CP-154,526: a potent and selective nonpeptide antagonist of corticotropin releasing factor receptors.

Here we describe the properties of CP-154,526, a potent and selective nonpeptide antagonist of corticotropin (ACTH) releasing factor (CRF) receptors. CP-154,526 binds with high affinity to CRF receptors (Ki < 10 nM) and blocks CRF-stimulated adenylate cyclase activity in membranes prepared from rat cortex and pituitary. Systemically administered CP-154,526 antagonizes the stimulatory effects of exogenous CRF on plasma ACTH, locus coeruleus neuronal firing and startle response amplitude. Potential anxiolytic activity of CP-154,526 was revealed in a fearpotentiated startle paradigm. These data are presented in the context of clinical findings, which suggest that CRF is hypersecreted in certain pathological states. We propose that a CRF antagonist such as CP-154,526 could affirm the role of CRF in certain psychiatric diseases and may be of significant value in the treatment of these disorders.

Insulin-secreting non-islet cells are resistant to autoimmune destruction.

Transgenic nonobese diabetic mice were created in which insulin expression was targeted to proopiomelanocortin-expressing pituitary cells. Proopiomelanocortin-expressing intermediate lobe pituitary cells efficiently secrete fully processed, mature insulin via a regulated secretory pathway, similar to islet beta cells. However, in contrast to the insulin-producing islet beta cells, the insulin-producing intermediate lobe pituitaries are not targeted or destroyed by cells of the immune system. Transplantation of the transgenic intermediate lobe tissues into diabetic nonobese diabetic mice resulted in the restoration of near-normoglycemia and the reversal of diabetic symptoms. The absence of autoimmunity in intermediate lobe pituitary cells engineered to secrete bona fide insulin raises the potential of these cell types for beta-cell replacement therapy for the treatment of insulin-dependent diabetes mellitus.

An essential regulatory role for macrophage migration inhibitory factor in T-cell activation.

The protein known as macrophage migration inhibitory factor (MIF) was one of the first cytokines to be discovered and was described 30 years ago to be a T-cell-derived factor that inhibited the random migration of macrophages in vitro. A much broader role for MIF has emerged recently as a result of studies that have demonstrated it to be released from the anterior pituitary gland in vivo. MIF also is the first protein that has been identified to be secreted from monocytes/macrophages upon glucocorticoid stimulation. Once released, MIF acts to "override" or counter-regulate the suppressive effects of glucocorticoids on macrophage cytokine production. We report herein that MIF plays an important regulatory role in the activation of T cells induced by mitogenic or antigenic stimuli. Activated T cells produce MIF and neutralizing anti-MIF antibodies inhibit T-cell proliferation and interleukin 2 production in vitro, and suppress antigen-driven T-cell activation and antibody production in vivo. T cells also release MIF in response to glucocorticoid stimulation and MIF acts to override glucocorticoid inhibition of T-cell proliferation and interleukin 2 and interferon gamma production. These studies indicate that MIF acts in concert with glucocorticoids to control T-cell activation and assign a previously unsuspected but critical role for MIF in antigen-specific immune responses.

Differential distribution of two ATP-gated channels (P2X receptors) determined by immunocytochemistry.

Several P2X receptor subunits were recently cloned; of these, one was cloned from the rat vas deferens (P2X1) and another from pheochromocytoma (PC12) cells differentiated with nerve growth factor (P2X2). Peptides corresponding to the C-terminal portions of the predicted receptor proteins (P2X1 391-399 and P2X2 460-472) were used to generate antisera in rabbits. The specificities of antisera were determined by staining human embryonic kidney cells stably transfected with either P2X1 or P2X2 receptors and by absorption controls with the cognate peptides. In the vas deferens and the ileal submucosa, P2X1 immunoreactivity (ir) was restricted to smooth muscle, whereas P2X2-ir was restricted to neurons and their processes. Chromaffin cells of the adrenal medulla and PC12 cells contained both P2X1- and P2X2-ir. P2X1-ir was also found in smooth muscle cells of the bladder, cardiac myocytes, and nerve fibers and terminals in the superficial dorsal horn of the spinal cord. In contrast, P2X2-ir was observed in scattered cells of the anterior pituitary, neurons in the hypothalamic arcuate and paraventricular nuclei, and catecholaminergic neurons in the olfactory bulb, the substantia nigra, ventral tegmental area, and locus coeruleus. A plexus of nerve fibers and terminals in the nucleus of the solitary tract contained P2X2-ir. This staining disappeared after nodose ganglionectomy, consistent with a presynaptic function. The location of the P2X1 subunit in smooth muscle is consistent with its role as a postjunctional receptor in autonomic transmission, while in neurons, these receptors appear in both postsynaptic and presynaptic locations.

Genetic separation of tumor growth and hemorrhagic phenotypes in an estrogen-induced tumor.

Chronic administration of estrogen to the Fischer 344 (F344) rat induces growth of large, hemorrhagic pituitary tumors. Ten weeks of diethylstilbestrol (DES) treatment caused female F344 rat pituitaries to grow to an average of 109.2 +/- 6.3 mg (mean +/- SE) versus 11.3 +/- 1.4 mg for untreated rats, and to become highly hemorrhagic. The same DES treatment produced no significant growth (8.9 +/- 0.5 mg for treated females versus 8.7 +/- 1.1 for untreated females) or morphological changes in Brown Norway (BN) rat pituitaries. An F1 hybrid of F344 and BN exhibited significant pituitary growth after 10 weeks of DES treatment with an average mass of 26.3 +/- 0.7 mg compared with 8.6 +/- 0.9 mg for untreated rats. Surprisingly, the F1 hybrid tumors were not hemorrhagic and had hemoglobin content and outward appearance identical to that of BN. Expression of both growth and morphological changes is due to multiple genes. However, while DES-induced pituitary growth exhibited quantitative, additive inheritance, the hemorrhagic phenotype exhibited recessive, epistatic inheritance. Only 5 of the 160 F2 pituitaries exhibited the hemorrhagic phenotype; 36 of the 160 F2 pituitaries were in the F344 range of mass, but 31 of these were not hemorrhagic, indicating that the hemorrhagic phenotype is not merely a consequence of extensive growth. The hemorrhagic F2 pituitaries were all among the most massive, indicating that some of the genes regulate both phenotypes.

Cytotoxic analogs of luteinizing hormone-releasing hormone containing doxorubicin or 2-pyrrolinodoxorubicin, a derivative 500-1000 times more potent.

Doxorubicin (DOX) and its daunosamine-modified derivative, 2-pyrrolino-DOX, which is 500-1000 times more active than DOX, were incorporated into agonistic and antagonistic analogs of luteinizing hormone-releasing hormone (LH-RH). The conjugation of DOX with LH-RH analogs was performed by using N-(9-fluorenylmethoxycarbonyl)-DOX-14-O-hemiglutarate, a dicarboxylic acid ester derivative of DOX. Coupling this derivative covalently to the epsilon-amino group of the D-Lys side chain of agonist [D-Lys6]LH-RH or antagonistic analog AC-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys-Leu-Arg-Pro-D-Ala-NH 2 [where Nal(2) = 3-(2-naphthyl)alanine, Pal(3) = 3-(3-pyridyl)alanine, and Phe(4CI) = 4-chlorophenylalanine] was followed by the removal of the 9-fluorenylmethoxycarbonyl protective group to yield cytotoxic derivatives of LH-RH analogs containing DOX. From these DOX containing LH-RH hybrids, intensely potent analogs with daunosamine-modified derivatives of DOX can be readily formed. Thus, cytotoxic LH-RH agonist containing DOX (AN-152) can be converted in a 66% yield by a reaction with a 30-fold excess of 4-iodobutyraldehyde in N,N-dimethylformamide into a derivative having 2-pyrrolino-DOX (AN-207). Hybrid molecules AN-152 and AN-207 fully preserve the cytotoxic activity of their radicals, DOX or 2-pyrrolino-DOX, respectively, in vitro, and also retain the high binding affinity of the peptide hormone portion of the conjugates to rat pituitary receptors for LH-RH. These highly potent cytotoxic analogs of LH-RH were designed as targeted anti-cancer agents for the treatment of various tumors that possess receptors for the carrier peptide. Initial in vivo studies show that the hybrid molecules are much less toxic than the respective cytotoxic radicals incorporated and significantly more active in inhibiting tumor growth.

Crystal structure at 2.6-A resolution of human macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF) was the first cytokine to be described, but for 30 years its role in the immune response remained enigmatic. In recent studies, MIF has been found to be a novel pituitary hormone and the first protein identified to be released from immune cells on glucocorticoid stimulation. Once secreted, MIF counterregulates the immunosuppressive effects of steroids and thus acts as a critical component of the immune system to control both local and systemic immune responses. We report herein the x-ray crystal structure of human MIF to 2.6 angstrom resolution. The protein is a trimer of identical subunits. Each monomer contains two antiparallel alpha-helices that pack against a four-stranded beta-sheet. The monomer has an additional two beta-strands that interact with the beta-sheets of adjacent subunits to form the interface between monomers. The three beta-sheets are arranged to form a barrel containing a solvent-accessible channel that runs through the center of the protein along a molecular 3-fold axis. Electrostatic potential maps reveal that the channel has a positive potential, suggesting that it binds negatively charged molecules. The elucidated structure for MIF is unique among cytokines or hormonal mediators, and suggests that this counterregulator of glucocorticoid action participates in novel ligand-receptor interactions.

Absence of opioid stress-induced analgesia in mice lacking beta-endorphin by site-directed mutagenesis.

A physiological role for beta-endorphin in endogenous pain inhibition was investigated by targeted mutagenesis of the proopiomelanocortin gene in mouse embryonic stem cells. The tyrosine codon at position 179 of the proopiomelanocortin gene was converted to a premature translational stop codon. The resulting transgenic mice display no overt developmental or behavioral alterations and have a normally functioning hypothalamic-pituitary-adrenal axis. Homozygous transgenic mice with a selective deficiency of beta-endorphin exhibit normal analgesia in response to morphine, indicating the presence of functional mu-opiate receptors. However, these mice lack the opioid (naloxone reversible) analgesia induced by mild swim stress. Mutant mice also display significantly greater nonopioid analgesia in response to cold water swim stress compared with controls and display paradoxical naloxone-induced analgesia. These changes may reflect compensatory upregulation of alternative pain inhibitory mechanisms.

Prolactin increases CD4/CD8 cell ratio in thymus-grafted congenitally athymic nude mice.

One distinctive effect on T-cell development was analyzed by selectively increasing serum prolactin (PRL) concentration in thymus-grafted congenitally athymic nude mice and by neutralizing PRL in suspension cultures of thymus from 1-day-old neonatal mice. Flow cytometric analysis of single-positive CD4+ and CD8+ cells derived from inguinal lymph nodes revealed a CD4/CD8 cell ratio of 2.2 +/- 0.18 (mean +/- SEM) in thymus-grafted nude mice that is similar to the ratio for immune-competent BALB/c mice (2.0 +/- 0.06). Addition of the pituitary to thymus-grafted nude mice significantly elevated serum PRL (P < 0.005) and increased the CD4/CD8 cell ratio (2.8 +/- 0.12; P < 0.005), demonstrating preferential stimulation of CD4+ cell development. T cells in nude mice receiving sham (submandibular salivary gland) or pituitary grafts alone were below detectable levels. Suspension cultures of neonatal thymus treated with anti-mouse PRL antiserum resulted in 20% and 30% decreases in double-positive CD4+8+ thymocytes and thymocyte viability, respectively. A 10-fold increase in double-negative CD4-8- thymocytes expressing the interleukin 2 receptor alpha chain, CD25, was also observed concurrently. Our findings illustrate an important way in which PRL may participate in two interrelated mechanisms: the regulation of peripheral single-positive cells and the maintenance of thymocyte viability during the double-positive stage of intrathymic differentiation.

cDNA structure, tissue distribution, and chromosomal localization of rat PC7, a novel mammalian proprotein convertase closest to yeast kexin-like proteinases.

By using reverse transcription-coupled PCR on rat anterior pituitary RNA, we isolated a 285-bp cDNA coding for a novel subtilisin/kexin-like protein convertase (PC), called rat (r) PC7. By screening rat spleen and PC12 cell lambda gt11 cDNA libraries, we obtained a composite 3.5-kb full-length cDNA sequence of rPC7. The open reading frame codes for a prepro-PC with a 36-amino acid signal peptide, a 104-amino acid prosegment ending with a cleavable RAKR sequence, and a 747-amino acid type I membrane-bound glycoprotein, representing the mature form of this serine proteinase. Phylogenetic analysis suggests that PC7 represents the most divergent enzyme of the mammalian convertase family and that it is the closest member to the yeast convertases krp and kexin. Northern blot analyses demonstrated a widespread expression with the richest source of rPC7 mRNA being the colon and lymphoid-associated tissues. In situ hybridization revealed a distinctive tissue distribution that sometimes overlaps with that of furin, suggesting that PC7 has widespread proteolytic functions. The gene for PC7 (Pcsk7) was mapped to mouse chromosome 9 by linkage analysis of an interspecific backcross DNA panel.

cAMP inducibility of transcriptional repressor ICER in developing and mature human T lymphocytes.

Stimulation of the cAMP-dependent signaling pathway exerts an inhibitory effect on the proliferation and effector functions of T cells. The ability of T cells to form high intracellular levels of cAMP is acquired during development in the human thymus and is retained by the majority of mature peripheral T lymphocytes. Here we show that elevated cAMP levels in T cells correlate with the expression of the potent transcriptional repressor ICER (inducible cAMP early repressor) previously described in the hypothalamic-pituitary-gonadal axis. Further, in transcriptional assays in vivo, ICER inhibits calcineurin-mediated expression of the interleukin 2 promoter as well as Tax-mediated transactivation of the human T-lymphotropic virus type I (HTLV-I) promoter. Thus, the induction of ICER in T cells may play an important role in the cAMP-induced quiescence and the persistent latency of HTLV-I.

A mechanism for the differential regulation of gonadotropin subunit gene expression by gonadotropin-releasing hormone.

The hypothalamic hormone gonadotropin-releasing hormone (GnRH) is released in a pulsatile fashion, with its frequency varying throughout the reproductive cycle. Varying pulse frequencies and amplitudes differentially regulate the biosynthesis and secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by pituitary gonadotropes. The mechanism by which this occurs remains a major question in reproductive physiology. Previous studies have been limited by lack of available cell lines that express the LH and FSH subunit genes and respond to GnRH. We have overcome this limitation by transfecting the rat pituitary GH3 cell line with rat GnRH receptor (GnRHR) cDNA driven by a heterologous promoter. These cells, when cotransfected with regulatory regions of the common alpha, LH beta, or FSH beta subunit gene fused to a luciferase reporter gene, respond to GnRH with an increase in luciferase activity. Using this model, we demonstrate that different cell surface densities of the GnRHR result in the differential regulation of LH and FSH subunit gene expression by GnRH. This suggests that the differential regulation of gonadotropin subunit gene expression by GnRH observed in vivo in rats may, in turn, be mediated by varying gonadotrope cell surface GnRHR concentrations. This provides a physiologic mechanism by which a single ligand can act through a single receptor to regulate differentially the production of two hormones in the same cell.

Mice deficient in the orphan receptor steroidogenic factor 1 lack adrenal glands and gonads but express P450 side-chain-cleavage enzyme in the placenta and have normal embryonic serum levels of corticosteroids.

The orphan nuclear receptor steroidogenic factor 1 (SF-1) is expressed in the adrenal cortex and gonads and regulates the expression of several P450 steroid hydroxylases in vitro. We examined the role of SF-1 in the adrenal glands and gonads in vivo by a targeted disruption of the mouse SF-1 gene. All SF-1-deficient mice died shortly after delivery. Their adrenal glands and gonads were absent, and persistent Mullerian structures were found in all genotypic males. While serum levels of corticosterone in SF-1-deficient mice were diminished, levels of adrenocorticotropic hormone (ACTH) were elevated, consistent with intact pituitary corticotrophs. Intrauterine survival of SF-1-deficient mice appeared normal, and they had normal serum level of corticosterone and ACTH, probably reflecting transplacental passage of maternal steroids. We tested whether SF-1 is required for P450 side-chain-cleavage enzyme (P450scc) expression in the placenta, which expresses both SF-1 and P450scc, and found that in contrast to its strong activation of the P450scc gene promoter in vitro, the absence of SF-1 had no effect on P450scc mRNA levels in vivo. Although the region targeted by our disruption is shared by SF-1 and by embryonal long terminal repeat-binding protein (ELP), a hypothesized alternatively spliced product, we believe that the observed phenotype reflects absent SF-1 alone, as PCR analysis failed to detect ELP transcripts in any mouse tissue, and sequences corresponding to ELP are not conserved across species. These results confirm that SF-1 is an important regulator of adrenal and gonadal development, but its regulation of steroid hydroxylase expression in vivo remains to be established.

Potent, structurally constrained agonists and competitive antagonists of corticotropin-releasing factor.

Predictive methods, physicochemical measurements, and structure activity relationship studies suggest that corticotropin-releasing factor (CRF; corticoliberin), its family members, and competitive antagonists (resulting from N-terminal deletions) usually assume an alpha-helical conformation when interacting with the CRF receptor(s). To test this hypothesis further, we have scanned the whole sequence of the CRF antagonist [D-Phe12,Nle21,38]r/hCRF-(12-41) (r/hCRF, rat/human CRF; Nle, norleucine) with an i-(i + 3) bridge consisting of the Glu-Xaa-Xaa-Lys scaffold. We have found astressin [cyclo(30-33)[D-Phe12,Nle21,38,Glu30,Lys33]r/ hCRF(12-41)] to be approximately 30 times more potent than [D-Phe12,Nle21,38]r/hCRF-(12-41), our present standard, and 300 times more potent than the corresponding linear analog in an in vitro pituitary cell culture assay. Astressin has low affinity for the CRF binding protein and high affinity (Ki = 2 nM) for the cloned pituitary receptor. Radioiodinated [D-125I-Tyr12]astressin was found to be a reliable ligand for binding assays. In vivo, astressin is significantly more potent than any previously tested antagonist in reducing hypophyseal corticotropin (ACTH) secretion in stressed or adrenalectomized rats. The cyclo(30-33)[Ac-Pro4,D-Phe12,Nle21,38,Glu30,Lys33++ +]r/hCRF-(4-41) agonist and its linear analog are nearly equipotent, while the antagonist astressin and its linear form vary greatly in their potencies. This suggests that the lactam cyclization reinstates a structural constraint in the antagonists that is normally induced by the N terminus of the agonist.

Thyroid-stimulating hormone (TSH)-directed induction of the CREM gene in the thyroid gland participates in the long-term desensitization of the TSH receptor.

Thyroid gland function is regulated by the hypothalamic-pituitary axis via the secretion of TSH, according to environmental, developmental, and circadian stimuli. TSH modulates both the secretion of thyroid hormone and gland trophism through interaction with a specific guanine nucleotide-binding protein-coupled receptor (TSH receptor; TSH-R), which elicits the activation of the cAMP-dependent signaling pathway. After TSH stimulation, the levels of TSH-R RNA are known to decrease dramatically within a few hours. This phenomenon ultimately leads to homologous long-term desensitization of the TSH-R. Here we show that TSH drives the induction of the inducible cAMP early repressor (ICER) isoform of the cAMP response element (CRE) modulator gene both in rat thyroid gland and in the differentiated thyroid cell line FRTL-5. The kinetics of ICER protein induction mirrors the down-regulation of TSH-R mRNA. ICER binds to a CRE-like sequence in the TSH-R promoter and represses its expression. Thus, ICER induction by TSH in the thyroid gland represents a paradigm of the molecular mechanism by which pituitary hormones elicit homologous long-term desensitization.