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We demonstrate an electrical control of an interfacial trapping effect for hot electrons injected in silicon by studying a magnetic tunnel transistor on wafer bonded Si substrate. Below 25 K, hot electrons are trapped at the Cu/Si interface, resulting in collector current suppression through scattering in both parallel and antiparallel magnetic configurations. Consequently, the magneto-current ratio strongly decreases from 300% at 27K to 30% at 22 K. The application of a relatively small electric field (∼333 V/cm) across the Cu/Si interface is enough to strip the trapped electrons and restore the magneto-current ratio at low temperature. We also present a model taking into account the effects of both electric field and temperature that closely reproduces the experimental results and allows extraction of the trapping binding energy (∼1.6meV) [ABSTRACT FROM AUTHOR]

The PIP 3 /PI3K network is a central regulator of metabolism and is frequently activated in cancer, commonly by loss of the PIP 3 /PI(3,4)P 2 phosphatase, PTEN. Despite huge research investment, the drivers of the PI3K network in normal tissues and how they adapt to overactivation are unclear. We find that in healthy mouse prostate PI3K activity is driven by RTK/IRS signaling and constrained by pathway feedback. In the absence of PTEN, the network is dramatically remodeled. A poorly understood YXXM- and PIP 3 /PI(3,4)P 2 -binding PH domain-containing adaptor, PLEKHS1, became the dominant activator and was required to sustain PIP 3 , AKT phosphorylation, and growth in PTEN-null prostate. This was because PLEKHS1 evaded pathway-feedback and experienced enhanced PI3K- and Src-family kinase-dependent phosphorylation of Y 258 XXM, eliciting PI3K activation. hPLEKHS1 mRNA and activating Y 419 phosphorylation of hSrc correlated with PI3K pathway activity in human prostate cancers. We propose that in PTEN-null cells receptor-independent, Src-dependent tyrosine phosphorylation of PLEKHS1 creates positive feedback that escapes homeostasis, drives PIP 3 signaling, and supports tumor progression., Competing Interests: Declaration of interests S.C. is an employee of AZ. We, the authors, have a patent related to this work (patent application number 2304156.9, granted by United Kingdom Patent Office, covering “modulating PLEKHS1 activity in a cell”)., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Knowledge of a protein's spatial dynamics at the subcellular level is key to understanding its function(s), interactions, and associated intracellular events. Indoleamine 2,3-dioxygenase 1 (IDO1) is a cytosolic enzyme that controls immune responses via tryptophan metabolism, mainly through its enzymic activity. When phosphorylated, however, IDO1 acts as a signaling molecule in plasmacytoid dendritic cells (pDCs), thus activating genomic effects, ultimately leading to long-lasting immunosuppression. Whether the two activities-namely, the catalytic and signaling functions-are spatially segregated has been unclear. We found that, under conditions favoring signaling rather than catabolic events, IDO1 shifts from the cytosol to early endosomes. The event requires interaction with class IA phosphoinositide 3-kinases (PI3Ks), which become activated, resulting in full expression of the immunoregulatory phenotype in vivo in pDCs as resulting from IDO1-dependent signaling events. Thus, IDO1's spatial dynamics meet the needs for short-acting as well as durable mechanisms of immune suppression, both under acute and chronic inflammatory conditions. These data expand the theoretical basis for an IDO1-centered therapy in inflammation and autoimmunity., (© 2020 The Authors.)

The PI3Kγ isoform is activated by Gi-coupled GPCRs in myeloid cells, but the extent to which the two endogenous complexes of PI3Kγ, p101/p110γ and p84/p110γ, receive direct regulation through Gβγ or indirect regulation through RAS and the sufficiency of those inputs is controversial or unclear. We generated mice with point mutations that prevent Gβγ binding to p110γ (RK552DD) or to p101 (VVKR777AAAA) and investigated the effects of these mutations in primary neutrophils and in mouse models of neutrophilic inflammation. Loss of Gβγ binding to p110γ substantially reduced the activation of both p101/p110γ and p84/p110γ in neutrophils by various GPCR agonists. Loss of Gβγ binding to p101 caused more variable effects, depending on both the agonist and cellular response, with the biggest reductions seen in PIP 3 production by primary neutrophils in response to LTB4 and MIP-2 and in the migration of neutrophils during thioglycolate-induced peritonitis or MIP2-induced ear pouch inflammation. We also observed that p101 VVKR777AAAA neutrophils showed enhanced p84-dependent ROS responses to f MLP and C5a, suggesting that competition may exist between p101/p110γ and p84/p110γ for Gβγ subunits downstream of GPCR activation. GPCRs did not activate p110γ in neutrophils from mice lacking both the p101 and p84 regulatory subunits, indicating that RAS binding to p110γ is insufficient to support GPCR activation in this cell type. These findings define a direct role for Gβγ subunits in activating both of the endogenous PI3Kγ complexes and indicate that the regulatory PI3Kγ subunit biases activation toward different GPCRs., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Circulating neutrophils are, by necessity, quiescent and relatively unresponsive to acute stimuli. In regions of inflammation, mediators can prime neutrophils to react to acute stimuli with stronger proinflammatory, pathogen-killing responses. In neutrophils G protein-coupled receptor (GPCR)-driven proinflammatory responses, such as reactive oxygen species (ROS) formation and accumulation of the key intracellular messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ), are highly dependent on PI3K-γ, a Ras-GTP, and Gβγ coincidence detector. In unprimed cells, the major GPCR-triggered activator of Ras is the Ras guanine nucleotide exchange factor (GEF), Ras guanine nucleotide releasing protein 4 (RasGRP4). Although priming is known to increase GPCR-PIP 3 signaling, the mechanisms underlying this augmentation remain unclear. We used genetically modified mice to address the role of the 2 RasGEFs, RasGRP4 and son of sevenless (SOS)1/2, in neutrophil priming. We found that following GM-CSF/TNFα priming, RasGRP4 had only a minor role in the enhanced responses. In contrast, SOS1/2 acquired a substantial role in ROS formation, PIP 3 accumulation, and ERK activation in primed cells. These results suggest that SOS1/2 signaling plays a key role in determining the responsiveness of neutrophils in regions of inflammation., (©2019 Society for Leukocyte Biology.)

Due to the difficulty of growing high-quality semiconductors on ferromagnetic metals, the study of spin diffusion transport in Si was limited to lateral geometry devices. In this work, by using an ultrahigh-vacuum wafer-bonding technique, we have successfully fabricated metal-semiconductor-metal CoFeB/MgO/Si/Pt vertical structures. We hereby demonstrate pure spin-current injection and transport in the perpendicular current flow geometry over a distance larger than 2 μm in n-type Si at room temperature. In those experiments, a pure propagating spin current is generated via ferromagnetic resonance spin pumping and converted into a measurable voltage by using the inverse spin Hall effect occurring in the top Pt layer. A systematic study varying both Si and MgO thicknesses reveals the important role played by the localized states at the MgO-Si interface for the spin-current generation. Proximity effects involving indirect exchange interactions between the ferromagnet and the MgO-Si interface states appears to be a prerequisite to establishing the necessary out-of-equilibrium spin population in Si under the spin-pumping action.

Class I phosphoinositide 3-kinases (PI3Ks) are important regulators of neutrophil migration in response to a range of chemoattractants. Their primary lipid products PtdIns(3,4,5)P3 and PtdIns(3,4)P2 preferentially accumulate near to the leading edge of migrating cells and are thought to act as an important cue organizing molecular and morphological polarization. We have investigated the distribution and accumulation of these lipids independently in mouse neutrophils using eGFP-PH reportersand electron microscopy (EM). We found that authentic mouse neutrophils rapidly polarized their Class I PI3K signalling, as read-out by eGFP-PH reporters, both at the up-gradient leading edge in response to local stimulation with fMLP as well as spontaneously and randomly in response to uniform stimulation. EM studies revealed these events occurred at the plasma membrane, were dominated by accumulation of PtdIns(3,4,5)P3, but not PtdIns(3,4)P2, and were dependent on PI3Kγ and its upstream activation by both Ras and Gβγs., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The molecular mechanisms by which receptors regulate the Ras Binding Domains of the PIP3-generating, class I PI3Ks remain poorly understood, despite their importance in a range of biological settings, including tumorigenesis, activation of neutrophils by pro-inflammatory mediators, chemotaxis of Dictyostelium and cell growth in Drosophila. We provide evidence that G protein-coupled receptors (GPCRs) can stimulate PLCb2/b3 and diacylglycerol- dependent activation of the RasGEF, RasGRP4 in neutrophils. The genetic loss of RasGRP4 phenocopies knock-in of a Ras-insensitive version of PI3Kc in its effects on PI3Kc-dependent PIP3 accumulation, PKB activation, chemokinesis and reactive oxygen species (ROS) formation. These results establish a new mechanism by which GPCRs can stimulate Ras, and the broadly important principle that PLCs can control activation of class I PI3Ks.

PI3Ks play important roles in the signaling pathways used by a wide variety of cell surface receptors on neutrophils. Class IB PI3K plays a major role in the initial generation of PtdIns(3,4,5)P₃ by Gi-coupled G-protein coupled receptors (GPCRs) (e.g., receptors for fMLP, C5a, LTB₄). Class IA PI3Ks generate PtdIns(3,4,5)P₃ downstream of receptors which directly or indirectly couple to protein tyrosine kinases such as integrins, FcγRs, cytokine receptors, and GPCRs. The PtdIns(3,4,5)P₃ made by Class I PI3Ks regulates the activity of several different effector proteins, many of which are plasma membrane GEFs or GAPs for small GTPases. Class III PI3K generates PtdIns(3)P in the phagosome membrane and plays an important role in efficient assembly of the NADPH oxidase at this location. Much still remains to be discovered about the molecular details that govern activation of PI3Ks and the mechanisms by which these enzymes regulate complex cellular processes, such as neutrophil spreading, chemotaxis, phagocytosis, and killing of pathogens. However, it is clear from recent use of transgenic mouse models and isoform-selective PI3K inhibitors that these pathways are important in regulating neutrophil recruitment to sites of infection and damage in vivo. Thus, PI3K pathways may present novel opportunities for selective inhibition in some inflammatory pathologies.

We describe a novel approach to the relative quantification of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)] and its application to measure, in neutrophils, the activation of phosphoinositide 3-kinase (PI3K). This protein-lipid overlay-based assay allowed us to confirm and extend the observations, first, that N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation of primed human neutrophils leads to a transient and biphasic increase in PtdIns(3,4,5)P(3) levels and, second, that the ability of fMLP to stimulate PtdIns(3,4,5)P(3) accumulation in neutrophils isolated from mice carrying a Ras-insensitive ('DASAA') knock-in of PI3Kgamma (p110gamma(DASAA/DASAA)) is substantially dependent on the Ras binding domain of PI3Kgamma.

Through their ability to regulate production of the key lipid messenger PtdIns(3,4,5)P(3), the class I phosphatidylinositol-3-OH kinases (PI(3)Ks) support many critical cell responses. They, in turn, can be regulated by cell-surface receptors through signals acting on either their adaptor subunits (for example, through phosphotyrosine or Gbetagammas) or their catalytic subunits (for example, through GTP-Ras). The relative significance of these controlling inputs is undefined in vivo. Here, we have studied the roles of Gbetagammas, the adaptor p101, Ras and the Ras binding domain (RBD) in the control of the class I PI(3)K, PI(3)Kgamma, in mouse neutrophils. Loss of p101 leads to major reductions in the accumulation of PtdIns(3,4,5)P(3), activation of protein kinase B (PKB) and in migration towards G-protein activating ligands in vitro, and to an aseptically inflamed peritoneum in vivo. Loss of sensitivity of PI(3)Kgamma to Ras unexpectedly caused similar reductions, but additionally caused a substantial loss in production of reactive oxygen species (ROS). We conclude that Gbetagammas, p101 and the Ras-RBD interaction all have important roles in the regulation of PI(3)Kgamma in vivo and that they can simultaneously, but differentially, control distinct PI(3)Kgamma effectors.

A variety of genetic and inhibitor studies have shown that phosphoinositide 3-kinase gamma (PI3Kgamma) plays an essential role in a number of physiological responses, including neutrophil chemotaxis, mast cell degranulation, and cardiac function []. PI3Kgamma is currently thought to be composed of a p110gamma catalytic subunit and a single regulatory subunit, p101. The binding of p110gamma to p101 dramatically increases the activation of the complex by Gbetagamma subunits and, hence, is thought to be critical for the coupling of PI3Kgamma to G protein coupled receptors []. Here, we characterize a new regulatory subunit for PI3Kgamma. p84 is present in human, mouse, chicken, frog, and fugu genomes and is located beside the p101 locus. It is broadly expressed in cells of the murine immune system. Both recombinant and endogenous p84 bind p110gamma specifically and with high affinity. Binding of p84 to p110gamma substantially increases the ability of Gbetagamma to stimulate phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P(3)) production both in vitro and in vivo. However, the p84/p110gamma heterodimer is approximately 4-fold less sensitive to Gbetagammas than p101/p110gamma. Endogenous murine p84 expression is substantially reduced in the absence of p110gamma expression. We conclude that p110gamma has two potential regulatory subunits in vivo, p84 and p101.

Background: Type I phosphoinositide 3-kinases are responsible for the hormone-sensitive synthesis of the lipid messenger phosphatidylinositol(3,4,5)-trisphosphate. Type IA and IB subfamily members contain a Ras binding domain and are stimulated by activated Ras proteins both in vivo and in vitro. The mechanism of Ras activation of type I PI3Ks is unknown, in part because no robust in vitro assay of this event has been established and characterized. Other Ras effectors, such as Raf and phosphoinositide-phospholipase Cepsilon, have been shown to be translocated into the plasma membrane, leading to their activation., Results: We show that posttranslationally lipid-modified, activated N-, H-, K-, and R-Ras proteins can potently and substantially activate PI3Kgamma when using a stripped neutrophil membrane fraction as a source of phospholipid substrate. We have found GTPgammaS-loaded Ras can significantly (6- to 8-fold) activate PI3Kgamma when using artificial phospholipid vesicles containing their substrate, and this effect is a result of both a decrease in apparent Km for phosphatidylinositol(4,5)-bisphosphate and an increase in the apparent Vmax. However, neither in vivo nor in the two in vitro assays of Ras activation of PI3Kgamma could we detect any evidence of a Ras-dependent translocation of PI3Kgamma to its source of phospholipid substrate., Conclusions: Our data suggest that Ras activate PI3Kgamma at the level of the membrane, by allosteric modulation and/or reorientation of the PI3Kgamma, implying that Ras can activate PI3Kgamma without its membrane translocation. This view is supported by structural work that has suggested binding of Ras to PI3Kgamma results in a change in the structure of the catalytic pocket.

The equine conceptus is surrounded by a fibrous capsule that persists until about day 20 of pregnancy, whereupon the capsule is lost, the conceptus attaches to the endometrium and placentation proceeds. Before attachment, the endometrium secretes in abundance a protein of the lipocalin family, uterocalin. The cessation of secretion coincides with the end of the period during which the conceptus is enclosed in its capsule, suggesting that uterocalin is essential for the support of the embryo before direct contact between maternal and foetal tissues is established. Using recombinant protein and fluorescence-based assays, we show that equine uterocalin binds the fluorescent fatty acids 11-(dansylamino)undecanoic acid, dansyl-D,L-alpha-amino-octanoic acid and cis-parinaric acid, and, by competition, oleic, palmitic, arachidonic, docosahexaenoic, gamma-linolenic, cis-eicosapentaenoic and linoleic acids. Uterocalin also binds all-trans-retinol, the binding site for which is coincident or interactive with that for fatty acids. Molecular modelling and intrinsic fluorescence analysis of the wild-type protein and a Trp-->Glu mutant protein indicated that uterocalin has an unusually solvent-exposed Trp side chain projecting from its large helix directly into solvent. This feature is unusual among lipocalins and might relate to binding to, and uptake by, the trophoblast. Uterocalin therefore has the localization and binding activities for the provisioning of the equine conceptus with lipids including those essential for morphogenesis and pattern formation. The possession of a fibrous capsule surrounding the conceptus might be an ancestral condition in mammals; homologues of uterocalin might be essential for early development in marsupials and in eutherians in which there is a prolonged preimplantation period.

Ras activation of phosphoinositide 3-kinase (PI3K) is important for survival of transformed cells. We find that PI3Kgamma is strongly and directly activated by H-Ras G12V in vivo or by GTPgammaS-loaded H-Ras in vitro. We have determined a crystal structure of a PI3Kgamma/Ras.GMPPNP complex. A critical loop in the Ras binding domain positions Ras so that it uses its switch I and switch II regions to bind PI3Kgamma. Mutagenesis shows that interactions with both regions are essential for binding PI3Kgamma. Ras also forms a direct contact with the PI3Kgamma catalytic domain. These unique Ras/PI3Kgamma interactions are likely to be shared by PI3Kalpha. The complex with Ras shows a change in the PI3K conformation that may represent an allosteric component of Ras activation.

Horses, donkeys, and therefore, probably all equids, secrete a nonglycosylated, progesterone-dependent, 19-kDa protein (P19) into the uterine lumen during early pregnancy, and significant quantities of it are taken up by the developing conceptus. Sequence analysis and structural modelling have identified P19 as a lipocalin with greatest similarity to the murine major urinary protein lipocalins. However, lack of strong identity with any particular group of lipocalins and several unusual structural features, including a unique amino acid triplet within one of the invariant domains and an unusual external tryptophan residue, classify it as a new member of the lipocalin family. P19 is therefore likely to be a transport protein involved in supporting early embryonic development. Preliminary evidence using recombinant-derived P19 and fluorescently tagged ligands suggests that it may transport a fatty acid or retinol-like molecule. Although an initial search failed to identify homologues of P19 in other mammals, they may nevertheless exist but are synthesised and secreted in much smaller quantities, making them difficult to detect. Equids appear to need particularly large quantities of the protein during early pregnancy because of the unusually late implantation in this species and the presence of a capsule surrounding the conceptus until about day 23 of gestation.

One of the major progesterone-dependent endometrial proteins in the mare is a novel 19 kDa lipocalin (P19). This protein is secreted by the endometrial glands and is readily detectable in uterine secretions during the luteal phase of the oestrous cycle and early pregnancy. The function of P19 is unknown, but since most lipocalins act as carriers of small hydrophobic molecules, it probably transports a maternal factor to the conceptus during pregnancy. In this study, a high titre antiserum raised against recombinant-derived P19 was used to detect by immunohistochemistry the protein in endometrial biopsies from normal healthy mares and mares with endometrosis. Immunoreactive P19 was undetectable in prepubertal fillies and in anoestrous mares and was barely detectable in oestrous mares. However, it was present in large amounts during dioestrus and very early pregnancy, and in moderate amounts after day 20 of gestation. In six subfertile mares with endometrosis, an abnormal pattern was found at all stages of the reproductive cycle, especially in the 'gland nests'. These showed either an absence of P19 or an accumulation of the protein due to a lack of secretion, thus indicating asynchronous glandular activity. These results identified a high proportion of abnormal, asynchronous endometrial glands in a group of subfertile mares and indicated that abnormal secretion of P19 may be one of the reasons for the reduced fertility rates observed in aged mares with endometrosis.

One of the major, progesterone-dependent proteins secreted into the uterine lumen of the mare is a 19-kDa lipocalin (P19). It associates strongly with the embryonic capsule that envelops the young horse conceptus in early gestation, suggesting that it may be involved in sustaining early development. However, it was not known whether the protein was transported through the capsule and/or trophoblast layer and into the yolk sac cavity. To address this question, polyclonal antisera were raised against a C-terminal peptide (based on the deduced amino acid sequence of P19) and a recombinant-derived P19 fusion protein. The antiserum raised against the C-terminal peptide recognized P19 on Western blots of denatured uterine secretions (subjected to SDS-PAGE), but it did not bind to the protein in tissue sections. However, the antiserum raised against the recombinant-derived fusion protein recognized P19 both on Western blots and in histological sections. Western blot analysis of tissues and fluids collected from early-pregnant mares demonstrated significant quantities of P19 in the endometrium and uterine secretions and in the embryonic capsule, the chorion, and the yolk sac fluid, showing that the protein is transferred through to the developing embryo. Concentrations of immunoreactive P19 declined during gestation so that, by Day 30, it had virtually disappeared from both maternal and fetal tissues and fluids. Immunohistochemical staining of endometrial biopsies collected during early pregnancy localized P19 to the glandular and luminal epithelia and to the lumina of the endometrial glands. The capsule and the trophoblast layer of the chorion from early (Days 16-17) horse conceptuses also stained positively with localization of P19 to the apical surface of the trophoblast cells. There was no detectable staining either in or on the embryonic disc. The presence of P19 in both the trophoblast layer and the yolk sac fluid suggests that P19 passes into the yolk sac fluid via trophoblast cells.

Transferrin secretion and expression were studied in cultured Sertoli cells recovered from fats at days 10 and 17 postpartum. Transferrin biosynthesis as measured by radioimmunoassay showed a dramatic, 3.9-fold increase between days 10 and 17. The majority of the transferrin was secreted, and a kinetic study revealed that the production was four times higher at day 17 than at day 10. This difference was not the result of altered transferrin degradation as the protein was shown to be very stable at both ages. To determine if this regulation was at the transcriptional or translation level, Northern blot analysis, nuclear run-on assays, mRNA stability (half-life) measurements, and mRNA intracytoplasmic distribution analyses were carried out. The Northern blots analysis, the nuclear run-on assays, and the half-life measurements revealed that transferrin mRNA levels, gene transcription rates, and mRNA stability were indistinguishable at both ages. Interestingly, the intracytoplasmic mRNA distribution analyses showed that most of the transferrin mRNA (80%) was associated with the 40 S and 60 S protein particles at both ages and was, therefore, theoretically untranslatable. However, approximately twice as much transferrin in RNA was found to be associated with polysomes at day 17 as compared to day 10. We have shown that the increase in transferrin biosynthesis by rat Sertoli cells during testicular development is not due to an increase in the amount of transferrin mRNA or an increase in its half-life, but appears to be due to an increase in translation rate of the transferrin mRNA.

The transcription of the transferrin (Tf) gene is induced by follitropin via cAMP in rat Sertoli cells. We previously demonstrated that the cAMP-responsive-element-binding protein (CREB) interacts on the proximal region II (PRII) of the human Tf promoter (Suire et al., 1995). The PRII region is identified as essential for cAMP inducibility of the Tf promoter and contains a CCAAT box. This unexpected result led us to study the relation that exists between CREB and the PRII site. In the liver, CCAAT/enhancer-binding (C/EBP) proteins act at the PRII site. Although these factors are absent in Sertoli cells, their overexpression in Sertoli cells disturbs basal and induced transcription. C/EBP alpha and delta were able to stimulate the basal transcription driven by the -100 to +39 region, placed upstream of the chloramphenicol acetyltransferase (CAT) gene. However, only C/EBP alpha allowed the cAMP-inducible expression. The Ka of CREB bZIP (254-327), a deleted form of CREB, for the CRE site (3.92 x 10(8)M-1) and for the PRII site (1.38 x 10(8)M-1) were determined using the surface plasmon resonance (SPR) method. The Ka values were similar, although the derived kinetics were different: higher ka and kd of CREB for the PRII site were found compared with the CRE site. Since we observed important dissociation kinetics, we hypothesized that the binding of CREB to the PRII site is stabilized by CREB-binding protein (CBP) or by chicken-ovalbumin-upstream-promoter transcription factor (COUP-TF) binding to PRI site near to PRII. However, we observed that the overexpression of CBP in Sertoli cells did not potentiate the basal and cAMP-stimulated activity of CREB of the -100 to +39Tf-CAT construct. In basal and cAMP-stimulated conditions, COUP-TF appeared to repress the transcription driven by the -100 to +39 region in a specific manner. These results demonstrate a direct action of CREB on hTf promoter, which is antagonized by COUP-TF and may explain the transcriptional regulation of Tf by follitropin, via cAMP.

FSH is a major regulator of transferrin (Tf) production in the testis. FSH effects on Sertoli cell Tf production are believed to be mediated, at least in part, via cAMP second messenger system. Previously, it has been shown that FSH and (Bu)2cAMP stimulate Tf mRNA levels. This study examines the effect of cAMP and FSH on Tf gene transcription using run-on assays. These data demonstrate rapid induction of Tf gene by (Bu)2cAMP (2.3-fold) and FSH (2.8-fold) within 30 min and 2 h, respectively. Furthermore, the ability of (Bu)2cAMP and FSH to drive the transcription of chimeric constructs containing a 0.6-kilobase segment of the 5'-regulatory region of the human Tf gene coupled to a chloramphenicol acetyltransferase (CAT) was examined. Deletion analysis indicated that the sequence -100/-52 base pairs is required for the cAMP-dependent transcription. This sequence shows no homology to that of the consensus cAMP-regulatory element (CRE). However, cotransfection experiments with a CRE-binding protein (CREB) expression vector revealed a basal induction of the Tf transcriptional activity as well as a synergistic activation of CREB and (Bu)2cAMP. Expression of KCREB, a dominant negative mutant form of CREB, completely blocked the cAMP induction of the -100+39Tf-CAT construct. This region contains two functional regions PRI and PRII. Gel shift assay with nuclear proteins from Sertoli cells using the PRII and PRI probes showed that the band shifts formed by PRII were competitive complexes with CRE, and a CREB antiserum retarded the migration of nuclear Sertoli cells proteins. We conclude that CREB is implicated in the FSH regulation on the Tf gene in Sertoli cells.