GnRH regulation of Jun and Atf3 requires calcium, calcineurin, and NFAT.

GnRH binds to its receptor on gonadotropes and activates multiple members of the MAPK signaling family that in turn regulates the expression of several immediate early genes (IEGs) including Jun, Fos, Atf3, and Egr1. These IEGs confer hormonal responsiveness to gonadotrope-specific genes including Gnrhr, Cga, Fshb, and Lhb. In this study we tested the hypothesis that GnRH specifically regulates the accumulation of Jun and Atf3 mRNA through a pathway that includes intracellular Ca²âº, calcineurin, and nuclear factor of activated T cells (NFAT). Our results indicate that pretreatment of murine LßT2 cells with 1, 2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)-ester, a Ca²âº chelator, reduced the expression of all the IEGs to varying degrees, whereas treatment with thapsigargin, an intracellular Ca²âº protein pump inhibitor, increased the expression of the IEG. Furthermore, cyclosporin A, a calcineurin-specific inhibitor, reduced the ability of GnRH to regulate accumulation of Jun and Atf3 mRNA and to a lesser extent Fos. In contrast, Egr1 mRNA was unaffected. NFATs are transcription factors regulated by calcineurin and were detected in LßT2 cells. GnRH increased luciferase activity of an NFAT-dependent promoter reporter that was dependent on intracellular Ca²âº and calcineurin activity. Additionally, although small interfering RNA specific for Nfat4 only marginally reduced GnRH regulation of Jun, Fos, and Atf3 mRNA accumulation, activity of an activator protein-1-responsive reporter construct was reduced by 48%. Together these data suggest that calcineurin and NFAT are new members of the gonadotrope transcriptional network that confer hormonal responsiveness to several key genes required for gonadotropin synthesis and secretion.

GnRH-regulated expression of Jun and JUN target genes in gonadotropes requires a functional interaction between TCF/LEF family members and beta-catenin.

GnRH regulates gonadotrope function through a complex transcriptional network that includes three members of the immediate early gene family: Egr1, Jun, and Atf3. These DNA-binding proteins act alone or in pairs to confer hormonal responsiveness to Cga, Lhb, Fshb, and Gnrhr. Herein we suggest that the transcriptional response of Jun requires a functional interaction between the T-cell factor (TCF)/lymphoid enhancer factor (LEF) family of DNA-binding proteins and beta-catenin (officially CTNNB1), a coactivator of TCF/LEF. Supporting data include demonstration that GnRH increases activity of TOPflash, a TCF/LEF-dependent luciferase reporter, in LbetaT2 cells, a gonadotrope-derived cell line. Additional cotransfection experiments indicate that a dominant-negative form of TCF7L2 (TCFDN) that binds DNA, but not beta-catenin, blocks GnRH induction of TOPflash. Overexpression of AXIN, an inhibitor of beta-catenin, also reduces GnRH stimulation of TOPflash. Transduction of LbetaT2 cells with TCFDN adenoviruses diminishes GnRH stimulation of Jun mRNA without altering expression of Egr1 and Atf3, two other immediate early genes that confer GnRH responsiveness. Reduction of beta-catenin in LbetaT2 cells, through stable expression of short hairpin RNA, also selectively compromises GnRH regulation of Jun expression and levels of JUN protein. Finally, overexpression of TCFDN attenuates GnRH regulation of Cga promoter activity, a known downstream target of JUN. Together, these results indicate that GnRH regulation of Jun transcription requires a functional interaction between TCF/LEF and beta-catenin and that alteration of either impacts expression of JUN downstream targets such as Cga.

Maximal activity of the luteinizing hormone beta-subunit gene requires beta-catenin.

GnRH regulates expression of LHB via transcriptional regulation of early growth response 1 (EGR1), an immediate early gene that encodes a zinc-finger DNA-binding protein. EGR1 interacts functionally with the orphan nuclear receptor steroidogenic factor 1 (SF1) and pituitary homeobox 1, a member of the paired-like homeodomain family. The functional synergism of this tripartite interaction defines the maximal level of LHB transcription that can occur in response to GnRH. Results presented herein provide new evidence that the interaction between SF1 and EGR1 also requires beta-catenin, a transcriptional coactivator and member of the canonical Wnt signaling pathway. For instance, targeted reduction of beta-catenin attenuates activity of a GnRH-primed LHB promoter. Additional gene reporter assays indicate that overexpression of beta-catenin, or its targeted reduction by small interfering RNA, modulates activity of both SF1 and EGR1 as well as their functional interaction. beta-Catenin coimmunoprecipitates with SF1. Moreover, an SF1 mutant that lacks a beta-catenin binding domain has compromised transcriptional activity and fails to interact synergistically with EGR1. Finally, GnRH promotes beta-catenin colocalization with SF1 and EGR1 on the endogenous mouse Lhb promoter-regulatory region. Taken together, these data suggest that beta-catenin binds to SF1 and that this interaction is required for subsequent functional interaction with EGR1. Thus, these data identify beta-catenin as a new and required member of the basal transcriptional complex that allows the LHB promoter to achieve maximal activity in response to GnRH.

Follicle-stimulating hormone/cAMP regulation of aromatase gene expression requires beta-catenin.

Estrogens profoundly influence the physiology and pathology of reproductive and other tissues. Consequently, emphasis has been placed on delineating the mechanisms underlying regulation of estrogen levels. Circulating levels of estradiol in women are controlled by follicle-stimulating hormone (FSH), which regulates transcription of the aromatase gene (CYP19A1) in ovarian granulosa cells. Previous studies have focused on two downstream effectors of the FSH signal, cAMP and the orphan nuclear receptor steroidogenic factor-1 (NR5A1). In this report, we present evidence for beta-catenin (CTNNB1) as an essential transcriptional regulator of CYP19A1. FSH induction of select steroidogenic enzyme mRNAs, including Cyp19a1, is enhanced by beta-catenin. Additionally, beta-catenin is present in transcription complexes assembled on the endogenous gonad-specific CYP19A1 promoter, as evidenced by chromatin immunoprecipitation assays. Transient expression and RNAi studies demonstrate that FSH- and cAMP-dependent regulation of this promoter is sensitive to alterations in the level of beta-catenin. The stimulatory effect of beta-catenin is mediated through functional interactions with steroidogenic factor-1 that involve four acidic residues within its ligand-binding domain, mutation of which attenuates FSH/cAMP-induced Cyp19a1 mRNA accumulation. Together, these data demonstrate that beta-catenin is essential for FSH/cAMP-regulated gene expression in the ovary, identifying a central and previously unappreciated role for beta-catenin in estrogen biosynthesis, and a potential broader role in other aspects of follicular maturation.

Closing of the nucleotide pocket of kinesin-family motors upon binding to microtubules.

We have used adenosine diphosphate analogs containing electron paramagnetic resonance (EPR) spin moieties and EPR spectroscopy to show that the nucleotide-binding site of kinesin-family motors closes when the motor.diphosphate complex binds to microtubules. Structural analyses demonstrate that a domain movement in the switch 1 region at the nucleotide site, homologous to domain movements in the switch 1 region in the G proteins [heterotrimeric guanine nucleotide-binding proteins], explains the EPR data. The switch movement primes the motor both for the free energy-yielding nucleotide hydrolysis reaction and for subsequent conformational changes that are crucial for the generation of force and directed motion along the microtubule.

A novel restricted photoaffinity spin-labeled non-nucleoside ATP analogue as a covalently attached reporter group of the active site of Myosin subfragment 1.

The photoaffinity spin-labeled non-nucleoside ATP analogue, 2-(4-azido-2-nitrophenyl)amino-2,2-(1-oxyl-2,2,6,6-tetramethyl-4-piperidylidene)di(oxymethylene)ethyl triphosphate (SSL-NANTP), has been shown to be a substrate for skeletal mysoin subfragment 1 (S1) that can be photoincorporated at the active site of S1 [Chen, X., et al. (2000) Bioconjugate Chem. 11, 725-733]. Electron paramagnetic resonance spectroscopy shows that the probe undergoes restricted motion with respect to the protein. The parent compound, NANTP (2-[(4-azido-2-nitrophenyl)amino]ethyl triphosphate), is specifically photoincorporated at Trp-130 on the amino-terminal 23 kDa tryptic fragment in rabbit skeletal myosin. Surprisingly, amino acid sequence analysis shows that SSL-NANTP is photoincorporated on the carboxy-terminal 20 kDa tryptic fragment at Lys-681 on the side opposite Trp-130 in the nucleotide pocket. This is the first direct evidence showing that this residue in the 20 kDa tryptic fragment is close enough to the active site to be photolabeled by trapped ATP analogues. After actin treatment in the presence of MgATP, SSL-NANDP-labeled myosin S1 had normal ATPase activity, indicating that photolabeling did not significantly alter the enzymatic properties of S1. Photoincorporated SSL-NANDP was bound inside the nucleotide site of S1, with an effective concentration of 20 mM as judged by the concentration of MgADP needed to displace it. Molecular dynamics simulations suggest that the ability of NANTP and SSL-NANTP to photolabel different sites results from different orientations of the phenyl ring in the active site. For SSL-NANTP, the p-azido group on the phenyl ring points toward Lys-681. For NANTP, it points in the opposite direction toward Trp-130.