Adrienne B. Neithardt

Role of EGF receptor transactivation in phosphoinositide 3-kinase-dependent activation of MAP kinase by GPCRs.

Many G protein coupled receptors (GPCRs) cause phosphorylation of MAP kinases through transactivation of the epidermal growth factor receptor (EGF‐R), leading to increased cell survival and growth, motility, and migration. Phosphoinositide 3‐kinase (PI3K) is one of the important cell survival signaling molecules activated by EGF‐R stimulation. However, the extent to which EGF‐R transactivation is essential for GPCR agonist‐stimulated PI3K activation is not known. Here we examined the mechanism of PI3K activation that elicits GPCR‐mediated ERK1/2 activation by pathways dependent and/or independent of EGF‐R transactivation in specific cell types. Immortalized hypothalamic neurons (GT1‐7 cells) express endogenous gonadotropin‐releasing hormone receptors (GnRH‐R) and their stimulation causes marked phosphorylation of ERK1/2 and Akt (Ser 473) through transactivation of the EGF‐R and recruitment of PI3K. In C9 hepatocytes, agonist activation of AT1 angiotensin II (AT1‐R), lysophosphatidic acid (LPA), and EGF receptors caused phosphorylation of Akt through activation of the EGF‐R in a PI3K‐dependent manner. However, ERK1/2 activation by these agonists in these cells was independent of PI3K activation. In contrast, agonist stimulation of HEK 293 cells stably expressing AT1‐R caused ERK1/2 phosphorylation that was independent of EGF‐R transactivation but required PI3K activation. LPA signaling in these cells showed partial and complete dependence on EGF‐R and PI3K, respectively. These data indicate that GPCR‐induced ERK1/2 phosphorylation is dependent or independent of PI3K in specific cell types, and that the involvement of PI3K during ERK1/2 activation is not dependent solely on agonist‐induced transactivation of the EGF‐R. Published 2005 Wiley‐Liss, Inc.

Should embryos developing to blastocysts on day 7 be cryopreserved and transferred: an analysis of pregnancy and implantation rates

OBJECTIVE To compare pregnancy rates (PRs) using blastocysts cryopreserved on day 7 with those cryopreserved on days 5 and 6. DESIGN Retrospective observational cohort study. SETTING Infertility center performing IVF. PATIENT(S) Eight hundred women with infertility undergoing frozen ET. INTERVENTION(S) Blastocysts cryopreserved on days 5, 6, and 7 after retrieval were thawed and transferred. MAIN OUTCOME MEASURE(S) Ongoing PRs (pregnancy developing appropriately into the second trimester). Thaw survival, implantation rates, and clinical PRs were also calculated. RESULT(S) A total of 1,406 embryos were thawed with a survival of 90.7% for day 5, 83.7% for day 6, and 78.7% for day 7. Implantation rates were 43.3%, 28.9%, and 28.9%, respectively. Ongoing PRs were 43.9%, 32.9%, and 26.7%, respectively. CONCLUSION(S) Blastocysts cryopreserved on day 7 have a lower, but clinically important potential. Embryos that do not achieve blastocyst stage on day 6 should not be universally discarded, but should be observed in culture 1 more day as 27% may result in an ongoing pregnancy.

Dependence of GnRH-induced phosphorylation of CREB and BAD on EGF receptor transactivation in GT1-7 neuronal cells.

The hypothalamic neuropeptide, gonadotropin releasing hormone (GnRH), is a primary regulatory factor in the neuroendocrine control of reproduction. The GnRH decapeptide is released in an episodic manner from hypothalamic GnRH neurons, which are known to express GnRH receptors. Here we examined the signaling pathways by which autocrine GnRH stimulation generates cell survival and proliferative signals in hypothalamic GT1‐7 cells. Both GnRH and epidermal growth factor (EGF) caused rapid phosphorylation of cyclic AMP response element binding protein (CREB) and BAD. The selective epidermal growth factor receptor (EGF‐R) antagonist, AG1478, attenuates the phosphorylation of these proteins by GnRH and EGF. Inhibition of PKC and Src abolished the stimulatory effects of GnRH, but not that of EGF, consistent with a critical role of these signaling molecules upstream of the EGF‐R. All of these effects of GnRH were mimicked by phorbol 12 myristate 13‐acetate (PMA). Consistent with the prosurvival and mitogenic effects of phosphoinositide 3‐kinase/Akt (PI3‐K/Akt) downstream of the EGF‐R, inhibition of PI3‐K diminished the activation of these proteins following stimulation with GnRH, EGF, and PMA. Overexpression of dominant negative Akt attenuated agonist‐induced phosphorylation of BAD, but not that of ERK1/2 and CREB. Moreover, overexpression of wild‐type RSK‐1 resulted in enhanced basal as well as agonist‐induced phosphorylation of CREB and BAD, indicating a critical role of RSK‐1 in activating cytosolic as well as nuclear proteins. These data reveal novel signaling mechanisms of GnRH‐induced phosphorylation of CREB and BAD in GT1‐7 neurons through transactivation of the EGF‐R. J. Cell. Physiol. 208: 586–593, 2006. Published 2006 Wiley‐Liss, Inc.