Effectiveness of recombinant human FSH: recombinant human LH combination treatment versus recombinant human FSH alone for assisted reproductive technology in women aged 35-40 years.

RESEARCH QUESTION: According to real-world data, is recombinant human FSH (r-hFSH) combined with recombinant human LH (r-hLH) or r-hFSH alone more effective for women of advanced maternal age (AMA) in terms of live birth? DESIGN: Non-interventional study comparing the effectiveness of r-hFSH and recombinant r-hLH (2:1 ratio) versus r-hFSH alone for ovarian stimulation during ART treatment in women aged 35-40 years, using real-world data from the Deutsches IVF-Register. RESULTS: Overall clinical pregnancy (29.8%, 95% CI 28.2 to 31.6 versus 27.8%, 95% CI 26.5 to 29.2) and live birth (20.3%, 95% CI 18.7 to 21.8 versus 18.0%, 95% CI 16.6 to 19.4) rates were not significantly different between the combined r-hFSH and r-hLH group and the r-hFSH alone group (P = 0.269 and P = 0.092, respectively). Treatment effect was significantly higher for combined r-hFSH and r-hLH compared with r-hFSH alone for clinical pregnancy (33.1%, 95% CI 31.0 to 35.0 versus 28.5%, 95% CI 26.6 to 30.4; P = 0.001, not adjusted for multiplicity) and live birth (22.5%, 95% CI 20.5 to 24.2 versus 19.4%, 95% CI 17.6 to 20.9; P = 0.014, not adjusted for multiplicity) in a post-hoc analysis of women with five to 14 oocytes retrieved (used as a surrogate for normal ovarian reserve), highlighting the potential benefits of combined r-hFSH and r-hLH for ovarian stimulation in women aged 35-40 years with normal ovarian reserve. CONCLUSIONS: Women of AMA with normal ovarian response benefit from treatment with combined r-hFSH and r-hLH in a 2:1 ratio versus r-hFSH alone in terms of live birth rate. The effectiveness of treatments is best assessed by RCTs; however, real-world data are valuable for examining the effectiveness of fertility treatment, especially among patient groups that are not well represented in clinical trials.

Calcium-Mediated Interactions Regulate the Subcellular Localization of Extracellular Signal-Regulated Kinases (ERKs).

BACKGROUND/AIMS: The subcellular localization of ERK1 and ERK2 (ERKs) in cells, which is important for proper signaling, may be regulated through protein-protein interactions. However, the proteins involved and the way they are regulated to affect localization is not entirely understood. METHODS: In order to identify the interacting proteins upon varying conditions, we used co-immunoprecipitation of ERK, active ERK and its binding CRS mutant. In addition, we examined the effect of intracellular calcium on the binding using calcium chelators and ionophores, analyzing the binding using silver stain, mass spectrometry and immunoblotting. The effect of calcium on ERK localization was examined using immunofluorescent staining and Western blotting. RESULTS: We found that inactive ERK2 interacts with a large number of proteins through its CRS/CD domain, whereas the phospho-ERK2 interacts with only few substrates. Varying calcium concentrations significantly modified the repertoire of ERK2-interacting proteins, of which many were identified. The effect of calcium on ERKs' interactions influenced also the localization of ERKs, as calcium chelators enhanced nuclear translocation, while elevated calcium levels prevented it. This effect of calcium was also apparent upon the physiological lysophosphatidic acid stimulation, where ERKs translocation was delayed compared to that induced by EGF in a calcium-dependent manner. In vitro translocation assay revealed that high calcium concentrations affect ERKs' translocation by preventing the shuttling machinery through the nuclear envelope, probably due to higher binding to nuclear pore proteins such as NUP153. These results are consistent with a model in which ERKs in quiescent cells are bound to several cytoplasmic proteins. CONCLUSION: Upon stimulation, ERKs are phosphorylated and released from their cytoplasmic anchors to allow shuttling into the nucleus. This translocation is delayed when calcium levels are increased, and this modifies the localization of ERKs and therefore also their spatiotemporal regulation. Thus, calcium regulates ERKs localization, which is important for the compartmentalization of ERKs with their proper substrates, and thereby their signaling specificity.

Pigment epithelium derived factor as a novel multi-target treatment for uterine fibroids.

RESEARCH QUESTION: Does recombinant pigment epithelium derived factor (PEDF) have potential in treating uterine fibroids? DESIGN: In-vitro models that used human leiomyoma and Eker rat uterine leiomyoma (ELT-3) cell lines. The ELT-3 cell line was used to examine cellular targets after adding recombinant PEDF to the culture media. Athymic nude female mice were used as an in-vivo model. They were injected with ELT-3 cells to induce ectopic fibroid lesions, then treated with recombinant PEDF. RESULTS: RNA expression of PEDF and its receptors was found in both leiomyoma cell lines, as well as the expression of PEDF receptors. Addition of recombinant PEDF to the culture medium of leiomyoma cell lines activated ERK in a time-dependent manner, induced down-regulation of vascular endothelial growth factor mRNA and protein, as well as the mRNAs of oestrogen receptors alpha and beta and inhibited cellular proliferation. Treatment of mice-bearing fibroids with recombinant PEDF reduced fibroid growth rate and resulted in smaller tumours. CONCLUSIONS: This study suggests that recombinant PEDF is a putative novel potent physiological treatment for uterine fibroids. It targets several cornerstones of fibroid pathobiology in parallel, including vascular endothelial growth factor and oestrogen receptors, which are needed for vascularization, and restricts fibroid growth and final size in an animal model.

Nuclear ERK Translocation is Mediated by Protein Kinase CK2 and Accelerated by Autophosphorylation.

BACKGROUND/AIMS: The extracellular signal-regulated kinases (ERK) 1 and 2 (ERK1/2) are members of the mitogen-activated protein kinase (MAPK) family. Upon stimulation, these kinases translocate from the cytoplasm to the nucleus, where they induce physiological processes such as proliferation and differentiation. The mechanism of translocation of this kinase involves phosphorylation of two Ser residues within a nuclear translocation signal (NTS), which allows binding to importin7 and a subsequent penetration via nuclear pores. However, the regulation of this process and the protein kinases involved are not yet clear. METHODS: To answer this point we developed specific anti phospho-SPS antibody, used this and other antibodies in Western blots and crystalized the phospho-mimetic mutated ERK. RESULTS: Here we show that the phosphorylation of both Ser residues is mediated mainly by casein kinase 2 (CK2) and that active ERK may assist in the phosphorylation of the N-terminal Ser. We also demonstrate that the phosphorylation is dependent on the release of ERK from cytoplasmic anchoring proteins. Crystal structure of the phosphomimetic ERK revealed that the NTS phosphorylation creates an acidic patch in ERK. Our model is that in resting cells ERK is bound to cytoplasmic anchors, which prevent its NTS phosphorylation. Upon stimulation, phosphorylation of the ERK TEY domain releases ERK and allows phosphorylation of its NTS by CK2 and active ERK to generate a negatively charged patch in ERK, binding to importin 7 and nuclear translocation. CONCLUSION: These results provide an important role of CK2 in regulating nuclear ERK activities.

Interleukin-1 deficiency prolongs ovarian lifespan in mice.

Oocyte endowment dwindles away during prepubertal and adult life until menopause occurs, and apoptosis has been identified as a central mechanism responsible for oocyte elimination. A few recent reports suggest that uncontrolled inflammation may adversely affect ovarian reserve. We tested the possible role of the proinflammatory cytokine IL-1 in the age-related exhaustion of ovarian reserve using IL-1α and IL-1ß-KO mice. IL-1α-KO mice showed a substantially higher pregnancy rate and litter size compared with WT mice at advanced age. The number of secondary and antral follicles was significantly higher in 2.5-mo-old IL-1α-KO ovaries compared with WT ovaries. Serum anti-Müllerian hormone, a putative marker of ovarian reserve, was markedly higher in IL-1α-KO mice from 2.5 mo onward, along with a greater ovarian response to gonadotropins. IL-1ß-KO mice displayed a comparable but more subtle prolongation of ovarian lifespan compared with IL-1α-KO mice. The protein and mRNA of both IL-1α and IL-1ß mice were localized within the developing follicles (oocytes and granulosa cells), and their ovarian mRNA levels increased with age. Molecular analysis revealed decreased apoptotic signaling [higher B-cell lymphoma 2 (BCL-2) and lower BCL-2-associated X protein levels], along with a marked attenuation in the expression of genes coding for the proinflammatory cytokines IL-1ß, IL-6, and TNF-α in ovaries of IL-1α-KO mice compared with WT mice. Taken together, IL-1 emerges as an important participant in the age-related exhaustion of ovarian reserve in mice, possibly by enhancing the expression of inflammatory genes and promoting apoptotic pathways.

Pigment epithelium-derived factor regulation by human chorionic gonadotropin in granulosa cells.

Human chorionic gonadotropin (hCG) is a known trigger of ovarian hyperstimulation syndrome (OHSS), a potentially life-threatening complication of assisted reproduction. Administration of hCG results in the release of vascular endothelial growth factor (VEGF) from the ovary. We have previously shown that expression of pigment epithelium-derived factor (PEDF) in granulosa cell line is regulated by hCG, reciprocally to VEGF, and that the PEDF-VEGF balance is impaired in OHSS. Our aim was to explore the signaling network by which hCG downregulates the expression of PEDF mRNA and protein in granulosa cells. We applied specific chemical inhibitors and stimuli to human primary granulosa cells and rat granulosa cell line. We found that PKA and protein kinase C, as well as EGFR, ERK1/2 and PI3K, participate in the signaling network. The finding that hCG-induced PEDF downregulation and VEGF upregulation are mediated by similar signaling cascades emphasizes the delicate regulation of ovarian angiogenesis.

A novel regulatory pathway in granulosa cells, the LH/human chorionic gonadotropin-microRNA-125a-3p-Fyn pathway, is required for ovulation.

Granulosa cells support the developing oocytes and serve as transducers of the ovulatory stimulus induced by LH surge. Fyn kinase is expressed in granulosa cells, though its role in these cells has not been studied. In human embryonic kidney 293T cells, microRNA (miR)-125a-3p down-regulates Fyn expression, causing a decrease in cells' migratory ability. Our aim was to explore the role of miR-125a-3p and Fyn in granulosa cells toward ovulation, focusing on migration as a possible mechanism. We demonstrate expression of miR-125a-3p and Fyn in mouse mural granulosa cells of preovulatory follicles and miR-125a-3p-induced down-regulation of Fyn expression in a granulosa cell line (rat). Administration of human chorionic gonadotropin (hCG; LH analog) caused a 75% decrease in the in vivo miR-125a-3p:Fyn mRNA ratio, followed by a 2-fold increased migratory ability of mural granulosa cells. In the hCG-treated granulosa cell line, miR-125a-3p expression was decreased, followed by Fyn up-regulation and phosphorylation of focal adhesion kinase and paxillin, enabling cell migration. An in vivo interference with miR-125a-3p:Fyn mRNA ratio in granulosa cells by intrabursal injections of Fyn small interfering RNA or miR-125a-3p mimic caused a 33 or 55% decrease in the number of ovulated oocytes, respectively. These observations reveal a new regulatory pathway in mural granulosa cells under the regulation of LH/hCG. Modulation of cell migration may account for the significance of the LH/hCG-miR-125a-3p-Fyn pathway to ovulation.

Identification and characterization of a general nuclear translocation signal in signaling proteins.

Upon stimulation, many proteins translocate into the nucleus in order to regulate a variety of cellular processes. The mechanism underlying the translocation is not clear since many of these proteins lack a canonical nuclear localization signal (NLS). We searched for an alternative mechanism in extracellular signal-regulated kinase (ERK)-2 and identified a 3 amino acid domain (SPS) that is phosphorylated upon stimulation to induce nuclear translocation of ERK2. A 19 amino acid stretch containing this phosphorylated domain inserts nondiffusible proteins to the nucleus autonomously. The phosphorylated SPS acts by binding to importin7 and the release from nuclear pore proteins. This allows its functioning both in passive and active ERK transports. A similar domain appears in many cytonuclear shuttling proteins, and we found that phosphorylation of similar sequences in SMAD3 or MEK1 also induces their nuclear accumulation. Therefore, our findings show that this phosphorylated domain acts as a general nuclear translocation signal (NTS).

microRNA-125a-3p reduces cell proliferation and migration by targeting Fyn.

Fyn, a member of the Src family kinases (SFKs), has a pivotal role in cell adhesion, proliferation, migration and survival, and its overexpression is associated with several types of cancer. MicroRNAs (miRNAs) play a major role in post-transcriptional repression of protein expression. In light of the significant functions of Fyn, together with studies demonstrating miR-125a as a tumor-suppressing miRNA that is downregulated in several cancer cell types and on our bioinformatics studies presented here, we chose to examine the post-transcription regulation of Fyn by miR-125a-3p in the HEK 293T cell line. We show that Fyn expression can be dramatically reduced by elevated levels of miR-125a-3p. Following this reduction, the activity of proteins downstream of Fyn, such as FAK, paxillin and Akt (proteins known to be overexpressed in various tumors), is also reduced. On a broader level, we show that miR-125a-3p causes an arrest of the cell cycle at the G2/M stage and decreases cell viability and migration, probably in a Fyn-directed manner. The results are reinforced by control experiments conducted using Fyn siRNA and anti-miR-125a-3p, as well as by the fact that numerous cancer cell lines show a significant downregulation of Fyn after mir-125a-3p overexpression. Collectively, we conclude that miR-125a-3p has an important role in the regulation of Fyn expression and of its signaling pathway, which implies that it has a therapeutic potential in overexpressed Fyn-related diseases.

Role of pigment epithelium-derived factor in the reproductive system.

The physiological function of the female reproductive organs is hormonally controlled. In each cycle, the reproductive organs undergo tissue modifications that are accompanied by formation and destruction of blood vessels. Proper angiogenesis requires an accurate balance between stimulatory and inhibitory signals, provided by pro- and anti-angiogenic factors. As with many other tissues, vascular endothelial growth factor (VEGF) appears to be one of the major pro-angiogenic factors in the female reproductive organs. Pigment epithelium-derived factor (PEDF) is a non-inhibitory member of the serine protease inhibitors (serpin) superfamily, possessing potent physiologic anti-angiogenic activity that negates VEGF activity. The role of PEDF in decreasing abnormal neovascularization by exerting its anti-angiogenic effect that inhibits pro-angiogenic factors, including VEGF, has been investigated mainly in the eye and in cancer. This review summarizes the function of PEDF in the reproductive system, showing its hormonal regulation and its anti-angiogenic activity. Furthermore, some pathologies of the female reproductive organs, including endometriosis, ovarian hyperstimulation syndrome, polycystic ovary syndrome, and others, are associated with a faulty angiogenic process. This review illuminates the role of PEDF in their pathogenesis and treatment. Collectively, we can conclude that although PEDF seems to play an essential role in the physiology and pathophysiology of the reproductive system, its full role and mechanism of action still need to be elucidated.

Hormonal regulation of pigment epithelium-derived factor (PEDF) in granulosa cells.

Angiogenesis is critical for the development of ovarian follicles. Blood vessels are abrogated from the follicle until ovulation, when they invade it to support the developing corpus luteum. Granulosa cells are known to secrete anti-angiogenic factors that shield against premature vascularization; however, their molecular identity is yet to be defined. In this study we address the physiological role of pigment epithelium-derived factor (PEDF), a well-known angiogenic inhibitor, in granulosa cells. We have shown that human and mouse primary granulosa cells express and secrete PEDF, and characterized its hormonal regulation. Stimulation of granulosa cells with increasing doses of estrogen caused a gradual decrease in the PEDF secretion, while stimulation with progesterone caused an abrupt decrease in its secretion. Moreover, We have shown, by time- and dose-response experiments, that the secreted PEDF and vascular endothelial growth factor (VEGF) were inversely regulated by hCG; namely, PEDF level was nearly undetectable under high doses of hCG, while VEGF level was significantly elevated. The anti-angiogenic nature of the PEDF secreted from granulosa cells was examined by migration, proliferation and tube formation assays in cultures of human umbilical vein endothelial cells. Depleting PEDF from primary granulosa cells conditioned media accelerated endothelial cells proliferation, migration and tube formation. Collectively, the dynamic expression of PEDF that inversely portrays VEGF expression may imply its putative role as a physiological negative regulator of follicular angiogenesis.

A physiological approach for treating endometriosis by recombinant pigment epithelium-derived factor (PEDF).

STUDY QUESTION: Is pigment epithelium-derived factor (PEDF) expressed in the rodent endometrium and can it be utilized to treat endometriosis without negatively affecting reproductive parameters? SUMMARY ANSWER: PEDF is dynamically expressed in rat endometrium throughout the estrous cycle in a reciprocal manner to vascular endothelial growth factor (VEGF); it possesses potent therapeutic properties for endometriosis that do not compromise the reproductive parameters. WHAT IS KNOWN ALREADY: Endometriosis pathogenesis depends mainly on neovascularization, with a high local level of VEGF. PEDF, a 50 kDa secreted glycoprotein with a potent anti-angiogenic activity, negates several strong pro-angiogenic factors, such as VEGF. STUDY DESIGN, SIZE, DURATION: Rat endometrial samples were collected at various days of the estrous cycle (n = 5 rats/day) and mRNA of VEGF and PEDF was determined. Endometriosis was induced by transplanting uterine pieces onto the inner surface of the abdominal wall of recipient rats, resulting in proliferation of the endometrial transplants. Recipient rats were randomly injected intravenously (IV), every third day for the next 3 weeks, with either Tris ('control'; n = 7) or recombinant PEDF (rPEDF; 2 mg/kg/day; 'PEDF prevention'; n = 7), while others were IV injected every third day starting from Day 9 after grafting until the end of 3 weeks, with rPEDF (2 mg/kg/day; 'PEDF treatment'; n = 6). The effect of rPEDF on the duration of the estrous cycle and on the number of ovulated oocytes was evaluated in rats that were randomly divided into four groups and were injected with either Tris or rPEDF every third day for 3 weeks: naive rats (n = 6); rPEDF-treated rats (n = 5); endometriosis-induced rats (n = 5); or endometriosis + rPEDF rats (n = 6). MATERIALS, SETTING, METHODS: Reproductive parameters: the estrous cycle was evaluated by daily vaginal smears, and the number of ovulated oocytes in the oviductal ampullae of estrus rats was counted. The efficiency of endometriosis induction and treatment was evaluated on the third week after endometrial transplantation, on the day of pro-estrus. Endometrial transplants were isolated and weighted. PEDF and VEGF were monitored by quantitative PCR and immunohistochemistry using confocal microscopy. MAIN RESULTS AND THE ROLE OF CHANCE: PEDF mRNA and protein were dynamically expressed in the endometrium all throughout the estrous cycle, reciprocally to VEGF; VEGF was highly expressed during estrus while PEDF expression was low, and vice versa at metestrus II. The weight of the endometrial transplants was significantly reduced after PEDF administration (13% of control for 'PEDF treatment' rats; 7% of control for 'PEDF prevention' rats; P < 0.001). Histology of the transplants' remnants showed a complete loss of their endometrial characteristics. Furthermore, the level of VEGF mRNA in the transplants of PEDF-administered rats was significantly lower (P < 0.05) than in transplants of control rats. Administration of rPEDF had no effect on the estrous cycle or ovulation rate of naive rats, while it had a significantly beneficial effect on the low ovulation rate of endometriosis-induced rats (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: The experiments were performed in a rat model. WIDER IMPLICATIONS OF THE FINDINGS: The endometriosis therapeutic potency of PEDF that is exerted reciprocally to VEGF and does not compromise reproductive parameters offers a rational for using PEDF as a treatment for endometriosis with a potential of treating other reproductive angiogenic-related pathologies.

De novo synthesis of protein phosphatase 1A, magnesium dependent, alpha isoform (PPM1A) during oocyte maturation.

Oocyte maturation in mammals is a multiple-stage process that generates fertilizable oocytes. Ovarian oocytes are arrested at prophase of the first meiotic division characterized by the presence of a germinal vesicle. Towards ovulation, the oocytes resume meiosis and proceed to the second metaphase in a process known as maturation; they undergo nuclear and cytoplasmic changes that are accompanied by translation and degradation of mRNA. Protein phosphatase 1A, magnesium dependent, alpha isoform (PPM1A), which belongs to the metal-dependent serine/threonine protein phosphatase family, is highly conserved during evolution. PPM1A plays a significant role in many cellular functions such as cell cycle progression, apoptosis and cellular differentiation. It works through diverse signaling pathways, including p38 MAP kinase JNK and transforming growth factor beta (TGF-ß). Herein we report that PPM1A is expressed in mouse oocytes and that its mRNA level rises during oocyte maturation. Using quantitative real-time polymerase chain reaction (qPCR) and western blot analysis, we found that PPM1A mRNA is synthesized at the beginning of the maturation process and remains elevated in the mature oocytes, promoting the accumulation of PPM1A protein. Since PPM1A function is mainly affected by its level, we propose that it might have an important role in oocyte maturation.

Effect of Genetic Variants of Gonadotropins and Their Receptors on Ovarian Stimulation Outcomes: A Delphi Consensus.

Background: A Delphi consensus was conducted to evaluate the influence of single nucleotide polymorphisms (SNPs) in genes encoding gonadotropin and gonadotropin receptors on clinical ovarian stimulation outcomes following assisted reproductive technology (ART) treatment. Methods: Nine experts plus two Scientific Coordinators discussed and amended statements plus supporting references proposed by the Scientific Coordinators. The statements were distributed via an online survey to 36 experts, who voted on their level of agreement or disagreement with each statement. Consensus was reached if the proportion of participants agreeing or disagreeing with a statement was >66%. Results: Eleven statements were developed, of which two statements were merged. Overall, eight statements achieved consensus and two statements did not achieve consensus. The statements reaching consensus are summarized here. (1) SNP in the follicle stimulating hormone receptor (FSHR), rs6166 (c.2039A>G, p.Asn680Ser) (N=5 statements): Ser/Ser carriers have higher basal FSH levels than Asn/Asn carriers. Ser/Ser carriers require higher amounts of gonadotropin during ovarian stimulation than Asn/Asn carriers. Ser/Ser carriers produce fewer oocytes during ovarian stimulation than Asn/Asn or Asn/Ser carriers. There is mixed evidence supporting an association between this variant and ovarian hyperstimulation syndrome. (2) SNP of FSHR, rs6165 (c.919G>A, p.Thr307Ala) (N=1 statement): Few studies suggest Thr/Thr carriers require a shorter duration of gonadotropin stimulation than Thr/Ala or Ala/Ala carriers. (3) SNP of FSHR, rs1394205 (-29G>A) (N=1 statement): Limited data in specific ethnic groups suggest that A/A allele carriers may require higher amounts of gonadotropin during ovarian stimulation and produce fewer oocytes than G/G carriers. (4) SNP of FSH ß-chain (FSHB), rs10835638 (-211G>T) (N=1 statement): There is contradictory evidence supporting an association between this variant and basal FSH levels or oocyte number. (5) SNPs of luteinizing hormone ß-chain (LHB) and LH/choriogonadotropin receptor (LHCGR) genes (N=1 statement): these may influence ovarian stimulation outcomes and could represent potential future targets for pharmacogenomic research in ART, although data are still very limited. Conclusions: This Delphi consensus provides clinical perspectives from a diverse international group of experts. The consensus supports a link between some variants in gonadotropin/gonadotropin receptor genes and ovarian stimulation outcomes; however, further research is needed to clarify these findings.

Interaction with MEK causes nuclear export and downregulation of peroxisome proliferator-activated receptor gamma.

The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) cascade plays a central role in intracellular signaling by many extracellular stimuli. One target of the ERK cascade is peroxisome proliferator-activated receptor gamma (PPARgamma), a nuclear receptor that promotes differentiation and apoptosis. It was previously demonstrated that PPARgamma activity is attenuated upon mitogenic stimulation due to phosphorylation of its Ser84 by ERKs. Here we show that stimulation by tetradecanoyl phorbol acetate (TPA) attenuates PPARgamma's activity in a MEK-dependent manner, even when Ser84 is mutated to Ala. To elucidate the mechanism of attenuation, we found that PPARgamma directly interacts with MEKs, which are the activators of ERKs, but not with ERKs themselves, both in vivo and in vitro. This interaction is facilitated by MEKs' phosphorylation and is mediated by the basic D domain of MEK1 and the AF2 domain of PPARgamma. Immunofluorescence microscopy and subcellular fractionation revealed that MEK1 exports PPARgamma from the nucleus, and this finding was supported by small interfering RNA knockdown of MEK1 and use of a cell-permeable interaction-blocking peptide, which prevented TPA-induced export of PPARgamma from the nucleus. Thus, we show here a novel mode of downregulation of PPARgamma by its MEK-dependent redistribution from the nucleus to the cytosol. This unanticipated role for the stimulation-induced nuclear shuttling of MEKs shows that MEKs can regulate additional signaling components besides the ERK cascade.

Filamin A is a novel caveolin-1-dependent target in IGF-I-stimulated cancer cell migration.

Caveolin-1 is an essential structural constituent of caveolae which is involved in regulation of mitogenic signaling and oncogenesis. Caveolin-1 has been implicated in cell migration but its exact role and mechanism of action in this process remained obscure. We have previously reported that expression of caveolin-1 in stably transfected MCF-7 human breast cancer (MCF-7/Cav1) cells up-regulates phosphorylation of a putative Akt substrate protein, designated pp340 [D. Ravid, S. Maor, H. Werner, M. Liscovitch, Caveolin-1 inhibits cell detachment-induced p53 activation and anoikis by upregulation of insulin-like growth factor-I receptors and signaling, Oncogene 24 (2005) 1338-1347.]. We now show, using differential detergent extraction, SDS-PAGE and mass spectrometry, that the major protein in the pp340 band is the actin filament cross-linking protein filamin A. The identity of pp340 as filamin A was confirmed by immunoprecipitation of pp340 with specific filamin A antibodies. RT-PCR, flow cytometry and Western blot analyses show that filamin A mRNA and protein levels are respectively 3.5- and 2.5-fold higher in MCF-7/Cav1 cells than in MCF-7 cells. Basal filamin A phosphorylation on Ser-2152, normalized to total filamin A levels, is 7.8-fold higher in MCF-7/Cav1 than in MCF-7 cells. Insulin-like growth factor-I (IGF-I) stimulates phosphorylation of filamin A on Ser-2152 in MCF-7 cells and further enhances Ser-2152 phosphorylation over its already high basal level in MCF-7/Cav1 cells. The effect of IGF-I is inhibited by the PI3K inhibitor wortmannin, indicating that IGF-I-stimulated phosphorylation of filamin A occurs via the PI3K/Akt pathway. Co-immunoprecipitation experiments have confirmed a previous report showing that filamin A and caveolin-1 co-exist in a complex and have revealed the presence of active phospho-Akt in this complex. Ser-2152 phosphorylation of filamin A has been implicated in cancer cell migration. Accordingly, caveolin-1 expression dramatically enhances IGF-I-dependent MCF-7 cell migration. These data indicate that caveolin-1 specifies filamin A as a novel target for Akt-mediated filamin A Ser-2152 phosphorylation thus mediating the effects of caveolin-1 on IGF-I-induced cancer cell migration.

The Dual Role of PEDF in the Pathogenesis of OHSS: Negating Both Angiogenic and Inflammatory Pathways.

CONTEXT: Ovarian hyperstimulation syndrome (OHSS) is a potentially life-threatening complication of assisted reproductive technologies. This complex syndrome is known to involve massive angiogenesis and inflammation. We have previously established the anti-angiogenic involvement of pigment epithelium-derived factor (PEDF) in the pathophysiology and treatment of OHSS. OBJECTIVE: Evaluate the anti-inflammatory role of PEDF in OHSS. DESIGN: In vivo mouse OHSS model and in vitro cultures of granulosa cells. MAIN OUTCOME: Changes in the expression of PEDF, IL-6, IL-8, and vascular endothelial growth factor (VEGF) were measured by quantitative PCR and ELISA; OHSS symptoms were recorded (body and ovarian weight gain and peritoneal vascular leakage quantified by the modified Miles's assay). RESULTS: Rat granulosa cell-line stimulated with lysophosphatidic acid (LPA), exhibited a significant increase in IL-6 expression, concomitantly with a decrease in PEDF level (P < .01). Co-stimulation with recombinant PEDF (rPEDF) decreased the expression of IL-6 significantly (P < .05). Furthermore, the expression of IL-6 and IL-8 increased in LPA-stimulated human primary granulosa cells (P < .01). Co-stimulation with rPEDF decreased the expression of LPA-induced IL-6 and IL-8 mRNA and protein by 4- and 2- to 5-fold, respectively. IL-8-stimulated human primary granulosa cells exhibited increased expression of VEGF mRNA; co-stimulation with hCG induced a significantly higher increase in the expression of VEGF mRNA (P < .001), which was counteracted by rPEDF. Subcutaneous injection of 0.5 mg/kg rPEDF to OHSS-induced mice reduced the increased expression of IL-6 in the ovary (P < .01) and alleviated the severity of all OHSS parameters. CONCLUSIONS: Our findings provide a framework that correlates down-regulation of OHSS symptoms caused by PEDF with both angiogenic and inflammatory pathways.

Protein-protein interactions in the regulation of the extracellular signal-regulated kinase.

The extracellular signal-regulated kinase (ERK) cascade is a central intracellular signaling pathway that is activated by a variety of extracellular stimuli, and thereby regulates cellular processes such as proliferation, differentiation, and oncogenic transformation. To execute these functions, the signals of those stimuli are transmitted to the cytosolic and nuclear targets in a rapid and specific manner. In the last few years it has become clear that the specificity and the rapid function of the ERK cascade is largely determined by protein-protein interactions with various signaling components and substrates. This review describes interactions of ERK with its immediate regulators, scaffold proteins, substrates, and localizing proteins, and shows their involvement in the functioning of the ERK cascade. Understanding the full scope of ERK-interactions is important for the development of new drugs for the treatment of cancer and other diseases.

GnRH Agonist Triggering Modulates PEDF to VEGF Ratio Inversely to hCG in Granulosa Cells.

CONTEXT: GnRH agonist (GnRH-a) triggering is associated with a reduced risk of ovarian hyperstimulation syndrome (OHSS) compared with human chorionic gonadotropin (hCG) in assisted reproduction technology cycles. We have shown that ovarian pigment epithelium derived factor (PEDF), a potent antiangiogenic factor, counteracts vascular endothelial growth factor (VEGF) expression and that OHSS is correlated with hCG-induced impaired PEDF to VEGF ratio. OBJECTIVE: The objective of the study was to explore whether GnRH-a triggering could directly modulate PEDF/VEGF balance in granulosa cells. DESIGN: The design of the study was a mouse model and cultured granulosa cells. MAIN OUTCOME: Changes in PEDF and VEGF were measured by quantitative PCR and Western blot analysis. OHSS symptoms were recorded by changes in body weight and in peritoneal vascular leakage, quantified by the modified Miles vascular permeability assay. RESULTS: GnRH-a stimulation significantly increased PEDF and decreased VEGF mRNA and protein levels both in rat granulosa cell line and human primary granulosa cells in vitro. GnRH-a and hCG triggering inversely modulated PEDF mRNA and protein level in human granulosa cells in vivo. In the GnRH-a triggering mouse model, we showed similar increase in PEDF to VEGF ratio as in the in vitro results. OHSS-predisposed mice did not develop OHSS parameters after GnRH-a triggering, opposed to hCG-triggered mice. Finally, GnRH-a triggering of OHSS-predisposed mice significantly increased ovarian PEDF to VEGF ratio compared with hCG-triggered mice and control mice. CONCLUSIONS: GnRH-a triggering induces a direct effect on PEDF/VEGF balance in granulosa cells inversely to hCG. Our results suggest a novel elucidation to the GnRH-a triggering-mediated risk reduction of OHSS and may clarify the pros and cons of this triggering method.