Strategies for construction of luteinizing hormone beta subunit analogs with carboxyl terminal extensions in non-primate, non-equid mammalian species.

Chorionic gonadotropins (CG) are unique because they have a carboxyl terminal peptide (CTP) extension on their beta subunits that prolongs circulatory survival. CGbeta genes from the human being and horse have evolved from ancestral luteinizing hormone (LH) beta genes by different pathways that involve deletions that change the reading frames and yield a CTP. Here we further review our previous analysis, aimed at determining whether LHbeta genes in non-primate, non-equid species inherently possess DNA sequences that encode CTP-like domains. In multiple mammalian species, simple frame-shift mutations using either the human or equine CGbeta gene as a model can be used to construct LHbeta analogs with putative CTP domains. Furthermore, DNA sequences from mammalian LHbeta genes can be aligned to maximize similarity with CGbeta genes in order to devise more refined strategies for construction of CTP-bearing LHbeta analogs as exemplified in the bovine case. Thus, mammalian LHbeta genes have DNA sequences that can be potentially expressed in order to construct CTP-bearing glycoprotein hormone analogs.

Homologous and heterologous carboxyl terminal peptide (CTP) linker sequences enhance the secretion of bioactive single-chain bovine LH analogs.

Single chain variants of the heterodimeric gonadotropins were engineered by tethering the genes of the individual subunits into one polypeptide. In tethered human (h) gonadotropins, the carboxyl terminal peptide (CTP) of the choriogonadotropin (CG) beta subunit serves as an effective linker to enhance the secretion of the analogs compared to variants lacking the CTP. The gonadotropin subunits of non-primate, non-equid species lack a CTP domain that precludes the use of a homologous CTP in tethered analogs in many species. Here we used the bovine LH as a model to examine the impact of the CTP domain of the hCGbeta subunit (denoted as huCTP) and of a previously untranslated CTP-like sequence decoded from the bovine LHbeta gene on the secretion and bioactivity of tethered analogs. This cryptic CTP (designated boCTP) was incorporated into the bovine LHbeta reading frame by deletion frame-shift mutations analogous to these that presumably occurred in primates and equids. We genetically engineered single chain variants in which the beta and alpha subunit domains were linked directly or via the heterologous huCTP or the homologous boCTP sequences and expressed them in CHO cells. The data suggest that the tethered analogs were expressed and N-glycosylated, but unlike the huCTP, the boCTP appears as devoid of mucin O-glycans. The incorporation of the boCTP or huCTP linkers enhanced by about 3fold the rate and efficiency of secretion from the transfected cells. The tether variants were bioactive, as estimated by induction of steroid production in immortalized granulosa cells expressing the rat LH receptor. Furthermore, the variants were about equally potent, as judged by their EC50s (0.7-0.9 ng/ml). Thus, the hCGbeta CTP maintains pro-secretory determinants without inhibiting receptor activation when applied as a linker in tethered bovine LH, implying that these CTP features are preserved when the domain is incorporated into non-primate single chain analogs. The study suggests that the boCTP and huCTP domains are advantageous for the secretion of tethered bovine gonadotropins, and also demonstrates strategies for the design of bioactive LH analogs in ruminant species.

Hormone binding to the follicle-stimulating hormone receptor--crystal clear!

The receptors for the trophic hormones luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyrotropin (TSH) play a central role in endocrinology. These receptors face the challenge to accommodate large heterodimeric glycoprotein ligands within their extracellular hormone-binding domain. Until recently, the mechanism of hormone binding and consequently the mode of receptor activation remained enigmatic. By solving the crystal structure of human follicle-stimulating hormone bound to the receptor's hormone binding domain, it has become clear that the follicle-stimulating hormone receptor grabs the glycoprotein hormone in a hand-clasp mode resulting in a hormone orientation perpendicular to the long axis of the ligand-binding domain. These findings have important ramifications for our understanding of the molecular mechanism of receptor activation and may provide a rational basis for the development of small, non-peptidic FSH receptor ligands.

Single chain variants of the glycoprotein hormones and their receptors as tools to study receptor activation and for analogue design.

One of the crucial steps in the biosynthesis of multisubunit proteins is their assembly. The glycoprotein hormone, thyroid-stimulating hormone, and the gonadotropins, luteinizing hormone, follicle-stimulating hormone and chorionic gonadotropin, are noncovalent heterodimers. Their assembly is critical for bioactivity because the heterodimers, but not the monomeric subunits, efficiently bind to and activate the cognate heptahelical receptor. Occasionally, mutated subunits cannot combine into a functional hormone, or the bioactivity of the assembled, yet modified, heterodimer is suboptimal.

The position of the alpha and beta subunits in a single chain variant of human chorionic gonadotropin affects the heterodimeric interaction of the subunits and receptor-binding epitopes.

The glycoprotein hormone family represents a class of heterodimers, which include the placental hormone human chorionic gonadotropin (CG) and the anterior pituitary hormones follitropin, lutropin, and thyrotropin. They are composed of common alpha subunit and a hormone-specific beta subunit. Based on the CG crystal structure, it was suggested that the quaternary subunit interactions are crucial for biological activity. However, recent observations using single chain glycoprotein hormone analogs, where the beta and alpha subunits are linked (NH(2)-CGbeta-alpha; CGbetaalpha orientation), implied that the heterodimeric-like quaternary configuration is not a prerequisite for receptor binding/signal transduction. To study the heterodimeric alignment of the two subunit domains in a single chain and its role in the intracellular behavior and biological action of the hormone, a single chain CG variant was constructed in which the carboxyl terminus of alpha was fused to the CGbeta amino terminus (NH(2)-alpha-CGbeta; alphaCGbeta orientation). The secretion rate of alphaCGbeta from transfected Chinese hamster ovary cells was less than that seen for CGbetaalpha. The alphaCGbeta tether was not recognized by dimer-specific monoclonal antibodies and did not bind to lutropin/CG receptor. To define if one or both subunit domains were modified in alphaCGbeta, it was co-transfected with a monomeric alpha or CGbeta gene. In each case, alphaCGbeta/alpha and alphaCGbeta/CGbeta complexes were formed indicating that CG dimer-specific epitopes were established. The alphaCGbeta/alpha complex bound to receptor indicating that the beta domain in the alphaCGbeta tether was still functional. In contrast, no significant receptor binding of alphaCGbeta/CGbeta was observed indicating a major perturbation in the alpha domain. These results suggest that although dimeric-like determinants are present in both alphaCGbeta/alpha and alphaCGbeta/CGbeta complexes, the receptor binding determinants in the alpha domain of the tether are absent. These results show that generating heterodimeric determinants do not necessarily result in a bioactive molecule. Our data also indicate that the determinants for biological activity are distinct from those associated with intracellular behavior.

Synthesis of multi-subunit domain gonadotropin complexes: a model for alpha/beta heterodimer formation.

The human glycoprotein hormones chorionic gonadotropin (CG), thyrotropin (TSH), lutropin (LH), and follitropin (FSH) are heterodimers, composed of a common alpha subunit assembled to a hormone-specific beta subunit. The subunits combine noncovalently early in the secretory pathway and exist as heterodimers, but not as multimers. Little information is available regarding the steps associated with the assembly reaction. It is unclear if the initial alpha beta engagement results either in the formation of only mature heterodimer or if the nascent complex is reversible and can undergo an exchange of subunits or combine transiently with an additional subunit. This is relevant for the case of LH and FSH, because both are synthesized in the same cell (i.e., pituitary gonadotrophs) and several of the alpha subunit sequences required for association with either the LH beta or FSH beta subunits are different. Such features could favor the generation of short-lived, multi-subunit forms prior to completion of assembly. Previously, we showed that the CG beta or FSH beta subunit genes can be genetically fused to the alpha gene to produce biologically active single chains, CG beta alpha and F beta alpha, respectively. Studies using monoclonal antibodies sensitive to the conformation of the hCG subunits suggested that in contrast to the highly compact heterodimer, the interactions between the beta and alpha domains in the single chain are in a more relaxed configuration. That the tethered domains do not interact tightly predicts that they could combine with an additional subunit to form triple domain complexes. We tested this point by cotransfecting CHO cells with the genes encoding F beta alpha and the CG beta subunit or the CG beta alpha and FSH beta monomer. The CG beta subunit combined noncovalently with F beta alpha to form a F beta alpha/CG beta complex. Ternary complex formation was not restricted to a specific set of single chain/monomeric subunit, because a CG beta alpha/FSH beta complex was also detected implying that triple domain intermediates could be transiently generated along the secretory pathway. Monoclonal antibodies specific for the CG heterodimer recognized the F beta alpha/CG beta complex, which suggests that the epitopes unique for dimeric CG were established. In addition, media containing F beta alpha/CG beta displayed high-affinity binding to both CG and FSH receptors. The presence of CG activity is presumptive for the existence of a functional F beta alpha/CG beta complex, because neither F beta alpha nor the uncombined CG beta subunit binds to CG receptor. These data show that the alpha subunit of the tether, although covalently linked to the FSH beta domain, can functionally interact with a different beta subunit implying that the contacts in the nascent alpha beta dimer are reversible. The formation of a functional single chain/subunit complex was not restricted to the FSH single chain/CG beta subunit since CG single chain interacts with the monomeric FSH beta subunit and exhibits FSH activity. The presence of the triple domain configuration does not abolish bioactivity, suggesting that although the gonadotropins are heterodimers, the cognate receptor is capable of recognizing a larger ligand composed of three subunit domains.

Genetic fusion of an alpha-subunit gene to the follicle-stimulating hormone and chorionic gonadotropin-beta subunit genes: production of a bifunctional protein.

The human glycoprotein hormones, hCG, TSH, LH, and FSH, are composed of a common alpha-subunit assembled to a hormone-specific beta-subunit. The subunits combine noncovalently early in the secretory pathway and exist as heterodimers but not as multimers. LH/FSH are synthesized in the pituitary gonadotrophs, and several of the alpha-subunit sequences required for association with either the LHbeta or FSHbeta subunits are different. Thus, it is intriguing that no ternary complexes are observed for LH and FSH in vivo (e.g. two different beta-assembled to a single alpha-subunit). To examine whether the alpha-subunit can interact with more than one beta-subunit, and to study the conformational relationships between the ligand and the receptor, we constructed a vector encoding two tandemly arranged beta-subunits fused to a single alpha-subunit gene (FSHbeta-CGbeta-alpha). This approach permitted structure-function analyses of alpha/beta domain complexes without the possibility of subunit dissociation. We reported previously that the CGbeta or FSHbeta subunit gene can be genetically fused to the alpha-gene and the resulting single chains (CGbetaalpha and FSHbetaalpha, respectively) were biologically active. Here we demonstrate that a triple-domain single chain bearing the configuration FSHbeta-CGbeta-alpha is efficiently secreted from transfected Chinese hamster ovary (CHO) cells and exhibits high-affinity receptor binding to both FSH and LH/hCG receptors, comparable to the native heterodimers. These results indicate that the alpha-subunit can interact with each beta-subunit in the same complex and that an alpha-domain fused to a beta-domain can still interact with an additional beta-subunit. The data also demonstrate the remarkable flexibility of the receptor to accommodate the increased bulkiness of the triple-domain ligand. In addition, the formation of intrachain FSH- and CG-like complexes observed in a triple-domain single chain suggests that the alpha-subunit can resonate, i.e. shuttle between alpha-beta heterodimeric intermediates during the early stages of synthesis and accumulation in the endoplasmic reticulum. Such model compounds could be useful as substrates to generate a new class of analogs in which the ratio of the LH/FSH activity is varied. This could aid in the design of analogs that could be used to mimic the in vivo hormonal profiles.

Glycoprotein hormone structure-function and analog design.

Human chorionic gonadotropin (hCG), luteinizing hormone, follicle-stimulating hormone (FSH), and thyrotropin (TSH) are hormones that share a common alpha subunit but differ in their beta subunits. Recombinant DNA techniques, valuable tools for structure-function analyses, provide an approach for designing therapeutic analogs. FSH is used clinically to stimulate the ovarian follicles for in vitro fertilization and to initiate follicular maturation in women with infertility problems. The CG beta subunit contains a carboxy-terminal extension (CTP) with four serine O-linked oligosaccharides, which is important for the long half-life of hCG. A clinical problem of FSH is its relatively short half-life in circulation. Fusing CTP to the FSH beta coding sequence increased the in vivo potency of the resulting FSH dimer over three-fold. Analogs of the other hormones containing CTP also increase their biologic half-life. Subunit assembly is vital to the function of these hormones. To address whether alpha and beta subunits can be synthesized as one chain and also maintain biological activity, a chimera comprised of the hCG beta subunit genetically fused to the alpha subunit was constructed. The resulting polypeptide was efficiently secreted and displayed an increased biologic activity in vitro and in vivo. Similarly, the single-chain form of FSH also retained in vivo activity. Since subunit dissociation inactivates the activity of the heterodimer, single-chain analogs should have longer biological half-lives. These analogs represent suitable substrates for engineering potent and stable agonists and antagonists.

Conversion of thyrotropin heterodimer to a biologically active single-chain.

TSH and the gonadotropins, FSH, LH, and CG are a family of heterodimeric glycoprotein hormones composed of a common alpha-subunit noncovalently linked to a hormone specific beta-subunit. Assembly of alpha- and beta-subunits is essential for hormone-specific posttranslational modifications, receptor binding, and bioactivity. Structure-function studies of TSH and gonadotropins using site-directed mutagenesis can often affect folding, assembly, and secretion of the hormone. To circumvent these difficulties, recently, the gonadotropin heterodimers were converted to single chains. Here we converted the hTSH heterodimer to a biologically active single chain by genetically fusing the amino terminal end of the common alpha-subunit to the carboxyl terminal end of hTSHbeta in the presence or absence of hCGbeta carboxyl terminal peptide (CTP), which was used as a linker. Wild-type hTSH and the single chains were expressed in Chinese hamster ovary (CHO) cells, and they were efficiently secreted. Although the secretion rate of the single chain was 3-fold higher than that of hTSH wild-type. Moreover, the secretion of the single chain in the presence of the CTP linker was dramatically increased. On the other hand, receptor binding and in vitro bioactivity of the single chains were similar to that of hTSH wild-type. These data indicate the potential of the single chain approach to further investigate structure-function relationships of TSH.

Cystine knot of the gonadotropin alpha subunit is critical for intracellular behavior but not for in vitro biological activity.

The common alpha subunit of glycoprotein hormones contains five disulfide bonds. Based on the published crystal structure, the assignments are 7-31, 59-87, 10-60, 28-82, and 32-84; the last three comprise the cystine knot, a structure also seen in a variety of growth factors. Previously, we demonstrated that the efficiency of secretion and the ability to form heterodimers by alpha subunits bearing single cysteine residue mutants in the cystine knot were significantly reduced. These results suggested that the cystine knot is critical for the intracellular integrity of the subunit. To assess if the presence of the free thiol affected the secretion kinetics, we constructed paired cysteine mutants of each disulfide bond of the alpha subunit. The secretion rate for these monomers was comparable with wild type except for the alpha-10-60 mutant, which was 40% lower. The recovery of the alpha7-31 and alpha59-87 mutants was greater than 95%, whereas for the cystine knot mutants, it was 20-40%. Co-expression of the wild-type chorionic gonadotropin beta subunit with double cysteine mutants did not enhance the recovery of alpha mutants in the media. Moreover, compared with wild-type, the efficiency of heterodimer formation of the alpha10-60 or alpha32-84 mutants was less than 5%. Because subunit assembly is required for biological activity, studies on the role of these disulfide bonds in signal transduction were not possible. To bypass the assembly step, we exploited the single chain model, where the alpha and beta subunits are genetically fused. The recovery of secreted tethered gonadotropins bearing mutations in the cystine knot was increased significantly. Although dimer-specific monoclonal antibodies discriminated the conformation of single chain alpha10-60 and alpha32-84 mutants from the native heterodimer, these mutants were nevertheless biologically active. Thus, individual bonds of cystine knot are important for secretion and heterodimer formation but not for in vitro bioactivity. Moreover, the data suggest that the native heterodimer configuration is not a prerequisite for receptor binding or signal transduction.

The biologic action of single-chain choriogonadotropin is not dependent on the individual disulfide bonds of the beta subunit.

Disrupting disulfide loops in the human chorionic gonadotropin beta subunit (CGbeta) inhibits combination with the alpha subunit. Because the bioactivity requires a heterodimer, studies on the role of disulfide bonds on receptor binding/signal transduction have previously been precluded. To address this problem, we bypassed the assembly step and genetically fused CGbeta subunits bearing paired cysteine mutations to a wild-type alpha (WTalpha) subunit. The changes altered secretion of the single-chain mutants which parallel that seen for the CGbeta monomeric subunit. Despite conformational changes in CG disulfide bond mutants (assayed by gel electrophoresis and conformationally sensitive monoclonal antibodies), the variants bind to the lutropin/CG receptor and activated adenylate cyclase in vitro. The data show that the structural requirements for secretion and bioactivity are not the same. The results also suggest that the extensive native subunit interactions determined by the cystine bonds are not required for signal transduction. Moreover, these studies demonstrate that the single-chain model is an effective approach to structure-activity relationships of residues and structural domains associated with assembly of multisubunit ligands.

Expression of biologically active fusion genes encoding the common alpha subunit and either the CG beta or FSH beta subunits: role of a linker sequence.

The gonadotropin/thyrotropin hormone family is characterized by a heterodimeric structure composed of a common alpha subunit non-covalently linked to a hormone-specific beta subunit. The conformation of the heterodimer is essential for controlling secretion, hormone-specific post-translational modifications and signal transduction. Structure-function studies of FSH and the other glycoprotein hormones are often hampered by mutagenesis induced defects in subunit combination. Thus, the ability to overcome the limitation of subunit assembly would expand the range of structure activity relationships that can be performed on these hormones. Here we converted the FSH heterodimer to a single chain by genetically fusing the carboxyl end of the FSH beta subunit to the amino end of the alpha subunit in the presence or absence of a natural linker sequence. In the absence of the CTP linker, the secretion rate was decreased over three fold. (The CTP sequence is the last 28 amino acids of the CG beta sequence and contains four serine-linked oligosaccharides). Unexpectedly however receptor binding/signal transduction was unaffected by absence of the linker. Molecular modelling of the tethers lacking the linker sequence show that the alignment of the alpha/beta domains in the single chain differ substantially from that seen in the heterodimer. These data show that the single chain FSH was secreted efficiently and is biologically active and that the conformation determinants required for secretion and biologic activity are not the same.

Arachidonic acid and lipoxygenase products stimulate protein kinase C beta mRNA levels in pituitary alpha T3-1 cell line: role in gonadotropin-releasing hormone action.

The cross-talk of arachidonic acid (AA) and its lipoxygenase products with protein kinase C beta (PKC beta) mRNA levels during the action of gonadotropin-releasing hormone (GnRH) was investigated in the pituitary alpha T3-1 cell line. The addition of AA or its 5-lipoxygenase products 5-hydroxyeicosatetraenoic acid (5-HETE) or leukotriene C4 (LTC4) for 30 or 60 min stimulated PCK beta, but not PKC alpha mRNA levels (3-5-fold); PCK gamma is not expressed by the cells. Other HETEs or leukotrienes tested showed no significant effect. The range of effective concentration for LTC4 and 5-HETE (around 10(-10) M) is the range found in GnRH-stimulated pituitary cells. Although PKC beta mRNA levels were preferentially elevated by LTC4 and 5-HETE at early time points, PKC alpha mRNA levels were elevated at 6-12 h of incubation when PKC beta mRNA levels returned to basal levels. The addition of the phospholipase A2 inhibitor 4-bromophenacyl bromide or the selective 5-lipoxygenase inhibitor L-656,224 abolished [D-Trp6]GnRH (GnRH-A) elevation of PKC beta mRNA levels, whereas PKC alpha mRNA levels were not increased by this neurohormone. The cyclo-oxygenase inhibitor indomethacin elevated basal PKC beta mRNA levels and potentiated the GnRH-A response. Cross-talk exists between AA and some of its lipoxygenase products and PKC beta gene expression during cell signalling. AA, 5-HETE and LTC4 participate in the rapid stimulation of PKC beta mRNA levels by GnRH.

Expression of biologically active fusion genes encoding the common alpha subunit and the follicle-stimulating hormone beta subunit. Role of a linker sequence.

The gonadotropin/thyrotropin hormone family is characterized by a heterodimeric structure composed of a common alpha subunit noncovalently linked to a hormone-specific beta subunit. The conformation of the heterodimer is essential for controlling secretion, hormone-specific post-translational modifications, and signal transduction. Structure-function studies of follicle-stimulating hormone (FSH) and the other glycoprotein hormones are often hampered by mutagenesis-induced defects in subunit combination. Thus, the ability to overcome the limitation of subunit assembly would expand the range of structure-activity relationships that can be performed on these hormones. Here we converted the FSH heterodimer to a single chain by genetically fusing the carboxyl end of the FSH beta subunit to the amino end of the alpha subunit in the presence or absence of a linker sequence. In the absence of the CTP linker, the secretion rate was decreased over 3-fold. Unexpectedly, however, receptor binding/signal transduction was unaffected by the absence of the linker. These data show that the single-chain FSH was secreted efficiently and is biologically active and that the conformation determinants required for secretion and biologic activity are not the same.

Converting heterodimeric gonadotropins to genetically linked single chains: new approaches to structure activity relationships and analogue design.

One of the distinguishing features of the gonadotropin and thyrotropin hormone family is their heterodimeric structure; the subunits combine early in the secretory pathway and only the dimers are capable of binding to receptors. Therefore, assembly is rate limiting in the production of functional heterodimers, a problem encountered when removing the carbohydrates from one or both subunits as discussed in this review. If the heterodimers can be expressed as single chains, this might avoid mutagenesis-induced defects in secretion and combination of individual subunits for structure-function studies and analogue design. Here we discuss the feasibility of this approach for such problems.

Mechanism of action of gonadotropin-releasing hormone upon gonadotropin alpha-subunit mRNA levels in the alpha T3-1 cell line: role of Ca2+ and protein kinase C.

Addition of [D-Trp6]gonadotropin-releasing hormone (GnRHa) to alpha T3-1 cells induced a very rapid response upon gonadotropin alpha-subunit mRNA which was detected after 30-60 min and was abolished by pretreatment with actinomycin D. A similar response was obtained with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA), or the Ca2+ ionophore, ionomycin. GnRHa (10 nM) also stimulated a secondary rise in alpha-subunit mRNA levels between 12 and 24 h of incubation. No additivity was obtained (at 60 min) upon the combined addition of GnRHa and PMA, GnRHa and ionomycin, or PMA and ionomycin. The effect of GnRHa upon alpha-subunit mRNA was blocked by the PKC inhibitors staurosporine or GF 109203X. Down-regulation of endogenous PKC activity resulted in inhibition of the stimulatory effect of gonadotropin-releasing hormone (GnRH), PMA and ionomycin. Removal of extra-cellular Ca2+ abolished the effect of GnRHa and PMA upon alpha-subunit mRNA levels. Interestingly PMA and ionomycin had no effect on alpha-subunit mRNA levels at 24 h of incubation; however, the combined addition of the drugs mimicked the late phase of GnRHa (10 nM) action. The data provide evidence that PKC and Ca2+ are involved in mediating the early and the late responses of GnRHa upon alpha-subunit mRNA elevation and that differential cross-talk exists between the messengers.

Biosynthesis of a biologically active single peptide chain containing the human common alpha and chorionic gonadotropin beta subunits in tandem.

One of the distinguishing features of the gonadotropin and thyrotropin hormone family is their heterodimeric structure, consisting of a common alpha subunit and a hormone-specific beta subunit. Subunit assembly is vital to the function of these hormones: The conformation of the heterodimer is essential for controlling secretion, hormone-specific posttranslational modifications, and signal transduction. To address whether alpha and beta subunits can be synthesized as one chain and also maintain biological activity, a chimera composed of the human chorionic gonadotropin (hCG) beta subunit genetically fused to the alpha subunit was constructed. The resulting polypeptide hCG molecule not only was efficiently secreted but also displayed an increased biological activity in vitro and in vivo. These data show that the alpha and hCG beta subunits encoded as a single chain retain a biologically active conformation similar to that seen in the heterodimer. This approach can be used to investigate structure-function relationships of the glycoprotein hormone family that were previously not tractable because of the absolute dependence on assembly for the biological response. Moreover, other bioactive multisubunit ligands can be engineered where the combination efficiency and specificity of heterodimers and homodimers are otherwise difficult to control.

Activation of protein kinase C beta gene expression by gonadotropin-releasing hormone in alpha T3-1 cell line. Role of Ca2+ and autoregulation by protein kinase C.

The gonadotroph-derived alpha T3-1 cell line was used to investigate the effect of gonadotropin-releasing hormone (GnRH) upon conventional protein kinase C sub-types (cPKCs) gene expression. Addition of the stable analog [D-Trp6]GnRH (GnRH-A, 0.1 nM) resulted in a rapid increase (30 min) of the steady state levels of PKC beta, but not PKC alpha, mRNA levels, while PKC gamma is not expressed in the cells. The rapid stimulatory effect of GnRH-A was blocked by pretreatment with actinomycin D or with the GnRH antagonist (D-pGlu1, pC1Phe2,D-Trp3,6)GnRH and was not mimicked by thyrotropin-releasing hormone. Addition of the PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted also in a rapid (30 min) and selective increase in PKC beta, but not PKC alpha, mRNA levels. In contrast, the calcium ionophore, ionomycin, increased rapidly (30 min) both PKC alpha and PKC beta mRNA levels, and its stimulatory effect on PKC beta was not additive with that of TPA. The rapid stimulatory effect of GnRH-A was blocked by the PKC inhibitor bisindolylmaleimide (GF 109203X) or by down-regulation of endogenous PKC. Similarly, the rapid effect of GnRH-A was abolished by the intracellular Ca2+ chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) or by removal of extracellular Ca2+. Stimulation of PKC beta mRNA levels by ionomycin was only reduced by GF 109203X and was not affected by down-regulation of PKC. In contrast the effect of TPA on PKC beta mRNA levels was reduced by BAPTA and abolished by removal of Ca2+. We conclude that Ca2+ and PKC act sequentially during GnRH-A-induced PKC beta gene expression and that PKC beta gene expression induced by GnRH-A is autoregulated by PKC.

Arachidonic acid and lipoxygenase products stimulate gonadotropin alpha-subunit mRNA levels in pituitary alpha T3-1 cell line: role in gonadotropin releasing hormone action.

The role of arachidonic acid (AA) and its lipoxygenase metabolites in gonadotropin releasing hormone (GnRH) induced alpha-subunit gene expression was investigated in the transformed gonadotroph cell line alpha T3-1. The stable analog [D-Trp6]GnRH (GnRHa) stimulated [3H]AA release from prelabeled cells after a lag of 1-2 min. Addition of AA stimulated alpha-subunit mRNA levels in a dose-dependent manner, a significant effect being detected at 5 microM AA. Among various lipoxygenase metabolites of AA, only the 5-lipoxygenase products 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene C4 (LTC4) stimulated alpha-subunit mRNA levels. However, while 5-HETE and LTC4 (0.1 nM each) were active already after 30 min of incubation, similar to GnRHa, AA (20 microM) stimulated alpha-mRNA levels after 1 h of incubation. Addition of the phospholipase A2 inhibitor 4-bromophenacyl bromide (BPB) or the selective 5-lipoxygenase inhibitor L-656,224 inhibited GnRHa elevation of alpha-subunit mRNA by 65%, while the cyclooxygenase inhibitor indomethacin had no effect. Addition of AA (20 microM) or LTC4 (0.1 nM) to normal cultured rat pituitary cells mimicked the rapid (30 min) stimulatory effect of GnRH (1 nM) upon alpha-subunit, LH beta, and FSH beta mRNA levels, while 5-HETE (0.1 nM) stimulated only FSH beta mRNA levels at this time point. Thus AA and selected 5-lipoxygenase products, in particular LTC4, participate in GnRHa-induced alpha-subunit mRNA elevation.

Developmental expression of protein kinase C subspecies in rat brain-pituitary axis.

We have examined the neonatal developmental expression of protein kinase C subspecies (PKCs) in rat brain, pituitary glands and cells by enzymatic activity assays, immunohistochemistry and Western blot analysis with type-specific antibodies. A very large increase (455%) was noticed in brain PKC activity during the first week of life with the particulate fraction (22% of total enzyme activity on day 1) increasing dramatically (900%) during the first week to 50% of enzyme activity. In contrast, the pituitary gland showed high activity on day 1 that decreased progressively to reach the lowest levels at 1 year of age. Paradoxically, the number of pituitary cells immunolabeled for PKC increases as a function of age. Western blot analysis showed only small changes in PKC alpha, PKC beta and PKC epsilon when brains from 6-day-old and 3-month-old female rats were compared, whereas PKC tau and PKC delta increased markedly during this period. On the other hand, brain PKC zeta decreased between 6 days and 3 months of age. Western blot analysis showed no major changes in pituitary PKC alpha, PKC beta and PKC zeta when 6-day-old and 3-month-old female rats were compared, while PKC tau was not detected. The major band of pituitary PKC delta (76 kDa) decreased markedly between 6 days and 3 months of age whereas the minor band (68 kDa) did not change.(ABSTRACT TRUNCATED AT 250 WORDS)