James L. Roberts

In situ cDNA: mRNA hybridization: Development of a technique to measure mRNA levels in individual cells

In this article we have described our protocol for in situ cDNA:mRNA hybridization and a variety of methodological considerations we have entertained to optimize the procedure. It should be stressed that each different system will have its own special characteristics and require optimization to obtain maximal and reproducible signals. We believe that by following a methodological logic as outlined here, researchers should be able to establish the in situ cDNA:mRNA hybridization protocol with their probes and tissue systems.

The proopiocortin (adrenocorticotropin/β-lipotropin) gene is located on chromosome 2 in humans

The proopiocortin gene is located on chromosome 2 in humans. A 13-kb DNA fragment containing proopiocortin gene sequences was identified in human cells while proopiocortin-related gene sequences of 9.8 and 6.2 kb were present in mouse cells. In human-mouse cell hybrids which contained reduced numbers of human chromosomes and a complete set of mouse chromosomes, the 9.8- and 6.2-kb fragments were always present while the 13-kb fragment segregated with human chromosome 2 and the chromosome 2 enzyme markers acid phosphatase-1 (ACP1), malate dehydrogenase-1 (MDH1), and isocitrate dehydrogenase-1 (IDH1). Analysis of a single cell hybrid with a broken chromosome 2 indicates that the proopiocortin andACP1 genes are closely linked and in the distal region of the short arm of chromosome 2.

Glucocorticoid regulation of proopiomelanocortin gene expression in rodent pituitary.

Publisher Summary This chapter discusses glucocorticoid regulation of proopiomelanocortin gene expression in rodent pituitary. The pituitary peptide hormones, adrenocorticotropin (ACTH), endorphin, and melanocyte stimulating hormone (MSH) are derived from a larger precursor protein designated as proopiomelanocortin (POMC). The three MSH peptides contained within POMC, α, β, and γ are each located in a different region of the precursor. POMC is produced in many different tissues, particularly, the anterior and intermediate lobes of the pituitary which are the major sites of its synthesis. Radioimmunoassay and immunohistochemical evidence have identified POMC related peptides in the central nervous system tissue, intestinal tissue, and in certain ectopic tumors. POMC is processed to its end product hormones by several posttranslational events, some of which are tissue specific. Differential processing of the POMC precursor is best characterized in the anterior and intermediate lobes of the pituitary. Although the POMC protein appears to be the same in the two lobes, proteolytical processing results in distinctly different final peptide products. The anterior lobe of the pituitary is the source of the bioactive ACTH which is secreted into the blood stream. There are three basic mechanisms by which glucocorticoids could specifically decrease anterior pituitary content of POMC-related peptides in conjunction with a decrease in secretory rate: (1) increase the rate of intracellular degradation, (2) block the processing pathway, or (3) decrease the rate of synthesis.

Presence of a pre-sequence (signal sequence) in the common precursor to ACTH and endorphin and the role of glycosylation in processing of the precursor and secretion of ACTH and endorphin.

Adrenocorticotropin, /3-endorphin, and aand /3-melanocyte-stimulating hormones are peptides with markedly different biological activities yet they are synthesized from a common precursor protein in mouse pituitary tumor cells- and in the anterior and intermediate lobes of the Although the two lobes start with similar forms of the precursor, they process these forms to different end produ c t ~ . ~ The release of these hormones is also regulated differently in the two lobes of the pituitary. In order to understand how the levels of these hormones are regulated in the blood, it is necessary to understand how their production is regulated in each lobe of the pituitary. To accomplish this, we have used the AtT-U)/D,,. mouse pituitary tumor cell line as a model system for studying the mechanisms of expression of the gene for the common precursor in the pituitary because this cell line closely mimics the behavior of anterior pituitary cells in culture6. and is very favorable material for biochemical studies. We have attempted to define the steps involved in synthesis and post-translational processing of the precursor protein in AtT-U) cells and then apply this knowledge to similar studies with monolayer cultures of anterior and intermediate lobe cells of mouse and rat pituitary. In this paper we will describe some of the studies we have done on the early events in processing of the precursor in AtT-20 culture systems and briefly summarize results obtained with cultures of normal pituitary cells.?

Evidence that AVP receptors in AtT-20 corticotrophs are not coupled to secretion of POMC-derived peptides

This study reports the presence in AtT-20 corticotrophs of high affinity-low capacity receptors for arginine-vasopressin (AVP), whose binding capacity was considerably enhanced by the divalent metal ion nickel. These binding sites, when analyzed in the presence of nickel, showed high affinity for AVP, vasotocin and oxytocin, but recognized to a lesser extent the V2-agonist 1-deamino-AVP, as well as V1-antagonists. Surprisingly, AVP failed to alter secretion of proopiomelanocortin (POMC)-derived peptides from the cells or corticotropin-releasing factor (CRF)-induced cAMP synthesis, as reported in normal corticotrophs. Exposure of cells to CRF elicited an increase in mRNAPOMC levels, while, in contrast, AVP was without significant effect. It thus appears that in AtT-20 tumor cells, the AVP receptors are not coupled to either the biochemical or biological cellular response.

Functional Assessment of Intrahypothalamic Implants of Immortalized Gonadotropin-Releasing Hormone-Secreting Cells in Female Hypogonadal Mice

The hypogonadal (HPG) mouse is a mutant that lacks a functional gonadotropin-releasing hormone (GnRH) gene. In this study, female HPG mice received bilateral intrahypothalamic implants of an immortalized GnRH-secreting cell line (GT1-7). Nine mice were tested 42-65 days after implantation to determine whether these cells could support spontaneous and/or N-methyl-D, L,-aspartic acid (NMDA)-stimulated luteinizing hormone (LH) secretion. When sampled via intravenous catheters, four mice had measurable LH secretion. Three of these mice responded to NMDA challenges with significant increases in circulating LH. GnRH immunocytochemistry revealed that GT1-7 cells were present in these four mice and three others in which LH values were not detectable. There were about 1200 GnRH cells dispersed within the piriform cortex and olfactory tubercle, and no tumor found in one of the HPG mice that responded to NMDA, whereas the other NMDA responders had large bilateral hypothalamic tumors. The presence or absence of such tumors did not predict the capacity to respond to the NMDA challenge with alterations in LH secretion. This study provides the first evidence that intrahypothalamic GT1-7 cells can support LH release in the HPG mouse, and that this secretion can be modified by pharmacological agents.

The Regulation of Proopiomelanocortin Gene Expression by Estrogen in the Rat Hypothalamus

Estrogen is known to the decrease the level of β-endorphin, a pro- opiomelanocortin (POMC) derived peptide, in the rat hypothalamus. We have used a POMC cDNA probe to measure the levels of POMC mRNA in the arcuate region of the brain in ovariectomized female rats with and without estrogen replacement. Our results show that estrogen causes a time dependent decrease in POMC mRNA of approximately 40% in rats ovariectomized for 2 wk. Preliminary studies using a nuclear transcription system have indicated that estrogen may decrease POMC mRNA levels by having an inhibitory effect on transcription of the POMC gene. It is not clear from these results, however, whether estrogen is acting directly on POMC neurons, or whether it is producing it inhibitory effect through an interneuron system.

Does the kallikrein-like enzyme gene family code for a group of peptide hormone-processing enzymes?

The cloning of genes which code for a family of kallikrein-like enzymes may provide a basis for understanding the substrate specificity of peptide-processing enzymes.

Peptide hormone gene expression in heterogeneous tissues The pro-opiomelanocortin system

Abstract The pro-opiomelanocortin (POMC) gene which codes for a polyhormone protein is regulated differentially in the lobes of the pituitary. The expression of the POMC gene can be modulated at the level of transcription, processing of heteronuclear RNA, and stabilization of messenger RNA. With recombinant DNA technology and the newly developed in-situ hybridization histochemistry, this system can be used advantageously in the study of peptide hormone gene regulation. Data obtained thus far point out two different types of regulation: gene activation or inactivation in corticotrophs and/or the change in number of POMC-producing cells. This paper will focus on the new approaches which may enable one to distinguish between the regulation of gene expression and the differentiation of cells due to hormonal induction.

USE OF IN SITU HYBRIDIZATION HISTOCHEMISTRY TO ANALYZE GENE TRANSCRIPTION IN INDIVIDUAL CEllS

Recent evidence from a variety of laboratories has shown that there is a coupling between the physiological stimulation of release of a peptide hormone and transcription of the gene encoding that peptide hormone. Although the exact mechanism of this coupling is unclear, it is believed that a product of the metabolic cascade elicited upon activation of plasma membrane receptors by catecholamines or peptide hormones is subsequently able to directly influence the rates of transcription of specific genes within the nucleus, including the genes encoding secreted proteins. We have studied the rat pituitary proopiomelanocortin (POMC) gene, which encodes the precursor protein to the pituitary peptide hormones ACTH, beta-endorphin, and melanotropin (1). Although the same POMC gene is expressed in both the intermediate and anterior lobes of the rat pituitary, a large body of evidence has now demonstrated that POMC peptide secretion and gene expression is regulated by different factors in these two distinct cell types. For example, hypothalamic corticotropin releasing hormone (CRH) positively regulates POMC secretion and gene transcription in anterior lobe corticotrophs, but is much less effective in regulating intermediate lobe melanotrophs (2–4). Glucocorticoids, whose production from the adrenal cortex is stimulated by ACTH, inhibit POMC transcription and secretion in the corticotroph, but have no effect on the melanotroph (5, 6).