraventricular balloon, and by producing hemodynamic overload using an aortocaval shunt. In all cases, HIF-1alpha accumulated in the nuclei of cardiac myocytes in the early phase, and this was followed by VEGF induction. Phosphatidylinositol 3-kinase (PI3K)-dependent Akt phosphorylation was found to be activated by mechanical stress and completely blocked by wortmannin (a PI3K inhibitor). Moreover, the stress-mediated induction of HIF-1alpha and VEGF was suppressed by gadolinium (a stretch-activated channel inhibitor), wortmannin, and rapamycin (a FRAP inhibitor). Our results suggest that HIF-1alpha plays an important role in the induction of VEGF in nonischemic and mechanically stressed myocardium, and that this is regulated by stretch-activated channels and the PI3K/Akt/FRAP pathway. Moreover, this signaling pathway, which induces HIF-1alpha, seems to play an important role in the adaptation of the myocardium to stresses. The full text of this article is available at http://www.circresaha.org.     

Requirement of Ras-dependent pathways for activation of the transforming growth factor beta3 promoter by estradiol

It has been previously observed that the transforming growth factor beta3 (TGFbeta3) gene can be activated by both estradiol (E(2)) and selective estrogen receptor modulators (SERMs) in vivo but that only SERMs have a potent stimulatory effect on the TGFbeta3 promoter in cultured cells. We demonstrate in this report that E(2) can act also as a potent inducer of the TGFbeta3 promoter via a novel and specific estrogen receptor (ER)alpha-mediated mechanism. Our results show that treatment with epidermal growth factor or transfection of a constitutively active Ras mutant allows E(2) to induce the TGFbeta3 promoter via ERalpha in cotransfected HeLa and osteosarcoma MG63 cells. Both protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) inhibitors can block the combined stimulatory effect of E(2) and epidermal growth factor/Ras. However, E(2) induction of the TGFbeta3 promoter was found to be unaffected by mutation of ERalpha serine 118, a well-characterized target of MAPK. Progressive deletion analysis of the ERalpha amino-terminal region delineated three separate domains modulating the E(2)/activated Ras response, revealing a complex functional organization of the ERalpha A/B domain required for regulation of the TGFbeta3 promoter. In addition, PKC and MAPK inhibitors had no effect on the induction of TGFbeta3 promoter activity by the SERM EM-652. These results indicate that induction of the TGFbeta3 promoter by the E(2)/ERalpha complex requires the concomitant activation of PKC and MAPK signaling and provide a novel framework for the design of more effective estrogen-based therapeutic strategies.     

TGF-β effects on airway smooth muscle cell proliferation, VEGF release and signal transduction pathways

Background and objective:   Airway smooth muscle (ASM) cell hyperplasia is a key feature of airway remodelling. Mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) are key components in signal transduction associated with cell proliferation; MAPK consists of the extracellular signal-regulated kinase (ERK), p38MAPK and c-Jun NH₂-terminal kinase (JNK). The effect of transforming growth factor (TGF)-β on the proliferation of ASM cells, the release of vascular endothelial growth factor (VEGF) by ASM cells and relevant signal transduction pathways were investigated.
Methods:   ASM cells were growth-arrested for 48 h then stimulated with platelet-derived growth factor (PDGF), TGF-β and dexamethasone. ASM cells were also treated with specific inhibitors of MAPK (PD98059), PI3K (wortmannin) and JNK (SP600125). Cell proliferation and VEGF concentrations were measured.
Results:   TGF-β neither augmented ASM cell proliferation nor showed a synergistic effect on PDGF-mediated ASM cell proliferation. Dexamethasone did not suppress ASM cell proliferation. VEGF release was augmented by TGF-β stimulation in a time-dependent manner, and was further enhanced by co-stimulation with PDGF and TGF-β. Dexamethasone suppressed VEGF release significantly. TGF-β enhanced PI3K phosphorylation, while PDGF augmented both ERK and PI3K phosphorylation. Wortmannin inhibited both TGF-β- and PDGF-stimulated VEGF release.
Conclusions:   TGF-β may facilitate airway remodelling by promoting VEGF release through the PI3K pathway, rather than via ASM cell proliferation.